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Michal Mauda-Havakuk,1 Andrew S Mikhail,1 Matthew F Starost,2 Elizabeth C Jones,3 Baktiar Karim,4 David E Kleiner,5 Ari Partanen,1 Juan A Esparza-Trujillo,1 Ivane Bakhutashvili,1 Paul G Wakim,6 Michael T Kassin,1 Andrew L Lewis,7 John W Karanian,1 Bradford J Wood,8 William F Pritchard1 1Center for Interventional Oncology, Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, Bethesda, MD, USA; 2Division of Veterinary Resources, National Institutes of Health, Bethesda, MD, USA; 3Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, Bethesda, MD, USA; 4National Cancer Institute, National Institutes of Health, Frederick, MD, USA; 5Center for Cancer Research, Clinical Center, National Cancer Institute, Bethesda, MD, USA; 6Biostatistics and Clinical Epidemiology Service, National Institutes of Health Clinical Center, Bethesda, MD, USA; 7Biocompatibles UK Ltd (a BTG International Group Company), Camberley, UK; 8Center for Interventional Oncology, Radiology and Imaging Sciences, Clinical Center, National Institute of Biomedical Imaging and Bioengineering and National Cancer Institute Center for Cancer Research, National Institutes of Health, Bethesda, MD, USACorrespondence: William F PritchardCenter for Interventional Oncology, Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 10 Center Drive, Room 3N320B, MSC 1182, Bethesda, MD, 20892, USATel +1 240-760-0153Fax +1 301-496-9933Email william.pritchard@nih.govBackground: Woodchucks chronically infected with woodchuck hepatitis virus (WHV), which resembles human hepatitis B virus, develop spontaneous hepatic tumors and may be an important biological and immunological model for human HCC. Nonetheless, this model requires further validation to fully realize its translational potential.Methods: Woodchucks infected at birth with WHV that had developed HCC (n=12) were studied. Computed tomography, ultrasound, and magnetic resonance imaging were performed under anesthesia. LI-RADS scoring and correlative histologic analysis of sectioned tissues were performed. For immune characterization of tumors, CD3 (T cells), CD4 (T helpers), NCAM (Natural killers), FOXP3 (T-regulatory), PDL-1 (inhibitory checkpoint protein), and the human hepatocellular carcinoma (HCC) biomarker alpha-fetoprotein (AFP) immunohistochemical stains were performed.Results: Forty tumors were identified on imaging of which 29 were confirmed to be HCC with 26 categorized as LR-4 or 5. The remainder of the tumors had benign histology including basophilic foci, adenoma, and lipidosis as well as pre-malignant dysplastic foci. LR-4 and LR-5 lesions showed high sensitivity (90%) and specificity (100%) for malignant and pre-malignant tumors. Natural killers count was found to be 2– 5 times lower in tumors relative to normal parenchyma while other immune cells were located in the periphery of tumors. Tumors expressed AFP and did not express PD-L1.Conclusion: Woodchucks chronically infected with WHV developed diverse hepatic tumor types with diagnostic imaging, pathology, and immune patterns comparable to that in humans. This unique animal model may provide a valuable tool for translation and validation of novel image-guided and immune-therapeutic investigations.Keywords: carcinoma, hepatocellular, radiology, interventional oncology, immunotherapy, models, animal, tumor microenvironment

Anastomotic leaks are a serious complication associated with Ivor Lewis esophagectomies. Endoluminal negative pressure vacuum devices create a possible treatment alternative to conventional surgical intervention. Ten pigs had an intrathoracic esophageal anastomosis with a 1-cm defect. The experimental group had the device placed intraoperatively across the defect, whereas the control group did not. Once treatment was completed, a contrast fluoroscopic study and necropsy was performed. All control pigs had contrast extravasation on fluoroscopy and contamination on necropsy. The experimental group had no radiologic leak and no contamination on necropsy. The P value for leak is 0.03. This study demonstrated that endoluminal negative pressure vacuum therapy is tolerated in the swine model and is successful in facilitating the healing of anastomotic leaks. Endoluminal negative pressure vacuum therapy has potential clinical benefits, including decreased morbidity and length of hospital stay. [ABSTRACT FROM AUTHOR]

Background: Flavocoxid is a prescription medical food that is used to treat osteoarthritis. It is a proprietary blend of 2 flavonoids, baicalin and catechins, which are derived from the botanicals Scutellaria baicalensis and Acacia catechu, respectively.Objective: To describe characteristics of patients with acute liver injury suspected of being caused by flavocoxid.Design: Case series.Setting: Drug-Induced Liver Injury Network Prospective Study ongoing at multiple academic medical centers since 2004.Patients: Four adults with liver injury.Measurements: Clinical characteristics, liver biochemistry values, and outcomes.Results: Among 877 patients enrolled in the prospective study, 4 had liver injury suspected to have been caused by flavocoxid. All were women; ages ranged from 57 to 68 years. All developed symptoms and signs of liver injury within 1 to 3 months after initiating flavocoxid. Liver injury was characterized by marked elevations in levels of alanine aminotransferase (mean peak, 1268 U/L; range, 741 to 1540 U/L), alkaline phosphatase (mean peak, 510 U/L; range, 286 to 770 U/L), and serum bilirubin (mean peak, 160.7 µmol/L [9.4 mg/dL]; range, 34.2 to 356 µmol/L [2.0 to 20.8 mg/dL]). Liver biochemistry values decreased to the normal range within 3 to 12 weeks after flavocoxid was stopped, and all patients recovered without experiencing acute liver failure or chronic liver injury. Causality was adjudicated as highly likely in 3 patients and as possible in 1 patient.Limitation: The frequency and mechanism of liver injury could not be assessed.Conclusion: Flavocoxid can cause clinically significant liver injury, which seems to resolve within weeks after cessation. [ABSTRACT FROM AUTHOR]

Objective: To evaluate ribavirin, an oral antiviral agent, as therapy for chronic hepatitis C.Design: Randomized, double-blind, placebo-controlled study.Setting: Clinical Center of the National Institutes of Health, a tertiary referral research hospital.Patients: 29 patients with chronic hepatitis C who received oral ribavirin (600 mg twice daily) for 12 months and 29 controls with chronic hepatitis C who received placebo for 12 months.Measurements: Effects of therapy were evaluated by measuring serum aminotransferase and hepatitis C virus (HCV) RNA levels before, during, and for 6 months after therapy and by histologic examination of liver specimens before and at the end of treatment.Results: Patients treated with ribavirin had a prompt decrease in serum aminotransferase levels (54% overall) compared with levels before treatment and levels in controls (5% decrease). Serum aminotransferase levels became normal or nearly normal in 10 patients treated with ribavirin (35% [95% CI, 18% to 54%]) but in no controls (0% [CI, 0% to 12%]). Aminotransferase levels remained normal in only 2 patients after ribavirin therapy was discontinued (7% [CI, 1% to 23%]). Serum HCV RNA levels did not change during or after therapy. Liver biopsy specimens showed a decrease in hepatic inflammation and necrosis among ribavirin-treated patients whose aminotransferase levels became normal.Conclusions: Ribavirin has beneficial effects on serum aminotransferase levels and histologic findings in the liver in patients with chronic hepatitis C, but these effects are not accompanied by changes in HCV RNA levels and are not sustained when ribavirin therapy is discontinued. Thus, ribavirin alone for periods as long as 12 months is unlikely to be of value as therapy for chronic hepatitis C. [ABSTRACT FROM AUTHOR]

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is common in people with HIV (PWH). The morphological spectrum of MASLD compared to matched controls and of the correlation between the NAFLD activity score (NAS) and fibrosis stage in PWH remains unknown., Methods: Overall, 107 liver biopsies from PWH with MASLD (MASLD-PWH) were matched to 107 biopsies from individuals with MASLD and without HIV (MASLD controls) on age at biopsy, race/ethnicity, sex, type 2 diabetes, body mass index (BMI) and alanine aminotransferase (ALT) level. Biopsies were scored using NAS., Results: Compared to MASLD-controls, MASLD-PWH had lower steatosis grade (OR: 0.65, 95% CI: (0.47-0.90), p = 0.01), lower lobular inflammation grade (OR: 0.55, 95% CI: (0.34-0.89), p = 0.02), less portal inflammation (OR: 0.42, 95% CI: (0.25-0.72), p = 0.002) and less ballooned hepatocytes (OR: 0.60, 95% CI: (0.41-0.88), p = 0.01). Thus, NAS was lower in MASLD-PWH (OR: 0.69, 95% CI: (0.56-0.85), p < 0.001) than in MASLD controls. There was a trend towards lower prevalence of steatohepatitis in MASLD-PWH (OR: 0.84, 95% CI: (0.68-1.03), p = 0.09). A multivariate analysis demonstrated that MASLD-PWH cases had significantly less steatosis (OR: 0.66, p = 0.03), portal inflammation (OR: 0.34, p = 0.001) and ballooned hepatocytes (OR: 0.55, p = 0.01), yet higher stage fibrosis (OR: 1.42, p = 0.03) compared to MASLD controls., Conclusion: The NAS and histological drivers of fibrosis (e.g. inflammation and hepatocyte ballooning) are less pronounced in MASLD-PWH, and yet fibrosis stage was generally higher when compared to matched controls with MASLD without HIV. This suggests HIV-specific factors beyond hepatic necroinflammation may contribute to fibrosis progression in MASLD-PWH., (© 2024 The Author(s). Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Liver biopsies have consistently contributed to our understanding of the pathogenesis and aetiologies of acute liver disease. As other diagnostic modalities have been developed and refined, the role of biopsy in the management of patients with acute liver failure (ALF), acute-on-chronic liver failure (ACLF) and acute hepatitis, including acute liver injury (ALI), has changed. Liver biopsy remains particularly valuable when first-line diagnostic algorithms fail to determine aetiology. Despite not being identified as a mandatory diagnostic tool in recent clinical guidelines for the management of ALF or ACLF, many centres continue to undertake biopsies given the relative safety of transjugular biopsy in this setting. Several studies have demonstrated that liver biopsy can provide prognostic information, particularly in the context of so-called indeterminate hepatitis, and is extremely useful in excluding conditions such as metastatic tumours that would preclude transplantation. In addition, its widespread use of percutaneous biopsies in cases of less severe acute liver injury, for example in the establishment of a diagnosis of acute presentation of autoimmune hepatitis or confirmation of a probable or definite drug-induced liver injury (DILI), has meant that many centres have seen a shift in the ratio of specimens they are receiving from patients with chronic to acute liver disease. Histopathologists therefore need to be equipped to deal with these challenging specimens. This overview provides an insight into the contemporary role of biopsies (as well as explant and autopsy material) in diagnosing acute liver disease. It outlines up-to-date clinical definitions of liver injury and considers recent recommendations for the diagnosis of AIH and drug-induced, autoimmune-like hepatitis (DI-AIH)., (© 2024 The Author(s). Histopathology published by John Wiley & Sons Ltd.)

Background & Aims: The clinical significance of change in liver stiffness measurement (LSM) by vibration-controlled transient elastography (VCTE) in patients with non-alcoholic fatty liver disease (NAFLD) is not well-understood. We prospectively defined rates of progression to and regression from LSM-defined compensated advanced chronic liver disease (cACLD) and their associations with liver-related events (LREs)., Methods: Participants in the NASH Clinical Research Network-led NAFLD Database 2 and 3 studies were included. Progression to cACLD was defined as reaching LSM ≥10 kPa in participants with LSM <10 kPa on initial VCTE; regression from cACLD was defined as reaching LSM <10 kPa in participants with baseline LSM ≥10 kPa. LREs were defined as liver-related death, liver transplant, hepatocellular carcinoma, MELD >15, development of varices, or hepatic decompensation. Univariate and multivariable interval-censored Cox regression analyses were used to compare the cumulative LRE probability by LSM progression and regression status., Results: In 1,403 participants, 89 LREs developed over a mean follow-up of 4.4 years, with an annual incidence rate for LREs of 1.5 (95% CI 1.2-1.8). In participants at risk, progression to LSM ≥10 or ≥15 kPa occurred in 29% and 17%, respectively, whereas regression to LSM <10 or <15 kPa occurred in 44% and 49%, respectively. Progressors to cACLD (≥10 kPa) experienced a higher cumulative LRE rate vs. non-progressors (16% vs. 4%, adjusted hazard ratio 4.0; 95% (1.8-8.9); p <0.01). Regressors from cACLD (to LSM <10 kPa) experienced a lower LRE rate than non-regressors (7% vs. 32%, adjusted hazard ratio 0.25; 95% CI 0.10-0.61; p <0.01)., Conclusions: Change in LSM over time is independently and bi-directionally associated with risk of LRE and is a non-invasive surrogate for clinical outcomes in patients with NAFLD., Impact and Implications: The prognostic value of change in LSM in patients with NAFLD is not well understood. In this large prospective study of patients with NAFLD and serial vibration-controlled transient elastography exams, baseline and dynamic changes in LSM were associated with the risk of developing liver-related events. LSM is a useful non-invasive surrogate of clinical outcomes in patients with NAFLD., (Copyright © 2024 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Liver involvement is an increasingly recognised complication of common variable immunodeficiency (CVID). Nodular regenerative hyperplasia (NRH), a subgroup of porto-sinusoidal vascular disorder, and manifestations of portal hypertension (PH) unrelated to cirrhosis are the most common findings. Nonetheless, the evolution of liver disease over time remains unknown., Methods: Retrospective review of patients followed at the National Institutes of Health with CVID-related liver disease and liver biopsy from 1990 to 2020. Clinical, imaging and histological follow-up were recorded as part of clinical research protocols., Results: Forty patients were included, with a median age of 37.5 years at initial biopsy, 73% presenting with clear evidence of NRH, and a median fibrosis stage of 1. At biopsy, median platelet count was 100 × 10 9 /L, spleen size 19.5 cm, hepatic venous pressure gradient 9.5 mmHg and 37.5% of patients had signs of PH. Cumulative incidence of PH was 65% at 5 years. In a subgroup of 16 patients, a follow-up liver biopsy, performed at a median time of 3 years after the index biopsy, revealed an increase in fibrosis by ≥2 stages in 31% of cases and an increase to an overall stage of 2.2 (p = 0.001). No clinical or histological factors were associated with progression of fibrosis., Conclusions: In this CVID cohort, NRH is the most common initial histological finding; however, unexpectedly fibrosis progresses over time in a subgroup of patients. A better understanding of the underlying causal process of liver disease CVID might lead to improved outcomes., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

Drug-induced liver injury (DILI) presents unique challenges in clinical practice. While some types of DILI are mild and resolve quickly after removing the drug, other situations are more complex, with competing aetiologies or underlying liver disease. Guidelines from professional societies agree that the liver biopsy retains a role in understanding and managing DILI in certain situations. Liver biopsy allows characterization of the histological pattern of injury as well as assessment of severity. Inflammatory infiltrates, bile duct injury or loss and vascular injury are all revealed by liver biopsy. Communication between the hepatopathologist and clinical team with clinicopathological correlation of the findings is necessary for the best determination of causality and differentiation from other diseases of exclusion, like autoimmune hepatitis and graft-versus-host disease. This review highlights important aspects of the role of liver biopsy in DILI evaluation., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Liver International published by John Wiley & Sons Ltd.)

Background and Aims: Metabolic dysfunction-associated steatohepatitis (MASH) with bridging fibrosis is a critical stage in the evolution of fatty liver disease. Second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) microscopy with artificial intelligence (AI) provides sensitive and reproducible quantitation of liver fibrosis. This methodology was applied to gain an in-depth understanding of intra-stage fibrosis changes and septa analyses in a homogenous, well-characterised group with MASH F3 fibrosis., Methods: Paired liver biopsies (baseline [BL] and end of treatment [EOT]) of 57 patients (placebo, n = 17 and tropifexor n = 40), with F3 fibrosis stage at BL according to the clinical research network (CRN) scoring, were included. Unstained sections were examined using SHG/TPEF microscopy with AI. Changes in liver fibrosis overall and in five areas of liver lobules were quantitatively assessed by qFibrosis. Progressive, regressive septa, and 12 septa parameters were quantitatively analysed., Results: qFibrosis demonstrated fibrosis progression or regression in 14/17 (82%) patients receiving placebo, while the CRN scoring categorised 11/17 (65%) as 'no change'. Radar maps with qFibrosis readouts visualised quantitative fibrosis dynamics in different areas of liver lobules even in cases categorised as 'No Change'. Measurement of septa parameters objectively differentiated regressive and progressive septa (p < .001). Quantitative changes in individual septa parameters (BL to EOT) were observed both in the 'no change' and the 'regression' subgroups, as defined by the CRN scoring., Conclusion: SHG/TPEF microscopy with AI provides greater granularity and precision in assessing fibrosis dynamics in patients with bridging fibrosis, thus advancing knowledge development of fibrosis evolution in natural history and in clinical trials., (© 2024 The Author(s). Liver International published by John Wiley & Sons Ltd.)

Background: Azithromycin (AZ) is a widely used antibiotic. The aim of this study was to characterise the clinical features, outcomes, and HLA association in patients with drug-induced liver injury (DILI) due to AZ., Methods: The clinical characteristics of individuals with definite, highly likely, or probable AZ-DILI enrolled in the US Drug-Induced Liver Injury Network (DILIN) were reviewed. HLA typing was performed using an Illumina MiSeq platform. The allele frequency (AF) of AZ-DILI cases was compared to population controls, other DILI cases, and other antibiotic-associated DILI cases., Results: Thirty cases (4 definite, 14 highly likely, 12 probable) of AZ-DILI were enrolled between 2004 and 2022 with a median age of 46 years, 83% white, and 60% female. Median duration of AZ treatment was 5 days. Latency was 18.5 days. 73% were jaundiced at presentation. The injury pattern was hepatocellular in 60%, cholestatic in 27%, and mixed in 3%. Ten cases (33%) were severe or fatal; 90% of these were hepatocellular. Two patients required liver transplantation. One patient with chronic liver disease died of hepatic failure. Chronic liver injury developed in 17%, of which 80% had hepatocellular injury at onset. HLA-DQA1*03:01 was significantly more common in AZ-DILI versus population controls and amoxicillin-clavulanate DILI cases (AF: 0.29 vs. 0.11, p = 0.001 and 0.002, respectively)., Conclusion: Azithromycin therapy can lead to rapid onset of severe hepatic morbidity and mortality in adult and paediatric populations. Hepatocellular injury and younger age were associated with worse outcomes. HLA-DQA1*03:01 was significantly more common in AZ cases compared to controls., (© 2024 The Author(s). Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd.)

Introduction: The aim of this study is to describe the presenting features, genetic factors, and outcomes of 23 adults who developed liver injury after coronavirus disease 2019 (COVID-19) mRNA vaccination., Methods: Patients with suspected COVID-19 vaccine hepatitis were enrolled into the Drug-Induced Liver Injury Network. Causality was assessed using the Drug-Induced Liver Injury Network expert opinion score. High-resolution HLA sequencing was undertaken using Illumina platform., Results: Amongst the 16 high causality cases, median time to onset was 16 days, median age was 63 years, and 75% were female. The injury was hepatocellular in 75% with a median alanine aminotransferase of 497 U/L, and 37% had jaundice. An antinuclear antibody and smooth muscle antibody were detectable in 27% and 36%, but only 12% had an elevated immunoglobulin G level. During follow-up, 37% received a short course of corticosteroids, and 88% fully recovered by 6 months with no deaths observed. HLA alleles associated with autoimmune hepatitis were not overrepresented compared with controls, but an ERAP-2 variant (rs1263907) and the ERAP-1 Hap6 haplotype were significantly overrepresented in the high causality cases vs controls ( P = 0.026 and 5 × 10 -5 , respectively)., Discussion: Acute liver injury may arise within 8 weeks of COVID-19 mRNA vaccination that is generally mild and self-limited in most patients. The absence of an association with the AIH HLA alleles combined with the significant ERAP-2 and ERAP-1 Hap6 haplotype associations implicates a unique but very rare host immune response to vaccine-derived antigens in the pathogenesis of COVID-19 vaccine hepatotoxicity., (Copyright © 2024 by The American College of Gastroenterology.)

Objective: Gene therapy by convection-enhanced delivery of type 2 adeno-associated virus-glial cell derived neurotrophic factor (AAV2-GDNF) to the bilateral putamina seeks to increase GDNF gene expression and treat Parkinson's disease (PD)., Methods: A 63-year-old man with advanced PD received AAV2-GDNF in a clinical trial. He died from pneumonia after anterior cervical discectomy and fusion 45 months later. An autopsy included brain examination for GDNF transgene expression. Putaminal catecholamine concentrations were compared to in vivo 18 F-Fluorodopa ( 18 F-FDOPA) positron emission tomography (PET) scanning results before and 18 months after AAV2-GDNF infusion., Results: Parkinsonian progression stabilized clinically. Postmortem neuropathology confirmed PD. Bilateral putaminal regions previously infused with AAV2-GDNF expressed the GDNF gene. Total putaminal dopamine was 1% of control, confirming the striatal dopaminergic deficiency suggested by baseline 18 F-DOPA-PET scanning. Putaminal regions responded as expected to AAV2-GDNF., Conclusion: After AAV2-GDNF infusion, infused putaminal regions showed increased GDNF gene expression, tyrosine hydroxylase immunoreactive sprouting, catechol levels, and 18 F-FDOPA-PET signal, suggesting the regenerative potential of AAV2-GDNF in PD., (© 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Background and Aims: There are limited data on the progression of liver stiffness measurement (LSM) by vibration-controlled transient elastography (VCTE) in people with type 2 diabetes mellitus (T2DM) versus those without T2DM in biopsy-proven metabolic dysfunction-associated steatotic liver disease. We examined LSM progression in participants with T2DM versus those without T2DM in a large, prospective, multicenter cohort study., Approach and Results: This study included 1231 adult participants (62% female) with biopsy-proven metabolic dysfunction-associated steatotic liver disease who had VCTEs at least 1 year apart. LSM progression and regression were defined by a ≥20% increase and an upward or downward change, respectively, in the LSM category in the Baveno VII categories for compensated advanced chronic liver disease, compared between participants with T2DM (n = 680) versus no T2DM (n = 551) at baseline. The mean (±SD) age and body mass index were 51.8 (±12.0) years and 34.0 (±6.5) kg/m 2 , respectively. The median (IQR) time between the first and last VCTE measurements was 4.1 (2.5-6.5) years. Participants with T2DM had higher LSM progression at 4 years (12% vs. 10%), 6 years (23% vs. 16%), and 8 years (50% vs. 39%), p = 0.04. Using a multivariable Cox proportional hazards model adjusted for multiple confounders, the presence of T2DM remained an independent predictor of LSM progression (adjusted HR: 1.35, 95% CI: 1.01-1.81, p = 0.04). T2DM was not associated with LSM regression ( p = 0.71). Mean HbA1c was significantly associated with LSM progression ( p = 0.003) and regression ( p = 0.02)., Conclusions: Using serial VCTE data from a multicenter study of participants with biopsy-proven metabolic dysfunction-associated steatotic liver disease, we demonstrate that T2DM and HbA1c are associated with LSM progression., (Copyright © 2024 American Association for the Study of Liver Diseases.)

Objectives: Inflammatory cytokines that signal through the Janus kinases-signal transducer and activator of transcription (JAK-STAT) pathway, especially interferons (IFNs), are implicated in Sjögren's disease (SjD). Although inhibition of JAKs is effective in other autoimmune diseases, a systematic investigation of IFN-JAK-STAT signalling and the effect of JAK inhibitor (JAKi) therapy in SjD-affected human tissues has not been fully investigated., Methods: Human minor salivary glands (MSGs) and peripheral blood mononuclear cells (PBMCs) were investigated using bulk or single-cell (sc) RNA sequencing (RNAseq), immunofluorescence (IF) microscopy and flow cytometry. Ex vivo culture assays on PBMCs and primary salivary gland epithelial cell (pSGEC) lines were performed to model changes in target tissues before and after JAKi., Results: RNAseq and IF showed activated JAK-STAT pathway in SjD MSGs. Elevated IFN-stimulated gene (ISGs) expression associated with clinical variables (eg, focus scores, anti-SSA positivity). scRNAseq of MSGs exhibited cell type-specific upregulation of JAK-STAT and ISGs; PBMCs showed similar trends, including markedly upregulated ISGs in monocytes. Ex vivo studies showed elevated basal pSTAT levels in SjD MSGs and PBMCs that were corrected with JAKi. SjD-derived pSGECs exhibited higher basal ISG expressions and exaggerated responses to IFN-β, which were normalised by JAKi without cytotoxicity., Conclusions: SjD patients' tissues exhibit increased expression of ISGs and activation of the JAK-STAT pathway in a cell type-dependent manner. JAKi normalises this aberrant signalling at the tissue level and in PBMCs, suggesting a putative viable therapy for SjD, targeting both glandular and extraglandular symptoms. Predicated on these data, a phase Ib/IIa randomised controlled trial to treat SjD with tofacitinib was initiated., Competing Interests: Competing interests: BMW has Cooperative Research Award and Development Agreements (CRADA) from Pfizer and Mitobridge (a subsidiary of Astellas Pharma). NIAMS has CRADAs with AstraZeneca and Bristol Myers Squibb. These CRADA did not financially support the experimental results presented herein., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Sjögren's Disease (SjD) is a systemic autoimmune disease without a clear etiology or effective therapy. Utilizing unbiased single-cell and spatial transcriptomics to analyze human minor salivary glands in health and disease we developed a comprehensive understanding of the cellular landscape of healthy salivary glands and how that landscape changes in SjD patients. We identified novel seromucous acinar cell types and identified a population of PRR4+CST3+WFDC2 - seromucous acinar cells that are particularly targeted in SjD. Notably, GZMK +CD8 T cells, enriched in SjD, exhibited a cytotoxic phenotype and were physically associated with immune-engaged epithelial cells in disease. These findings shed light on the immune response's impact on transitioning acinar cells with high levels of secretion and explain the loss of this specific cell population in SjD. This study explores the complex interplay of varied cell types in the salivary glands and their role in the pathology of Sjögren's Disease.

Background: Bile acids mediate gut-liver cross-talk through bile acid receptors. Serum, hepatic, and microbial bile acid metabolism was evaluated in HCV-compensated chronic liver disease., Methods: Patients underwent liver biopsy; portal and peripheral blood were obtained before (HCVi), and 6 months after sustained virologic response (SVR), splenic blood was obtained only after SVR. The fecal microbiome and liver transcriptome were evaluated using RNA-Seq. Twenty-four bile acids were measured in serum, summed as free, taurine-conjugated bile acids (Tau-BAs), and glycine-conjugated bile acids., Results: Compared to SVR, HCVi showed elevated conjugated bile acids, predominantly Tau-BA, compounded in HCVi cirrhosis. In the liver, transcription of bile acids uptake, synthesis, and conjugation was decreased with increased hepatic spillover into systemic circulation in HCVi. There was no difference in the transcription of microbial bile acid metabolizing genes in HCVi. Despite an overall decrease, Tau-BA remained elevated in SVR cirrhosis, mainly in splenic circulation. Only conjugated bile acids, predominantly Tau-BA, correlated with serum proinflammatory markers and hepatic proinflammatory pathways, including NLRP3 and NFKB. Among hepatic bile acid receptors, disease-associated conjugated bile acids showed the strongest association with hepatic spingosine-1-phosphate receptor 2 (S1PR2)., Conclusions: Enhanced expression of hepatic S1PR2 in HCVi and HCVi-cirrhosis and strong associations of S1PR2 with Tau-BAs suggest pathological relevance of Tau-BA-hepatic S1PR2 signaling in chronic liver disease. These findings have therapeutic implications in chronic liver diseases.

Background: Thymoma presents with several autoimmune manifestations and is associated with secondary autoimmune regulator (AIRE) deficiency. Pneumonitis has recently been described as an autoimmune manifestation associated with thymoma presenting with similar clinical, radiographic, histological, and autoantibody features as seen in patients with inherited AIRE deficiency who suffer from Autoimmune PolyEndocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED) syndrome., Objectives: To treat two patients with biopsy-proven thymoma-associated pneumonitis with lymphocyte-directed immunomodulation., Methods: Two patients with thymoma were enrolled on IRB-approved protocols at the NIH Clinical Center. We performed history and physical examination; laboratory, radiographic, histologic and pulmonary function evaluations; and measurement of the lung-directed autoantibodies KCNRG and BPIFB1 prior to and at 1- and 6-months following initiation of lymphocyte-directed immunomodulation with azathioprine with or without rituximab., Results: Combination T- and B-lymphocyte-directed immunomodulation resulted in improvement of clinical, functional, and radiographic parameters at 6-month follow-up evaluations in both patients with sustained remission up to 12-36 months following treatment initiation., Conclusion: Lymphocyte-directed immunomodulation remitted autoimmune pneumonitis in two patients with thymoma., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Histological subtyping of hepatocellular carcinoma (HCC) is challenging in the presence of histological heterogeneity, where distinctly different morphological patterns are present within the same tumor. Current approaches rely on percent cut-offs. We hypothesized that morphologic intratumor heterogeneity is a non-random biological feature and that incorporating recurrent patterns would improve histological subtyping of HCC. Resected HCC were studied and the overall frequency of morphologic intratumor heterogeneity was 45% in 242 specimens. Steatohepatitic HCC (SH-HCC) had the highest frequency of morphologic intratumor heterogeneity (91%); this was confirmed in additional cohorts of SH-HCC from different medical centers (overall frequency of 78% in SH-HCC). Morphologic intratumor heterogeneity in SH-HCC showed distinct and recurrent patterns that could be classified as early, intermediate, and advanced. Incorporating these patterns into the definition of SH-HCC allowed successful resolution of several persistent challenges: the problem of the best cut-off for subtyping SH-HCC, the problem of the relationship between SH-HCC and scirrhous HCC, and the classification for HCC with abundant microvesicular steatosis. This approach also clarified the relationship between SH-HCC and CTNNB1 mutations, showing that CTNNB1 mutations occur late in a subset of SH-HCC. In summary, there is a high frequency of morphologic intratumor heterogeneity in HCC. Incorporating this finding into histological subtyping resolved several persistent problems with the SH-HCC subtype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Identifying cell types and states remains a time-consuming, error-prone challenge for spatial biology. While deep learning is increasingly used, it is difficult to generalize due to variability at the level of cells, neighborhoods, and niches in health and disease. To address this, we developed TACIT, an unsupervised algorithm for cell annotation using predefined signatures that operates without training data. TACIT uses unbiased thresholding to distinguish positive cells from background, focusing on relevant markers to identify ambiguous cells in multiomic assays. Using five datasets (5,000,000-cells; 51-cell types) from three niches (brain, intestine, gland), TACIT outperformed existing unsupervised methods in accuracy and scalability. Integrating TACIT-identified cell types with a novel Shiny app revealed new phenotypes in two inflammatory gland diseases. Finally, using combined spatial transcriptomics and proteomics, we discovered under- and overrepresented immune cell types and states in regions of interest, suggesting multimodality is essential for translating spatial biology to clinical applications., Competing Interests: Conflict of interest: The authors had access to the study data and reviewed and approved the final manuscript. Although the authors view each of these as noncompeting financial interests, KMB, QTE, BFM, and BMW are all active members of the Human Cell Atlas; furthermore, KMB is a scientific advisor at Arcato Laboratories. All other authors declare no competing interests.

Saliva contains antimicrobial peptides considered integral components of host innate immunity, and crucial for protection against colonizing microbial species. Most notable is histatin-5 which is exclusively produced in salivary glands with uniquely potent antifungal activity against the opportunistic pathogen Candida albicans . Recently, SARS-CoV-2 was shown to replicate in salivary gland acinar cells eliciting local immune cell activation. In this study, we performed mechanistic and clinical studies to investigate the implications of SARS-CoV-2 infection on salivary histatin-5 production and Candida colonization. Bulk RNA-sequencing of parotid salivary glands from COVID-19 autopsies demonstrated statistically significant decreased expression of histatin genes. In situ hybridization, coupled with immunofluorescence for co-localization of SARS-CoV-2 spike and histatin in salivary gland cells, showed that histatin was absent or minimally present in acinar cells with replicating viruses. To investigate the clinical implications of these findings, salivary histatin-5 levels and oral Candida burden in saliva samples from three independent cohorts of mild and severe COVID-19 patients and matched healthy controls were evaluated. Results revealed significantly reduced histatin-5 in SARS-CoV-2 infected subjects, concomitant with enhanced prevalence of C. albicans . Analysis of prospectively recovered samples indicated that the decrease in histatin-5 is likely reversible in mild-moderate disease as concentrations tended to increase during the post-acute phase. Importantly, salivary cytokine profiling demonstrated correlations between activation of the Th17 inflammatory pathway, changes in histatin-5 concentrations, and subsequent clearance of C. albicans in a heavily colonized subject. The importance of salivary histatin-5 in controlling the proliferation of C. albicans was demonstrated using an ex vivo assay where C. albicans was able to proliferate in COVID-19 saliva with low histatin-5, but not with high histatin-5. Taken together, the findings from this study provide direct evidence implicating SARS-CoV-2 infection of salivary glands with compromised oral innate immunity, and potential predisposition to oral candidiasis.

Objective: To describe patients with NSAID-DILI, including genetic factors associated with idiosyncratic DILI., Methods: In DILIN, subjects with presumed DILI are enrolled and followed for at least 6 months. Causality is adjudicated by a Delphic approach. HLA sequencing of multiethnic NSAID-DILI patients and HLA allele imputation of matching population controls were performed following overall, class and drug-based association analysis. Significant results were tested in a non-Hispanic White (NHW) case-control replication cohort., Results: Between September 2004 and March 2022, causality was adjudicated in 2498, and 55 (41 [75%] women) were assessed as likely due to NSAIDs. Median age at onset was 55 y (range 22-83 y). Diclofenac was the causative drug in 29, celecoxib in 7, ibuprofen in 5, etodolac and meloxicam each in 4. Except for meloxicam and oxaprozin (n = 2), the liver injury was hepatocellular with median R 15-25. HLA-DRB1*04:03 and HLA-B*35:03 were significantly more frequent in NSAID-DILI patients than in non-NSAID DILI controls. Interestingly, 85% of the HLA-DRB1*04:03 carriers developed DILI due to the use of acetic acid derivative NSAIDs, supporting the hypothesis that HLA-DRB1*04:03 could be a drug and/or class risk factor. HLA-B*35:03 but not HLA-DRB1*04:03 association was confirmed in the independent NHW replication cohort, which was largely driven by diclofenac., Conclusions: Despite prevalent use, NSAID-DILI is infrequent in the United States. Diclofenac is the most commonly implicated, and adherence to warnings of risk and close observation are recommended. The increased frequency of HLA-B*35:03 and DRB1*04:03, driven by diclofenac, suggests the importance of immune-mediated responses., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Non-cirrhotic portal hypertension (NCPH) is a spectrum of liver diseases, including porto-sinusoidal vascular disorder, with portal hypertension (PH) in the absence of cirrhosis. The natural history and diagnostic approach to NCPH are not well understood., Aim: We aimed to evaluate disease progression and outcomes in NCPH., Methods: Patients with or at risk for NCPH were enrolled in a single centre prospective study; two groups were formed based on the presence of specific features of PH, such as varices, collaterals, portal hypertensive gastropathy or portal hypertensive bleeding. All participants underwent a baseline liver biopsy. Liver stiffness measurement (LSM), and imaging were repeated every 6-12 months., Results: Fifteen patients without specific features of PH (Group I), and 35 patients with specific features (Group II) were enrolled. The median follow-up time was 50 months. Group II had higher hepatic venous pressure gradients, non-invasive measures of PH and a lower platelet count (PLT) when compared to Group I. Rates of survival and decompensation were similar in both groups. Patients with PLT ≤100 K/mcL had lower survival compared to those with PLT >100 K/mcL. Patients with LSM ≥10 kPa had lower survival and survival without decompensation when compared to patients with LSM <10 kPa., Conclusions: Patients irrespective of specific features of PH had similar survival or survival without decompensation. Patients without specific features are at risk for disease progression and should be monitored closely. Thrombocytopenia and increased LSM are associated with severe forms of liver disease, which are strongly associated with outcomes., (© 2024 John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Background: Autoimmune polyendocrine syndrome type 1 (APS-1) is a life-threatening, autosomal recessive syndrome caused by autoimmune regulator (AIRE) deficiency. In APS-1, self-reactive T cells escape thymic negative selection, infiltrate organs, and drive autoimmune injury. The effector mechanisms governing T-cell-mediated damage in APS-1 remain poorly understood., Methods: We examined whether APS-1 could be classified as a disease mediated by interferon-γ. We first assessed patients with APS-1 who were participating in a prospective natural history study and evaluated mRNA and protein expression in blood and tissues. We then examined the pathogenic role of interferon-γ using Aire -/- Ifng -/- mice and Aire -/- mice treated with the Janus kinase (JAK) inhibitor ruxolitinib. On the basis of our findings, we used ruxolitinib to treat five patients with APS-1 and assessed clinical, immunologic, histologic, transcriptional, and autoantibody responses., Results: Patients with APS-1 had enhanced interferon-γ responses in blood and in all examined autoimmunity-affected tissues. Aire -/- mice had selectively increased interferon-γ production by T cells and enhanced interferon-γ, phosphorylated signal transducer and activator of transcription 1 (pSTAT1), and CXCL9 signals in multiple organs. Ifng ablation or ruxolitinib-induced JAK-STAT blockade in Aire -/- mice normalized interferon-γ responses and averted T-cell infiltration and damage in organs. Ruxolitinib treatment of five patients with APS-1 led to decreased levels of T-cell-derived interferon-γ, normalized interferon-γ and CXCL9 levels, and remission of alopecia, oral candidiasis, nail dystrophy, gastritis, enteritis, arthritis, Sjögren's-like syndrome, urticaria, and thyroiditis. No serious adverse effects from ruxolitinib were identified in these patients., Conclusions: Our findings indicate that APS-1, which is caused by AIRE deficiency, is characterized by excessive, multiorgan interferon-γ-mediated responses. JAK inhibition with ruxolitinib in five patients showed promising results. (Funded by the National Institute of Allergy and Infectious Diseases and others.)., (Copyright © 2024 Massachusetts Medical Society.)

Context: Leptin replacement therapy with metreleptin improves metabolic abnormalities in patients with generalized lipodystrophy (GLD)., Objective: Determine how timing of metreleptin initiation in the clinical course of GLD affects long-term metabolic health., Methods: Retrospective analysis of patients ≥ 6 months old with congenital (n=47) or acquired (n=16) GLD treated with metreleptin at the National Institutes of Health since 2001. Least squares means (LSM) for HbA1c, insulin area under the curve (AUC) from oral glucose tolerance tests, triglycerides, urine protein excretion, platelets, transaminases, and aspartate aminotransferase (AST) to Platelet Ratio Index (APRI) for early and late treatment groups, defined by baseline metabolic health, were analyzed during median 72 (24, 108) months follow-up., Results: Compared to late groups, early groups based on metabolic status had higher mean±SEM insulin AUC (20831±1 vs 11948±1), lower HbA1c (5.3±0.3 vs 6.8±0.3%), triglycerides (101±1 vs 193±1 mg/dL), urine protein excretion (85±1.5 vs 404±1.4 mg/24 hr), ALT (30±1 vs 53±1 U/L), AST (23±1 vs 40±1 U/L), and APRI (0.22±1.3 vs 0.78±1.3), and higher platelets (257±24 vs 152±28 K/µL) during follow-up (P<0.05). Compared to patients ≥6 years old at baseline, patients <6 years had lower HbA1c (4.5±0.5 vs 6.4±0.2%) and higher AST (40±1vs 23±1 U/L) during follow (P<0.05)., Conclusion: Patients with GLD who initiated metreleptin before the onset of severe metabolic complications had better long-term control of diabetes, proteinuria, and hypertriglyceridemia. Early treatment may also result is less severe progression of liver fibrosis, but further histological studies are needed to determine the effects of metreleptin therapy on liver disease., (Published by Oxford University Press on behalf of the Endocrine Society 2024.)

Background: The long-term effect of bariatric surgery on adolescent non-alcoholic fatty liver disease is not clear., Objectives: To evaluate longitudinal change in serum alanine aminotransferase (ALT) levels and to determine the factors independently associated with this change over 2 years after bariatric surgery in adolescents with severe obesity., Setting: An observational prospective cohort from the Teen-LABS Consortium., Methods: We examined the relationship of longitudinal change in serum ALT (% change and normalization) to change in body mass index (BMI), homeostasis model assessment of insulin resistance (HOMA-IR), triglycerides (TG), high- (HDL) and low-density lipoprotein cholesterol, A1C and fasting glucose, accounting for age, sex, race-ethnicity, blood pressure, and baseline BMI in 219 adolescents during the first 2 years post-surgery., Results: Mean BMI declined from a baseline of 52.6 to 37.2 kg/m 2 at 2 years (P < .01). Alanine aminotransferase decreased significantly from baseline (36.5 [95% CI: 31.4, 41.7]) to 6 months (30.5 [95% CI: 25.4, 35.6]), and remained stable at 12 and 24 months, all P < .01 versus baseline. After adjustment, improvement in BMI, fasting glucose, HOMA-IR, triglycerides, TG/HDL ratio, and HDL were independently associated with reduced ALT at 6 months. These remained significantly associated with a decline in ALT after adjusting for BMI change. The %participants with elevated ALT decreased from 71% at baseline to 42% and 36% at 1 and 2 years post-surgery., Conclusions: Bariatric surgery resulted in significant and sustained improvement in ALT levels over 2 years. Although associated with weight loss, this decline was also associated with improved metabolic indices, independent of weight loss., (Copyright © 2024 American Society for Metabolic and Bariatric Surgery. All rights reserved.)

A granuloma is a discrete collection of activated macrophages and other inflammatory cells. Hepatic granulomas can be a manifestation of localized liver disease or be a part of a systemic process, usually infectious or autoimmune. A liver biopsy is required for the detection and evaluation of granulomatous liver diseases. The prevalence of granulomas on liver biopsy varies from 1% to 15%. They may be an incidental finding in an asymptomatic individual, or they may represent granulomatous hepatitis with potential to progress to liver failure, or in chronic disease, to cirrhosis. This review focuses on pathogenesis, histological features of granulomatous liver diseases, and most common etiologies, knowledge that is essential for timely diagnosis and intervention.

Introduction: Perturbations in aromatic (AAAs) and branched-chain amino acids (BCAAs) are seen in decompensated liver disease. The aim of this study was to evaluate the dynamic, postprandial relationship between hepatitis C virus-induced liver disease and amino acid concentrations in patients with compensated liver disease., Methods: Patients infected with hepatitis C virus underwent a baseline liver biopsy to determine Ishak Fibrosis Score and evaluate the liver transcriptome. Patients ate a standard meal and underwent peripheral vein sampling at defined intervals. Quantitative analysis of amino acids was performed using liquid chromatography-tandem mass spectrometry., Results: At baseline, there was no difference in AAA and BCAA concentrations between patients with cirrhosis and non-cirrhotic patients. After a standard meal, AAAs, but not BCAAs, were elevated in patients with cirrhosis compared with non-cirrhotic patients at every time point. The HepQuant SHUNT fraction was significantly higher in patients with cirrhosis and positively correlated with AAA concentration at all time points, but not BCAA. Analysis of the hepatic transcriptome demonstrated greater downregulation of the AAA degradation pathways than the BCAA degradation pathways., Discussion: At baseline, cirrhotic patients with compensated liver disease have adequate reserve liver function to metabolize AAAs and BCAAs. When faced with a metabolic stressor, such as a standard meal, patients with cirrhosis are less able to metabolize the increased load of AAAs. This impairment correlates with portosystemic shunting. Further evaluation of AAA levels in compensated liver disease might further the understanding of the liver-muscle axis and the role it may play in the development of sarcopenia in liver disease., (Copyright © 2024 Written work prepared by employees of the Federal Government as part of their official duties is, under the U.S. Copyright Act, a “work of the United States Government” for which copyright protection under Title 17 of the United States Code is not available. As such, copyright does not extend to the contributions of employees of the Federal Government.)

Background: Current standard of care for advanced biliary tract cancer (BTC) is gemcitabine, cisplatin plus anti-PD1/PD-L1, but response rates are modest. The purpose of this study was to explore the efficacy and safety of durvalumab (anti-PD-L1) and tremelimumab (anti-CTLA-4), with and without an interventional radiology (IR) procedure in advanced BTC., Methods: Eligible patients with advanced BTC who had received or refused at least one prior line of systemic therapy were treated with tremelimumab and durvalumab for four combined doses followed by monthly durvalumab alone with and without an IR procedure until the progression of disease or unacceptable toxicity. Objective response was assessed through CT or MRI by Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1) every 8 weeks. Adverse events (AEs) were recorded and managed. The primary endpoint was 6-month progression-free survival (PFS)., Results: Twenty-three patients with advanced BTC were enrolled; 17 patients were assigned to treatment with durvalumab and tremelimumab (Durva/Treme); and 6 patients were treated with the combination of durvalumab, tremelimumab plus IR procedure (Durva/Treme + IR). The best clinical responses in the Durva/Treme arm were partial response (n = 1), stable disease (n = 5), progressive disease (n = 5), and in the Durva/Treme + IR arm: partial response (n = 0), stable disease (n = 3), progressive disease (n = 3). The median PFS was 2.2 months (95% CI: 1.3-3.1 months) in the Durva/Treme arm and 2.9 months (95% CI: 1.9-4.7 months) in the Durva/Treme + IR arm (p = 0.27). The median OS was 5.1 months (95% CI: 2.5-6.9 months) in the Durva/Treme arm and 5.8 months (95% CI: 2.9-40.1 months) in the Durva/Treme + IR arm (p = 0.31). The majority of AEs were grades 1-2., Conclusion: Durva/Treme and Durva/Treme + IR showed similar efficacy. With a manageable safety profile. Larger studies are needed to fully characterize the efficacy of Durva/Treme ± IR in advanced BTC., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Causality assessment of suspected drug-induced liver injury (DILI) during metabolic dysfunction-associated steatohepatitis (MASH) clinical trials can be challenging, and liver biopsies are not routinely performed as part of this evaluation. While the field is moving away from liver biopsy as a diagnostic and prognostic tool, information not identified by non-invasive testing may be provided on histology., Aim: To address the appropriate utilisation of liver biopsy as part of DILI causality assessment in this setting., Methods: From 2020 to 2022, the Liver Forum convened a series of webinars on issues pertaining to liver biopsy during MASH trials. The Histology Working Group was formed to generate a series of consensus documents addressing these challenges. This manuscript focuses on liver biopsy as part of DILI causality assessment., Results: Expert opinion, guidance and recommendations on the role of liver biopsy as part of causality assessment of suspected DILI occurring during clinical trials for a drug(s) being developed for MASH are provided. Lessons learned from prior MASH programs are reviewed and gaps identified., Conclusions: Although there are no pathognomonic features, histologic evaluation of suspected DILI during MASH clinical trials may alter patient management, define the pattern and severity of injury, detect findings that favour a diagnosis of DILI versus MASH progression, identify prognostic features, characterise the clinicopathological phenotype of DILI, and/or define lesions that influence decisions about trial discontinuation and further development of the drug., (© 2023 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd.)

Hemophagocytic lymphohistiocytosis (HLH) is a rare disease with high mortality. Liver involvement is common (based on elevated liver function tests) with most patients demonstrating acute hepatitis. Liver biopsies are frequently obtained in the setting of suspected HLH for the purpose of identification of erythrophagocytosis, and if present, this finding is thought to suggest or support the diagnosis of HLH. However, there are problems with this approach; in particular, we do not know whether this finding is reproducible or whether it is specific to HLH. Therefore, we conducted a multi-institutional study in which experienced liver pathologists reviewed images taken from liver biopsies from patients with normal liver, acute hepatitis, possible HLH, and clinical HLH to determine if there was agreement about the presence or absence of erythrophagocytosis, and to ascertain whether the finding corresponds to a clinical diagnosis of HLH. Twelve liver pathologists reviewed 141 images in isolation (i.e., no clinical information or diagnosis provided). These came from 32 patients (five normal, 17 acute hepatitis, six HLH, four possible HLH). The pathologists classified each image as negative, equivocal, or positive for erythrophagocytosis. Kappa was .08 (no agreement) for case-level and 0.1 for image-level (1.4% agreement, based on two images which were universally considered negative). There was no difference in the proportion of pathologists who diagnosed erythrophagocytosis among those with different diagnoses at case or image-level (p = 0.82 and p = 0.82, respectively). Thus, erythrophagocytosis is an entirely unreliable histologic parameter in liver, as it is irreproducible and not demonstrably associated with a clinical disease (namely, HLH). Unless and until more reliable guidelines can be established, pathologists should refrain from commenting on the presence or absence of erythrophagocytosis in liver biopsy., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Histological assessment of nonalcoholic fatty liver disease (NAFLD) has anchored knowledge development about the phenotypes of the condition, their natural history and their clinical course. This fact has led to the use of histological assessment as a reference standard for the evaluation of efficacy of drug interventions for nonalcoholic steatohepatitis (NASH) - the more histologically active form of NAFLD. However, certain limitations of conventional histological assessment systems pose challenges in drug development. These limitations have spurred intense scientific and commercial development of machine learning and digital approaches towards the assessment of liver histology in patients with NAFLD. This research field remains an area in rapid evolution. In this Perspective article, we summarize the current conventional assessment of NASH and its limitations, the use of specific digital approaches for histological assessment, and their application to the study of NASH and its response to therapy. Although this is not a comprehensive review, the leading tools currently used to assess therapeutic efficacy in drug development are specifically discussed. The potential translation of these approaches to support routine clinical assessment of NAFLD and an agenda for future research are also discussed., (© 2023. Springer Nature Limited.)