Shah, Vijay H.

AbstractBackgroundAlcoholic hepatitis (AH) is a severe and life‐threatening alcohol‐associated liver disease. Only a minority of heavy drinkers acquires AH and severity varies among affected individuals, suggesting a genetic basis for the susceptibility to and severity of AH.MethodsA cohort consisting of 211 patients with AH and 176 heavy drinking controls was genotyped for five variants in five candidate genes that have been associated with chronic liver diseases: rs738409 in patatin‐like phospholipase domain‐containing protein 3 (PNPLA3), rs72613567 in hydroxysteroid 17‐beta dehydrogenase 13 (HSD17B13), rs58542926 in transmembrane 6 superfamily member 2 (TM6SF2), rs641738 in membrane bound O‐acyltransferase domain containing 7 (MBOAT7), and a copy number variant in the haptoglobin (HP) gene. We tested the effects of individual variants and the combined/interacting effects of variants on AH risk and severity.ResultsWe found significant associations between AH risk and the risk alleles of rs738409 (p = 0.0081) and HP (p = 0.0371), but not rs72613567 (p = 0.3132), rs58542926 (p = 0.2180), or rs641738 (p = 0.7630), after adjusting for patient's age and sex. A multiple regression model indicated that PNPLA3 rs738409:G [OR = 1.59 (95% CI: 1.15–2.22), p = 0.0055] and HP*2 [OR = 1.38 (95% CI: 1.04–1.82), p = 0.0245], when combined and adjusted for age and sex also had a large influence on AH risk among heavy drinkers. In the entire cohort, variants in PNPLA3 and HP were associated with increased total bilirubin and Model for End‐stage Liver Disease (MELD) score, both measures of AH severity. The HSD17B13 rs72613567:AA allele was not found to reduce risk of AH in patients carrying the G allele of PNPLA3 rs738409 (p = 0.0921).ConclusionPNPLA3 and HP genetic variants increase AH risk and are associated with total bilirubin and MELD score, surrogates of AH severity.

BackgroundHeavy alcohol consumption disrupts gut epithelial integrity, leading to increased permeability of the gastrointestinal tract and subsequent translocation of microbes. Regenerating islet‐derived protein 3α (REG3α) and Trefoil factor 3 (TFF3) are mainly secreted to the gut lumen by Paneth and Goblet cells, respectively, and are functionally linked to gut barrier integrity. Circulating levels of REG3α and TFF3 have been identified as biomarkers for gut damage in several human diseases. We examined whether plasma levels of REG3α and TFF3 were dysregulated and correlated with conventional markers of microbial translocation (MT) and pro‐inflammatory mediators in heavy drinkers with and without alcoholic hepatitis (AH).MethodsCross‐sectional and longitudinal studies were performed to monitor plasma levels of REG3α and TFF3 in 79 AH patients, 66 heavy drinkers without liver disease (HDC), and 46 healthy controls (HC) at enrollment and at 6‐ and 12‐month follow‐ups. Spearman correlation was used to measure the relationships of REG3α and TFF3 levels with MT, disease severity, inflammation, and effects of abstinence from alcohol.ResultsAt enrollment, AH patients had significantly higher levels of REG3α and TFF3 than HDC and HC. The elevated REG3α levels were positively correlated with the 30‐day fatality rate. Plasma levels of REG3α and TFF3 in AH patients differentially correlated with conventional MT markers (sCD14, sCD163, and LBP) and several highly up‐regulated inflammatory cytokines/chemokines/growth factors. At follow‐ups, although REG3α and TFF3 levels were decreased in AH patients with alcohol abstinence, they did not fully return to baseline levels.ConclusionsCirculating levels of REG3α and TFF3 were highly elevated in AH patients and differentially correlated with AH disease severity, MT, and inflammation, thereby serving as potential biomarkers of MT and gut epithelial damage in AH patients.

Background and Aims During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. Approach and Results CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC‐specific p300 deletion (p300 LSECΔ/Δ) or C‐C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)–p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild‐type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time‐of‐flight. Epigenetic histone marks were modified by CRISPR endonuclease‐deficient CRISPR‐associated 9‐fused with the Krüppel associated box domain (CRISPR‐dCas9‐KRAB)–mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300 LSECΔ/Δ mice compared to p300 fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300 LSECΔ/Δ mouse livers. Ccl2 was the most up‐regulated chemokine in injured LSECs, but its increase was abrogated in p300 LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB‐p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC‐specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR‐dCas9‐KRAB technology repressed TNFα‐induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. Conclusions In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.

Modern medical care produces large volumes of multimodal patient data, which many clinicians struggle to process and synthesize into actionable knowledge. In recent years, artificial intelligence (AI) has emerged as an effective tool in this regard. The field of hepatology is no exception, with a growing number of studies published that apply AI techniques to the diagnosis and treatment of liver diseases. These have included machine‐learning algorithms (such as regression models, Bayesian networks, and support vector machines) to predict disease progression, the presence of complications, and mortality; deep‐learning algorithms to enable rapid, automated interpretation of radiologic and pathologic images; and natural‐language processing to extract clinically meaningful concepts from vast quantities of unstructured data in electronic health records. This review article will provide a comprehensive overview of hepatology‐focused AI research, discuss some of the barriers to clinical implementation and adoption, and suggest future directions for the field.

Alcohol‐associated liver disease (ALD) is a major driver of global liver related morbidity and mortality. There are 2.4 billion drinkers (950 million heavy drinkers) and the lifetime prevalence of any alcohol use disorder (AUD) is 5.1%‐8.6%. In 2017, global prevalence of alcohol‐associated compensated and decompensated cirrhosis was 23.6 million and 2.5 million, respectively. Combined, alcohol‐associated cirrhosis and liver cancer account for 1% of all deaths worldwide with this burden expected to increase. Solutions for this growing epidemic must be multi‐faceted and focused on both population and patient‐level interventions. Reductions in ALD‐related morbidity and mortality require solutions that focus on early identification and intervention, reducing alcohol consumption at the population level (taxation, reduced availability and restricted promotion), and solutions tailored to local socioeconomic realities (unrecorded alcohol consumption, focused youth education). Simple screening tools and algorithms can be applied at the population level to identify alcohol misuse, diagnose ALD using non‐invasive serum and imaging markers, and risk‐stratify higher‐risk ALD/AUD patients. Novel methods of healthcare delivery and platforms are needed (telehealth, outreach, use of non‐healthcare providers, partnerships between primary and specialty care/tertiary hospitals) to proactively mitigate the global burden of ALD. An integrated approach that combines medical and AUD treatment is needed at the individual level to have the highest impact. Future needs include (1) improving quality of ALD data and standardizing care, (2) supporting innovative healthcare delivery platforms that can treat both ALD and AUD, (3) stronger and concerted advocacy by professional hepatology organizations, and (4) advancing implementation of digital interventions.

A number of cell culture approaches have been described for maintenance of primary hepatocytes. Forming hepatocytes into three-dimensional (3-D) spheroids is one well-accepted method for extending epithelial phenotype of these cells. Our laboratory has previously observed enhanced function of two-dimensional (2-D, monolayer) hepatocyte cultures in microfluidic devices due to increased production of several hepato-inductive growth factors, including hepatocyte growth factor (HGF). In the present study, we wanted to test a hypothesis that culturing hepatocyte spheroids (3-D) in microfluidic devices will also result in enhanced phenotype and function. To test this hypothesis, we fabricated devices with small and large volumes. Both types of devices included a microstructured floor containing arrays of pyramidal wells to promote assembly of hepatocytes into spheroids with individual diameters of ~100 µm. The hepatocyte spheroids were found to be more functional, as evidenced by higher level of albumin synthesis, bile acid production, and hepatic enzyme expression, in low-volume compared with large-volume devices. Importantly, high functionality of spheroid cultures correlated with elevated levels of HGF secretion. Although decay of hepatic function (albumin secretion) was observed over the course 3 wk, this behavior could be abrogated by inhibiting TGF-β1 signaling. With TGF-β1 inhibitor, microfluidic hepatocyte spheroid cultures maintained high and stable levels of albumin synthesis over the course of 4 wk. To further highlight utility of this culture platform for liver disease modeling, we carried out alcohol injury experiments in microfluidic devices and tested protective effects of interleukin-22: a potential therapy for alcoholic hepatitis.

Abstract Background: Accuracy of microbial community detection in 16S rRNA marker-gene and metagenomic studies suffers from contamination and sequencing errors that lead to either falsely identifying microbial taxa that were not in the sample or misclassifying the taxa of DNA fragment reads. Filtering is defined as removing taxa that are present in a small number of samples and have small counts in the samples where they are observed. This approach reduces extreme sparsity of microbiome data and has been shown to correctly remove contaminant taxa in cultured "mock" datasets, where the true taxa compositions are known. Although filtering is frequently used, careful evaluation of its effect on the data analysis and scientific conclusions remains unreported. Here, we assess the effect of filtering on the alpha and beta diversity estimation, as well as its impact on identifying taxa that discriminate between disease states. Results: The effect of filtering on microbiome data analysis is illustrated on four datasets: two mock quality control datasets where same cultured samples with known microbial composition are processed at different labs and two disease study datasets. Results show that in microbiome quality control datasets, filtering reduces the magnitude of differences in alpha diversity and alleviates technical variability between labs, while preserving between samples similarity (beta diversity). In the disease study datasets, DESeq2 and linear discriminant analysis Effect Size (LEfSe) methods were used to identify taxa that are differentially expressed across groups of samples, and random forest models to rank features with largest contribution towards disease classiffcation. Results reveal that filtering retains significant taxa and preserves the model classification ability measured by the area under the receiver operating characteristic curve (AUC). The comparison between filtering and contaminant removal method shows that they have complementary effects and are advised to be used in conjunction. Conclusions: Filtering reduces the complexity of microbiome data, while preserving their integrity in downstream analysis. This leads to mitigation of the classification methods' sensitivity and reduction of technical variability, allowing researchers to generate more reproducible and comparable results in microbiome data analysis.

BackgroundAlcoholic hepatitis (AH) is a severe inflammatory liver disease that develops in some heavy drinkers. AH patients have intense hepatic infiltration of leukocytes. Up‐regulation of cell adhesion molecules (CAMs) upon endothelial cell (EC) activation plays an important role in leukocyte transendothelial migration. CAMs can shed from EC surface and accumulate in the blood, serving as soluble markers for EC activation. In this study, we examined the impact of heavy drinking on expression of soluble forms of EC activation markers (CD146, ICAM‐1, VCAM‐1, and VEGF‐A) and the effect of alcohol abstinence on the reversal of these abnormalities in heavy drinkers with and without AH.MethodsELISA and multiplex immunoassays were used to measure soluble EC activation markers in plasma samples from 79 AH patients, 66 heavy drinkers without overt liver disease (HDC), and 44 healthy controls (HC) at baseline, 31 AH patients and 30 HDC at 6‐month follow‐up, and 18 AH patients and 25 HDC at 12‐month follow‐up.ResultsAt baseline, the 4 soluble markers were significantly up‐regulated in AH patients compared with HDC and HC, whereas only sVCAM‐1 was elevated in HDC relative to HC. At follow‐ups, plasma levels of CD146, VCAM‐1, and VEGF‐A remained higher in AH patients, even for those who stopped drinking. These dysregulated markers correlated with AH disease severity, clinical parameters, and several soluble inflammatory factors.ConclusionsThe levels of soluble CD146, ICAM‐1, VCAM‐1, and VEGF‐A were highly elevated in AH patients, and alcohol abstinence did not completely reverse these abnormalities.