Mallory Bodies Research Articles

Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation.

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory–Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62− total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-β-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.

Young Adult LEW.1WR1 Rats, a Model of Liver FAT10 Overexpression, Develop Insulin Resistance and Fatty Liver With Age

As human lifespan increases, comorbid conditions that impact quality of life have become a serious problem. FAT10 has been identified as a gene that when knocked out, improves age associated metabolic dysfunctions and increased longevity in mice (1). There is increased Fat10 expression in the liver in obesity (2,5). Providing evidence that fat10 expression may be important for triggering the transition to metabolic dysfunction in aging. Adult LEW.1WR1(1WR1) rats have increased body mass without excess abdominal fat mass compared to control rats (3). Yet, it was unclear where the excess mass was stored. We hypothesized that the 1WR1 rat would develop increased liver fat mass as a product of increased insulin resistance in response to increased liver fat10 expression over time. To test this hypothesis, we did insulin tolerance tests(7 weeks & 15 weeks), triglyceride assays, and histological analysis of the liver(23 weeks), in 1WR1 rats(n=7) and Wistar Furth (WF) rats(n=7) on control diets. We analyzed images using histological scoring for nonalcoholic fatty liver disease from the literature (4). We also assessed the slides for Mallory Denk bodies (MBs). The body mass of 1WR1 rats were increased compared to WF rat groups starting from the age of 7 weeks (391.4∓8.572g vs. 271.8∓11.62g; p <0.0006). 1WR1 rats became more insulin resistant with age, the 1WR1 rat group has increased AUC of 7 and 15 week Insulin Tolerance Tests (401.5∓23.54 vs. 245.3∓10.20 7w ITT1; p= 0.0728, 15w ITT2 328.2∓14.86 vs. 217.8∓9.; p <0.0003) compared to WF rats. 1WR1 rats have increased liver mass (11.85g∓0.7699g vs. 7.235g∓0.3864g; p=0.0006) liver triglyceride levels compared to WF rats (192.8∓21.8 mg/mL vs. 130.1∓13.075 mg/mL; p=0.0728). 1WR1 rats have increased steatosis scores(1.857∓0.2608 vs. 1.143∓0.1429;p= 0.0862) yet significantly reduced inflammatory foci level (2∓0.8165 vs. 3∓0;p= 0.007), most 1WR1 hepatocytes are enlarged (ballooned) and contained MBs compared to WF rats suggesting 1WR1 rats have already passed the early inflammation stage. Adult 1WR1 rats developed reduced insulin sensitivity and lipid accumulation in the liver. These data support our hypothesis that 1WR1 rats would develop increased liver fat and impaired insulin resistance in response to aging and show that this process may be inflammation driven. (1) Canaan et al.,PNAS. April 2014; 111 (14): 5313-5318.(2)Vidal et al., FASEB.2020, 34: 1-1,(3) Collins et al., J Endocr Soc. 2019;3(1),(4).Kleiner et al., Hepatology, 2005; 41 (6): 1313–1321,(5).Dali-Youcef et al., Hepatol Commun. 2019;3(9):1205-1220.

LEW.1WR1 Rat Shows an Increased Risk of Developing Malignant Forms of NAFLD

Non alcoholic Fatty Liver Disease(NAFLD) is a disease usually found alongside diabetes in patients and is known as a manifestation of the metabolic disorder. The disease describes a progression of increasingly malignant conditions affecting the liver starting from benign steatosis. Steatosis can progress to steatohepatitis, damaging cells and encouraging scar tissue formation. The LEW.1WR1(1WR1) rat overexpresses fat10, a gene which plays a role in age-related inflammation, insulin resistance, adiposity, and hepatocellular carcinoma. This gene has been shown to be essential in maintaining liver cell protein quality control and Mallory Denk body(MDB) formation(Jia, Ji, & French, 2020). 1WR1 rats have been shown to become more glucose intolerant as they age, developing insulin resistance increasing risk of Diabetes and NAFLD. They have also been shown to develop increased body mass while not having significantly different epididymal fat pad deposits and increased fat10 concentration in the liver compared to control animals (Collins, Clopp, Mercado, Gibson, & Love-Rutledge, 2019). Increasing understanding of fat10’s functions is important for future research in reducing disease susceptibility in NAFLD. For this project we hypothesized that because weight was not being added to the fat pads in the previous study it would be added to the organs; therefore these animals would develop increased liver mass or fatty liver as they age increasing risk of NAFLD. This project studied glucose intolerance, triglyceride levels, liver weight, MDBs, and NAFLD Activity score(NAS) of 1WR1 and WF/NHsd(WF) rat livers. These rats were subjected to a 7% sucrose or normal diet type by Research Diets from 5-7 weeks of age. At 21 weeks old a 120 minute human like glucose tolerance test was done after a fasting period of 8 hours. Triglyceride concentrations in the blood, liver, and muscles were measured. Sacrifice occurred at 18 weeks of study and the livers were weighed and formalin fixed. They were then sent to Histowiz for slide processing and digitalization. Scoring for MDBs and NAS were done on the resulting slides. The results of the glucose tolerance test confirmed these rats became more glucose intolerant during the study as their glucose levels were unable to return to baseline after 120 minutes. The Triglyceride assay showed significantly higher concentrations of liver and muscle triglyceride levels to the control. The liver mass measurements showed the 1WR1 rats had significantly heavier livers to the control. The MDB scoring showed their presence primarily in the 1WR1 rats. Finally, the results of our NAS scoring indicated that the 1WR1 rats developed steatosis at a higher rate than the control. The steatosis development at a higher rate correlates to a higher risk of steatohepatitis and further malignant forms of NAFLD. Fat10’s roles in age related glucose intolerance and liver function increases risk of development of later malignant NAFLD forms.

Hepatic Steatosis and Steatohepatitis in a Large North American Cohort of Adults With Chronic Hepatitis B.

Fatty liver disease (FLD) influences liver disease progression and liver cancer risk. We investigated the impact of FLD on liver disease severity in a large North American cohort with chronic hepatitis B virus (HBV). Liver biopsies from 420 hepatitis B surface antigen-positive adults enrolled in the Hepatitis B Research Network and who were not on HBV therapy in the previous month were evaluated for inflammation and fibrosis. Steatohepatitis was based on steatosis, hepatocyte ballooning ± Mallory-Denk bodies, and perisinusoidal fibrosis. Models evaluated factors associated with steatohepatitis, and the associations of steatohepatitis with fibrosis, and longitudinal alanine aminotransferase, aspartate aminotransferase, and Fibrosis-4. The median age was 42 years, 62.5% were male, and 79.5% were Asian. One hundred thirty-two (31.4%) patients had FLD (77 [18.3%] steatosis only, 55 [13.1%] steatohepatitis). Older age, overweight/obesity, and diabetes were associated with steatohepatitis. Steatohepatitis (vs no FLD) was associated with 1.68 times higher risk of advanced fibrosis at baseline (95% confidence interval, 1.12-2.51), and there was an indication of higher incident cirrhosis rate during follow-up. Steatohepatitis vs no FLD was also independently associated with, on average, 1.39 times higher alanine aminotransferase (P < 0.01) and 1.25 times higher Fibrosis-4 (P = 0.04) across 4 years. Coexisting steatosis occurred in nearly a third of adults (13% had steatohepatitis) with chronic HBV in this North American cohort who underwent liver biopsies. Steatohepatitis was associated with advanced fibrosis and higher biochemical measures of hepatic inflammation over time. Therefore, in addition to viral suppression, screening for and managing metabolic abnormalities is important to prevent disease progression in HBV.

Nonalcoholic Steatohepatitis: Case-Based Focused on Pediatric and Adult Guidelines

Nonalcoholic fatty liver disease (NAFLD) comprises a continuum of conditions associated with insulin resistance and obesity in the absence of secondary causes of hepatic steatosis (alcohol, medication, genetic disorders, hepatitis, etc.) The milder, benign form of NAFLD is simple fatty liver or steatosis. Fatty infiltration affects more than 5% of the liver.1 This is determined by histologic exam, direct quantification or imaging. The range progresses to nonalcoholic steatohepatitis (NASH) and culminates in fibrosis and cirrhosis. Histologic changes include ballooning degeneration of hepatocytes, the presence of Mallory bodies, macrovesicular steatosis, lobular or portal inflammation.2 &#x0D; NAFLD is now the foremost cause of childhood, adolescent and adult chronic hepatic disease. The pediatric and adult obesity epidemic makes NAFLD a potentially ubiquitous hepatic pathology amongst all patients.3,4,5 The increase of obese children and adults with obesity correlates to the rise of NAFLD cases. In adults, the rate of NAFLD increase is paired with the epidemics of obesity and Type 2 Diabetes (T2DM).4,5 Data proposes hepatic-related mortality may be due to NASH ultimately progressing to cirrhosis.3,4 NAFLD is linked to pediatric and adult cardiovascular risk and morbidity.4,6 Non-invasive biomarkers and the gold standard of liver biopsy not only diagnose but assist in targeted therapies.6 Most pharmacologic therapy for NAFLD is in trial stages for patients of all ages. Pioglitazone is favored in adults who have NAFLD/NASH and T2DM.7 The gut biome is also impactful. Lifestyle modifications of diet and exercise can reduce the public health burden of this disease.7,8,9,10

Primary Versus Secondary NAFLD: Perspective on Advanced Fibrosis

The presence of macrovesicular steatosis on liver biopsy is the commonest histopathological finding. Nonalcoholic fatty liver disease (NAFLD) is the presence of ≥5% macrovesicular steatosis without significant alcohol use. It is subdivided into primary and secondary NAFLD; information on their differences is limited. To determine the histopathological differences between primary and secondary NAFLD and establish whether the prevalence of advanced fibrosis varies between the two types. Three years of retrospective study of 90 liver biopsies with ≥5% macrovesicular steatosis. Age, gender, clinical history, serum transaminaselevels were noted. The biopsy was reviewed for steatosis, inflammation, and fibrosis. Differences between primary and secondary NAFLD for age, gender, AST/ALT ratio, histopathological features were determined. Descriptive statistical analysis, 2-tailed Student's t test, Chi-square test, Fisher's exact test were used, where p<0.05 was considered significant. Primary and secondary NAFLD were 42 (46.7%) and 48 (53.3%), respectively. Inflammation was noted in 50 (55.5%) and fibrosis in 31 (34.4%). The prevalence of advanced fibrosis was 24.4%. Primary and secondary NAFLD differed significantly on ballooning degeneration, Mallory Denk bodies (MDBs), glycogenated nuclei, and fibrosis stage (p < 0.05). There were no significant differences among AST/ALT ratio, steatosis, and inflammation grade. Primary NAFLD is a more severe type of liver disease. On histopathology, ballooning degeneration, MDBs, glycogenated nuclei, and advanced fibrosis was more prevalent in primary than secondary NAFLD.

A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation

Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NF-κB-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IκBα-loss with consequent NF-κB activation. Four known mechanisms of IκBα-loss i.e. the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBβ into insoluble aggregates, thereby releasing NF-κB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-κB subunit p65, which stably interacts with IκBα under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IκBα under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IκBα after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IκBα-nuclear import. The concurrent aggregation of IκBα, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IκBα and its consequent binding and termination of NF-κB activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.

Cardiac and vascular changes in the patho- and morphogenesis of alcohol use disorder

Aim. To reveal the patho- and morphogenesis of microvascular injury, cardiac and vascular changes in individuals with alcohol use disorder (AUD) and the role of cardiovascular pathology in the thanatogenesis of patients with chronic alcohol intoxication.Material and methods. The study was carried out using the data of 1118 autopsies of patients with AUD. We used histological, immuno-histochemical, electron microscopic and biochemical methods, as well as morphometrics with statistical processing of the results.Results. Most patients with binge drinking developed alcoholic cardiomyopathy, cardiac hypertrophy, myocardial fatty degeneration, microvascular arteriolosclerosis and hyalinosis, diffuse focal sclerosis, as well as mitochondrial swelling and fragmentation of cristae in cardio-myocytes (CMC). The proportion of damaged CMC was 30,9±0,6%. Sclerosis affected 20,1±0,9% of the stroma. If alcohol was abandoned or consumed within the basal metabolic rate, these changes were reversible. With alcoholism, alcoholic cardiomyopathy, arteriosclerosis and arteriolosclerosis progressed. Compared with the binge drinking, the proportion of damaged CMC (44,8±1,1%) and the cardiosclerosis area (28,1±0,5%) significantly increased. Mallory bodies with autoantigen properties were detected. Morphological changes became irreversible.Conclusion. The data obtained identified the need for AUD isolation as an independent nosological entity with stage-by-stage pathogenesis. In the stage of binge drinking, the compensatory and adaptive capabilities of the body are preserved. With appropriate treatment, this stage of AUD is treatable. In the stage of alcoholism, changes in organs and tissues are irreversible, and it is more advisable for addiction medicine physicians to deal with palliative treatment of such patients.

Altered regulation of LncRNA analysis of human alcoholic hepatitis with Mallory-Denk Bodies (MDBs) is revealed by RNA sequencing

Mallory-Denk Bodies (MDBs) are prevalent in a variety of liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. Long noncoding RNAs (lncRNAs) are considered as emerging new gene regulators, which participates in many functional activities through diverse mechanisms. We previously reported the mechanisms involved in the formation of liver MDBs in mouse model and in AH livers where MDBs had formed. To investigate the regulation of mRNAs expression and the probable role of lncRNAs in AH livers with MDBs, RNA-Seq analyses was further conducted to determine the mRNA and lncRNA expression profiles of the AH livers compared with the normal livers. It showed that different lncRNAs have different information contribution degrees by principal component analysis, and the integrated analysis of lncRNA-mRNA co-expression networks were linked to endocytosis, cell cycle, p53 signaling pathways in the human. Based on the co-expression networks, we identify 36 mRNAs that could be as potential biomarkers of alcoholic liver disease (ALD) and hepatocellular carcinoma (HCC). To our knowledge, this is the first report on the regulatory network of lncRNAs associated with liver MDB formation in human, and these results might offer new insights into the molecular mechanisms of liver MDB formation and the progression of AH to HCC.

Steatohepatitic hepatocellular carcinoma: imaging findings with clinicopathological correlation

To evaluate the clinicopathological and computed tomography (CT) and magnetic resonance imaging (MRI) findings of steatohepatitic hepatocellular carcinoma (SH-HCC). Clinicopathological and radiological features were evaluated in 20 patients with SH-HCC. The diagnosis of SH-HCC was made histologically if the tumour had four of the following five characteristics: steatosis (>5% tumour cells), ballooning, Mallory-Denk bodies, interstitial fibrosis, and inflammation. All patients underwent dynamic CT and MRI. CT and MRI images were reviewed for morphological features including tumour size, presence, and distribution of fat, and patterns and degree of contrast enhancement. Obesity, hypertension, and history of heavy alcohol intake were common clinical findings observed in 10 (50%), 13 (65%), and 11 (55%) of the 20 patients, respectively. Steatosis and steatohepatitis were pronounced in the background liver in 12 (60%) and 10 (50%) patients, respectively. SH-HCC was moderately differentiated in 18 patients (90%) and well differentiated in two (10%). Pathologically, steatohepatitic features were diffuse in 12 (60%) of the 20 tumours and focal in eight (40%). Tumour size and the percentage of intratumoural steatosis were not correlated (r=0.17, p=0.47). On CT, 16 (80%) patients showed arterial phase enhancement and delayed washout. On MRI, 16 (80%) of 20 tumours showed prominent fatty deposition (10 diffusely, six focally) with arterial phase enhancement. SH-HCC is likely to show prominent fatty deposits with arterial phase enhancement on CT and MRI. A hypervascular lesion with prominent fatty change should raise the diagnostic suspicion of SH-HCC.

S2404 Acute Fatty Liver of Pregnancy in the Early Second Trimester Masquerading as Autoimmune Hepatitis

INTRODUCTION: Acute fatty liver of pregnancy (AFLP) is a rare and potentially fatal condition characterized by microvesicular steatosis and liver failure. It typically presents in the third trimester or early postpartum period, but several cases have been described in the second trimester, with the earliest reported case at 18 weeks gestation. Diagnosis is based on the Swansea Criteria, though liver biopsy can be performed under certain circumstances. Treatment involves supportive care and prompt fetal delivery, with recovery expected over the ensuing days to weeks. We present the earliest known case of AFLP at 15 weeks gestation and detail how its clinical course masqueraded as autoimmune hepatitis (AH). CASE DESCRIPTION/METHODS: A 41-year-old G4P2103 with morbid obesity was transferred to our institution with jaundice, scleral icterus, and vomiting after laboratory testing revealed AST 51 IU/L, ALT 88 IU/L, ALP 191 IU/L, and TB 15 mg/dL. On admission she was alert and oriented with stable vital signs. Laboratory testing was notable for an ANA with >1:1280 titer. Additional autoantibodies, anti-cardiolipin, and ADAMTS13 were negative. Blood smear showed no hemolysis. On day 2 an abdominal ultrasound revealed intrauterine fetal demise, so dilation and evacuation were performed. There was no evidence of preeclampsia or hemolysis with elevated liver enzymes and low platelets, so she was treated with methylprednisolone out of concern for AH. She did not improve with steroids so liver biopsy was performed which revealed mixed micro- and macrovesicular steatosis consistent with AFLP and nonalcoholic fatty liver disease (NAFLD). She was transferred to a liver transplant center where she expired without further intervention. DISCUSSION: This case was challenging because AFLP has never been reported in the early second trimester and the presence of high ANA titers suggested an autoimmune etiology. The biopsy showed mixed micro- and macrovesicular steatosis with cytoplasmic ballooning, mild inflammation, and no fibrosis. AH was excluded due to the lack of interface hepatitis, rosettes, plasma cell inflammatory changes, or improvement with steroids. Although NAFLD is associated with autoantibodies it does not cause acute liver failure, which this patient experienced despite receiving the standard of care. Therefore, based on the patient's clinical course and biopsy we present the earliest case of AFLP complicated by concomitant NAFLD.Figure 1.: Low power, hematoxylin and eosin (H&E) stained section showing mild steatosis in a zonal pattern, primarily involving zone 3. The inflammation is mild with a lobular predominance, composed principally of lymphocytes and plasma cells with smaller populations of neutrophils and eosinophils.Figure 2.: High power, hematoxylin and eosin (H&E) stained section showing cytoplasmic ballooning of hepatocytes with the presence of Mallory's Hyaline (arrows).Figure 3.: High power, hematoxylin and eosin (H&E) stained section of microvesicular (arrowheads) and macrovesicular (asterisk) steatosis.

Histopathological findings of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is based on the concept of pathological morphology as well as clinical findings, and is broadly categorized into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). The differential diagnosis between NAFL and NASH is important because NASH has the potential to progress to cirrhosis and hepatocellular carcinoma. NAFL is simple hepatic steatosis without hepatocellular injury, while NASH is characterized by macrovesicular steatosis, inflammation, and ballooning hepatocytes with a predominantly centrilobular (zone 3) distribution. Liver biopsy is a useful test for diagnosing NAFLD, but it is invasive. Therefore, various noninvasive methods including diagnostic imaging have been developed in recent years. To verify their usefulness, it is necessary to clarify in detail how the pathological findings are reflected in the image findings as imaging and histopathological findings are closely related. We describe the main histological features of NAFLD, i.e., steatosis, inflammation, ballooning hepatocytes, Mallory-Denk bodies, and fibrosis, as well as the evolutional process to liver cirrhosis.

Hepatoid Teratoma, Hepatoid Yolk Sac Tumor, and Hepatocellular Carcinoma: A Morphologic and Immunohistochemical Study of 30 Cases.

Rare hepatoid teratomas (HTs) in testicular germ cell tumor patients mimic hepatoid yolk sac tumor (HYST) and hepatocellular carcinoma (HCC). We compared the features of 2 metastatic HTs, 12 HYSTs, and 16 HCCs. The mean ages were 36, 40, and 62.5 years, respectively. The HTs formed sheets of hepatocyte-like cells with macrovesicular fat arranged in vague lobules with intervening fibrous bands containing biliary ductule-like structures and abortive portal triads. HTs lacked basement membrane deposits, with hepatoid cells staining for glypican-3, arginase, and HepPar-1 (2/2), whereas stains for CK19 (2/2) and CK7 (1/2) highlighted ductules and for villin hepatoid cells and ductules (1/2). SALL4 and CDX2 stains were negative (0/2). HYSTs formed nests, trabeculae, cords, and occasional gland-like structures, and most (10/12; 83%) produced intercellular basement membrane. No Mallory-Denk bodies were seen. Stains for SALL4 (100%), glypican-3 (100%), CK19 (88%), CDX2 (88%), and villin (75%) were positive, whereas those for HepPar-1 highlighted rare tumor cells (70%) and for arginase were mostly negative (26%). All HCCs lacked basement membrane deposits, with Mallory-Denk bodies occurring in 50%. Stains for HepPar-1 (100%) and arginase (94%) were positive, glypican-3 infrequent (19%), and SALL4, CK19, villin, and CDX2 negative. In summary, HTs are distinguished from HYST by the formation of ductules and abortive portal tracts, lack of basement membrane deposits, more consistent staining for arginase and HepPar-1, and negativity for SALL4 and CDX2. Contrasting features of HCCs with HYSTs include negativity for SALL4, CK19, and CDX2, frequent Mallory-Denk bodies, and absence of basement membrane deposits.

Histology of Liver of the Deceased Due to Harmful Use of Alcohol.

To study types and incidence of histological changes in liver of people deceased due to harmful use of alcohol. A retrospective review of medico-legal autopsy of 236 adults who died in the years 2015-2016 due to harmful use of alcohol was done. Histopathological liver samples taken during autopsies were evaluated. Blood alcohol content was analyzed. Serological tests for hepatitis B surface antigen and anti-hepatitis C virus (HCV) were performed. The most common liver pathology (83.1%) was steatosis, mainly mixed type (50%); 66.9% had high-grade steatosis. Liver fibrosis was detected in 39.4% of cases, with fibrosis of higher than or equal to third grade in 14%, hepatitis in 44.5% and steatohepatitis in 19.1%. Toxic hepatocyte injury features (ballooning degeneration, Mallory-Denk bodies) were found in 20.8% cases and degenerative-damage changes in 41.1%. The correlation between the grade of steatosis and fibrosis (P=0.0005), toxic injury (0.00000101) and degenerative-traumatic changes (P=0.00000741) was found. The correlation was found between hepatitis and higher than or equal to third grade steatosis (P=0.037), cholestasis (P=0.0139), toxic injury features (P=2.58×10-13), degenerative-damage changes (P=7.9×10-12) and presence of anti-HCV (P=0.00723) and between progression of fibrosis and presence of toxic injury features (2.28×10-19), degenerative-damage changes (P=4.25×10-11) and anti-HCV (P=0.0263). Spectrum of histopathological liver changes is broad regardless of sex, and various traits are present in various patterns. Comorbidities have strong influence on the picture of changes in the liver. Exact evaluation how often and what histopathological changes will develop in alcohol liver disease is not possible by reason of variability of external factors.

The Role of FAT10 in Alcoholic Hepatitis Pathogenesis.

FAT10 expression is highly up-regulated by pro-inflammatory cytokines IFNγ and TNFα in all cell types and tissues. Increased FAT10 expression may induce increasing mitotic non-disjunction and chromosome instability, leading to tumorigenesis. In this review, we summarized others’ and our work on FAT10 expression in liver biopsy samples from patients with alcoholic hepatitis (AH). FAT10 is essential to maintain the function of liver cell protein quality control and Mallory–Denk body (MDB) formation. FAT10 overexpression in AH leads to balloon degeneration and MDB aggregation formation, all of which is prevented in fat10-/- mice. FAT10 causes the proteins’ accumulation, overexpression, and forming MDBs through modulating 26s proteasome’s proteases. The pathway that increases FAT10 expression includes TNFα/IFNγ and the interferon sequence response element (ISRE), followed by NFκB and STAT3, which were all up-regulated in AH. FAT10 was only reported in human and mouse specimens but plays critical role for the development of alcoholic hepatitis. Flavanone derivatives of milk thistle inhibit TNFα/IFNγ, NFκB, and STAT3, then inhibit the expression of FAT10. NFκB is the key nodal hub of the IFNα/TNFα-response genes. Studies on Silibinin and other milk thistle derivatives to treat AH confirms that overexpressed FAT10 is the major key molecule in these networks.

Trehalose alleviates oxidative stress-mediated liver injury and Mallor-Denk body formation via activating autophagy in mice.

Autophagy is a degradation pathway for long-lived cytoplasmic proteins or damaged organelles and also for many aggregate-prone and disease-causing proteins. Endoplasmic reticulum (ER) stress and oxidative stress are associated with the pathophysiology of various liver diseases. These stresses induce the accumulation of abnormal proteins, Mallory-Denk body (MDB) formation and apoptosis in hepatocytes. A disaccharide trehalose had been reported to induce autophagy and decrease aggregate-prone proteins and cytotoxicity in neurodegenerative disease models. But the effects of trehalose in hepatocytes have not been fully understood. We examined the effect of trehalose on autophagy, ER stress and oxidative stress-mediated cytotoxicity and MDB formation in hepatocytes using mice model with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treatment for 3months. We administered trehalose by intraperitoneal injection of water containing 10% trehalose (0.02mg/g body weight) every other day for 3months. Our results demonstrated that trehalose induced autophagy and reduced ER stress, oxidative stress, MDB formation and apoptosis in hepatocytes of DDC-fed mice by Western blotting and immunostaining analyses. Electron microscopy revealed that trehalose induced autolysosome formation, which located is close to the MDBs. Thus, our findings suggest that trehalose can become a therapeutic agent for oxidative stress-related liver diseases via activating autophagy.

Comparison of High Fructose Corn Syrup Versus Sucrose Consumption on Non-Alcoholic Fatty Liver Disease in Juvenile Iberian Pigs

Abstract Objectives Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic disorder and the most common liver disease in pediatric populations. Epidemiological studies have observed a parallel increase in fructose consumption and incidence of NAFLD among children. The objective of this study was to compare the relative effect of inclusion of isocaloric amounts of high fructose corn syrup (66.5% fructose, 33.5% glucose) versus sucrose (50% fructose, 50% glucose) in the diet for 16 weeks on endpoints of NAFLD and insulin resistance. Methods 30-d-old Iberian pigs were housed in pairs and randomly assigned to receive solid diets (g/kg body weight × d) of 1) control (CON; n = 6): 0 g HFCS, 0 g SUC, and 174.03 kcal metabolizable energy (ME), 2) high-fructose corn syrup (HFCS; n = 8): 31.20 g high-fructose corn syrup, 0 g sucrose and 261 kcal ME, and 3) Sucrose (SUC; n = 6): 0 g high-fructose corn syrup, 24.04 g sucrose and 261 kcal ME for 16 consecutive weeks. Results Compared to CON, both HFCS and SUC diets increased body weight gain (P ≤ 0.001), relative liver weight (P ≤ 0.01) and leptin levels (P ≤ 0.01), and decreased percentage of lean mass composition in the animals (P ≤ 0.001). In addition, HFCS increased fasting insulin levels compared to CON (P ≤ 0.05), and decreased percentage lean mass compared to SUC (P ≤ 0.05). 75% of HFCS and 66.6% of SUC pigs showed histopathological lesions consistent with microvesicular steatosis with periportal or diffuse distribution. Serum markers of liver injury did not differ between diets, and none of the animals developed inflammation, hepatocellular ballooning, Mallory hyaline or necrosis in the liver. Metabolomics analysis revealed liver sorbitol and monosaccharide concentrations were higher in both the HFCS and SUC groups versus CON (P ≤ 0.05), while adenosine monophosphate (AMP) were higher and adenosine diphosphate levels lower in the HFCS and SUC in comparison to CON (P ≤ 0.05). Numerous phosphatidylcholines and sphingomyelins were differentially changed in the HFCS group versus CON (P ≤ 0.05). Conclusions Feeding diets high in either sucrose or high fructose corn syrup promoted obesity and steatosis in the animals. Further research is needed to investigate the mechanisms leading to increased insulin resistance in the HFCS group. Funding Sources ARI #58,873, AcornSeekers, STRIDE.

Progressive Weakness and Encephalopathy in a 34-Year-Old Woman

A 34-year-old woman presented because of falls, weakness, and new-onset jaundice. Examination demonstrated mild encephalopathy and scleral icterus. Liver enzymes indicated aspartate aminotransferase (AST) 259 U/L, alanine aminotransferase (ALT) 76 U/L, total bilirubin 9.9 mg/dL, and international normalized ratio 1.6. She underwent a liver biopsy, which revealed macrovesicular steatosis with hepatocyte ballooning (solid black arrow) and Mallory bodies (dashed black arrow) shown in Figures A and B (H&E stain; original magnification, ×100 and ×200, respectively). Masson trichrome staining of the biopsy specimens demonstrated cirrhosis and pericellular fibrosis (black arrows, Figure C; original magnification, ×100). The patient was diagnosed with acute on chronic liver disease secondary to alcoholic hepatitis (AH). The prevalence of AH is difficult to estimate because of the disease spectrum. Alcohol is the strongest risk factor. Laboratory findings in AH reveal an AST/ALT ratio greater than 2. Pathologic findings include hepatic steatosis, Mallory bodies, and fibrosis. The Maddrey discriminant function (MDF) is used to stratify patients and guide treatment. Treatment consists of alcohol cessation and corticosteroids. If treatment is initiated, the Lille model can determine whether corticosteroids should be continued. Our patient had an MDF of 51 and was started on corticosteroids. Alcohol cessation was emphasized, and she remains abstinent.

The Role of FAT10 in Alcoholic Hepatitis Pathogenesis

BackgroundMorphologic features of Alcoholic Hepatitis (AH) balloon cells forming Mallory‐Denk bodies and bile duct metaplasia are two morphologic features consistently being found in AH. Increased FAT10 expression may induce increasing mitotic non‐disjunction and chromosome instability leading to tumorigenesis. FAT10 expression is highly up‐regulated by pro‐inflammatory cytokines IFNγ and TNFα in all cell types and tissues.ObjectiveTo clarify the role of FAT10 in alcoholic hepatitis pathogenesis, we studied FAT10 expression in liver biopsy samples from patients with AH.MethodsWe use gene and protein array to detect the potential changes during alcoholic hepatitis pathogenesis. The potential target, FAT10, were tested at protein expression level by using western blot, immunofluorescent (IF) staining and quantitation. And FAT10 was also studied by using knockout animal models.ResultsThe results show that FAT10 is essential to maintain the function of liver cell protein quality control and Mallory‐Denk body (MDB) formation. This is consistent with the finding with fat10−/− mice which fail to form MDBs. FAT10 also signals a switch in the 26s proteasome’s proteases to cause the proteins accumulation, overexpression, and then forming MDBs. FAT10 overexpression in AH causes an extensive change in protein content in the liver leading to balloon degeneration and MDB aggregation formation, all of which is prevented in fat10−/− mice. The pathway that increases FAT10 expression includes TNFα, IFNγ, followed by NFκB and STAT3, which were all up‐regulated in AH. Using proteins expression quantitation by measuring the intensity of immunofluorescent (IF) staining, it showed the FAT10 protein expression was 4.5 fold increased in the AH livers but not in the NASH or control livers.DiscussionMilk thistle derivatives were used to treat FAT10 overexpression experimentally. Flavanone derivatives of milk thistle inhibit IFNγ/TNFα, NFκB and STAT3 then inhibits the expression of FAT10. NFκB is the key nodal hub of the IFNα/TNFα‐response genes. Silibinin derived from milk thistle is reported to be a powerful antioxidant and has anti‐carcinoma effects including against TNFα‐induced tumor growth. Studies on Silibinin or other milk thistle derivatives to treat AH confirms that overexpressed FAT10 with NFκB and TNFα are the major key nodal molecules in these networks.ConclusionOverexpressed FAT10 with over‐activated NFκB and TNFα are the major key nodal molecules in alcoholic hepatitis pathogenesis. These molecules might be potential therapeutic targets.

Quantitative Histopathological Analysis of a Diet‐Induced NASH Mouse Model Using Artificial Intelligence

Non‐alcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease. It results in an accumulation of fat in the liver, and can progress to a more pathologically significant form of NAFLD known as non‐alcoholic steatohepatitis (NASH). Patients with NAFLD and NASH present on a spectrum of the disease, characterized by hepatitis (inflammation) and hepatocellular ballooning (cellular injury), which can lead to excessive fibrosis and scarring. Currently, diagnosis is confirmed by liver biopsies that are qualitatively analyzed by experienced pathologists who assign scores for each feature. However, documented inter‐pathologist variability in scoring and the semi‐quantitative nature of the scoring system itself highlight the need for new methods to differentiate NAFLD and NASH, and ensure the unbiased, consistent assessment of disease to better diagnose and stratify patients for treatment options. To support this effort we developed imageDx™: NASH, a collection of artificial intelligence (AI)‐based pathology models, to histopathologically validate and characterize NASH in a novel Diet Induced NASH BL6 Model. Livers from mice on a NASH‐inducing diet and a control diet were processed, sectioned, stained and digitally scanned to generate whole slide images. Quantification of each of the pathological features in mice was performed using imageDx™: NASH, including steatosis (macro‐ and micro‐vesicular), inflammation, hepatocellular ballooning/degeneration, fibrosis and the presence of Mallory bodies. These validated machine learning algorithms were developed with input from experienced pathologists with the goal of providing a more informative analysis of the pathology. The AI‐generated results were compared with semi‐quantitative scores provided by a board‐certified doctor of veterinary medicine. Using this diet‐induced NASH rodent model both with and without therapeutic interventions, these algorithms effectively identified small differences in tissue morphology that may not be easily discernible by visual examination. By developing highly reproducible and sensitive computational models, this study provides a foundation for a better understanding of disease mechanism and the development of effective therapeutics for NASH.