Hepatocyte Ballooning Research Articles

Hepatitic Variant of Graft-vs-Host Disease.

Graft-vs-host disease (GVHD) of the liver is a complication of allogeneic hematopoietic stem cell transplantation with hepatitic and classic variants. We determined the percentage of hepatitic variant cases, compared clinicopathologic features of the two groups, and assessed prognostic factors. Fifty liver biopsy specimens from 40 patients with GVHD were studied. Fifteen (30%) cases had moderate to marked lobular inflammation and were classified as a hepatitic variant. Bile duct damage was present in all cases. Ductular reaction, apoptosis. and endotheliitis were more commonly seen in the hepatitic variant. Hepatocyte ballooning was an independent poor prognostic factor. The median aspartate aminotransferase and alanine aminotransferase were higher in the hepatitic variant while alkaline phosphatase and bilirubin were higher in the classic group. Forty (80%) GVHD cases were more than 100 days after transplant, correlating to immunosuppression taper. There was response to treatment with increased immunosuppression in both groups, but time to normalization of liver function tests was higher in the hepatitic variant. Bile duct damage was the most consistent pathologic finding in our cohort and was present in all cases of GVHD. Moderate to marked lobular inflammation can be seen in GVHD in up to 30% of cases without any other coexisting cause. Hepatocyte ballooning is an independent poor prognostic factor.

Cajanus cajan ameliorated CCl4-induced oxidative stress in Wistar rats via the combined mechanisms of anti-inflammation and mitochondrial-membrane transition pore inhibition

Ethnopharmacological relevanceLiver diseases is a public health issue in sub-saharan Africa and has been reported to be the major cause of many hospital admissions. Oxidative stress, mitochondrial dysfunction and inflammation play important roles in several diseases including liver injury. Cajanus cajan is an indigenous medicinal plant useful in the traditional treatment of jaundice, inflammation and liver injury. Aim of studyThis study assessed the effects of methanol extract Cajanus cajan (MECC) on mitochondrial permeability transition (mPT) pore opening, biomarkers of oxidative stress and inflammation in CCl4-induced liver injury in rats. MethodsWistar albino rats (200–210g) were completely randomized into five (5) groups of six animals each. Group I (control) was given distilled water orally once daily. Animals in group II were administered CCl4 in parafin (1:1) at a dose of 0.5 mL/kg i.p on the seventh day. Animals in groups III, IV and V were administered methanol extract of Cajanus cajan (MECC) at doses of 100, 200 mg/kg and silymarin (100 mg/kg) respectively for 7 days prior to a single intraperitoneal dose of CCl4. After 24 h of CCl4 treatment, serum and liver tissues were collected. Mitochondrial permeability transition (mPT) pore opening, mitochondrial ATPase activities and biomarkers of oxidative stress were determined spectrophotometrically. Tumor necrosis factor (TNFα), NF-κB and COX-2 were determined by immunohistochemistry and the phytochemicals present in the extract were determined by GC-MS. ResultsLiver enzyme (AST, ALP, ALT and γGT) activities and MDA levels were significantly decreased in rats pretreated with MECC at the dose of 100, 200 and silymarin (100 mg/kg) when compared to the rats administered CCl4 alone (p < 0.05). GSH, GST, CAT and SOD increased and the expressions of TNFα, NF-κB and COX- 2 were also reduced when compared to the CCl4- treated animals.In addition, the liver histopathological analyses revealed that MECC markedly alleviated inflammatory cell infiltration, hepatic fibrosis, hepatocyte ballooning, necrosis and severe apoptosis of hepatocytes induced by CCl4. GC-MS analysis yielded 23 compounds including flavonoids, terpenoids and fatty acids. ConclusionCajanus cajan leaf extract elicited hepatoprotective action on CCl4-induced liver injury via inhibition of mPT pore opening, prevention of CCl4-induced hepatic oxidative stress and suppression of inflammatory response thus it may become useful for chemoprevention of liver injury. This supports its traditional use.

Triglyceride and glucose index: a useful tool for non-alcoholic liver disease assessed by liver biopsy in patients with metabolic syndrome?

Introduction: Non-alcoholic fatty liver disease (NAFLD) is associated with metabolic impairments, being a component of metabolic syndrome. Considering the involvement of fat accumulation and insulin resistance in NAFLD, triglyceride and glucose (TyG) index was proposed as a marker of NAFLD progression. The “gold standard” for the evaluation of liver lesions characteristic for NAFLD remains the liver biopsy. The aim of this study was to establish the links between TyG index, assessing insulin resistance, and histopathological lesions of liver samples obtained by liver biopsy in patients with metabolic syndrome. Patients, Materials and Methods: We conducted a study over a period of three years, including 113 adult patients with metabolic syndrome in whom hepatic disorders were assessed by liver biopsy and insulin resistance was evaluated by TyG index. Results and Discussions: In our study, steatosis had a frequency of 92.03%, being identified 26 cases with mild steatosis, 48 with moderate steatosis and 31 with severe steatosis. Regarding non-alcoholic steatohepatitis (NASH), the frequency of this disorder in our study group was 29.2% in the subjects with liver steatosis, while liver fibrosis had a frequency of 53.09%. When we analyzed the relationships between TyG index and the presence of each type of lesion necessary for NASH diagnosis, we obtained statistically significant differences for the presence of hepatocyte ballooning (p=0.01) and a high statistically significance for the NAFLD activity score (NAS) (p<0.0001). Conclusions: TyG index is a facile tool that can be used to identify patients at risk for advanced NAFLD lesions evaluated by liver biopsy.

Frailty in metabolic syndrome, focusing on nonalcoholic fatty liver disease.

In recent years, frailty has been increasingly recognized among researchers of distinct medical specialties worldwide. Frailty comprises a complex of multisystemic physiological decline, reduced physiologic reserve, and vulnerability to stressors. Frail people tend to have a shorter lifespan and greater disability, morbidity and mortality. In the field of hepatology, frailty is identified in nearly 50% of patients who have cirrhosis of any cause. The most predominant cause of chronic liver disease is nonalcoholic fatty liver disease (NAFLD), considered as the hepatic manifestation of the metabolic syndrome (MetS). Although it is viewed as a benign disease, it may progress to nonalcoholic steatohepatitis (NASH), characterized by the additional emergence of inflammation and hepatocyte ballooning, with or without fibrosis. During the progression of NAFLD to NASH and liver cirrhosis, NAFLD patients present sarcopenia along with lower skeletal muscle strength and function. Moreover, aging and the increased prevalence of comorbidities further exacerbate their physical performance. The aforementioned features are strongly associated with the frailty phenotype, implying that the latter could be associated with both MetS and NAFLD. Although it is a relatively new topic of research interest, in this review we aim to provide a synopsis of the current literature dealing with the interplay between frailty and MetS, and to shed more light on the association between NAFLD and frailty. Finally, we discuss the potential pathophysiological mechanisms linking the distinct features of MetS and NAFLD with aspects of the frailty phenotype.

A fast and accurate mouse model for inducing non-alcoholic steatohepatitis.

This study aimed to perform a head-to-head comparison of changes during NASH progression throughout 6-11 weeks of an experiment to supply a faster nutritional model in mimicking NASH to decrease the duration and cost of in vivo studies. New therapies are urgently needed because of the growing prevalence of non-alcoholic steatohepatitis (NASH) and the lack of an effective treatment approach. Currently, dietary interventions are the most efficient options. This study compared features of NASH in a murine model using protocol that combined special nutritional regimes based on the combination of 21.1% fat, 41% sucrose, and 1.25% cholesterol with weekly intraperitoneal injections of carbon tetrachloride (CCl4). Male C57BL/6J mice received either special compositions + CCl4 (NASH group) or standard chow diet (healthy control group) for 11 weeks. Liver histopathology based on hematoxylin and eosin (H&E) and Masson's Trichrome (TC) staining and biochemical analyses were used to assess disease progression. In C57BL/6J mice administered a high fat, high cholesterol, high sucrose diet and CCl4 for 8 weeks, steatohepatitis with pronounced hepatocyte ballooning, inflammation, steatosis, and fibrosis was observed. According to the NAFLD activity scoring system, the maximum NAS score was manifested after 8-9 weeks (NAS score: 6.75). Following this protocol also led to a significant increase in AST and ALT, total cholesterol, and total triglyceride serum levels in the NASH group. Following the special nutritional regime based on high fat, cholesterol, and sucrose in combination with CCL4 injections resulted in a NASH model using C57BL/6J mice in a shorter time compared to similar studies. The obtained histopathological NASH features can be advantageous for preclinical drug testing.

Hydroethanolic Extract of Defatted Buchholzia coriacea Seeds Alleviates Tamoxifen-Induced Hepatic Triglyceride Accumulation, Inflammation and Oxidative Distress in Rat.

Background: Tamoxifen (TMX) has proven to be effective in the prevention and treatment of breast cancer. However, long-term use of TMX is associated with hepatic steatosis, oxidative liver injury and hepatocarcinoma. Buchholzia coriacea seeds (BCS) have been widely applied in traditional medicine due to their nutritional and therapeutic potentials. This study investigates the protective effect of hydroethanolic extract of (defatted) B. coriacea seeds (HEBCS) against TMX–induced hepatotoxicity in rats. Methods: Thirty-six (36) male albino rats were divided into six groups (n = 6/group). Group I served as control. Group II received 50 mg/kg/day TMX orally (p.o.) (TMX) for 21 days, group III received TMX plus 125 mg/kg/d HEBCS p.o. (HEBCS 125) for 21 days, group IV received TMX plus 250 mg/kg/d HEBCS p.o. (HEBCS 250) for 21 days and rats in group V and VI received HEBCS 125 and HEBCS 250 respectively for 21 days. Results: Compared with the control, TMX caused a significant increase (p < 0.05) in serum hepatic function biomarkers: alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase by 57%, 60% and 68% respectively. TMX also caused a significant increase in hepatic triglycerides level by 166% when compared with control and a significant decrease in serum HDL-cholesterol level by 37%. Compared with control, hepatic marker of inflammation, tumour necrosis factor alpha (TNF-α) increased significantly by 220%, coupled with significant increase in expression of interleukin 6 and cyclooxygenase 2. There was also significant increase in levels of Biomarkers of oxidative stress, nitric oxide, malondialdehyde and protein carbonyls in the TMX group by 89%, 175% and 114% respectively when compared with the control. Hepatic antioxidants, reduced glutathione (GSH) level and activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GSH-Px) decreased significantly in the TMX group by 35%, 67%, 41%, 59% and 53% respectively when compared with the control. However, HEBCS at 250 mg/kg significantly protected against TMX–induced hepatotoxicity by decreasing hepatic triglyceride content, serum hepatic function biomarkers, hepatic inflammation and oxidative stress with significant improvement in hepatic antioxidant system. Histopathological findings show that HEBCS alleviate TMX–induced hepatocyte ballooning. Conclusions: Current data suggest that HEBCS protected against TMX–induced hepatotoxicity in rats. HEBCS may be useful in managing TMX–induced toxicities in breast cancer patients. It may also be helpful against other forms of liver injury involving steatosis, inflammation, free radicals, and oxidative damage.

Escherichia fergusonii Promotes Nonobese Nonalcoholic Fatty Liver Disease by Interfering With Host Hepatic Lipid Metabolism Through Its Own msRNA 23487.

Background & AimsGut microbiota and microbial factors regulate the pathogenesis of nonalcoholic fatty liver disease (NAFLD) in patients with obesity and metabolic abnormalities, but little is known about their roles in nonobese NAFLD. Expansion of Escherichia is associated with NAFLD pathogenesis. We aimed to investigate the pathogenic role of Escherichia fergusonii and its products in the development of nonobese NAFLD.MethodsWe characterized the intestinal microbiome signature in a cohort of NAFLD patients and healthy controls by 16S ribosomal RNA sequencing. The role of E fergusonii was estimated in rats after 16 weeks of administration, and features of NAFLD were assessed. E fergusonii–derived microRNA-sized, small RNAs (msRNAs) were analyzed by deep sequencing.ResultsWe detected an expansion of Escherichia_Shigella in NAFLD patients compared with healthy controls, and its increase was associated with disease severity independent of obesity. E fergusonii, a member of the genus Escherichia, induced the development of nonobese NAFLD characterized by hepatic steatosis and hepatocyte ballooning in rats without obesity. It disturbed host lipid metabolism by inhibiting hepatic lipid β-oxidation and promoting de novo lipogenesis. We also showed that E fergusonii caused the development of hepatic inflammation and fibrosis in a sizable fraction of animals at an advanced stage of NAFLD. Mechanistically, E fergusonii–derived msRNA 23487 down-regulated host hepatic peroxisome proliferator-activated receptor α expression, which could contribute to lipid accumulation in the liver.ConclusionsThese results suggest that E fergusonii promotes the pathogenesis of steatohepatitis and fibrosis in nonobese rats by secreting msRNA 23487, and it might be a potential biomarker for predicting steatohepatitis in nonobese NAFLD.

Abstract 10979: Liver-Humanized Mice Provide a New Platform to Study Human Cardiometabolic Diseases

Introduction: Conventional mouse models do not fully recapitulate human cardiometabolic diseases (CMD), e.g. , atherosclerosis and nonalcoholic steatohepatitis (NASH), due to species differences in metabolism. Liver-humanized mice (LHM), i.e. , mice repopulated with human hepatocytes, have been shown to be an improved translatable model to study human hepatic and lipoprotein metabolism. Hypothesis: As dietary challenge is often used to induce CMD in animal models, we sought to characterize lipoprotein and lipid metabolism in LHM challenged by different diets. Methods: Immune-deficient FRGN mice receiving hepatocytes from different human donors (LHM) or NOD mouse (liver-murinized mice, LMM) were fed a high-fat/high-sucrose diet (HFHSD) for 8 weeks or a high-fat/high-fructose/high-cholesterol diet (NASH-diet) for 8, 12, 16 or 20 weeks. Plasma lipids and lipoproteins were quantified after size-exclusion chromatography separation or by routine clinical assays. Cholesterol in the descending aorta was analyzed by GC-MS. Histologic analysis of hearts or livers was done using hematoxylin/eosin or Oil Red O staining, and anti-mouse CD68-antibody was used to detect murine macrophages. Results: Compared with LMM, LHM fed either HFHSD or NASH-diet accumulated plasma cholesterol and triglycerides within LDL and triglyceride-rich particles (VLDL and remnants). However, only the NASH-diet induced a notable hyperlipidemia and a substantial increase in cholesteryl esters within the aorta. NASH-diet also determined a clear accumulation of neutral lipids and CD68-positive cells in the aortic valves of LHM. Similarly, hepatocyte ballooning, liver steatosis and fibrosis were also induced by the NASH-diet. All these pathologic changes occurred during diet challenge at different time points. Conclusions: LHM developed severe hyperlipidemia, atherosclerosis and NASH when fed a NASH-diet, but not on HFHSD. The high quantity of cholesterol in the NASH-diet likely compensates for the high utilization of cholesterol of LHM due to their known increase in bile acid synthesis, prompting dyslipidemia and CMD. LHM fed a NASH-diet is an improved model for human CMD and for the development of therapeutic strategies rapidly translatable to the human condition.

Estimating Drug Efficacy with a Diet-Induced NASH Model in Chimeric Mice with Humanized Livers.

Nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) is the most common liver disorder in developed countries. Although many new therapeutics for NASH are present in the drug development pipeline, there are still no approved drugs. One of the reasons that makes NASH drug development challenging is the lack of appropriate animal NASH models that resolve issues arising from inter-species differences between humans and rodents. In the present study, we developed a choline-deficient, L-amino-acid-defined, high-fat-diet (CDAHFD)-induced human NASH model using human liver chimeric mice. We demonstrated human hepatocyte injury by an elevation of plasma human alanine aminotransferase 1 in mice fed CDAHFD. Histological analysis showed that CDAHFD feeding induced similar histological changes to human NASH patients, including ballooning, inflammation, apoptosis, regeneration of human hepatocytes, and pericellular and perisinusoidal fibrosis. The chimeric mice fed CDAHFD were treated with a peroxisome-proliferator-activated receptor α/δ agonist, Elafibranor. Elafibranor ameliorated steatosis, ballooning of hepatocytes, and preserved fibrosis progression. We developed a novel humanized NASH model that can elucidate pathophysiological mechanisms and predict therapeutic efficacy in human NASH. This model will be useful in exploring new drugs and biomarkers in the early stages of human NASH.

Emerging Roles of T Cells in the Pathogenesis of Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma.

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. A significant proportion of patients with NAFLD develop a progressive inflammatory condition termed nonalcoholic steatohepatitis (NASH), which may eventually advance to cirrhosis and hepatocellular carcinoma (HCC). NASH is characterized by steatosis, hepatocyte ballooning, and lobular inflammation. Heightened immune cell infiltration is a hallmark of NASH, yet the mechanisms whereby hepatic inflammation occurs in NASH and how it contributes to disease initiation and progression remain incompletely understood. Emerging evidence indicates that intrahepatic T cell immune mechanisms play an integral role in the pathogenesis of NASH and its transition to HCC. In this review, we summarize the current knowledge regarding the T cell-mediated mechanisms of inflammation in NASH. We highlight recent preclinical and human studies implicating various subsets of conventional and innate-like T cells in the onset and progression of NASH and HCC. Finally, we discuss the potential therapeutic strategies targeting T cell-mediated responses for the treatment of NASH.

A Novel Mouse Model of Nonalcoholic Steatohepatitis Suggests that Liver Fibrosis Initiates around Lipid-Laden Macrophages

While the interaction of cells such as macrophages and hepatic stellate cells is known to be involved in the generation of fibrosis in nonalcoholic steatohepatitis (NASH), the mechanism remains unclear. This study employed a high-fat/cholesterol/cholate (HFCC) diet to generate a model of NASH-related fibrosis to investigate the pathogenesis of fibrosis. Two mouse strains: C57BL/6J, the one susceptible to obesity, and A/J, the one relatively resistant to obesity, developed hepatic histologic features of NASH, including fat deposition, intralobular inflammation, hepatocyte ballooning, and fibrosis, after 9 weeks of HFCC diet. The severity of hepatic inflammation and fibrosis was greater in A/J mice than in the C57BL/6J mice. A/J mice fed HFCC diet exhibited characteristic CD204-positive lipid-laden macrophage aggregation in hepatic parenchyma. Polarized light was used to visualize the Maltese cross, cholesterol crystals within the aggregated macrophages. Fibrosis developed in a ring shape from the periphery of the aggregated macrophages such that the starting point of fibrosis could be visualized histologically. Matrix-assisted laser desorption/ionization mass spectrometry imaging analysis detected a molecule at m/z 772.462, which corresponds to the protonated ion of phosphatidylcholine [P-18:1 (11Z)/18:0] and phosphatidylethanolamine [18:0/20:2 (11Z, 14Z)], in aggregated macrophages adjacent to the fibrotic lesions. In conclusion, the HFCC diet-fed A/J model provides an ideal tool to study fibrogenesis and enables novel insights into the pathophysiology of NASH-related fibrosis.

OC-13Free Light Chains as a potential biomarker of inflammation in non-alcoholic steatohepatitis

Background: NAFLD is the most common chronic liver disease worldwide. Almost 20-30% of NAFLD patient develop steatohepatitis (NASH) characterized by hepatocyte ballooning, lobular inflammation and progressive fibrosis. Inflammation process in NASH is known to be associated with overexpression of pro-inflammatory biomarkers (IL1-beta, IL6, TNF-alfa).

S2905 Prolonged Cholestasis After COVID-19

Introduction: The coronavirus SARS-CoV-2 (COVID-19) is known to cause both pulmonary and hepatic injury. Cholangiocytes have an abundance of angiotensin-converting enzyme 2 receptors, which are the main mode of entry for the virus. Whether the liver injury is caused directly by the virus or as a result of the cytokine storm remains unknown. We present a case of prolonged cholestatic liver injury secondary to COVID-19. Case Description/Methods: A 55-year-old woman presented to our institution with sudden onset of right leg pain and swelling. She had hypertension, diabetes, obesity, and hypothyroidism. She denied any history of liver disease, toxic habits and was not taking any hepatotoxic medications or supplements. Two months prior to presentation, she was admitted to another hospital with severe COVID-19 pneumonia requiring mechanical ventilation. She had received Remdesivir, Convalescent Plasma, Extra Corporeal Membrane Oxygenation (ECMO), and hemodialysis for acute renal failure, before being discharged. During this admission, she was found to have a right lower extremity thrombus and was started on Enoxaparin. Her liver function tests were significant for a markedly elevated alkaline phosphatase level. An ultrasound abdomen was significant only for a fatty liver. CT scan of the abdomen with contrast and an MRCP did not reveal any hepatobiliary abnormalities. Viral hepatitis serology and autoimmune workup were negative. She underwent a liver biopsy which demonstrated cholestasis, mild lobular necroinflammation, and macrovesicular steatosis. There was no PCR evidence of SARS-CoV-2. Her hospital course was complicated by MRSA and pseudomonas pneumonia. She suffered a cardiac arrest and ultimately succumbed to her infection 2 months later. Her Alkaline phosphatase remained elevated throughout her hospital stay. Discussion: Although our patient’s liver biopsy did not demonstrate PCR evidence of SARS-COV-2, it was consistent with histological features of COVID-19 related hepatic injury. Macrovesicular steatosis is the most common histological finding, followed by lobular necroinflammation, according to a case series of 40 patients who died of COVID-19. PCR for SARS-COV-2 in liver biopsy was positive in only 55% of patients. Recent reports have identified ECMO as a cause of secondary sclerosing cholangitis in critically ill patients, which remains a possibility in our case. However, a normal bilirubin level and MRCP did not support this diagnosis in our patient.Figure 1.: (A) 4 x magnification, (B) 10 x magnification, and (C) 40 x magnification of a liver biopsy specimen demonstrating hepatocellular cholestasis with feathery degeneration of hepatocytes, mild lobular neuroinflammation, and mild macrovesicular steatosis. There is sinusoidal dilatation and glycogenated nuclei. Portal tracts show mild to moderate mixed cellular infiltration, including eosinophils, endothelilitis, ductular reaction, and focal mild bile duct damage. There is no pericellular fibrosis to indicate non-alcoholic chronic steatohepatitis, although rare perivenular ballooned hepatocytes are noted on the H&E slide. CK7 immunostain shows moderate to marked ductular reaction, rare cholestatic hepatocytes, and damaged bile ducts. Findings are those of subacute cholestatic hepatitis.Table 1.: Laboratory values from the previous hospitalization, on admission day of this hospitalization, at 1-month of hospitalization, and prior to death.

METTL3 Regulates Liver Homeostasis, Hepatocyte Ploidy, and Circadian Rhythm–Controlled Gene Expression in Mice

N6-methyladenosine (m6A), the most abundant internal modifier of mRNAs installed by the methyltransferase 13 (METTL3) at the (G/A)(m6A)C motif, plays a critical role in the regulation of gene expression. METTL3 is essential for embryonic development, and its dysregulation is linked to various diseases. However, the role of METTL3 in liver biology is largely unknown. In this study, METTL3 function was unraveled in mice depleted of Mettl3 in neonatal livers (Mettl3fl/fl; Alb-Cre). Liver-specific Mettl3 knockout (M3LKO) mice exhibited global decrease in m6A on polyadenylated RNAs and pathologic features associated with nonalcoholic fatty liver disease (eg, hepatocyte ballooning, ductular reaction, microsteatosis, pleomorphic nuclei, DNA damage, foci of altered hepatocytes, focal lobular and portal inflammation, and elevated serum alanine transaminase/alkaline phosphatase levels). Mettl3-depleted hepatocytes were highly proliferative, with decreased numbers of binucleate hepatocytes and increased nuclear polyploidy. M3LKO livers were characterized by reduced m6A and expression of several key metabolic transcripts regulated by circadian rhythm and decreased nuclear protein levels of the core clock transcription factors BMAL1 and CLOCK. A significant decrease in total Bmal1 and Clock mRNAs but an increase in their nuclear levels were observed in M3LKO livers, suggesting impaired nuclear export. Consistent with the phenotype, methylated (m6A) RNA immunoprecipitation coupled with sequencing and RNA sequencing revealed transcriptome-wide loss of m6A markers and alterations in abundance of mRNAs involved in metabolism in M3LKO. Collectively, METTL3 and m6A modifications are critical regulators of liver homeostasis and function.

Evidence-based clinicalpractice guidelines for nonalcoholic fatty liver disease/nonalcoholic steatohepatitis 2020.

Nonalcoholic fatty liver disease (NAFLD) has become a serious public health issue not only in Western countries but also in Japan. Within the wide spectrum of NAFLD, nonalcoholic steatohepatitis (NASH) is a progressive form of disease that often develops into liver cirrhosis and increases the risk of hepatocellular carcinoma (HCC). While a definite diagnosis of NASH requires liver biopsy to confirm the presence of hepatocyte ballooning, hepatic fibrosis is the most important prognostic factor in NAFLD. With so many NAFLD patients, it is essential to have an effective screening method for NAFLD with hepatic fibrosis. As HCC with non-viral liver disease has increased markedly in Japan, effective screening and surveillance of HCC are also urgently needed. The most common death etiology in NAFLD patients is cardiovascular disease event. Gastroenterologists must, therefore, pay close attention to CVD when examining NAFLD patients. In the updated guidelines, we propose screening and follow-up methods for hepatic fibrosis, HCC, and CVD in NAFLD patients. Several drug trials are ongoing for NAFLD/NASH therapy, however, there is currently no specific drug therapy for NAFLD/NASH. In addition to vitamin E and thiazolidinedione derivatives, recent trials have focused on sodium glucose co-transporter 2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1) analogues, and effective therapies are expected to be developed. These practical guidelines for NAFLD/NASH were established by the Japanese Society of Gastroenterology in conjunction with the Japan Society of Hepatology. Clinical evidence reported internationally between 1983 and October 2018 was collected, and each clinical and background question was evaluated using the Grades of Recommendation Assessment, Development and Evaluation (GRADE) system. This English summary pro- vides the core essentials of these clinical practice guidelines, which include the definition and concept, screening systems for hepatic fibrosis, HCC and CVD, and current therapies for NAFLD/NASH in Japan.

Comparison of ADAPT, FIB-4 and APRI as non-invasive predictors of liver fibrosis and NASH within the CENTAUR screening population

The development of accurate non-invasive tests to detect and measure the extent of fibrosis and disease activity in patients with non-alcoholic steatohepatitis (NASH) - the progressive phenotype of non-alcoholic fatty liver disease (NAFLD) - is of great clinical importance. Herein, we aimed to validate the performance of PRO-C3 and ADAPT for the detection of moderate/severe fibrosis within the CENTAUR screening population. PRO-C3 was assessed in plasma from the screening population of the phase IIb CENTAUR study (NCT02217475) in adults with NASH and liver fibrosis. The relation between PRO-C3 and histologic features of NASH was evaluated, as well as the demographics of patients with high and low levels of PRO-C3. The diagnostic ability of PRO-C3, as a standalone marker or incorporated into ADAPT, to identify patients with F≥2 and NASH was estimated using receiver-operating characteristic analysis and logistic regression models. A total of 517 individuals with matched biopsy and PRO-C3 measurements were included. Patients with PRO-C3 levels ≥20.2 ng/ml showed increased levels of insulin, HOMA-IR, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, and platelet count compared to patients with low PRO-C3 (p <0.05). PRO-C3 increased stepwise with increasing liver fibrosis, lobular inflammation, hepatocyte ballooning, steatosis, and NAFLD activity score (p <0.05), and could distinguish between NAFL and NASH (p <0.0001). PRO-C3 was independently associated with fibrosis and NASH when adjusted for clinical confounders. ADAPT outperformed Fibrosis-4, AST-to-platelet ratio index, and AST/ALT ratio as a predictor of advanced fibrosis and NASH (p <0.001). PRO-C3 was associated with NAFLD activity score and fibrosis. ADAPT outperformed other non-invasive scores for detecting NASH. These data support the use of PRO-C3 and ADAPT as diagnostic tools to identify patients with NASH eligible for inclusion in clinical trials. NCT02217475 LAY SUMMARY: PRO-C3 is a serological biomarker associated with liver disease activity and fibrosis. Its performance for the detection of disease activity and fibrosis is improved when it is incorporated into the ADAPT score. Herein, we showed that ADAPT was better at selecting patients with non-alcoholic steatohepatitis for inclusion in clinical trials than other non-invasive scores.

Establishment of a murine model of acute-on-chronic liver failure with multi-organ dysfunction

Acute-on-chronic liver failure (ACLF) is a distinct clinical entity with high probability of organ failure and mortality. Since patients generally present late, experimental models are needed to understand the pathophysiology and natural course of the disease. To reproduce the syndrome of ACLF, chronic liver disease was induced in C57BL6 mice (6-8weeks; approximately 20-24g weight) by intraperitoneal administration of carbon tetrachloride (CCl4) for 10weeks followed by an acute injury with acetaminophen (APAP) and lipopolysaccharide (LPS). Blood, ascitic fluid, and organs were collected to study cell death, regeneration, and fibrosis. At 24h post-APAP/LPS infusion, the liver tissue showed increased hepatocyte ballooning and endothelial cell TUNEL positivity. This was followed by progressive hepatocyte necrosis from perivascular region at day 7 to lobular region by day 11. ACLF (day 7 and day 11) animals showed increase in bilirubin (p < 0.05), prothrombin time (p < 0.0001), blood ammonia (p < 0.001), and portal pressure post-acute hepatocellular injury similar to human ACLF. Ascites was noticed by day 11 with median serum-ascites albumin gradient of 1.2 (1.1-1.3)g/dL. In comparison to cirrhosis, ACLF group (day 7 and day 11) showed significant decrease in Sirius red (p ≤ 0.0001), collagen1 (p < 0.0001), and a-SMA proportionate area (p < 0.0001) with loss of hepatocytes regeneration (p < 0.005). At day 11, ACLF animals also showed significant increase in serum creatinine (p < 0.05) and acute tubular necrosis suggestive of organ failure, compared to cirrhotic animals. The CCL4/APAP/LPS (CALPS) model of ACLF mimics the clinical, biochemical, and histological features of ACLF with demonstrable progressive hepatocellular necrosis, liver failure, impaired regeneration, development of portal hypertension, and organ dysfunction in an animal with chronic liver disease.

Critical Role for Hepatocyte-Specific eNOS in NAFLD and NASH.

Regulation of endothelial nitric oxide synthase (eNOS) in hepatocytes may be an important target in nonalcoholic fatty liver disease (NAFLD) development and progression to nonalcoholic steatohepatitis (NASH). In this study, we show genetic deletion and viral knockdown of hepatocyte-specific eNOS exacerbated hepatic steatosis and inflammation, decreased hepatic mitochondrial fatty acid oxidation and respiration, increased mitochondrial H2O2 emission, and impaired the hepatic mitophagic (BNIP3 and LC3II) response. Conversely, overexpressing eNOS in hepatocytes in vitro and in vivo increased hepatocyte mitochondrial respiration and attenuated Western diet-induced NASH. Moreover, patients with elevated NAFLD activity score (histology score of worsening steatosis, hepatocyte ballooning, and inflammation) exhibited reduced hepatic eNOS expression, which correlated with reduced hepatic mitochondrial fatty acid oxidation and lower hepatic protein expression of mitophagy protein BNIP3. The current study reveals an important molecular role for hepatocyte-specific eNOS as a key regulator of NAFLD/NASH susceptibility and mitochondrial quality control with direct clinical correlation to patients with NASH.

Ballooning overview of liver histopathology of adolescent rat models with type 2 diabetes mellitus induced by high-fat diet and Streptozotocin

Background and Aims: Type 2 diabetes mellitus (T2DM) becomes the top 10 non-communicable diseases. Patients with T2DM usually have a risk of having non-alcoholic steatohepatitis (NASH). It begins from insulin resistance, leading to triglyceride accumulation and a higher risk of atherosclerosis. In histopathology, there are several overviews in this condition, and one of them is a ballooning of hepatocytes. This study aims to observe correlations between T2DM with ballooning overviews of liver histopathology in adolescent rats.

High frequency and long persistency of ballooning hepatocyte were associated with glucose intolerance in patients with severe obesity

Nonalcoholic steatohepatitis (NASH) and glucose intolerance are associated with an increased risk of mortality in patients with severe obesity; however, whether histological findings of the liver are related to glucose intolerance in these patients remain unknown. Sixty-nine consecutive patients who underwent metabolic surgery between June 2008 and February 2020 were included; histological findings of the liver and laboratory data were analyzed. Twenty patients with biopsy-proven NASH were chronologically evaluated using sequential biopsies; data before metabolic surgery was considered as the baseline. Glucose intolerance—demonstrated by an increased area under the curve (AUC) for blood sugar (BS) during the 75-g oral glucose tolerance test—and increased homeostatic model assessment for insulin resistance (HOMA-IR) correlated with the grade of hepatocyte ballooning in patients. Patients with persistent ballooning at the follow-up biopsy had a higher HOMA-IR, high AUC for BS, and lower adiponectin level than those in patients in whom ballooning was eliminated, while there was no significant difference in body weight. We concluded that glucose intolerance was associated with the grade of hepatocyte ballooning; additionally, persistent hepatocyte ballooning sustained glucose intolerance, while elimination of hepatocyte ballooning improved the condition. Glucose intolerance may, thus, mediate balloon formation of the hepatocyte.