Paraneoplastic Kelch-like protein 11 antibody-associated cerebellar and limbic encephalitis caused by metastatic "burned-out" seminoma - A scar(r)y phenomenon.

INTRODUCTION: The diagnosis of paraneoplastic neurologic syndromes is challenging when the primary tumor masquerades as scar tissue (i.e. "burned-out"). METHODS: Case report. RESULTS: A 45-year-old male patient presented with progressive cerebellar symptoms and hearing loss. Initial screening for malignancy and extensive testing of paraneoplastic and autoimmune neuronal antibodies gave negative results. Repeated whole-body FDG-PET CT revealed a single paraaortic lymphadenopathy, metastasis of a regressed testicular seminoma. Anti-Kelch-like protein-11 (KLHL11) encephalitis was finally diagnosed. CONCLUSION: Our case highlights the importance of continued efforts to find an often burned-out testicular cancer in patients with a highly unique clinical presentation of KLHL11 encephalitis.

HunCRC: annotated pathological slides to enhance deep learning applications in colorectal cancer screening.

Histopathology is the gold standard method for staging and grading human tumors and provides critical information for the oncoteam's decision making. Highly-trained pathologists are needed for careful microscopic analysis of the slides produced from tissue taken from biopsy. This is a time-consuming process. A reliable decision support system would assist healthcare systems that often suffer from a shortage of pathologists. Recent advances in digital pathology allow for high-resolution digitalization of pathological slides. Digital slide scanners combined with modern computer vision models, such as convolutional neural networks, can help pathologists in their everyday work, resulting in shortened diagnosis times. In this study, 200 digital whole-slide images are published which were collected via hematoxylin-eosin stained colorectal biopsy. Alongside the whole-slide images, detailed region level annotations are also provided for ten relevant pathological classes. The 200 digital slides, after pre-processing, resulted in 101,389 patches. A single patch is a 512 × 512 pixel image, covering 248 × 248 µm2 tissue area. Versions at higher resolution are available as well. Hopefully, HunCRC, this widely accessible dataset will aid future colorectal cancer computer-aided diagnosis and research.

Chronic alcohol-induced neuroinflammation involves CCR2/5-dependent peripheral macrophage infiltration and microglia alterations.

BACKGROUND: Chronic alcohol consumption is associated with neuroinflammation, neuronal damage, and behavioral alterations including addiction. Alcohol-induced neuroinflammation is characterized by increased expression of proinflammatory cytokines (including TNFα, IL-1ß, and CCL2) and microglial activation. We hypothesized chronic alcohol consumption results in peripheral immune cell infiltration to the CNS. Since chemotaxis through the CCL2-CCR2 signaling axis is critical for macrophage recruitment peripherally and centrally, we further hypothesized that blockade of CCL2 signaling using the dual CCR2/5 inhibitor cenicriviroc (CVC) would prevent alcohol-induced CNS infiltration of peripheral macrophages and alter the neuroinflammatory state in the brain after chronic alcohol consumption. METHODS: C57BL/6J female mice were fed an isocaloric or 5% (v/v) ethanol Lieber DeCarli diet for 6 weeks. Some mice received daily injections of CVC. Microglia and infiltrating macrophages were characterized and quantified by flow cytometry and visualized using CX3CR1eGFP/+ CCR2RFP/+ reporter mice. The effect of ethanol and CVC treatment on the expression of inflammatory genes was evaluated in various regions of the brain, using a Nanostring nCounter inflammation panel. Microglia activation was analyzed by immunofluorescence. CVC-treated and untreated mice were presented with the two-bottle choice test. RESULTS: Chronic alcohol consumption induced microglia activation and peripheral macrophage infiltration in the CNS, particularly in the hippocampus. Treatment with CVC abrogated ethanol-induced recruitment of peripheral macrophages and partially reversed microglia activation. Furthermore, the expression of proinflammatory markers was upregulated by chronic alcohol consumption in various regions of the brain, including the cortex, hippocampus, and cerebellum. Inhibition of CCR2/5 decreased alcohol-mediated expression of inflammatory markers. Finally, microglia function was impaired by chronic alcohol consumption and restored by CVC treatment. CVC treatment did not change the ethanol consumption or preference of mice in the two-bottle choice test. CONCLUSIONS: Together, our data establish that chronic alcohol consumption promotes the recruitment of peripheral macrophages into the CNS and microglia alterations through the CCR2/5 axis. Therefore, further exploration of the CCR2/5 axis as a modulator of neuroinflammation may offer a potential therapeutic approach for the treatment of alcohol-associated neuroinflammation.

Development and Initial Testing of a Modified UroVysion-Based Fluorescence In Situ Hybridization Score for Prediction of Progression in Bladder Cancer.

OBJECTIVES: Our aim was to predict progression of non-muscle-invasive bladder urothelial carcinomas (NMIUCs) into muscle-invasive disease by assessing cytogenetic abnormality of tumors with a new UroVysion scoring system. METHODS: Seventy-five bladder cancer cases (including 57 NMIUCs) were classified according to the quantitatively assessed degree of UroVysion-detected chromosomal abnormalities into urine fluorescence in situ hybridization score (UFS) groups: UFS I, II, and III. Cox time-to-event, Kaplan-Meier, and C-statistics analyses were performed. RESULTS: UFS proved to be an independent prognostic factor of progression-free survival (PFS) and time to progression (TTP). NMIUCs with UFS III had a 34.05-fold increased hazard for progression to muscle-invasive cancer (TTP; 95% confidence interval, 5.841-198.5; P < .001) in comparison with UFS I to II cases. The addition of UFS to conventional risk scores increased the C-index for PFS and TTP. CONCLUSIONS: UFS can indicate an increased risk for progression into muscle-invasive disease in patients with NMIUC and improves prognostic accuracy of the current clinical risk assessment systems.

Pharmacological Inhibition of CCR2/5 Signaling Prevents and Reverses Alcohol-Induced Liver Damage, Steatosis, and Inflammation in Mice.

Kupffer cell and macrophage (MØ) activation contributes to steatosis, inflammation, and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells, and expression of C-C chemokine receptor type 2 (Ccr2) and C-C chemokine receptor type 5 (Ccr5) in the livers of patients with ALD, and increased circulating chemokines, C-C chemokine ligand types 2 (CCL2), and C-C chemokine ligand types 5 (CCL5) in patients with alcoholic hepatitis. We hypothesized that inhibition of CCL2 signaling with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate ALD. In a mouse model of ALD, liver injury (alanine aminotransferase [ALT]) and steatosis were prevented by CVC whether administered as "prevention" throughout the alcohol feeding or as "treatment" started after the development of ALD. Alcohol-induced increases in early liver fibrosis markers (sirius red, hydroxyproline, and collagen-1) were normalized by both modes of CVC administration. We found that prevention and treatment with CVC reversed alcohol-related increases in liver mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and CCL2. CVC administration regimens prevented the increase in infiltrating MØ (F4/80lo CD11bhi ) and reduced proinflammatory Ly6Chi MØ in livers of alcohol-fed mice. CVC increased liver T-cell numbers and attenuated Il-2 expression without an effect on CD69+ or CD25+ T-cell expression. In vitro, CVC inhibited CCL2-induced increases in hepatocyte fatty acid synthase (Fasn) and adipose differentiation-related protein (Adrp), whereas it augmented acyl-coenzyme A oxidase 1 (Acox-1), proliferator-activated receptor gamma co-activator alpha (Pgc1α) and uncoupling protein 2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to lipopolysaccharide-induced liver injury (TNF-α, ALT, and lactate dehydrogenase release). Alcohol feeding induced apoptosis (poly ADP-ribose polymerase [PARP] and caspase-3 [CASP-3] cleavage) and pyroptosis (gasdermin D [GSDMD] cleavage) in livers, and CVC prevented both of these forms of cell death. Conclusion: Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol-induced steatohepatitis and liver damage.

MicroRNA Expression in Focal Nodular Hyperplasia in Comparison with Cirrhosis and Hepatocellular Carcinoma.

The liver disease focal nodular hyperplasia (FNH) has several histological features that resemble hepatic cirrhosis. Since cirrhosis may develop further into hepatocellular carcinoma (HCC) contrary to FNH, the aim of the present study was to identify microRNAs (miRNA), which, by their altered expression levels, may be associated with the benign, tumor-like nature of FNH. Altogether 106 surgically removed formalin-fixed paraffin-embedded liver samples were selected, including 22 FNH, 45 cirrhosis, 24 HCC and 15 normal liver tissues. Etiology of the cases of cirrhosis and HCC includes hepatitis C and alcoholism and the HCC cases developed in cirrhotic livers. Relative expression levels of 14 miRNAs were determined using TaqMan MicroRNA Assays. In comparison to normal liver, the levels of miR-34a and miR-224 were elevated not only in FNH but also in cirrhosis and HCC, while the expression of miR-17-5p, miR-18a and miR-210 was decreased in FNH. Further, the levels of miR-21 and miR-222 were increased in cirrhosis and HCC but were decreased in FNH and the expression of miR-17-5p, miR-18a, miR-195 and miR-210 was decreased in FNH as compared with cirrhosis and/or HCC. In conclusion, the elevation of miR-34a and miR-224 may be associated with both benign and malignant proliferative processes, nevertheless the increased expression of oncomiRs miR-21 and miR-222 in cirrhosis and HCC but not in FNH may be related to malignant processes of the liver. The decreased levels of miR-18a, miR-195 and miR-210 may further differentiate FNH from cirrhosis, reflecting the different pathogenesis of these two entities contrary to some histologically similar features.

Reduced gut microbiome protects from alcohol-induced neuroinflammation and alters intestinal and brain inflammasome expression.

BACKGROUND: The end-organ effects of alcohol span throughout the entire body, from the gastrointestinal tract to the central nervous system (CNS). In the intestine, alcohol use changes the microbiome composition and increases gut permeability allowing translocation of microbial components into the circulation. Gut-derived pathogen-associated signals initiate inflammatory responses in the liver and possibly elsewhere in the body. Because previous studies showed that the gut microbiome contributes to alcohol-induced liver disease, we hypothesized that antibiotic administration to reduce the gut microbiome would attenuate alcohol-induced inflammation in the brain and small intestine (SI). METHODS: Six- to 8-week-old C57BL/6J female mice were fed alcohol in a liquid diet or a calorie-matched control diet for 10 days with an acute alcohol binge or sugar on the final day (acute-on-chronic alcohol administration). Some mice were treated with oral antibiotics daily to diminish the gut microbiome. We compared serum levels of TNFα, IL-6, and IL-1ß by ELISA; expression of cytokines Tnfα, Mcp1, Hmgb1, Il-17, Il-23, Il-6, and Cox2; and inflammasome components Il-1ß, Il-18, Casp1, Asc, and Nlrp3 in the CNS and SI by qRT-PCR. Microglial morphology was analyzed using immunohistochemical IBA1 staining in the cortex and hippocampus. RESULTS: Antibiotics dramatically reduced the gut microbiome load in both alcohol- and pair-fed mice. Alcohol-induced neuroinflammation and increase in SI cytokine expression were attenuated in mice with antibiotic treatment. Acute-on-chronic alcohol did not induce serum TNFα, IL-6, and IL-1ß. Alcohol feeding significantly increased the expression of proinflammatory cytokines such as Tnfα, Mcp1, Hmgb1, Il-17, and Il-23 in the brain and intestine. Reduction in the gut bacterial load, as a result of antibiotic treatment, attenuated the expression of all of these alcohol-induced proinflammatory cytokines in both the brain and SI. Alcohol feeding resulted in microglia activation and morphologic changes in the cortex and hippocampus characterized by a reactive phenotype. These alcohol-induced changes were abrogated following an antibiotic-induced reduction in the gut microbiome. Unexpectedly, antibiotic treatment increased the mRNA expression of some inflammasome components in both the brain and intestine. CONCLUSIONS: Our data show for the first time that the acute-on-chronic alcohol administration in mice induces both neuroinflammation and intestinal inflammation and that reduction in the intestinal bacterial load can attenuate alcohol-associated CNS and gut inflammation. Gut microbiome-derived signals contribute to neuroinflammation in acute-on-chronic alcohol exposure.

Abnormal neutrophil traps and impaired efferocytosis contribute to liver injury and sepsis severity after binge alcohol use.

BACKGROUND & AIMS: Neutrophil extracellular traps (NETs) are an important strategy utilized by neutrophils to immobilize and kill invading microorganisms. Herein, we studied NET formation and the process of neutrophil cell death (NETosis), as well as the clearance of NETs by macrophages (MΦ) (efferocytosis) in acute sepsis following binge drinking. METHODS: Healthy volunteers consumed 2 ml of vodka/kg body weight, before blood endotoxin and 16 s rDNA were measured. Peripheral neutrophils were isolated and exposed to alcohol followed by phorbol 12-myristate 13-acetate (PMA) stimulation. Mice were treated with three alcohol binges and intraperitoneal lipopolysaccharide (LPS) to assess the dynamics of NET formation and efferocytosis. In vivo, anti-Ly6G antibody (IA8) was used for neutrophil depletion. RESULTS: Inducers of NETs (endotoxin and bacterial DNA) significantly increased in the circulation after binge alcohol drinking in humans. Ex vivo, alcohol alone increased NET formation, but upon PMA stimulation alcohol attenuated NET formation. Binge alcohol in mice resulted in a biphasic response to LPS. Initially, binge alcohol reduced LPS-induced NET formation and resulted in a diffuse distribution of neutrophils in the liver compared to alcohol-naïve mice. Moreover, indicators of NET formation including citrullinated histone H3, neutrophil elastase, and neutrophil myeloperoxidase were decreased at an early time point after LPS challenge in mice receiving binge alcohol, suggesting decreased NET formation. However, in the efferocytosis phase (15 h after LPS) citrullinated histone-H3 was increased in the liver in alcohol binge mice, suggesting decreased clearance of NETs. In vitro alcohol treatment reduced efferocytosis and phagocytosis of NETotic neutrophils and promoted expression of CD206 on MΦ. Finally, depletion of neutrophils prior to binge alcohol ameliorated LPS-induced systemic inflammation and liver injury in mice. CONCLUSIONS: Dysfunctional NETosis and efferocytosis following binge drinking exacerbate liver injury associated with sepsis. LAY SUMMARY: Disease severity in alcoholic liver disease (ALD) is associated with a significant presence of neutrophils (a type of immune cell) in the liver. It remains unknown how alcohol affects the capacity of neutrophils to control infection, a major hallmark of ALD. We found that binge alcohol drinking impaired important strategies used by neutrophils to contain and resolve infection, resulting in increased liver injury during ALD.

MicroRNA 122, Regulated by GRLH2, Protects Livers of Mice and Patients From Ethanol-Induced Liver Disease.

BACKGROUND & AIMS: Chronic, excessive alcohol consumption leads to alcoholic liver disease (ALD) characterized by steatosis, inflammation, and eventually cirrhosis. The hepatocyte specific microRNA 122 (MIR122) regulates hepatocyte differentiation and metabolism. We investigated whether an alcohol-induced decrease in level of MIR122 contributes to development of ALD. METHODS: We obtained liver samples from 12 patients with ALD and cirrhosis and 9 healthy individuals (controls) and analyzed them by histology and immunohistochemistry. C57Bl/6 mice were placed on a Lieber-DeCarli liquid diet, in which they were fed ethanol for 8 weeks, as a model of ALD, or a control diet. These mice were also given injections of CCl4, to increase liver fibrosis, for 8 weeks. On day 28, mice with ethanol-induced liver disease and advanced fibrosis, and controls, were given injections of recombinant adeno-associated virus 8 vector that expressed the primary miR-122 transcript (pri-MIR122, to overexpress MIR122 in hepatocytes) or vector (control). Two weeks before ethanol feeding, some mice were given injections of a vector that expressed an anti-MIR122, to knock down its expression. Serum and liver tissues were collected; hepatocytes and liver mononuclear cells were analyzed by histology, immunoblots, and confocal microscopy. We performed in silico analyses to identify targets of MIR122 and chromatin immunoprecipitation quantitative polymerase chain reaction analyses in Huh-7 cells. RESULTS: Levels of MIR122 were decreased in liver samples from patients with ALD and mice on the Lieber-DeCarli diet, compared with controls. Transgenic expression of MIR122 in hepatocytes of mice with ethanol-induced liver disease and advanced fibrosis significantly reduced serum levels of alanine aminotransferase (ALT) and liver steatosis and fibrosis, compared with mice given injections of the control vector. Ethanol feeding reduced expression of pri-MIR122 by increasing expression of the spliced form of the transcription factor grainyhead like transcription factor 2 (GRHL2) in liver tissues from mice. Levels of GRHL2 also were increased in liver tissues from patients with ALD, compared with controls; increases correlated with decreases in levels of MIR122 in human liver. Mice given injections of the anti-MIR122 before ethanol feeding had increased steatosis, inflammation, and serum levels of alanine aminotransferase compared with mice given a control vector. Levels of hypoxia-inducible factor 1 alpha (HIF1α) mRNA, a target of MIR122, were increased in liver tissues from patients and mice with ALD, compared with controls. Mice with hepatocyte-specific disruption of Hif1α developed less-severe liver injury following administration of ethanol, injection of anti-MIR122, or both. CONCLUSIONS: Levels of MIR122 decrease in livers from patients with ALD and mice with ethanol-induced liver disease, compared with controls. Transcription of MIR122 is inhibited by GRHL2, which is increased in livers of mice and patients with ALD. Expression of an anti-MIR122 worsened the severity of liver damage following ethanol feeding in mice. MIR122 appears to protect the liver from ethanol-induced damage by reducing levels of HIF1α. These processes might be manipulated to reduce the severity of ALD in patients.

Recovery of ethanol-induced Akkermansia muciniphila depletion ameliorates alcoholic liver disease.

OBJECTIVE: Alcoholic liver disease (ALD) is a global health problem with limited therapeutic options. Intestinal barrier integrity and the microbiota modulate susceptibility to ALD. Akkermansia muciniphila, a Gram-negative intestinal commensal, promotes barrier function partly by enhancing mucus production. The aim of this study was to investigate microbial alterations in ALD and to define the impact of A. muciniphila administration on the course of ALD. DESIGN: The intestinal microbiota was analysed in an unbiased approach by 16S ribosomal DNA (rDNA) sequencing in a Lieber-DeCarli ALD mouse model, and faecal A. muciniphila abundance was determined in a cohort of patients with alcoholic steatohepatitis (ASH). The impact of A. muciniphila on the development of experimental acute and chronic ALD was determined in a preventive and therapeutic setting, and intestinal barrier integrity was analysed. RESULTS: Patients with ASH exhibited a decreased abundance of faecal A. muciniphila when compared with healthy controls that indirectly correlated with hepatic disease severity. Ethanol feeding of wild-type mice resulted in a prominent decline in A. muciniphila abundance. Ethanol-induced intestinal A. muciniphila depletion could be restored by oral A. muciniphila supplementation. Furthermore, A. muciniphila administration when performed in a preventive setting decreased hepatic injury, steatosis and neutrophil infiltration. A. muciniphila also protected against ethanol-induced gut leakiness, enhanced mucus thickness and tight-junction expression. In already established ALD, A. muciniphila used therapeutically ameliorated hepatic injury and neutrophil infiltration. CONCLUSION: Ethanol exposure diminishes intestinal A. muciniphila abundance in both mice and humans and can be recovered in experimental ALD by oral supplementation. A. muciniphila promotes intestinal barrier integrity and ameliorates experimental ALD. Our data suggest that patients with ALD might benefit from A. muciniphila supplementation.

Correction: Alcohol-related changes in the intestinal microbiome influence neutrophil infiltration, inflammation and steatosis in early alcoholic hepatitis in mice.

[This corrects the article DOI: 10.1371/journal.pone.0174544.].

Alcohol-related changes in the intestinal microbiome influence neutrophil infiltration, inflammation and steatosis in early alcoholic hepatitis in mice.

BACKGROUND: Alcohol-induced intestinal dysbiosis disrupts homeostatic gut-liver axis function and is essential in the development of alcoholic liver disease. Here, we investigate changes in enteric microbiome composition in a model of early alcoholic steatohepatitis and dissect the pathogenic role of intestinal microbes in alcohol-induced liver pathology. MATERIALS AND METHODS: Wild type mice received a 10-day diet that was either 5% alcohol-containing or an isocaloric control diet plus a single binge. 16S rDNA sequencing defined the bacterial communities in the cecum of alcohol- and pair-fed animals. Some mice were treated with an antibiotic cocktail prior to and throughout alcohol feeding. Liver neutrophils, cytokines and steatosis were evaluated. RESULTS: Acute-on-chronic alcohol administration induced shifts in various bacterial phyla in the cecum, including increased Actinobacteria and a reduction in Verrucomicrobia driven entirely by a reduction in the genus Akkermansia. Antibiotic treatment reduced the gut bacterial load and circulating bacterial wall component lipopolysaccharide (LPS). We found that bacterial load suppression prevented alcohol-related increases in the number of myeloperoxidase- (MPO) positive infiltrating neutrophils in the liver. Expression of liver mRNA tumor necrosis factor alpha (Tnfα), C-X-C motif chemokine ligand 1 (Cxcl1) and circulating protein monocyte chemoattractant protein-1 (MCP-1) were also reduced in antibiotic-treated alcohol-fed mice. Alcohol-induced hepatic steatosis measured by Oil-Red O staining was significantly reduced in antibiotic treated mice. Genes regulating lipid production and storage were also altered by alcohol and antibiotic treatment. Interestingly, antibiotic treatment did not protect from alcohol-induced increases in serum aminotransferases (ALT/AST). CONCLUSIONS: Our data indicate that acute-on-chronic alcohol feeding alters the microflora at multiple taxonomic levels and identifies loss of Akkermansia as an early marker of alcohol-induced gut dysbiosis. We conclude that gut microbes influence liver inflammation, neutrophil infiltration and liver steatosis following alcohol consumption and these data further emphasize the role of the gut-liver axis in early alcoholic liver disease.

Hepatocellular carcinoma is accelerated by NASH involving M2 macrophage polarization mediated by hif-1αinduced IL-10.

Obesity-related inflammation promotes cancer development. Tissue resident macrophages affect tumor progression and the tumor micro-environment favors polarization into alternatively activated macrophages (M2) that facilitate tumor invasiveness. Here, we dissected the role of western diet-induced NASH in inducing macrophage polarization in a carcinogen initiated model of hepatocellular carcinoma (HCC). Adult C57BL/6 male mice received diethyl nitrosamine (DEN) followed by 24 weeks of high fat-high cholesterol-high sugar diet (HF-HC-HSD). We assessed liver MRI and histology, serum ALT, AFP, liver triglycerides, and cytokines. Macrophage polarization was determined by IL-12/TNFα (M1) and CD163/CD206 (M2) expression using flow cytometry. Role of hif-1α-induced IL-10 was dissected in hepatocyte specific hif-1αKO and hif-1αdPA (over-expression) mice. The western diet-induced features of NASH and accelerated HCC development after carcinogen exposure. Liver fibrosis and serum AFP were significantly increased in DEN + HF-HC-HSD mice compared to controls. Western diet resulted in macrophage (F4/80+CD11b+) infiltration to liver and DEN + HF-HC-HSD mice showed preferential increase in M2 macrophages. Isolated hepatocytes from western diet fed mice showed significant upregulation of the hypoxia-inducible transcription factor, hif-1α, and livers from hif-1α over-expressing mice had increased proportion of M2 macrophages. Primary hepatocytes from wild-type mice treated with DEN and palmitic acid in vitro showed activation of hif-1α and induction of IL-10, a M2 polarizing cytokine. IL-10 neutralization in hepatocyte-derived culture supernatant prevented M2 macrophage polarization and silencing hif-1α in macrophages blocked their M2 polarization. Therefore, our data demonstrate that NASH accelerates HCC progression via upregulation of hif-1α mediated IL-10 polarizing M2 macrophages.

Endoplasmic Reticulum Stress-induced Hepatocellular Death Pathways Mediate Liver Injury and Fibrosis via Stimulator of Interferon Genes.

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl4 administration to WT and IRF3-, Toll/Interleukin-1R (TIR) domain-containing adapter-inducing interferon-ß (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4 Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4 In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.

[Vitamin D metabolism and signaling in human hepatocellular carcinoma and surrounding non-tumorous liver]. / A D-vitamin metabolizmusa humán hepatocellularis carcinomában és az azt körülvevo tumormentes májszövetben.

INTRODUCTION: 1,25-Dihydroxy vitamin D3 mediates antitumor effects in hepatocellular carcinoma. AIM: We examined mRNA and protein expression differences in 1,25-Dihydroxy vitamin D3-inactivating CYP24A1, mRNA of activating CYP27B1 enzymes, and that of VDR between human hepatocellular carcinoma and surrounding non-tumorous liver. METHODS: Snap-frozen tissues from 13 patients were studied for mRNA and protein expression of CYP24A1. Paraffin-embedded tissues from 36 patients were used to study mRNA of VDR and CYP27B1. mRNA expression was measured by RT-PCR, CYP24A1 protein was detected by immunohistochemistry. RESULTS: Expression of VDR and CYP27B1 was significantly lower in hepatocellular carcinoma compared with non-tumorous liver (p<0.05). The majority of the HCC samples expressed CYP24A1 mRNA, but neither of the non-tumorous liver. The gene activation was followed by CYP24A1 protein synthesis. CONCLUSIONS: The presence of CYP24A1 mRNA and the reduced expression of VDR and CYP27B1 mRNA in human hepatocellular carcinoma samples indicate decreased bioavailability of 1,25-Dihydroxy vitamin D3, providing an escape mechanism from the anti-tumor effect. Orv. Hetil., 2016, 157(48), 1910-1918.

Inhibition of spleen tyrosine kinase activation ameliorates inflammation, cell death, and steatosis in alcoholic liver disease.

UNLABELLED: The spectrum of alcoholic liver disease (ALD) is a major cause of mortality with limited therapies available. Because alcohol targets numerous signaling pathways in hepatocytes and in immune cells, the identification of a master regulatory target that modulates multiple signaling processes is attractive. In this report, we assessed the role of spleen tyrosine kinase (SYK), a nonreceptor tyrosine kinase, which has a central modulatory role in multiple proinflammatory signaling pathways involved in the pathomechanism of ALD. Using mouse disease models that represent various phases in the progression of human ALD, we found that alcohol, in all of these models, induced SYK activation in the liver, both in hepatocytes and liver mononuclear cells. Furthermore, significant SYK activation also occurred in liver samples and peripheral blood mononuclear cells of patients with ALD/alcoholic hepatitis compared to controls. Functional inhibition of SYK activation in vivo abrogated alcohol-induced hepatic neutrophil infiltration, resident immune cell activation, as well as inflammasome and extracellular signal-regulated kinase 1 and 2-mediated nuclear factor kappa B activation in mice. Strikingly, inhibition of SYK activation diminished alcohol-induced hepatic steatosis and interferon regulatory factor 3-mediated apoptosis. CONCLUSION: Our data demonstrate a novel, functional, and multicellular role for SYK phosphorylation in modulating immune cell-driven liver inflammation, hepatocyte cell death, and steatosis at different stages of ALD. These novel findings highlight SYK as a potential multifunctional target in the treatment of alcoholic steatohepatitis. (Hepatology 2016;64:1057-1071).

Therapeutic Benefits of Spleen Tyrosine Kinase Inhibitor Administration on Binge Drinking-Induced Alcoholic Liver Injury, Steatosis, and Inflammation in Mice.

BACKGROUND: Binge drinking is increasingly recognized as an important cause of liver disease with limited therapeutic options for patients. Binge alcohol use, similar to chronic alcohol consumption, induces numerous deregulated signaling events that drive liver damage, steatosis, and inflammation. In this article, we evaluated the role of spleen tyrosine kinase (SYK), which modulates numerous signaling events previously identified linked in the development alcohol-induced liver pathology. METHODS: A 3-day alcohol binge was administered to C57BL/6 female mice, and features of alcoholic liver disease were assessed. Some mice were treated daily with intraperitoneal injections of a SYK inhibitor (R406; 5 to 10 mg/kg body weight) or drug vehicle control. Liver and serum samples were collected and were assessed by Western blotting, biochemical, ELISA, electrophoretic mobility shift assays, real-time quantitative polymerase chain reaction, and histopathological analysis. RESULTS: We found that binge drinking induced significant SYK activation (SYK(Y525/526) ) with no change in total SYK expression in the liver. Functional inhibition of SYK activation using a potent SYK inhibitor, R406, was associated with a significant decrease in alcohol-induced hepatic inflammation as demonstrated by decreased phospho-nuclear factor kappa beta (NF-κB) p65, NF-κB nuclear binding, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 mRNA in the liver. Compared to vehicle controls, SYK inhibitor treatment decreased alcohol binge-induced hepatocyte injury indicated by histology and serum alanine aminotransferase. Strikingly, SYK inhibitor treatment also resulted in a significant reduction in alcohol-induced liver steatosis. CONCLUSIONS: Our novel observations demonstrate the role of SYK, activation in the pathomechanism of binge drinking-induced liver disease highlighting SYK a potential multifaceted therapeutic target.

Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease.

AIM: To establish a mouse model of alcohol-driven hepatocellular carcinoma (HCC) that develops in livers with alcoholic liver disease (ALD). METHODS: Adult C57BL/6 male mice received multiple doses of chemical carcinogen diethyl nitrosamine (DEN) followed by 7 wk of 4% Lieber-DeCarli diet. Serum alanine aminotransferase (ALT), alpha fetoprotein (AFP) and liver Cyp2e1 were assessed. Expression of F4/80, CD68 for macrophages and Ly6G, MPO, E-selectin for neutrophils was measured. Macrophage polarization was determined by IL-1ß/iNOS (M1) and Arg-1/IL-10/CD163/CD206 (M2) expression. Liver steatosis and fibrosis were measured by oil-red-O and Sirius red staining respectively. HCC development was monitored by magnetic resonance imaging, confirmed by histology. Cellular proliferation was assessed by proliferating cell nuclear antigen (PCNA). RESULTS: Alcohol-DEN mice showed higher ALTs than pair fed-DEN mice throughout the alcohol feeding without weight gain. Alcohol feeding resulted in increased ALT, liver steatosis and inflammation compared to pair-fed controls. Alcohol-DEN mice had reduced steatosis and increased fibrosis indicating advanced liver disease. Molecular characterization showed highest levels of both neutrophil and macrophage markers in alcohol-DEN livers. Importantly, M2 macrophages were predominantly higher in alcohol-DEN livers. Magnetic resonance imaging revealed increased numbers of intrahepatic cysts and liver histology confirmed the presence of early HCC in alcohol-DEN mice compared to all other groups. This correlated with increased serum alpha-fetoprotein, a marker of HCC, in alcohol-DEN mice. PCNA immunostaining revealed significantly increased hepatocyte proliferation in livers from alcohol-DEN compared to pair fed-DEN or alcohol-fed mice. CONCLUSION: We describe a new 12-wk HCC model in adult mice that develops in livers with alcoholic hepatitis and defines ALD as co-factor in HCC.

Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome activation and protects from alcoholic steatohepatitis in mice.

BACKGROUND & AIMS: The inflammasome is a well-characterized inducer of inflammation in alcoholic steatohepatitis (ASH). Inflammasome activation requires two signals for mature interleukin (IL)-1ß production. Here we asked whether metabolic danger signals trigger inflammasome activation in ASH. METHODS: Wild-type mice, ATP receptor 2x7 (P2rx7)-KO mice, or mice overexpressing uricase were fed Lieber-DeCarli ethanol or control diet. We also implemented a pharmacological approach in which mice were treated with probenecid or allopurinol. RESULTS: The sterile danger signals, ATP and uric acid, were increased in the serum and liver of alcohol-fed mice. Depletion of uric acid or ATP, or lack of ATP signaling attenuated ASH and prevented inflammasome activation and its major downstream cytokine, IL-1ß. Pharmacological depletion of uric acid with allopurinol provided significant protection from alcohol-induced inflammatory response, steatosis and liver damage, and additional protection was achieved in mice treated with probenecid, which depletes uric acid and blocks ATP-induced P2rx7 signaling. We found that alcohol-damaged hepatocytes released uric acid and ATP in vivo and in vitro and that these sterile danger signals activated the inflammasome in LPS-exposed liver mononuclear cells. CONCLUSIONS: Our data indicate that the second signal in inflammasome activation and IL-1ß production in ASH results from the endogenous danger signals, uric acid and ATP. Inhibition of signaling triggered by uric acid and ATP may have therapeutic implications in ASH.

Pretreatment MicroRNA Level and Outcome in Sorafenib-treated Hepatocellular Carcinoma.

Sorafenib represents the first effective targeted therapy for advanced stage hepatocellular carcinoma (HCC); however, adequate patient stratification regarding sorafenib-responsiveness is still missing. Our aim was to analyse the association between the pretreatment microRNA profile of HCC and patient survival under sorafenib treatment. Total RNA was extracted from diagnostic fine-needle aspiration biopsy (FNAB) cytological smears of 20 advanced stage HCC patients collected between June 2008 and July 2012. All patients underwent sorafenib administration after FNA. Clinicopathological and survival data were recorded. Fourteen frequently deregulated miRNAs in HCC (miR-17-5p, miR-18a, miR-21, miR-34a, miR-122, miR-195, miR-210, miR-214, miR-221, miR-222, miR-223, miR-224, miR-140, miR-328) were tested by qRT-PCR. NormFinder software was used to select proper miR (mir-140) as a reference. Satisfactory amount of total RNA was obtained from all the considered samples (mean 10.8 ± 9.3 µg, range 0.2-32.2 µg). Among the analysed miRNAs, high miR-214 expression was associated with smaller tumor size (p=0.019), whereas high miR-17-5p expression correlated with better Eastern Cooperative Oncology Group performance status (p=0.003). The survival analysis revealed that high miR-224 expression was associated with increased progression-free and overall survival (PFS p=0.029; OS p=0.012). Pretreatment microRNA profiling, especially miR-224 expression, might serve as an ancillary tool for the better assessment of expected survival rates for patients under sorafenib treatment.