Paraffin-embedded Liver Sections Research Articles

Detection of Chlamydia sp. by fluorescence in situ hybridization (FISH) in histologic sections of the liver from exotic and native avian species

Chlamydiosis is a zoonotic disease that affects several animal species. Therefore, reliable detection techniques are essential for efficient control of the disease. Fluorescence in situ hybridization (FISH) for Chlamydia sp. was applied to 137 formalin-fixed, paraffin-embedded liver sections from native and exotic birds. The samples were divided into two groups: retrospective (n=57) and prospective (n=80). The probe was designed based on an annealing sequence that targets the Major Outer Membrane Protein coding gene. Livers previously confirmed for Chlamydia psittaci by polymerase chain reaction (PCR) and immunohistochemistry (IHC) were used as positive controls. Also, 47 randomly selected samples from the prospective group were submitted to PCR for Chlamydia psittaci for confirmation. From all 137 samples, 67% (92/137) were positive for Chlamydia sp. through FISH, from which 39% (36/57) and 61% (56/80) were from the retrospective and prospective groups, respectively. From the samples of the prospective group submitted for PCR confirmation, 83% (39/47) of them had a positive correlation with FISH results. When considering the number of targeted microorganisms per 400x field, 42,39% (39/92) of the samples had up to five microorganisms, 14,13% (13/92) had from six to ten, and 43,47% (40/92) of the samples had 11 or more microorganisms per field. FISH is a specific and reliable method to identify Chlamydia sp. in histologic sections of the liver, providing an additional tool for detecting avian chlamydiosis.

Altered DNA methylation pattern characterizes the peripheral immune cells of patients with autoimmune hepatitis.

Little is known about the impact of DNA methylation modifications on autoimmune hepatitis (AIH) pathogenesis and therapeutic response. We investigated the potential alterations of DNA methylation in AIH peripheral lymphocytes at diagnosis and remission. Ten AIH patients at diagnosis (time-point 1; AIH-tp1), 8/10 following biochemical response (time-point 2; AIH-tp2), 9 primary biliary cholangitis (PBC) and 10 healthy controls (HC) were investigated. Peripheral CD19(+) and CD4(+) cells were isolated. Global DNA methylation (5m C)/hydroxymethylation (5hm C) was studied by ELISAs. mRNA of DNA methylation (DNMT1/3A/3B) and their counteracting hydroxymethylation enzymes (TET1/2/3) was determined by quantitative RT-PCR. Epigenome wide association study (EWAS) was performed in CD4(+) cells (Illumina HumanMethylation 850K array) in AIH and HC. Total 5m C/5hm C was also assessed by immunohistochemistry (IHC) on paraffin-embedded liver sections. Reduced TET1 and increased DNMT3A mRNA levels characterized CD19(+) and CD4(+)-lymphocytes from AIH-tp1 compared to HC and PBC, respectively, without affecting global DNA 5m C/5hm C. In AIH-tp1, CD4(+) DNMT3A expression was negatively correlated with serum IgG (P=.03). In remission, DNMT3A decreased in both CD19(+) and CD4(+) cells compared to AIH-tp1 (P=.02, P=.03 respectively). EWAS in CD4(+) cells from AIH patients confirmed important modifications in genes implicated in immune responses (HLA-DP, TNF, lnRNAs and CD86). IHC showed increased 5hm C staining of periportal infiltrating lymphocytes in AIH-tp1 compared to HC and PBC. Altered TET1 and DNMT3A expressions, characterize peripheral lymphocytes in AIH. DNMT3A was associated with disease activity and decreased following remission. Gene DNA methylation modifications affect immunological pathways that may play an important role in AIH pathogenesis.

Liver fibrosis and biliary proliferation are enhanced in the rat model of inflammatory bowel disease

BackgroundPatients who suffer from Inflammatory Bowel Disease (IBD) often experience concurrent conditions such as lymphatic dysfunction and bone loss. Additionally, liver and biliary abnormalities are frequently observed, including primary sclerosing cholangitis (PSC), autoimmune hepatitis, and cholelithiasis. The pathogenesis is unknown but is presumed to be secondary to chronic inflammation, which drives intrahepatic inflammation and fibrogenesis. The lymphatic system, via transport of inflammatory stimuli, is a likely route by which the regional inflammatory changes in the bowel effect distal organs such as the liver. Therefore, the aim of this study was to evaluate changes in biliary damage and senescence, ductular reaction (DR), and liver inflammation and fibrosis in an experimental rat model of inflammatory bowel disease.MethodsStudies were performed in male rats receiving a 2,4,6‐trinitrobenzene sulfonic acid (TNBS) enemas at 30mg/kg of body weight, once a week, for 4 weeks. Lymph, livers, isolated cholangiocytes, and hepatic stellate cells were collected. Ductular reaction was evaluated by immunohistochemistry (IHC) for CK19 in paraffin embedded liver sections and cellular senescence was determined by SA‐β‐gal staining. Gene expression was measured using qPCR for p16, CCL2 and p21 in total liver and laser‐captured microscopy (LCM) isolated cholangiocytes. Liver fibrosis was assessed via Sirius Red staining and qPCR for the following genes; α‐SMA, TGFβ‐1, FN‐1, and collagen1a1 in total liver, HSCs, and pure cholangiocytes. To determine the level of inflammation, IHC for CD68 and MHCII was performed, while gene expression was measured using qPCR for inflammatory/senescence markers (IL‐1β, IL‐6, IL‐33 and TNF‐α) in total liver and LCM isolated cholangiocytes. Immunofluorescence was used to visualize the expression of TLR4 in the intact bile ducts of total liver sections. Lymph was used for proteomic analysis post‐instillation at peak inflammation, to identify potential inflammatory signals that may precipitate the changes that were noted.ResultsRats treated with TNBS had increased DR, collagen deposition, and fibrotic gene expression in both cholangiocytes and HSCs; enhanced liver inflammation with increased CD68 positive macrophages in liver sections; increased biliary senescence and biliary expression of senescence markers; and increased biliary expression of TLR4. There was a strong correlation between the increase in inflammatory pathways in the lymph from IBD animals, with marked increases in pathways associated with bacterial translocation and lipid metabolism.ConclusionWe observed increased biliary ductular reaction that was associated with increased liver inflammation/fibrosis similar to the co‐morbidity of PSC reported in IBD patients.Support or Funding InformationNIH RO1 DK110035

Assessment of the Mode of Action Underlying the Effects of GenX in Mouse Liver and Implications for Assessing Human Health Risks.

GenX is an alternative to environmentally persistent long-chain perfluoroalkyl and polyfluoroalkyl substances. Mice exposed to GenX exhibit liver hypertrophy, elevated peroxisomal enzyme activity, and other apical endpoints consistent with peroxisome proliferators. To investigate the potential role of peroxisome proliferator-activated receptor alpha (PPARα) activation in mice, and other molecular signals potentially related to observed liver changes, RNA sequencing was conducted on paraffin-embedded liver sections from a 90-day subchronic toxicity study of GenX conducted in mice. Differentially expressed genes were identified for each treatment group, and gene set enrichment analysis was conducted using gene sets that represent biological processes and known canonical pathways. Peroxisome signaling and fatty acid metabolism were among the most significantly enriched gene sets in both sexes at 0.5 and 5 mg/kg GenX; no pathways were enriched at 0.1 mg/kg. Gene sets specific to the PPARα subtype were significantly enriched. These findings were phenotypically anchored to histopathological changes in the same tissue blocks: hypertrophy, mitoses, and apoptosis. In vitro PPARα transactivation assays indicated that GenX activates mouse PPARα. These results indicate that the liver changes observed in GenX-treated mice occur via a mode of action (MOA) involving PPARα, an important finding for human health risk assessment as this MOA has limited relevance to humans.

Kupffer Cell and Monocyte-Derived Macrophage Identification by Immunofluorescence on Formalin-Fixed, Paraffin-Embedded (FFPE) Mouse Liver Sections.

Kupffer cells are the liver-resident macrophages and represent the first line of defense between the pathogens circulating from the intestines through the portal vein and systemic circulation. Recent works have highlighted the complex heterogeneity of macrophage functions and origins, thus raising awareness on the need for a better characterization of macrophage populations. The immunohistochemistry method here described, allows for a rapid distinction between Kupffer cells and monocyte-derived macrophages present on formalin-fixed, paraffin-embedded mouse liver samples. This protocol has been optimized for its reproducibility, reliability, and simplicity.

Hepatic sonic hedgehog protein expression measured by computer assisted morphometry significantly correlates with features of non-alcoholic steatohepatitis

BackgroundHepatic expression of Sonic Hedgehog (SHH) is associated with Non-alcoholic fatty liver disease (NAFLD) and development of Non-alcoholic steatohepatitis (NASH). Hepatic SHH detection increases with the diagnosis of NASH. This pilot study was designed to confirm that staining for SHH is useful in NASH diagnosis and determine whether quantification of staining by computer assisted morphometry (CAM) can be used to assess severity of ballooning degeneration.MethodsSHH was detected by immunohistochemistry (IHC) on paraffin-embedded liver sections in subjects (N = 69) with biopsy proven NAFLD and no liver disease (control). Serum samples were also available for these subjects. Post-staining, a digitized image of the section was acquired and an area quantification algorithm was used to quantify the degree of SHH expression. Additionally, circulating M30, M65, and SHH were measured by ELISA.ResultsNotably, hepatic SHH expression correlated with histologic ballooning degeneration (rho = 0.62, p < 0.0001), steatosis grade (rho = 0.554, P < 0.001), Mallory-Denk bodies (rho = 0.54, P < 0.001), pericellular fibrosis (rho = 0.527, P < 0.001), and lymphocytic infiltration (rho = 0.435, P < 0.0002). Additionally, hepatic SHH expression correlated with circulating M65 (rho = 0.588, p < 0.0001), and circulating M30 (rho = 0.375, p = 0.001), as well as AST and ALT (rho = 0.43, p = 0.0004, and rho = 0.27, p = 0.03, respectively). Further, serum M30 was almost twice as high in NASH patients compared to non-NASH (539.1 ± 290.8 U/L vs. 287.6 ± 190.5 U/L; p = 0.0002), while M65 was almost three times higher in NASH patients compared to non-NASH (441.2 ± 464.2 U/L vs. 162.8 ± 353.1 U/L, P = 0.0006). Logistic modeling indicates hepatic SHH expression and presence of type 2 diabetes as independent predictors of advanced fibrosis (defined as portal and pericellular fibrosis > 2: OR = 1.986, p = 0.01, and OR = 3.280, p = 0.03, respectively).ConclusionThus, our findings show quantitation of SHH expression by CAM can provide a tool for quantifying changes in hepatocyte injury and assist in unambiguous staging/grading of NASH. Our study showed minimal interobserver variability using CAM based quantification. Once validated, CAM assessment of hepatic SHH could benefit clinical trials or long term outcomes studies of NASH subjects.

Utility of Serum miR-122, Liver Enzymes, and Hepatic Histopathology in Response to Hepatotoxicants in Sprague-Dawley Rats.

More than 80,000 chemicals are in commercial use worldwide. Hepatic metabolism to toxic intermediates is often a key mechanism leading to tissue damage and organ dysfunction. Effective treatment requires prompt detection of hepatotoxicity, ideally with rapid, minimally invasive diagnostic assays. In this study, archetypal histologic features of chemically induced hepatic injury were compared with clinical chemistries (including liver enzymes) and serum concentrations of microRNA-122 (miR-122, the processed form miR-122-5p), a biomarker of liver injury. The hepatotoxicants 4,4'-methylenedianiline (4,4'-MDA), allyl alcohol (AA), or carbon tetrachloride (CCl4) were orally administered to male Sprague-Dawley rats for 1, 5, 14, or 28 days to induce liver damage. Formalin-fixed, paraffin-embedded liver sections were evaluated histologically for inflammation, fibrosis, necrosis, and lipid accumulation. Liver enzymes were measured in serum, and serum miR-122 concentrations were assessed by quantitative polymerase chain reaction (qPCR). Histologic features of hepatic injury dose-dependently increased in both severity and frequency. Increases in liver enzymes and bilirubin were more pronounced in response to AA or 4,4'-MDA than to CCl4 at early time points. Elevated serum miR-122 levels in animals administered CCl4, AA, or 4,4'-MDA were more strongly associated with degree of hepatic histopathology than with dosage. Given this sensitive expression pattern postexposure, liver-specific miR-122 may improve the diagnostic accuracy of early hepatic injury.

PDE4 Inhibitor Rolipram Mitigates Liver Ischemia Reperfusion Injury in a Rat Model by Preventing Neutrophil Accumulation

BACKGROUND/INTRODUCTIONIschemia‐reperfusion (IR) injury is caused by a transient loss of blood flow (ischemia) followed by return of blood flow (reperfusion), which initiates an inflammatory response. IR injury occurs in organ transplantation, sepsis, respiratory failure, hemorrhage, trauma, stroke, cardiac arrest, and shock. Ischemia reperfusion, as a primary cause of post graft non‐function, is a limiting factor in the success of liver transplantation. Due to a crucial shortage of donor livers, there are about 17,000 people on the waiting list for a liver transplant, and 1,500 a year will die while waiting for an organ (American Liver Foundation). Pharmacological attenuation of the IR‐induced inflammatory response could improve graft function and survival. We investigated the potential of using the phosphodiesterase (PDE) inhibitor, rolipram, in hepatic ischemia reperfusion injury. PDEs are the only enzymes that can hydrolyze cyclic adenosine monophosphate (cAMP). Higher cAMP levels can initiate and sustain anti‐inflammatory signaling. Therefore, we hypothesized that suppression of the inflammatory response by rolipram would reduce liver damage following IR injury.METHODSMale Sprague‐Daley rats were randomized to six groups (n=6–8 per group): 1) sham, 2) sham + 3 mg/kg rolipram, 3) 40% ischemia reperfusion, 4) 40% ischemia reperfusion + 3 mg/kg rolipram, 5) 70% ischemia reperfusion, and 6) 70% ischemia reperfusion + 3 mg/kg rolipram. Two different levels of severity of IR injury were achieved by clamping either 40% (left lobe) or 70% (left and median lobes) of the liver. In both cases, occlusion was for 1 hour followed by 3 hours of reperfusion. Treatment was given I.V. at the end of the ischemic period. Blood and tissue were collected. Blood chemistry was assayed on a Vetscan 2 (Abaxis). Paraffin embedded liver sections were stained for H&amp;E (morphology), intercellular adhesion molecule 1 (ICAM‐1) (neutrophil adhesion molecule), and chloroacetate esterase (CAE, stains neutrophils). Liver myeloperoxidase (MPO, from neutrophils) and tumor necrosis factor alpha (TNF‐α) and serum hyaluronate (a marker of vascular damage) were assessed by ELISA.RESULTSBlood chemistry revealed that IR significantly (p=0.01) elevated serum alanine aminotransferase (ALT), an indicator of liver damage, in both the 40% and 70% models. Rolipram lowered serum ALT levels in the 40% model. Inflammation‐induced neutrophil infiltration is a major source of IR damage. Histology showed areas of coagulative necrosis and increased neutrophil infiltration, which were ameliorated by rolipram, in both 40% and 70% models. Liver ICAM‐1 was reduced by rolipram treatment in both models. MPO was significantly increased by 70% but not 40% IR, and rolipram did not lower it. Liver TNF‐α showed no difference between sham and IR groups, but rolipram seemed to lower it independently of IR. Rolipram treatment showed a trend towards decreasing serum hyaluronan in both models.CONCLUSIONMechanisms still remain to be determined, but overall, these data suggest that inhibition of PDE reduces hepatic IR injury by decreasing neutrophil recruitment. We conclude that rolipram might have therapeutic potential in ischemia reperfusion injury.Support or Funding InformationSupported by grants from NIH and Veterans AdministrationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 2096: Mechanistic insights into the pathogenesis of anti-DLL4-related hepatic sinusoidal dilatation

Abstract Background: The Delta-like 4 (DLL4)-mediated NOTCH signaling pathway is an attractive therapeutic target in cancer. However, chronic blockade of DLL4 signalling has been observed to result in vascular toxicities, such as hepatic sinusoidal dilatation. As the underlying pathogenesis is unclear, the current study was undertaken to interrogate gene expression changes and potential safety biomarkers associated with anti-DLL4 related hepatic sinusoidal dilatation. Methods: Formalin-fixed paraffin-embedded (FFPE) liver sections were derived from male and female cynomolgus monkeys administered FDLL8566 (recombinant humanized anti-DLL4 F(ab’)2 antibody) by intravenous injection once weekly for 8 weeks at dose levels of 0, 5, 15 and 50 mg/kg/week (n = 3/sex/group). RNA was extracted from whole liver sections and laser capture-microdissected (LCM) hepatic regions with or without sinusoidal dilatation. mRNA expression was quantified using a high-throughput RT-qPCR approach, with 96 pre-validated, species-specific TaqMan gene expression assays. A non-parametric statistical test was used to assess differential gene expression between sinusoidal dilatation-affected and non-affected liver tissues. The Benjamini-Hochberg multiple testing comparison error-based False-Discovery-Rate (FDR) method was applied to calculate the adjusted p-values for each probe set. Results: Fourteen candidate genes were differentially expressed between sinusoidal dilatation-affected and non-affected cynomolgus monkey livers with an FDR adjusted p value &amp;lt;0.05. The presence of sinusoidal dilatation could be discriminated based on hierarchical clustering and principal component analysis (PCA). Modulation of expression in genes associated with the VEGF/NOTCH pathway, vascular remodeling, and inflammation were related to anti-DLL4-induced sinusoidal dilatation. Conclusions: This work highlights the involvement of the NOTCH pathway in the maintenance of hepatic sinusoidal homeostasis in the nonhuman primate (NHP). As preclinical toxicities in NHPs have translated to patients for other anti-DLL4 inhibitors, the changes in candidate genes and potential mechanism of toxicity identified in this study are likely to be relevant to humans. The phenotypic characteristics of hepatic sinusoidal dilatation associated with anti-DLL4 resemble the microscopic and molecular features observed in the liver following oxaliplatin treatment of patients (including involvement of angiogenesis), suggesting that similar molecular mechanisms of hepatic toxicity may exist between anti-DLL4 and oxaliplatin. This work was supported by an EU funded Industry Academia Pathways and Partnerships Marie Curie Award (AngioTox) Grant Number 251528. Citation Format: Monika A. Jarzabek, Rupal Desai, Yuda Zhu, Christina Z. de Zafra, Joe Beyer, Gary Cain, Rajiv Raja, Annette T. Byrne, Priti Hegde, Jacqueline M. Tarrant. Mechanistic insights into the pathogenesis of anti-DLL4-related hepatic sinusoidal dilatation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2096.

Elemental bioimaging of platinum in mouse tissues by laser ablation-inductively coupled plasma-mass spectrometry for the study of localization behavior of structurally similar complexes

In the last decade, the number of applications of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) in bioimaging has been increasing. To further extend its capability in drug development, in this study, we used this bioimaging tool to visualize deposition behavior of chemically different metallo-complexes, including two that differed only subtly in structure. A systematic approach with in vitro study and ICP-MS elemental analysis were included to supplement the findings.Two chemically distinct platinum complexes (Pt-1 and Pt-2) were synthesized; their potencies were investigated first on different healthy and cancer cell lines and then on mice. The commercialized anti-cancer drug, cis-platin was used as a reference. In animal studies, the mice were given 4mg/kg of the complex via intraperitoneal injection and sacrificed 24h post-injection. ICP-MS analysis was performed on six organs to study the bioavailability of the complexes. Pt accumulated in the organs, from greatest to least, from liver>kidney>lung>testis>heart>brain. Among the complexes, the bioavailability showed a general trend of Pt–2>cis-platin>Pt-1.In LA-ICP-MS bioimaging analysis of paraffin-embedded mouse liver and kidney sections, a spatial resolution of 50μm was adopted. Deposition trends matched the findings obtained in elemental analysis. In addition, differential deposition of Pt was observed in the kidney sections of mice treated with different complexes. The LA biomaps successfully distinguished the differential distribution of two structurally similar platinum complexes (Pt-1 and Pt-2) in mice liver and kidney. This information is of particular interest because these two Pt-based complexes can potentially be developed into anti-cancer drugs. This work demonstrates that LA-ICP-MS imaging is a valuable tool for therapeutic drug development, especially in assisting molecular modification of metal-containing complexes.

Phenotypic Characterization of Transgenic Japanese Medaka (Oryzias latipes) That Express a Red Fluorescent Protein in Hepatocytes

Transgenic organisms that express fluorescent proteins are used frequently for in vivo visualization of proteins and cells. The phenotype of a transgenic medaka (Oryzias latipes) strain that expresses a red fluorescent protein (RFP) in hepatocytes was characterized using light and fluorescence microscopy, immunohistochemistry, and transmission electron microscopy (TEM). Expression of RFP was first detected by confocal fluorescence microscopy in the location of the liver bud of live medaka embryos at 60 hr postfertilization (developmental stage 27). Subsequently, RFP signal was observed exclusively in hepatocytes throughout life using fluorescence microscopy in live fish and immunohistochemistry in formalin-fixed, paraffin-embedded liver sections. As the fish aged, prominent intracytoplasmic eosinophilic inclusions immunoreactive for RFP were observed by light microscopy and were correlated with membrane-bound electron dense inclusions on TEM. These results define the onset and location of RFP expression in the Tg(zf.L-fabp:DsRed) medaka and characterize a histologic phenotype that results from RFP expression in hepatocytes.

Failure of Fibrotic Liver Regeneration in Mice Is Linked to a Severe Fibrogenic Response Driven by Hepatic Progenitor Cell Activation

Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.

Disfunction of endosome prevents developing alcoholic fatty liver disease via inhibition of TLR4, TLR7, and TLR9 signaling pathways

The pathogenesis of alcoholic fatty liver disease has never been established. It has been that TLR‐signaling pathway plays an important role in the development. What primes TLR‐signaling pathway and any prevention remains unclear. Endosome includes TLR4, TLR7 and TLR9, which may contribute to innate immune response in macrophage. We hypothesized that disturbance of endosome reduces alcoholic fatty liver disease. To clarify the hypothesis, we evaluated fatty liver injury in alcohol‐fed rats after treatment with endosome inhibitors. Male Wistar rats were fed with Lieber‐DeCarli alcohol liquid diet for 4 weeks. Omeprazole or lansoprazole, endosome inhibitors, was treated to alcohol liquid diet‐fed rats. Immunohistochemical staining for TLR4, TLR7 and TLR9 was performed in formalin‐fixed, paraffin‐embedded liver sections. ER stress markers were analyzed by quantitative realtime PCR. Alcohol feeding caused expression of TLR4, TLR7 and TLR9 and accumulation of ER stress. Inhibition of endosome results in reduction of fatty liver change, especially lipid droplets inflammation in hepatocytes and no staining for TLR7 and TLR9. However, accumulation of ER stress was not changed by inhibition of proton pump. Therefore, chronic alcohol consumption causes developing fatty liver disease via TLR7 and TLR9, which expression can be reduced by inhibition of endosome.

Application of Interleukin-22 Mediates Protection in Experimental Acetaminophen-Induced Acute Liver Injury

Acetaminophen (APAP, paracetamol)-induced hepatotoxicity, although treatable by timely application of N-acetylcysteine, can be fatal. Because it is among the common causes of acute liver failure in intensive care units and in light of its gradually increasing incidence, the need for novel therapeutic strategies aimed at severe intoxication is apparent. Recently, it has been shown that IL-22, a STAT3-activating cytokine, has the capability to mediate liver protection. Herein, the protective potential of IL-22 in murine APAP-induced hepatotoxicity was assessed. Intravenous administration of prophylactic IL-22 significantly reduced serum alanine aminotransferase levels and histopathologic damage in APAP-induced liver injury, a process that coincided with increased hepatocyte proliferation invivo. Concomitant gene expression analysis revealed hepatic induction of genes prototypically up-regulated by the IL-22/STAT3 axis, among others suppressor of cytokine signaling-3, lipocalin-2, and α1-antichymotrypsin. Notably, in a translational setting of therapeutic treatment 2 hours after APAP, IL-22 supported protection in the context of suboptimal N-acetylcysteine dosing. IL-22 likewise connected to augmented hepatocyte proliferation in this experimental setting. As detected by analysis of inflammatory cytokine production, systemically applied IL-22 did not display acute immunomodulation/stimulation in otherwise untreated or endotoxemic mice. Those latter observations clearly confirm acute tolerability of systemically applied IL-22. Observations presented altogether suggest that therapeutic IL-22 administration is a conceivable tissue-protective regimen aimed at hard-to-treat patients with severe APAP-induced hepatotoxicity.

Presence of disease specific autoantibodies against liver sinusoidal cells in primary biliary cirrhosis

To investigate the presence of autoantibodies directed against liver sinusoidal cells in primary biliary cirrhosis (PBC). Liver biopsies from 21 PBC patients were studied and compared with 12 liver biopsies from disease controls [3 patients with hepatitis B (HBV) virus, 3 patients with hepatitis C virus (HCV), 3 patients with non-alcoholic steatohepatitis and 3 patients with acute alcoholic hepatitis (AAH)]. As healthy controls, we used tissue specimens adjacent to metastatic liver adenocarcinoma. Normal serum was taken from staff members of the unit. The determination of the cell type targeted by autoantibodies present in the patients sera was performed by indirect immunofluorescence (IIF) analysis using paraffin-embedded liver sections as a substrate. Sera from homologous or heterologous PBC patients or sera from the disease control group were used as primary antibodies. The presence of autoantibodies was identified using confocal microscopy. In total, 18/21 (85.7%) PBC patients exhibited positive staining in the sinusoidal cells, 10/21 (47.6%) in lymphocytes, 8/21 (38%) in cholangiocytes and 7/21 (33.3%) in hepatocytes, when homologous serum and fluorescein isothiocyanate-conjugated immunoglobulin type G (IgG) secondary antibody were used. PBC sections incubated with heterologous PBC serum showed reduced staining (20% for sinusoidal cells, 20% for lymphocytes, 20% for cholangiocytes and 13.3% for hepatocytes). When IgM immunoglobulin, instead of IgG, was used as secondary antibody, positive staining was observed in 75% of lymphocytes, 62.5% of cholangiocytes, 37.5% of hepatocytes and 50% of the sinusoidal cells with a much stronger staining intensity. No staining was observed when either normal or PBC sera were used as a primary antibody on liver sections from the disease control group. When PBC sera were incubated with healthy control sections, weak positive staining of cholangiocytes was observed in 3/21 (14.3%) PBC serum samples. Steatohepatitis serum on PBC sections gave a positive staining of some hepatocytes and lymphocytes but no staining on viral hepatitis sections. Incubation with HBV sera stained some hepatocytes, cholangiocytes and intra-sinusoidal or portal lymphocytes of PBC, HBV and AAH patients but not HCV patients. In this study, for the first time in diseased liver tissue, we have demonstrated that a large proportion of PBC patients have disease specific autoantibodies against liver sinusoidal cells.

Transfer of HBV Genomes Using Low Doses of Adenovirus Vectors Leads to Persistent Infection in Immune Competent Mice

Studies of mechanisms responsible for the persistence of hepatitis B virus (HBV) infection have been hindered by a lack of appropriate animal models. HBV genomes can be delivered to livers of mice using hydrodynamic injection or high doses of an adenoviral vector; these lead to clearance of HBV. We found that infection of immunocompetent mice with low doses of an adenoviral vector resulted in persistent HBV infection; the mice neither underwent seroconversion to production of antibodies against HBV nor developed a strong HBV-specific effector T-cell response. As in patients with chronic HBV infection, DNA vaccination failed to generate T cells that cleared infection. This model of persistent HBV infection could be used to study the pathogenesis of chronic HBV infection and develop new therapeutic strategies.

Hepatic Akt expression correlates with advanced fibrosis in patients with chronic hepatitis C infection

Hepatitis C virus (HCV) proteins can activate the PI3K/Akt pathway which is involved in multiple cellular functions such as inflammatory cell activation and liver fibrosis. The aim of the present study was to elucidate the correlation between Akt expression and liver fibrosis staging in chronic hepatitis C (CHC) patients. Paraffin-embedded liver sections from 133 consecutive treatment-naïve CHC patients were recruited. The expression features of Akt were analyzed using immunohistochemical methods and the results were compared with histological, virological and biochemical profiles. The 73 patients with high Akt expression carried higher histological activity index scores (6.52 ± 2.5 vs 5.62 ± 2.4, P = 0.04) and advanced fibrosis (72.7% vs 26.3%, P < 0.01) than other 60 patients with low Akt expression. The high Akt expression showed a significant incremental trend dependent on fibrosis stages, from 33.3% of F0 to 85.7% of F4 (P = 0.005). Akt expression was not correlated with degrees of steatosis and virological features of HCV infection, such as viral load and genotypes. Multivariate logistic regression analysis showed advanced fibrosis was the most significant factor associated with high Akt expression (odds ratio = 3.16). Hepatic Akt expression correlated with advanced liver fibrosis in CHC patients.

A method for MS E differential proteomic analysis of archival formalin-fixed celloidin-embedded human inner ear tissue

Proteomic analysis of cadaveric formalin-fixed, celloidin-embedded (FFCE) temporal bone tissue has the potential to provide new insights into inner ear disorders. We have developed a liquid chromatography–mass spectrometry (LC–MS) method for tissue sections embedded with celloidin. Q-TOF (Quadrupole-time of flight mass spectrometry) MS E (mass spectrometry where E represents collision energy) and Identity E™ were used in conjunction with nano-UPLC (capillary ultrahigh pressure liquid chromatography) for robust identification and quantification of a large number of proteins. Formalin-fixed paraffin-embedded (FFPE) mouse liver sections were used to evaluate formalin de-cross-linking by five different methods. Unfixed fresh mouse liver tissue was used as a control. Five different methods for preparation of FFPE tissue for MS analysis were compared, as well as four methods for celloidin removal with FFCE mouse liver tissue. The methods judged best were applied to FFCE 20 μm sections of mouse inner ear samples, and FFCE 20 μm human inner ear and human otic capsule bone sections. Three of the five-tissue extraction methods worked equally in detecting peptides and proteins from FFPE mouse liver tissue. The modified Liquid Tissue kit protocol was chosen for further studies. Four different celloidin removal methods were compared and the acetone removal method was chosen for further analysis. These two methods were applied to the analysis of FFCE inner ear and otic capsule sections. Proteins from all major cellular components were detected in the FFCE archival human temporal bone sections. This newly developed technique enables the use of FFCE tissues for proteomic studies.

Accumulation of miscoding etheno-DNA adducts and highly expressed DNA repair during liver fluke-induced cholangiocarcinogenesis in hamsters

Infection by Opisthorchis viverrini, a risk factor for cholangiocarcinoma (CCA) may act through chronic inflammation, oxidative stress and lipid peroxidation (LPO)-related damage and growth stimuli. 1, N 6-Etheno-2′-deoxyadenosine (ɛdA), and 3, N 4-etheno-2′-deoxycytidine (ɛdC), markers for LPO-derived DNA damage were highly increased in white blood cell and urine of O. viverrini-infected Thai patients. In order to investigate tissue specificity etheno adducts were measured in a cholangiocarcinogenesis model, in O. viverrini-infected hamsters that had received N-nitrosodimethylamine (NDMA, 12.5 ppm in dw) for 2 months. ɛdA- and ɛdC-levels were analyzed in paraffin-embedded liver sections by a novel immunohistochemical method, from 21 up to 180 days post- O. viverrini-infection. In inflamed areas of the liver, etheno adducts were localized in the nuclei of inflammatory cells and in the epithelial lining of the bile duct. Semi-quantitative image analysis showed higher adduct levels in the liver of O. viverrini-infected hamsters, treated with or w/o NDMA when compared with untreated controls. Levels were found highest in the liver of O. viverrini-infected plus NDMA-treated hamsters. Adducts increased in an age-dependent manner from O. viverrini-infection until CCA development. Increased adduct formation paralleled histopathological changes in plasma alkaline phosphatase (ALP) activity, bile duct hyperplasia, dysplasia, precancerous lesions, and CCA appearance. Also elevated expression of alkyladenine DNA glycosylase (AAG), which excises 1, N 6-ethenoadenine (ɛA) was linked to higher adduct formation, suggesting imbalanced repair. Our results implicate accumulation of inflammation-related, promutagenic DNA damage in target tissue and possibly imbalanced repair in the onset of cholangiocarcinogenesis.

559 Galectin-1 expression in Hodgkin-lymphoma cells

The metabolism of CCl4 initiates the peroxidation of polyunsaturated fatty acids producing α,β-unsaturated aldehydes, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). The facile reactivity of these electrophilic aldehydic products suggests they play a role in the toxicity of compounds like CCl4. To determine the rate at which CCl4-initiated lipid peroxidation results in the formation of 4-HNE and/or MDA hepatic protein adducts, rats were given an intragastric dose of CCl4 (1.0 ml/kg) and euthanized 0–72 h after administration. Rabbit polyclonal antisera directed toward 4-HNE- or MDA-protein epitopes were employed in immuno-histochemical and immuno-precipitation/Western analyses to detect 4-HNE and MDA-protein adducts in paraffin-embedded liver sections and liver homogenates. As early as 6 h post CCl4 exposure, 4-HNE and MDA adducts were detected immuno-histochemically in hepatocytes localized to zone 2 of the hepatic acinus. Liver injury was progressive to 24 h as lipid peroxidation and hepatocellular necrosis increased. The hallmark of CCl4 hepatotoxicity, zone 3 necrosis, was observed 24 h after CCl4 administration and immuno-positive hepatocytes were observed in zone 2 as well as zone 3. Immuno-positive cells were no longer visible by 36 to 72 h post CCl4 administration. From 6 to 48 h after CCl4 administration, at least four adducted proteins were immuno-precipitated from liver homogenates with the anti-MDA or anti-4HNE serum, which corresponded to molecular weights of 80, 150, 205, and greater than 205 kDa. These results demonstrate that 4-HNE and MDA alkylate specific hepatic proteins in a time-dependent manner, which appears to be associated with hepatocellular injury following CCl4 exposure.