Modelling the impact of deferring HCV treatment on liver-related complications in HIV coinfected men who have sex with men

BACKGROUND AND AIMS Hepatitis C (HCV) is a leading cause of morbidity and mortality in people who live with HIV. In many countries, access to direct acting antiviral agents to treat HCV is restricted to individuals with advanced liver disease (METAVIR stage F3 or F4). Our goal was to estimate the long term impact of deferring HCV treatment for men who have sex with men (MSM) who are coinfected with HIV and often have multiple risk factors for liver disease progression. METHODS We developed an individual-based model of liver disease progression in HIV/HCV coinfected men who have sex with men. We estimated liver-related morbidity and mortality as well as the median time spent with replicating HCV infection when individuals were treated in liver fibrosis stages F0, F1, F2, F3 or F4 on the METAVIR scale. RESULTS The percentage of individuals who died of liver-related complications was 2% if treatment was initiated in F0 or F1. It increased to 3% if treatment was deferred until F2, 7% if it was deferred until F3 and 22% if deferred until F4. The median time individuals spent with replicating HCV increased from 5 years if treatment was initiated in F2 to almost 15 years if it was deferred until F4. CONCLUSIONS Deferring HCV therapy until advanced liver fibrosis is established could increase liver-related morbidity and mortality in HIV/HCV coinfected individuals, and substantially prolong the time individuals spend with replicating HCV infection.

Intraoperative image-guided navigation system: development and applicability in 65 patients undergoing liver surgery

Background Image-guided systems have recently been introduced for their application in liver surgery.We aimed to identify and propose suitable indications for image-guided navigation systems in the domain of open oncologic liver surgery and,more specifically, in the setting of liver resection with and without microwave ablation. Method Retrospective analysis was conducted in patients undergoing liver resection with and without microwave ablation using an intraoperative image-guided stereotactic system during three stages of technological development (accuracy: 8.4 ± 4.4 mm in phase I and 8.4 ± 6.5 mm in phase II versus 4.5 ± 3.6 mm in phase III). It was evaluated, in which indications image-guided surgery was used according to the different stages of technical development. Results Between 2009 and 2013, 65 patients underwent image-guided surgical treatment, resection alone (n=38), ablation alone (n =11), or a combination thereof (n =16). With increasing accuracy of the system, image guidance was progressively used for atypical resections and combined microwave ablation and resection instead of formal liver resection (p<0.0001). Conclusion Clinical application of image guidance is feasible, while its efficacy is subject to accuracy. The concept of image guidance has been shown to be increasingly efficient for selected indications in liver surgery. While accuracy of available technology is increasing pertaining to technological advancements, more and more previously untreatable scenarios such as multiple small, bilobar lesions and so-called vanishing lesions come within reach.

Hepatoprotective effect of tumour necrosis factor alpha blockade in psoriatic arthritis: a cross-sectional study

Objective To evaluate the impact of tumour necrosis factor α (TNFα) blockers on the presence of liver fibrosis in patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA) treated with methotrexate (MTX). Methods Participants were consecutive patients with RA and PsA who had undergone MTX treatment for at least 1 year ± TNF blockade for over 6 months. Liver fibrosis was assessed using non-invasive transient elastography (FibroScan). Regression models were used to compare FibroScan values of patients with RA and patients with PsA receiving TNFα blockers with those who were not. Results FibroScan assessments were performed on 51 patients with RA and 43 patients with PsA. Compared to patients with RA, those with PsA were predominantly young men, received lower cumulative dosages of MTX and exhibited a higher incidence of liver steatosis and hyperlipidaemia. An abnormal result was observed in 7.1% of the anti-TNFα-naïve and in 13% of the anti-TNFα-treated patients in the RA group and in 30% of the anti-TNFα-naïve and 4.3% of the anti-TNFα-treated patients in the PsA group (OR=0.11, 95% CI 0.02 to 0.98). Results of the PsA group were robust when adjusted for baseline characteristics. Conclusion The results suggest a protective effect of TNFα inhibitors against the development of liver fibrosis in patients with PsA.

Quercetin aglycone is bioavailable in murine pancreas and pancreatic xenografts

Quercetin is a potential chemopreventive and chemotherapeutic agent for pancreatic and other cancers. This study examined the distribution of quercetin in plasma, lung, liver, pancreas, and pancreatic cancer xenografts in a murine in vivo model and the uptake of quercetin in pancreatic cancer MiaPaCa-2 cells in a cellular in vitro model. Mice were randomly allocated to control or 0.2 and 1% quercetin diet groups utilizing the AIN93G-based diet (n = 12 per group) for 6 weeks. In addition, 6 mice from each group were injected weekly with the chemotherapeutic drug gemcitabine (120 mg/kg mouse, ip). MiaPaCa cells were collected from culture medium after cells were exposed to 30 muM quercetin for 0.5, 1, 2, 4, 8, and 24 h. Levels of quercetin and 3-O'-methylquercetin in mouse tissues and MiaPaCa-2 cells were measured by high-pressure liquid chromatography following enzymatic hydrolysis and then extraction. The study showed that quercetin is accumulated in pancreatic cancer cells and is absorbed in the circulating system, tumors, and tissues of pancreas, liver, and lung in vivo. A higher proportion of total quercetin found in tumors and pancreas is aglycones. Gemcitabine cotreatment with quercetin reduced absorption of quercetin in the mouse circulatory system and liver. Results from the study provide important information on the interpretation of the chemotherapeutic efficacy of quercetin.

Tumor necrosis factor sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. CONCLUSION: Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid.

Rapidly excised and cryofixed rat tissue

All preparation efforts of biological samples in electron microscopy are focused to preserve structures as close as possible to the native state. To achieve this goal with tissues, it is of advantage to have a very short time between excision and fixation. The most common approach is chemical fixation: cross-linking of the tissue samples with aldehydes followed by postfixation with osmium tetroxide. Here, the fastest approach for tissue samples is perfusion. However, the diffusion of the fixation solution from blood vessels into the depth of the tissue is still slow and does not allow an overall instant fixation of a single cell. As a result, osmotic effects become evident (swelling or shrinkage of cell organelles). Another possibility is to take a tissue sample from the experimental animal. Excision of tissue can last quite some time, which results in even more pronounced autolytic induced osmotic effects. Furthermore, the animal does not survive the procedure in most cases. Alternatively, microbiopsies are an elegant technique to rapidly excise small quantities of tissue. Some tissues, such as liver and muscle, may be obtained using a non-lethal approach. To avoid the artifacts introduced by chemical fixation, high-pressure freezing of microbiopsies (brain, liver, kidney, and muscle) is a powerful alternative to chemical fixation. Here, we describe the microbiopsy method, and high-pressure freezing/freeze-substitution (HPF/FS) as a follow-up procedure. Cryosectioning of high-pressure frozen samples is optimally preserving the ultrastructure; however, it is not considered to be a routine approach yet.

Optimizing heroin-assisted treatment (HAT): Assessment of the contribution of direct ethanol metabolites in identifying hazardous and harmful alcohol use

Heavy alcohol consumption may accelerate the progression of hepatitis C-related liver disease and/or limit efforts at antiviral treatment in opioid-dependent patients receiving heroin-assisted treatment (HAT). Our study aims to assess alcohol intake among HAT patients by self-reports compared to direct ethanol metabolites.

Deposition, imaging, and clearance: what remains to be done?

Deposition and clearance studies are used during product development and in fundamental research. These studies mostly involve radionuclide imaging, but pharmacokinetic methods are also used to assess the amount of drug absorbed through the lungs, which is closely related to lung deposition. Radionuclide imaging may be two-dimensional (gamma scintigraphy or planar imaging), or three-dimensional (single photon emission computed tomography and positron emission tomography). In October 2009, a group of scientists met at the "Thousand Years of Pharmaceutical Aerosols" conference in Reykjavik, Iceland, to discuss future research in key areas of pulmonary drug delivery. This article reports the session on "Deposition, imaging and clearance." The objective was partly to review our current understanding, but more importantly to assess "what remains to be done?" A need to standardize methodology and provide a regulatory framework by which data from radionuclide imaging methods could be compared between centers and used in the drug approval process was recognized. There is also a requirement for novel radiolabeling methods that are more representative of production processes for dry powder inhalers and pressurized metered dose inhalers. A need was identified for studies to aid our understanding of the relationship between clinical effects and regional deposition patterns of inhaled drugs. A robust methodology to assess clearance from small conducting airways should be developed, as a potential biomarker for therapies in cystic fibrosis and other diseases. The mechanisms by which inhaled nanoparticles are removed from the lungs, and the factors on which their removal depends, require further investigation. Last, and by no means least, we need a better understanding of patient-related factors, including how to reduce the variability in pulmonary drug delivery, in order to improve the precision of deposition and clearance measurements.

Aberrant lipid metabolism in hepatocellular carcinoma revealed by plasma metabolomics and lipid profiling

There has been limited analysis of the effects of hepatocellular carcinoma (HCC) on liver metabolism and circulating endogenous metabolites. Here, we report the findings of a plasma metabolomic investigation of HCC patients by ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS), random forests machine learning algorithm, and multivariate data analysis. Control subjects included healthy individuals as well as patients with liver cirrhosis or acute myeloid leukemia. We found that HCC was associated with increased plasma levels of glycodeoxycholate, deoxycholate 3-sulfate, and bilirubin. Accurate mass measurement also indicated upregulation of biliverdin and the fetal bile acids 7α-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochol-4,6-dien-24-oic acid in HCC patients. A quantitative lipid profiling of patient plasma was also conducted by ultraperformance liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (UPLC-ESI-TQMS). By this method, we found that HCC was also associated with reduced levels of lysophosphocholines and in 4 of 20 patients with increased levels of lysophosphatidic acid [LPA(16:0)], where it correlated with plasma α-fetoprotein levels. Interestingly, when fatty acids were quantitatively profiled by gas chromatography-mass spectrometry (GC-MS), we found that lignoceric acid (24:0) and nervonic acid (24:1) were virtually absent from HCC plasma. Overall, this investigation illustrates the power of the new discovery technologies represented in the UPLC-ESI-QTOFMS platform combined with the targeted, quantitative platforms of UPLC-ESI-TQMS and GC-MS for conducting metabolomic investigations that can engender new insights into cancer pathobiology.

[Non-alcoholic steatohepatitis - from NAFLD to MAFLD]

Non-alcoholic steatohepatitis (NASH) as one entity of non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and accompanies the rise in the prevalence of obesity, diabetes mellitus, hypertension and hyperlipidemia in the western world. It is not known why some patients progress in the disease and develop inflammation in the liver, whereas others remain in the stage of simple steatosis, which generally has a benign course. However, NASH can progress to fibrosis and cirrhosis as well as hepatocellular carcinoma. Therefore, it is important to determine the stage of the disease in patients presenting with the metabolic syndrome and abnormal liver function tests, suggesting NAFLD. Liver biopsy is the only tool that allows for reliable detection, grading and staging of liver disease. The main strategies in the treatment of NASH are correction of risk factors (lifestyle modifications, insuline sensitizer) and anti-oxidants (ursodeoxycholic acid, vitamin E) which both have been shown to improve liver histology as well as liver enzymes. Patients wih alcoholic fatty liver disease (AFLD) present the same liver histology and often also metabolic alterations similar to metabolic syndrome. Therefore, MAFLD (metabolic syndrome-associated fatty liver disease) might describe both patient populations more accurately and also describes the pathophysiological characteristics.

Genetic variation in the PNPLA3 gene is associated with alcoholic liver injury in caucasians

A recent genome-wide study revealed an association between variation in the PNPLA3 gene and liver fat content. In addition, the PNPLA3 single-nucleotide polymorphism rs738409 (M148I) was reported to be associated with advanced alcoholic liver disease in alcohol-dependent individuals of Mestizo descent. We therefore evaluated the impact of rs738409 on the manifestation of alcoholic liver disease in two independent German cohorts. Genotype and allele frequencies of rs738409 (M148I) were determined in 1,043 alcoholic patients with or without alcoholic liver injury and in 376 at-risk drinkers from a population-based cohort. Relative to alcoholic patients without liver damage (n = 439), rs738409 genotype GG was strongly overrepresented in patients with alcoholic liver cirrhosis (n = 210; OR 2.79; P(genotype) = 1.2 × 10(-5) ; P(allelic) = 1.6 × 10(-6) ) and in alcoholic patients without cirrhosis but with elevated alanine aminotransferase levels (n = 219; OR 2.33; P(genotype) = 0.0085; P(allelic) = 0.0042). The latter, biochemically defined association was confirmed in an independent population-based cohort of at-risk drinkers with a median alcohol intake of 300 g/week (OR 4.75; P(genotype) = 0.040; P(allelic) = 0.022), and for aspartate aminotransferase (AST) levels. Frequencies of allele PNPLA3 rs738409(G) in individuals with steatosis and normal alanine aminotransferase (ALT) and AST levels were lower than in alcoholics without steatosis and normal ALT/AST (P(combined) = 0.03). The population attributable risk of cirrhosis in alcoholic carriers of allele PNPLA3 rs738409(G) was estimated at 26.6%. CONCLUSION: Genotype PNPLA3 rs738409(GG) is associated with alcoholic liver cirrhosis and elevated aminotransferase levels in alcoholic Caucasians.

Elevated systemic monocyte chemoattractrant protein-1 in hepatic steatosis without significant hepatic inflammation

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and the metabolic syndrome. It encompasses a clinico-pathologic spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine monocyte chemoattractant protein 1 (MCP-1) plays an important role in the progression of hepatic inflammation and fibrosis, and both increased hepatic expression and circulating serum levels have been described in NASH. Here, we aimed to investigate MCP-1 expression in simple hepatic steatosis. Upon feeding a high-fat diet mice developed hepatic steatosis in the absence of significant hepatic inflammation, but elevated hepatic MCP-1 expression compared to control mice fed a standard chow. Interestingly, high-fat diet fed mice had significantly higher MCP-1 serum levels, and MCP-1 mRNA expression was significantly increased in visceral adipose tissue. Furthermore, MCP-1 serum levels were also elevated in patients with ultrasound-diagnosed NAFLD and correlated with the body-mass index and fasting glucose. In conclusion, our data indicate both the liver and adipose tissue as cellular sources of elevated circulating MCP-1 levels already in the early phase of hepatic steatosis. Since MCP-1 derived from visceral adipose tissue reaches the liver via portal circulation at high concentrations it may significantly contribute to the progression of simple steatosis to NASH.

Outcome of patients with ischemic-like cholangiopathy with secondary sclerosing cholangitis after liver transplantation

Sclerosing cholangitis in critically ill patients (SC-CIP) with sepsis and acute respiratory distress syndrome (ARDS) is a cholestatic liver disease with a rapid progression to liver cirrhosis and hepatic failure. Data on outcome of these patients after liver transplantation (LT) are sparse.

Impaired hepatic removal of interleukin-6 in patients with liver cirrhosis

Systemic concentrations of interleukin-6 (IL-6) are elevated in patients with liver cirrhosis, and impaired hepatic uptake of IL-6 was suggested to contribute to higher levels in these patients. To test this hypothesis IL-6 was measured in portal venous serum (PVS), hepatic venous serum (HVS) and systemic venous serum (SVS) of 41 patients with liver cirrhosis and four patients with normal liver function. IL-6 was higher in PVS than HVS of all blood donors and about 43% of portal vein derived IL-6 was extracted by the healthy liver, and 6.3% by the cirrhotic liver demonstrating markedly impaired removal of IL-6 by the latter. Whereas in patients with CHILD-PUGH stage A IL-6 in HVS was almost 25% lower than in PVS, in patients with CHILD-PUGH stage C IL-6 was similarly abundant in the two blood compartments. Ascites is a common complication in cirrhotic patients and was associated with higher IL-6 levels in all blood compartments without significant differences in hepatic excretion. Hepatic venous pressure gradient did not correlate with the degree of hepatic IL-6 removal excluding hepatic shunting as the principal cause of impaired IL-6 uptake. Furthermore, patients with alcoholic liver cirrhosis had higher IL-6 in all blood compartments than patients with cryptogenic liver cirrhosis. Aetiology of liver cirrhosis did not affect hepatic removal rate indicating higher IL-6 synthesis in patients with alcoholic liver cirrhosis. In summary, the current data provide evidence that impaired hepatic removal of IL-6 is explained by hepatic shunting and liver dysfunction in patients with liver cirrhosis partly explaining higher systemic levels.

Relationship of porcine IGF2 imprinting status to DNA methylation at the H19 DMD and the IGF2 DMRs 1 and 2

BACKGROUND: Porcine IGF2 and the H19 genes are imprinted. The IGF2 is paternally expressed, while the H19 gene is maternally expressed. Extensive studies in mice established a boundary model indicating that the H19 differentially methylated domain (DMD) controls, upon binding with the CTCF protein, reciprocal imprinting of the IGF2 and the H19 genes. IGF2 transcription is tissue and development specific involving the use of 4 promoters. In the liver of adult Large White boars IGF2 is expressed from both parental alleles, whereas in skeletal muscle and kidney tissues we observed variable relaxation of IGF2 imprinting. We hypothesized that IGF2 expression from both paternal alleles and relaxation of IGF2 imprinting is reflected in differences in DNA methylation patterns at the H19 DMD and IGF2 differentially methylated regions 1 and 2 (DMR1 and DMR2). RESULTS: Bisulfite sequencing analysis did not show any differences in DNA methylation at the three porcine CTCF binding sites in the H19 DMD between liver, muscle and kidney tissues of adult pigs. A DNA methylation analysis using methyl-sensitive restriction endonuclease SacII and 'hot-stop' PCR gave consistent results with those from the bisulfite sequencing analysis. We found that porcine H19 DMD is distinctly differentially methylated, at least for the region formally confirmed by two SNPs, in liver, skeletal muscle and kidney of foetal, newborn and adult pigs, independent of the combined imprinting status of all IGF2 expressed transcripts. DNA methylation at CpG sites in DMR1 of foetal liver was significantly lower than in the adult liver due to the presence of hypomethylated molecules. An allele specific analysis was performed for IGF2 DMR2 using a SNP in the IGF2 3'-UTR. The maternal IGF2 DMR2 of foetal and newborn liver revealed a higher DNA methylation content compared to the respective paternal allele. CONCLUSIONS: Our results indicate that the IGF2 imprinting status is transcript-specific. Biallelic IGF2 expression in adult porcine liver and relaxation of IGF2 imprinting in porcine muscle were a common feature. These results were consistent with the IGF2 promoter P1 usage in adult liver and IGF2 promoter P2, P3 and P4 usages in muscle. The results showed further that bialellic IGF2 expression in liver and relaxation of imprinting in muscle and kidney were not associated with DNA methylation variation at and around at least one CTCF binding site in H19 DMD. The imprinting status in adult liver, muscle and kidney tissues were also not reflected in the methylation patterns of IGF2 DMRs 1 and 2.