The antioxidant effect of β-caryophyllene protects rat liver from carbon tetrachloride-induced fibrosis by inhibiting hepatic stellate cell activation.

Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce the risk of developing chronic diseases. β-Caryophyllene (CAR) is a common constituent of the essential oil of numerous plants, vegetables, fruits and medicinal herbs, and has been used as a flavouring agent since the 1930 s. Here, we report the antioxidant activity of CAR, its protective effect on liver fibrosis and its inhibitory capacity on hepatic stellate cell (HSC) activation. CAR was tested for the inhibition of lipid peroxidation and as a free radical scavenger. CAR had higher inhibitory capacity on lipid peroxidation than probucol, α-humulene and α-tocopherol. Also, CAR showed high scavenging activities against hydroxyl radical and superoxide anion. The activity of 5-lipoxygenase, an enzyme that actively participates in fibrogenesis, was significantly inhibited by CAR. Carbon tetrachloride-treated rats received CAR at 2, 20 and 200 mg/kg. CAR significantly improved liver structure, and reduced fibrosis and the expression of Col1a1, Tgfb1 and Timp1 genes. Oxidative stress was used to establish a model of HSC activation with overproduction of extracellular matrix proteins. CAR (1 and 10 μm) increased cell viability and significantly reduced the expression of fibrotic marker genes. CAR, a sesquiterpene present in numerous plants and foods, is as a natural antioxidant that reduces carbon tetrachloride-mediated liver fibrosis and inhibits hepatic cell activation.

Irinotecan loaded in eluting beads: preclinical assessment in a rabbit VX2 liver tumor model.

PURPOSE: The purpose of this study was to study the pharmacokinetics of irinotecan injected intravenously, intra-arterially, or loaded onto a delivery platform. MATERIAL AND METHODS: Fifty-four New Zealand White rabbits with VX2 liver tumor, divided in 3 groups of 17 rabbits, each received irinotecan either by intravenous (IV) route, intra-arterial hepatic (IA) route, or loaded on drug-eluting beads (DEBIRI). Animals were killed at 1, 6, and 24 h. Irinotecan and SN-38 concentrations were measured at different time points in serum, tumor, and normal liver. RESULTS: Twelve milligrams of irinotecan were injected IV and IA, whereas 6-16.5 mg were injected loaded onto DEBIRI. Normalized serum irinotecan reached a peak of 333 ng/ml (range 198.8-502.5) for IV, 327.1 ng/ml (range 277.1-495.6) for IA, and 189.7 ng/ml (range 111.1-261.9) for DEBIRI (P < 0.001) delivery. The area-under-the-curve value from 10 to 60 min of serum irinotecan concentration was significantly lower for DEBIRI (P = 0.0009). Tumor irinotecan levels for IV, IA, and DEBIRI (in ng/200 mg of tissue followed by ranges in parentheses) were, respectively, 23.6 (0.3-24.9), 36.5 (7.7-1914.1), and 20.2 (2.9-319) at 1 h; 4.2 (1-27.9), 99.3 (46.6-159.5), and 42.1 (11.3-189) at 6 h; and 2.7 (2.5-6.9), 18.3 (1.5-369.1), and 174.4 (3.4-5147.3) at 24 h (P = 0.02). At 24 h, tumor necrosis was 25% (10-30), 60% (40-91.25), and 95% (76.25-95) for IV, IA, and DEBIRI, respectively (P = 0.03). CONCLUSION: Compared with IV or IA, DEBIRI induces lower early serum levels of irinotecan, a high and prolonged intratumoral level of irinotecan, and a greater rate of tumor necrosis at 24 h. Further evaluation of the clinical benefit of DEBIRI is warranted.

Long-term Outcome after Liver Resection for Hepatocellular Carcinoma Larger than 10 cm.

BACKGROUND: The purpose of the present study was to analyze long-term survival and disease-free survival after liver resection for giant hepatocellular carcinoma (HCC) ≥ 10 cm compared to HCC < 10 cm in diameter. The surgical approach in the treatment of giant HCC may achieve long-term survival and disease-free survival comparable to treatment of smaller lesions. METHODS: This retrospective analysis was a monocentric study conducted in a tertiary university center. It included 101 patients from 114 consecutive liver resections for HCC, separated into two groups: those with tumors less than 10 cm in diameter (small HCC; n = 79) and those with tumors larger than 10 cm (giant HCC; n = 22). The main outcome measures were overall five-year survival, five-year disease-free survival, recurrence rate, perioperative mortality at 30 days, surgical complication rate, and re-intervention rate. RESULTS: The two groups were homogeneously distributed, apart from cirrhosis, which was found more frequently in the group with small HCC (77 vs. 41 %; p = 0.0013). Both median survival (24 vs. 27 months; p = 0.0085) and overall 5-year survival (21 vs. 45; p = 0.04) were significantly poorer in the small HCC group compared to the giant HCC group. There were no differences en terms of recurrence rate, pattern, and timing. CONCLUSIONS: Liver resection for HCC larger than 10 cm is a valuable option in selected patients, one that provides overall survival and disease-free survival comparable to smaller lesions. Functional reserves of the liver, more than the size of the lesion, may be important in patient selection for surgical resection.

Performance of diagnostic biomarkers in predicting liver fibrosis among hepatitis C virus-infected Egyptian children

The aim of the present study was to identify specific markers that mirror liver fibrosis progression as an alternative to biopsy when biopsy is contraindicated, especially in children. After liver biopsies were performed, serum samples from 30 hepatitis C virus (HCV) paediatric patients (8-14 years) were analysed and compared with samples from 30 healthy subjects. All subjects were tested for the presence of serum anti-HCV antibodies. Direct biomarkers for liver fibrosis, including transforming growth factor-β1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), hyaluronic acid (HA), procollagen type III amino-terminal peptide (PIIINP) and osteopontin (OPN), were measured. The indirect biomarkers aspartate and alanine aminotransferases, albumin and bilirubin were also tested. The results revealed a significant increase in the serum marker levels in HCV-infected children compared with the healthy group, whereas albumin levels exhibited a significant decrease. Significantly higher levels of PIIINP, TIMP-1, OPN and HA were detected in HCV-infected children with moderate to severe fibrosis compared with children with mild fibrosis (p < 0.05). The diagnostic accuracy of these direct biomarkers, represented by sensitivity, specificity and positive predictive value, emphasises the utility of PIIINP, TIMP-1, OPN and HA as indicators of liver fibrosis among HCV-infected children.

Incidence of liver damage of uncertain origin in HIV patients not co-infected with HCV/HBV.

BACKGROUND AND AIMS Several studies have reported that a significant number of HIV patients not co-infected with HCV/HBV develop liver damage of uncertain origin (LDUO). The objective of our study was to evaluate the incidence of and risk factors for the development of LDUO in HIV infected patients not co-infected with HCV/HBV. METHODS Prospective longitudinal study that included HIV-infected patients free of previous liver damage and viral hepatitis B or C co-infections. Patients were followed up at 6-monthly intervals. Liver stiffness was measured at each visit. Abnormal liver stiffness (ALS) was defined as a liver stiffness value greater than 7.2 kPa at two consecutive measurements. For patients who developed ALS, a protocol was followed to diagnose the cause of liver damage. Those patients who could not be diagnosed with any specific cause of liver disease were diagnosed as LDUO and liver biopsy was proposed. RESULTS 210 patients matched the inclusion criteria and were included. 198 patients completed the study. After a median (Q1-Q3) follow-up of 18 (IQR 12-26) months, 21 patients (10.6%) developed ALS. Of these, fifteen patients were diagnosed as LDUO. The incidence of LDUO was 7.64 cases/100 patient-years. Histological studies were performed on ten (66.6%) patients and all showed liver steatosis. A higher HOMA-IR value and body mass index were independently associated with the development of LDUO. CONCLUSION We found a high incidence of LDUO in HIV-infected patients associated with metabolic risk factors. The leading cause of LDUO in our study was non-alcoholic fatty liver disease.

Stringent V beta requirement for the development of NK1.1+ T cell receptor-alpha/beta+ cells in mouse liver.

The liver of C57BL/6 mice contains a major subset of CD4+8- and CD4-8- T cell receptor (TCR)-alpha/beta+ cells expressing the polymorphic natural killer NK1.1 surface marker. Liver NK1.1+TCR-alpha/beta+ (NK1+ T) cells require interaction with beta2-microglobulin-associated, major histocompatibility complex I-like molecules on hematopoietic cells for their development and have a TCR repertoire that is highly skewed to Vbeta8.2, Vbeta7, and Vbeta2. We show here that congenic C57BL/6.Vbeta(a) mice, which lack Vbeta8- expressing T cells owing to a genomic deletion at the Vbeta locus, maintain normal levels of liver NK1+ T cells owing to a dramatic increase in the proportion of cells expressing Vbeta7 and Vbeta2 (but not other Vbetas). Moreover, in C57BL/6 congenic TCR-V Vbeta3 and -Vbeta8.1 transgenic mice (which in theory should not express other Vbeta, owing to allelic exclusion at the TCR-beta locus), endogenous TCR-Vbeta8.2, Vbeta7, and Vbeta2 (but not other Vbetas) are frequently expressed on liver NK1+T cells but absent on lymph node T cells. Finally, when endogenous V beta expression is prevented in TCR-Vbeta3 and Vbeta8.1 transgenic mice (by introduction of a null allele at the C beta locus), the development of liver NK1+T cells is totally abrogated. Collectively, our data indicate that liver NK1+T cells have a stringent requirement for expression of TCR-Vbeta8.2, Vbeta7, or Vbeta2 for their development.

Selectively impaired development of intestinal T cell receptor gamma delta+ cells and liver CD4+ NK1+ T cell receptor alpha beta+ cells in T cell factor-1-deficient mice.

T cell factor-1 (Tcf-1) is a transcription factor that binds to a sequence motif present in several T cell-specific enhancer elements. In Tcf-1-deficient (Tcf-1-/-) mice, thymocyte development is partially blocked at the transition from the CD4-8+ immature single-positive stage to the CD4+8+ double-positive stage, resulting in a marked decrease of mature peripheral T cells in lymph node and spleen. We report here that the development of most intestinal TCR gamma delta+ cells and liver CD4+ NK1.1+TCR alpha beta+ (NK1+T) cells, which are believed to be of extrathymic origin, is selectively impaired in Tcf-1-/- mice. In contrast, thymic and thymus-derived (splenic) TCR gamma delta+ cells are present in normal numbers in Tcf-1-/- mice, as are other T cell subsets in intestine and liver. Collectively, our data suggest that Tcf-1 is differentially required for the development of some extrathymic T cell subsets, including intestinal TCR gamma delta+ cells and liver CD4+ NK1+T cells.

CYR61 and alphaVbeta5 integrin cooperate to promote invasion and metastasis of tumors growing in preirradiated stroma.

Radiotherapy is widely used to treat human cancer. Patients locally recurring after radiotherapy, however, have increased risk of metastatic progression and poor prognosis. The clinical management of postradiation recurrences remains an unresolved issue. Tumors growing in preirradiated tissues have an increased fraction of hypoxic cells and are more metastatic, a condition known as tumor bed effect. The transcription factor hypoxia inducible factor (HIF)-1 promotes invasion and metastasis of hypoxic tumors, but its role in the tumor bed effect has not been reported. Here, we show that tumor cells derived from SCCVII and HCT116 tumors growing in a preirradiated bed, or selected in vitro through repeated cycles of severe hypoxia, retain invasive and metastatic capacities when returned to normoxia. HIF activity, although facilitating metastatic spreading of tumors growing in a preirradiated bed, is not essential. Through gene expression profiling and gain- and loss-of-function experiments, we identified the matricellular protein CYR61 and alphaVbeta5 integrin as proteins cooperating to mediate these effects. The anti-alphaV integrin monoclonal antibody 17E6 and the small molecular alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 suppressed invasion and metastasis induced by CYR61 and attenuated metastasis of tumors growing within a preirradiated field. These results represent a conceptual advance to the understanding of the tumor bed effect and identify CYR61 and alphaVbeta5 integrin as proteins that cooperate to mediate metastasis. They also identify alphaV integrin inhibition as a potential therapeutic approach for preventing metastasis in patients at risk for postradiation recurrences.

The use of liver biopsy evaluation in discrimination of idiopathic autoimmune hepatitis versus drug-induced liver injury.

Distinguishing drug-induced liver injury (DILI) from idiopathic autoimmune hepatitis (AIH) can be challenging. We performed a standardized histologic evaluation to explore potential hallmarks to differentiate AIH versus DILI. Biopsies from patients with clinically well-characterized DILI [n = 35, including 19 hepatocellular injury (HC) and 16 cholestatic/mixed injury (CS)] and AIH (n = 28) were evaluated for Ishak scores, prominent inflammatory cell types in portal and intra-acinar areas, the presence or absence of emperipolesis, rosette formation, and cholestasis in a blinded fashion by four experienced hepatopathologists. Histologic diagnosis was concordant with clinical diagnosis in 65% of cases; but agreement on final diagnosis among the four pathologists was complete in only 46% of cases. Interface hepatitis, focal necrosis, and portal inflammation were present in all evaluated cases, but were more severe in AIH (P < 0.05) than DILI (HC). Portal and intra-acinar plasma cells, rosette formation, and emperiopolesis were features that favored AIH (P < 0.02). A model combining portal inflammation, portal plasma cells, intra-acinar lymphocytes and eosinophils, rosette formation, and canalicular cholestasis yielded an area under the receiver operating characteristic curve (AUROC) of 0.90 in predicting DILI (HC) versus AIH. All Ishak inflammation scores were more severe in AIH than DILI (CS) (P ≤ 0.05). The four AIH-favoring features listed above were consistently more prevalent in AIH, whereas portal neutrophils and intracellular (hepatocellular) cholestasis were more prevalent in DILI (CS) (P < 0.02). The combination of portal inflammation, fibrosis, portal neutrophils and plasma cells, and intracellular (hepatocellular) cholestasis yielded an AUC of 0.91 in predicting DILI (CS) versus AIH. Conclusion: Although an overlap of histologic findings exists for AIH and DILI, sufficient differences exist so that pathologists can use the pattern of injury to suggest the correct diagnosis.

Drug-induced autoimmune liver disease: A diagnostic dilemma of an increasingly reported disease.

The aetiology of autoimmune hepatitis (AIH) is uncertain but the disease can be triggered in susceptible patients by external factors such as viruses or drugs. AIH usually develops in individuals with a genetic background mainly consisting of some risk alleles of the major histocompatibility complex (HLA). Many drugs have been linked to AIH phenotypes, which sometimes persist after drug discontinuation, suggesting that they awaken latent autoimmunity. At least three clinical scenarios have been proposed that refers to drug- induced autoimmune liver disease (DIAILD): AIH with drug-induced liver injury (DILI); drug induced-AIH (DI-AIH); and immune mediated DILI (IM-DILI). In addition, there are instances showing mixed features of DI-AIH and IM-DILI, as well as DILI cases with positive autoantibodies. Histologically distinguishing DILI from AIH remains a challenge. Even more challenging is the differentiation of AIH from DI-AIH mainly relying in histological features; however, a detailed standardised histologic evaluation of large cohorts of AIH and DI-AIH patients would probably render more subtle features that could be of help in the differential diagnosis between both entities. Growing information on the relationship of drugs and AIH is being available, being drugs like statins and biologic agents more frequently involved in cases of DIAILD. In addition, there is some evidence on the fact that patients diagnosed with DIAILD may have had a previous episode of hepatotoxicity. Further collaborative studies in DIAILD will strengthen the knowledge and understanding of this intriguing and complex disorder which might represent different phenotypes across the spectrum of disease.

Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection.

Coxiella burnetii as a possible cause of autoimmune liver disease: a case report.

INTRODUCTION: Q fever is a zoonotic infection that may cause severe hepatitis. Q-fever hepatitis has not yet been associated with autoimmune hepatitis and/or primary biliary cirrhosis. CASE PRESENTATION: We describe a 39-year-old man of Sri Lankan origin with chronic Q-fever hepatitis who developed autoantibodies compatible with autoimmune hepatitis/primary biliary cirrhosis overlap syndrome. Ursodeoxycholic acid in addition to antibiotic therapy markedly improved hepatic enzyme levels suggesting that autoimmunity, potentially triggered by the underlying infection, was involved in the pathogenesis of liver damage. CONCLUSION: We suggest that Coxiella burnetii might trigger autoimmune liver disease. Patients with Q-fever hepatitis who respond poorly to antibiotics should be investigated for serological evidence of autoimmune hepatitis, primary biliary cirrhosis or overlap syndrome, as these patients could benefit from adjunctive therapy with ursodeoxycholic acid. Conversely, C. burnetii serology might be necessary in patients with autoimmune liver disease in order to exclude underlying Coxiella infection.

Evaluation of the antitumoral effect sunitinib eluting beads in VX2 liver tumour in a rabbit model.

Background: We demonstrated that DC Bead (Biocompatibles UK, Ltd) could be loaded with sunitinib and injected intra-arterially in the rabbit without unexpected toxicity. The purpose of this study is to evaluate the antitumoral effect of sunitinib eluting beads in the VX2 tumor model of liver cancer. Methods: VX2 tumors were implanted in the left liver lobe of New-Zealand white rabbits. Animals were assigned to 3 groups: Group 1 (n=6) received 1.5mg of sunitinib loaded in 0.05ml of 100-300um DC Bead, group 2 (n=5) received 0.05ml of 100-300um DC Bead, group 3 (n=5) received 0.05ml NaCl 0.9% in the left hepatic artery. One animal in each group was sacrificed at 24 hours and the others were followed for survival until day 15. Liver enzymes were measured daily. In group 1, plasmatic sunitinib concentration were measured daily by LC MS/MS tandem mass spectroscopy. At day 15 all living animals were sacrificed. After sacrifice, the livers were harvested for determination of the VEGF receptor tyrosine kinase activity by western blot and histopathological examination. Results: In group 1, no animals died during follow-up. In group 2, 2 animals died during follow-up on day x. In control group 3, 3 animals died during follow up on day x. In group 1 plasmatic sunitinib levels remained under therapeutic concentration throughout the experiment. Very high concentrations of sunitinib were measured in the liver tissue 24 and 15 days after embolization. Inhibition of the phosphorylation of the RTK was demonstrated at 24h and 15 days in groups 1. Sunitinib eluting beads seemed to penetrate in the tumor more effectively and there was more necrosis around the beads than their bland counterparts. Conclusions: Administration of sunitinib eluting beads in VX2 carrying rabbits resulted invery high drug concentrations at the site of embolization with minimal systemic passage. Despite the very high tissular sunitinib concentration we did not observe any additional toxicity with loaded beads. Sunitinib eluting beads inhibit the activation of RTK's triggered by ischemia and seem to prolong survival of the treated animals. Therefore we consider that local treatment with sunitinib may provide a promising approach for the treatment of liver cancer.