Human pancreatic cancer patients with Epithelial-to-Mesenchymal Transition and an aggressive phenotype show a disturbed balance in Protein Phosphatase Type 2A expression and functionality.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a low survival, its incidence is rising and little therapeutic improvements are expected in the near future. It has been observed that Epithelial-to-Mesenchymal transition (EMT) contributes (including in PDAC) to a more aggressive cancer phenotype. Additionally, largely unexplored, studies indicate a mechanistic interplay between Protein Phosphatase Type 2A (PP2A) enzymes and EMT that could offer treatment opportunities. The aim was to investigate the relation of a PP2A expression signature (encompassing all PP2A subunits, endogenous inhibitors and activators) with EMT and aggressive pancreatic cancer, and to discuss possible implications. METHODS: We retrieved different PDAC expression datasets from NCBI to capture the variation in patients, and analyzed these using datamining, survival analysis, differential gene and protein expression. We determined genes highly associated with aggressive PDAC. For in vitro evaluation, Panc-1 cells were treated with the pharmacologic PP2A inhibitor Okadaic Acid (OA). Additionally, two OA-resistant Panc-1 clones were developed and characterized. RESULTS: In patients, there is a strong correlation between EMT and aggressive PDAC, and between aggressive PDAC and PP2A, with a significant upregulation of PP2A inhibitor genes. Several PP2A genes significantly correlated with decreased survival. In vitro, short-term exposure to OA induced EMT in Panc-1 cells. This shift towards EMT was further pronounced in the OA-resistant Panc-1 clones, morphologically and by pathway analysis. Proteomic analysis and gene sequencing showed that the advanced OA-resistant model most resembles the clinical PDAC presentation (with EMT signature, and with several specific PP2A genes upregulated, and others downregulated). CONCLUSIONS: We demonstrated a strong association between EMT, altered PP2A expression and aggressive PDAC in patients. Also, in vitro, PP2A inhibition induces EMT. Overall, statistics suggests the mechanistic importance of PP2A dysregulation for PDAC progression. Translationally, our observations indicate that pharmacologic restoration of PP2A activity could be an attractive therapeutic strategy to block or reverse progression.

Autophagy-Related Chemoprotection against Sorafenib in Human Hepatocarcinoma: Role of FOXO3 Upregulation and Modulation by Regorafenib.

Early acquisition of sorafenib resistance is responsible for the dismal prognosis of advanced hepatocarcinoma (HCC). Autophagy, a catabolic process involved in liver homeostasis, has been associated with chemosensitivity modulation. Forkhead box O3 (FOXO3) is a transcription factor linked to HCC pathogenesis whose role on autophagy-related sorafenib resistance remains controversial. Here, we unraveled the linkage between autophagy and sorafenib resistance in HCC, focusing on the implication of FOXO3 and its potential modulation by regorafenib. We worked with two HepG2-derived sorafenib-resistant HCC in vitro models (HepG2S1 and HepG2S3) and checked HCC patient data from the UALCAN database. Resistant cells displayed an enhanced basal autophagic flux compared to HepG2, showing higher autophagolysosome content and autophagy markers levels. Pharmacological inhibition of autophagy boosted HepG2S1 and HepG2S3 apoptosis and subG1 cells, but reduced viability, indicating the cytoprotective role of autophagy. HCC samples displayed higher FOXO3 levels, being associated with shorter survival and autophagic genes expression. Consistently, chemoresistant in vitro models showed significant FOXO3 upregulation. FOXO3 knockdown suppressed autophagy and caused resistant cell death, demonstrating that overactivation of such pro-survival autophagy during sorafenib resistance is FOXO3-dependent; a cytoprotective mechanism that the second-line drug regorafenib successfully abolished. Therefore, targeting FOXO3-mediated autophagy could significantly improve the clinical efficacy of sorafenib.

Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity.

OBJECTIVE: Acute-on-chronic liver failure (ACLF) is associated with dysfunctional circulating monocytes whereby patients become highly susceptible to bacterial infections. Here, we identify the pathways underlying monocyte dysfunction in ACLF and we investigate whether metabolic rewiring reinstates their phagocytic and inflammatory capacity. DESIGN: Following phenotypic characterisation, we performed RNA sequencing on CD14+CD16- monocytes from patients with ACLF and decompensated alcoholic cirrhosis. Additionally, an in vitro model mimicking ACLF patient-derived features was implemented to investigate the efficacy of metabolic regulators on monocyte function. RESULTS: Monocytes from patients with ACLF featured elevated frequencies of interleukin (IL)-10-producing cells, reduced human leucocyte antigen DR isotype (HLA-DR) expression and impaired phagocytic and oxidative burst capacity. Transcriptional profiling of isolated CD14+CD16- monocytes in ACLF revealed upregulation of an array of immunosuppressive parameters and compromised antibacterial and antigen presentation machinery. In contrast, monocytes in decompensated cirrhosis showed intact capacity to respond to inflammatory triggers. Culturing healthy monocytes in ACLF plasma mimicked the immunosuppressive characteristics observed in patients, inducing a blunted phagocytic response and metabolic program associated with a tolerant state. Metabolic rewiring of the cells using a pharmacological inhibitor of glutamine synthetase, partially restored the phagocytic and inflammatory capacity of in vitro generated- as well as ACLF patient-derived monocytes. Highlighting its biological relevance, the glutamine synthetase/glutaminase ratio of ACLF patient-derived monocytes positively correlated with disease severity scores. CONCLUSION: In ACLF, monocytes feature a distinct transcriptional profile, polarised towards an immunotolerant state and altered metabolism. We demonstrated that metabolic rewiring of ACLF monocytes partially revives their function, opening up new options for therapeutic targeting in these patients.

Protective effects of Mentha piperita L. leaf essential oil against CCl4 induced hepatic oxidative damage and renal failure in rats.

BACKGROUND: Mentha piperita L. is a flowering plant belonging to the Lamiaceae family. Mentha plants constitute one of the main valuable sources of essential oil used in foods and for medicinal purposes. METHODS: The present study aimed to investigate the composition and in vitro antioxidant activity of Mentha piperita leaf essential oil (MpEO). A single dose of CCl4 was used to induce oxidative stress in rats, which was demonstrated by a significant rise of serum enzyme markers. MpEO was administrated for 7 consecutive days (5, 15, 40 mg/kg body weight) to Wistar rats prior to CCl4 treatment and the effects on serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ -glutamyl transpeptidase (γ-GT) levels, as well as the liver and kidney superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity and thiobarbituric acid reactive substances (TBARS) levels were evaluated. In addition, histopathological examinations of livers and kidneys was performed. RESULTS: The in vitro antioxidant activity of MpEO was lower than that of silymarin. Pretreatment of animals with MpEO at a dose of 5 mg/kg did not have a significant effect on ALT, AST, ALP, LDH, γGT, urea or creatinine levels in CCl4-induced stress. Whereas pretreatment with MpEO at doses of 15 and 40 mg/kg prior to CCl4, significantly reduced stress parameters (ALT, AST, ALP, LDH, γGT, urea and creatinine) compared to the CCl4-only group. Moreover, a significant reduction in hepatic and kidney lipid peroxidation (TBARS) and an increase in antioxidant enzymes SOD, CAT and GPx was also observed after treatment with MpEO (40 mg/kg) compared to CCl4-treated rats. Furthermore, pretreatment with MpEO at 40 mg/kg can also markedly ameliorate the histopathological hepatic and kidney lesions induced by administration of CCl4. CONCLUSIONS: We could demonstrate with this study that MpEO protects liver and kidney from CCl4-induced oxidative stress and thus substantiate the beneficial effects attributed traditionally to this plant.

Hepatocellular uptake of cyclodextrin-complexed curcumin during liver preservation: A feasibility study.

The increasing demand for donor organs and the decreasing organ quality is prompting research toward new methods to reduce ischemia reperfusion injury (IRI). Several strategies have been proposed to protect preserved organs from this injury. Before curcumin/dextrin complex (CDC), a potent antioxidant and anti-inflammatory agent, can be used clinically we need to better understand the intracellular uptake under hypothermic conditions on a rat model of liver donation after circulatory death (DCD) and brain death (DBD). To be able to use the fluorescence of CDC for quantification the stability of CDC in different preservation solutions at 4°C or 37°C was investigated. Livers from Wistar rats were procured after being flushed-out through the portal vein using CDC-enriched preservation solutions and stored at 4°C for variable periods. The CDC signal was stable in different preservation solutions over a period of 4 h and allowed the rapid and lasting uptake of curcumin into cells. After 4 h of preservation, CDC was no longer visible microscopically, and HPLC analysis showed very low to non-detectable tissue levels of CDC, proving metabolization during preservation. However, the distribution of CDC was not affected by warm ischemia damage (p = 0.278) nor by flushing the livers before or after 4 h of cold storage and without a warm preflush. Finally, curcumin reduced oxidative stress, lowered histological injury and did not change gene expression after WI/cold storage. Therefore, the use of CDC flush solution for the initial organ flush can offer a promising approach to the enhancement of liver preservation and the maintenance of its quality.

Hepatocyte MyD88 affects bile acids, gut microbiota and metabolome contributing to regulate glucose and lipid metabolism.

OBJECTIVE: To examine the role of hepatocyte myeloid differentiation primary-response gene 88 (MyD88) on glucose and lipid metabolism. DESIGN: To study the impact of the innate immune system at the level of the hepatocyte and metabolism, we generated mice harbouring hepatocyte-specific deletion of MyD88. We investigated the impact of the deletion on metabolism by feeding mice with a normal control diet or a high-fat diet for 8 weeks. We evaluated body weight, fat mass gain (using time-domain nuclear magnetic resonance), glucose metabolism and energy homeostasis (using metabolic chambers). We performed microarrays and quantitative PCRs in the liver. In addition, we investigated the gut microbiota composition, bile acid profile and both liver and plasma metabolome. We analysed the expression pattern of genes in the liver of obese humans developing non-alcoholic steatohepatitis (NASH). RESULTS: Hepatocyte-specific deletion of MyD88 predisposes to glucose intolerance, inflammation and hepatic insulin resistance independently of body weight and adiposity. These phenotypic differences were partially attributed to differences in gene expression, transcriptional factor activity (ie, peroxisome proliferator activator receptor-α, farnesoid X receptor (FXR), liver X receptors and STAT3) and bile acid profiles involved in glucose, lipid metabolism and inflammation. In addition to these alterations, the genetic deletion of MyD88 in hepatocytes changes the gut microbiota composition and their metabolomes, resembling those observed during diet-induced obesity. Finally, obese humans with NASH displayed a decreased expression of different cytochromes P450 involved in bioactive lipid synthesis. CONCLUSIONS: Our study identifies a new link between innate immunity and hepatic synthesis of bile acids and bioactive lipids. This dialogue appears to be involved in the susceptibility to alterations associated with obesity such as type 2 diabetes and NASH, both in mice and humans.

The release of microRNA-122 during liver preservation is associated with early allograft dysfunction and graft survival after transplantation.

Early allograft dysfunction (EAD) after liver transplantation (LT) is associated with inferior graft survival. EAD is more prevalent in grafts from donation after circulatory death (DCD). However, accurate prediction of liver function remains difficult because of the lack of specific biomarkers. Recent experimental and clinical studies highlight the potential of hepatocyte-derived microRNAs (miRNAs) as sensitive, stable, and specific biomarkers of liver injury. The aim of this study was to determine whether miRNAs in graft preservation fluid are predictive for EAD after clinical LT and in an experimental DCD model. Graft preservation solutions of 83 liver grafts at the end of cold ischemia were analyzed for miRNAs by reverse transcription polymerase chain reaction. Of these grafts, 42% developed EAD after transplantation. Results were verified in pig livers (n = 36) exposed to different lengths of warm ischemia time (WIT). The absolute miR-122 levels and miR-122/miR-222 ratios in preservation fluids were significantly higher in DCD grafts (P = 0.001) and grafts developing EAD (P = 0.004). In concordance, the miR-122/miR-222 ratios in perfusion fluid correlate with serum transaminase levels within the first 24 hours after transplantation. Longterm graft survival was significantly diminished in grafts with high miR-122/miR-222 ratios (P = 0.02). In the porcine DCD model, increased WIT lead to higher absolute miR-122 levels and relative miR-122/miR-222 ratios in graft perfusion fluid (P = 0.01 and P = 0.02, respectively). High miR-122/miR-222 ratios in pig livers were also associated with high aspartate aminotransferase levels after warm oxygenated reperfusion. In conclusion, both absolute and relative miR-122 levels in graft preservation solution are associated with DCD, EAD, and early graft loss after LT. As shown in a porcine DCD model, miRNA release correlated with the length of WITs. Liver Transplantation 23 946-956 2017 AASLD.

Dietary intervention, but not losartan, completely reverses non-alcoholic steatohepatitis in obese and insulin resistant mice.

BACKGROUND: Dietary intervention is the cornerstone of non-alcoholic steatohepatitis (NASH) treatment. However, histological evidence of its efficacy is limited and its impact on hepatic pathways involved in NASH is underreported. The efficacy of the angiotensin receptor type 1 blocker losartan is controversial because of varying results in a few animal and human studies. We evaluated the effect of dietary intervention versus losartan on NASH and associated systemic metabolic features in a representative mouse model. METHODS: Male C57BL/6 J mice with high fat-high sucrose diet (HF-HSD) induced NASH, obesity, insulin resistance and hypercholesterolemia were subjected to dietary intervention (switch from HF-HSD to normal chow diet (NCD)) (n = 9), continuation HF-HSD together with losartan (30 mg/kg/day) (n = 9) or continuation HF-HSD only (n = 9) for 8 weeks. 9 mice received NCD during the entire experiment (20 weeks). We assessed the systemic metabolic effects and performed a detailed hepatic histological and molecular profiling. A P-value of < 0.05, using the group with continuation of HF-HSD only as control, was considered as statistically significant. RESULTS: Dietary intervention normalized obesity, insulin resistance, and hypercholesterolemia (for all P < 0.001), and remarkably, completely reversed all histological features of pre-existent NASH (for all P < 0.001), including fibrosis measured by quantification of collagen proportional area (P < 0.01). At the hepatic molecular level, dietary intervention targeted fibrogenesis with a normalization of collagen type I alpha 1, transforming growth factor ß1, tissue inhibitor of metalloproteinase 1 mRNA levels (for all P < 0.01), lipid metabolism with a normalization of fatty acid translocase/CD36, fatty acid transport protein 5, fatty acid synthase mRNA levels (P < 0.05) and markers related to mitochondrial function with a normalization of hepatic ATP content (P < 0.05) together with sirtuin1 and uncoupling protein 2 mRNA levels (for both P < 0.001). Dietary intervention abolished p62 accumulation (P < 0.01), suggesting a restoration of autophagic flux. Losartan did not significantly affect obesity, insulin resistance, hypercholesterolemia or any histological NASH feature. CONCLUSIONS: Dietary intervention, and not losartan, completely restores the metabolic phenotype in a representative mouse model with pre-existent NASH, obesity, insulin resistance and hypercholesterolemia.

Association of Adipose Tissue Inflammation With Histologic Severity of Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: The prevalence of nonalcoholic fatty liver disease (NAFLD) has increased with the obesity pandemic. We analyzed the transcriptional profiles of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT), and phenotypes and functional characteristics of adipocyte tissue macrophages (ATMs), in obese patients undergoing bariatric surgery. METHODS: We collected anthropometric data; plasma samples; and SAT, VAT, and liver tissues from 113 obese patients undergoing bariatric surgery at academic hospitals in Europe (Antwerp and Leuven) and South Africa. Based on clinical and histologic features, patients were assigned to the following groups: obese, NAFLD, nonalcoholic steatohepatitis (NASH), or NASH with fibrosis. Microarray analyses were performed to identify genes expressed differentially among groups. We measured levels of cytokines and chemokines in plasma samples and levels of RNAs in adipose tissues by quantitative reverse-transcription polymerase chain reaction. ATMs were isolated from patients and 13 lean individuals undergoing cholecystectomy (controls), analyzed by flow cytometry, and cultured; immunophenotypes and levels of cytokines and chemokines in supernatants were determined. RESULTS: We observed increased expression of genes that regulate inflammation in adipose tissues from patients with NAFLD and NASH; expression of these genes increased as disease progressed from NAFLD to NASH. We found 111 genes associated with inflammation that were expressed differentially between VAT and SAT. Serum levels of interleukin 8, chemokine (C-C motif) ligand 3, and tumor necrosis factor-α correlated with liver inflammation and NAFLD activity score. We developed 2 models that could be used to determine patients' liver histology based on gene expression in VAT and SAT. Flow cytometry showed increased proportions of CD11c+CD206+ and CCR2+ macrophages in VAT from patients with NASH, and supernatants of cultured macrophages had increased levels of cytokines and chemokines compared with controls. CONCLUSIONS: VAT and SAT from patients with NAFLD and NASH have an increased expression of genes that regulate inflammation, and ATM produce increased levels of inflammatory cytokines, compared with adipose tissues from controls. We identified an expression profile of 5 genes in SAT that accurately predict liver histology in these patients. Transcript profiling: accession numbers: GSE58979 and GSE59045.

Successful application of endoscopic ultrasound-guided fine needle biopsy to establish pancreatic patient-derived tumor xenografts: a pilot study.

Background and study aim: Typically, pancreatic patient-derived tumor xenografts (PDXs) are established by transplanting large tumor biopsies obtained through invasive surgery approaches into immunocompromised mice. We aimed to develop pancreatic PDXs by transplanting tumor tissue acquired by endoscopic ultrasound (EUS)-guided fine needle biopsies (FNB), assess take rates compared to surgery-derived PDXs, and demonstrate the histological and genetic resemblance to the original tumor. Patients and methods: Biopsies of untreated pancreatic carcinoma were collected at surgery and during EUS and processed to generate PDXs. Results: By centrifugation of FNB-derived tissue prior to engraftment, we achieved an engraftment rate of 60 % (6/10). Despite a decrease in stromal tissue, the general morphology of FNB-derived PDXs was conserved as assessed by histopathology. At the genetic level, somatic mutation and copy number profiles were largely similar to the primary tumor. Conclusion: We show that it is technically feasible to establish pancreatic PDXs using a minimally invasive sampling technique, such as EUS-FNB. Although only a limited amount of tumor tissue was acquired, we obtained results similar to those from surgery-derived PDXs.

Roux-en-y gastric bypass attenuates hepatic mitochondrial dysfunction in mice with non-alcoholic steatohepatitis.

OBJECTIVE: No therapy for non-alcoholic steatohepatitis (NASH) has been approved so far. Roux-en-y gastric bypass (RYGB) is emerging as a therapeutic option, although its effect on NASH and related hepatic molecular pathways is unclear from human studies. We studied the effect of RYGB on pre-existent NASH and hepatic mitochondrial dysfunction-a key player in NASH pathogenesis-in a novel diet-induced mouse model nicely mimicking human disease. DESIGN: C57BL/6J mice were fed a high-fat high-sucrose diet (HF-HSD). RESULTS: HF-HSD led to early obesity, insulin resistance and hypercholesterolaemia. HF-HSD consistently induced NASH (steatosis, hepatocyte ballooning and inflammation) with fibrosis already after 12-week feeding. NASH was accompanied by hepatic mitochondrial dysfunction, characterised by decreased mitochondrial respiratory chain (MRC) complex I and IV activity, ATP depletion, ultrastructural abnormalities, together with higher 4-hydroxynonenal (HNE) levels, increased uncoupling protein 2 (UCP2) and tumour necrosis factor-α (TNF-α) mRNA and free cholesterol accumulation. In our model of NASH and acquired mitochondrial dysfunction, RYGB induced sustained weight loss, improved insulin resistance and inhibited progression of NASH, with a marked reversal of fibrosis. In parallel, RYGB preserved hepatic MRC complex I activity, restored ATP levels, limited HNE production and decreased TNF-α mRNA. CONCLUSIONS: Progression of NASH and NASH-related hepatic mitochondrial dysfunction can be prevented by RYGB. RYGB preserves respiratory chain complex activity, thereby restoring energy output, probably by limiting the amount of oxidative stress and TNF-α. These data suggest that modulation of hepatic mitochondrial function contributes to the favourable effect of RYBG on established NASH.

The plant decapeptide OSIP108 prevents copper-induced apoptosis in yeast and human cells.

We previously identified the Arabidopsis thaliana-derived decapeptide OSIP108, which increases tolerance of plants and yeast cells to oxidative stress. As excess copper (Cu) is known to induce oxidative stress and apoptosis, and is characteristic for the human pathology Wilson disease, we investigated the effect of OSIP108 on Cu-induced toxicity in yeast. We found that OSIP108 increased yeast viability in the presence of toxic Cu concentrations, and decreased the prevalence of Cu-induced apoptotic markers. Next, we translated these results to the human hepatoma HepG2 cell line, demonstrating anti-apoptotic activity of OSIP108 in this cell line. In addition, we found that OSIP108 did not affect intracellular Cu levels in HepG2 cells, but preserved HepG2 mitochondrial ultrastructure. As Cu is known to induce acid sphingomyelinase activity of HepG2 cells, we performed a sphingolipidomic analysis of OSIP108-treated HepG2 cells. We demonstrated that OSIP108 decreased the levels of several sphingoid bases and ceramide species. Moreover, exogenous addition of the sphingoid base dihydrosphingosine abolished the protective effect of OSIP108 against Cu-induced cell death in yeast. These findings indicate the potential of OSIP108 to prevent Cu-induced apoptosis, possibly via its effects on sphingolipid homeostasis.

Lanreotide Reduces Liver Volume, But Might Not Improve Muscle Wasting or Weight Loss, in Patients With Symptomatic Polycystic Liver Disease.

BACKGROUND & AIMS: Polycystic liver disease (PCLD) can induce malnutrition owing to extensive hepatomegaly and patients might require liver transplantation. Six months of treatment with the somatostatin analogue lanreotide (120 mg) reduces liver volume. We investigated the efficacy of a lower dose of lanreotide and its effects on nutritional status. METHODS: We performed an 18-month prospective study at 2 tertiary medical centers in Belgium from January 2011 through August 2012. Fifty-nine patients with symptomatic PCLD were given lanreotide (90 mg, every 4 weeks) for 6 months. Patients with reductions in liver volume of more than 100 mL (responders, primary end point) continued to receive lanreotide (90 mg) for an additional year (18 months total). Nonresponders were offered increased doses, up to 120 mg lanreotide, until 18 months. Liver volume and body composition were measured by computed tomography at baseline and at months 6 and 18. Patients also were assessed by the PCLD-specific complaint assessment at these time points. RESULTS: Fifty-three patients completed the study; 21 patients (40%) were responders. Nineteen of the responders (90%) continued as responders until 18 months. At this time point, they had a mean reduction in absolute liver volume of 430 ± 92 mL. In nonresponders (n = 32), liver volume increased by a mean volume of 120 ± 42 mL at 6 months. However, no further increase was observed after dose escalation in the 24 patients who continued to the 18-month end point. All subjects had decreased scores on all subscales of the PCLD-specific complaint assessment, including better food intake (P = .04). Subjects did not have a mean change in subcutaneous or visceral fat mass, but did have decreases in mean body weight (2 kg) and total muscle mass (1.06 cm(2)/h(2)). Subjects also had a significant mean reduction in their level of insulin-like growth factor 1, from 19% below the age-adjusted normal range level at baseline to 50% at 18 months (P = .002). CONCLUSIONS: In a prospective study, we observed that low doses of lanreotide (90 mg every 4 weeks) reduced liver volumes and symptoms in patients with PCLD. However, patients continued to lose weight and muscle mass. The effects of somatostatin analogues on sarcopenia require investigation. Clinicaltrials.gov: NCT01315795.

Neurotoxicity in rats induced by the poisonous dreamfish (Sarpa salpa).

CONTEXT: Consumption of Sarpa salpa Linn. (Sparidae) in certain periods of the year is inadvisable because it can cause central nervous system disorders resulting in sea food poisoning. AIMS: The present study assesses the cytotoxic effects of compounds, not-yet identified, present in the organ extracts of S. salpa, collected in autumn, the period corresponding to the peak in human health problems. MATERIALS AND METHODS: The toxicity was assessed by mouse bioassay of aqueous extract of the fish organs. Wistar rats received daily extracts of different organs of S. salpa by gastric gavage for 7 d (0.3 mL of extract/100 g body weight BW). The dose of tissue extracts of viscera, liver, brain, and flesh of S. salpa administered to rats was as follows: 172, 313, 2050, and 2660 mg/kg BW, respectively. No deaths occurred during the period of treatment. RESULTS: The lethal dose (LD50) determined for the crude ciguatoxin (neurotoxins) extracts of viscera, liver, brain, and flesh of S. salpa was as follows: 1.2, 2.2, 14.4, and 18.6 g/kg mouse, respectively. Changes in locomotor activity during the first 2 h and failure in breathing and no evident signs of gastrointestinal problems were recorded. We observed (1) induction of oxidative stress, indicated by an increase in lipid peroxidation (TBARS) in groups that received extracts of liver (+425%) or viscera (+433%), and a significant decrease in antioxidant enzyme activities (SOD, CAT, and GPx) in cerebral cortex tissue by 13%, 25%, and 25% (LT: animals receiving liver extracts) and by 16%, 26%, and 27% (VT: animals receiving viscera extracts), respectively. In contrast, the administration of extracts of flesh and brain induced an increase in antioxidant enzyme activities (SOD, CAT, and GPx) in cerebral cortex tissue by 26%, 23%, and 44% (FT: flesh extract) and 28%, 24%, and 46% (BT: brain extract), respectively; (2) a significant decrease for acetylcholinesterase (AChE) activity in cerebral cortex was recorded in FT, BT, LT, and VT by 27, 34, 58, and 78%, respectively. Moreover, a significant decrease of AChE activity in plasma was recorded in FT, BT, LT, and VT by 16, 21, 38, and 48%, respectively; (3) the histological findings confirmed the biochemical results. CONCLUSIONS: Liver and especially the visceral part of S. salpa presented toxicity, which clearly indicates the danger of using this fish as food.

Protective effect of kombucha on rats fed a hypercholesterolemic diet is mediated by its antioxidant activity.

CONTEXT: Kombucha (KT) is claimed to have various beneficial effects on human health, but there is very little scientific evidence available in the literature. OBJECTIVE: The present study investigates the effects of Camellia sinensis (GT) Linn. (Theaceae) and KT, two natural drinks, on cholesterol and antioxidant status using a hypercholesterolemia rat model. MATERIALS AND METHODS: The present study compared the free-radical scavenging abilities and polyphenol levels of GT and KT. Wistar rats fed cholesterol-rich diets were given KT or GT (5 mL/kg body weight per day, po) for 16 weeks, then fasted overnight and sacrificed. The plasma lipid levels, thiobarbituric acid reactive substances (TBARS) and aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transpeptidase (GGT) serum levels, antioxidant activities of superoxide dismutase (SOD) and catalase (CAT), and creatinine and urea rats were examined. RESULTS: KT had a phenolic compound of 955 ± 0.75 mg GAE/g) followed, by GT (788.92 ± 0.02 mg GAE/g). The free radical scavenging activity of KT was higher than GT. Compared with GT, KT induced lowered serum levels of TC, TG, VLDL-C, and LDL-C by 26, 27, 28, and 36%, respectively, and increased the serum level of high-density lipoprotein cholesterol (HDL-C). KT induced a 55% decrease of TBARS level in liver and 44% in kidney, compared with those of rats fed a cholesterol-rich diet alone. Moreover, CAT and SOD activities were reduced by 29 and 33%, respectively, in liver and 31 and 35%, respectively, in kidney, after oral administration of KT, compared with those of HCD-fed rats. CONCLUSION: The findings revealed that KT administration induced attractive curative effects on hypercholesterolemic, particularly in terms of liver-kidney functions in rats. Its effect on humans needs to be studied further.

Protective effects of Hammada scoparia flavonoid-enriched fraction on liver injury induced by warm ischemia/reperfusion.

CONTEXT: Hepatic ischemia/reperfusion injury (IRI) is a major cause of liver damage during liver surgery and transplantation. Plants have historically been used in treating liver damage, and Hammada scoparia (Pomel) (Chenopodiaceae) has been reported to possess a broad spectrum of pharmacological and therapeutic activities. OBJECTIVE: In this study, a flavonoid-enriched fraction was used before the warm ischemia (WI) process as pharmacological preconditioning and in combination with technical postconditioning to evaluate their protective effects. MATERIALS AND METHODS: The rats were divided into five groups: a sham group; a control group (Control-IR) that was submitted to 60 min WI; a Pharmacological Preconditioning group (PreC-IR) that received flavonoid-enriched fraction (200 mg/kg body weight); a Postconditioning group (PostC) and a PreC + PostC group. RESULTS: The use of the flavonoid-enriched fraction was noted to significantly (p < 0.05) reduce liver injury, as evidenced by the decrease in liver transaminase activities (AST and ALT) and lactic dehydrogenase (LDH), alkaline phosphatase (ALP), and lipid peroxidation (TBARS), levels as well as the enhancement of antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) responses. The results also indicated that, compared with the separate application of pharmacological preconditioning and postconditioning, the combination of both treatments was more effective in reducing tissue oxidative stress levels through modulating SOD, GSH-PX, and CAT activities. Furthermore, the combined protocol further decreased the liver morphological score compared with solo treatment. DISCUSSION AND CONCLUSION: Overall, the results indicate that the H. scoparia flavonoid-enriched fraction could be a promising candidate for future application as a pharmacological preconditioning agent against hepatic IRI.

Development and validation of a polycystic liver disease complaint-specific assessment (POLCA).

BACKGROUND & AIMS: Polycystic liver disease (PCLD) may lead to extensive hepatomegaly and invalidating complaints. Therapeutic decisions, including somatostatin-analogues (SAs) and (non)-transplant surgery are besides the existence of hepatomegaly, also guided by the severity of complaints. We developed and validated a self-report instrument to capture the presence and severity of disease specific complaints for PCLD. METHODS: The study population consisted of 129 patients. Items for the PCLD-complaint-specific assessment (POLCA) were developed based on the chart review of symptomatic PCLD patients (n=68) and literature, and discussed during expert-consensus-meetings. 61 patients who needed therapy were asked to complete the POLCA and the short form health survey version 2 (SF36V2) at baseline and after 6 months of SA-treatment. CT-scans were used to calculate liver volumes (LV). Factor analysis was conducted to identify subscales and remove suboptimal items. Reliability was assessed by Cronbach's alpha. Convergent, criterion validity and responsiveness were tested using prespecified hypotheses. RESULTS: In the validation group (n=61), 47 received lanreotide (LAN) and 14 were offered LAN as bridge to liver transplantation (LTx). Factor analysis identified four subscales, which correlated with the physical component summary (PCS). Baseline POLCA scores were significantly higher in LTx-listed patients. In contrast to SF36V2, POLCA-paired observations in 47 patients demonstrated that 2 subscales were lowered significantly and 2 borderline. LV reduction of ⩾ 120 ml resulted in a numerical, more pronounced relative decrease of all scores. CONCLUSIONS: In contrast to SF36V2, the POLCA shows good validity and responsiveness to measure complaint severity in PCLD.

Assessing warm ischemic injury of pig livers at hypothermic machine perfusion.

BACKGROUND: Livers originating from donation after circulatory death (DCD) donors are exposed to warm ischemia (WI) before liver transplantation (LTx). Currently, there are no objective tests to evaluate the damage sustained before LTx. This study aims to identify surrogate markers for liver injury that can be assessed during hypothermic machine perfusion (HMP) preservation. In addition, we want to use mathematical equation modeling combining these markers to improve our assessment of DCD livers for transplantation. MATERIALS AND METHODS: Porcine livers were exposed to incremental periods of WI (0-120 min) and subsequently HMP preserved for 4 h. Biochemical and hemodynamic parameters were repeatedly measured in the perfusate during HMP. Subsequently, to mimic LTx, normothermic isolated-liver perfusion was applied for 2 h and the injury assessed using a morphological score. RESULTS: With increasing WI periods, the perfusate became more acidotic, and levels of aspartate aminotransferase (AST), liver fatty acid binding protein, redox-active iron, and arterial vascular resistance increased. A damage index, combining AST and pH (damage index = 2 - 37 × ß(AST) - 257 × ß(pH)) based on multifactorial analysis of the changing pattern of these markers, had increased sensitivity and specificity to reflect WI and reperfusion injury. CONCLUSIONS: This proof of concept study demonstrated the potential role for objective evaluation of DCD porcine livers during HMP and the advantage to use multifactorial analysis on the markers' changing pattern.

Transcatheter arterial chemoembolization with doxorubicin-eluting superabsorbent polymer microspheres in the treatment of hepatocellular carcinoma: midterm follow-up.

PURPOSE: To investigate prospectively the safety, tolerability, and efficacy of transarterial chemoembolization using superabsorbent polymer (SAP) microspheres loaded with doxorubicin for the treatment of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: During the years 2006-2011, 64 patients underwent 144 transarterial chemoembolization with SAP microspheres procedures. Most of the patients were staged as Barcelona Clinic Liver Cancer class B (65%). The most frequent underlying liver diseases were hepatitis C (35%) and alcoholic liver disease (28%) resulting in Child-Pugh A (73.4%) or Child-Pugh B (17%) liver cirrhosis. Tumor response was assessed using modified Response Evaluation Criteria in Solid Tumors with magnetic resonance (MR) imaging performed 4-6 weeks after each procedure. RESULTS: Serious adverse events (n = 9) were ischemic or infectious in nature. Transarterial chemoembolization with SAP microspheres resulted in objective response rates of 67.5%, 44.5%, and 25% after first, second, and third sessions. There were 16 patients (25%) who underwent orthotopic liver transplantation after transarterial chemoembolization with SAP microspheres, of whom 2 experienced recurrent disease. During a median follow-up time of 14 months (range, 2-55 mo), 26 patients (40.5%) died. Median overall and transplant-free survivals were 20.5 months (95% confidence interval, 13.2-27.7) and 18 months (95% confidence interval, 14.2-21.8), respectively. CONCLUSIONS: Transarterial chemoembolization with SAP microspheres has an excellent safety profile in cirrhotic patients, even in the presence of advanced liver disease (Child-Pugh B) or advanced stages of HCC. This treatment produced meaningful tumor response rates as assessed by MR imaging.

Diversity of cultivable halophilic archaea and bacteria from superficial hypersaline sediments of Tunisian solar salterns.

Prokaryotes in the superficial sediments are ecologically important microorganisms that are responsible for the decomposition, mineralization and subsequent recycling of organic matter. The aim of this study was to explore the phylogenetic and functional diversity of halophilic archaea and bacteria isolated from the superficial sediments of solar salterns at Sfax, Tunisia. Sixty four strains were isolated from crystallizer (TS18) and non-crystallizer (M1) ponds and submitted to genotypic characterization and evaluation by amplified ribosomal RNA restriction analysis (ARDRA) techniques. Our findings revealed that the archaeal diversity observed for 29 isolates generated five distinct patterns from the non-crystallizer M1 pond, with Halorubrum chaoviator as the most prevalent cultivable species. However, in the TS18 crystallizer pond, ten restriction patterns were observed, with the prevalence of haloarchaea EB27K, a not yet identified genotype. The construction of a neighbour-joining tree of 16S rRNA gene sequences resulted in the division of the potential new species into two major groups, with four strains closely related to the sequence of the unculturable haloarchaeon EB27K and one strain to the recently described Halovenus aranensis strain. The 35 bacterial strains observed in this work were present only in the non-crystallizer pond (M1) and presented two distinct ARDRA patterns. These strains belonged to the γ-proteobacteria subdivision, with members of Salicola marasensis (83%) being the most predominant species among the isolates. 16S rRNA gene sequencing revealed that Salicola strains displayed different degrees of homogeneity. The results from pulsed field gel electrophoresis assays showed that the Salicola isolates could be clustered in two distinct groups with different genome sizes.