Photodynamic therapy for cholangiocarcinoma: overview and new developments

PURPOSE OF REVIEW: Photodynamic therapy (PDT) with hematoporphyrins has emerged as promising treatment for nonresectable cholangiocarcinoma in several prospective observational studies and two randomized studies. This review describes the mechanism of action of PDT, gives an overview of clinical experience in cholangiocarcinoma and summarizes the results published in 2007 and 2008. RECENT FINDINGS: The mechanism of action of PDT has been further elucidated. PDT induces an apoptotic, antiangiogenic as well as an immunomodulatory response. Interleukin-6, a bile duct epithelium growth factor correlating with tumor burden, decreases after PDT. The efficacy of PDT was confirmed in a comparative study in the United States. Patients with no visible mass on imaging studies, high serum albumin levels and treatment immediately after diagnosis seem to benefit most from PDT. Although it is recommended to perform PDT in bile ducts without stents in place, illumination through metal stents is possible if the light dose is adjusted. Meso-tetrahydroxyphenyl chlorine is a new potent photosensitizer for PDT of cholangiocarcinoma. SUMMARY: In advanced nonresectable cholangiocarcinoma, PDT is the only evidence-based treatment that improves survival when compared with stenting. Therefore, PDT should be offered to those who are unsuitable for surgery.

High protein intake reduces intrahepatocellular lipid deposition in humans

BACKGROUND: High sugar and fat intakes are known to increase intrahepatocellular lipids (IHCLs) and to cause insulin resistance. High protein intake may facilitate weight loss and improve glucose homeostasis in insulin-resistant patients, but its effects on IHCLs remain unknown. OBJECTIVE: The aim was to assess the effect of high protein intake on high-fat diet-induced IHCL accumulation and insulin sensitivity in healthy young men. DESIGN: Ten volunteers were studied in a crossover design after 4 d of either a hypercaloric high-fat (HF) diet; a hypercaloric high-fat, high-protein (HFHP) diet; or a control, isocaloric (control) diet. IHCLs were measured by (1)H-magnetic resonance spectroscopy, fasting metabolism was measured by indirect calorimetry, insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp, and plasma concentrations were measured by enzyme-linked immunosorbent assay and gas chromatography-mass spectrometry; expression of key lipogenic genes was assessed in subcutaneous adipose tissue biopsy specimens. RESULTS: The HF diet increased IHCLs by 90 +/- 26% and plasma tissue-type plasminogen activator inhibitor-1 (tPAI-1) by 54 +/- 11% (P < 0.02 for both) and inhibited plasma free fatty acids by 26 +/- 11% and beta-hydroxybutyrate by 61 +/- 27% (P < 0.05 for both). The HFHP diet blunted the increase in IHCLs and normalized plasma beta-hydroxybutyrate and tPAI-1 concentrations. Insulin sensitivity was not altered, whereas the expression of sterol regulatory element-binding protein-1c and key lipogenic genes increased with the HF and HFHP diets (P < 0.02). Bile acid concentrations remained unchanged after the HF diet but increased by 50 +/- 24% after the HFHP diet (P = 0.14). CONCLUSIONS: Protein intake significantly blunts the effects of an HF diet on IHCLs and tPAI-1 through effects presumably exerted at the level of the liver. Protein-induced increases in bile acid concentrations may be involved. This trial was registered at www.clinicaltrials.gov as NCT00523562.

Maternal cholestasis during pregnancy programs metabolic disease in offspring

The intrauterine environment is a major contributor to increased rates of metabolic disease in adults. Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy that affects 0.5%-2% of pregnant women and is characterized by increased bile acid levels in the maternal serum. The influence of ICP on the metabolic health of offspring is unknown. We analyzed the Northern Finland birth cohort 1985-1986 database and found that 16-year-old children of mothers with ICP had altered lipid profiles. Males had increased BMI, and females exhibited increased waist and hip girth compared with the offspring of uncomplicated pregnancies. We further investigated the effect of maternal cholestasis on the metabolism of adult offspring in the mouse. Females from cholestatic mothers developed a severe obese, diabetic phenotype with hepatosteatosis following a Western diet, whereas matched mice not exposed to cholestasis in utero did not. Female littermates were susceptible to metabolic disease before dietary challenge. Human and mouse studies showed an accumulation of lipids in the fetoplacental unit and increased transplacental cholesterol transport in cholestatic pregnancy. We believe this is the first report showing that cholestatic pregnancy in the absence of altered maternal BMI or diabetes can program metabolic disease in the offspring.

Cholestasis in a patient with gallstones and a normal gamma-glutamyl transferase

Cholestasis with normal gamma glutamyl transferase characterizes functional deficiencies in the gene ABCB11, which encodes the bile salt export pump (BSEP), a liver-specific adenosine triphosphate (ATP)-binding cassette transporter. Here we report the case of a patient presenting with features of benign recurrent intrahepatic cholestasis associated with a heterozygous mutation in the ABCB11 gene. Immunohistochemistry showed a gradual decrease of BSEP from zone 1 to zone 3 of the liver lobule, suggesting that the mutation identified here may predispose patients to cholestasis through a delocalization process of BSEP at the lobular level. (HEPATOLOGY 2013;57:2539-2541).

Infected pancreatic necrosis increases the severity of experimental necrotizing pancreatitis in mice

OBJECTIVE Infection of pancreatic necrosis in necrotizing pancreatitis increases the lethality of patients with acute pancreatitis. To examine mechanisms underlying this clinical observation, we developed and tested a model, in which primary infection of necrosis is achieved in taurocholate-induced pancreatitis in mice. METHODS Sterile necrosis of acute necrotizing pancreatitis was induced by retrograde injection of 4% taurocholate into the common bile duct of Balb/c mice. Primary infection of pancreatic necrosis was induced by coinjecting 10 colony-forming units of Escherichia coli. Animals were killed after 6, 12, 24, 48, and 120 hours, and pancreatic damage and pancreatitis-associated systemic inflammatory response were assessed. RESULTS Mice with pancreatic acinar cell necrosis had an increased bacterial concentration in all tissues and showed sustained bacteremia. Acute pancreatitis was induced only by coinjection of taurocholate and not by bacterial infection alone. Infection of pancreatic necrosis increased pancreatic damage and the pulmonary vascular leak. Serum glucose concentrations serving as a parameter of hepatic function were reduced in mice with infected pancreatic necrosis. CONCLUSIONS Primary infection of pancreatic necrosis with E. coli increases both pancreatic damage and pulmonary and hepatic complications in acute necrotizing pancreatitis in mice.

Vascular cell adhesion molecule 1 expression by biliary epithelium promotes persistence of inflammation by inhibiting effector T-cell apoptosis

Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM-1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding a4b1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM-1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM-1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM-1 promotes the survival of intrahepatic a4b1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova-bil antigen-driven murine model of biliary inflammation. VCAM-1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM-1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin-17. Liver-derived T cells adhered to cholangiocytes in vitro by a4b1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen-activated protein kinase phosphorylation. This led to increased mitochondrial B-cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM-1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 Tcell survival. Conclusions: VCAM-1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to a4b1 expressing proinflammatory T lymphocytes in CLD.

Isolation and {\it in vitro}-immunosuppressive characterization of a novel cyclosporine metabolite

Cyclosporine (CsA) has shown great benefit to organ transplant recipients, as an immunosuppressive drug. To optimize CsA immunosuppressive therapy, pharmacodynamic evaluation of serial patient serum samples after CsA administration, using mixed lymphocyte culture (MLC) assays, revealed in vitro serum immunosuppressive activity of a CsA-like, ether-extractable component, associated with good clinical outcome in vivo. Since the in vitro immunosuppressive CsA metabolites, M-17 and M-1, are erythrocyte-bound, the immunosuppressive activity demonstrated in patient serum suggests that other immunosuppressive metabolites need exist. To test this hypothesis and obtain CsA metabolites for study, ether-extracted bile from tritiated and nonradioactive CsA-treated pigs was processed by novel high performance liquid and thin-layer chromatography (HPLC and HPTLC) techniques. Initial MLC screening of potential metabolites revealed a component, designated M-E, to have immunosuppressive activity. Pig bile-derived M-E was characterized as a CsA metabolite, by radioactive CsA tracer studies, by 56% crossreactivity in CsA radioimmunoassay, and by mass spectrometric (MS) analysis. MS revealed a CsA ring structure, hydroxylated at a site other than at amino acid one. M-E was different than M-1 and M-17, as demonstrated by different retention properties for each metabolite, using HPTLC and a novel rhodamine B/ $\alpha$-cyclodextrin stain, and using HPLC, performed by Sandoz, that revealed M-E to be different than previously characterized metabolites. The immunosuppressive activity of M-E was quantified by determination of mean metabolite potency ratio in human MLC assays, which was found to be 0.79 $\pm$ 0.23 (CsA, 1.0). Similar to parent drug, M-E revealed inter-individual differences in its immunosuppressive activity. M-E demonstrates inhibition of IL-2 production by concanavalin A stimulated C3H mouse spleen cells, similar to CsA, as determined with an IL-2 dependent mouse cytotoxic T-cell line. ^

Toward individualized cholesterol-lowering treatment in end-stage renal disease.

There is broad evidence that lowering low-density lipoprotein (LDL) cholesterol will reduce cardiovascular risk. However, in patients on maintenance hemodialysis treatment, lowering LDL cholesterol is not as effective in preventing cardiovascular complications as in the general population. Cholesterol is either endogenously synthesized or absorbed from the intestine. It has been suggested that the benefit of using statins to prevent atherosclerotic complications is less pronounced in people with high absorption of cholesterol. Recent data indicate that patients on hemodialysis have high absorption of cholesterol. Therefore, these patients may benefit from dietary counseling to reduce cholesterol intake, from functional foods containing plant sterols and stanols, and from drugs that interfere with intestinal absorption of sterols (i.e., ezetimibe, bile acid resins, and sevelamer). This review discusses cholesterol homeostasis and the perspective of personalized treatment of hypercholesterolemia in hemodialysis.

The metabolomic window into hepatobiliary disease

The emergent discipline of metabolomics has attracted considerable research effort in hepatology. Here we review the metabolomic data for non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), alcoholic liver disease (ALD), hepatitis B and C, cholecystitis, cholestasis, liver transplantation, and acute hepatotoxicity in animal models. A metabolomic window has permitted a view into the changing biochemistry occurring in the transitional phases between a healthy liver and hepatocellular carcinoma or cholangiocarcinoma. Whether provoked by obesity and diabetes, alcohol use or oncogenic viruses, the liver develops a core metabolomic phenotype (CMP) that involves dysregulation of bile acid and phospholipid homeostasis. The CMP commences at the transition between the healthy liver (Phase 0) and NAFLD/NASH, ALD or viral hepatitis (Phase 1). This CMP is maintained in the presence or absence of cirrhosis (Phase 2) and whether or not either HCC or CCA (Phase 3) develops. Inflammatory signalling in the liver triggers the appearance of the CMP. Many other metabolomic markers distinguish between Phases 0, 1, 2 and 3. A metabolic remodelling in HCC has been described but metabolomic data from all four Phases demonstrate that the Warburg shift from mitochondrial respiration to cytosolic glycolysis foreshadows HCC and may occur as early as Phase 1. The metabolic remodelling also involves an upregulation of fatty acid β-oxidation, also beginning in Phase 1. The storage of triglycerides in fatty liver provides high energy-yielding substrates for Phases 2 and 3 of liver pathology. The metabolomic window into hepatobiliary disease sheds new light on the systems pathology of the liver.

Evidence for a role of sterol 27-hydroxylase in glucocorticoid metabolism in vivo

The intracellular availability of glucocorticoids is regulated by the enzymes 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) and 11β-hydroxysteroid dehydrogenase 2 (HSD11B2). The activity of HSD11B1 is measured in the urine based on the (tetrahydrocortisol+5α-tetrahydrocortisol)/tetrahydrocortisone ((THF+5α-THF)/THE) ratio in humans and the (tetrahydrocorticosterone+5α-tetrahydrocorticosterone)/tetrahydrodehydrocorticosterone ((THB+5α-THB)/THA) ratio in mice. The cortisol/cortisone (F/E) ratio in humans and the corticosterone/11-dehydrocorticosterone (B/A) ratio in mice are markers of the activity of HSD11B2. In vitro agonist treatment of liver X receptor (LXR) down-regulates the activity of HSD11B1. Sterol 27-hydroxylase (CYP27A1) catalyses the first step in the alternative pathway of bile acid synthesis by hydroxylating cholesterol to 27-hydroxycholesterol (27-OHC). Since 27-OHC is a natural ligand for LXR, we hypothesised that CYP27A1 deficiency may up-regulate the activity of HSD11B1. In a patient with cerebrotendinous xanthomatosis carrying a loss-of-function mutation in CYP27A1, the plasma concentrations of 27-OHC were dramatically reduced (3.8 vs 90-140 ng/ml in healthy controls) and the urinary ratios of (THF+5α-THF)/THE and F/E were increased, demonstrating enhanced HSD11B1 and diminished HSD11B2 activities. Similarly, in Cyp27a1 knockout (KO) mice, the plasma concentrations of 27-OHC were undetectable (<1 vs 25-120 ng/ml in Cyp27a1 WT mice). The urinary ratio of (THB+5α-THB)/THA was fourfold and that of B/A was twofold higher in KO mice than in their WT littermates. The (THB+5α-THB)/THA ratio was also significantly increased in the plasma, liver and kidney of KO mice. In the liver of these mice, the increase in the concentrations of active glucocorticoids was due to increased liver weight as a consequence of Cyp27a1 deficiency. In vitro, 27-OHC acts as an inhibitor of the activity of HSD11B1. Our studies suggest that the expression of CYP27A1 modulates the concentrations of active glucocorticoids in both humans and mice and in vitro.

Linking Ah receptor mediated effects of sediments and impacts on fish to key pollutants in the Yangtze Three Gorges Reservoir, China – A comprehensive perspective

The Three Gorges Reservoir (TGR), created in consequence of the Yangtze River's impoundment by the Three Gorges Dam, faces numerous anthropogenic impacts that challenge its unique ecosystem. Organic pollutants, particularly aryl hydrocarbon receptor (AhR) agonists, have been widely detected in the Yangtze River, but only little research was yet done on AhR-mediated activities. Hence, in order to assess effects of organic pollution, with particular focus on AhR-mediated activities, several sites in the TGR area were examined applying the "triad approach". It combines chemical analysis, in vitro, in vivo and in situ investigations to a holistic assessment. Sediments and the benthic fish species Pelteobagrus vachellii were sampled in 2011/2012, respectively, to identify relevant endpoints. Sediment was tested in vitro with the ethoxyresorufin-O-deethylase (EROD) induction assay, and in vivo with the Fish Embryo Toxicity Test and Sediment Contact Assay with Danio rerio. Activities of phase I (EROD) and phase II (glutathione-S-transferase) biotransformation enzymes, pollutant metabolites and histopathological alterations were studied in situ in P. vachellii. EROD induction was tested in vitro and in situ to evaluate possible relationships. Two sites, near Chongqing and Kaixian city, were identified as regional hot-spots and further investigated in 2013. The sediments induced in the in vitro/in vivo bioassays AhR-mediated activities and embryotoxic/teratogenic effects - particularly on the cardiovascular system. These endpoints could be significantly correlated to each other and respective chemical data. However, particle-bound pollutants showed only low bioavailability. The in situ investigations suggested a rather poor condition of P. vachellii, with histopathological alterations in liver and excretory kidney. Fish from Chongqing city exhibited significant hepatic EROD induction and obvious parasitic infestations. The polycyclic aromatic hydrocarbon (PAH) metabolite 1-hydroxypyrene was detected in bile of fish from all sites. All endpoints in combination with the chemical data suggest a pivotal role of PAHs in the observed ecotoxicological impacts.

Real-Time In Vivo Characterization of Primary Liver Tumors With Diffuse Optical Spectroscopy During Percutaneous Needle Interventions: Feasibility Study in Woodchucks

OBJECTIVE This study presents the first in vivo real-time optical tissue characterization during image-guided percutaneous intervention using near-infrared diffuse optical spectroscopy sensing at the tip of a needle. The goal of this study was to indicate transition boundaries from healthy tissue to tumors, namely, hepatic carcinoma, based on the real-time feedback derived from the optical measurements. MATERIALS AND METHODS Five woodchucks with hepatic carcinoma were used for this study. The woodchucks were imaged with contrast-enhanced cone beam computed tomography with a flat panel detector C-arm system to visualize the carcinoma in the liver. In each animal, 3 insertions were performed, starting from the skin surface toward the hepatic carcinoma under image guidance. In 2 woodchucks, each end point of the insertion was confirmed with pathologic examination of a biopsy sample. While advancing the needle in the animals under image guidance such as fluoroscopy overlaid with cone beam computed tomography slice and ultrasound, optical spectra were acquired at the distal end of the needles. Optical tissue characterization was determined by translating the acquired optical spectra into clinical parameters such as blood, water, lipid, and bile fractions; tissue oxygenation levels; and scattering amplitude related to tissue density. The Kruskal-Wallis test was used to study the difference in the derived clinical parameters from the measurements performed within the healthy tissue and the hepatic carcinoma. Kurtoses were calculated to assess the dispersion of these parameters within the healthy and carcinoma tissues. RESULTS Blood and lipid volume fractions as well as tissue oxygenation and reduced scattering amplitude showed to be significantly different between the healthy part of the liver and the hepatic carcinoma (P < 0.05) being higher in normal liver tissue. A decrease in blood and lipid volume fractions and tissue oxygenation as well as an increase in scattering amplitude were observed when the tip of the needle crossed the margin from the healthy liver tissue to the carcinoma. The kurtosis for each derived clinical parameter was high in the hepatic tumor as compared with that in the healthy liver indicating intracarcinoma variability. CONCLUSIONS Tissue blood content, oxygenation level, lipid content, and tissue density all showed significant differences when the needle tip was guided from the healthy tissue to the carcinoma and can therefore be used to identify tissue boundaries during percutaneous image-guided interventions.

Significant lethality following liver resection in A20 heterozygous knockout mice uncovers a key role for A20 in liver regeneration

Hepatic expression of A20, including in hepatocytes, increases in response to injury, inflammation and resection. This increase likely serves a hepatoprotective purpose. The characteristic unfettered liver inflammation and necrosis in A20 knockout mice established physiologic upregulation of A20 as integral to the anti-inflammatory and anti-apoptotic armamentarium of hepatocytes. However, the implication of physiologic upregulation of A20 in modulating hepatocytes' proliferative responses following liver resection remains controversial. To resolve the impact of A20 on hepatocyte proliferation and the liver's regenerative capacity, we examined whether decreased A20 expression, as in A20 heterozygous knockout mice, affects outcome following two-third partial hepatectomy. A20 heterozygous mice do not demonstrate a striking liver phenotype, indicating that their A20 expression levels are still sufficient to contain inflammation and cell death at baseline. However, usually benign partial hepatectomy provoked a staggering lethality (>40%) in these mice, uncovering an unsuspected phenotype. Heightened lethality in A20 heterozygous mice following partial hepatectomy resulted from impaired hepatocyte proliferation due to heightened levels of cyclin-dependent kinase inhibitor, p21, and deficient upregulation of cyclins D1, E and A, in the context of worsened liver steatosis. A20 heterozygous knockout minimally affected baseline liver transcriptome, mostly circadian rhythm genes. Nevertheless, this caused differential expression of >1000 genes post hepatectomy, hindering lipid metabolism, bile acid biosynthesis, insulin signaling and cell cycle, all critical cellular processes for liver regeneration. These results demonstrate that mere reduction of A20 levels causes worse outcome post hepatectomy than full knockout of bona fide liver pro-regenerative players such as IL-6, clearly ascertaining A20's primordial role in enabling liver regeneration. Clinical implications of these data are of utmost importance as they caution safety of extensive hepatectomy for donation or tumor in carriers of A20/TNFAIP3 single nucleotide polymorphisms alleles that decrease A20 expression or function, and prompt the development of A20-based liver pro-regenerative therapies.

The Gut Microbiome and Cirrhosis: Basic Aspects

In this chapter the basic aspects helping to understand the microbiome in terms of quantity, diversity, complexity, function, and interaction with the host are discussed. First the nomenclature, definitions of taxa, and measures of diversity as well as methods to unravel this kingdom are outlined. A brief summary on its physiological relevance for general health and the functions exerted specifically by the microbiome is presented. Differences in the composition of the microbiome along the gastrointestinal tract and across the gut wall and its interindividual variations, enterotypes, and stability are highlighted. The reader will be familiarized with all different modulators impacting on the microbiome, namely, intrinsic and extrinsic factors. Intrinsic factors include gastrointestinal secretions (gastric acid, bile, pancreatic juice, mucus), antimicrobial peptides, motility, enteric nervous system, and host genotype. Extrinsic factors are mainly dietary choices, hygiene, stress, alcohol consumption, exercise, and medications. The second part of the chapter focuses on quantitative and qualitative changes in microbiome in liver cirrhosis. The mechanisms contributing to dysbiosis, small intestinal bacterial overgrowth, and bacterial translocation are delineated underscoring their role for the liver-gut axis.

ENaC activity in collecting ducts modulates NCC in cirrhotic mice.

Cirrhosis is a frequent and severe disease, complicated by renal sodium retention leading to ascites and oedema. A better understanding of the complex mechanisms responsible for renal sodium handling could improve clinical management of sodium retention. Our aim was to determine the importance of the amiloride-sensitive epithelial sodium channel (ENaC) in collecting ducts in compensate and decompensate cirrhosis. Bile duct ligation was performed in control mice (CTL) and collecting duct-specific αENaC knockout (KO) mice, and ascites development, aldosterone plasma concentration, urinary sodium/potassium ratio and sodium transporter expression were compared. Disruption of ENaC in collecting ducts (CDs) did not alter ascites development, urinary sodium/potassium ratio, plasma aldosterone concentrations or Na,K-ATPase abundance in CCDs. Total αENaC abundance in whole kidney increased in cirrhotic mice of both genotypes and cleaved forms of α and γ ENaC increased only in ascitic mice of both genotypes. The sodium chloride cotransporter (NCC) abundance was lower in non-ascitic KO, compared to non-ascitic CTL, and increased when ascites appeared. In ascitic mice, the lack of αENaC in CDs induced an upregulation of total ENaC and NCC and correlated with the cleavage of ENaC subunits. This revealed compensatory mechanisms which could also take place when treating the patients with diuretics. These compensatory mechanisms should be considered for future development of therapeutic strategies.