Effects of trientine and penicillamine on intestinal copper uptake: A mechanistic 64 Cu PET/CT study in healthy humans.

BACKGROUND AND AIMS: Trientine (TRI) and D-penicillamine (PEN) are used to treat copper overload in Wilson disease. Their main mode of action is thought to be through the facilitation of urinary copper excretion. In a recent study, TRI was noninferior to PEN despite lower 24-hour urinary copper excretion than PEN. We tested whether TRI and/or PEN also inhibit intestinal copper absorption. APPROACH AND RESULTS: Sixteen healthy volunteers were examined with positron emission tomography (PET)/CT 1 and 15 hours after an oral Copper-64 ( 64 Cu) dose. They then received 7 days of either PEN or TRI (trientine tetrahydrochloride), after which the 64 Cu PET/CT scans were repeated. Venous blood samples were also collected. Pretreatment to posttreatment changes of the hepatic 64 Cu uptake reflect the effect of drugs on intestinal absorption. 64 Cu activity was normalized to dose and body weight and expressed as the mean standard uptake value. TRI (n=8) reduced hepatic 64 Cu activity 1 hour after 64 Cu dose from 6.17 (4.73) to 1.47 (2.97) standard uptake value, p <0.02, and after 15 hours from 14.24 (3.09) to 6.19 (3.43), p <0.02, indicating strong inhibition of intestinal 64 Cu absorption. PEN (n=8) slightly reduced hepatic standard uptake value at 15 hours, from 16.30 (5.63) to 12.17 (1.44), p <0.04. CONCLUSIONS: In this mechanistic study, we show that TRI inhibits intestinal copper absorption, in addition to its cupriuretic effect. In contrast, PEN has modest effects on the intestinal copper absorption. This may explain why TRI and PEN are equally effective although urinary copper excretion is lower with TRI. The study questions whether the same therapeutic targets for 24-hour urinary excretion apply to both drugs.

Similar Insulin Regulation of Splanchnic FFA and VLDL-TG in Men with Nonalcoholic Hepatic Steatosis and Steatohepatitis.

This study aimed to determine whether obese men with nonalcoholic fatty liver disease (NAFLD) display differences between those with simple steatosis vs. steatohepatitis (NASH) in splanchnic and hepatic FFA and VLDL-triglycerides (VLDL-TG) balances. The study involved 17 obese men with biopsy-proven NAFLD (9 with NASH and 8 with simple steatosis). We used hepatic vein catheterization in combination with [3H]palmitate and [14C]VLDL-TG tracers to measure splanchnic palmitate and VLDL-TG uptake and release rates during basal and hyperinsulinemic conditions. Indocyanine green was used to measure splanchnic plasma flow. Splanchnic palmitate uptake was similar in the two groups and significantly reduced during hyperinsulinemia (NASH: 62 (48-77) vs. 38 (18-58) µmol/min; simple steatosis: 62 (46-78) vs. 45 (25-65) µmol/min, mean (95% CI), basal vs. clamp periods, respectively, p = 0.02 time-effect). Splanchnic palmitate release was also comparable between groups and non-significantly diminished during hyperinsulinemia. The percent palmitate delivered to the liver originating from visceral adipose tissue (VAT) lipolysis was similar and unchanged by hyperinsulinemia. Splanchnic uptake and release of VLDL-TG were similar between groups. Hyperinsulinemia suppressed VLDL-TG release (p <0.05 time-effect) in both groups. Insulin mediated glucose disposal was similar in the two groups (p = 0.54). IN CONCLUSIONS: Obese men with NASH and simple steatosis have similar splanchnic uptake and release of FFA and VLDL-TG and a similar proportion of FFA from VAT lipolysis delivered to the liver. These results suggest that FFA and VLDL-TG splanchnic balances are unaffected by NAFLD severity.

Effects of tetrathiomolybdate on copper metabolism in healthy volunteers and in patients with Wilson disease.

BACKGROUND & AIMS: In Wilson disease (WD), copper accumulates in the liver and brain causing disease. Bis-choline tetrathiomolybdate (TTM) is a potent copper chelator that may be associated with a lower risk of inducing paradoxical neurological worsening than conventional therapy for neurologic WD. To better understand the mode of action of TTM, we investigated its effects on copper absorption and biliary excretion. METHODS: In a double-blind randomized setting, hepatic 64Cu activity was examined after orally administered 64Cu by PET/CT in 16 healthy volunteers before and after seven days of TTM treatment (15 mg/d) or placebo. Oral 64Cu was administered one hour after the final TTM dose. Changes in hepatic 64Cu activity reflected changes in intestinal 64Cu uptake. Additionally, in four patients with WD, the distribution of 64Cu in venous blood, liver, gallbladder, kidney, and brain was followed after i.v. 64Cu dosing for up to 68 hours before and after seven days of TTM (15 mg/day), using PET/MRI. Increased gallbladder 64Cu activity was taken as evidence of increased biliary 64Cu excretion. RESULTS: In healthy volunteers, TTM reduced intestinal 64Cu uptake by 82% 15 hours after the oral 64Cu dose. In patients with WD, gallbladder 64Cu activity was negligible before and after TTM, while TTM effectively retained 64Cu in the blood, significantly reduced hepatic 64Cu activity at all time-points and significantly reduced cerebral 64Cu activity two hours after the intravenous 64Cu dose. CONCLUSIONS: While we did not show an increase in biliary excretion of 64Cu following TTM administration, we demonstrated that TTM effectively inhibited most intestinal 64Cu uptake and retained 64Cu in the blood stream, limiting the exposure of organs like the liver and brain to 64Cu. IMPACT AND IMPLICATIONS: Bis-choline tetrathiomolybdate (TTM) is an investigational copper chelator being developed for the treatment of Wilson disease. In animal models of Wilson disease, TTM has been shown to facilitate biliary copper excretion. In the present human study, TTM surprisingly did not facilitate biliary copper excretion but instead reduced intestinal copper uptake to a clinically significant degree. Our study builds on our understanding of human copper metabolism and the mechanism of action of TTM.

ARBM101 (Methanobactin SB2) Drains Excess Liver Copper via Biliary Excretion in Wilson's Disease Rats.

BACKGROUND & AIMS: Excess copper causes hepatocyte death in hereditary Wilson's disease (WD). Current WD treatments by copper-binding chelators may gradually reduce copper overload; they fail, however, to bring hepatic copper close to normal physiological levels. Consequently, lifelong daily dose regimens are required to hinder disease progression. This may result in severe issues due to nonadherence or unwanted adverse drug reactions and also due to drug switching and ultimate treatment failures. This study comparatively tested bacteria-derived copper binding agents-methanobactins (MBs)-for efficient liver copper depletion in WD rats as well as their safety and effect duration. METHODS: Copper chelators were tested in vitro and in vivo in WD rats. Metabolic cage housing allowed the accurate assessment of animal copper balances and long-term experiments related to the determination of minimal treatment phases. RESULTS: We found that copper-binding ARBM101 (previously known as MB-SB2) depletes WD rat liver copper dose dependently via fecal excretion down to normal physiological levels within 8 days, superseding the need for continuous treatment. Consequently, we developed a new treatment consisting of repetitive cycles, each of ∼1 week of ARBM101 applications, followed by months of in-between treatment pauses to ensure a healthy long-term survival in WD rats. CONCLUSIONS: ARBM101 safely and efficiently depletes excess liver copper from WD rats, thus allowing for short treatment periods as well as prolonged in-between rest periods.

The pathophysiology of Wilson's disease visualized: A human 64 Cu PET study.

BACKGROUND AND AIMS: Wilson's disease (WD) is a genetic disease with systemic accumulation of copper that leads to symptoms from the liver and brain. However, the underlying defects in copper transport kinetics are only partly understood. We sought to quantify hepatic copper turnover in patients with WD compared with heterozygote and control subjects using PET with copper-64 (64 Cu) as a tracer. Furthermore, we assessed the diagnostic potential of the method. APPROACH AND RESULTS: Nine patients with WD, 5 healthy heterozygote subjects, and 8 healthy controls were injected with an i.v. bolus of 64 Cu followed by a 90-min dynamic PET scan of the liver and static whole-body PET/CT scans after 1.5, 6, and 20 h. Blood 64 Cu concentrations were measured in parallel. Hepatic copper retention and redistribution were evaluated by standardized uptake values (SUVs). At 90 min, hepatic SUVs were similar in the three groups. In contrast, at 20 h postinjection, the SUV in WD patients (mean ± SEM, 31 ± 4) was higher than in heterozygotes (24 ± 3) and controls (21 ± 4; p < 0.001). An SUV-ratio of hepatic 64 Cu concentration at 20 and 1.5 h completely discriminated between WD patients and control groups (p < 0.0001; ANOVA). By Patlak analysis of the initial 90 min of the PET scan, the steady-state hepatic clearance of 64 Cu was estimated to be slightly lower in patients with WD than in controls (p = 0.04). CONCLUSIONS: 64 Cu PET imaging enables visualization and quantification of the hepatic copper retention characteristic for WD patients. This method represents a valuable tool for future studies of WD pathophysiology, and may assist the development of therapies, and accurate diagnosis.

Designing Clinical Trials in Wilson's Disease.

BACKGROUND AND AIMS: Wilson's disease (WD) is an autosomal-recessive disorder caused by ATP7B gene mutations leading to pathological accumulation of copper in the liver and brain. Adoption of initial treatments for WD was based on empirical observations. These therapies are effective, but there are still unmet needs for which treatment modalities are being developed. An increase of therapeutical trials is anticipated. APPROACH AND RESULTS: The first Wilson Disease Aarhus Symposium (May 2019) included a workshop on randomized clinical trial design. The authors of the article were organizers or presented during this workshop, and this article presents their consensus on the design of clinical trials for WD, addressing trial population, treatment comparators, inclusion and exclusion criteria, and treatment endpoints. To achieve adequate recruitment of patients with this rare disorder, the study groups should include all clinical phenotypes and treatment-experienced as well as treatment-naïve patients. CONCLUSIONS: The primary study endpoint should be clinical or a composite endpoint until appropriate surrogate endpoints are validated. Standardization of clinical trials will permit pooling of data and allow for better treatment comparisons, as well as reduce the future numbers of patients needed per trial.

The galactose elimination capacity test to monitor liver disease course in patients with Wilson's disease.

BACKGROUND: The prognosis of Wilson's disease (WD) has changed radically since the introduction of medical therapy with chelators and zinc. However, there is an unmet need for methods to evaluate the long-term treatment response and the liver disease progression in order to identify treatment failures. The galactose elimination capacity test (GEC) is a physiological measure of the total metabolic capacity of the liver, and a strong predictor of long- and short-term mortality in patients with liver cirrhosis. Our aim was to investigate if the GEC test is useful for evaluation of treatment response and prediction of treatment failures in WD patients. METHODS: We included all patients with WD in Denmark from 1992 through 2017 and retrieved data on GEC along with data on transplantation and death. RESULTS: In total, 37 patients had completed one or more GEC tests. Of these, 31 were alive (three transplanted) and six were dead (two transplanted). A total of 24 patients had completed more than one GEC test. All 18 alive, nontransplanted patients showed improvement in GEC values following onset of treatment, except one patient, who was clinically confirmed with treatment failure. All six patients who underwent liver transplantation or died had a prior decline in their GEC. The difference in GEC development between patients alive and not transplanted and patients dead or transplanted was significant (p < .001). Index GEC values could not predict transplantation or death (p = .26). CONCLUSION: The GEC test is a promising tool for monitoring treatment response and identifying treatment failures in patients with WD.

Psychometric methods for diagnosing and monitoring minimal hepatic encephalopathy -current validation level and practical use.

Hepatic encephalopathy (HE) is cerebral dysfunction caused by liver failure and inflicts 30-40% of patients with liver cirrhosis during their disease course. Clinically manifest HE is often preceded by minimal HE (MHE) - a clinically undetectable cognitive disturbance closely associated with loss of quality of life. Accordingly, detecting and treating MHE improve the patients' daily functioning and prevent HE-related hospital admissions. The scope of this review article is to create an overview of the validation level and usage of psychometric tests used to detect MHE: Portosystemic hepatic encephalopathy test, continuous reaction time test, Stroop EncephalApp, animal naming test, critical flicker frequency test, and inhibitory control test. Our work is aimed at the clinician or scientist who is about to decide on which psychometric test would fit best in their clinic, cohort, or study. First, we outline psychometric test validation obstacles and requirements. Then, we systematically approach the literature on each test and select well-conducted studies to answer the following questions:• Which percentage of patients with cirrhosis does the test deem as having MHE?• Is the test able to predict clinically manifest HE?• Is there a well-known test-retest variation and inter-observer variation?• Is the test able to detect a treatment response?• Is the test result affected by age, educational level, gender, or comorbidities?

The Prevalence of Wilson's Disease: An Update.

BACKGROUND AND AIMS: In 1984, Scheinberg and Sternlieb estimated the prevalence of Wilson's disease to be 1:30,000 based on the limited available data. This suggested a large number of overlooked cases with potentially fatal consequences. The "Scheinberg-Sternlieb Estimate" is still widely used, although more recent clinical and genetic studies of higher quality are now available. In the present study, we included these data to update the prevalence estimate. APPROACH AND RESULTS: A MEDLINE Ovid, Science Citation Index Expanded, and PubMed systematic search for all relevant studies on the prevalence of Wilson's disease was conducted. In total, 59 studies (50 clinical and 9 population-based genetic) were included in the final analysis. We identified 4 recent clinical studies based on nationwide databases of high quality, providing prevalence estimates from 1:29,000 to 1:40,000. Higher frequency populations do exist because of frequent first-cousin marriages and/or a higher mutation frequency. When calculating prevalence from the incidence related to number of births, estimates were 1:40,000-1:50,000. Clinical screening studies, including examination for Kayser-Fleischer rings or ceruloplasmin, did not improve these estimates because of insufficient sample size or selection biases. Population-based genetic studies in US and UK populations were not in disagreement with the clinically based estimates. At the same time, studies from France and Sardinia suggested that the genetic prevalence may be 3-4 times higher than the clinical disease prevalence. This raises the question whether the penetrance is indeed 100% as generally assumed. CONCLUSIONS: The original prevalence estimate from 1984 of 1:30,000-1:50,000 still appears valid, at least for the United States, Europe, and Asia. In some population-based studies, the genetic prevalence was 3-4 times higher than clinically based estimates. The question of penetrance needs further evaluation.

Interferon lambda 4 genotype and pathway in alcoholic hepatitis.

OBJECTIVES: Single nucleotide polymorphisms within the interferon lambda 4 (IFNL4) gene influence liver inflammation and fibrosis in chronic liver disease. We investigated whether this is also the case during acute liver disease, alcoholic hepatitis. We, therefore, related variants within the IFNL4 gene to the clinical course of acute alcoholic hepatitis, and characterized the activation state of the IFN lambda system in these patients. METHODS: In this pilot study, 58 patients with alcoholic hepatitis were genotyped for the rs368234815IFNL4 single nucleotide polymorphism (deltaG, deltaG/TT: IFN lambda 4 positive, TT/TT: IFN lambda 4 negative). The genotypes were related to mortality, infection and inflammation and expression of the IFNL receptor 1 and IFN inducible genes were measured in liver and peripheral leukocytes. RESULTS: Amongst the alcoholic hepatitis patients who died, the IFN negative patients live longer after diagnosis, and also the IFN negative patients tended to have an overall short-term survival benefit compared to IFN lambda positive patients (p = .058). The IFN lambda 4 negative patients at diagnosis had fewer circulating monocytes and lower plasma soluble CD163. The patients with alcoholic hepatitis had reduced expression of the IFNL receptor 1in both liver and blood compared with healthy controls. In blood, the expression of IFN stimulated genes was lower than in healthy controls and most so in the patients, who died. CONCLUSIONS: The IFN lambda 4 pathway seems involved in the acute disease processes of alcoholic hepatitis and patients without IFN lambda expression seem to have a short-term survival benefit.

Cognitive impairment in stable Wilson disease across phenotype.

In Wilson disease (WD), mutations in the gene encoding the ATP7B copper transport protein causes accumulation of copper especially in liver and brain. WD typically presents with hepatic and/or neuropsychiatric symptoms. Impaired cognition is a well-described feature in patients with neurological WD, while the reports on cognition in hepatic WD patients are fewer and less conclusive. We examined cognition in a cohort of WD patients with both phenotypes. In this cross-sectional pilot study, we investigated cognition in 28 stable Danish WD patients by the PortoSystemic Encephalopathy (PSE) and the Continuous Reaction Time (CRT) tests. Half of the patients were female, and their median age was 35.5 years (IQR 24.5). Their phenotype was hepatic in 14 (50%), neurologic in 10 (36%) and mixed in 4 (14%). The duration of treatment was > 2 year in all patients, and their condition was stable as judged by urinary copper excretion, liver enzymes, and clinical assessment. The hepatic patients did not show signs of liver failure. In total, 16 (57%) patients performed worse than normal in the PSE and/or the CRT tests. The two tests were correlated (rho = 0.60, p = 0.0007), but neither correlated with phenotype, MELD-, Child-Pugh score, 24 h-U-Cu, or treatment type. Measurable cognitive impairment was present in more than half of the stable WD patients independent of phenotype. Thus, our data questions the existence of a purely hepatic phenotype.

Early normalization of reduced urea synthesis capacity after direct-acting antiviral therapy in hepatitis C cirrhosis.

Laursen TL, Sandahl TD, Kazankov K, Eriksen PL, Kristensen LH, Holmboe CH, Laursen AL, Vilstrup H, Grønbæk H. Early normalization of reduced urea synthesis capacity after direct-acting antiviral therapy in hepatitis C cirrhosis. Am J Physiol Gastrointest Liver Physiol 319: G151-G156, 2020. First published June 29, 2020; doi:10.1152/ajpgi.00128.2020.-Effects of direct-acting antiviral (DAA) treatment of chronic hepatitis C (CHC) cirrhosis on metabolic liver function are unknown but important for prognosis. Ureagenesis is an essential metabolic liver function involved in whole body nitrogen homeostasis. We aimed to investigate the ureagenesis capacity before and immediately after DAA therapy and relate the findings to hepatic inflammation and structural changes. In an observational before-and-after intervention study, the ureagenesis capacity was quantified by functional hepatic nitrogen clearance (FHNC) in 9 CHC patients with cirrhosis and 10 healthy volunteers. Hepatic inflammation was evaluated by alanine aminotransferase (ALT) and the macrophage activation markers sCD163 and sMR. Structural changes were estimated as liver stiffness and by portal hypertension as the hepatic venous pressure gradient (HVPG). Before treatment, the FHNC in the patients was half of the controls [16.4 L/h (8.2-24.5) vs. 33.4 (29.2-37.6), P = 0.0004]; after successful DAA treatment, it normalized [28.4 (15.9-40.9), P = 0.008 vs. baseline]. DAA treatment normalized ALT (P < 0.0001) and decreased the elevated sCD163 from 5.6 mg/L (3.5-7.7) to 3.4 (2-0-4.8) (P < 0.001) and sMR from 0.35 mg/L (0.21-0.49) to 0.31 (0.17-0.45) (P < 0.01). Liver stiffness fell by 30% (P < 0.05) but remained over the cirrhosis threshold. HVPG was not affected (P = 0.59). DAA treatment restored the severely reduced ureagenesis capacity, along with amelioration of hepatic inflammation but without normalization of other cirrhosis characteristics. Our findings indicate that the anti-inflammatory effect of virus eradication independent of hepatic structural effects rapidly improves metabolic dysfunction. We suggest this effect to be an important early onset part of the expected clinical DAA treatment benefit.NEW & NOTEWORTHY Antiviral treatment of chronic hepatitis C restores the liver's reduced capacity to produce urea along with an improvement in liver inflammation without immediate effects on structural liver changes. The effect is suggested to be an important early onset part of the expected clinical treatment benefit.

Time-dependent improvement of liver inflammation, fibrosis and metabolic liver function after successful direct-acting antiviral therapy of chronic hepatitis C.

Sofosbuvir-based direct-acting antiviral (DAA) therapy generally cures chronic hepatitis C (CHC) infections, however, the effects on the underlying liver disease and the potential rate of recovery are unclear. We aimed to investigate the effects of DAA therapy on liver inflammation, fibrosis, metabolic function and cognitive function and the time course in CHC patients with advanced liver disease. Seventy-one CHC patients with advanced liver disease were studied before, during and one year after successful sofosbuvir-based DAA therapy. Liver inflammation was assessed by plasma sCD163 and sMR levels (ELISA), fibrosis by liver stiffness (transient elastography), function by galactose elimination capacity (GEC) and cognitive performance by continuous reaction time (CRT). During DAA therapy, we observed a rapid sCD163 decline from baseline to end of treatment (6.9 vs 3.8 mg/L, P < .0001), whereas the change in sMR was more subtle (0.37 vs 0.30 mg/L, P < .0001). Liver stiffness decreased by 20% at end of treatment (17.8 vs 14.3 kPa, P < .0001), together suggesting rapid resolution of liver inflammation. One year after treatment, liver stiffness decreased by an additional 15% (P < .0001), suggestive of fibrosis regression. The GEC improved at follow-up (all: 1.74 vs 1.98 mmol/min), mainly at 12 weeks post-treatment, both in patients with cirrhosis (n = 56) and those with advanced liver fibrosis (n = 15) (P < .001). The CRT improved at one-year follow-up (1.86 vs 2.09, P = .04). In conclusion, successful DAA therapy of CHC proves beneficial in advanced liver disease, with an initial rapid resolution of liver inflammation and a subsequent gradual but steady improvement in liver fibrosis, metabolic liver function and reaction time.

Extracellular vesicle-associated soluble CD163 and CD206 in patients with acute and chronic inflammatory liver disease.

Background: Extracellular vesicles (EVs) are implicated in intercellular communication in liver diseases. An EV-associated fraction of the macrophage biomarker soluble CD163, denoted EV-CD163, was recently identified. EV-CD163 may be released during later phases of the inflammatory response as opposed to the acute shedding of CD163 ectodomain (Ecto-CD163). Total sCD163 is a well-described biomarker in liver inflammation, and we investigated the distribution of CD163 fractions along with EV-associated soluble CD206 (EV-CD206) in patients with acute and chronic alcoholic liver inflammation.Methods: Patients with acute alcoholic hepatitis (AH) (n = 48) and alcoholic cirrhosis (AC) (n = 26) were enrolled. Patients with AH were followed for 30 days after diagnosis. Healthy blood donors (n = 30) served as a reference group. Fractions of sCD163 and sCD206 were separated using ExoQuick™ and measured by ELISA.Results: We demonstrated a possible EV-associated fraction of CD206 in plasma, correlating with levels of EV-CD163 (rs = 0.46, p < .001). The distribution of biomarker fractions was skewed toward EVs in chronic cirrhosis for both biomarkers (median: 35.8% EV-CD163, 58.8% EV-CD206) as compared to AH patients (median: 26.2% EV-CD163 p < .0001, 48.8% EV-CD206, p < .01). In AH patients, total sCD163 and Ecto-CD163 at inclusion were related to survival, whereas EV-CD163 was not.Conclusion: Extracellular vesicles of macrophage origin associated with membrane receptors CD163 and CD206 are present in liver disease. We observed a shift in the distribution towards an increased EV fraction in chronic liver cirrhosis. These data support that Ecto and EV fractions may be markers of different inflammatory processes, possibly resulting from a switch in macrophage phenotype.

Computing the Pathogenicity of Wilson's Disease ATP7B Mutations: Implications for Disease Prevalence.

Genetic variations in the gene encoding the copper-transport protein ATP7B are the primary cause of Wilson's disease. Controversially, clinical prevalence seems much smaller than the prevalence estimated by genetic screening tools, causing fear that many people are undiagnosed, although early diagnosis and treatment is essential. To address this issue, we benchmarked 16 state-of-the-art computational disease-prediction methods against established data of missense ATP7B mutations. Our results show that the quality of the methods varies widely. We show the importance of optimizing the threshold of the methods used to distinguish pathogenic from nonpathogenic mutations against data of clinically confirmed pathogenic and nonpathogenic mutations. We find that most methods use thresholds that predict too many ATP7B mutations to be pathogenic. Thus, our findings explain the current controversy on Wilson's disease prevalence because meta-analysis and text search methods include many computational estimates that lead to higher disease prevalence than clinically observed. As proteins and diseases differ widely, a one-size-fits-all threshold cannot distinguish pathogenic and nonpathogenic mutations efficiently, as shown here. We also show that amino acid changes with small evolutionary substitution probability, mainly due to amino acid volume, are more associated with the disease, implying a pathological effect on the conformational state of the protein, which could affect copper transport or adenosine triphosphate recognition and hydrolysis. These findings may be a first step toward a more quantitative genotype-phenotype relationship of Wilson's disease.

Rapid and persistent decline in soluble CD163 with successful direct-acting antiviral therapy and associations with chronic hepatitis C histology.

BACKGROUND AND AIM: Soluble CD 163 (sCD163) is released from activated liver macrophages in chronic viral hepatitis C (HCV) and serum levels reflect liver disease severity. The impact of direct-acting antiviral (DAA)-therapy on sCD163-levels and the ability of sCD163 to predict the presence of liver fibrosis remain unclear. In a combined observational and prospective study, we aimed to investigate changes in sCD163 with DAA-treatment, to investigate associations between sCD163 and histopathological activity and fibrosis and to validate the sCD163-based fibrosis score in HCV-patients. METHODS: We examined three groups of patients: an Australian (n = 28) treated with pegylated-interferon and a first-generation DAA, a Danish (n = 38) treated with sofosbuvir-based DAA-regimens and a Japanese (n = 562) assessed for activity and fibrosis (Metavir scoring system) in liver biopsies. Serum sCD163-levels were quantified by ELISA. RESULTS: Thirteen (46%) of the Australian patients achieved sustained virological response (SVR) and only these patients had significant decreases in sCD163-levels (2.7 (95%CI:1.9-3.6) vs. 4.1(2.9-5.7) mg L - 1, p = .008). In the Danish group, 37 (97%) patients achieved SVR at 12-weeks post-treatment with 32% reduction in sCD163-levels (5.0 (4.3-5.8) vs. 7.4 (6.3-8.7), p < .001). The decline was rapid and persisted 12 months after treatment cessation (p < .007). sCD163 levels increased in parallel with inflammatory activity and fibrosis (p < .001). The sCD163-based fibrosis score outperformed established fibrosis scores for significant fibrosis (areas under the receiver operating characteristics curves (AUROCs): 0.79 (0.75-0.83) vs. aspartate aminotransferase to platelet ratio index (APRI) 0.73 (0.69-0.77), Fibrosis-4 (FIB-4) 0.74 (0.70-0.78), p < .001). CONCLUSION: sCD163-levels decline rapidly with successful DAA therapy and are associated with histological inflammatory activity and fibrosis, confirming a key role for macrophages in HCV inflammation and fibrosis and supporting sCD163 as a biomarker of treatment response.

Soluble CD163 and mannose receptor associate with chronic hepatitis B activity and fibrosis and decline with treatment.

BACKGROUND AND AIM: Liver macrophages are activated in chronic hepatitis B virus (CHB) infection and play a pivotal role in hepatic inflammation and fibrosis. However, their role during antiviral treatment is unclear. The soluble (s) macrophage activation markers, sCD163 and mannose receptor (sMR), are released during liver damage, and their serum levels reflect liver disease severity and portal hypertension. We aimed to investigate associations between sCD163 and sMR and histopathological activity and fibrosis and changes in sCD163, sMR, and hepatic CD163-expression following antiviral treatment in CHB patients. METHODS: We assessed Ishak histological necroinflammatory activity and fibrosis scores in liver biopsies from 254 CHB patients and serially in 71 patients before and after nucleoside-analogue treatment. Liver CD163-expression was semi-quantitatively determined by immunohistochemistry and serum sCD163 and sMR measured by enzyme-linked immunosorbent assays. RESULTS: Before treatment, the mean levels of sCD163 and sMR were 3.57 (SD 1.72) mg/L and 0.35 (0.12) mg/L. sCD163 and sMR increased with histological inflammatory activity (sCD163: r = 0.46, P < 0.00001; sMR: r = 0.48, P < 0.00001) and correlated positively with fibrosis (sCD163: OR 1.16, 95% CI:1.03-1.31; sMR: OR 1.34, 95% CI:1.13-1.59); both were markers of fibrosis independent of other biochemical parameters and risk factors. Antiviral treatment significantly reduced sCD163 (3.76 [1.46] vs 2.31 [0.95], P < 0.00001), sMR (0.37 [0.1] vs 0.29 [0.07], P < 0.00001) and hepatic CD163-expression (P = 0.0002). CONCLUSION: The macrophage activation markers sCD163 and sMR were associated with activity and fibrosis in liver biopsies from CHB patients. Both serum markers decreased with antiviral treatment, along with decreased hepatic CD163 expression.

Combination of radiofrequency ablation with transarterial chemoembolization for treatment of hepatocellular carcinoma: experience from a Danish tertiary liver center.

BACKGROUND: Recent studies have shown that the combination of radiofrequency ablation (RFA) and transarterial chemoembolization (TACE) for unresectable hepatocellular carcinoma (HCC) may offer a survival advantage compared to monotherapy. PURPOSE: To study the effectiveness of combination therapy with RFA and TACE compared to that of TACE alone in a Scandinavian tertiary liver cancer center. MATERIAL AND METHODS: A retrospective study of the patients treated with combination therapy vis-à-vis TACE alone from June 2007 to November 2012 was performed. Eighteen patients were treated with a combination of RFA and TACE with an interval of 1-4 days between the treatments. For comparison, a group of 18 patients treated with TACE as monotherapy in the same time period was matched with the combination group by demographic data, tumor characteristics, biochemical and clinical parameters, and performance status (PS). RESULTS: Each group consisted of 14 patients with cirrhosis and four without. There were no significant differences between the groups regarding age, gender, tumor characteristics, causes of cirrhosis, levels of bilirubin, creatinine, prothrombin time, Child Pugh score, or World Health Organization (WHO) performance status. The median survival of patients in the RFA + TACE combination group was 586 days compared to 296 days in the control group. The difference was not statistically significant (P = 0.26). However, when we stratified the data for cirrhosis and WHO performance status, patients in the combination group had significantly better survival (P = 0.024). CONCLUSION: Combination therapy with RFA and TACE for unresectable HCC, compared to TACE alone, may offer a survival benefit for a selected group of patients with HCC.

Cytotoxic T lymphocytes and natural killer cells display impaired cytotoxic functions and reduced activation in patients with alcoholic hepatitis.

The dynamics and role of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and NKT cells in the life-threatening inflammatory disease alcoholic hepatitis is largely unknown. These cells directly kill infected and damaged cells through, e.g., degranulation and interferon-γ (IFNγ) production, but cause tissue damage if overactivated. They also assist tissue repair via IL-22 production. We, therefore, aimed to investigate the frequency, functionality, and activation state of such cells in alcoholic hepatitis. We analyzed blood samples from 24 severe alcoholic hepatitis patients followed for 30 days after diagnosis. Ten healthy abstinent volunteers and 10 stable abstinent alcoholic cirrhosis patients were controls. Using flow cytometry we assessed cell frequencies, NK cell degranulation capacity following K562 cell stimulation, activation by natural killer group 2 D (NKG2D) expression, and IL-22 and IFNγ production. In alcoholic hepatitis we found a decreased frequency of CTLs compared with healthy controls (P < 0.001) and a similar trend for NK cells (P = 0.089). The NK cell degranulation capacity was reduced by 25% compared with healthy controls (P = 0.02) and by 50% compared with cirrhosis patients (P = 0.04). Accordingly, the NKG2D receptor expression was markedly decreased on NK cells, CTLs, and NKT cells (P < 0.05, all). The frequencies of IL-22-producing CTLs and NK cells were doubled compared with healthy controls (P < 0.05, all) but not different from cirrhosis patients. This exploratory study for the first time showed impaired cellular cytotoxicity and activation in alcoholic hepatitis. This is unlikely to cause hepatocyte death but may contribute toward the severe immune incompetence. The results warrant detailed and mechanistic studies.

Circulating mannan-binding lectin, M-, L-, H-ficolin and collectin-liver-1 levels in patients with acute liver failure.

BACKGROUND & AIMS: The complement system is activated in liver diseases including acute liver failure (ALF); however, the role of the lectin pathway of complement has scarcely been investigated in ALF. The pathway is initiated by soluble pattern recognition molecules: mannan-binding lectin (MBL), M-, L-, and H-ficolin and collectin-liver-1 (CL-L1), which are predominantly synthesized in the liver. We aimed to study lectin levels in ALF patients and associations with clinical outcome. METHODS: Serum samples from 75 patients enrolled by the US ALF Study Group were collected on days 1 and 3. We included 75 healthy blood donors and 20 cirrhosis patients as controls. Analyses were performed using sandwich-type immunoassays (ELISA, TRIFMA). RESULTS: At day 1, the MBL level in ALF patients was 40% lower compared with healthy controls {[median (interquartile range) 0.72 µg/ml(0.91) vs. 1.15 (1.92)(P = 0.02]}, and increased significantly by day 3 [0.83 µg/ml(0.94)(P = 0.01)]. The M-ficolin level was 60% lower [0.54 µg/ml(0.50) vs. 1.48(1.01)(P < 0.0001)]. The CL-L1 level at day 1 was slightly higher compared with healthy controls [3.20 µg/ml(2.37) vs. 2.64(0.72)(P = 0.11)]; this was significant at day 3 [3.35(1.84)(P = 0.006)]. H- and L-ficolin levels were similar to healthy controls. Spontaneous ALF survivors had higher levels of MBL at day 1 [0.96 µg/ml(1.15) vs. 0.60(0.60)(P = 0.02)] and lower levels of L-ficolin by day 3 compared with patients who died or were transplanted [1.61 µg/ml(1.19) vs. 2.17(2.19)(P = 0.02)]. CONCLUSION: We observed significant dynamics in lectin levels in ALF patients, which may suggest they play a role in ALF pathogenesis. High MBL and low L-ficolin levels are associated with survival.