Clinical significance of serum Ck18-M65 and M30 levels in patients with chronic hepatitis B combined with nonalcoholic steatohepatitis and liver fibrosis.

In this study, we aimed to explore the clinical significance of serum CK18-M65 and CK18-M30 levels in patients with chronic hepatitis B (CHB) complicated by nonalcoholic steatohepatitis (NASH) and liver fibrosis. The observation and control groups comprised 133 patients with CHB complicated by NASH and 50 healthy patients from our hospital, respectively. Liver function indices, including alanine aminotransferase, glutamic aminotransferase, γ-glutamyltransferase, total bilirubin, total protein, and total cholesterol, were determined using an automatic biochemical analyzer. Hyaluronic acid, type III procollagen, type IV collagen, laminin, and CK18-M65 and M30 levels were detected using ELISA. Serum CK18-M65 and M30 levels in patients with CHB complicated by NASH were positively correlated with the liver fibrosis stage (P < .05). While serum CK18-M65 demonstrated a low diagnostic value for liver fibrosis in the S0-1 stage, it exhibited good diagnostic value for S2-3 stage liver fibrosis. Serum CK18-M30 displayed good diagnostic value for S0-1 and S2-3 hepatic fibrosis, particularly for S2-3 hepatic fibrosis. Elevated serum CK18-M65 and CK18-M30 levels in patients with CHB complicated with NASH suggest their potential utility in evaluating the progression of liver fibrosis in this population. In particular, CK18-M30 exhibits superior diagnostic efficiency.

Preliminary study on the diagnostic value of LEAP-2 and CK18 in biopsy-proven MAFLD.

Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD) has become the leading cause of chronic liver disease. Liver biopsy, as the diagnostic gold standard, is invasive and has sampling bias, making it particularly important to search for sensitive and specific biomarkers for diagnosis. Cytokeratin 18 (CK18) M30 and M65 are products of liver cell apoptosis and necrosis, respectively, and liver-expressed antimicrobial peptide 2 (LEAP-2) is a related indicator of glucose and lipid metabolism. Correlation studies have found that all three indicators positively correlate with the liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Through comparison of diagnostic values, it was found that CK18 M65 can better distinguish between healthy individuals and MAFLD; LEAP-2 can effectively distinguish MAFLD from other liver diseases, especially ALD.

Clinical Utility of Cytokeratins for Accurate Diagnosis of Hepatocellular Carcinoma Among Hepatitis C Virus High-Risk Patients.

OBJECTIVES: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and a global health problem. It is often diagnosed at advanced stage where hopeless for effective therapies. Identification of more reliable biomarkers for early detection of HCC is urgently needed. Cytokeratins are a marker of hepatic progenitor cells and act as a key player in tumor invasion. Herein, we sought to develop a novel score based on the combination of cytokeratin 18 (CK18) and cytokeratin 19 (CK19) with routine laboratory tests for accurate detection of HCC. MATERIAL & METHODS: Serum CK18, CK 19, α-fetoprotein, albumin and platelets count were assayed in HCC patients (75), liver cirrhosis patients (55) and healthy control (20). Areas under receiving operating curve (AUCs) were calculated and used for construction on novel score. A novel score named CK-HCC = CK 19 (ng/ml)×0.001+ CK18 (ng/ml)×0.004 + AFP (U/L)×5.4 - Platelets count (×109)/L×0.003 - Albumin (g/L)×0.27-36 was developed. CK-HCC score produces AUC of 0.919 for differentiating patients with HCC from those with liver cirrhosis with sensitivity and specificity of a cut-off 1.3 (i.e., less than 1.3 the case is considered cirrhotic, whereas above 1.3 it is considered HCC. CONCLUSION: CK-HCC score could replace AFP during screening of HCV patients and early detection of HCC.

Cytokeratin-18 is a sensitive biomarker of alanine transaminase increase in a placebo-controlled, randomized, crossover trial of therapeutic paracetamol dosing (PATH-BP biomarker substudy).

Drug-induced liver injury (DILI) is a challenge in clinical medicine and drug development. Highly sensitive novel biomarkers have been identified for detecting DILI following a paracetamol overdose. The study objective was to evaluate biomarker performance in a 14-day trial of therapeutic dose paracetamol. The PATH-BP trial was a double-blind, placebo-controlled, crossover study. Individuals (n = 110) were randomized to receive 1 g paracetamol 4× daily or matched placebo for 2 weeks followed by a 2-week washout before crossing over to the alternate treatment. Blood was collected on days 0 (baseline), 4, 7, and 14 in both arms. Alanine transaminase (ALT) activity was measured in all patients. MicroRNA-122 (miR-122), cytokeratin-18 (K18), and glutamate dehydrogenase (GLDH) were measured in patients who had an elevated ALT on paracetamol treatment (≥50% from baseline). ALT increased in 49 individuals (45%). All 3 biomarkers were increased at the time of peak ALT (K18 paracetamol arm: 18.9 ± 9.7 ng/ml, placebo arm: 11.1 ± 5.4 ng/ml, ROC-AUC = 0.80, 95% CI 0.71-0.89; miR-122: 15.1 ± 12.9fM V 4.9 ± 4.7fM, ROC-AUC = 0.83, 0.75-0.91; and GLDH: 24.6 ± 31.1U/l V 12.0 ± 11.8U/l, ROC-AUC = 0.66, 0.49-0.83). All biomarkers were correlated with ALT (K18 r = 0.68, miR-122 r = 0.67, GLDH r = 0.60). To assess sensitivity, biomarker performance was analyzed on the visit preceding peak ALT (mean 3 days earlier). K18 identified the subsequent ALT increase (K18 ROC-AUC = 0.70, 0.59-0.80; miR-122 ROC-AUC = 0.60, 0.49-0.72, ALT ROC-AUC = 0.59, 0.48-0.70; GLDH ROC-AUC = 0.70, 0.50-0.90). Variability was lowest for ALT and K18. In conclusion, K18 was more sensitive than ALT, miR-122, or GLDH and has potential significant utility in the early identification of DILI in trials and clinical practice.

Serum keratin-18 detects hepatic inflammation and predicts progression in compensated alcohol-associated liver disease.

Alcohol-associated liver fibrosis accumulates over decades, driven by hepatic inflammation and cell death. We investigated the diagnostic accuracy of keratin-18 degradation, measured using serum M30 and M65 levels, and the ActiTest for hepatic inflammatory activity in patients with compensated alcohol-associated liver disease (ALD). Furthermore, we evaluated the prognostic accuracy of markers for liver-related events and all-cause mortality. All findings were compared with routine liver function tests: Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase. Our prospective, biopsy-controlled, single-center study included 265 patients with ongoing or prior excessive alcohol intake, representing the full spectrum of compensated ALD. We defined hepatic inflammatory activity as a combined score of lobular inflammation and ballooning. For severe hepatic inflammatory activity (n = 40), we found excellent diagnostic accuracy for M30 (area under the receiver operating characteristics curve [AUROC] = 0.90), M65 (AUROC = 0.86), and AST (AUROC = 0.86). Elevated M30 (M30 > 240 U/L) had the highest positive predictive value (PPV) and specificity, significantly higher than M65, ActiTest and ALT, but not AST (M30: sensitivity = 83%, specificity = 82%, positive predictive value = 45%, negative predictive value = 95%). Patients were followed up for 1445 patient-years. All markers, except for ALT, significantly predicted liver-related events and all-cause mortality. After adjusting for advanced fibrosis, drinking behavior and body mass index, M30 and M65 remained significant predictors of liver-related events, whereas M30 and AST were significant predictors of all-cause mortality. Conclusion: M30 and AST accurately detect severe hepatic inflammatory activity in patients with compensated ALD. M30 was the only significant predictor of both liver-related events and all-cause mortality after adjusting for advanced fibrosis, body mass index, and drinking behavior at inclusion.

A new detection system for serum fragmented cytokeratin 18 as a biomarker reflecting histologic activities of human nonalcoholic steatohepatitis.

Caspase-generated fragmented cytokeratin 18 (fCK18) is recognized as a useful noninvasive biomarker in the diagnosis of nonalcoholic fatty liver disease (NAFLD), particularly nonalcoholic steatohepatitis (NASH). However, fCK18 measurement is not applied clinically due to widely variable cut-off values under the current enzyme-linked immunosorbent assay platform. Therefore, we developed a highly sensitive chemiluminescent enzyme immunoassay using newly developed monoclonal antibodies against fCK18 and investigated its relevance in NASH diagnosis. Serum fCK18 levels were measured in the derivation and validation cohort. The correlation between serum fCK18 levels and NAFLD activity score (NAS), fibrosis stage, and liver function was examined. Serum fCK18 levels were significantly correlated with alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase. Serum fCK18 levels were significantly associated with NAS, Brunt's grade/stage, Matteoni's classification, portal inflammation, and fat accumulation in the liver. Notably, hepatocyte ballooning was the only independent variable significantly associated with serum fCK18 in the multivariate linear regression analysis. Serum fCK18 levels were significantly elevated in patients with NAFLD and nonalcoholic fatty liver (NAFL) compared to healthy individuals. They were also significantly elevated in patients with NAFL compared to NASH defined by NAS or Matteoni's classification, with area under the curve values being 0.961 (NAFLD vs. healthy), 0.913 (NAFL vs. healthy), 0.763 (NASH vs. NAFL), and 0.796 (NASH type 3-4 vs. NAFL type 1-2). These results were confirmed by a validation cohort. Notably, changes over time in serum fCK18 levels were significantly correlated with changes in ALT, AST, and the fibrosis-4 index in 25 patients who underwent lifestyle modification. Serum fCK18 levels were significantly correlated with liver damage associated with NASH pathology. Serum fCK18 levels are accurate in distinguishing patients with NAFL or NASH from healthy individuals and may be useful to monitor NASH over time.

Diagnostic and prognostic significance of cell death markers in patients with cirrhosis and acute decompensation.

BACKGROUND: The transition from compensated to decompensated liver cirrhosis is a hallmark of disease progression, however, reliable predictors to assess the risk of decompensation in individual patients from routine diagnostics are lacking. Here, we characterize serum levels of cell death-associated markers and routine biochemistry from patients with chronic liver disease with and without decompensation. METHODS: A post-hoc analysis was based on prospectively collected clinical data from 160 patients with chronic liver disease, stably compensated or decompensated at baseline or during follow-up, over a median period of 721 days. Serum levels of damage-associated molecular patterns (DAMPs) and routine biochemistry are quantified at baseline (for all patients) and during follow-up (for patients with acute decompensation). The panel of DAMPs assessed in this study comprises high-mobility group-box protein 1 (HMGB1), cytochrome C (cyt C), soluble Fas-ligand (sFasL), interleukin 6 (IL-6), soluble cytokeratin-18 (CK18-M65) and its caspase-cleaved fragment CK18-M30. RESULTS: In this cohort study, 80 patients (50%) were diagnosed with alcoholic liver cirrhosis, 60 patients (37.5%) with hepatitis C virus- and 20 patients (13.5%) with hepatitis B virus-related liver cirrhosis. At baseline, 17 patients (10.6%) showed decompensated liver disease and another 28 patients (17.5%) developed acute decompensation during follow-up (within 24 months). One hundred fifteen patients showed stable liver disease (71.9%). We found DAMPs significantly elevated in patients with decompensated liver disease versus compensated liver disease. Patients with acute decompensation during follow-up showed higher baseline levels of IL-6, sFasL, CK18-M65 and-M30 (P<0.01) compared to patients with stably compensated liver disease. In multivariate analyses, we found an independent association of baseline serum levels of sFasL (P = 0.02; OR = 2.67) and gamma-glutamyl transferase (GGT) (P<0.001; OR = 2.1) with acute decompensation. Accuracy of the marker combination for predicting acute decompensation was high (AUC = 0.79). Elevated aminotransferase levels did not correlate with decompensated liver disease and acute decompensation. CONCLUSIONS: DAMPs are elevated in patients with decompensated liver disease and patients developing acute decompensation. The prognostic value of a marker combination with soluble Fas-ligand and GGT in patients with liver cirrhosis should be further evaluated.

Short-Term Safety of Repeated Acetaminophen Use in Patients With Compensated Cirrhosis.

Current guidelines recommend restricting acetaminophen (APAP) use in patients with cirrhosis, but evidence to support that recommendation is lacking. Prior studies focused on pharmacokinetics (PK) of APAP in cirrhosis but did not rigorously examine clinical outcomes, sensitive biomarkers of liver damage, or serum APAP-protein adducts, which are a specific marker of toxic bioactivation. Hence, the goal of this pilot study was to test the effects of regularly scheduled APAP dosing in a well-defined compensated cirrhosis group compared to control subjects without cirrhosis, using the abovementioned outcomes. After a 2-week washout, 12 subjects with and 12 subjects without cirrhosis received 650 mg APAP twice per day (1.3 g/day) for 4 days, followed by 650 mg on the morning of day 5. Patients were assessed in-person at study initiation (day 1) and on days 3 and 5. APAP-protein adducts and both conventional (alanine aminotransferase) and sensitive (glutamate dehydrogenase [GLDH], full-length keratin 18 [K18], and total high-mobility group box 1 protein) biomarkers of liver injury were measured in serum on the mornings of days 1, 3, and 5, with detailed PK analysis of APAP, metabolites, and APAP-protein adducts throughout day 5. No subject experienced adverse clinical outcomes. GLDH and K18 were significantly different at baseline but did not change in either group during APAP administration. In contrast, clearance of APAP-protein adducts was dramatically delayed in the cirrhosis group. Minor differences for other APAP metabolites were also detected. Conclusion: Short-term administration of low-dose APAP (650 mg twice per day, <1 week) is likely safe in patients with compensated cirrhosis. These data provide a foundation for future studies to test higher doses, longer treatment, and subjects who are decompensated, especially in light of the remarkably delayed adduct clearance in subjects with cirrhosis.

Development of a highly sensitive chemiluminescent enzyme immunoassay for fragmented cytokeratin 18 using new antibodies.

Fragmented cytokeratin 18 (fCK18) released from epithelial cells undergoing apoptosis is widely studied in various diseases. However, fCK18 measurement is not utilized in clinical practice due to imprecise disease-state cutoff values. Therefore, we set out to generate new monoclonal antibodies (mAbs) and a recombinant fCK18 (rfCK18) calibrator in an effort to develop a highly sensitive chemiluminescent enzyme immunoassay (CLEIA). New capture mAb (K18-624) had a high binding ability compared to the current commercial antibody. New detection mAb (K18-328) recognized 323S-340G of CK18. A rfCK18 was expressed in the soluble fraction of E. coli when the N-terminal region (260 amino acid residues) of CK18 was truncated. Analysis of performance and measurement of human fCK18 were evaluated using K18-624 and K18-328 in a highly sensitive CLEIA. The coefficients of variation (CV) for within-run and between-day repeatability were below 10% and the recoveries were in the range of 15%. The detection sensitivity was 0.056 ng/mL. Serum fCK18 levels were significantly increased in non-alcoholic steatohepatitis (NASH) patients when compared to healthy individuals. Our new fCK18 mAbs showed high affinity and sensitivity. CLEIA using our new antibodies will be useful in measuring fCK18 in human blood thereby generating accurate clinical diagnoses of human liver diseases.

The application of cytokeratin-18 as a biomarker for drug-induced liver injury.

Drug-induced liver injury (DILI) is a frequent and dangerous adverse effect faced during preclinical and clinical drug therapy. DILI is a leading cause of candidate drug attrition, withdrawal and in clinic, is the primary cause of acute liver failure. Traditional diagnostic markers for DILI include alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Yet, these routinely used diagnostic markers have several noteworthy limitations, restricting their sensitivity, specificity and accuracy in diagnosing DILI. Consequently, new biomarkers for DILI need to be identified.A potential biomarker for DILI is cytokeratin-18 (CK18), an intermediate filament protein highly abundant in hepatocytes and cholangiocytes. Extensively researched in a variety of clinical settings, both full length and cleaved forms of CK18 can diagnose early-stage DILI and provide insight into the mechanism of hepatocellular injury compared to traditionally used diagnostic markers. However, relatively little research has been conducted on CK18 in preclinical models of DILI. In particular, CK18 and its relationship with DILI is yet to be characterised in an in vivo rat model. Such characterization of CK18 and ccCK18 responses may enable their use as translational biomarkers for hepatotoxicity and facilitate management of clinical DILI risk in drug development. The aim of this review is to discuss the application of CK18 as a biomarker for DILI. Specifically, this review will highlight the properties of CK18, summarise clinical research that utilised CK18 to diagnose DILI and examine the current challenges preventing the characterisation of CK18 in an in vivo rat model of DILI.

Total Keratin-18 (M65) as a Potential, Early, Non-Invasive Biomarker of Hepatocyte Injury in Alcohol Intoxicated Adolescents-A Preliminary Study.

BACKGROUND: Underage drinking is associated with health risk behaviors. Serum keratin-18 (CK18) levels are increased in liver diseases and may be biomarkers of outcome. The purpose of this study was to determine if the total CK18 (M65) or caspase-cleaved CK18 (M30) levels were different in adolescents admitted to hospital because of alcohol intoxication and controls with excluded liver diseases. METHODS: A prospective study included 57 adolescents after alcohol use and 23 control subjects. The concentrations of M30 and M65 in the serum samples were evaluated using an enzyme-linked immunosorbent assay. RESULTS: The median age was 15 (14-17) years and 49% were male. There were significant differences in M65 levels between the study and control groups (p = 0.03). The concentrations of M30 and M65 were insignificant in adolescents divided into subgroups according to blood alcohol concentrations (BAC). Significant positive correlations were found between BAC and M65 levels (p = 0.038; r = 0.3). In receiver operating characteristic (ROC) analysis M65 (cut-off = 125.966 IU/l, Se = 70.2%, Sp = 43.5%) allowed to differentiate between patients with and without alcohol intoxication (AUC = 0.66, p = 0.03). CONCLUSION: M65 appears to be a promising non-invasive biomarker of hepatocyte injury during alcohol intoxication in adolescents. Moreover, a higher concentration of M65 may indicate early organ injury before the increase in the activity of liver enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

Role of Bacterial Infection in the Development of Acute Liver Failure in Patients with Decompensated Alcoholic Liver Cirrhosis.

We examined 74 patients with acute decompensation of alcoholic liver cirrhosis: 34 (45.9%) with bacterial infection (group 1) and 40 (54.1%) without bacterial infection (group 2). The degree and index of acute-on-chronic liver failure (ACLF) were determined using an on-line CLIF-C ACLF Calculator and the levels of cytokeratin-18 fragments, TNFα, IL-1ß, IL-4, IL-6, and IL-8. In group 1, AST, cytokeratin-18, TNFα, IL-1ß, IL-6, degree and score of ACLF were significantly higher than in group 2. ACLF developed in 18 (52.9%) patients in group 1 and in 11 (27.5%) (p<0.05) patients in group 2. Within 1 month, 10 (29.4%) patients of group 1 and 2 (5%) patients of group 2 died (p<0.05). Patients with bacterial infection showed a more severe course of alcoholic liver cirrhosis and ACLF than those without bacterial infection.

Plasma Cytokeratin 18 and fecal Alpha-1 Antitrypsin concentrations in dogs with osteosarcoma receiving carboplatin chemotherapy.

Gastrointestinal (GI) toxicosis is a common side effect of cytotoxic chemotherapy treatment in humans and dogs. Measurement of cytokeratin 18 (CK18), an intracellular structural protein released during epithelial apoptosis, and Alpha1-Antitrypsin (A1AT) in faeces provides a mechanism for evaluating damage to the intestinal mucosa secondary to cytotoxic chemotherapy. Our goal was to evaluate the clinical utility of plasma CK18 and faecal A1-AT levels as non-invasive biomarkers of cytotoxic chemotherapy induced GI toxicity. We conducted a prospective cohort study in dogs (N = 10) with osteosarcoma undergoing amputation followed by carboplatin chemotherapy. We hypothesized that plasma CK18 and faecal A1-AT levels would increase following carboplatin administration due to drug-induced GI epithelial damage/apoptosis, and that plasma CK18 and faecal A1-AT levels would correlate with severity of GI toxicity. Mean baseline plasma CK18 concentration was variable amongst patients; however, CK18 concentration prior to carboplatin chemotherapy treatment was not significantly different from CK18 levels after treatment. There was significant intra and inter-patient variability in mean faecal A1-AT levels at baseline. Mean A1-AT concentration did not change significantly from day 0 to day 21. Gastrointestinal toxicity was minimal; therefore, we were unable to determine the association of plasma CK18 and faecal A1-AT concentrations with development of GI toxicosis. In this study population, plasma CK18 and faecal A1-AT concentration were not clinically useful biomarkers for the detection of GI toxicosis secondary to carboplatin administration. Further prospective evaluation of CK18 and A1-AT as biomarkers of drug-induced GI toxicity is warranted in a larger cohort of dogs receiving cytotoxic chemotherapy. AVMA clinical trial registration number: AAHSD004827.

Investigating the Levels of Brain-Specific Proteins in Hydrocephalus Patients.

BACKGROUND: Hydrocephalus, a common brain disorder in children, can cause permanent brain damage. A timely diagnosis of this disorder is crucial. OBJECTIVE: The aim of this study was to evaluate the levels of S-100, CK-18, and NSE brainspecific proteins in patients with hydrocephalus. We examined the levels of these proteins in the blood samples of hydrocephalic patients. METHODS: The study was conducted on the hydrocephalus (n = 31) patients and a healthy control group (n = 30). A Receiver Operating Characteristic (ROC) curve was used to assess the validity of the NSE, CK-18, and S100B to differentiate between the hydrocephalus and the control groups. The suitability of the data to the normal distribution was tested with the Shapiro Wilk test, and the Student t-test was used to compare the characteristics of the normal distribution in two independent groups. The individuals in the hydrocephalus and control groups had similar values in terms of age, height, and weight. RESULTS: It was observed that NSE, CK-18, and S100B mean values of the individuals in the hydrocephalus group were significantly higher than NSE, CK-18, and S100B mean values of the control group. CONCLUSION: Experiments have shown that the levels of these proteins increase significantly in hydrocephalus patients compared to the healthy group. These three parameters can be considered as important markers in the diagnosis of hydrocephalus.

In Severe Alcoholic Hepatitis, Serum Keratin-18 Fragments Are Diagnostic, Prognostic, and Theragnostic Biomarkers.

INTRODUCTION: Up to 40% of patients with severe alcoholic hepatitis (AH) die within 6 months of presentation, making prompt diagnosis and appropriate treatment essential. We determined the associations between serum keratin-18 (K18) and histological features, prognosis, and differential response to prednisolone in patients with severe AH. METHODS: Total (K18-M65) and caspase-cleaved K18 (K18-M30) were quantified in pretreatment sera from 824 patients enrolled in the Steroids or Pentoxifylline for Alcoholic Hepatitis trial (87 with suitable histological samples) and disease controls. RESULTS: K18 fragments were markedly elevated in severe AH and strongly predicted steatohepatitis (alcoholic steatohepatitis) on biopsy (area under receiver operating characteristics: 0.787 and 0.807). Application of published thresholds to predict alcoholic steatohepatitis would have rendered biopsy unnecessary in 84% of all AH cases. K18-M30 and M65 were associated with 90-day mortality, independent of age and Model for End-stage Liver Disease score in untreated patients. The association for K18-M65 was independent of both age and Model for End-stage Liver Disease in prednisolone-treated patients. Modelling of the effect of prednisolone on 90-day mortality as a function of pretreatment serum K18 levels indicated benefit in those with high serum levels of K18-M30. At low pretreatment serum K18 levels, prednisolone was potentially harmful. A threshold of K18-M30 5 kIU/L predicted therapeutic benefit from prednisolone above this level (odds ratio: 0.433, 95% confidence interval: 0.19-0.95, P = 0.0398), but not below (odds ratio: 1.271, 95% confidence interval: 0.88-1.84, P = 0.199). Restricting prednisolone usage to the former group would have reduced exposure by 87%. DISCUSSION: In a large cohort of patients with severe AH, serum K18 strongly correlated with histological severity, independently associated with 90-day mortality, and predicted response to prednisolone therapy. Quantification of serum K18 levels could assist in clinical decision-making.

Non-invasive screening for subclinical liver graft injury in adults via donor-specific anti-HLA antibodies.

The majority of liver grafts exhibit abnormal histological findings late after transplantation, even when liver enzymes are normal. Such subclinical graft injuries were associated with rejection and fibrosis progression in recent studies. The identification of non-invasive biomarkers for subclinical graft injury might help to individualize immunosuppression. Therefore, graft injury was assessed in 133 liver biopsies with normal/near normal liver enzymes from a prospective liver biopsy program. Cytokeratin-18 cell death marker (M65) and donor specific anti-HLA antibodies (DSA) were measured as non-invasive markers in paired plasma samples in addition to routine parameters. M65 was associated with subclinical graft injury but this association was too weak for reasonable clinical application. DSA positivity was associated with more graft inflammation (OR = 5.4) and more fibrosis (OR = 4.2). Absence of DSA excluded fibrosis in 87-89%, while presence of DSA excluded histological criteria for immunosuppression minimization attempts in 92-97%. While CK18 cell death marker had no diagnostic value for the detection of subclinical liver graft injury, DSA testing can help to preselect patients for immunosuppression reduction in case of DSA negativity, while DSA positivity should prompt elastography or liver biopsy for the assessment of subclinical graft injury.

Autophagy alleviates amiodarone-induced hepatotoxicity.

Amiodarone is a widely used antiarrhythmic drug that can cause the development of steatohepatitis as well as liver fibrosis and cirrhosis. The molecular mechanisms of amiodarone-mediated liver injury remain largely unknown. We therefore analyzed amiodarone-mediated hepatocellular injury in patients with chronic heart failure, in primary hepatocytes and HepG2 cells. We found that amiodarone-treated patients with chronic heart failure revealed significantly higher serum levels of caspase-cleaved keratin-18, an apoptosis biomarker, compared to healthy individuals or patients not receiving amiodarone. Furthermore, amiodarone treatment of hepatocytes resulted in apoptosis associated with lipid accumulation and ER-stress induction. Liver cell steatosis was accompanied by enhanced de novo lipogenesis which, after reaching peak levels, declined together with decreased activation of ER stress. The decline of amiodarone-mediated lipotoxicity was associated with protective autophagy induction. In contrast, in hepatocytes treated with the autophagy inhibitor chloroquine as well as in autophagy gene (ATG5 or ATG7)-deficient hepatocytes, amiodarone-triggered toxicity was increased. In conclusion, we demonstrate that amiodarone induces lipid accumulation associated with ER stress and apoptosis in hepatocytes, which is mirrored by increased keratin-18 fragment serum levels in amiodarone-treated patients. Autophagy reduces amiodarone-mediated lipotoxicity and could provide a therapeutic strategy for protection from drug-induced liver injury.

Simple resistance exercise decreases cytokeratin 18 and fibroblast growth factor 21 levels in patients with nonalcoholic fatty liver disease: A retrospective clinical study.

Cytokeratin 18 (CK18) and fibroblast growth factor 21 (FGF21) are elevated in patients with nonalcoholic fatty liver disease (NAFLD) and are useful markers for identifying or monitoring outcomes. Exercise therapy is one of the established treatments for NAFLD; however, few studies have investigated the effectiveness of exercise therapy on CK18 and FGF21 levels. Therefore, the aim of the present study was to assess the effects of 12 weeks of simple resistance exercise on CK18 and FGF21 levels in patients with NAFLD.Fifty patients with NAFLD were assigned to a resistance exercise group (n = 23) or a control group (n = 27) for a trial period of 12 weeks. During the study, the resistance exercise group performed two exercises (push-ups and squats) three times a week on nonconsecutive days, whereas the control group proceeded with regular physical activities under a restricted diet. We then compared serum levels of CK18 fragments (M65) and FGF21 between groups just before and after the 12-week period.Serum M65 levels (880.0 ±â€Š503.6 vs 648.9 ±â€Š450.2 U/L; P < .01) were significantly decreased in the exercise group. However, no significant differences were observed in body mass index or skeletal muscle. The decreases in serum M65 (-231.1 ±â€Š354.7 vs 56.2 ±â€Š375.0 U/L; P = .02), and FGF21 levels (-41.7 ±â€Š98.2 vs. 33.2 ±â€Š127.6 pg/mL; P = .03) were significantly greater in the exercise than in the control group. Changes in M65 levels in the exercise group were significantly correlated with changes in alanine aminotransferase levels (r = 0.618, P < .01).Simple resistance exercise reduced CK18 and FGF21 levels in patients with NAFLD. These findings suggest that resistance exercise consisting of push-ups and squats helps prevent the progression of NAFLD.

Elevated Serum Level of Cytokeratin 18 M65ED Is an Independent Indicator of Cardiometabolic Disorders.

BACKGROUND: Recent studies have suggested that cell death might be involved in the pathophysiology of metabolic disorders. The cytokeratin 18 (CK18) fragment, as a cell death marker, plays an important role in nonalcoholic fatty liver disease (NAFLD). However, only a limited number of studies have found elevated serum levels of CK18 in patients with type 2 diabetes. Moreover, no studies have been conducted yet to investigate the role of CK18 in hypertension or dyslipidemia. In particular, CK18 M65ED is a more sensitive marker of cell death, and its role in cardiometabolic disorders has not been revealed yet. METHODS: A total of 588 subjects were enrolled from the local communities of Shanghai. Serum CK18 M65ED were determined using the enzyme-linked immunosorbent assay. A cardiometabolic disorder was identified by the presence of at least one of the components including overweight or central obesity, diabetes, dyslipidemia, and hypertension. RESULTS: Subjects with cardiometabolic disorders exhibited significantly higher serum levels of CK18 M65ED than those without cardiometabolic disorders (197.36 (121.13-354.50) U/L versus 83.85 (52.80-153.75) U/L, respectively, P < 0.001). Increased serum CK18 M65ED quartiles were associated with the increased prevalence of cardiometabolic disorders and its components (P < 0.001 for all components). Multiple stepwise regression analysis also revealed that diastolic blood pressure, glycated hemoglobin A1c, alanine transaminase, and high-density lipoprotein cholesterol were independently correlated with serum CK18 M65ED levels (all P < 0.01). In addition, logistic regression analysis showed that the serum CK18 M65ED levels were positively correlated with cardiometabolic disorders and in an independent manner. Further, CK18 M65ED was revealed to be an indicator of cardiometabolic disorders in a NAFLD-independent manner. CONCLUSIONS: Elevated levels of CK18 M65ED, a sensitive cell death marker, were independently and positively correlated with cardiometabolic disorders, even after the adjustment for the presence of NAFLD and other cardiovascular risk factors.