ABSTRACT
BACKGROUND: Type 1 diabetes mellitus (T1DM) is characterized by immune and metabolic dysregulation. Apo1/Fas is implicated in maintaining homeostasis of the immune system. Cytokeratin-18 (cCK-18) is a predictive marker of liver disorders in T2DM. Intercellular adhesion molecule-1 (ICAM-1) is considered to increase susceptibility to diabetes mellitus. All three markers are associated with endothelial function, apoptosis and diabetes-related complications. The possible role of Apo1/Fas, cCK-18 and ICAM-1 was investigated in children and adolescents with T1DM. METHOD: Forty-nine (49) children and adolescents with T1DM and 49 controls were included in the study. Somatometric measurements were obtained and the Body Mass Index (BMI) of the participants was calculated. Biochemical parameters were measured by standard laboratory methods and Apo1/Fas, cCK-18 and ICAM-1 were measured using appropriate ELISA kits. The statistical analysis was performed using the IBM SPSS Statistics 23 program. RESULTS: Apo1/Fas (p = 0.001), cCK-18 (p < 0.001) and ICAM-1 (p < 0.001) were higher in patients with T1DM compared to the controls. Apo1Fas was negatively correlated with glucose (p = 0.042), uric acid (p = 0.026), creatinine (p = 0.022), total cholesterol (p = 0.023) and LDL (p = 0.005) in the controls. In children and adolescents with T1DM, Apo1/Fas was positively correlated with total cholesterol (p = 0.013) and LDL (p = 0.003). ICAM-1 was negatively correlated with creatinine (p = 0.019) in the controls, whereas in patients with T1DM it was negatively correlated with HbA1c (p = 0.05). CONCLUSIONS: Apo1/Fas, cCK-18 and ICAM-1 may be useful as serological markers for immune and metabolic dysregulation in children and adolescents with T1DM. Also, Apo1/Fas may have a protective role against metabolic complications in healthy children.
Subject(s)
Biomarkers , Diabetes Mellitus, Type 1 , Intercellular Adhesion Molecule-1 , Humans , Diabetes Mellitus, Type 1/blood , Intercellular Adhesion Molecule-1/blood , Child , Adolescent , Male , Female , Biomarkers/blood , Case-Control Studies , Keratin-18/blood , fas Receptor/blood , Apoptosis , Apolipoprotein A-I/bloodABSTRACT
Liver resection (LR) is the primary treatment for hepatic tumors, yet posthepatectomy liver failure (PHLF) remains a significant concern. While the precise etiology of PHLF remains elusive, dysregulated inflammatory processes are pivotal. Therefore, we explored the theragnostic potential of extracellular high-mobility-group-box protein 1 (HMGB1), a key damage-associated molecular pattern (DAMP) released by hepatocytes, in liver recovery post LR in patients and animal models. Plasma from 96 LR patients and liver tissues from a subset of 24 LR patients were analyzed for HMGB1 levels, and associations with PHLF and liver injury markers were assessed. In a murine LR model, the HMGB1 inhibitor glycyrrhizin, was administered to assess its impact on liver regeneration. Furthermore, plasma levels of keratin-18 (K18) and cleaved cytokeratin-18 (ccK18) were quantified to assess suitability as predictive biomarkers for PHLF. Patients experiencing PHLF exhibited elevated levels of intrahepatic and circulating HMGB1, correlating with markers of liver injury. In a murine LR model, inhibition of HMGB1 improved liver function, reduced steatosis, enhanced regeneration and decreased hepatic cell death. Elevated levels of hepatic cell death markers K18 and ccK18 were detected in patients with PHLF and correlations with levels of circulating HMGB1 was observed. Our study underscores the therapeutic and predictive potential of HMGB1 in PHLF mitigation. Elevated HMGB1, K18, and ccK18 levels correlate with patient outcomes, highlighting their predictive significance. Targeting HMGB1 enhances liver regeneration in murine LR models, emphasizing its role in potential intervention and prediction strategies for liver surgery.
Subject(s)
HMGB1 Protein , Hepatectomy , Liver Failure , Aged , Animals , Female , Humans , Male , Mice , Middle Aged , Biomarkers , Cell Death , Disease Models, Animal , Glycyrrhizic Acid/pharmacology , Hepatectomy/adverse effects , Hepatocytes/metabolism , HMGB1 Protein/metabolism , HMGB1 Protein/blood , Keratin-18/metabolism , Keratin-18/blood , Liver/metabolism , Liver/pathology , Liver Failure/etiology , Liver Failure/metabolism , Liver Failure/pathology , Liver Regeneration , Mice, Inbred C57BLABSTRACT
Cytokeratin-18 (CK-18) is a marker of hepatic cell death. Serum CK-18 could serve as a prognostic marker for patients with advanced liver disease (ALD). This meta-analysis aims to explore the association between total CK-18 (M65) and caspase-cleaved CK-18 (M30) levels with the prognosis of ALD patients. Relevant longitudinal observational studies were identified through comprehensive searches of the Medline, Web of Science, and Embase databases. A random-effects model was utilized to synthesize the findings, accommodating heterogeneity among studies. The analysis included 14 datasets from 11 studies. Elevated serum CK-18 levels at admission were linked to a higher risk of death or liver transplantation during follow-up. This association was consistent for both M65 (risk ratio [RR] 1.99, 95% confidence interval [CI] 1.65 to 2.40, p < 0.001; I2 = 43%) and M30 (RR 1.94, 95% CI 1.57 to 2.40, p < 0.001; I2 = 46%). Subgroup analysis revealed that the relationship between serum M65 levels and adverse outcomes was attenuated in studies using multivariate analysis compared to those using univariate analysis (RR 1.78 vs. 2.80, p for subgroup difference = 0.03). Further subgroup analyses indicated that the prognostic significance of CK-18 for ALD patients was not significantly influenced by study design, methods of determining CK-18 cutoff values, or follow-up durations. Elevated serum CK-18 levels at admission indicate a poor prognosis in patients with ALD. This finding holds for both M65 and M30.
Subject(s)
Biomarkers , Keratin-18 , Keratin-18/blood , Humans , Prognosis , Biomarkers/blood , Liver Diseases/blood , Liver Diseases/diagnosis , Liver Diseases/mortality , Liver Transplantation , Peptide FragmentsABSTRACT
BACKGROUND: At present, liver transplantation (LT) is one of the best treatments for hepatocellular carcinoma (HCC). Accurately predicting the survival status after LT can significantly improve the survival rate after LT, and ensure the best way to make rational use of liver organs. AIM: To develop a model for predicting prognosis after LT in patients with HCC. METHODS: Clinical data and follow-up information of 160 patients with HCC who underwent LT were collected and evaluated. The expression levels of alpha-fetoprotein (AFP), des-gamma-carboxy prothrombin, Golgi protein 73, cytokeratin-18 epitopes M30 and M65 were measured using a fully automated chemiluminescence analyzer. The best cutoff value of biomarkers was determined using the Youden index. Cox regression analysis was used to identify the independent risk factors. A forest model was constructed using the random forest method. We evaluated the accuracy of the nomogram using the area under the curve, using the calibration curve to assess consistency. A decision curve analysis (DCA) was used to evaluate the clinical utility of the nomograms. RESULTS: The total tumor diameter (TTD), vascular invasion (VI), AFP, and cytokeratin-18 epitopes M30 (CK18-M30) were identified as important risk factors for outcome after LT. The nomogram had a higher predictive accuracy than the Milan, University of California, San Francisco, and Hangzhou criteria. The calibration curve analyses indicated a good fit. The survival and recurrence-free survival (RFS) of high-risk groups were significantly lower than those of low- and middle-risk groups (P < 0.001). The DCA shows that the model has better clinical practicability. CONCLUSION: The study developed a predictive nomogram based on TTD, VI, AFP, and CK18-M30 that could accurately predict overall survival and RFS after LT. It can screen for patients with better postoperative prognosis, and improve long-term survival for LT patients.
Subject(s)
Biomarkers, Tumor , Carcinoma, Hepatocellular , Liver Neoplasms , Liver Transplantation , Nomograms , alpha-Fetoproteins , Humans , Carcinoma, Hepatocellular/surgery , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/blood , Liver Neoplasms/surgery , Liver Neoplasms/mortality , Liver Neoplasms/pathology , Liver Neoplasms/blood , Male , Liver Transplantation/adverse effects , Middle Aged , Female , Risk Factors , alpha-Fetoproteins/analysis , Biomarkers, Tumor/blood , Biomarkers, Tumor/analysis , Prognosis , Adult , Retrospective Studies , Aged , Treatment Outcome , Keratin-18/blood , Keratin-18/analysis , Decision Support TechniquesABSTRACT
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 (Pâ <â .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.
Subject(s)
Biomarkers , Hepatitis B, Chronic , Keratin-18 , Liver Cirrhosis , Non-alcoholic Fatty Liver Disease , Humans , Non-alcoholic Fatty Liver Disease/blood , Non-alcoholic Fatty Liver Disease/complications , Non-alcoholic Fatty Liver Disease/diagnosis , Hepatitis B, Chronic/blood , Hepatitis B, Chronic/complications , Male , Female , Keratin-18/blood , Liver Cirrhosis/blood , Liver Cirrhosis/diagnosis , Adult , Middle Aged , Biomarkers/blood , Peptide Fragments/blood , Liver Function Tests/methods , Case-Control Studies , Clinical RelevanceABSTRACT
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.
Subject(s)
Antimicrobial Cationic Peptides , Biomarkers , Keratin-18 , Adult , Female , Humans , Male , Middle Aged , Alanine Transaminase/blood , Antimicrobial Cationic Peptides/blood , Aspartate Aminotransferases/blood , Biomarkers/blood , Biopsy , Fatty Liver/diagnosis , Fatty Liver/pathology , Fatty Liver/blood , Keratin-18/blood , Liver/pathology , Peptide Fragments , Sensitivity and SpecificityABSTRACT
Animal models of COVID-19 facilitate the development of vaccines and antivirals against SARS-CoV-2. The efficacy of antivirals or vaccines may differ in different animal models with varied degrees of disease. Here, we introduce a mouse model expressing human angiotensin-converting enzyme 2 (ACE2). In this model, ACE2 with the human cytokeratin 18 promoter was knocked into the Hipp11 locus of C57BL/6J mouse by CRISPR - Cas9 (K18-hACE2 KI). Upon intranasal inoculation with high (3 × 105 PFU) or low (2.5 × 102 PFU) dose of SARS-CoV-2 wildtype (WT), Delta, Omicron BA.1, or Omicron BA.2 variants, all mice showed obvious infection symptoms, including weight loss, high viral loads in the lung, and interstitial pneumonia. 100% lethality was observed in K18-hACE2 KI mice infected by variants with a delay of endpoint for Delta and BA.1, and a significantly attenuated pathogenicity was observed for BA.2. The pneumonia of infected mice was accompanied by the infiltration of neutrophils and pulmonary fibrosis in the lung. Compared with K18-hACE2 Tg mice and HFH4-hACE2 Tg mice, K18-hACE2 KI mice are more susceptible to SARS-CoV-2. In the antivirals test, REGN10933 and Remdesivir had limited antiviral efficacies in K18-hACE2 KI mice upon the challenge of SARS-CoV-2 infections, while Nirmatrelvir, monoclonal antibody 4G4, and mRNA vaccines potently protected the mice from death. Our results suggest that the K18-hACE2 KI mouse model is lethal and stable for SARS-CoV-2 infection, and is practicable and stringent to antiviral development.
Subject(s)
Angiotensin-Converting Enzyme 2 , Antiviral Agents , COVID-19 , Disease Models, Animal , Mice, Inbred C57BL , SARS-CoV-2 , Animals , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Mice , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/drug effects , Antiviral Agents/pharmacology , Humans , Lung/virology , Lung/pathology , COVID-19 Drug Treatment , Keratin-18/genetics , Viral Load , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/pharmacology , Gene Knock-In Techniques , Antibodies, Viral/immunology , Antibodies, Viral/blood , FemaleABSTRACT
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.
Subject(s)
Biomarkers, Tumor , Carcinoma, Hepatocellular , Hepacivirus , Keratin-18 , Keratin-19 , Liver Neoplasms , alpha-Fetoproteins , Humans , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/virology , Liver Neoplasms/diagnosis , Liver Neoplasms/blood , Liver Neoplasms/virology , Biomarkers, Tumor/blood , Female , Male , Middle Aged , Keratin-18/blood , Hepacivirus/isolation & purification , Keratin-19/blood , Case-Control Studies , alpha-Fetoproteins/analysis , alpha-Fetoproteins/metabolism , Liver Cirrhosis/diagnosis , Liver Cirrhosis/blood , Liver Cirrhosis/virology , Hepatitis C/diagnosis , Hepatitis C/virology , Hepatitis C/blood , Hepatitis C/complications , Prognosis , Follow-Up Studies , Adult , AgedABSTRACT
Early embryonic mortality resulting from insufficient interaction between the embryo and the uterus leads to the failure of pregnancy in livestock animals. Thus, it is imperative to comprehend the multifaceted process of implantation at molecular levels, which requires synchronized feto-maternal interaction. The in-vitro models serve as valuable tools to investigate the specific stages of implantation. The present study was undertaken to develop a simple method to isolate and culture the primary buffalo endometrial epithelial cells (pBuEECs), followed by proteome profiling of the proliferating cells. Collagenase I was used to separate uterine epithelial cells (UECs) from the ipsilateral uterine horn, and then the cells were separated using a cell strainer. After being seeded on culture plates, UECs developed colonies with characteristic epithelial shape and expressed important markers such as cytokeratin 18 (KRT18), progesterone receptor (PGR), ß-estrogen receptor (ESR1), and leukemia inhibitory factor (LIF), which were confirmed by PCR. The purity of epithelial cells was assessed using cytokeratin 18 immunostaining, which indicated approximately 99% purity in cultured cells. The proteome profiling of pBuEECs via high-throughput tandem mass spectrometry (MS), identified a total of 3383 proteins. Bioinformatics analysis revealed enrichment in various biological processes, including cellular processes, metabolic processes, biological regulation, localization, signaling, and developmental processes. Moreover, the KEGG pathway analysis highlighted associations with the ribosome, proteosome, oxidative phosphorylation, spliceosome, and cytoskeleton regulation pathways. In conclusion, these well characterized cells offer valuable in-vitro model to enhance the understanding of implantation and uterine pathophysiology in livestock animals, particularly buffaloes.
Subject(s)
Buffaloes , Keratin-18 , Pregnancy , Female , Animals , Buffaloes/physiology , Keratin-18/metabolism , Proteome/metabolism , Proteomics/methods , Endometrium/metabolism , Embryo Implantation/physiology , Epithelial Cells/metabolismABSTRACT
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.
Subject(s)
Acetaminophen , Alanine Transaminase , Biomarkers , Chemical and Drug Induced Liver Injury , Cross-Over Studies , Keratin-18 , Humans , Alanine Transaminase/blood , Biomarkers/blood , Male , Chemical and Drug Induced Liver Injury/blood , Chemical and Drug Induced Liver Injury/etiology , Female , Double-Blind Method , Keratin-18/blood , Adult , Middle Aged , MicroRNAs/blood , Young Adult , Glutamate Dehydrogenase/blood , Analgesics, Non-NarcoticABSTRACT
BACKGRUOUND: We aimed to evaluate whether composite blood biomarkers including aldo-keto reductase family 1 member B10 (AKR1B10) and cytokeratin 18 (CK-18; a nonalcoholic steatohepatitis [NASH] marker) have clinically applicable performance for the diagnosis of NASH, advanced liver fibrosis, and high-risk NASH (NASH+significant fibrosis). METHODS: A total of 116 subjects including healthy control subjects and patients with biopsy-proven nonalcoholic fatty liver disease (NAFLD) were analyzed to assess composite blood-based and imaging-based biomarkers either singly or in combination. RESULTS: A composite blood biomarker comprised of AKR1B10, CK-18, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) showed excellent performance for the diagnosis of, NASH, advanced fibrosis, and high-risk NASH, with area under the receiver operating characteristic curve values of 0.934 (95% confidence interval [CI], 0.888 to 0.981), 0.902 (95% CI, 0.832 to 0.971), and 0.918 (95% CI, 0.862 to 0.974), respectively. However, the performance of this blood composite biomarker was inferior to that various magnetic resonance (MR)-based composite biomarkers, such as proton density fat fraction/MR elastography- liver stiffness measurement (MRE-LSM)/ALT/AST for NASH, MRE-LSM+fibrosis-4 index for advanced fibrosis, and the known MR imaging-AST (MAST) score for high-risk NASH. CONCLUSION: Our blood composite biomarker can be useful to distinguish progressive forms of NAFLD as an initial noninvasive test when MR-based tools are not available.
Subject(s)
Biomarkers , Keratin-18 , Non-alcoholic Fatty Liver Disease , Humans , Non-alcoholic Fatty Liver Disease/blood , Keratin-18/blood , Biomarkers/blood , Male , Female , Middle Aged , Adult , Aldo-Keto Reductases/blood , Liver Cirrhosis/blood , Liver Cirrhosis/diagnosis , Liver Cirrhosis/diagnostic imaging , ROC Curve , Case-Control Studies , Aldehyde Reductase/blood , Alanine Transaminase/blood , Aspartate Aminotransferases/blood , Disease Progression , Liver/diagnostic imaging , Liver/pathology , AgedABSTRACT
INTRODUCTION: The effect of antiretroviral therapy (ART), particularly integrase strand transfer inhibitors (INSTIs), on non-alcoholic fatty liver disease (NAFLD) in people with HIV remains unclear. We evaluated the effect of switching non-INSTI backbone antiretroviral medications to raltegravir on NAFLD and metabolic parameters. MATERIALS AND METHODS: This was a single-centre, phase IV, open-label, randomized controlled clinical trial. People living with HIV with NAFLD and undetectable viral load while receiving a non-INSTI were randomized 1:1 to the switch arm (raltegravir 400 mg twice daily) or the control arm (continuing ART regimens not containing INSTI). NAFLD was defined as hepatic steatosis by controlled attenuation parameter ≥238 dB/m in the absence of significant alcohol use and viral hepatitis co-infections. Cytokeratin 18 was used as a biomarker of non-alcoholic steatohepatitis. Changes over time in outcomes were quantified as standardized mean differences (SMDs), and a generalized linear mixed model was used to compare outcomes between study arms. RESULTS: A total of 31 people with HIV (mean age 54 years, 74% male) were randomized and followed for 24 months. Hepatic steatosis improved between baseline and end of follow-up in both the switch (SMD -43.4 dB/m) and the control arm (-26.6 dB/m); the difference between arms was not significant. At the end of follow-up, aspartate aminotransferase significantly decreased in the switch arm compared with the control arm (SMD -9.4 vs. 5.5 IU/L). No changes in cytokeratin 18, body mass index, or lipids were observed between study arms. DISCUSSION: Switching to a raltegravir-based regimen improved aspartate aminotransferase but seemed to have no effect on NAFLD, body weight, and lipids compared with remaining on any other ART.
Subject(s)
HIV Infections , Non-alcoholic Fatty Liver Disease , Male , Humans , Middle Aged , Female , Raltegravir Potassium/therapeutic use , HIV Infections/complications , HIV Infections/drug therapy , Non-alcoholic Fatty Liver Disease/drug therapy , Keratin-18 , Anti-Retroviral Agents/therapeutic use , Lipids , Aspartate AminotransferasesABSTRACT
Keratins are epithelial intermediate filament proteins that play a crucial role in cellular stress protection, with K8 being the most abundant in the colon. The intestinal epithelial-specific K8-deficient mouse model (K8flox/flox;Villin-Cre) exhibits characteristics of inflammatory bowel disease, including diarrhea, crypt erosion, hyperproliferation, and decreased barrier function. Nevertheless, the order in which these events occur and whether they are a direct cause of K8 loss or a consequence of one event inducing another remains unexplored. Increased knowledge about early events in the disruption of colon epithelial integrity would help to understand the early pathology of inflammatory and functional colon disorders and develop preclinical models and diagnostics of colonic diseases. Here, we aimed to characterize the order of physiological events after Krt8 loss by utilizing K8flox/flox;Villin-CreERt2 mice with tamoxifen-inducible Krt8 deletion in intestinal epithelial cells, and assess stool analysis as a noninvasive method to monitor real-time gene expression changes following Krt8 loss. K8 protein was significantly decreased within a day after induction, followed by its binding partners, K18 and K19 from day 4 onward. The sequential colonic K8 downregulation in adult mice leads to immediate diarrhea and crypt elongation with activation of proliferation signaling, followed by crypt loss and increased neutrophil activity within 6-8 days, highlighting impaired water balance and crypt elongation as the earliest colonic changes upon Krt8 loss. Furthermore, epithelial gene expression patterns were comparable between colon tissue and stool samples, demonstrating the feasibility of noninvasive monitoring of gut epithelia in preclinical research utilizing Cre-LoxP-based intestinal disease models.NEW & NOTEWORTHY Understanding the order in which physiological and molecular events occur helps to recognize the onset of diseases and improve their preclinical models. We utilized Cre-Lox-based inducible keratin 8 deletion in mouse intestinal epithelium to characterize the earliest events after keratin 8 loss leading to colitis. These include diarrhea and crypt elongation, followed by erosion and neutrophil activity. Our results also support noninvasive methodology for monitoring colon diseases in preclinical models.
Subject(s)
Colitis , Keratin-8 , Animals , Mice , Colitis/genetics , Diarrhea , Keratin-18/genetics , Keratin-8/genetics , Keratin-8/metabolism , Keratins/chemistry , Keratins/geneticsABSTRACT
Hepatocellular carcinoma (HCC) is commonly associated with disturbances in glucose metabolism and enhanced glycolysis. However, a controversial role for gluconeogenesis was reported to be tumor-promoting and tumor-suppressive. We investigated novel anti-HCC treatments through either the simultaneous inhibition of glycolysis and gluconeogenesis by "phloretin" and "sodium meta-arsenite", respectively (Combination 1); or the concurrent inhibition of glycolysis and induction of gluconeogenesis by phloretin and dexamethasone, respectively, (combination 2). A total of 110 Swiss albino mice were divided into eleven groups, HCC was induced by N, N-dimethyl-4-aminoazobenzene. We have measured the expression of the glucose transporter 2 (GLUT2), Phosphoenolpyruvate carboxykinases (PEPCK), Caspase-3, Beclin 1, Cyclin D1, and cytokeratin 18 genes; blood glucose and ATP levels; alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. Furthermore, in silico molecular docking was performed to investigate the potential drug-receptor interactions. Histologically, the phloretin-based combinations resulted in a significant regression of malignant tissue compared to various treatments. GLUT2 and PEPCK mRNA analysis indicated successful off/on modulation of glycolysis and gluconeogenesis. Docking confirmed the potent binding between phloretin, sodium meta-arsenite, and dexamethasone with GLUT2, PEPCK, and Retinoid X Receptor Alpha, respectively. Molecularly, Combination 2 resulted in the highest reduction in cyclin D1, cytokeratin 18, and Beclin 1 expression contemporaneously with the upregulation in Caspase-3 levels. Biochemically, both combinations caused a significant reduction in ATP levels, ALT, and AST activity compared to the other groups. In conclusion, we propose two novel phloretin-based combinations that can be used in treating HCC through the regulation of glucose metabolism and ATP production.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Mice , Animals , Carcinoma, Hepatocellular/genetics , Caspase 3 , Cyclin D1 , Keratin-18 , Liver Neoplasms/genetics , Molecular Docking Simulation , Phloretin/pharmacology , Beclin-1 , Glucose/metabolism , Adenosine Triphosphate/metabolism , DexamethasoneABSTRACT
Background: Since most patients with oral cancer do not benefit from current treatments, new therapeutic strategies or drugs must be developed to improve patient prognosis. Qing Yan Li Ge Tang (QYLGT), a Chinese herbal medicine, is known for its anticancer activity. This study aimed to investigate whether QYLGT has anticancer effects on human OEC-M1 oral cancer cells. Methods: To evaluate whether QYLGT affects viability, morphology, and colony formation ability of the OEC-M1 cells, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, morphology study, and colony formation assay were performed, respectively. Each assay was carried out in triplicate, and the whole set of experiments was performed three times independently. To investigate whether QYLGT induces apoptotic effects in OEC-M1 cells, the enzyme-linked immunosorbent (ELISA) was carried out to quantify cytokeratin 18 fragment (an apoptosis marker). Each assay was carried out in triplicate, and the whole set of experiments was performed three times independently. The immunoblotting assay was performed to detect the protein expression after QYLGT treatment. The whole set of experiments was performed two times independently. Results: The results from the MTT and colony formation assays indicate that QYLGT inhibited the cell viability and clonogenic growth capacity of OEC-M1 cells. The morphology study shows that QYLGT increased plasma membrane blebbing in OEC-M1 clles. The results of ELISA and an immunoblotting assay show that QYLGT increased cytokeratin 18 fragment release and poly ADP-ribose polymerase cleavage (another apoptosis marker) in OEC-M1 cells. In addition, the results from immunoblotting assay show that QYLGT also activated apoptotic executor proteins, including caspase-8, caspase-9, and caspase-3, and the results of ELISA indicate that treatment with the pan-caspase inhibitor, Z-VAD-FMK, inhibited QYLGT-induced cytokeratin 18 fragment release. These results indicate that QYLGT inhibited cell viability in OEC-M1 cells and induced OEC-M1 apoptosis through caspase activation. Additionally, QYLGT-activated c-Jun N-terminal kinase, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and nuclear factor-kappa B (NF-κB), and the related inhibitors, including SP600125, PD184352, SB202190, and Bay11-7082, were used to confirm which signaling was involved in QYLGT-induced apoptosis. Moreover, only Bay11-7082, the NF-κB inhibitor, could suppress QYLGT-induced the release of cytokeratin 18 fragments from OEC-M1 cells. Conclusions: QYLGT induced apoptosis in OEC-M1 cells via the NF-κB pathway.
Subject(s)
Mouth Neoplasms , NF-kappa B , Humans , NF-kappa B/metabolism , NF-kappa B/pharmacology , Keratin-18/pharmacology , Apoptosis , Mouth Neoplasms/drug therapy , Cell Line, TumorABSTRACT
Female infertility is a worldwide concern that impacts the quality of life and well-being of affected couples. Failure of embryo implantation is a major cause of early pregnancy loss and is precisely regulated by a programmed molecular mechanism. Recent studies have shown that proper trophoblast adhesion and invasion are essential for embryo implantation. However, the potential regulatory mechanism involved in trophoblast adhesion and invasion has yet to be fully elucidated. KRT18 has been reported to play a critical role in early embryonic development, but its physiological function in embryo implantation remains unclear. In the present study, we revealed that KRT18 was highly expressed in trophoblast cells and that knockdown of KRT18 in mouse embryos inhibited embryo adhesion and implantation. In vitro experiments further showed that silencing KRT18 disturbed trophoblast migration and invasion. More importantly, we provide evidence that KRT18 directly binds to and stabilizes cell surface E-cadherin in trophoblast cells through microscale thermophoresis (MST) analysis and molecular biology experiments. In brief, our data reveal that KRT18, which is highly expressed in trophoblast cells, plays an important role in the regulation of trophoblast invasion and adhesion during embryo implantation by directly binding to E-cadherin.
Subject(s)
Embryo Implantation , Keratin-18 , Trophoblasts , Animals , Female , Mice , Pregnancy , Cadherins , Embryonic Development , Keratin-18/metabolismABSTRACT
BACKGROUND/AIM: Cisplatin is a drug for treating oral cancer. However, several previous studies indicate that oral cancer cells can develop resistance to cisplatin, which may result in a poor prognosis for patients with oral cancer. Fucoidan, a natural health product extracted from brown seaweed, has anticancer abilities against various types of cancer cell. This study evaluated whether fucoidan can enhance the sensitivity of oral cancer cells to cisplatin and explored the underlying mechanism. MATERIALS AND METHODS: SCC-25 cells were used in the present study and treated with 0.3125 mg/ml fucoidan, 12.5 µg/ml cisplatin, or 0.3125 mg/ml fucoidan plus 12.5 µg/ml cisplatin for 48 h, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, enzyme-linked immunosorbent, and immunoblotting assays were performed to evaluate cell survival, cytokeratin-18 fragment release, and expression of markers of apoptosis and autophagy, respectively. RESULTS: Cotreatment with fucoidan enhanced cisplatin-induced reduction of SCC-25 cell survival compared to cisplatin alone. In addition, cotreatment also increased the expression of apoptosis markers, including activated caspase-8, activated caspase-9, activated caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP), but did not increase the expression of the two autophagy markers studied, beclin and autophagy-related 12-autophagy-related 5 conjugate. Fucoidan significantly inhibited cisplatin-induced AKT serine/threonine kinase 1 activation, which promoted PARP cleavage, caspase-3 activation, and cytokeratin-18 fragment expression in SCC-25 cells. CONCLUSION: Fucoidan promoted cisplatin-induced effects by inhibiting phosphatidylinositol 4,5 bisphosphate 3 kinase/AKT serine/threonine kinase 1 activation induced by cisplatin. The results of this study may provide a basis for the possible application of the combination of fucoidan and cisplatin in the clinical treatment of oral cancer in the future to improve the prognosis of patients with oral cancer.
Subject(s)
Cisplatin , Mouth Neoplasms , Humans , Caspase 3 , Cisplatin/pharmacology , Keratin-18 , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Poly(ADP-ribose) Polymerase Inhibitors , Mouth Neoplasms/drug therapy , SerineABSTRACT
Alterations in apoptosis, as reflected by circulating Cytokeratin 18 (CK18), are involved in the progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis and atherogenesis. We aimed to explore the discriminant accuracy of Cytokeratin 18 (CK18, including M65 and M30 forms) for an elevated fatty liver index (FLI) as a validated proxy of NAFLD, and cardiovascular disease (CVD) risk in the general population. Both serum CK18 forms were measured using a commercial immunoassay in randomly selected samples from 312 participants of the PREVEND general population cohort. FLI ≥ 60 was used to indicate NAFLD. Framingham Risk Score (FRS) and the SCORE2 were used to estimate the 10-year risk of CVD. The Receiver Operating Characteristic (ROC) curve, linear/logistic regression models, and Spearman's correlations were used. Intricate associations were found between CK18, FLI, and CVD risk scores. While M30 was the only independent predictor of FLI ≥ 60, M65 best discriminated NAFLD individuals at very-high 10-year CVD risk according to SCORE2 (AUC: 0.71; p = 0.001). Values above the predefined manufacturer cutoff (400 U/L) were associated with an independent 5-fold increased risk (adjusted odds ratio: 5.44, p = 0.01), with a negative predictive value of 93%. Confirming that NAFLD is associated with an increased CVD risk, our results in a European general population-based cohort suggest that CK18 M65 may represent a candidate biomarker to identify NAFLD individuals at low CVD risk.
Subject(s)
Cardiovascular Diseases , Non-alcoholic Fatty Liver Disease , Humans , Biomarkers , Cardiovascular Diseases/diagnosis , Keratin-18 , Non-alcoholic Fatty Liver Disease/complications , Prognosis , Risk FactorsABSTRACT
Visceral obesity is linked to the progression of fatty liver to nonalcoholic steatohepatitis (NASH). Cytokeratin-18 (CK18) epitopes M30 (CK18M30) and M65 (CK18M65) represent accurate markers for detecting NASH. The aim of this study was to evaluate the association of CK18M30 and CK18M65 levels with anthropometric and metabolic characteristics, liver stiffness, and liver indices of steatosis and fibrosis in a cohort of subjects with visceral obesity; in this cross-sectional study, transient elastography (TE-Fibroscan®), anthropometric measurements, metabolic parameters, High Sensitivity C-Reactive Protein (hsCRP), and CK18M30 and CK18M65 levels (Apoptosense ELISA, PEVIVA, Germany) were evaluated. Fatty Liver Index (FLI), Fibrosis 4 (FIB-4), and Aspartate transaminase (AST)-platelet ratio index (APRI) were calculated; among 48 subjects, 47.2% presented metabolic syndrome, 93.8% hepatic steatosis, 60.4% high liver stiffness, and 14.6% hypertransminasemia, while FIB-4 and APRI were normal. CK18M30 and CK18M65 levels were significantly correlated with waist circumference, AST, ALT, HoMA-IR, liver stiffness, and APRI (p < 0.001). Subjects with CK18 fragments above the median values showed significantly higher waist circumference, HbA1c, AST, ALT, HoMA-IR, FLI, and APRI compared to those with values below the median; CK18M30 and CK18M65 levels correlated well with anthropometric and metabolic characteristics, representing good biomarkers for early identification of NASH in subjects with visceral obesity.
Subject(s)
Non-alcoholic Fatty Liver Disease , Humans , Non-alcoholic Fatty Liver Disease/metabolism , Obesity, Abdominal/metabolism , Keratin-18/metabolism , Cross-Sectional Studies , Liver/metabolism , Fibrosis , Biomarkers/metabolism , Liver Cirrhosis/metabolismABSTRACT
BACKGROUND & AIMS: In patients with compensated alcohol-related cirrhosis, reliable prognostic biomarkers are lacking. Keratin-18 and hepatocyte-derived large extracellular vesicle (lEV) concentrations reflect disease activity, but their ability to predict liver-related events is unknown. METHODS: We measured plasma keratin-18 and hepatocyte lEV concentrations in 500 patients with Child-Pugh class A alcohol-related cirrhosis. The ability of these hepatocyte-derived biomarkers, alone or combined with model for end-stage liver disease (MELD) and FibroTest scores, to predict liver-related events at 2 years was analyzed, taking into account the alcohol consumption at inclusion and during follow-up. RESULTS: Keratin-18 and hepatocyte lEV concentrations increased with alcohol consumption. In patients without active alcohol consumption at enrollment (n = 419), keratin-18 concentration predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both keratin-18 concentrations >285 U/L and FibroTest >0.74 had a 24% cumulative incidence of liver-related events at 2 years, vs. 5% to 14% in other groups of patients. Similar results were obtained when combining keratin-18 concentrations >285 U/L with MELD >10. In patients with active alcohol consumption at enrollment (n = 81), hepatocyte lEVs predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both hepatocyte lEV concentrations >50 U/L and FibroTest >0.74 had a 62% cumulative incidence of liver-related events at 2 years, vs. 8% to 13% in other groups of patients. Combining hepatocyte lEV concentrations >50 U/L with MELD >10 had a lower discriminative ability. Similar results were obtained when using decompensation of cirrhosis, defined according to Baveno VII criteria, as an endpoint. CONCLUSION: In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers with FibroTest or MELD scores identifies patients at high risk of liver-related events, and could be used for risk stratification and patient selection in clinical trials. IMPACT AND IMPLICATIONS: In patients with compensated alcohol-related cirrhosis, reliable predictors of outcome are lacking. In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers (keratin-18 and hepatocyte-large extracellular vesicles) with FibroTest or MELD scores identifies those at high risk of liver-related events at 2 years. The identified patients at high risk of liver-related events are the target-of-choice population for intensive surveillance (e.g., referral to tertiary care centers; intensive control of risk factors) and inclusion in clinical trials.