Liver-specific Enzyme Research Articles

Characterization and Proteomic Profiling of Hepatocyte-Like Cells Derived from Human Wharton’s Jelly Mesenchymal Stromal Cells: De Novo Expression of Liver-Specific Enzymes

End-stage liver disease (ESLD), affecting millions worldwide, represents a challenging issue for clinical research and global public health. Liver transplantation is the gold standard therapeutic approach but shows some drawbacks. Hepatocyte transplantation could be a reliable alternative for patient treatment. Mesenchymal stromal cells derived from Wharton’s jelly of the umbilical cord (WJ-MSCs) can differentiate into hepatocyte-like cells (HLCs) and show immunomodulatory functions. Due to the increasing demand for fully characterized cell therapy vehicles warranting both the safety and efficacy of treatments, in this work, we extensively characterized WJ-MSCs before and after the application of a hepatocyte-directed differentiation protocol. HLCs exhibited a morphology resembling that of hepatocytes, expressed early and late hepatic markers (α-fetoprotein, albumin, CK18, HNF4-α), and acquired hepatic functions (glycogen synthesis, xenobiotics detoxification), as also revealed by the shotgun proteomics approach. HLCs maintained the same pattern of immunomodulatory molecule expression and mesenchymal markers, other than displaying specific enzymes, suggesting these cells as promising candidates for cellular therapy of ESLD. Our work shed new light on the basic biology of HLCs, suggesting new therapeutic approaches to treat ESLD.

Investigation of Hepatozoon Canis Related Alterations in the Haemato Biochemical Profile of Dogs

Aims: Hepatozoonosis, caused by a protozoan parasite Hepatozoon canis, is important for canine health due to its ability to cause chronic, debilitating disease, and the challenges it presents in terms of diagnosis and management. The present study was conducted to see the effect of H. canis infection on various haematological and biochemical parameters as compared to the normal healthy dogs in order to understand the disease pattern and suspecting the disease. Study Design: The clinically affected dogs that come to the veterinary hospital with symptoms of hepatozoonosis were included in the study. Haematological and biochemical parameters of dogs found positive for hepatozoonosis were analysed. The values of these parameters were subsequently compared to healthy animals in order to identify any significant difference caused by H. canis. Place and Duration of Study: The studied was conducted at Department of Veterinary Medicine, DUVASU Mathura, India. Total 212 blood samples were collected from the dogs that were brought for the treatment in between February 2023 to March 2024. Methodology: Total 212 dogs were included in the present study out of this 46 dogs were found positive with H. canis using PCR based molecular test. These positive animals were subjected to study the changes in biochemical parameters like blood urea nitrogen, creatinine, alkaline phosphatase, alanine transaminase, aspartate amino transferase, total leucocyte count, total erythrocyte count, haemoglobin, haematocrit, erythrocyte indices (MCV, MCH, MCHC), differential leucocyte count, platelet count and platelet indices (MPV, PDW) using autoanalyzer. Student's t-test in Graphpad Prism was used to compare these values to healthy animals. Results: The study confirms that Hepatozoonosis leads to significant haematological and biochemical changes in dogs. Infected dogs have lower Hb, TEC, and PCV than healthy animals, indicating anaemia. Thrombocytopenia and leukocytosis were also observed in the infected animals. Non-significant differences in MCV, MCH, and MCHC indicate normocytic, normochromic anaemia. Anisocytosis and non-regenerative anaemia is indicated by significant RDW-CV increases with decrease in MCV values. Increased liver-specific enzymes like ALT, AST, and ALP and kidney-related enzymes like BUN and CRT suggest hepatic and renal involvement in disease pathophysiology. Conclusion: In hepatozoonosis, haemato- biochemical parameters like non-regenerative anaemia. anisocytosis, thrombocytopenia, leukocytosis, low serum protein concentration and increased ALT, AST, ALP and BUN values may be used as non-specific biomarkers. The results can be used in making clinical decisions and monitoring therapeutic responses in cases of hepatozoonosis.

Cannabidiol Modulation of Nicotine‐Induced Toxicity: Assessing Effects on Behavior, Brain‐Derived Neurotrophic Factor, and Oxidative Stress in C57BL/6 Male Mice

ABSTRACTHigh doses of nicotine administered to rodents serve as a model for studying anxiety and test compounds' potential anxiolytic effects. At these doses, anxiety in rodents is accompanied by disruption of brain‐derived neurotrophic factor (BDNF). The endocannabinoids and nicotine modulate several central nervous system processes via their specific receptors, impacting locomotion, anxiety, memory, nociception, and reward. Cannabidiol (CBD), an active ingredient of Cannabis sativa L., is devoid of psychoactive actions and has gained attention for its anxiolytic, antioxidant, and anti‐inflammatory properties, among others. This work aims to examine the potential anxiety‐reducing properties of CBD in a well‐established experimental mouse model of anxiety‐like behavior induced by high doses of nicotine on male C57BL/6 mice. In this context, the open‐field behavioral test was specially conducted to assess CBD's effects on anxiety‐like behavior and locomotion. Brain neuronal plasticity, modulated by BDNF, along with a diverse array of blood's metabolic markers, was examined as a means of evaluating systemic toxicity under various treatments. Finally, oxidative stress was evaluated through the measurement of glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA), while pro‐inflammatory cytokine assessments were conducted to evaluate redox status and immune system function. Our research suggests that CBD shows potential in reducing anxiety‐like behaviors induced by high doses of nicotine, by mitigating changes in BDNF protein levels in cerebral hemispheres and cerebellum. At the same time, CBD targets specific liver enzymes, maintains tissue's systemic toxicity (i.e., renal, kidney, and pancreatic), balances redox status (SOD, GSH, and MDA), and regulates the secretion of pro‐inflammatory cytokines (TNF‐alpha and IL‐6).

Bilirubin, once a toxin but now an antioxidant alleviating non-alcoholic fatty liver disease in an autophagy-dependent manner in high-fat diet-induced rats: a molecular and histopathological analysis.

As an endogenous antioxidant, bilirubin has surprisingly been inversely correlated with the risk of non-alcoholic fatty liver disease (NAFLD). Thereupon, the current evaluation was designed to assess the positive effects of bilirubin on the autophagy flux, as well as the other pathogenic processes and parameters involved in the expansion of NAFLD. Thirty adult male rats weighing 150-200 g with free access to sucrose solution (18%) were randomly subdivided into 5 groups (n = 6). Subsequently, the animals were euthanized, and their blood specimens and liver tissue samples were collected to measure serum biochemical indices, liver histopathological changes, intrahepatic triglycerides content, and tissue stereological alterations. Furthermore, the expression levels of autophagy-related genes (Atgs) were measured to assess the state of the autophagy flux. Fasting blood glucose, body weight, as well as liver weight, liver-specific enzyme activity, and serum lipid profile indices markedly decreased in rats that underwent a six-week bilirubin treatment compared to the control group. In addition, histopathological studies showed that hepatic steatosis, fibrosis, inflammation, and necrosis significantly decreased in the groups that received bilirubin compared to the control animals. Bilirubin also caused significant alterations in the expression levels of the Atgs, as well as the Beclin- 1 protein. Bilirubin may have potential ameliorative effects on NAFLD-associated liver damage. Moreover, the beneficial effects of bilirubin on intrahepatic lipid accumulation and steatosis were comparable with the group that did not ever receive bilirubin.

Expanding the Genetic Spectrum of AGXT Gene Variants in Egyptian Patients with Primary Hyperoxaluria Type I.

Introduction: Approximately 80% of primary hyperoxaluria cases are caused by primary hyperoxaluria type 1 (PH1, OMIM# 259900), which is characterized by pathogenic variants in the AGXT gene, resulting in deficiency of the liver-specific enzyme alanine-glyoxylate aminotransferase (AGT). This leads to increased production of oxalate, which cannot be effectively eliminated from the body, resulting in its accumulation primarily in the kidneys and other organs. Subjects and Methods: This study included 17 PH1 Egyptian patients from 12 unrelated families, recruited from the Inherited Kidney Disease Outpatient Clinic and the Dialysis Units, Cairo University Hospitals, during the period from January 2018 to December 2019, aiming to identify the pathogenic variants in the AGXT gene. Results: Six different variants were detected. These included three frameshift and three missense variants, all found in homozygosity within the respective families. The most common variant was c.121G>A;p.(Gly41Arg) detected in four families, followed by c.725dup;p.(Asp243GlyfsTer12) in three families, c.33dup;p.(Lys12Glnfs156) in two families, and c.731T >C;p.(Ile244Thr), c.33delC;p.(Lys12Argfs34), and c.568G>A;p.(Gly190Arg) detected in one family each. Conclusion: Consanguineous Egyptian families with history of renal stones or renal disease suspicious of primary hyperoxaluria should undergo AGXT genetic sequencing, specifically targeting exons 1 and 7, as variants in these two exons account for >75% of disease-causing variants in Egyptian patients with confirmed PH1.

Compromised very-low density lipoprotein induced polyunsaturated triglyceride accumulation in N-nitrosodiethylamine-induced hepatic steatosis

N-Nitrosodiethylamine (NDEA), a carcinogen in some foods and medications, is linked to liver damage similar to non-alcoholic fatty liver disease (NAFLD). This study explores how NDEA disrupts liver lipid metabolism. Sprague-Dawley rats were given two doses of NDEA (100 mg/kg) orally, 24 h apart. Liver response was assessed through tissue staining, blood tests, and biochemical markers, including fatty acids, lipid peroxidation, and serum very-low density lipoprotein (VLDL) levels. Additionally, lipidomic analysis of liver tissues and serum was performed. The results indicated significant hepatic steatosis (fat accumulation in the liver) following NDEA exposure. Blood analysis showed signs of inflammation and liver damage. Biochemical tests revealed decreased liver protein synthesis and specific enzyme alterations, suggesting liver cell injury but maintaining mitochondrial function. Increased fatty acid levels without a rise in lipid peroxidation were observed, indicating fat accumulation. Lipidomic analysis showed increased polyunsaturated triglycerides in the liver and decreased serum VLDL, implicating impaired VLDL transport in liver dysfunction. In conclusion, NDEA exposure disrupts liver lipid metabolism, primarily through the accumulation of polyunsaturated triglycerides and impaired fat transport. These findings provide insight into the mechanisms of NDEA-induced liver injury and its progression to hepatic steatosis.

The assessment of liver function test and fertility hormones in Saudi athletes using anabolic androgenic steroids

BackgroundA growing number of athletes are using synthetic anabolic–androgenic steroids (AAS), comprised of testosterone and other derivatives, to enhance athletic performance and muscle mass. Over the years, numerous reports elucidated the side effects of the illegal use of AAS, such as infertility, and liver disorders. The effect of AAS on the hepatic and reproductive systems in Saudi athletes has not yet been studied. Therefore, this study examined the liver function and sex hormone parameters of AAS users as compared to non-users. MethodsFasting blood samples were collected from 16 male Saudi athletes, 10 AAS-users (cases) and 6 non-users (controls) to measure liver function tests (ALT, AST, GGT, ALP, total protein, albumin, direct and total bilirubin) and muscle enzymes (CK, LDH), Fertility hormones (LH, FSH, total testosterone, estradiol, and prolactin) were included also. Furthermore, a self-reported questionnaire was obtained to identify the type of AAS used, the dosage, and the length of the course before sample collection. ResultsThe results show a statistically significant increase in ALT (P < 0.001), AST (P < 0.001), CK (P < 0.05), and a significant decrease (P < 0.05) in albumin (P < 0.001) and total bilirubin levels (P < 0.01) in AAS-users. Total testosterone increased significantly among AAS (P < 0.05), along with a significant decrease in LH (P < 0.01), and FSH (P < 0.001) levels, while serum prolactin and estradiol levels were significantly increased (P < 0.05). ConclusionAAS can enhance physical performance and appearance, its potential adverse effects on the hepatic and reproductive systems necessitate careful consideration. Our research demonstrates an increase in the liver-specific enzyme ALT in AAS users relative to non-users and the possibility that short-term AAS usage increases the risk of liver injury.

LEAP2 is Associated with Cardiometabolic Markers, but is Unchanged by Antidiabetic Treatment in People with Prediabetes.

Aims To examine whether fasting plasma liver-expressed antimicrobial peptide 2 (FP-LEAP2) is associated with markers of cardiometabolic disease susceptibility in a cohort with prediabetes and overweight/obesity and whether antidiabetic interventions affect FP-LEAP2 levels. Materials and methods The analysis included 115 individuals with prediabetes (hemoglobin A1c (HbA1c) 39-47 mmol/mol, 5.7-6.4%) and overweight/obesity (BMI ≥ 25 kg/m2) from a randomized controlled trial. Changes in FP-LEAP2 levels were assessed in relation to treatment with dapagliflozin (10 mg once daily), metformin (1700 mg daily) or interval-based exercise (5 days/week, 30 min/session) compared to control (habitual lifestyle) after 6 and 13 weeks of treatment. Results FP-LEAP2 levels were associated with (standardized beta-coefficient (95%-CI)): BMI 0.22 (0.03:0.41), p=0.027; body weight 0.27 (0.06:0.48), p=0.013; fat mass 0.2 (0.00:0.4), p=0.048; lean mass 0.47 (0.13:0.8), p=0.008; HbA1c 0.35 (0.17:0.53), p<0.001; fasting plasma glucose (FPG) 0.32 (0.12:0.51), p=0.001; fasting serum insulin 0.28 (0.09:0.47), p=0.005; insulin sensitivity -0.22 (-0.41:-0.03), p=0.022; total cholesterol 0.19 (0.01:0.38), p=0.043; triglycerides: 0.31 (0.13:0.5), p<0.001; transaminases and Fatty Liver Index (standardized beta-coefficients 0.23-0.32), all p<0.020; and eGFR -0.34 (-0.56:-0.12), p=0.003. FP-LEAP2 levels were not associated with fat distribution or body fat percentage, fasting glucagon, post-load glucose, beta cell function or low-density lipoprotein. The interventions were not associated with changes in FP-LEAP2. Conclusion FP-LEAP2 is associated with body mass, impaired insulin sensitivity, liver specific enzymes and kidney function. The findings highlight the importance of LEAP2 in obesity, type 2 diabetes, and non-alcoholic fatty liver disease. FP-LEAP2 was not affected by metformin, dapaglifloxin, or exercise in this population.

The effect of the chelated form of trace elements in diet on weight gain, production traits, egg specific gravity, immune system, blood parameters, liver enzymes, and progesterone hormone in Ross 308 broiler breeder chickens

This experiment was conducted in order to investigate the effect of the chelated form of trace elements in diet on the production traits, immunity, and blood parameters of Ross 308 broiler breeder chickens. Treatments included Treatment 1 (Negative control): Mn, Se, Cu, Fe and Zn provided as non-chelated form; Treatment 2 (Positive control): Mn, Se, Cu, Fe and Zn provided as chelated form; Treatment 3: Mn, Se, Cu, Fe and Zn provided as 50% chelated form and 50% non-chelated form; Treatment 4: Zn provided as chelated form; Treatment 5: Cu provided as chelated form; Treatment 6: Fe provided as chelated form; Treatment 7: Mn provided as chelated form; Treatment 8: Se provided as chelated form. The results showed that over the entire production period egg production and egg mass were affected by the experimental treatments (p < 0.05) and that the highest levels were observed in treatments 2 and 3. The chelated form of trace elements had a significant effect, increasing the number of settable eggs and decreasing the unsettable eggs over the entire production period (p < 0.05). Average egg weight over the entire period was not significantly changed (p > 0.05). The highest percentage of 2-yolk, thin shell, broken and small eggs was observed in 61st week of age significantly (p < 0.05). The experimental treatments had a significant effect on the immunity and the antibody titre against Newcastle disease showed a significant increase in treatments 2 and 8 (p < 0.05). The concentration of glucose, triglyceride, progesterone, AST and ALT were affected by the experimental treatments (p < 0.05). In conclusion, the use of a basic diet containing Zn, Fe, Cu, Se and Mn in the chelated form or the use of a basic diet containing mineral elements of Zn, Fe, Cu, Se and Mn with 50% non-chelated form and 50% in the chelated form improves the performance of production traits, immunity and blood parameters of Ross 308 broiler breeder chickens. Highlights Chelated form of trace elements in the diet led to improvement in productive traits, immune response and blood parameters compared to the mineral form Basic diet containing mineral elements of Zn, Fe, Cu, Se, and Mn, 50% in the non-chelated form and 50% in the chelated form led to a significant improvement in egg production, egg mass, egg weight and egg specific gravity Chelated form of trace elements in the diet led to a decrease in the number of abnormal eggs including double-yolk, thin shell, broken, deformed, and small eggs

Abstract 684: Hepatic Oxalate Overproduction As A Driver And Therapeutic Target In NASH And Atherosclerosis

Affecting one third of the global population, with no pharmacotherapy available, nonalcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease. Surprisingly, the major cause of death in patients with NAFLD, particularly in those with the more severe nonalcoholic steatohepatitis (NASH), is atherosclerotic cardiovascular disease (CVD). Thus, there is a critical need to identify targetable pathways for concurrent treatment of NASH and atherosclerotic CVD, which has been hampered by limited understanding of the pathophysiology and metabolic pathways linking these two diseases. Recently, we and others uncovered oxalate metabolism commonly dysregulated in NAFLD and CVD. Using unbiased transcriptomics, we identified suppression of genes that limit oxalate production in livers from humans and mice with NASH. Alanine-glyoxylate aminotransferase (AGXT), a liver-specific enzyme that detoxifies glyoxylate, the oxalate precursor, was reduced and oxalate was markedly increased in correlation with NASH severity. Remarkably, oxalate was also increased both in patients and mice with atherosclerosis. In our mouse models of hepatic oxalate overproduction ( Agxt -/- and Agxt -/- / Apoe -/- ), both NASH and atherosclerosis were increased with suppressed hepatic fatty acid β-oxidation (FAO) and induction of proinflammatory pathways. In hepatocytes, oxalate induced mitochondrial dysfunction and lipid accumulation while downregulating peroxisome proliferator-activated receptor α (PPARα), inhibiting FAO and upregulating C-C motif chemokine ligand 5 (CCL5). Importantly, limiting hepatic oxalate production via hepatocyte-specific overexpression of AGXT (AAV8-TBG-AGXT) reduced NASH and atherosclerosis in mice by stimulating FAO and attenuating proinflammatory responses. Together, our findings uncover hepatic oxalate overproduction as a dysregulated metabolic pathway linking NASH and atherosclerotic CVD, highlighting the potential of oxalate reduction for concurrent treatment of these two prominent diseases.

Target-specificity of different amyrin subunits in impeding HCV influx mechanism inside the human cells considering the quantum tunnel profiles and molecular strings of the CD81 receptor: a combined in silico and in vivo study.

HCV is a hepatotropic RNA virus recognized for its frequent virulence and fatality worldwide. Despite many vaccine development programs underway, researchers are on a quest for natural bioactive compounds due to their multivalent efficiencies against viral infections, considering which the current research aimed to figure out the target-specificity and therapeutic potentiality of α, β, and δ subunits of amyrin, as novel bioactive components against the HCV influx mechanism. Initially, the novelty of amyrin subunits was conducted from 203 pharmacophores, comparing their in-silico pharmacokinetic and pharmacodynamic profiles. Besides, the best active site of CD81 was determined following the quantum tunneling algorithm. The molecular dynamic simulation was conducted (100ns) following the molecular docking steps to reveal the parameters- RMSD (Å); Cα; RMSF (Å); MolSA (Å2); Rg (nm); PSA (Å); SASA (Å2), and the MM-GBSA dG binding scores. Besides, molecular strings of CD81, along with the co-expressed genes, were classified, as responsible for encoding CD81-mediated protein clusters during HCV infection, resulting in the potentiality of amyrins as targeted prophylactics in HCV infection. Finally, in vivo profiling of the oxidative stress marker, liver-specific enzymes, and antioxidant markers was conducted in the DMN-induced mice model, where β-amyrin scored the most significant values in all aspects.

Glycolate oxidase inhibition by lumasiran varies between patients with primary hyperoxaluria type 1

Glycolate oxidase inhibition by lumasiran varies between patients with primary hyperoxaluria type 1

A Novel Variant in the LIPA Gene Associated with Distinct Phenotype.

Deficiency of lysosomal acid lipase (LAL-D) is caused by biallelic pathogenic variants in the LIPA gene. Spectrum of LAL-D ranges from early onset of hepatosplenomegaly and psychomotor regression (Wolman disease) to a more chronic course (cholesteryl ester storage disease - CESD). The diagnosis is based on lipid and biomarker profiles, specific liver histopathology, enzyme deficiency, and identification of causative genetic variants. Biomarker findings are a useful for diagnostics of LAL-D, including high plasma concentration of chitotriosidase as well as elevated oxysterols. Current treatment options include enzyme replacement therapy (sebelipase-alpha), statins, liver transplantation, and stem cell transplantation. We present two pairs of siblings from Serbia with a distinctive phenotype resembling LAL-D with a novel variant of unknown significance (VUS) detected in the LIPA gene and residual LAL activity. All patients presented with hepatosplenomegaly at early childhood. In siblings from family 1, compound heterozygosity for a pathogenic c.419G>A (p.Trp140Ter) variant and a novel VUS c.851C>T (p.Ser284Phe) was detected. Patients from family 2 were homozygous for c.851C>T VUS and both have typical histopathologic findings for LAL-D in the liver. Enzyme activity of LAL was tested in three patients and reported as sufficient, and therefore enzyme replacement therapy could not be approved. When confronted with a challenge of diagnosing an inherited metabolic disorder, several aspects are taken into consideration: clinical manifestations, specific biomarkers, enzyme assay results, and molecular genetic findings. This report brings cases to light which have a considerable discrepancy between those aspects, namely the preserved LAL enzyme activity in presence of clinical manifestations and rare variants in the LIPA gene.

Construction of a spheroid array culture system on a suspended permeable hydrogel membrane scaffold for improving the expression of a liver-specific drug-metabolizing enzyme of HepG2 cells.

Herein, we constructed a spheroid array culture system on a flexible hydrogel membrane suspended in the culture medium. When we applied this culture system to HepG2 cells, the results suggested that an aerobic culture environment was implemented, and the gene expression of a liver-specific drug-metabolizing enzyme was improved in comparison with that of the conventional immobilized monolayer culture.

Sinapic acid attenuates cyclophosphamide-induced liver toxicity in mice by modulating oxidative stress, NF-κB, and caspase-3.

Cyclophosphamide (CP) as an antineoplastic drug is widely used in cancer patients, and liver toxicity is one of its complications. Sinapic acid (SA) as a natural phenylpropanoid has anti-oxidant, anti-inflammatory, and anti-cancer properties. The purpose of the current study was to determine the protective effect of SA versus CP-induced liver toxicity. In this research, BALB/c mice were treated with SA (5 and 10 mg/kg) orally for one week, and CP (200 mg/kg) was injected on day 3 of the study. Oxidative stress markers, serum liver-specific enzymes, histopathological features, caspase-3, and nuclear factor kappa-B cells were then checked. CP induced hepatotoxicity in mice and showed structural changes in liver tissue. CP significantly increased liver enzymes and lipid peroxidation, and decreased glutathione. The immunoreactivity of caspase-3 and nuclear factor kappa-B cells was significantly increased. Administration of SA significantly maintained histochemical parameters and liver function enzymes in mice treated with CP. Immunohistochemical examination showed SA reduced apoptosis and inflammation. The data confirmed that SA with anti-apoptotic, anti-oxidative, and anti-inflammatory activities was able to preserve CP-induced liver injury in mice.

Chronic Graft Versus Host Disease Detected By Tissue-Specific Cell-Free DNA Methylation Biomarkers

Accurate detection of graft versus host disease (GVHD) is a major challenge in the management of patients that undergo hematopoietic stem cell transplantation (HCT). Current diagnosis of chronic GVHD (cGVHD) in bone marrow transplant patients is based on inaccurate, operator-dependent clinical markers, and less often on biopsies. These methods are time consuming, costly, invasive and yield late-stage diagnoses that negatively affect morbidity and mortality. Here we demonstrate the use of circulating cell-free DNA (cfDNA) for detection of chronic GVHD and damage to specific organs. We established a cocktail of tissue-specific DNA methylation markers, and used it to determine the concentration of cfDNA molecules derived from the liver, skin, lungs, colon and specific immune cells in 203 samples from 82 HCT patients(Fig 1). Patients with active cGVHD show elevated concentration of cfDNA, as well as tissue-specific and immune cell driven methylation markers that agree with the 2014 National Institute of Health (NIH) clinical scores. Interestingly transplanted patients with no clinical symptoms show abnormally high levels of cfDNA and tissue-specific markers, suggesting hidden tissue turnover even in the absence of evident pathology. We used machine learning to create a model, which can aid predicting the likelihood that a patient has cGVHD. We compared XG boost and random forest classifiers on our data set. Algorithms were tested on data that included pre-selected specific measures (cfDNA of skin, lung, liver, GI, neutrophils, monocyte, eosinophil, T lymphocytes, CD8+ cells, Treg cells and B lymphocytes, as well as white blood cell (WBC) counts, BIL, AST, ALT and ALP liver enzyme levels). The data was randomly divided to a training set consisting of 67% of the samples and a testing set consisting of the remaining 33%. The model was constructed based on the training set alone that was later verified on the testing set. As the XG boost model had both higher accuracy as well as more robust results by cross-validation, we applied XG boost for further analyses. A total of 179 samples were divided between the training and test set. One hundred nineteen samples were randomly chosen for training the machine learning classifier, while 60 samples were used solely for testing the model. Each sample was intended either for the training or test sets. The integrative model was able to correctly identify GVHD with a sensitivity of 91.49% and precision of 89%% (AUC of 0.9, Fig 2). Our study shows that tissue-specific DNA methylation patterns can serve as plasma biomarkers for detection of tissue damage in cGVHD. To our knowledge, this is the first report of potential tissue-specific cfDNA utility in the context of chronic GVHD. Larger cohorts will be needed to assess the prognostic potential of methylation-based biomarkers. Tissue-specific cfDNA markers can open a window into uncovering underlying tissue dynamics in patients who underwent allogeneic stem cell transplantation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intrabiliary infusion of naked DNA vectors targets periportal hepatocytes in mice

Hydrodynamic tail vein injection (HTV) is the "gold standard" for delivering naked DNA vectors to mouse liver, thereby transfecting predominately perivenous hepatocytes. While HTV corrects metabolic liver defects such as phenylketonuria or cystathionine β-synthase deficiency, correction of spf ash mice with ornithine transcarbamylase (OTC) deficiency was not possible despite overexpression in the liver, as the OTC enzyme is primarily expressed in periportal hepatocytes. To target periportal hepatocytes, we established hydrodynamic retrograde intrabiliary injection (HRII) in mice and optimized minicircle (MC) vector delivery using luciferase as a marker gene. HRII resulted in a transfection efficiency below 1%, 100-fold lower than HTV. While HRII induced minimal liver toxicity compared with HTV, overexpression of luciferase by both methods, but not of a natural liver-specific enzyme, elicited an immune response that led to the elimination of luciferase expression. Further testing of MC vectors delivered via HRII in spf ash mice did not result in sufficient therapeutic efficacy and needs further optimization and/or selection of the corrected cells. This study reveals that luciferase expression is toxic for the liver. Furthermore, physical delivery of MC vectors via the bile duct has thepotential to treat defects restricted to periportal hepatocytes, which opens new doors for non-viral liver-directed gene therapy.

LiMAx Prior to Radioembolization for Hepatocellular Carcinoma as an Additional Tool for Patient Selection in Patients with Liver Cirrhosis.

Simple SummaryRadioembolization is a well-established therapeutic option for patients with advanced hepatocellular carcinoma. However, patients with advanced tumor disease and presence of liver cirrhosis often present a borderline liver function and are at risk for post-interventional hepatic decompensation. The aim of our study was to evaluate the ability of the LiMAx®, a non-invasive test for liver function assessment, in predicting post-therapeutic hepatic deterioration and thus improve patient selection prior radioembolization.Background and Aims: Radioembolization (RE) has recently demonstrated a non-inferior survival outcome compared to systemic therapy for advanced hepatocellular carcinoma (HCC). Therefore, current guidelines recommend RE for patients with advanced HCC and preserved liver function who are unsuitable for transarterial chemoembolization (TACE) or systemic therapy. However, despite the excellent safety profile of RE, post-therapeutic hepatic decompensation remains a serious complication that is difficult to predicted by standard laboratory liver function parameters or imaging modalities. LiMAx® is a non-invasive test for liver function assessment, measuring the maximum metabolic capacity for 13C-Methacetin by the liver-specific enzyme CYP 450 1A2. Our study investigates the potential of LiMAx® for predicting post-interventional decompensation of liver function. Patients and methods: In total, 50 patients with HCC with or without liver cirrhosis and not amenable to TACE or systemic treatments were included in the study. For patients prospectively enrolled in our study, LiMAx® was carried out one day before RE (baseline) and 28 and 90 days after RE. Established liver function parameters were assessed at baseline, day 28, and day 90 after RE. The relationship between baseline LiMAx® and pre-and post-interventional liver function parameters, as well as the ability of LiMAx® to predict hepatic decompensation, were analyzed. Results: We observed a strong association between baseline LiMAx® and bilirubin, albumin, ALBI grade, and MELD score. Patients presenting with Child–Pugh score B 28 days after RE or with a deterioration in Child–Pugh score by at least one point had a significantly lower baseline LiMAx® compared to those with Child–Pugh score A or with stable Child–Pugh score. The ability of LiMAx® to predict hepatic decompensation after RE was determined using ROC curve analysis and was compared to MELD score and ALBI grade. LiMAx® achieved a substantial AUC of 0.8117, comparable to MELD score and ALBI grade. Conclusion: Patients with lower LiMAx® values at baseline have a significantly increased risk for hepatic decompensation after RE, despite being categorized as Child–Pugh A. Therefore, LiMAx® can be used as an additional tool to identify patients at high risk of post-interventional hepatic failure.

Anti-apoptotic properties of N-Acetyl cysteine and its effects on of Liver X receptor and Sirtuin 1 expression in the liver of rats exposed to Lead

BackgroundThis study aimed to evaluate the expression of Liver X receptor (Lxr), Sirtuin 1 (Sirt1), apoptotic-related genes, and the protective role of N-acetylcysteine (NAC) in the liver of rats treated with Lead (Pb). MethodsRats were randomly divided into 5 groups, including G1 (control), G2 (single dose of Pb), G3 (continuous dose of Pb), G4 (single dose of Pb + NAC), and G5 (continuous dose of Pb + NAC). Lipid profiles and liver specific enzymes were assessed. Expression of Lxr, Sirt1, Bax and Caspase-3 genes was considered using RT-PCR. ResultsExposure to Pb caused a significant accumulation of Pb in the blood and liver tissue, increase in serum AST, ALT and ALP enzymes, as well as lipid profiles. Chronic exposure to Pb caused a significant decrease in Lxr (3.15-fold; p < 0.001) and Sirt1 (2.78-fold; p = 0.009), but significant increase in expression of Bax (4.49-fold; p < 0.001) and Caspase-3 (4.10-fold; p < 0.001) genes when compared to the control. Combined therapy with Pb + NAC in rats caused a significant decrease in AST, ALT and ALP values (28.93%, 20.80% and 28.86%, respectively) in the blood as compared to rats treated with Pb alone. Co-treated with Pb + NAC significantly increased the expression of Lxr (1.72-fold; p = 0.043) and Sirt1 (2.45-fold; p = 0.008), but decreased the expression of Bax (1.96-fold; p = 0.03) and Caspase 3 (2.22-fold; p = 0.029) genes when compared to rats treated with Pb alone. ConclusionChronic exposure to Pb is strongly associated with accumulation of Pb in the blood and liver, hepatic cells apoptosis, down-expression of Lxr and Sirt1 genes and consequently liver injury and abnormal lipid profiles. NAC reversed the Pb-induced toxicity on the liver tissue.

HAO1 negatively regulates liver macrophage activation via the NF-κB pathway in alcohol-associated liver disease

Inflammation is a key factor contributing to the progression of alcohol-associated liver disease (ALD). Accumulating data have shown that ethyl alcohol (EtOH) induced liver macrophages activation along with an inflammatory response that contributes to the development of ALD. The liver-specific peroxisomal enzyme hydroxyacid oxidase 1 (HAO1) has been found to be associated with chronic liver disease. But the role of HAO1 remains unknown in ALD. In our study, HAO1 was found to be decreased in ALD patients and EtOH-fed mice. Interestingly, HAO1 expression was reduced in primary hepatocytes, whereas HAO1 was elevated in peripheral blood monocytes from ALD patients and EtOH-fed mice liver macrophages as well as LPS-treated RAW264.7 cells. Moreover, HAO1 knockdown exacerbated the inflammatory response, while HAO1 overexpression inhibited inflammation in LPS-stimulated RAW264.7 cells. Additionally, overexpression or silencing of HAO1 in vitro significantly affected NF-κB signaling pathway. Collectively, the results revealed a key role of HAO1-mediated macrophage activation and may provide a potential target for treating ALD.