Transcriptomic analysis reveals a novel regulatory factor of ECHDC1 involved in lipid metabolism of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the highest incidence of chronic liver disease worldwide characterized by lipid accumulation in the liver. The full understanding of the lipogenesis of NAFLD is extreme importance. Here, whole-genome transcriptome analysis was performed on liver tissues of NAFLD patients and healthy controls to identify the differentially expressed genes and find new pathways and target genes related to the lipogenesis of NAFLD. Combined with the Gene Expression Omnibus (GEO) database, we found 86 overlapping genes, many of which are related to lipid metabolism of NAFLD. ECHDC1 is one of 86 overlapping genes, and its role in NAFLD has not been reported. The expression of ECHDC1 was significantly increased in liver tissue of patients with NAFLD than that of healthy controls, and oil Red O intensity was positively correlated with the expression levels of ECHDC1. Inhibition of ECHDC1 expression in HepG2 cells by RNAi significantly reduced intracellular lipid droplet number in vitro. In summary, this study analyzed pathogenic factors related to NAFLD at the whole-genome level and demonstrated that ECHDC1 may be involved in the occurrence and development of NAFLD by regulating hepatic lipid metabolism.

Cell-free DNA methylation markers for differential diagnosis of hepatocellular carcinoma.

BACKGROUND: Aberrant DNA methylation may offer opportunities in revolutionizing cancer screening and diagnosis. We sought to identify a non-invasive DNA methylation-based screening approach using cell-free DNA (cfDNA) for early detection of hepatocellular carcinoma (HCC). METHODS: Differentially, DNA methylation blocks were determined by comparing methylation profiles of biopsy-proven HCC, liver cirrhosis, and normal tissue samples with high throughput DNA bisulfite sequencing. A multi-layer HCC screening model was subsequently constructed based on tissue-derived differentially methylated blocks (DMBs). This model was tested in a cohort consisting of 120 HCC, 92 liver cirrhotic, and 290 healthy plasma samples including 65 hepatitis B surface antigen-seropositive (HBsAg+) samples, independently validated in a cohort consisting of 67 HCC, 111 liver cirrhotic, and 242 healthy plasma samples including 56 HBsAg+ samples. RESULTS: Based on methylation profiling of tissue samples, 2321 DMBs were identified, which were subsequently used to construct a cfDNA-based HCC screening model, achieved a sensitivity of 86% and specificity of 98% in the training cohort and a sensitivity of 84% and specificity of 96% in the independent validation cohort. This model obtained a sensitivity of 76% in 37 early-stage HCC (Barcelona clinical liver cancer [BCLC] stage 0-A) patients. The screening model can effectively discriminate HCC patients from non-HCC controls, including liver cirrhotic patients, asymptomatic HBsAg+ and healthy individuals, achieving an AUC of 0.957(95% CI 0.939-0.975), whereas serum α-fetoprotein (AFP) only achieved an AUC of 0.803 (95% CI 0.758-0.847). Besides detecting patients with early-stage HCC from non-HCC controls, this model showed high capacity for distinguishing early-stage HCC from a high risk population (AUC=0.934; 95% CI 0.905-0.963), also significantly outperforming AFP. Furthermore, our model also showed superior performance in distinguishing HCC with normal AFP (< 20ng ml-1) from high risk population (AUC=0.93; 95% CI 0.892-0.969). CONCLUSIONS: We have developed a sensitive blood-based non-invasive HCC screening model which can effectively distinguish early-stage HCC patients from high risk population and demonstrated its performance through an independent validation cohort. TRIAL REGISTRATION: The study was approved by the ethic committee of The Second Xiangya Hospital of Central South University (KYLL2018072) and Chongqing University Cancer Hospital (2019167). The study is registered at ClinicalTrials.gov(# NCT04383353 ).

Identification of CYP46A1 as a new regulator of lipid metabolism through CRISPR-based whole-genome screening.

Abnormal lipid droplet (LD) metabolism causes a variety of disorders, especially to nonalcoholic fatty liver disease (NAFLD). But the mechanism of abnormal aggregation of LD is still not fully elucidated. Here, Genome-wide CRISPR-Cas9 knockout (GeCKO) screening was employed to identify candidate genes regulating LD metabolism in L02 cell. We analyzed simultaneously the transcriptomics of liver tissues of NAFLD to find potential genes involved in pathogenesis of NAFLD. After integration these data, we found that the expression of 43 candidate genes from the GeCKO screening was also decreased in tissues of NAFLD patients. Many of these 43 overlapping genes have been reported to play an important role in the formation of LD. Subsequently, we focused on CYP46A1, one of 43 candidate genes and mitochondria-related genes. We confirmed that the protein expression of CYP46A1 is deceased in tissues of NAFLD patients. Downregulation or overexpression of CYP46A1 affected LD accumulation in vitro. Deficiency of CYP46A1 impaired mitochondrial morphology and function, which may be responsible for the accumulation of LD. In summary, this study explored regulatory factors of LD accumulation at the whole-genome level, and demonstrated that CYP46A1 regulated LD formation involving in NAFLD pathogenesis. It provides new clues for studying the molecular mechanisms of diseases related to abnormal lipid metabolism.

Prognostic factors influencing outcome in adult liver transplantation using hypernatremic organ donation after brain death.

BACKGROUND: Hypernatremic donors was regarded as the expanded criteria donors in liver transplantation. The study was to investigate the effects of donor hypernatremia on the outcomes of liver transplantation and identify the prognostic factors possibly contributing to the poor outcomes. METHODS: Donor serum sodium levels before procurement were categorized as normal sodium (< 155 mmol/L), moderate high sodium (155-170 mmol/L), and severe high sodium (≥ 170 mmol/L). Furthermore, we subdivided the 142 hypernatremic donors (≥ 155 mmol/L) into two subgroups: subgroup A, the exposure time of liver grafts from hypernatremia to reperfusion was < 36 h; and subgroup B, the exposure time was ≥ 36 h. The outcomes included initial graft function, survival rates of grafts and recipients, graft loss and early events within the first year following liver transplantation. RESULTS: There were no significant differences in the 1-year survival rates of grafts and recipients, 1-year graft loss rates and early events among the normal, moderate high and severe high sodium groups. However, the overall survival rates of grafts and recipients in subgroup A were significantly higher than those in subgroup B. Cox model showed that the exposure time (HR = 1.117; 95% CI: 1.053-1.186; P < 0.001), cold ischemia time (HR = 1.015; 95% CI: 1.006-1.024; P = 0.001) and MELD (HR = 1.061; 95% CI: 1.003-1.121; P = 0.037) were the important prognostic factors contributing to the poor outcomes of recipients with hypernatremic donors. CONCLUSIONS: The level of donor sodium immediately before organ procurement does not have negative effects on the early outcomes following adult liver transplantation. For hypernatremia liver donors, minimization of the exposure time from hypernatremia to reperfusion is critical to prevent graft loss.

MicroRNA-146a-5p enhances radiosensitivity in hepatocellular carcinoma through replication protein A3-induced activation of the DNA repair pathway.

Hepatocellular carcinoma (HCC) is known for its high mortality rate worldwide. Based on intensive studies, microRNA (miRNA) expression functions in tumor suppression. Therefore, we aimed to evaluate the contribution of miR-146a-5p to radiosensitivity in HCC through the activation of the DNA damage repair pathway by binding to replication protein A3 (RPA3). First, the limma package of R was performed to differentially analyze HCC expression chip, and regulative miRNA of RPA3 was predicted. Expression of miR-146a-5p, RPA3, and DNA damage repair pathway-related factors in tissues and cells was determined. The effects of radiotherapy on the expression of miR-146a-5p and RPA3 as well as on cell radiosensitivity, proliferation, cell cycle, and apoptosis were also assessed. The results showed that there exists a close correlation between miR-146a and the radiotherapy effect on HCC progression through regulation of RPA3 and the DNA repair pathway. The positive rate of ATM, pCHK2, and Rad51 in HCC tissues was higher when compared with that of the paracancerous tissues. SMMC-7721 and HepG2 cell proliferation were significantly inhibited following 8 Gy 6Mv dose. MiR-146a-5p restrained the expression of RPA3 and promoted the expression of relative genes associated with the DNA repair pathway. In addition, miR-146a-5p overexpression suppresses cell proliferation and enhances radiosensitivity and cell apoptosis in HCC cells. In conclusion, the present study revealed that miR-146a-5p could lead to the restriction of proliferation and the promotion of radiosensitivity and apoptosis in HCC cells through activation of DNA repair pathway and inhibition of RPA3.

Foxp3-modified bone marrow mesenchymal stem cells promotes liver allograft tolerance through the generation of regulatory T cells in rats.

BACKGROUND: The transcription factor forkhead box P3 (Foxp3) is a master regulatory gene necessary for the development and function of CD4(+)CD25(+) regulatory T cells (Tregs). Mesenchymal stem cells (MSC) have recently emerged as promising candidates for cell-based immunosuppression/tolerance induction protocols. Thus, we hypothesized that MSC-based Foxp3 gene therapy would improve immunosuppressive capacity of MSC and induce donor-specific allograft tolerance in rat's liver allograft model. METHODS: The present study utilized a lentivirus vector to overexpress the therapeutic gene Foxp3 on MSC. In vivo, Injections of 2 × 10(6) MSC, FUGW-MSC or Foxp3-MSC into the portal vein were carried out immediately after liver transplantation. RESULTS: Successful gene transfer of Foxp3 in MSC was achieved by lentivirus carrying Foxp3 and Foxp3-MSC engraftment in liver allograft was confirmed by fluorescence microscopy. Foxp3-MSC treatment significantly inhibited the proliferation of allogeneic ACI CD4(+) T cells to splenocytes (SC) from the same donor strain or third-party BN rat compared with MSC. Foxp3-MSC suppressive effect on the proliferation of CD4(+) T cells is contact dependent and associated with Programmed death ligand 1(PD-L1) upregulation in MSC. Co-culture of CD4(+) T cells with Foxp3-MSC results in a shift towards a Tregs phenotype. More importantly, Foxp3-MSC monotherapy achieved donor-specific liver allograft tolerance and generated a state of CD4(+)CD25(+)Foxp3(+) Tregs-dependent tolerance. CONCLUSION: Foxp3-engineered MSC therapy seems to be a promising and attractive cell therapy approach for inducing immunosuppression or transplant tolerance.

Splenic artery trunk embolization reduces the surgical risk of liver transplantation.

BACKGROUND: Portal hypertension is one of the most important clinical conditions that cause intraoperative intensive hemorrhage in cirrhotic patients undergoing liver transplantation. Pre-transplant portal decompression may reduce the intraoperative bleeding during liver transplantation. METHODS: Splenic artery trunk embolization (SATE) was performed one month prior to liver transplantation. Platelet count, prealbumin, international normalized ratio, and blood flow in the portal vein and hepatic artery were monitored before and one month after SATE. The measurements above were collected on admission and before surgery in the non-SATE patients, who served as controls. We also recorded the intraoperative blood loss, operating time, required transfusion, post-transplant ascites, and complications within three months after operation in all patients. RESULTS: SATE significantly reduced portal venous blood flow, increased hepatic arterial blood flow, normalized platelet count, and improved prealbumin and international normalized ratio in the patients before liver transplantation. Compared to the non-SATE patients, the pre-transplant SATE significantly decreased the operating time, intraoperative bleeding, post-transplant ascites and severe surgical complications. CONCLUSION: Pre-transplant SATE decreases portal pressure, improves liver function reserve, and reduces the surgical risk of liver transplantation effectively in patients with severe portal hypertension.

Unveiling the role of disulfidptosis-related genes in the pathogenesis of non-alcoholic fatty liver disease.

Backgrounds: Non-alcoholic fatty liver disease (NAFLD) presents as a common liver disease characterized by an indistinct pathogenesis. Disulfidptosis is a recently identified mode of cell death. This study aimed to investigate the potential role of disulfidptosis-related genes (DRGs) in the pathogenesis of NAFLD. Methods: Gene expression profiles were obtained from the bulk RNA dataset GSE126848 and the single-cell RNA dataset GSE136103, both associated with NAFLD. Our study assessed the expression of DRGs in NAFLD and normal tissues. Weighted gene co-expression network analysis (WGCNA) and differential expression analysis were employed to identify the key NAFLD-specific differentially expressed DRGs (DE-DRGs). To explore the biological functions and immune regulatory roles of these key DE-DRGs, we conducted immune infiltration analysis, functional enrichment analysis, consensus clustering analysis, and single-cell differential state analysis. Finally, we validated the expression and biological functions of DRGs in NAFLD patients using histology and RNA-sequencing transcriptomic assays with human liver tissue samples. Results: Through the intersection of WGCNA, differentially expressed genes, and DRGs, two key DE-DRGs (DSTN and MYL6) were identified. Immune infiltration analysis indicated a higher proportion of macrophages, T cells, and resting dendritic cells in NAFLD compared to control liver samples. Based on the key DE-DRGs, Two disulfidptosis clusters were defined in GSE126848. Cluster 1, with higher expression of the key DE-DRGs, exhibited increased immune infiltration abundance and was closely associated with oxidative stress and immune regulation compared to cluster 2. High-resolution analysis of mononuclear phagocytes highlighted the potential role of MYL6 in intrahepatic M1 phenotype Kupffer cells in NAFLD patients. Our transcriptome data revealed that the expression levels of the majority of DRGs were significantly increased in NAFLD patients. NAFLD patients exhibit elevated MYL6 correlating with inflammation, oxidative stress, and disease severity, offering promising diagnostic specificity. Conclusion: This comprehensive study provides evidence for the association between NAFLD and disulfidptosis, identifying potential target genes and pathways in NAFLD. The identification of MYL6 as a possible treatment target for NAFLD provided a novel understanding of the disease's development.

Alterations of axis inhibition protein 1 (AXIN1) in hepatitis B virus-related hepatocellular carcinoma and overexpression of AXIN1 induces apoptosis in hepatocellular cancer cells.

Axis inhibition protein 1 (AXIN1) is a negative regulator of Wnt/beta-catenin signaling via regulating the level of beta-catenin. However, the role of AXIN1 in the tumorigenesis and progression of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is less clear. PCR sequence analysis, immunohistochemistry, and Western blot were performed on 22 HBV-related HCC samples and corresponding nontumor liver tissues to detect variants in AXIN1 gene and the expression level of AXIN1. Human hepatoma cell lines SNU475 and SNU423 were transfected with pCDNA3.1-AXIN1-myc or AXIN1 G425S-myc mutant. The growth curve and apoptosis rate of cell lines, phosphorylation of beta-catenin, and cell cycle regulatory proteins depending on beta-catenin transcriptional activity were detected. We identified four mutations of AXIN1 in 22 primary HBV-related HCCs and demonstrated a lower expression of AXIN1 in HBV-related HCC tissues than that in paired adjacent nontumor tissues. Overexpression of AXIN1 wild-type but not AXIN1 mutant inhibited the growth of HCC cell lines, accelerated their apoptosis, and negatively regulated beta-catenin-dependent transcriptional activity. Our study revealed that alterations of AXIN1 were involved in HBV-related HCC. Overexpression of AXIN1 but not AXIN1 mutant negatively regulated beta-catenin-dependent transcriptional activity and downregulated the level of cell cycle regulatory proteins, suggesting that AXIN1 may be a potential target for gene therapy of primary HCC.

Pathogenic factors of cognitive dysfunction after liver transplantation: an observational study.

OBJECTIVES: Neurocognitive complications significantly reduce long-term health-related quality of life in patients undergoing liver transplantation; however, few studies have focused on their perioperative cognitive status. The authors designed a prospective observational study to determine the incidence and risk factors of posttransplant cognitive dysfunction. METHODS: This study included patients with end-stage liver disease who were on the liver transplantation waiting list. We performed an investigation with a neuropsychological battery before and 1 week after the successful transplant, analyzed the changes, and further explored the complicated perioperative factors that contribute to cognitive dysfunction. RESULTS: A total of 132 patients completed all the investigations. Compared with healthy controls and preoperative cognitive performance, 54 patients experienced deterioration, 50 patients remained unchanged, and 28 patients showed rapid improvement. Logistic regression analysis showed that age [odds ratio (OR) = 1.15, 95% confidence interval (CI, 1.07-1.22), P  < 0.001], the model for end-stage liver disease (MELD) score [OR = 1.07, 95% CI (1.03-1.13), P  = 0.038], systemic circulation pressure [OR = 0.95, 95% CI (0.91-0.99), P  = 0.026] within the first 30 min after portal vein opening, and total bilirubin concentration [OR = 1.02, 95% CI (1.01-1.03), P  = 0.036] on the seventh day post-transplant were closely related to the deterioration of cognitive function. CONCLUSION: The incidences of deterioration, maintenance, and improvement in cognitive function were 40.9%, 37.9%, and 21.2%, respectively. Increasing age, higher MELD score, lower perfusion pressure in the early stage of the new liver, and higher total bilirubin concentration postoperatively may be independent pathogenic factors.

Factors Prognostic of Survival in Liver Transplant Recipients with Hepatitis B Virus Related Acute-on-Chronic Liver Failure.

Objectives: Factors prognostic of survival in liver transplant (LT) recipients with hepatitis B virus related acute-on-chronic liver failure (HBV-ACLF) remain unclear. This study evaluated risk factors for survival in LT recipients with HBV-ACLF and determined the scoring system optimal for assessing patient prognosis. Methods: This retrospective study included 323 HBV-ACLF related patients undergoing LT, including 112, 146, and 65 patients with HBV-ACLF grades 1, 2, and 3, respectively. Overall survival (OS) was estimated by the Kaplan-Meier method, and factors associated with survival were analysed by multivariate Cox proportional hazards models. Pretransplant prognostic scoring systems were compared by receiver operating characteristic (ROC) curve analysis. Results: The one-year survival rate was significantly lower in HBV-ACLF grade 3 (80.0%) than in grades 1 (93.8%) and 2 (91.8%) recipients (p=0.0063). Cox multivariate analysis showed that age >53 years (hazard ratio (HR) 3.731; 95% confidence interval (CI) 1.640-8.407), WBC count >8.6 × 109/L (HR 4.544; 95% CI 1.140-18.107), HBV-ACLF 3 (HR 2.729; 95% CI 1.050-7.096), and cold ischaemia time >8.5 hours (HR 2.867; 95% CI, 1.38-5.921) were independently prognostic of 1-year survival. Comparisons of pretransplant scoring systems showed that chronic liver failure-consortium ACLF score (CLIF-C ACLFs) was superior to COSSH-ACLF, MELD-Na, and MELD scores in predicting 1-year OS in these patients. Conclusions: Age >53 years, WBC counts >8.6 × 109/L, HBV-ACLF grade 3, and cold ischaemia time >8.5 hours are independently prognostic of OS in LT recipients with HBV-ACLF. CLIF-C ACLFs is superior to other scoring methods in predicting 1-year OS in these patients.

Hemodynamic alterations with large spontaneous splenorenal shunt ligation during adult deceased donor liver transplantation.

Background: A large spontaneous splenorenal shunt (SRS) will greatly impact portal inflow to the graft during liver transplantation (LT). Direct ligation of a large SRS is an uncommon surgical procedure and the hemodynamic consequences of this procedure are unknown. Methods: In this retrospective study, we described our technique for direct ligation of a large SRS and the consequent hemodynamic changes during LT. 3-Dimensional computed tomography and Doppler ultrasonography were used to evaluate SRS and portal vein blood flow volume (PFV). Results: A total of 22 recipients had large SRS including 13 with PFV <85 ml/min/100 g (ligation group) and 9 with PFV ≥85 ml/min/100 g (no ligation group). The diameter of SRS was significantly larger in the ligation group than in the non-ligation group (22.92 ± 4.18 vs. 16.24 ± 3.60 mm; p = 0.0009). In all ligation patients, the SRS was easily identified and isolated, it was located just below the distal pancreas and beside the inferior mesenteric vein. PV flow increased significantly from 68.74 ± 8.77 to 116.80 ± 16.50 ml/min/100 g (p < 0.0001) after ligation; this was followed by a reduction in peak systolic velocity of the hepatic artery from 58.17 ± 14.87 to 46.67 ± 13.28 cm/s (p = 0.0013). Conclusions: Direct ligation of large SRS was an effective and safe surgical procedure to overcome the problem of portal hypoperfusion during LT.

PIAS1 Alleviates Hepatic Ischemia-Reperfusion Injury in Mice through a Mechanism Involving NFATc1 SUMOylation.

Recently, attentions have come to the alleviatory effect of protein inhibitor of activated STAT1 (PIAS1) in hepatic ischemia-reperfusion injury (HIRI), but the underlying molecular mechanistic actions remain largely unknown, which were illustrated in the present study. Microarray-based analysis predicted a possible regulatory mechanism involving the PIAS1/NFATc1/HDAC1/IRF-1/p38 MAPK signaling axis in HIRI. Then, growth dynamics of hypoxia/reoxygenation- (H/R-) exposed hepatocytes and liver injury of HIRI-like mice were delineated after the alteration of the PIAS1 expression. We validated that PIAS1 downregulation occurred in H/R-exposed hepatocytes and HIRI-like mice, while the expression of NFATc1, HDAC1, and IRF-1 and phosphorylation levels of p38 were increased. PIAS1 inactivated p38 MAPK signaling by inhibiting HDAC1-mediated IRF-1 through NFATc1 SUMOylation, thereby repressing the inflammatory response and apoptosis of hepatocytes in vitro, and alleviated liver injury in vivo. Collectively, the NFATc1/HDAC1/IRF-1/p38 MAPK signaling axis is highlighted as a promising therapeutic target for potentiating hepatoprotective effects of PIAS1 against HIRI.

IRX3 plays an important role in the pathogenesis of metabolic-associated fatty liver disease by regulating hepatic lipid metabolism.

Metabolic-associated fatty liver disease (MAFLD) affects approximately a quarter of the global population. Identification of the key genes and pathways involved in hepatic lipid metabolism is of the utmost importance for the diagnosis, treatment, and prevention of MAFLD. In this study, differentially expressed genes were identified through whole-genome transcriptional analysis of liver tissue from MAFLD patients and healthy controls, and a series of lipid metabolism-related molecules and pathways were obtained through pathway analysis. Subsequently, we focused on Iroquois homeobox protein 3 (IRX3), one of 13 transcription factors that were screened from the 331 differentially expressed genes. The transcription factor IRX3 was significantly decreased in the liver tissue of patients with MAFLD when compared with healthy controls. Pearson's correlation analysis showed that the expression levels of IRX3 in liver tissue were negatively correlated with serum total cholesterol, triglycerides, low-density lipoprotein cholesterol, and uric acid levels. The overexpression and interference of IRX3 induced the increased and decreased lipid droplet accumulation in vitro, respectively. Moreover, interference of IRX3 expression increased mitochondrial fragmentation and reduced the activity of the mitochondrial respiratory chain complex IV. In summary, the study demonstrated that IRX3 regulated hepatic lipid metabolism of MAFLD, and also revealed the effect of IRX3 on mitochondria might be an important mechanism by which IRX3 regulated hepatic lipid metabolism of MAFLD.

Risk Factors of Early Allograft Dysfunction in Patients With Hepatitis B Virus-Related Acute-on-Chronic Liver Failure After Deceased Donor Liver Transplant.

OBJECTIVES: Hepatitis B virus-related acute-on-chronic liver failure remains a life-threatening syndrome, and transplant is the definitive treatment. Early allograft dysfunction is a postoperative complication and affects morbidity and mortality. We studied the risk factors associated with early allograft dysfunction in livertransplantrecipients with hepatitis B virus-related acute-on-chronic liver failure. MATERIALS AND METHODS: This single-center retrospective study of early allograft dysfunction is based on data from January 2015 to June 2020 for 323 recipients with hepatitis B virus-related acute-on-chronic liver failure and 445 with only hepatitis B virus infection (control group). Data that correlated with early allograft dysfunction and outcome were analyzed. RESULTS: Incidence of early allograft dysfunction in patients with hepatitis B virus-related acute-on-chronic liver failure was significantly higher versus the control group (39.3% vs 21.1%; P < .001). Transplant recipients with hepatitis B virus-related acute-onchronic liver failure who developed early allograft dysfunction had lower 90-day, 180-day, and 360-day patient survival rates versus patients with no early allograft dysfunction (89.0% vs 98.0%, 82.7% vs 97.5%, and 80.3% vs 96.4%, respectively; P < .001). Pretransplant kidney failure (odds ratio, 2.644; 95% CI, 1.019-6.864; P = .046), pretransplant coagulation failure (odds ratio, 2.162; 95% CI, 1.291-3.621; P = .003), and operative time (odds ratio, 1.005; 95% CI, 1.002-1.008; P = .003) were independent risk factors for early allograft dysfunction in liver transplant recipients with hepatitis B virus-related acute-onchronic liver failure. There was a synergistic effect of early allograft dysfunction and preoperative kidney/coagulation failure on survival rates of liver transplant recipients with hepatitis B virus-related acute-on-chronic liver failure. CONCLUSIONS: Preoperative kidney/coagulation failure and operative time were independent risk factors of early allograft dysfunction in deceased donor liver transplant recipients with hepatitis B virus-related acute-on-chronic liver failure. The combination of early allograft dysfunction and preoperative kidney/coagulation failure was significantly associated with lower survival of these recipients.

[Effect of interleukin-17 and T helper cell 17 on acute heart allograft rejection in mice].

OBJECTIVE: To investigate the role of T helper cell 17(Th17) cells and their cytokines IL-17 in heart allograft rejection in mice. METHODS: The heart transplantation models were randomly divided into 2 groups: a allograft group (n=12) and an isograft group (n=12).On the post-operative day (POD) 3 and 7, serum IL-17 level was tested by enzyme-linked immunosorbent assay, and Th17 cells in heart grafts were measured by flow cytometry. The heart grafts were harvested and saved in 10% formalin, and then embedded in paraffin. RESULTS: Compared with the iso-graft group, the allograft group had a higher serum level of IL-17 on POD3 and POD7 (P<0.05), and the level of IL-17 was higher on POD7 than that on POD3 (P<0.05). The allograft group had more Th17 cells infiltrating in grafts on POD3 and POD7 (P<0.05) and there were more Th17 cells infiltrating on POD7 than that on POD3 (P<0.05). Histological examination showed that the prolongation of post transplantation time resulted in more rejection pathological changes. CONCLUSION: Th17 cells may play an important role in the development of heart transplant rejection. IL-17 may serve as a predictive parameter for allograft rejection in the future.

IL-17 in the early diagnosis of acute renal allograft rejection in mice.

OBJECTIVE: To investigate the expression of T helper (Th) 17 cells and the related interleukin 17 (IL-17) in acute renal allograft rejection in mice and its significance. METHODS: We established a mouse renal allograft model, in which mice were randomly divided into a renal isograft group and an acute renal allograft rejection group. Three and 7 d after the transplantation, the serum interferon (IFN)-γ and IL-17 levels in the mice were determined by enzyme-linked immunosorbent assay, the percentage of Th1 and Th17 cells in the total kidney-infiltrating lymphocytes was investigated by flow cytometry, and the transplanted kidney species were given routine pathological examination after fixation with 10% formalin. RESULTS: Compared with the isograft group, the allograft mice showed a significantly higher content of IL-17 (P<0.05) but not IFN-γ in the serum 3 d after transplantation, and showed significantly higher serum IL-17 and IFN-γ contents 7 d after transplantation (P<0.05). Also, compared with the isograft group, the allograft mice exhibited significantly higher percentage of Th1 and Th17 cells on both day 3 and day 7 (P<0.05). In the allograft group, the contents of serum IFN-γ and IL-17 and the percentage of Th1 and Th17 cells were significantly higher on day 7 than on day 3 (P<0.05). Routine pathological examination indicated that, as time passed, the allograft mice showed gradually stronger rejection responses. CONCLUSION: Th17 cells might play an important role in the development of acute renal allograft rejection, and IL-17 can be used as an early indicator of acute rejection.

Sequencing of Cell-Free DNA to Monitor Cytomegalovirus Infection After Liver Transplant.

OBJECTIVES: In the present study, we investigated donor-derived cell-free DNA dynamics and assessed the diagnostic efficacy of 2 tests: the sequencing of cytomegalovirus-derived cell-free DNA and the quantitative nucleic acid amplification test in cytomegalovirus infection following liver transplant. MATERIALS AND METHODS: We first examined 6 patients who were identified with active cytomegalovirus DNAemia by both quantitative nucleic acid amp-lification test and next-generation sequencing of cytomegalovirus-derived cell-free DNA and then performed a receiver operating characteristic analysis to evaluate the efficacy of cell-free DNA sequencing and establish a cutoff for this assay. Further validation of the next-generation sequencing method was also performed in 84 liver transplant recipients. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and the Declaration of Istanbul. RESULTS: In the first 6 patients, there was no significant correlation between the cytomegalovirus infection and donor-derived cell-free DNA. We determined that the levels of cytomegalovirus-derived cell-free DNA sequencing directly correlate with the results of the quantitative nucleic acid amplification test (area under the curve 0.982) and obtained a value of 0.015% as a cutoff for the cell-free DNA sequencing assay. In the validation cohort composed of 84 liver transplant recipients, next-generation sequencing of cell-free DNA revealed the occurrence of cytomegalovirus infection that remains otherwise undetected by the quantitative nucleic acid amplification test. CONCLUSIONS: Cytomegalovirus infections that do not cause direct graft injury (cytomegalovirus-related hepatitis) did not result in elevations of donor-derived cell-free DNA. Next-generation sequencing of cytomegalovirus-derived cell-free DNA provides a potential tool for detection of cytomegalovirus infection that remains undetected by the quantitative nucleic acid amplification test.

Combined Analysis of Expression Profiles in a Mouse Model and Patients Identified BHMT2 as a New Regulator of Lipid Metabolism in Metabolic-Associated Fatty Liver Disease.

Metabolic associated fatty liver disease (MAFLD) is associated with obesity, type 2 diabetes mellitus, and other metabolic syndromes. Farnesoid X receptor (FXR, NR1H4) plays a prominent role in hepatic lipid metabolism. This study combined the expression of liver genes in FXR knockout (KO) mice and MAFLD patients to identify new pathogenic pathways for MAFLD based on genome-wide transcriptional profiling. In addition, the roles of new target genes in the MAFLD pathogenic pathway were also explored. Two groups of differentially expressed genes were obtained from FXR-KO mice and MAFLD patients by transcriptional analysis of liver tissue samples. The similarities and differences between the two groups of differentially expressed genes were analyzed to identify novel pathogenic pathways and target genes. After the integration analysis of differentially expressed genes, we identified 134 overlapping genes, many of which have been reported to play an important role in lipid metabolism. Our unique analysis method of comparing differential gene expression between FXR-KO mice and patients with MAFLD is useful to identify target genes and pathways that may be strongly implicated in the pathogenesis of MAFLD. The overlapping genes with high specificity were screened using the Gene Expression Omnibus (GEO) database. Through comparison and analysis with the GEO database, we determined that BHMT2 and PKLR could be highly correlated with MAFLD. Clinical data analysis and RNA interference testing in vitro confirmed that BHMT2 may a new regulator of lipid metabolism in MAFLD pathogenesis. These results may provide new ideas for understanding the pathogenesis of MAFLD and thus provide new targets for the treatment of MAFLD.

Induction of lymphocyte apoptosis in rat liver allograft by adenoviral gene transfection of human interleukin-10.

BACKGROUND/AIMS: Gene therapy can provide a possible avenue in organ transplantation to treat acute allograft rejection. This study was designed to investigate the effect of adenovirus-mediated human IL-10 (hIL-10) gene transfer on the apoptosis of infiltrating lymphocytes and examine the efficacy of hIL-10 gene transfer in combination with subtherapeutic doses of cyclosporine A (CsA) in a rat liver transplantation model. METHODS: Inbred male DA and LEW rats were used for liver donors and recipients, respectively. The rats were divided into saline, Ad-lacZ, CsA, Ad-hIL-10 and Ad-hIL-10 + CsA groups. Graft survival, histopathological, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction and flow cytometry were performed in liver specimens obtained from different time points after transplantation in the 5 groups. RESULTS: Ad-hIL-10 pretreatment inhibited allograft rejection, prolonged the survival of hepatic allografts, and downregulated the expression of IFN-gamma and IL-2 mRNA, with simultaneous upregulation of IL-4 mRNA. In addition, Ad-hIL-10 pretreatment upregulated the expression of Fas mRNA in the isolated graft-infiltrating lymphocytes and induced graft-infiltrating lymphocyte apoptosis. A single subtherapeutic dose of CsA acted synergistically with it. CONCLUSION: hIL-10 gene therapy induced alloreactive lymphocyte apoptosis via Fas/FasL pathway. hIL-10 gene transfection in combination with subtherapeutic doses of CsA facilitates the long-term survival of liver grafts.