Direct Observation of the Mutual Coupling Effect in the Protein-Water-Glycerol Mixture by Combining Neutron Scattering and Selective Deuteration.

Numerous studies have discussed the impact of cosolvents on the structure, dynamics, and stability of proteins in aqueous solutions. However, the dynamics of cosolvents in the protein-water-cosolvent ternary system is largely unexplored in experiments due to technical difficulty. Consequently, a comprehensive understanding of the interplay among proteins, water, and cosolvents is still lacking. Here, we employed selective deuteration and neutron scattering techniques to characterize the individual motions of each component in the protein/water/glycerol (GLY) mixture across various temperatures. The consistent dynamic onset temperatures and the correlation between the MSD of the protein and the viscosity of solvents revealed the mutual coupling effects among the three components. Furthermore, our experimental and simulation results showed that the hydrogen bond relaxation energy barrier in the ternary system is ∼43 kJ/mol, whereas in the protein-water binary system it is merely ∼35 kJ/mol. Therefore, we suggest that GLY can enhance hydrogen bond interactions in the ternary system through the mutual coupling effect, thereby serving as one of the protective mechanisms of protein preservation by GLY.

Landscape of Immune Cells Heterogeneity in Liver Transplantation by Single-Cell RNA Sequencing Analysis.

Rejection is still a critical barrier to the long-term survival of graft after liver transplantation, requiring clinicians to unveil the underlying mechanism of liver transplant rejection. The cellular diversity and the interplay between immune cells in the liver graft microenvironment remain unclear. Herein, we performed single-cell RNA sequencing analysis to delineate the landscape of immune cells heterogeneity in liver transplantation. T cells, NK cells, B cells, and myeloid cell subsets in human liver and blood were enriched to characterize their tissue distribution, gene expression, and functional modules. The proportion of CCR6+CD4+ T cells increased within an allograft, suggesting that there are more memory CD4+ T cells after transplantation, in parallel with exhausted CTLA4+CD8+ T and actively proliferating MKI67+CD8+ T cells increased significantly, where they manifested heterogeneity, distinct function, and homeostatic proliferation. Remarkably, the changes of CD1c+ DC, CADM+ DC, MDSC, and FOLR3+ Kupffer cells increase significantly, but the proportion of CD163+ Kupffer, APOE+ Kupffer, and GZMA+ Kupffer decreased. Furthermore, we identified LDLR as a novel marker of activated MDSC to prevent liver transplant rejection. Intriguingly, a subset of CD4+CD8+FOXP3+ T cells included in CTLA4+CD8+ T cells was first detected in human liver transplantation. Furthermore, intercellular communication and gene regulatory analysis implicated the LDLR+ MDSC and CTLA4+CD8+ T cells interact through TIGIT-NECTIN2 signaling pathway. Taken together, these findings have gained novel mechanistic insights for understanding the immune landscape in liver transplantation, and it outlines the characteristics of immune cells and provides potential therapeutic targets in liver transplant rejection.

[A twenty-year review of clinical liver transplantation].

OBJECTIVE: To review the development of adult and pediatric liver transplantation in Tianjin First Center Hospital, and to enhance academic exchanges, improve technological innovation, and jointly promote the progress and maturity in the field of liver transplantation. METHODS: The development of liver transplantation in Tianjin First Center Hospital was analyzed. The clinical data of adult and pediatric liver transplantation from September 1998 to September 2018 were collected. The important events and technological innovation achievements of liver transplantation during the 20 years were summarized. RESULTS: The first clinical liver transplantation was attempted in Tianjin First Central Hospital in April 1980. The first long-term survival adult liver transplantation in China was completed in 1994 (11 years survival after the operation). The specialized team of liver transplantation was formally established in September 1998. The 20-year clinical exploration and progress reflected the characteristics of era changes and technological innovation during the rapid development of liver transplantation in China. Our center performed liver re-transplantation in January 1999, reduced-size pediatric liver transplantation in August 2000. In May 2001, we organized the formulation for the preventive and treatment plan for hepatitis B recurrence after liver transplantation. We performed combined liver and kidney transplantation in July 2002, split liver transplantation (SLT) in April 2004, the first domino liver transplantation (DLT) in August 2005. Pediatric living donor liver transplantation (LDLT) was initiated in October 2006, adult LDLT was carried out in August 2007. In September 2007, the first living donor combined liver and kidney transplantation from the same donor in Asia was performed. The first domino+living donor double grafts liver transplantation in the world was performed in January 2009. In March 2011, we performed laparoscopically assisted right hepatic lobe liver transplantation (LDLT) with middle hepatic vein. In May 2014, living donor laparoscopic left lateral lobe procurement was successfully established. In April 2016, simultaneous liver, pancreas and kidney multi-organ transplantation was completed. Domino donor-auxiliary liver transplantation was performed in February 2017. In December 2017, extracorporeal membrane oxygenation (ECMO)-supported liver transplantation in a patient with severe pulmonary hypertension was successfully completed. Liver transplantation combined with partial splenectomy was established in April 2018. Cross-domino liver transplantation (hypersensitive kidney transplantation with auxiliary liver transplantation+pediatric liver transplantation) was performed in May 2018. During the 20 years, the team has performed or assisted other centers in Beijing, Shanghai, Guangzhou and Shenzhen to carry out more than 10 000 cases of liver transplantations. A total of 7 043 cases of various types of liver transplantation were performed in the single center of the hospital (6 005 adult liver transplantations and 1 038 pediatric liver transplantations). Concerning adult liver transplantation, the cumulative 1-year, 3-year and 5-year survival rate from September 1998 to March 2003 were 83.1%, 73.0% and 69.0%, from April 2003 to March 2009 were 85.3%, 76.2% and 72.1% and from April 2009 to September 2018 were 87.5%, 79.2% and 75.1%, respectively. The cumulative 1-year, 3-year and 5-year survival rate for pediatric liver transplantation were 93.5%, 92.2% and 90.2%, respectively. The nucleoside (acid) analogue combined with low dose hepatitis B immunoglobulin (HBIG) was developed to prevent the recurrence of hepatitis B after liver transplantation, this plan has reduced the recurrence rate of hepatitis B and the 5-year re-infection rate of hepatitis B virus (HBV) after liver transplantation significantly. The risk assessment system for tumor recurrence after liver transplantation was established and individual treatment method was established based on this assessment system. Continuous exploration and improvement of liver transplantation for liver cancer, liver re-transplantation, liver transplantation with portal vein thrombosis, SLT, DLT and multi-organ combined transplantation have significantly improved the clinical efficacy of patients and the post-operative survival rate. CONCLUSIONS: The liver transplantation team of Tianjin First Center Hospital has carried out a scientific and technological exploration on the key problems and technical difficulties of clinical liver transplantation. This work strongly has initiated and promoted the rapid development of liver transplantation in China. The restrictive barrier of hepatitis B recurrence after liver transplantation has been overcome. The risk prevention and control system of tumor recurrence after liver transplantation has been established. A series of innovative achievements that can be popularized have been achieved in the field of complex liver transplantation and expansion of donor liver source. The iterative progress and sustainable development of liver transplantation have been realized.

A six-gene-based prognostic signature for hepatocellular carcinoma overall survival prediction.

AIMS: The purpose of this study was to propose a pipeline to identify prognostic signature for HCC overall survival (OS) prediction based on HCC gene expression datasets from The Cancer Genome Atlas (TCGA). RESULTS: Differential expression analysis identified 3573 genes aberrantly expressed (DEGs) in HCC samples. Univariate cox regression analysis obtained 1605 and 1067 HCC OS and relapse free survival (RFS) related genes, which are abbreviated as OS-Gene and RFS-Gene respectively. Besides, there are 55 overlaps among DEGs, OS-Genes and RFS-Genes. Further prioritization of the 55 overlapping genes through Sure Independence Screening (SIS) resulted in 6 genes, including SRL, TTC26, CPSF2, TAF3, C16orf46 and CSN1S1, and the prognostic signature is the weighted combination of their expression values. Kaplan-Meier analysis based on the prognostic score (PS) of every sample indicates higher PS is associated with better HCC OS. Robustness of the prognostic signature was evaluated through another HCC gene expression datasets from the Gene Expression Omnibus (GEO). What's more, univariate and multivariate cox regression analysis indicate significant associations between stage/PS and HCC OS. CONCLUSIONS: Our study provides a pipeline for the identification of prognostic signature for HCC OS prediction, which should also be suit for other types of cancers.

Cobalt-protoporphyrin enhances heme oxygenase 1 expression and attenuates liver ischemia/reperfusion injury by inhibiting apoptosis.

The aim of the present study was to investigate the preconditioning effect and underlying mechanisms of cobalt-protoporphyrin (CoPP) in a mouse model of liver ischemia­reperfusion (I/R) injury. Mice were divided into five groups: Sham­operated (control), I/R, I/R + CoPP, I/R + CoPP and zinc­protoporphyrin (ZnPP) and I/R + ZnPP. Serum levels of aspartate transaminase (AST) and alanine aminotransferase (ALT) were detected using commercial kits. The expression of the pro­apoptotic protein caspase­3 was detected by immunohistochemistry and the expression levels of the anti­apoptotic protein B­cell lymphoma 2 (Bcl­2) and heme oxygenase 1 (HO­1) were analyzed by western blotting. Sections of liver tissue were stained with hematoxylin and eosin to observe pathologic alterations. Furthermore, hepatocyte apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. AST and ALT levels of the CoPP preconditioned group were significantly reduced compared with the IR injury group (P<0.05) and liver damage was attenuated. The expression levels of the pro­apoptotic protein caspase3 was inhibited and those of HO­1 and Bcl­2 were increased in the CoPP group compared with the I/R group; the opposite results were observed in the ZnPP group. Furthermore, the percentage of apoptotic cells as detected by TUNEL was significantly decreased in the CoPP group compared with the I/R group (P<0.05); these protective effects were abrogated by ZnPP. In conclusion, the results of the present study suggested that CoPP may induce HO­1 overexpression and produce anti­apoptotic effects in liver I/R injury.

The correlation between the expression of genes involved in drug metabolism and the blood level of tacrolimus in liver transplant receipts.

Immunosuppressive medications, such as tacrolimus and mycophenolate mofetil, are commonly used for reducing the risk of organ rejection in receipts of allogeneic organ transplant. The optimal dosages of these drugs are required for preventing rejection and avoiding toxicity to receipts. This study aimed to identify the correlation between the expression profiling of genes involved in drug metabolism and the blood level of tacrolimus in liver transplant receipts. Sixty-four liver transplant receipts were enrolled in this retrospective study. Receipts were divided into low (2-5.9 ng/ml) and high (6-15 ng/ml) tacrolimus groups. Clinical assessment showed that the blood level of tacrolimus was inversely correlated with the liver function evaluated by blood levels of total bilirubin and creatinine. Compared to the high tacrolimus group, expression levels of six cytochrome P450 enzymes, CYP1A1, CYP2B6, CYP3A5, CYP4A11, CYP19A1, and CYP17A1 were significantly higher in the low tacrolimus group. The expression levels of these genes were negatively correlated with the tacrolimus blood level. Enzyme assays showed that CYP3A5 and CYP17A1 exerted direct metabolic effects on tacrolimus and mycophenolate mofetil, respectively. These results support clinical application of this expression profiling of genes in drug metabolism for selection of immunosuppressive medications and optimal dosages for organ transplant receipts.

Clonal Origin of Hepatocellular Carcinoma and Recurrence After Liver Transplantation.

BACKGROUND This study aimed to determine whether patterns of tumor clonal origin in pluri-nodular hepatocellular carcinoma (PNHC) could serve as an indicator of tumor recurrence following liver transplantation. MATERIAL AND METHODS Tumor tissue samples from 60 PNHC patients who underwent liver transplantation were examined. The diagnosis of patients conformed to the University of California San Francisco (UCSF) standards for pluri-nodular hepatocellular carcinoma. We performed loss of heterozygosity tests at multiple microsatellite sites to determine the clonal origins of the tumors. Clinical information, pathological data, preoperative serum alpha-feto protein (AFP) and postoperative follow-ups were obtained and correlations between the clonal origin of the tumor, tumor-free survival, pathological characteristics, and AFP levels in serum were studied. RESULTS A total of 165 tumor nodules were collected. Tumor clonal origins were identified as intrahepatic metastasis (IM; 41.67%), multicentric occurrence (MO; 55%) or unidentified (3.33%). Three-year tumor-free survival for the IM group was 48% compared to 75.76% in the MO group (p<0.05), while the occurrence of microscopic tumor thrombus was 100% and 3.03% (p<0.05) for these groups, respectively. The degree of tumor differentiation was 80% for the IM group and 18.18% for the MO group (p<0.05), while the mean AFP concentration for these groups was 226.80 µg/L (2.78-3000 µg/L) and 24.59 µg/L (1.16-531. 30 µg/L; p<0.05), respectively. CONCLUSIONS Clonal origin patterns can serve as important indicators to predict the recurrence of PNHC following liver transplantation. Taken together with pathological characteristics and preoperative serum AFP levels, the risk of recurrence can be established in advance.

Impact of non-oncological factors on tumor recurrence after liver transplantation in hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC) is the most common primary neoplasm of the liver and is one of the leading causes of cancer-related death worldwide. Liver transplantation (LT) has become one of the best curative therapeutic options for patients with HCC, although tumor recurrence after LT is a major and unaddressed cause of mortality. Furthermore, the factors that are associated with recurrence are not fully understood, and most previous studies have focused on the biological properties of HCC, such as the number and size of the HCC nodules, the degree of differentiation, the presence of hepatic vascular invasion, elevated serum levels of alpha-fetoprotein, and the tumor stage outside of the Milan criteria. Thus, little attention has been given to factors that are not directly related to HCC (i.e., "non-oncological factors"), which have emerged as predictors of tumor recurrence. This review was performed to assess the effects of non-oncological factors on tumor recurrence after LT. The identification of these factors may provide new research directions and clinical strategies for the prophylaxis and surveillance of tumor recurrence after LT, which can help reduce recurrence and improve patient survival.

Global MicroRNA Expression Profiling of Mouse Livers following Ischemia-Reperfusion Injury at Different Stages.

Hepatic ischemia-reperfusion injury is a dynamic process consisting of two stages: ischemia and reperfusion, and triggers a cascade of physiological and biochemical events. Given the important role of microRNAs in regulating gene expression, we analyzed gene expression changes in mouse livers at sham control, ischemia stage, and reperfusion stage. We generated global expression profiles of microRNA and mRNA genes in mouse livers subjected to ischemia-reperfusion injury at the three stages, respectively. Comparison analysis showed that reperfusion injury had a distinct expression profile whereas the ischemia sample and the sham control were clustered together. Consistently, there are 69 differentially expressed microRNAs between the reperfusion sample and the sham control whereas 28 differentially expressed microRNAs between the ischemia sample and the sham control. We further identified two modes of microRNA expression changes in ischemia-reperfusion injury. Functional analysis of both the differentially expressed microRNAs in the two modes and their target mRNAs revealed that ischemia injury impaired mitochondrial function, nutrient consumption, and metabolism process. In contrast, reperfusion injury led to severe tissue inflammation that is predominantly an innate-immune response in the ischemia-reperfusion process. Our staged analysis of gene expression profiles provides new insights into regulatory mechanisms of microRNAs in mouse hepatic IR injury.

Application of nucleoside analogues to liver transplant recipients with hepatitis B.

Hepatitis B is a common yet serious infectious disease of the liver, affecting millions of people worldwide. Liver transplantation is the only possible treatment for those who advance to end-stage liver disease. Donors positive for hepatitis B virus (HBV) core antibody (HBcAb) have previously been considered unsuitable for transplants. However, those who test negative for the more serious hepatitis B surface antigen can now be used as liver donors, thereby reducing organ shortages. Remarkable improvements have been made in the treatment against HBV, most notably with the development of nucleoside analogues (NAs), which markedly lessen cirrhosis and reduce post-transplantation HBV recurrence. However, HBV recurrence still occurs in many patients following liver transplantation due to the development of drug resistance and poor compliance with therapy. Optimized prophylactic treatment with appropriate NA usage is crucial prior to liver transplantation, and undetectable HBV DNA at the time of transplantation should be achieved. NA-based and hepatitis B immune globulin-based treatment regimens can differ between patients depending on the patients' condition, virus status, and presence of drug resistance. This review focuses on the current progress in applying NAs during the perioperative period of liver transplantation and the prophylactic strategies using NAs to prevent de novo HBV infection in recipients of HBcAb-positive liver grafts.

[The risk factors of splenic arterial steal syndrome after orthotopic liver transplantation].

OBJECTIVE: To discuss the risk factors of splenic arterial steal syndrome (SASS) after orthotopic liver transplantation. METHODS: Twenty-four cases who confirmed SASS after liver transplantation in Tianjin First Central Hospital between June 2005 and June 2013 were analyzed retrospectively. Another 96 cases were selected randomly from those patients of the same time with no complication of SASS patients postoperatively as control group. Clinical data of two groups including diameter of splenic artery and hepatic artery preoperatively, weight of graft, weight of recipients, cold/warm ischemia time, an hepatic period and operation time and so on were collected. Others including hepatic artery peak systolic velocity (PSV), end diastolic velocity (EDV), blood flow resistance index and portal vein average velocity (PVF) on the first day after liver transplantation, the day before diagnosis, the day when diagnosed, the 1, 3, 7 days after treatment in SASS group and on 1, 3, 7, 9, 11, 14 days after liver transplantation in control group. Statistical analysis were made between two groups. RESULTS: The splenic artery/hepatic artery ratio preoperatively and weight of donor liver,and the GRWR in SASS group and control group were 1.26 and 1.00, 1 032 g and 1 075 g, (1.40±0.30)% and (1.82±0.21)% respectively, with significantly statistical differences (Z=-6.40, Z=-2.22, t=-6.50; all P<0.05). The warm ischemia time, the cold ischemia time, the anhepatic period and operation time in SASS group and control group were 3.5 minutes and 4.0 minutes, 10.25 hours and 10.10 hours, 43 minutes and 45 minutes, 8.7 hours and 8.7 hours, with no significantly statistical differences (all P>0.05). RI of hepatic went up gradually in the early time after transplantation while dropped obviously when spleen artery spring coils embolization was received (P<0.01) and trended to stable two weeks later. CONCLUSIONS: Splenic artery/hepatic artery ratio and GRWR are the positive and negative risk factors respectively for SASS. The gradual rising of hepatic RI in the early time after transplantation may be the warning signal SASS and spleen artery spring coils embolization is the effective strategy for SASS after liver transplantation.

Glypican-3 expression and its relationship with recurrence of HCC after liver transplantation.

AIM: To investigate the diagnostic value of glypican-3 (GPC3) and its relationship with hepatocellular carcinoma (HCC) recurrence after liver transplantation. METHODS: HCC tissue samples (n = 31) obtained from patients who had undergone liver transplantation were analyzed. GPC3 mRNA and protein expression were analyzed by TaqMan real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Correlation between the GPC3 expression and clinicopathological features was analyzed. The potential prognostic value of GPC3 was investigated by comparing recurrence-free survival between HCC patients with and without GPC3 expression. RESULTS: Using a cutoff value of 3.5 × 10⁻², 20 of 31 cancerous tissues had expression values of > 3.5 × 10⁻², whereas 3 of 31 adjacent non-neoplastic parenchyma and 0 of 20 control liver tissues had expression values of > 3.5 × 10⁻² (P < 0.001). GPC3 protein was immunoexpressed in 68% of cancerous tissues, but not in adjacent non-neoplastic parenchyma and control liver tissues. Vascular invasion was significantly related to GPC3 expression (P < 0.05). Recurrence-free survival was significantly longer for patients without GPC3 mRNA overexpression (> 3.5 × 10⁻²) and those without vascular invasion (P < 0.05 for both). CONCLUSION: GPC3 expression may serve as a valuable diagnostic marker for HCC. GPC3 mRNA overexpression may be an adverse indicator for HCC patients after liver transplantation.

Diagnostic and therapeutic progress of multi-drug resistance with anti-HBV nucleos(t)ide analogues.

Nucleos(t)ide analogues (NA) are a breakthrough in the treatment and management of chronic hepatitis B. NA could suppress the replication of hepatitis B virus (HBV) and control the progression of the disease. However, drug resistance caused by their long-term use becomes a practical problem, which influences the long-term outcomes in patients. Liver transplantation is the only choice for patients with HBV-related end-stage liver disease. But, the recurrence of HBV after transplantation often caused by the development of drug resistance leads to unfavorable outcomes for the recipients. Recently, the multi-drug resistance (MDR) has become a common issue raised due to the development and clinical application of a variety of NA. This may complicate the antiviral therapy and bring poorly prognostic outcomes. Although clinical evidence has suggested that combination therapy with different NA could effectively reduce the viral load in patients with MDR, the advent of new antiviral agents with high potency and high genetic barrier to resistance brings hope to antiviral therapy. The future of HBV researches relies on how to prevent the MDR occurrence and develop reasonable and effective treatment strategies. This review focuses on the diagnostic and therapeutic progress in MDR caused by the anti-HBV NA and describes some new research progress in this field.