K33 only mutant ubiquitin augments bone marrow-derived dendritic cell-mediated CTL priming via PI3K-Akt pathway.

To explore the effect of K33 only mutant ubiquitin (K33O) on bone marrow-derived dendritic cells' (BMDCs') maturity, antigen uptake capability, surface molecule expressions and BMDC-mediated CTL priming, and further investigate the role of PI3K-Akt engaged in K33O-increased BMDC maturation, antigen uptake and presentation, surface molecule expressions and BMDC-based CTL priming. BMDCs were conferred K33O and other ubiquitin mutants (K33R, K48R, K63R-mutant ubiquitin) incubation or LY294002 and wortmannin pretreatment. PI3K-Akt phosphorylation, antigen uptake, antigenic presentation and CD86/MHC class I expression in BMDC were determined by western blot or flow cytometry. BMDC-based CTL proliferation and priming were determined by in vitro mixed lymphocyte reaction (MLR), ex vivo enzyme-linked immunospot assay (Elispot) and flow cytometry with intracellular staining, respectively. The treatment with K33O effectively augmented PI3K-Akt phosphorylation, BMDCs' antigen uptake, antigenic presentation, CD86/MHC class I and CD11c expressions. MLR, Elispot and flow cytometry revealed that K33O treatment obviously enhanced CTL proliferation, CTL priming and perforin/granzyme B expression. The pretreatment with PI3K-Akt inhibitors efficiently abrogated K33O's effects on BMDC. The replenishment of K33 only mutant ubiquitin augments BMDC-mediated CTL priming in bone marrow-derived dendritic cells via PI3K-Akt signalling.

Validation and performance of three scoring systems for predicting primary non-function and early allograft failure after liver transplantation.

BACKGROUND: Primary non-function (PNF) and early allograft failure (EAF) after liver transplantation (LT) seriously affect patient outcomes. In clinical practice, effective prognostic tools for early identifying recipients at high risk of PNF and EAF were urgently needed. Recently, the Model for Early Allograft Function (MEAF), PNF score by King's College (King-PNF) and Balance-and-Risk-Lactate (BAR-Lac) score were developed to assess the risks of PNF and EAF. This study aimed to externally validate and compare the prognostic performance of these three scores for predicting PNF and EAF. METHODS: A retrospective study included 720 patients with primary LT between January 2015 and December 2020. MEAF, King-PNF and BAR-Lac scores were compared using receiver operating characteristic (ROC) and the net reclassification improvement (NRI) and integrated discrimination improvement (IDI) analyses. RESULTS: Of all 720 patients, 28 (3.9%) developed PNF and 67 (9.3%) developed EAF in 3 months. The overall early allograft dysfunction (EAD) rate was 39.0%. The 3-month patient mortality was 8.6% while 1-year graft-failure-free survival was 89.2%. The median MEAF, King-PNF and BAR-Lac scores were 5.0 (3.5-6.3), -2.1 (-2.6 to -1.2), and 5.0 (2.0-11.0), respectively. For predicting PNF, MEAF and King-PNF scores had excellent area under curves (AUCs) of 0.871 and 0.891, superior to BAR-Lac (AUC = 0.830). The NRI and IDI analyses confirmed that King-PNF score had the best performance in predicting PNF while MEAF served as a better predictor of EAD. The EAF risk curve and 1-year graft-failure-free survival curve showed that King-PNF was superior to MEAF and BAR-Lac scores for stratifying the risk of EAF. CONCLUSIONS: MEAF, King-PNF and BAR-Lac were validated as practical and effective risk assessment tools of PNF. King-PNF score outperformed MEAF and BAR-Lac in predicting PNF and EAF within 6 months. BAR-Lac score had a huge advantage in the prediction for PNF without post-transplant variables. Proper use of these scores will help early identify PNF, standardize grading of EAF and reasonably select clinical endpoints in relative studies.

Diagnosis and treatment of biliary mucinous cystic neoplasms: A single-center experience.

BACKGROUND: Biliary mucinous cystic neoplasms (BMCNs) are rare hepatobiliary cystic tumors, which can be divided into noninvasive and invasive types. This study aimed to investigate the diagnosis, treatment, and prognosis of BMCNs in a large single center. METHODS: We analyzed 49 patients with BMCNs confirmed by postoperative pathology at the First Affiliated Hospital, Zhejiang University School of Medicine between January 2007 and December 2021. RESULTS: Among the 49 patients, 37 were female (75.5%), and the average age was 57.04 years. Common symptoms included abdominal discomfort, jaundice and fever, while 22 patients (44.9%) had no symptoms. Serum carbohydrate antigen (CA) 19-9 and CA125 concentrations were elevated in 34.8% and 19.6% of patients, respectively. Forty-eight patients had tumors in the intrahepatic bile ducts and only one had a tumor in the extrahepatic bile duct. Forty-eight patients with noninvasive intrahepatic BMCNs were further analyzed in terms of pathological features: 34 (70.8%) had low-grade intraepithelial neoplasms (LGINs), and 14 (29.2%) had high-grade intraepithelial neoplasms (HGINs). The potential immunohistochemical markers of BMCNs were cytokeratin (CK) 19, CK7, estrogen receptor and progesterone receptor. Follow-up data for 37 patients with intrahepatic BMCNs were obtained. The median overall survival (OS) of BMCNs was not reached. The longest survival time was 137 months.The 5- and 10-year OS rates were 100% and 85.4%, respectively. The 5- and 10-year recurrence-free survival (RFS) rates were 93.9% and 80.2%, respectively. CONCLUSIONS: BMCNs are rare cystic neoplasms that commonly occur in middle-aged females. BMCNs can only be diagnosed and classified by postoperative pathology, as there are no specific clinical presentations, serological indicators or imaging modalities for preoperative diagnosis. Complete surgical resection is necessary for BMCNs, and the postoperative prognosis is favorable.

Exosome is involved in liver graft protection after remote ischemia reperfusion conditioning.

BACKGROUND: Remote ischemic perconditioning (RIPerC) has been demonstrated to protect grafts from hepatic ischemia-reperfusion injury (IRI). This study investigated the role of exosomes in RIPerC of liver grafts in rats. METHODS: Twenty-five rats (including 10 donors) were randomly divided into five groups (n = 5 each group): five rats were used as sham-operated controls (Sham), ten rats were for orthotopic liver transplantation (OLT, 5 donors and 5 recipients) and ten rats were for OLT + RIPerC (5 donors and 5 recipients). Liver architecture and function were evaluated. RESULTS: Compared to the OLT group, the OLT + RIPerC group exhibited significantly improved liver graft histopathology and liver function (P < 0.05). Furthermore, the number of exosomes and the level of P-Akt were increased in the OLT + RIPerC group. CONCLUSIONS: RIPerC effectively improves graft architecture and function, and this protective effect may be related to the increased number of exosomes. The upregulation of P-Akt may be involved in underlying mechanisms.

K48- and K27-mutant ubiquitin regulates adaptive immune response by affecting cross-presentation in bone marrow precursor cells.

K48-linked ubiquitination determines antigen degradation and plays vital roles in the process of cross-presentation of bone marrow precursor cell (BMPC)-derived dendritic cells (DCs). Although previous studies revealed that K48 and K27-linked ubiquitination regulates innate immunity, the exact roles of K48 and K27-linked ubiquitination in cross-presentation and BMPC-based adaptive immunity are still uncertain. In this study, we investigated the effects of K48- and K27-mutant ubiquitin (Ub) on BMPC-based adaptive immune response by observing the effects of MG132, Ub deficiency, and K48/K27-mutant Ub on cross-presentation, T cell proliferation, IFN-γ secretion, BMPC-based CTL priming, and thereby the efficiency of cytolytic capacity of BMPC-activate T cells. We demonstrated that MG132, Ub deficiency, and K48-mutant Ub impair cross-presentation, T cell proliferation, IFN-γ secretion, BMPC-based CTL priming, and the cytolytic capacity of BMPC-activated T cells. Moreover, although K27-only Ub decreases cross-presentation, the replenishment of K27-mutant Ub restores Ub deficiency impaireds the abilities of T cell proliferation, IFN-γ secretion, CTL priming, and the cytolytic capacity of BMPC-activated T cells. Thus, these data suggest that K48- and K27-linked ubiquitination contributes to BMPC-mediated adaptive immune response by affecting BMPC cross-presentation and the cytolytic capacity by up-regulating both perforin and granzyme B in BMPC-activated T cells. K48- and K27-mutant Ub might have potential clinical therapeutic function in adaptive immune response-associated diseases.

Erratum to: Exosome-derived galectin-9 may be a novel predictor of rejection and prognosis after liver transplantation.

Erratum to: J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2019 20(7):605-612. https://doi.org/10.1631/jzus.B1900051. The original version of this article unfortunately contained a mistake. In p.605, the number of the Zhejiang Provincial Natural Science Foundation of China (No. Y17H160118) in Funding is incorrect. The correct number should be LY17H160026, which is the approval number of the project, whereas Y17H160118 is the application number of the project.

Exosome-derived galectin-9 may be a novel predictor of rejection and prognosis after liver transplantation.

Acute cellular rejection (ACR) remains a major concern after liver transplantation. Predicting and monitoring acute rejection by non-invasive methods are very important for guiding the use of immunosuppressive drugs. Many studies have shown that exosomes and their contents are potential biomarkers for various liver diseases. Here, we identify and validate the role of exosomes and galectin-9 in ACR after liver transplantation. Exosomes were isolated from three sets of paired patients, with and without ACR, and the proteins within the exosomes were isolated and identified. Candidate proteins were then validated using a tissue microarray containing resected liver samples from 73 ACR and 63 non-rejection patients. Finally, protein expression and clinical manifestations were included in Kaplan-Meier survival and Cox regression analyses. Circulating exosomes were isolated from ACR and non-rejection patients and characterized using transmission electron microscopy and western blotting for CD63/CD81. Western blotting experiments revealed higher levels of galectin-9 protein in circulating exosomes from ACR recipients. Immunohistochemical analysis of the tissue microarray showed that the expression of galectin-9 in resected liver was significantly higher in the ACR group than in the non-rejection group (P<0.05). Higher levels of galectin-9 expression in resected livers were associated with poorer prognosis (P<0.05). Exosome-derived galectin-9 may be a novel predictor of rejection and prognosis after liver transplantation.

Graft protection of the liver by hypothermic machine perfusion involves recovery of graft regeneration in rats.

OBJECTIVE: This study was performed to evaluate the impact and underlying mechanisms of hypothermic machine perfusion (HMP) on half-size liver graft regeneration. METHODS: Forty rats were randomly assigned to five groups: two in vitro groups (static cold storage [SCS] and HMP) and three in vivo groups (orthotopic liver transplantation, SCS, and HMP). Perfusates and plasma samples were collected for analysis of hepatic enzymes. Liver tissue was obtained for evaluation of histology, immunohistochemistry (Ki67 and proliferating cell nuclear antigen [PCNA]), and the regeneration rate. Cell cycle genes were analyzed by quantitative real-time polymerase chain reaction, and cyclin D1 and cyclin E1 were semiquantified by western blot. RESULTS: HMP improved histopathological outcomes and decreased hepatic enzyme release. The expression of Ki67 and PCNA demonstrated a greater proliferation activity in the HMP than SCS group, and the expression of almost all cell cycle genes was elevated following HMP. Western blot results showed higher protein levels of cyclin D1 and cyclin E1 in the HMP than SCS group. CONCLUSIONS: Our findings suggest for the first time that half-size liver graft protection by HMP involves recovery of graft regeneration.

Machine perfusion for liver transplantation: A concise review of clinical trials.

BACKGROUND: With the increased use of extended-criteria donors, static cold storage has failed to provide optimal preservation of liver grafts, resulting in early allograft dysfunction and long-term complications. Machine perfusion (MP) is a beneficial alternative preservation strategy for donor livers, particularly for those considered to be of suboptimal quality, and could expand the limited donor pool. DATA SOURCES: A comprehensive search in PubMed, EMBASE, Ovid databases and ClinicalTrials.gov website was conducted using the medical subject heading terms "machine perfusion", "machine preservation", "liver transplantation", combined with free text terms such as "hypothermic", "normothermic" and "subnormothermic". The deadline for the search was September 30, 2017. RESULTS: MP can be classified as hypothermic, subnormothermic, and normothermic with the temperature maintained at 0-12 °C, 25-34 °C and 35-38 °C, respectively. Twelve clinical trials of MP have been reported in recent years. MP effectively decreased AST/ALT level and the incidence of early allograft dysfunction. However, the graft and patient survival rate after MP were similar to static cold storage. The detailed clinical characteristics such as liver function, graft survival, patient survival and early allograft dysfunction were reviewed. CONCLUSIONS: Clinical trial results showed that MP improves delayed graft function, primary non-function and biliary strictures. However, MP still requires validation in large clinical trials and the key parameters during MP still require optimization.

Optimal immunosuppressor induces stable gut microbiota after liver transplantation.

AIM: To study the influence of different doses of tacrolimus (FK506) on gut microbiota after liver transplantation (LT) in rats. METHODS: Specific pathogen-free Brown Norway (BN) rats and Lewis rats were separated into five groups: (1) Tolerance group (BN-BN LT, n = 8); (2) rejection group (Lewis-BN LT, n = 8); (3) high dosage FK506 (FK506-H) group (Lewis-BN LT, n = 8); (4) middle dosage FK506 (FK506-M) group (Lewis-BN LT, n = 8); and (5) low dosage FK506 (FK506-L) group (Lewis-BN LT, n = 8). FK506 was administered to recipients at a dose of 1.0 mg/kg, 0.5 mg/kg, and 0.1 mg/kg body weight for 29 d after LT to the FK506-H, FK506-M, and FK506-L groups, respectively. On the 30th day after LT, all rats were sampled and euthanized. Blood samples were harvested for liver function and plasma endotoxin testing. Hepatic graft and ileocecal tissues were collected for histopathology observation. Ileocecal contents were used for DNA extraction, Real-time quantitative polymerase chain reaction (RT-PCR) and digital processing of denaturing gradient gel electrophoresis (DGGE) profiles and analysis. RESULTS: Compared to the FK506-H and FK506-L groups, FK506-M was optimal for maintaining immunosuppression and inducing normal graft function; the FK506-M maintained gut barrier integrity and low plasma endotoxin levels; furthermore, DGGE results showed that FK506-M induced stable gut microbiota. Diversity analysis indicated that FK506-M increased species richness and rare species abundance, and cluster analysis confirmed the stable gut microbiota induced by FK506-M. Phylogenetic tree analysis identified crucial bacteria associated with FK506-M; seven of the nine bacteria that were decreased corresponded to Bacteroidetes, while increased bacteria were of the Bifidobacterium species. FK506-M increased Faecalibacterium prausnitzii and Bifidobacterium spp. and decreased Bacteroides-Prevotella and Enterobacteriaceae, as assessed by RT-PCR, which confirmed the crucial bacterial alterations identified through DGGE. CONCLUSION: Compared to the low or high dosage of FK506, an optimal dosage of FK506 induced immunosuppression, normal graft function and stable gut microbiota following LT in rats. The stable gut microbiota presented increased probiotics and decreased potential pathogenic endotoxin-producing bacteria. These findings provide a novel strategy based on gut microbiota for immunosuppressive dosage assessment for recipients following LT.

Partial Inhibition of HO-1 Attenuates HMP-Induced Hepatic Regeneration against Liver Injury in Rats.

We found better liver graft regeneration with hypothermic machine perfusion (HMP) compared with static cold storage (SCS) for the first time in our pilot study, but the underlying mechanisms are unknown. Upregulated heme oxygenase- (HO-) 1 expression has been reported to play a pivotal role in promoting hepatocyte proliferation. Here, we evaluated the novel role of HO-1 in liver graft protection by HMP. Rats with a heterozygous knockout of HO-1 (HO-1+/-) were generated and subjected to 3 h of SCS or HMP pre-half-size liver transplantation (HSLT) in vivo or 6 h of SCS or HMP in vitro; control rats were subjected to the same conditions (HO-1+/+). We found that HSLT induced significant elevation of the HO-1 protein level in the regenerated liver and that HO-1 haplodeficiency resulted in decreased proliferation post-HSLT. Compared with SCS, HMP induced significant elevation of the HO-1 protein level along with better liver recovery, both of which were reduced by HO-1 haplodeficiency. HO-1 haplodeficiency-induced decreased proliferation was responsible for the attenuated regenerative ability of HMP. Mechanistically, HO-1 haploinsufficiency resulted in suppression of hepatocyte growth factor (HGF)/Akt activity. Our results suggest that inhibition of HO-1 mitigates HMP-induced liver recovery effects related to proliferation, in part, by downregulating the HGF-Akt axis.

Optimized postconditioning algorithm protects liver graft after liver transplantation in rats.

BACKGROUND: Ischemia reperfusion injury (IRI) causes postoperative complications and influences the outcome of the patients undergoing liver surgery and transplantation. Postconditioning (PostC) is a known manual conditioning to decrease the hepatic IRI. Here we aimed to optimize the applicable PostC protocols and investigate the potential protective mechanism. METHODS: Thirty Sprague-Dawley rats were randomly divided into 3 groups: the sham group (n = 5), standard orthotopic liver transplantation group (OLT, n = 5), PostC group (OLT followed by clamping and re-opening the portal vein for different time intervals, n = 20). PostC group was then subdivided into 4 groups according to the different time intervals: (10 s × 3, 10 s × 6, 30 s × 3, 60 s × 3, n = 5 in each subgroup). Liver function, histopathology, malondialdehyde (MDA), myeloperoxidase (MPO), expressions of p-Akt and endoplasmic reticulum stress (ERS) related genes were evaluated. RESULTS: Compared to the OLT group, the grafts subjected to PostC algorithm (without significant prolonging the total ischemic time) especially with short stimulus and more cycles (10 s × 6) showed significant alleviation of morphological damage and graft function. Besides, the production of reactive oxidative agents (MDA) and neutrophil infiltration (MPO) were significantly depressed by PostC algorithm. Most of ERS related genes were down-regulated by PostC (10 s × 6), especially ATF4, Casp12, hspa4, ATF6 and ELF2, while p-Akt was up-regulated. CONCLUSIONS: PostC algorithm, especially 10 s × 6 algorithm, showed to be effective against rat liver graft IRI. These protective effects may be associated with its antioxidant, inhibition of ERS and activation of p-Akt expression of reperfusion injury salvage kinase pathway.

Mitofusin-2 mediated mitochondrial Ca2+ uptake 1/2 induced liver injury in rat remote ischemic perconditioning liver transplantation and alpha mouse liver-12 hypoxia cell line models.

AIM: To investigate the protective mechanism of mitofusin-2 (Mfn2) in rat remote ischemic perconditioning (RIC) models and revalidate it in alpha mouse liver-12 (AML-12) hypoxia cell lines. METHODS: Sprague-Dawley rats were divided into three groups (n = 6 each): sham, orthotopic liver transplantation and RIC. After operation, blood samples were collected to test alanine aminotransferase and aspartate aminotransferase. The liver lobes were harvested for histopathological examination, western blotting (WB) and quantitative real-time (qRT)-PCR. AML-12 cell lines were then subjected to normal culture, anoxic incubator tank culture (hypoxia) and anoxic incubator tank culture with Mfn2 knockdown (hypoxia + Si), and data of qRT-PCR, WB, mitochondrial membrane potential (ΔΨm), apoptosis, endoplasmic reticulum Ca2+ concentrations and mitochondrial Ca2+ concentrations were collected. RESULTS: Both sham and normal culture groups showed no injury during the experiment. The RIC group showed amelioration of liver function compared with the orthotopic liver transplantation group (P < 0.05). qRT-PCR and WB confirmed that Mfn2-mitochondrial Ca2+ uptake 1/2 (MICUs) axis was changed (P < 0.005). In AML-12 cell lines, compared with the hypoxia group, the hypoxia + Si group attenuated the collapse of ΔΨm and apoptosis (P < 0.005). The endoplasmic reticulum Ca2+ decrease and mitochondrial Ca2+ overloading observed in the hypoxia group were also attenuated in the hypoxia + Si group (P < 0.005). Finally, qRT-PCR and WB confirmed the Mfn2-MICUs axis change in all the groups (P < 0.005). CONCLUSION: Mfn2 participates in liver injury in rat RIC models and AML-12 hypoxia cell lines by regulating the MICUs pathway.

Remote ischemic perconditioning prevents liver transplantation-induced ischemia/reperfusion injury in rats: Role of ROS/RNS and eNOS.

AIM: To investigate the underlying mechanisms of the protective role of remote ischemic perconditioning (RIPerC) in rat liver transplantation. METHODS: Sprague-Dawley rats were subjected to sham, orthotopic liver transplantation (OLT), ischemic postconditioning (IPostC) or RIPerC. After 3 h reperfusion, blood samples were taken for measurement of alanine aminotransferase, aspartate aminotransferase, creatinine (Cr) and creatinine kinase-myocardial band (CK-MB). The liver lobes were harvested for the following measurements: reactive oxygen species (ROS), H2O2, mitochondrial membrane potential (ΔΨm) and total nitric oxide (NO). These measurements were determined using an ROS/H2O2, JC1 and Total NOx Assay Kit, respectively. Endothelial NO synthase (eNOS) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting, and peroxynitrite was semi-quantified by western blotting of 3-nitrotyrosine. RESULTS: Compared with the OLT group, the grafts subjected to RIPerC showed significantly improved liver and remote organ functions (P < 0.05). ROS (P < 0.001) including H2O2 (P < 0.05) were largely elevated in the OLT group as compared with the sham group, and RIPerC (P < 0.05) reversed this trend. The collapse of ΔΨm induced by OLT ischemia/reperfusion (I/R) injury was significantly attenuated in the RIPerC group (P < 0.001). A marked increase of NO content and phosphoserine eNOS, both in protein and mRNA levels, was observed in liver graft of the RIPerC group as compared with the OLT group (P < 0.05). I/R-induced 3-nitrotyrosine content was significantly reduced in the RIPerC group as compared with the OLT group (P < 0.05). There were no significant differences between the RIPerC and IPostC groups for all the results except Cr. The Cr level was lower in the RIPerC group than in the IPostC group (P < 0.01). CONCLUSION: Liver graft protection by RIPerC is similar to or better than that of IPostC, and involves inhibition of oxidative stress and up-regulation of the PI3K/Akt/eNOS/NO pathway.