Dynamic immune recovery process after liver transplantation revealed by single-cell multi-omics analysis.

Elucidating the temporal process of immune remodeling under immunosuppressive treatment after liver transplantation (LT) is critical for precise clinical management strategies. Here, we performed a single-cell multi-omics analysis of peripheral blood mononuclear cells (PBMCs) collected from LT patients (with and without acute cellular rejection [ACR]) at 13 time points. Validation was performed in two independent cohorts with additional LT patients and healthy controls. Our study revealed a four-phase recovery process after LT and delineated changes in immune cell composition, expression programs, and interactions along this process. The intensity of the immune response differs between the ACR and non-ACR patients. Notably, the newly identified inflamed NK cells, CD14+RNASE2+ monocytes, and FOS-expressing monocytes emerged as predictive indicators of ACR. This study illuminates the longitudinal evolution of the immune cell landscape under tacrolimus-based immunosuppressive treatment during LT recovery, providing a four-phase framework that aids the clinical management of LT patients.

Corrigendum: Case report: Reconstruction of distal medial tibial epiphysis using iliac crest apophyseal autograft.

[This corrects the article DOI: 10.3389/fped.2022.950211.].

Genotype-guided model significantly improves accuracy of tacrolimus initial dosing after liver transplantation.

Background: The initial dose of tacrolimus after liver transplantation (LT) is critical for rapidly achieving the steady state of the drug concentration, minimizing the potential adverse reactions and warranting long-term patient prognosis. We aimed to develop and validate a genotype-guided model for determining personalized initial dose of tacrolimus. Methods: By combining pharmacokinetic modeling, pharmacogenomic analysis and multiple statistical methods, we developed a genotype-guided model to predict individualized tacrolimus initial dose after LT in the discovery (n = 150) and validation cohorts (n = 97) respectively. This model was further validated in a prospective, randomized and single-blind clinical trial from August, 2021 to February, 2022 (n = 40, ChiCTR2100050288). Findings: Our model included donor's and recipient's genotypes, recipient's weight and total bilirubin, which achieved an area under the curve of receiver operating characteristic curve (AUC of ROC) of 0.88 and 0.79 in the discovery and validation cohorts, respectively. We found that patients who were given tacrolimus within the recommended concentration range (RCR) (4-10 ng/mL), the new-onset metabolic syndromes are lower, especially for new-onset diabetes (p = 0.043). In the clinical trial, compared to those in experience-based (EB) group, patients in the model-based (MB) group were more likely to achieving the RCR (75% vs 40%, p = 0.025) with a more variable individualized dose (0.023-0.096 mg/kg/day vs 0.045-0.057 mg/kg/day). Moreover, significantly fewer medication adjustments were required for the MB group than the EB group (2.75 ± 2.01 vs 6.05 ± 3.35, p = 0.001). Interpretation: Our genotype-based model significantly improved the initial dosing accuracy of tacrolimus and reduced the number of medication adjustments, which are critical for improving the prognosis of LT patients. Funding: National Natural Science Foundation of China, Shanghai three-year action plan, National Science and Technology Major Project of China.

Case report: Reconstruction of distal medial tibial epiphysis using iliac crest apophyseal autograft.

Background: Salter-Harris type VI physeal fracture is a rare injury. This case study aims to present a novel method for treating a rare entity of Salter-Harris type Salter-Harris VI physeal injury of the medial malleolus. Case presentation: A 6-year-old boy with Salter-Harris type VI physeal injury was successfully treated using the two-stage procedure. In the first stage, the patient was treated with intravenous antibiotics, a series of debridement and lavage followed by a skin graft that left a defect in the medial malleolus. In the second stage, an autogenous iliac crest apophyseal graft was transplanted to reconstruct the medial malleolus, and the ankle joint was stabilized by an external fixator. An additional anticipatory Langenskiold procedure was performed for the physeal bar resection. Although the complete radiological development of medial malleolus compared to the contralateral side was not evident at the last follow-up, the functional and cosmetic outcomes were satisfactory. Conclusion: The reconstruction of medial malleolus using an autologous iliac crest apophyseal graft and stabilization of the ankle joint with an external fixator is a novel reconstruction technique in treating Salter-Harris type VI physeal injury of the medial malleolus. This technique provides satisfactory functional and cosmetic outcomes in such a fracture pattern; however, a further clinical study using a larger sample size is warranted in order to find the definitive outcome of the technique.

Hepatic pannexin-1 mediates ST2+ regulatory T cells promoting resolution of inflammation in lipopolysaccharide-induced endotoxemia.

Sepsis remains the most lethal infectious disease and substantially impairs patient prognosis after liver transplantation (LT). Our previous study reported a role of the pannexin 1 (PANX1)-interleukin-33 (IL-33) axis in activating innate immunity to protect against methicillin-resistant Staphylococcus aureus infection; however, the role of PANX1 in regulating adaptive immunity in sepsis and the underlying mechanism are unclear. In this study, we examined the role of the PANX1-IL-33 axis in protecting against sepsis caused by a gram-negative bacterial infection in an independent LT cohort. Next, in animal studies, we assessed the immunological state of Panx1-/- mice with lipopolysaccharide (LPS)-induced endotoxemia and then focused on the cytokine storm and regulatory T cells (Tregs), which are crucial for the resolution of inflammation. To generate liver-specific Panx1-deficient mice and mimic clinical LT procedures, a mouse LT model was established. We demonstrated that hepatic PANX1 deficiency exacerbated LPS-induced endotoxemia and dysregulated the immune response in the mouse LT model. In hepatocytes, we confirmed that PANX1 positively regulated IL-33 synthesis after LPS administration. We showed that the adenosine triphosphate-P2X7 pathway regulated the hepatic PANX1-IL-33 axis during endotoxemia in vitro and in vivo. Recombinant IL-33 treatment rescued LPS-induced endotoxemia by increasing the numbers of liver-infiltrating ST2+ Tregs and attenuating the cytokine storm in hepatic PANX1-deficient mice. In conclusion, our findings revealed that the hepatic PANX1-IL-33 axis protects against endotoxemia and liver injury by targeting ST2+ Tregs and promoting the early resolution of hyperinflammation.

Reduced pannexin 1-IL-33 axis function in donor livers increases risk of MRSA infection in liver transplant recipients.

Liver transplantation patients are at increased risk for methicillin-resistant Staphylococcus aureus (MRSA) infection, but the molecular mechanism remains unclear. We found that genetic predisposition to low pannexin 1 (PANX1) expression in donor livers was associated with MRSA infection in human liver transplantation recipients. Using Panx1 and Il-33-knockout mice for liver transplantation models with MRSA tail vein injection, we demonstrated that Panx1 deficiency increased MRSA-induced liver injury and animal death. We found that decreased PANX1 expression in the liver led to reduced release of adenosine triphosphate (ATP) from hepatocytes, which further reduced the activation of P2X2, an ATP-activating P2X receptor. Reduced P2X2 function further decreased the NLRP3-mediated release of interleukin-33 (IL-33), reducing hepatic recruitment of macrophages and neutrophils. Administration of mouse IL-33 to Panx1-/- mice significantly (P = 0.011) ameliorated MRSA infection and animal death. Reduced human hepatic IL-33 protein abundance also associated with increased predisposition to MRSA infection. Our findings reveal that genetic predisposition to reduced PANX1 function increases risk for MRSA infection after liver transplantation by decreasing hepatic host innate immune defense, which can be attenuated by IL-33 treatment.

Association between donor and recipient Interleukin-18 gene polymorphisms and the risk of infection after liver transplantation.

PURPOSE: The purpose of this study was to retrospectively evaluate the association between Interleukin-18 (IL-18) gene polymorphisms of the donor and recipient in liver transplant patients with bacterial infections. METHODS: Five single nucleotide polymorphisms (SNPs) (rs7106524, rs5744247, rs1946518, rs549908 and rs187238) of the IL-18 gene from the donors were genotyped and their association with post-operative bacterial infections was evaluated in liver transplant patients (N=113). A second independent group of liver transplant patients from a different organ transplant centre was also recruited for validation purposes (N=44). RESULTS: IL-18 mRNA mean expression levels and protein levels were significantly lower in liver transplant patients with bacterial infections. For the donor SNP rs1946518, more recipients carried the A allele in the bacterial-infected group than the uninfected group (61.4% vs 39.7%; P ≤0.002). The mean IL-18 mRNA expression and protein levels were significantly lower in the transplanted livers of recipients carrying the rs1946518 AA genotype compared with those from recipients with CC genotype (3.64, 3.33 vs. 2.75, P≤0.048). The A allele of rs1946518 also resulted in lower luciferase activity than the C allele in a reporter assay. The area under ROC curve indicated that the rs1946518 SNP genotype in the donor liver predicted an increased risk of bacterial infection after liver transplantation (AUROC>0.82). CONCLUSIONS: These findings indicate that the IL-18 rs1946518 SNP in the donor liver is a risk factor for developing bacterial infection after liver transplantation.

MicroRNA-191 acts as a tumor promoter by modulating the TET1-p53 pathway in intrahepatic cholangiocarcinoma.

Current treatment of intrahepatic cholangiocarcinoma (ICC) remains ineffective because knowledge of ICC carcinogenesis is unclear. Increasing evidence suggests that microRNAs (miRNAs), including miR-191, play an important role in tumorigenesis; but expression and biological functions of miR-191 in ICC remain to be established. This study investigated the functions and underlying mechanisms of miR-191 in ICC. ICC miRNA profiles were generated in five pairs of ICC and matched to normal bile duct tissues by next-generation sequencing technology; ICC miRNA profiles were verified in 18 pairs of ICC tissues and normal bile duct tissues by quantitative RT-PCR. The miR-191-associated mechanisms in ICC were investigated in vitro and in vivo, and clinical outcomes associated with miR-191 were correlated in 84 patients. Our results showed that miR-191 expression was significantly increased in ICC compared with the adjacent normal bile duct tissues (P < 0.001). Overexpression of miR-191 promoted proliferation, invasion, and migration of cholangiocarcinoma cells in vitro and in vivo. The elevated miR-191 expression reduced the expression level of ten-eleven translocation 1 (TET1)-a direct target gene of miR-191 in ICC, which catalyzes demethylation. The reduced TET1 expression level allowed the methylated CpG-rich regions at the p53 gene transcription start site stay methylated, leading to reduced p53 expression level, which compromises p53's anticancer vigor. Finally, miR-191 was found to be an independent risk factor for poor prognosis in patients with ICC (overall survival, hazard ratio = 3.742, 95% confidence interval 2.080-6.733, P < 0.001; disease-free survival, hazard ratio = 2.331, 95% confidence interval 1.346-4.037, P = 0.003). CONCLUSION: Our results suggest that overexpressed miR-191 is associated with ICC progression through the miR-191/TET1/p53 pathway. (Hepatology 2017;66:136-151).

Prognosis for recipients with hepatocellular carcinoma of salvage liver transplantation versus those of primary liver transplantation: a retrospective single-center study.

PURPOSE: The prognosis for recipients with hepatocellular carcinoma (HCC) of salvage liver transplantation (SLT) versus those of primary liver transplantation (PLT) remains controversial. The objective of this study was to evaluate the clinical features and survival rate of SLT recipients. METHODS: Three hundred seventy-one patients with HCC transplanted at Shanghai General Hospital, China, between October 2001 and October 2011 were separated into PLT (n = 295) and SLT (n = 76) groups. Patient characteristics and survival curves were studied by univariate and multivariate analysis. A Milan criteria-stratified survival analysis was conducted. RESULTS: The proportions of reoperation (11.8 vs. 5.4 %, P = 0.047) and early postoperative mortality (11.8 vs. 4.7 %, P = 0.032) were higher in the SLT group than in the PLT group. Recurrence free survival (RFS) rate and overall survival (OS) rate had no statistically significant differences after stratification using Milan criteria between the PLT group and SLT group. Alphafetoprotein >400 ng/mL (P = 0.011), microscopic vascular invasion (MVI) (P < 0.001), tumor node metastasis (TNM) staging (P = 0.006), and out of Milan criteria (P < 0.001) were independent risk factors for RFS, while MVI (P < 0.001), TNM staging (P = 0.009), and out of Milan criteria (P = 0.003) were factors for OS. In the multivariate logistic regression analysis, HCC recurrence was associated with MVI (OR = 4.196 [2.538-6.936], P < 0.001), and out of Milan criteria (OR = 2.704 [1.643-4.451], P < 0.001). CONCLUSIONS: Our retrospective, single-center study demonstrated that SLT increases surgical difficulty; however, it has good post-transplantation OS and is a feasible alternative after HCC recurrence within Milan criteria.

Mannose-binding lectin 2 rs11003123 polymorphism is associated with the development of hepatocellular carcinoma in patients with hepatitis B-related cirrhosis in the Chinese population.

BACKGROUND: Mannose-binding lectin 2 (MBL2) plays a key role in the host immune response, but whether it is associated with hepatocellular carcinoma (HCC) is not clear. The present study aimed to identify the association between MBL2 gene polymorphisms and HCC in patients with hepatitis B virus (HBV)-related cirrhosis in the Chinese population. METHODS: A single-nucleotide polymorphism of MBL2, rs11003123, was genotyped and analyzed in a case-control study of HBV-related cirrhotic patients with HCC (n=77) and without HCC (n=40). RESULTS: We found that Child-Pugh profiles, model for end-stage liver disease score, and the incidence of encephalopathy were all higher in the non-HCC group (P<0.05). A significant association between allele mutants and HCC occurrence was demonstrated by allele comparison (A vs G) (OR=0.34; 95% CI: 0.15-0.76; P=0.006). Heterozygous comparison (GA vs GG) revealed that the individuals with GA mutants had a reduced risk of HCC occurrence compared with those with GG wild type (adjusted OR=0.28; 95% CI: 0.10-0.80; P=0.004). In a dominant model (GA+AA vs GG), a decreased risk of HCC occurrence was observed in individuals with variant genotypes (GA and AA) compared with those with the wild type (adjusted OR=0.30; 95% CI: 0.11-0.85; P=0.004). However, no statistically significant associations were observed between rs11003123 and prognosis of patients with HCC after liver transplantation in both recurrence-free survival and overall survival (P=0.449 and P=0.384, respectively). CONCLUSION: MBL2 rs11003123 polymorphism may be a marker for the risk of HCC occurrence in patients with HBV-related cirrhosis in the Chinese population.

Prognostic factors in patients with recurrent hepatocellular carcinoma treated with salvage liver transplantation: a single-center study.

Although salvage liver transplantation (LT) has been widely adopted as a treatment for recurrent hepatocellular carcinoma(HCC), candidate selection criteria have not been established. This single-center study aimed to identify risk factors associated with HCC recurrence and survival following salvage LT. The study included 74 patients treated with salvage LT between October 2001 and February 2013. The median follow-up was 37.2 months after LT. There were 29 cases of HCC recurrence and 31 deaths following LT. Microvascular invasion at the time of liver resection, a time interval to post-LR HCC recurrence of ≤ 12months, an alpha-fetoprotein level at LT greater than 200 ng/mL, and having undergone LT outside of the UCSF criteria were independent risk factors for HCC recurrence after salvage LT. Patients with no more than one risk factor had a 5-year recurrence-free survival rate of 71.2% compared to 15.9% in patients with two or more risk factors. These findings suggest that to avoid post-LT HCC recurrence and a dismal prognosis, patients with no more than one risk factor for recurrence should be given priority for salvage LT. These criteria may improve the outcomes of patients treated with salvage LT and facilitate the effective use of limited organ supplies.

C7 genotype of the donor may predict early bacterial infection after liver transplantation.

Post-transplantation infection causes high mortality and remains a significant challenge. High clinical risk factors for bacterial infection in recipients are often found in critically ill patients. However, for some recipients, bacterial infections are inevitable. It is conceivable that this susceptibility may be related to the genetics of the donor and recipient. Using expression quantitative trait loci (eQTL) analysis, we found that the C7 rs6876739 CC genotypes and mannan-binding lectin (MBL2) gene polymorphisms of liver donors were significantly associated with bacterial infection in recipients. In an extended validation group of 113 patients, donor C7 rs6876739 genetic variation was an independent risk factor for bacterial infection. The donor C7 rs6876739 CC genotype was associated with lower levels of recipient C7 protein, soluble membrane attack complex (MAC), and IL-1ß expression compared with the donor C7 rs6876739 TT genotype. In vitro, the MAC significantly triggered NLRP3 inflammasome activation and IL-1ß release, suggesting that the mechanism by which C7 defends against bacteria may involve MAC formation, leading to NLRP3 inflammasome activation and IL-1ß release. Our findings may be helpful in identifying transplantation recipients at risk of bacterial infection prior to surgery and may contribute to novel infection prevention strategies and the improvement of postoperative outcomes.

Association between donor and recipient smoothened gene polymorphisms and the risk of hepatocellular carcinoma recurrence following orthotopic liver transplantation in a Han Chinese population.

Hepatocellular carcinoma (HCC) recurrence after orthotopic liver transplantation (OLT) is potential cause for the poor outcome. Smoothened (SMO) gene has been considered associating with HCC and HCC recurrence, but its association with HCC recurrence after OLT is not clear yet. In this study, we aim at evaluating the association between donor and recipient SMO gene polymorphisms and HCC recurrence after OLT. A total of 76 patients with HCC who had undergone OLT from July 2007 to August 2012 were included. A single nucleotide polymorphism (SNP), SMO rs3824, located at the 3'UTR region, was genotyped and analyzed in both donor and recipient. We demonstrated that recipient rs3824 polymorphism was significantly associated with HCC recurrence following OLT. In multivariate logistic regression analysis, TNM stage (p = 0.001), recipient SMO rs3824 genotype (CG vs. CC/GG p = 0.001), and histologic grade (p = 0.019) were identified as independent risk factors of HCC recurrence. Recurrence-free survival (RFS) and overall survival (OS) were significantly higher in the recipient CC/GG group than in the CG group (p = 0.003 and p = 0.011, respectively). Cox proportional hazards modeling revealed that TNM stage, recipient SMO rs3824 genotype, pre-OLT serum AFP level, and histologic grade were independent factors (p < 0.05) for patients' clinical outcomes. In conclusion, recipient SMO rs3824 polymorphism is associated with an increased risk of HCC recurrence following OLT and has a potential clinical value for the prognosis of HCC patients treated with OLT.

Impact of age on the prognosis after liver transplantation for patients with hepatocellular carcinoma: a single-center experience.

BACKGROUND: Liver transplantation (LT) offers the most effective treatment for hepatocellular carcinoma patients. Various preoperative variables are correlated with survival after LT, but the prognostic role of aging on LT remains controversial. METHODS: Between January 2001 and December 2011, 290 consecutive transplants for patients with hepatocellular carcinoma performed in Shanghai First People's Hospital (People's Republic of China) were analyzed retrospectively. We compared patient characteristics and survival curves between a younger group (less than 49 years, n=135) and an aged group (50 years or older, n=155). We then performed Cox multivariate regression analysis of the risk factors for survival in aged and younger patients. RESULTS: Younger age was associated with higher alpha-fetoprotein (P=0.014), larger tumor size (P=0.038), poorer differentiation (P=0.025), portal lymph node metastasis (P=0.001), and higher recurrence rate (P=0.038). Aged patients had significantly longer recurrence-free survival and overall survival (P=0.020 and P=0.014, respectively); however, there were no significant differences between the younger and aged patients who met the Milan criteria (P>0.05). The 1-, 3-, and 5-year recurrence-free survival rates were 59.7%, 44.5%, and 37.3%, respectively, in the younger group, and 67.9%, 55.3%, and 53.8%, respectively, in the aged group. The 1-, 3-, and 5-year overall survival rates were 68.4%, 45.5%, and 38.9%, respectively, in the younger group, and 76.1%, 59.7%, and 53.9%, respectively, in the aged group. Alpha-fetoprotein ≥400 ng/mL, microvascular invasion, and tumor size >5 cm were independent risk factors for prognosis in both groups. CONCLUSION: Younger patients in our center tended to present with more aggressive tumors and have a higher risk of recurrence. Our single-center experience suggests that younger patients should be assessed more rigorously before LT, while aged patients should be actively considered for LT after appropriate selection.