The impacts of CYP3A4 genetic polymorphism and drug interactions on the metabolism of lurasidone.

The aim of this study was to investigate the impacts of 24 variants of recombinant human CYP3A4 and drug interactions on the metabolism of lurasidone. In vitro, enzymatic reaction incubation system of CYP3A4 was established to determine the kinetic parameters of lurasidone catalyzed by 24 CYP3A4 variants. Then, we constructed rat liver microsomes (RLM) and human liver microsomes (HLM) incubation system to screen potential anti-tumor drugs that could interact with lurasidone and studied its inhibitory mechanism. In vivo, Sprague-Dawley (SD) rats were applied to study the interaction between lurasidone and olmutinib. The concentrations of the analytes were detected by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). As the results, we found that compared with the wild-type CYP3A4, the relative intrinsic clearances vary from 355.77 % in CYP3A4.15 to 14.11 % in CYP3A4.12. A series of drugs were screened based on the incubation system, and compared to without olmutinib, the amount of ID-14283 (the metabolite of lurasidone) in RLM and HLM were reduced to 7.22 % and 7.59 %, and its IC50 were 18.83 ± 1.06 µM and 16.15 ± 0.81 µM, respectively. At the same time, it exerted inhibitory effects both through a mixed mechanism. When co-administration of lurasidone with olmutinib in rats, the AUC(0-t) and AUC(0-∞) of lurasidone were significantly increased by 73.52 % and 69.68 %, respectively, while CLz/F was observably decreased by 43.83 %. In conclusion, CYP3A4 genetic polymorphism and olmutinib can remarkably affect the metabolism of lurasidone.

Metronomic capecitabine inhibits liver transplant rejection in rats by triggering recipients' T cell ferroptosis.

BACKGROUND: Capecitabine (CAP) is a classic antimetabolic drug and has shown potential antirejection effects after liver transplantation (LT) in clinical studies. Our previous study showed that metronomic CAP can cause the programmed death of T cells by inducing oxidative stress in healthy mice. Ferroptosis, a newly defined non-apoptotic cell death that occurs in response to iron overload and lethal levels of lipid peroxidation, is an important mechanism by which CAP induces cell death. Therefore, ferroptosis may also play an important role in CAP-induced T cell death and play an immunosuppressive role in acute rejection after trans-plantation. AIM: To investigate the functions and underlying mechanisms of antirejection effects of metronomic CAP. METHODS: A rat LT model of acute rejection was established, and the effect of metronomic CAP on splenic hematopoietic function and acute graft rejection was evaluated 7 d after LT. In vitro, primary CD3+ T cells were sorted from rat spleens and human peripheral blood, and co-cultured with or without 5-fluorouracil (5-FU) (active agent of CAP). The levels of ferroptosis-related proteins, ferrous ion concentration, and oxidative stress-related indicators were observed. The changes in mito-chondrial structure were observed using electron microscopy. RESULTS: With no significant myelotoxicity, metronomic CAP alleviated graft injury (Banff score 9 vs 7.333, P < 0.001), prolonged the survival time of the recipient rats (11.5 d vs 16 d, P < 0.01), and reduced the infiltration rate of CD3+ T cells in peripheral blood (6.859 vs 3.735, P < 0.001), liver graft (7.459 vs 3.432, P < 0.001), and spleen (26.92 vs 12.9, P < 0.001), thereby inhibiting acute rejection after LT. In vitro, 5-FU, an end product of CAP metabolism, induced the degradation of the ferritin heavy chain by upregulating nuclear receptor coactivator 4, which caused the accumulation of ferrous ions. It also inhibited nuclear erythroid 2 p45-related factor 2, heme oxygenase-1, and glutathione peroxidase 4, eventually leading to oxidative damage and ferroptosis of T cells. CONCLUSION: Metronomic CAP can suppress acute allograft rejection in rats by triggering CD3+ T cell ferroptosis, which makes it an effective immunosuppressive agent after LT.

Liver graft injury caused by de novo donor-specific HLA antibodies in pediatric liver transplant recipients with low, moderate, and high immunologic risk.

BACKGROUND: This study investigated the association between different risk levels of de novo donor-specific anti-human leukocyte antigen antibodies (dnDSAs) and liver graft injury after liver transplantation in pediatric patients. METHODS: This retrospective cohort study enrolled 130 patients after liver transplantation. Subjects were divided into the following 4 groups according to the mean fluorescence intensity (MFI) of dnDSAs: high risk group(MFI ≥10,000), medium risk group(4000 ≤ MFI <10,000), low risk group(500 ≤ MFI <4000), and negative group(<500). Liver function indices were examined along with liver puncture biopsy，and the relationship between dnDSA risk level and liver injury after transplantation was assessed. RESULTS: Pediatric liver transplant recipients showed significant differences in liver function (ALT, AST, GGT and Bilirubin) according to dnDSA risk level (P < 0.05), and no differences in cumulative incidences of rejection (P = 0.413) and liver fibrosis (P = 0.978) were observed among the number of dnDSAs group. There were differences in the cumulative incidences of antibody-mediated rejection (AMR) (P = 0.001) and T cell-mediated rejection （TCMR） (P = 0.003) across risk groups. The cumulative incidences of TCMR and liver fibrosis (P = 0.0001) were higher in the low-risk group than in the other 3 groups. There were no differences in graft survival rate (P = 0.846) across risk groups. CONCLUSION: DnDSAs in pediatric liver transplant recipients are associated with liver transplant rejection and fibrosis. The level of dnDSAs in low risk group should not be disregarded. Routine detection of dnDSAs has clinical utility for noninvasive risk stratification in this population.

Antibody-mediated rejection owing to donor-specific HLA-DQA1 antibodies after renal transplantation: A case report.

BACKGROUND: Donor-specific HLA antibodies are important risk factors in antibody-mediated rejection and graft loss after renal transplantation and are associated with higher rejection rates and lower graft survival. Most de novo donor specific antibodies (dnDSA) after renal transplantation are directed toward donor HLA-DQ antigens. An HLA-DQ antigen is a heterodimer consisting of an alpha and beta chain. Traditionally, HLA-DQA1 typing has not been part of pretransplant evaluation. Therefore, DQ alpha proteins are not usually considered in the interpretation of HLA-DQ antibody reactions. METHODS: The renal transplant recipient had a 0% panel reactive antibody pretransplant. Two years after transplantation, he developed symptoms of abdominal distension and bilateral lower extremity edema. Histopathological findings on renal puncture biopsy showed a combination of T-cell-mediated acute rejection type IIA and antibody-mediated rejection with a trend toward chronicity in the transplanted kidney. DSAs were investigated by HLA-I (HLA-A/B) and HLA-II (HLA-DRB1/DQA1/DQB1) single antigen bead (SAB) assay. HLA typing was performed to explain the antibody reactivity patterns by PCR-SSO and Sequencing-based typing (SBT). HLAMatchmaker analysis was performed to identify eplets that explain antibody reactivity patterns. RESULTS: HLA-II SAB analysis of the patient's serum at the time of rejection showed positive reactions with all DQB1*03:03-carrying beads with high mean fluorescence intensity (MFI). However, DQB1*03:03 was not a dnDSA antigen. High-resolution HLA typing revealed that HLA-DQA1*05:01 and DQA1*03:02 were mismatched donor antigens. HLA Matchmaker analysis demonstrated reactivity toward 130R and 116 V eplet on DQA1 and DQB1. CONCLUSIONS: Antibodies specific to DQα chains after renal transplantation were highlighted.

Comparison of Liver Transplantation and Liver Resection for Hepatocellular Carcinoma Patients with Portal Vein Tumor Thrombus Type I and Type II.

PURPOSE: The aim of this study was to compare the efficacy of liver transplantation (LT) and liver resection (LR) for hepatocellular carcinoma (HCC) patients with portal vein tumor thrombus (PVTT) and to investigate risk factors affecting prognosis. MATERIALS AND METHODS: A total of 94 HCC patients with PVTT type I (segmental PVTT) and PVTT type II (lobar PVTT) were involved and divided into LR (n=47) and LT groups (n=47). Recurrence-free survival (RFS) and overall survival (OS) were compared before and after inverse probability of treatment weighting (IPTW). Prognostic factors for RFS and OS were explored. RESULTS: Two treatment groups were well-balanced using IPTW. In the entire cohort, LT provided a better prognosis than LR. Among patients with PVTT type I, RFS was better with LT (p=0.039); OS was not different significantly between LT and LR (p=0.093). In subgroup analysis of PVTT type I patients with α-fetoprotein (AFP) levels >200 ng/mL, LT elicited significantly longer median RFS (18.0 months vs. 2.1 months, p=0.022) and relatively longer median OS time (23.6 months vs. 9.8 months, p=0.065). Among patients with PVTT type II, no significant differences in RFS and OS were found between LT and LR (p=0.115 and 0.335, respectively). Multivariate analyses showed treatment allocation (LR), tumor size (>5 cm), AFP and aspartate aminotransferase (AST) levels to be risk factors of RFS and treatment allocation (LR), AFP and AST as risk factors for OS. CONCLUSION: LT appeared to afford a better prognosis for HCC with PVTT type I than LR, especially in patients with AFP levels >200 ng/mL.

Long non-coding RNA highly up-regulated in liver cancer promotes exosome secretion.

BACKGROUND: Highly upregulated in liver cancer (HULC) is a long non-coding RNA (lncRNA) which has recently been identified as a key regulator in hepatocellular carcinoma (HCC) progression. However, its role in the secretion of exosomes from HCC cells remains unknown. AIM: To explore the mechanism by which HULC promotes the secretion of exosomes from HCC cells. METHODS: Serum and liver tissue samples were collected from 30 patients with HCC who had not received chemotherapy, radiotherapy, or immunotherapy before surgery. HULC expression in serum exosomes and liver cancer tissues of patients was measured, and compared with the data obtained from healthy controls and tumor adjacent tissues. The effect of HULC upregulation in HCC cell lines and the relationship between HULC and other RNAs were studied using qPCR and dual-luciferase reporter assays. Nanoparticle tracking analysis was performed to detect the quantity of exosomes. RESULTS: HULC expression in serum exosomes of patients with HCC was higher than that in serum exosomes of healthy controls, and HULC levels were higher in liver cancer tissues than in tumor adjacent tissues. The expression of HULC in serum exosomes and liver cancer tissues correlated with the tumor-node-metastasis (TNM) classification, and HULC expression in tissues correlated with that in serum exosomes. Upregulation of HULC promoted HCC cell growth and invasion and repressed apoptosis. Notably, it also facilitated the secretion of exosomes from HCC cells. Moreover, qPCR assays showed that HULC repressed microRNA-372-3p (miR-372-3p) expression. We also identified Rab11a as a downstream target of miR-372-3p. Dual-luciferase reporter assays suggested that miR-372-3p could directly bind both HULC and Rab11a. CONCLUSION: Our findings illustrate the importance of the HULC/miR-372-3p/Rab11a axis in HCC and provide new insights into the molecular mechanism regulating the secretion of exosomes from HCC cells.

PIK3C2A mRNA functions as a miR-124 sponge to facilitate CD151 expression and enhance malignancy of hepatocellular carcinoma cells.

Competing endogenous RNAs (ceRNAs) are RNA transcripts that can crosstalk with each other by competing for shared microRNAs (miRNAs) through miRNA response elements (MREs). Involved in ceRNA networks, the RNA transcripts may be in a balance, disruption of which could lead to tumorigenesis. Here we reveal a ceRNA interaction between PIK3C2A and CD151 mRNAs in hepatocellular carcinoma (HCC) cells. PIK3C2A is a candidate ceRNA of CD151 because mRNA 3' untranslated regions (3'UTRs) of these two genes contain miR-124 binding sites. miR-124 is downregulated, while PIK3C2A and CD151 are upregulated in HCC cells compared with normal hepatocytes. Direct and negative regulation of PIK3C2A and CD151 by miR-124 was confirmed in HCC cells. miR-124 and the two potential ceRNAs are all recruited to the RNA-induced silencing complex (RISC). In HCC cell lines QGY- 7703 and SMMC-7721, and normal hepatic cell line HL-7702, miR-124 plays a tumor suppressor role by targeting PIK3C2A and CD151. The MREs within PIK3C2A 3'UTR can independently stimulate CD151 expression level by acting as miR-124 decoys. PIK3C2A MREs enhance HCC cell malignancy by absorbing endogenous miR-124 and activating CD151 in HCC cells. We conclude that PIK3C2A 3'UTR functions as a trans activator to stimulate CD151 by competing for miR-124 binding in HCC cells. The collaboration of PIK3C2A and CD151 through ceRNA mechanism may be implicated in HCC initiation and development.

[A study on mutations of the overlapping hepatitis B virus surface and polymerase gene in patients with HBV reinfection after liver transplantations].

OBJECTIVE: To investigate the influence of combined hepatitis B immune globulin (HBIG) and lamivudine (LMV) treatment on hepatitis B virus (HBV) surface antigen and polymerase overlapping gene mutations in HBV reinfected liver transplant recipients. METHODS: From June 2002 to December 2003, 320 patients who underwent liver transplantations due to HBV-related end-stage liver diseases were followed-up for 1.5 to 3 years postoperatively. Fourteen patients developed HBV reinfection. They had LMV before their liver transplantations and had LMV and HBIG after the transplantations to prevent HBV infections. Their serum levels of HBV DNA were measured by polymerase chain reaction. Gene sequencing method was used to analyze HBV genotype and mutations of the S gene. Micro-particle enzyme immunoassay was used to measure the serum concentration of HBIG. RESULTS: (1) There was no obvious difference in the number of amino acid mutation sites in S and P regions before and after the transplantations. (2) The HBV genotypes were B-type (n=2) and C-type (n=12) in the reinfected group before the transplantations, and genotypes after the transplantations remained the same. (3) HBIG concentrations were 0 U/L in 7 patients, less than 100 U/L in 5 patients, and more than 100 U/L in 2 patients. Mutations were detected as I126S, T131N, S143T and G145R in 'a' determinant and L110F, I113S, T160K in up- or down-stream of 'a' determinant. (4) Mutations in S gene caused missense mutation in the surface antigen region. These mutations also caused corresponding missense mutations in the polymerase region. The missense mutation in the polymerase region involved lamivudine mutation sites and other mutation sites. CONCLUSION: Immunosuppressant therapy has no obvious influence on the numbers of mutations, but it can influence the sites of the mutations. Besides 'a' determinant mutations, there exist mutations in up- or down-streams of 'a' determinant and they may cause HBV reinfection.

[A clinico-pathological analysis of HCV infection in post-liver transplantation patients].

OBJECTIVE: To investigate the pathological characteristics of HCV infection after liver transplantation. METHODS: This is a retrospective analysis of the clinico-pathological changes of 73 liver biopsies obtained from 61 patients who had HCV infection (including HCV recurrence and reinfection) after liver transplantation in our center from September 2000 to September 2006. RESULTS: Abnormal enzyme test results due to HCV infection happened on the 9th to the 1553rd post-transplantation surgery day. The serum HCV RNA level was higher than 10(5) copies/ml in 19 cases and between 10(2)-10(5) copies/ml in the other 42 cases. The histological changes in the transplanted livers were hepatocellular degeneration, necrosis and apoptosis, portal infiltrations and fibrosis. They were classified into two stages (early stage and late stage) according to the onset of fibrosis which appeared within 90 days or later after their transplantation in our study. The incidence of predominant portal infiltrates and liver fibrosis in early stage and late stage was 5.7% (2/35) and 94.7% (36/38) (chi2=54.34, P<0.01) and 2.9% (1/35) and 97.4% (37/38) (chi2=61.47, P<0.01) respectively. CONCLUSIONS: Pathological features of early stage and late stage hepatitis C infection in transplanted livers are different and they are also different from that in native livers. Liver biopsies are important in clinical staging, evaluation of the severity, and differential diagnosis of post-transplantation HCV infection.

[Clinical study of Milan criteria for liver transplantation].

OBJECTIVE: To investigate the value and deficiency of Milan criteria for liver transplantation in patients with hepatocellular carcinoma (HCC). METHODS: Between December 2001 and November 2005, 125 patients underwent orthotopic liver transplantation ( OLT), who measured up Milan criteria with preoperation computerized tomography (CT) scanning. The results of pre-transplant multidetector CT scan and post-transplant pathology were retrospectively analyzed, and survival rates were compared. RESULTS: Pathology examination demonstrated that 97 cases met Milan criteria (77.6%), 26 cases exceeded Milan criteria,and the other 2 cases were diagnosed as nodular cirrhosis. The 1-,2-,3-,4- and 5-year survival rates for those met pre-transplant multidetector CT scanning pre-transplant met Milan criteria vs. those met post-transplant pathology post-transplant criteria were 92.0% vs. 92.8%, 87.2% vs. 90.7%, 86.4% vs. 89.7%, 86.4% vs. 89.7%, and 86.4% vs. 89.7%, respectively. There was no statistic significant difference (P > 0.05). The 1-,2-,3-,4- and 5-year survival rates were 73.0%, 65.4%, 61.5%, 61.5% and 61.5%, for those pathology exceed Milan criteria respectively. The difference between this group and each of the above two were statistically significant (P < 0.05). CONCLUSIONS: The prognosis of OLT for HCC is good for those met Milan criteria by pre-transplant multidetector CT. Factors leading to poor prognosis such as portal vein tumor thrombi and lymphatic metastasis should be accurately evaluated avoiding for misjudgement.

[Clinicopathological analysis of biliary tract complications on post-liver transplantation patients].

OBJECTIVE: To analyze the pathohistological changes of the livers and the clinical features of patients with biliary tract complications after their orthotopic liver transplantations. METHODS: From Sept 1998 to June 2005 clinical and pathological data of 173 post-liver transplantation patients with biliary tract complications were analyzed. RESULTS: Biliary tract complications occurred within 3-2920 days after the transplantation operations. These complications occurred within 1-30 days, 31-90 days, 91-180 days, 180 days at rates of 49.71%, 17.92%, 4.62%, 27.74% respectively. The complications were of inflammatory nature in 171 cases, (72.25%), and of obstructive nature in 164 cases (27.74%). The main pathological changes were epithelium degeneration of interlobular bile ducts, inflammatory cell infiltration in portal areas, proliferation of interlobular bile ducts, fibrosis in portal areas, cholestasis in small bile ducts and hepatocytes. CONCLUSION: Many of the biliary tract complications of post-liver transplantation in our cases were of inflammatory nature and they often occurred within 30 days after the surgery. Obstructive nature complications often occurred in 90 days after the surgery and the prognosis of these cases was much poorer. The pathological changes of live tissues shown in liver biopsies are important for prognostic evaluation, differential diagnosis and categorization of biliary tract complications.

[Pathologic diagnosis of 1123 post-transplant liver biopsies from 665 liver transplant patients].

OBJECTIVE: To summarize the Chinese experience in pathologic diagnosis of liver biopsies after orthotopic liver transplantation (OLTx). METHODS: 1123 post-transplant liver biopsies from 665 OLTx patients from the Shanghai Eastern Hepatobiliary Surgery Hospital, Tianjin First Central Hospital, Guangzhou Sun Yat-sen University and Chongqing Southwest Hospital were retrospectively analyzed. All liver biopsies were stained with hematoxylin and eosin. Immunohistochemical studies for cytomegalovirus, HBsAg, CK19, CD4 and CD8 were also performed in selected examples. RESULTS: In the involved hospitals, 4 to 12 types of complications were encountered after OLTx. The number of liver biopsies performed for each patient ranged from 1 to 9 (mean = 2.2). The timing of these biopsies varied from the second to the 2877 th post-transplant day. The 5 most common complications were acute cellular rejection (35.6%), ischemic-reperfusion injury (13.4%), biliary stricture (5.6%), drug complication (5.0%) and chronic rejection (4.7%). The 5 earliest complications after OLTx were primary non-function (occurring at day 4.7 +/- 2.1), ischemic-reperfusion injury (occurring at day 14.0 +/- 4.0), acute cellular rejection (occurring at day 32.1 +/- 62.9), hepatic artery thrombosis / stricture (occurring at day 62.9 +/- 74.2) and cytomegalovirus infection (occurring at day 107.7 +/- 93.0). CONCLUSIONS: This study has evaluated the types, incidence and timing of major complications occurring after OLTx. The most important issue is the distinction between rejection and non-rejection pathology. Thorough understanding of atypical pathologic features of these complications is necessary. The Banff Schema (rejection activity index) for grading liver allograft rejection is useful for monitoring anti-rejection therapy and should be used routinely.