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.

Regulatory effect of rat bone marrow mesenchymal stem cells on Treg/Th17 immune balance in vitro.

Bone marrow mesenchymal stem cells (BM­MSCs) regulate the balance between regulatory T cells (Tregs) and T helper 17 (Th17) cells. However, the role of different factors on BM­MSCs­mediated regulation of the Treg/Th17 balance is unknown. BM­MSCs and CD4+ T lymphocytes were co­cultured with various treatments. The ratio of Treg/Th17 cells was calculated and the expression of different cytokines was measured. BM­MSCs were found to have a proliferative effect on Th17 cells at a basal concentration and at a 2­fold increase in the number of BM­MSCs. However, when the number of BM­MSCs used was increased 4­fold, they had an inhibitory effect on the Th17 cells. The effect of BM­MSCs on Tregs was inhibited by the addition of tacrolimus but not rapamycin. The effect of BM­MSCs on Th17 cells was inhibited by rapamycin. Additionally, the effect of BM­MSCs on Tregs were inhibited by the addition of a transforming growth factor­ß (TGF­ß) blocker, whereas these TGF­ß­blockers had no effect on Th17 cells. Addition of an interleukin (IL)­2 blocker reduced the proportion of Th17 cells when co­cultured with a high number of MSCs compared with the low concentration group and the proportion of Treg cells was significantly decreased when cells were treated with an IL­2 blocker compared with the control group. Together, these results showed the varying effects of MSCs on the ratio of Treg/Th17, its dependence on the number of MSCs and the effects of cytokines in inducing these changes in the balance.

The T-helper cells 17 instead of Tregs play the key role in acute rejection after pediatric liver transplantation.

Th17 and imbalance of Treg/Th17 might be one of the mechanisms of acute rejection. We aim to explore the role of Th17s in the balance of Treg/Th17 in acute rejection after LT in children diagnosed with BA. The ratios of Treg and Th17 in peripheral blood were detected by flow cytometry pre-LT, post-LT, and when rejection occurred. Treg proportion was higher before transplantation than at 2 weeks and 1 month after transplantation, with no statistical difference between 2 weeks and 1 month. However, Treg proportions were lower in pediatric recipients than healthy controls. The proportion of Tregs before anti-rejection treatment was lower than control group, with no statistical difference compared to the stable group and it showed no difference compared with that at 2 weeks and 1 month post-LT. The Th17 proportions were higher at 2 weeks and 1 month after transplantation than healthy controls. The Th17 proportion under the circumstances of rejection was higher than that in the stable group and control group; the proportion in stable group was higher than that in control group. After anti-rejection therapy, the proportions of Th17 were lower than those before therapy. In conclusion, the imbalance of Treg/Th17, especially Th17s instead of Tregs, may be one of the important mechanisms in acute rejection.

Different phenotypes of CD4+CD25+Foxp3+ regulatory T cells in recipients post liver transplantation.

CD4+ regulatory T cells (Tregs) play an important role in inducing immune tolerance in organ transplantation, which can be divided into CD45RA+Tregs (resting Tregs, rTregs) and CD45RO+Tregs (activated Tregs, aTregs). Currently, the expressions and phenotypic changes of Tregs in recipients after liver transplantation (LT) is unknown. We therefore investigated the expression and transformation of rTregs and aTregs in 83 cases of recipients with normal status post-LT. The percentages of CD45RA, CD45RO, CD31 in CD4+Tregs were detected by flow cytometry and the effective factors were analyzed. In LT recipients, the percentage of CD45RO+Tregs in CD4+Tregs was higher than that of CD45RA+Tregs. There was significant difference in the ratio of positive Foxp3 between CD45RA+Tregs and CD45RO+Tregs. Percentage of CD45RA+Tregs was higher in pediatric group than that in adult group, whereas percentage of CD45RO+Tregs was lower in the pediatric group. However, it was different only in CD45RO+Tregs in various survival periods post-LT. In conclusion, Tregs pool in human was heterogeneous post-LT and contained different subsets in phenotypes. Upon stimulation by donor graft, percentages of CD4+Tregs and CD45RO+Tregs were increased post-LT and most of rTregs was transformed into aTregs in peripheral blood, and rTregs and aTregs were both related to recipients' ages.

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.

Acute Wnt pathway activation positively regulates leptin gene expression in mature adipocytes.

Genome-wide association studies (GWAS) have revealed the implication of several Wnt signaling pathway components, including its effector transcription factor 7-like 2 (TCF7L2) in diabetes and other metabolic disorders. As TCF7L2 is expressed in adipocytes, we investigated its expression and function in rodent fat tissue and mature adipocytes. We found that TCF7L2 mRNA expression in C57BL/6 mouse epididymal fat tissue was up-regulated by feeding but down-regulated by intraperitoneal insulin injection. In high-fat diet (HFD) fed mice, db/db mice and Zucker (fa/fa) rats, epididymal fat TCF7L2 mRNA levels were lower than the corresponding controls. Treating rat adipocytes with 100nM insulin repressed TCF7L2 mRNA and protein levels, associated with the repression of leptin mRNA level. The treatment with 1nM insulin, however, stimulated TCF7L2 and leptin mRNA levels. This stimulation could be attenuated by iCRT14, an inhibitor of ß-catenin/TCF-responsive transcription. Wnt3a stimulated leptin mRNA level, which was also blocked by iCRT14 co-treatment. Utilizing the leptin-expressing cell line HTR8 as a tool, we defined an evolutionarily conserved CREB binding motif that mediated Wnt3a activation. Although Wnt activation is known to repress the differentiation of 3T3-L1 cells towards mature adipocytes, short-term Wnt3a treatment of differentiated 3T3-L1 cells stimulated leptin mRNA levels. Thus, wnt pathway plays a dual function in adipocytes, including the well-known repressive effect on adipogenesis and the stimulation of leptin production in mature adipocytes in response to nutritional status.

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.