Galectin-9 contributes to the survival of fully allogeneic skin grafts by modulating effector T cells and Tregs imbalance.

Allogeneic skin transplantation is an important clinical treatment for many diseases. Although Galectin-9 demonstrates multifaceted roles in modulating immune cell homeostasis and inflammation, its precise impact on balancing effector T cells and Tregs during allogeneic skin transplantation remains uncertain. This work was performed to evaluate the modulation of the survival time of allogeneic skin grafts by Gal-9 and to explore the critical mechanism involved in this process. Skin graft transplantation was conducted using C57BL/6 and BALB/c mice. Additionally, the levels of IL-2, IFN-γ, IL-4, and IL-17A were measured. Hematoxylin and eosin staining assay was performed to analyze the pathological conditions of skin grafts of experiment mice. The results revealed that Gal-9 noticeably prolonged the survival of the allogeneic skin graft and ameliorated the damage caused by acute immune rejection. Furthermore, Gal-9 resulted in selective reduction of effectors T cells such as Th1, and Th17. Simultaneously, Gal-9 didn't attenuate the protective function for allograft, which alleviated the immune rejection caused by abnormal immune imbalance. Gal-9 exhibited a therapeutic effect on the allogeneic skin graft by selectively reducing CD4+TIM-3+ T effector cells and inducing a shift from a Th1 to an anti-inflammatory Th2 response. Furthermore, Gal-9 did not attenuate the protective function. Our present study may represent a novel therapeutic candidate for modulating effector T cells and Tregs imbalance-based therapy of allograft transplantation.

LNCRNA XIST Inhibits miR-377-3p to Hinder Th17 Cell Differentiation through Upregulating ETS1.

Background: Th17 cell differentiation is involved in the development and progression of many diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Present study mainly focused on the role of LINC-XIST in Th17 cell differentiation. Methods: The naïve CD4+ T cells were isolated from human whole blood. Cells were cultured under Th17 cell-polarizing condition for 6 days. The expression of LINC-XIST and miR-153-3p was measured by qPCR. The relationship between LINC-XIST, miR-153-3p, and ETS1 was predicted by TargetScan website and authenticated by luciferase reporter assay. ELISA assays were conducted to evaluate the IL-17 concentration. Western blot was utilized to measure the protein expression of ETS1. Th17 cell frequency was examined by flow cytometry. Results: The expression of XIST markedly decreased and miR-153-3p expression markedly increased with Th17 cell differentiation. The mRNA expression of IL-17, IL-17 concentration, and Th17 cell frequency were observably decreased in overexpressed LINC-XIST group. Luciferase reporter assay authenticated that miR-153-5p was directly regulated by LINC-XIST. miR-153-3p inhibitor observably decreased IL-17 concentration, mRNA expression of IL-17, and Th17 cell frequency while si-XIST reversed this impact. ETS1 was confirmed to be regulated by miR-153-5p via luciferase reporter assay. In addition, ETS1 markedly decreased IL-17 mRNA expression, IL-17 concentration, and Th17 cell frequency while miR-153-5p mimic reversed this impact. Conclusion: LNCRNA XIST inhibited miR-377-3p to hinder Th17 cell differentiation through upregulating ETS1.

[To promote the kidney transplantation of sensitized recipients in China by multilateral cooperation].

Sensitized recipients have an increased risk of kidney transplantation due to the immune memory of human leucocyte antigen. They lag on the waiting list and have poorer outcomes of transplantation. The society should give every patient an equal opportunity for treatment. Facing this worldwide problem, Chinese transplant community has some practical difficulties under current status of transplantation development in our country. It requires the cooperation of transplant laboratories, organ procurement organizations, clinicians, and the national organ allocation system to explore a path of solution.

A prediction model of delayed graft function in deceased donor for renal transplant: a multi-center study from China.

BACKGROUND: Kidneys obtained from deceased donors increase the incidence of delayed graft function (DGF) after renal transplantation. Here we investigated the influence of the risk factors of donors with DGF, and developed a donor risk scoring system for DGF prediction. METHODS: This retrospective study was conducted in 1807 deceased kidney donors and 3599 recipients who received donor kidneys via transplants in 29 centers in China. We quantified DGF associations with donor clinical characteristics. A donor risk scoring system was developed and validated using an independent sample set. RESULTS: The incidence of DGF from donors was 19.0%. Six of the donor characteristics analyzed, i.e., age, cause of death, history of hypertension, terminal serum creatinine, persistence of hypotension, and cardiopulmonary resuscitation (CPR) time were risk factors for DGF. A 49-point scoring system of donor risk was established for DGF prediction and exhibited a superior degree of discrimination. External validation of DGF prediction revealed area under the receiver-operating characteristic (AUC) curves of 0.7552. CONCLUSIONS: Our study determined the deceased donor risk factors related to DGF after renal transplantation pertinent to the Chinese cohort. The scoring system developed here had superior diagnostic significance and consistency and can be used by clinicians to make evidence-based decisions on the quality of kidneys from deceased donors and guide renal transplantation therapy.

Reconstruction of the hepatic artery using the superior mesenteric artery for liver transplantation.

BACKGROUND: To investigate the application of the superior mesenteric artery (SMA) for the in vitro reconstruction of the hepatic artery for liver transplantation, and to improve the success rate and safety of donor liver transplantation. METHODS: The donor liver and the pancreas were obtained, and the SMA and its branches were used to reconstruct the hepatic artery. Liver transplantation was performed after reconstruction to understand the intraoperative situation after donor liver opening, as well as postoperative liver function. Color Doppler ultrasound of the transplanted liver was also performed. RESULTS: During the period from September 2016 to March 2020, a total of 98 pancreases were obtained. The common hepatic artery and gastroduodenal artery loop (CHA-GDA) were preserved to the donor pancreas, and only the proper hepatic artery (PHA) or left/right hepatic artery (LHA/RHA) were preserved to the donor liver. If the PHA of the donor liver was short or absent, the SMA was used for lengthening the PHA or in vitro reconstruction of the LHA/RHA, followed by implantation of the donor liver after reconstruction. A total of 17 cases of this type of donor liver required mesenteric artery lengthening or reconstruction. After opening, the donor liver was well-filled, bile secretion was normal, and liver function recovered as scheduled after surgery. Color Doppler ultrasound and CT angiography (CTA) of the transplanted liver revealed that hepatic arteries were normal without complications such as hepatic artery embolism. CONCLUSIONS: In vitro reconstruction of the hepatic artery with the SMA is an effective new method of vascular reconstruction, which ensures the blood flow of the hepatic artery, reduces the anastomosis difficulty of the arteries of the donor liver, and reduces the occurrence of vascular complications.

Effects of corn steep liquor on ß-poly(L-malic acid) production in Aureobasidium melanogenum.

ß-poly(L-malic acid) (PMLA) is a water-soluble biopolymer used in medicine, food, and other industries. However, the low level of PMLA biosynthesis in microorganisms limits its further application in the biotechnological industry. In this study, corn steep liquor (CSL), which processes high nutritional value and low-cost characteristics, was selected as a growth factor to increase the PMLA production in strain, Aureobasidium melanogenum, and its metabolomics change under the CSL addition was investigated. The results indicated that, with 3 g/L CSL, PMLA production, cell growth, and yield (Yp/x) were increased by 32.76%, 41.82%, and 47.43%, respectively. The intracellular metabolites of A. melanogenum, such as amino acids, organic acids, and key intermediates in the TCA cycle, increased after the addition of CSL, and the enrichment analysis showed that tyrosine may play a major role in the PMLA biosynthesis. The results presented in this study demonstrated that the addition of CSL would be an efficient approach to improve PMLA production.

The advantage of Sirolimus in amplifying regulatory B cells and regulatory T cells in liver transplant patients.

Sirolimus has been shown to ameliorate steroid-resistant rejection and induce long-term immune tolerance among liver transplant patients. However, the detailed mechanism of how Sirolimus achieve these advantages is still lacking. This study attempts to reveal some possible mechanisms by investigating regulatory B cells (Bregs), regulatory T cells (Tregs) and some cytokines in liver transplant recipients whose Tacrolimus was partially converted to Sirolimus. The results showed that CD19+CD24+CD38+Bregs and CD4+CD25+FoxP3+Tregs increased significantly during the first month after drug conversion (P < 0.01 and P < 0.05). The percentages of IL-10+Bregs and TGF-ß1+Bregs were also elevated (P < 0.05 and P < 0.01), and the same trend was observed in the levels of IL-10 and TGF-ß1 (P < 0.01 and P < 0.01). However, in the observation period, these investigated lymphocyte subsets and cytokines didn't change significantly in patients without Sirolimus usage. The incidence of biliary stenosis in the conversion group were significantly lower than that in the control group (P < 0.05). At the same time, in vitro experiments showed that Sirolimus could significantly amplify Bregs and Tregs (P < 0.01 and P < 0.01) while Tacrolimus did not show the amplifications effects. Sirolimus' function of amplifying Bregs was weakened, and its function of amplifying Tregs even disappeared after IL-10 and TGF-ß1 were neutralized. In conclusion, Sirolimus could amplify Bregs and Tregs among liver transplant recipient, which might be benefit to mitigate the immune response, decrease chances of rejection and alleviate biliary complication. IL-10 and TGF-ß1 may play important roles during this process.

Outcomes in kidney transplantation with mycophenolate mofetil-based maintenance immunosuppression in China: a large-sample retrospective analysis of a national database.

There is no large data analysis reporting the outcome of Chinese kidney transplant patients using mycophenolate mofetil (MMF). This study analyzed 6719 patients from the Chinese Scientific Registry of Kidney Transplantation using MMF, which included 1153 from donation after cardiac death (DCD), 1271 from donation after brain and cardiac death (DBCD), and 4295 from living donor (LD). Compared with the transplants from deceased donor (DD), better outcomes including 3-year graft survival probabilities (LD = 95.8% vs. DD = 91.3%), incidence of delayed graft function (DGF, LD = 2.4% vs. DD = 17.7%), infection (LD = 10.7% vs. DD = 20.7%), graft loss (LD = 2.3% vs. DD = 6.3), and death (LD = 1.3% vs. DD = 3.2%) were shown in the LD group, with similar incidences of acute rejection (AR, LD = 3.7% vs. DD = 4.7%), hyperuricemia (LD = 21.7% vs. DD = 22.2%) within postoperative 1 year, and serum creatinine (Scr) >133 µmol/l at 1 year (LD = 18.8% vs. DD = 18.6%). Nonsignificant differences were found between the DCD and DBCD group. The 5-year survival of patient and graft in the LD group were 97.5% and 93.0%. Adjusted Cox model for graft loss showed significant associations with DGF [hazard ratio 3.7 (95% CI: 2.4, 5.8)], AR [2.8 (1.7, 4.6)], Scr >133 µmol/l at 1 year [2.6 (1.5, 4.2)], hyperuricemia [2.3 (1.6, 3.3)], and DD [1.6 (1.1, 2.4)].

Follicular Helper T Cell Derived Exosomes Promote B Cell Proliferation and Differentiation in Antibody-Mediated Rejection after Renal Transplantation.

Follicular helper T cells (Tfh cells) are closely related to the occurrence and development of antibody-mediated rejection (AMR) after renal transplantation. Exosomes play a key role in the rejection after organ transplantation. However, whether Tfh-derived exosomes are involved in AMR has not been reported. We collected peripheral blood from 42 kidney transplant patients and found no significant differences in CD4+CXCR5+ and CD4+CXCR5+CXCR3+CCR6-exosomes between AMR and non-AMR groups, whereas the proportion of CD4+CXCR5+CXCR3-exosomes was significantly higher in AMR group than that in non-AMR group; CTLA-4 expression of CD4+CXCR5+exosomes was significantly lower in AMR group than that in non-AMR group. HLA-G expression was not significantly different between two groups. We further separated CD4+CXCR5+cells from patients by magnetic beads. Coculture experiments showed that Tfh cell-derived exosomes in AMR patients significantly promoted B cell proliferation and differentiation, compared with non-AMR group, the percentage of B cells and plasma cells increased by 87.52% and 110.2%, respectively. In conclusion, our study found that Tfh cell-derived exosomes could promote the proliferation and differentiation of B cells and they may play an important role in the development of AMR after renal transplantation.

Circulating NK cell subsets and NKT­like cells in renal transplant recipients with acute T­cell­mediated renal allograft rejection.

Emerging evidence indicates that natural killer (NK) cells and NKT­like cells may affect allograft outcomes following solid organ transplantation. However, the roles of these cells in allograft acceptance and dysfunction are controversial. To assess the changes in NK cell and CD3+CD56+ NKT­like cell frequency and phenotype in renal allograft recipients and to explore their associations with acute T­cell­mediated renal allograft rejection (ACR), longitudinal changes in NK and NKT­like cell frequency and phenotype were characterized using flow cytometry and immunohistochemistry in the peripheral blood and kidney allograft tissues in 142 recipients undergoing kidney transplantation. The serum concentrations of NK cell­associated cytokines were also detected by cytokine multiplex immunoassay. In contrast to the healthy controls, recipients with stable graft function exhibited increased proportions of CD56brightCD16dim subsets and decreased proportions of NKT­like cells in their peripheral blood mononuclear cells (PBMCs). Patients with ACR demonstrated increased proportions of NK cells, which were associated with increased CD3­CD56bright subsets and decreased CD3­CD56dim subsets, an increase in the CD56bright/CD56dim ratio in PBMCs and increased CD56+ NK cell infiltration in the kidney allograft, compared with the stable controls. In addition, there was a decreased proportion of NKT­like cells in patients with ACR, and an increased ratio of CD56bright/NKT­like cells compared with the stable controls. These differences appeared to be consistent with the increase in the serum concentrations of C­C motif chemokine 19 and the decrease in the serum concentrations of interleukin­15. These data indicate that CD56bright NK cells may promote the development of ACR, and that NKT­like cells may have immunoregulatory function. The results also imply that the CD56bright/CD56dim ratio may affect the ACR signatures.

CD4+ T Cell Immune Response to VP1 and VP3 in BK Virus Infected Recipients of Renal Transplantation.

OBJECTIVE: To investigate the characteristics of BK virus (BKV) specific cellular immune response in the recipients who have early infection with BKV after renal transplantation. METHODS: The recipients of renal allografts (n = 30) were divided into groups of BK virus nephropathy (BKVN), viruria, and viremia. The BKV load was observed with real-time fluorescence quantitative polymerase chain reaction in urine and blood every three months. The values of serum creatinine (SCr) were detected. The peripheral blood mononuclear cells (PBMCs) were cultivated with overlapping peptide pool containing BKV structural proteins VP1, VP2, and VP3, and regulatory proteins large tumor antigen (LT-Ag) and small tumor antigen (st-Ag), to stimulate in vitro specific cellular immunoresponse. Flow cytometry was used to measure the proliferation of CD3+/CD4+/CD8+ T and interferon [INF]-γ/interleukin [IL]-2/tumor necrosis factor [TNF]-α T cell subsets. RESULTS: The BKV infection increased SCr values in recipients of renal transplantation. CD4+ T cells were dominant (>90%) in the in vitro cellular immunoresponse to VP1, VP2, VP3, LT-Ag, and st-Ag. At the presence of viremia and BKVN, IL-2/IFN-γ+/TNF-α+ CD4+ T cells showed significantly decreased in vitro cellular immunoresponse to VP1, VP2, and VP3 (p < 0.05), but insignificantly changed to LT-Ag and st-Ag (p > 0.05). For the cases of viruria and viremia, IL-2/IFN-γ+/TNF-α+ CD4+ T cells showed significantly higher in vitro cellular immunoresponse to VP1, VP2, and VP3 than to LT-Ag and st-Ag (p < 0.05). The immunogenicity of VP1 and VP3 was significantly higher than that of VP2 (p < 0.05). CONCLUSIONS: The BKV infection increases SCr values, and CD4+ T cells are dominant in the in vitro BKV specific cellular immunoresponse in the recipients of renal transplantation. Viremia significantly decreased the immunoresponse to VP1, VP2, and VP3. There is the significantly stronger immunoresponse to VP1 and VP3 when compared with that to VP2, LT-Ag, and st-Ag, suggesting that VP1 and VP3 may be the major targets for the BKV specific immune response.

Ectopic expression of E3 ubiquitin-protein ligase 2 in glioma and enhances resistance to apoptosis through activating nuclear factor κ-light-chain-enhancer of B cells.

Nuclear factor κ-light-chain-enhancer of B cells (NF-κB) is one of the most important tumorigenic factors. Although it has been established that NF-κB is overly activated in human glioma cells, the molecular mechanisms that lead to the signal transduction to NF-κB and thereby the induction of resistance to apoptosis remain poorly understood. The present study demonstrated that mRNA and protein levels of E3 ubiquitin-protein ligase 2 (MIB2) were markedly upregulated in glioma cell lines and clinical samples. Immunohistochemical analysis also revealed high levels of MIB2 expression in glioma specimens. Ectopic overexpression of MIB2 was established in glioma cell lines to investigate its fundamental roles in the response of human glioma to apoptotic inducers. The results indicated that ultraviolet irradiation-induced cell apoptosis was inhibited with MIB2 overexpression in glioma cells. Notably, knockdown of MIB2 using RNA interference was able to increase the sensitivity of glioma cells to the pro-apoptotic agents. The present study identified that MIB2 induces NF-κB activation and facilitates the resistance of glioma cell to apoptosis. It was proposed that MIB2 may not only be an important hallmark to glioma disease progression, but that it may also offer novel clinical strategies to overcome resistance to cancer therapies.

The promotion of tissue engineering blood vessel patency by CGS21680 through regulating pro-inflammatory activities of endothelial progenitor cell.

The mobilization and homing of endothelial progenitor cells (EPCs) contribute to the rapid endothelialization of tissue engineering blood vessel (TEBV). Inflammation can affect TEBV patency, and monocytes/macrophages (MM) are the main effector cells. But it is not clear how EPCs interact with MM after TEBV transplantation. Our results showed acellular materials would not directly cause acute and severe inflammatory responses but activate E-selectin expression in homing EPCs, gradually promoting the polarization of MM to the M1. Adenosine A2a receptor agonist CGS21680 promoted the secretion of more proangiogenic factors from MM, inducing EPC migration and mobilization. CGS21680 could inhibit MM polarization to the M1 type through the down-regulation of EPC proinflammatory molecules, such as E-selectin. Chitosan/(2-hydroxypropyl)-ß-cyclodextrin nanoparticles were prepared to control the release of CGS-21680 and then modified to TEBVs through layer-by-layer assembly. Animal experiments showed that this TEBV can maintain patency for 6 months and good endothelialization was observed. In summary, our results showed the regulation of EPC pro-inflammatory activities is a new approach to enhance TEBV patency. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2634-2642, 2018.

mTOR masters monocyte development in bone marrow by decreasing the inhibition of STAT5 on IRF8.

Monocytes and macrophages play a key role in defending pathogens, removing the dead cells or cell debris, and wound healing. The mammalian target of rapamycin (mTOR) inhibitor rapamycin (RPM) is widely used in clinics to treat patients with organ transplantation or tumors. The role of mTOR in monocyte/macrophage development remains to be clarified. Here we found that mTOR intrinsically controls monocyte/macrophage development, as evidenced by the decreased percentages and cell numbers of CD11b+F4/80+ cells resulting from mTOR inhibition in SCID mice, mTOR-deficient mice, and mixed chimera mice, and the in vitro colony formation and monocyte/macrophage induction assays. However, Lyzs-mTOR knockout mice displayed normal levels of monocytes/macrophages, indicating that mTOR is not essential for the survival and maturation of monocytes/macrophages. Further studies showed that mTOR deficiency significantly reduced macrophage colony-stimulating factor receptor CD115 expression at the transcriptional and translational levels. The molecular mechanism studies indicate that the impaired monocyte/macrophage development caused by mTOR deficiency is mainly a result of the overactivated STAT5 and subsequent downregulation of IRF8, but not the altered cell metabolism and autophagy. Therefore, our work identifies that mTOR is an intrinsic master for monocyte/macrophage development at the early stages through regulating STAT5-IRF8-dependent CD115-expressing pathway. Long-term usage of RPM may cause a defect of myeloid progenitors in bone marrow.

Exosome-Modified Tissue Engineered Blood Vessel for Endothelial Progenitor Cell Capture and Targeted siRNA Delivery.

Instability and poor targeting causes the long-term patency of RNA-modified tissue engineering blood vessels (TEBVs) remaining unsatisfactory. RNA can be enriched in exosome and then delivered into targeted cells while whether exosome-modified TEBVs achieve RNA targeted delivery is unclear. Here, to promote the expression of klotho protein on the mesenchymal stem cell (MSC)-derived exosomes, klotho plasmids are first transfected into MSCs, and adenosine kinase (ADK) siRNA is then loaded into exosome (klotho/ADK siRNA-exosome) using electrotransfection. Flow chamber results show that klotho/ADK siRNA-exosome can effectively capture circulating endothelial progenitor cells (EPCs). Besides, the captured EPCs can endocytose this exosome, and then decompose it into klotho protein and ADK siRNA. Moreover, ADK siRNA promotes the paracrine of proangiogenic factors and adenosine from EPCs, which further facilitate proliferation and migration of endothelial cells. Based on polyethyleneimine-capped gold nanoparticles, exosome-modified TEBVs are constructed through layer-by-layer assembly. Animal experimental results show that klotho/ADK siRNA-exosome-modified TEBVs can maintain the patency up to one month, and good endothelialization is observed. In short, one exosome-modified TEBV is constructed, capture molecules on the surface of exosome capture the circulating EPCs, and the loaded RNA achieves its purpose of accurate treatment depending on the needs of patients.

Clinical significance of p16INK4A and p14ARF promoter methylation in renal cell carcinoma: a meta-analysis.

The inactivation of p16INK4A and p14ARF via promoter methylation has been investigated in various cancers. However, the clinical effects of p16INK4A and p14ARF promoter methylation on renal cell carcinoma (RCC) remain to be clarified. The pooled data were calculated and summarized. Finally, an investigation of 14 eligible studies with 1231 RCC patients and 689 control patients was performed. Methylated p16INK4A and p14ARF were observed to be significantly higher in RCC than in control subjects without malignancies (OR = 2.77, P = 0.005; OR = 11.73, P < 0.001, respectively). Methylated p16INK4A was significantly associated with the risk of RCC in the tissue subgroup, but not in the serum and urine subgroups. Methylated p16INK4A was significantly associated with tumor size. We did not find that p16INK4A promoter methylation was associated with sex, tumor grade, lymph node status, and tumor histology. Methylated p14ARF was significantly correlated with sex and tumor histology. Three studies reported that p16INK4A methylation was not significantly correlated with the prognosis of RCC. The results suggested that p16INK4A and p14ARF promoter methylation may be correlated with the carcinogenesis of RCC, and that methylated p14ARF , especially, can be a major susceptibility gene. We also found the different clinicopathological significance of 16INK4A and p14ARF in RCC. Additional studies with sufficient data are essential to further evaluate the clinical features and prognostic effect of p16INK4A and p14ARF promoter methylation in RCC.

Expression and genetic polymorphism of necroptosis related protein RIPK1 is correlated with severe hepatic ischemia-reperfusion injury and prognosis after hepatectomy in hepatocellular carcinoma patients.

OBJECTIVES: The goal of our study was to assess the prognostic impact of the necroptosis relative protein RIPK1 genetic polymorphism in ischemia-reperfusion injury and survival after hepatectomy in hepatocellular carcinoma (HCC) patients. METHODS: In this study, expression of RIPK1 and its genetic polymorphism(rs2272990) were examined in plasma of 44 HCC patients. All these patients were undergoing partial hepatectomy. The prognostic values of RIPK1 genetic polymorphism for tumor development and survival, and ischemia-reperfusion injury after hepatectomy were further determined. RESULTS: Plasma RIPK1 expressions were significantly increased in HCC patients, compared to the healthy control group. Totally 19 patients have the GA + AA genotype in the RIPK1 rs2272990 SNP site and 25 have GG genotype. There were no statistically significant intergroup differences observed in age, gender, AFP value, HBV positive, tumor size or cirrhosis. GG genotype had positive correlation with TNM classification (p= 0.033) and lymphatic metastasis (p= 0.027) and was significantly associated with severe hepatic ischemia-reperfusion injury and decreased survival rate after hepatectomy. CONCLUSION: In conclusion, the RIPK1 polymorphism is an indicator of hepatic injury and a novel prognostic biomarker for tumor development and survival of HCC recipients after hepatectomy.