Meeting report: Xenotransplantation Development Conference in Neijiang, China.

A conference on progress in the development of xenotransplantation in China was held in Neijiang, Sichuan, in May 2023, and was attended by approximately 100 established researchers and trainees. Progress in xenotransplantation research was reviewed by both Chinese and foreign experts. The topics discussed ranged from genetic engineering of pigs and the results of pig-to-nonhuman primate organ transplantation to the requirements for designated pathogen-free (DPF) pig facilities and regulation of xenotransplantation. This conference served as an opportunity to collectively advance the development of xenotransplantation in China and pave the way for its clinical application.

Suppression of allograft rejection by regulatory B cells induced via TLR signaling.

B lymphocytes have long been recognized for their critical contributions to adaptive immunity, providing defense against pathogens through cognate antigen presentation to T cells and Ab production. More recently appreciated is that B cells are also integral in securing self-tolerance; this has led to interest in their therapeutic application to downregulate unwanted immune responses, such as transplant rejection. In this study, we found that PMA- and ionomycin-activated mouse B cells acquire regulatory properties following stimulation through TLR4/TLR9 receptors (Bregs-TLR). Bregs-TLR efficiently inhibited T cell proliferation in vitro and prevented allograft rejection. Unlike most reported Breg activities, the inhibition of alloimmune responses by Bregs-TLR relied on the expression of TGF-ß and not IL-10. In vivo, Bregs-TLR interrupted donor-specific T cell expansion and induced Tregs in a TGF-ß-dependent manner. RNA-Seq analyses corroborated the involvement of TGF-ß pathways in Breg-TLR function, identified potential gene pathways implicated in preventing graft rejection, and suggested targets to foster Breg regulation.

Interleukin-27 in liver xenotransplantation: A rational target to mitigate ischemia reperfusion injury and increase xenograft survival.

Transplantation of xenogeneic organs is an attractive solution to the existing organ shortage dilemma, thus, securing a clinically acceptable prolongation of xenograft survival is an important goal. In preclinical transplantation models, recipients of liver, kidney, heart, or lung xenotransplants demonstrate significant graft damages through the release of pro-inflammatory molecules, including the C-reactive protein, cytokines, and histone-DNA complexes that all foster graft rejection. Recent studies have demonstrated that mitigation of ischemia reperfusion injury (IRI) greatly improves xenograft survival. Organ IRI develops primarily on a complex network of cytokines and chemokines responding to molecular cues from the graft milieu. Among these, interleukin 27 (IL-27) plays an immunomodulatory role in IRI onset due to graft environment-dependent pro- and anti- inflammatory activities. This review focuses on the impact of IL-27 on IRI of liver xenotransplants and provides insights on the function of IL-27 that could potentially guide genetic engineering strategies of donor pigs and/or conditioning of organs prior to transplantation.

Properties of regulatory B cells regulating B cell targets.

Regulatory B cells (Bregs) have shown promise as anti-rejection therapy applied to organ transplantation. However, less is known about their effect on other B cell populations that are involved in chronic graft rejection. We recently uncovered that naïve B cells, stimulated by TLR ligand agonists, converted into B cells with regulatory properties (Bregs-TLR) that prevented allograft rejection. Here, we examine the granular phenotype and regulatory properties of Breg-TLR cells suppressing B cells. Cocultures of Bregs-TLR with LPS-activated B cells showed a dose-dependent suppression of targeted B cell proliferation. Adoptive transfers of Bregs-TLR induced a decline in antibody responses to antigenically disparate skin grafts. The role of Breg BCR specificity in regulation was assessed using B cell-deficient mice replenished with transgenic BCR (OB1) and TCR (OT-II) lymphocytes of matching antigenic specificity. Results indicated that proliferation of OB1 B cells, mediated through help from CD4+ OT-II cells, was suppressed by OB1 Bregs of similar specificity. Transcriptomic analyses indicated that Bregs-TLR suppression is associated with a block in targeted B cell differentiation controlled by PRDM1 (Blimp1). This work uncovered the regulatory properties of a new brand of Breg cells and provided mechanistic insights into potential applications of Breg therapy in transplantation.

TGF-ß-secreting regulatory B cells: unsung players in immune regulation.

Regulatory B cells contribute to the regulation of immune responses in cancer, autoimmune disorders, allergic conditions and inflammatory diseases. Although most studies focus on regulatory B lymphocytes expressing interleukin-10, there is growing evidence that B cells producing transforming growth factor ß (TGF-ß) can also regulate T-cell immunity in inflammatory diseases and promote the emergence of regulatory T cells that contribute to the induction and maintenance of natural and induced immune tolerance. Most research on TGF-ß+ regulatory B cells has been conducted in models of allergy, cancer and autoimmune diseases, but there has, as yet, been limited scrutiny of their role in the transplant setting. Herein, we review recent investigations seeking to understand how TGF-ß-producing B cells direct the immune response in various inflammatory diseases and whether these regulatory cells may have a role in fostering tolerance in transplantation.

Author Correction: A novel microRNA signature predicts survival in liver hepatocellular carcinoma after hepatectomy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Bioinformatical identification of key pathways and genes in human hepatocellular carcinoma after CSN5 depletion.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. It has been previously reported that CSN5 depletion is an effective method in human HCC. In the current study, we aimed to uncover gene signatures and key pathways during HCC. Gene expression profiles of GSE26485 were downloaded from GEO database. Totally, 101 differentially expressed genes (DEGs) were up-regulated and 146 ones were down-regulated. Biological processes (BP) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis showed that the DEGs were mainly enriched in regulation of cell growth, oxidation-reduction process, mitotic cytokinesis, negative regulation of macroautophagy, endosome organization, lysosome, biosynthesis of antibiotics, small cell lung cancer and glutathione metabolism and so on (P < 0.05). Protein-protein interaction (PPI) network, Kaplan-Meier, log-rank method, western blot, immunohistochemistry and encyclopedia of DNA elements (ENCODE) analysis showed that CSN5 depletion took effects through down-regulation of SMAD5-related pathways which include EXO1, CENPA and NCAPG, resulting in the inactivation of H3K4me3 and H3K36me3. Those genes represent the promising targets for therapeutic intervention in HCC patients.

Droplet digital PCR-based circulating microRNA detection serve as a promising diagnostic method for gastric cancer.

BACKGROUND: Novel non-invasive biomarkers for gastric cancer (GC) are needed, because the present diagnostic methods for GC are either invasive or insensitive and non-specific in clinic. The presence of stable circulating microRNAs (miRNAs) in plasma suggested a promising role as GC biomarkers. METHODS: Based on the quantitative droplet digital PCR (ddPCR), four miRNAs (miR-21, miR-93, miR-106a and miR-106b) related to the presence of GC were identified in plasma from a training cohort of 147 participants and a validation cohort of 28 participants. RESULTS: All circulating miRNA levels were significantly higher in the plasma of GC patients compared to healthy controls (P < 0.05). Through a combination of four miRNAs by logistic regression model, receiver operating characteristic (ROC) analyses yielded the highest AUC value of 0.887 in discriminating GC patients from healthy volunteers. Furthermore, miR-21, miR-93 and miR-106b levels were significantly related to an advanced TNM stage in GC patients. ROC analyses of the combined miRNA panel also showed the highest AUC value of 0.809 in discriminating GC patients with TNM stage I and II from stage III and IV. Through combining four miRNAs and clinical parameters, a classical random forest model was established in the training stage. In the validation cohort, it correctly discriminated 23 out of 28 samples in the blinded phase (false rate, 17.8%). CONCLUSIONS: Using the ddPCR technique, circulating miR-21, miR-93, miR-106a and miR-106b could be used as diagnostic plasma biomarkers in gastric cancer patients.

A novel microRNA signature predicts survival in liver hepatocellular carcinoma after hepatectomy.

Liver hepatocellular carcinoma (LIHC) is the most common type of primary liver cancer. In the current study, genome-wide miRNA-Seq and mRNA profiles in 318 LIHC patients derived from The Cancer Genome Atlas (TCGA) were analysed to identify miRNA-based signatures for LIHC prognosis with survival analysis and a semi-supervised principal components (SPC) method. A seven-miRNA signature was confirmed for overall survival (OS) prediction by comparing miRNA profiles in paired primary tumour and solid tumour normal tissues. Thereafter, a linear prognostic model that consisted of seven miRNAs was established and used to divide patients into high- and low-risk groups according to prognostic scores. Subsequent Kaplan-Meier analysis revealed that the seven-miRNA signature correlated with a good predictive clinical outcome for 5-year survival in LIHC patients. Additionally, this miRNA-based prognostic model could also be used for OS prognosis of LIHC patients in early stages, which could guide the future therapy of those patients and promote the OS rate. Moreover, the seven-miRNA signature was an independent prognostic factor. In conclusion, this signature may serve as a prognostic biomarker and guide LIHC therapy, and it could even be used as an LIHC therapeutic target in the future.

Mangiferin induces islet regeneration in aged mice through regulating p16INK4a.

Previous studies by our group on mangiferin demonstrated that it exerts an anti­hyperglycemic effect through the regulation of cell cycle proteins in 3­month­old, partially pancreatectomized (PPx) mice. However, ß­cell proliferation is known to become severely restricted with advanced age. Therefore, it is unknown whether mangiferin is able to reverse the diabetic condition and retain ß­cell regeneration capability in aged mice. In the present study, 12­month­old C57BL/6J mice that had undergone PPx were subjected to mangiferin treatment (90 mg/kg) for 28 days. Mangiferin­treated aged mice exhibited decreased blood glucose levels and increased glucose tolerance, which was accompanied with higher serum insulin levels when compared with those in untreated PPx control mice. In addition, islet hyperplasia, elevated ß­cell proliferation and reduced ß­cell apoptosis were also identified in the mice that received mangiferin treatment. Further studies on the mRNA transcript and protein expression levels indicated comparatively increased levels of cyclins D1 and D2 and cyclin­dependent kinase 4 in mangiferin­treated mice, while the levels of p27Kip1 and p16INK4a were decreased relative to those in the untreated PPx controls. Of note, mangiferin treatment improved the proliferation rate of islet ß­cells in adult mice overexpressing p16INK4a, suggesting that mangiferin induced ß­cell proliferation via the regulation of p16INK4a. In addition, the mRNA transcription levels of critical genes associated with insulin secretion, including pancreatic and duodenal homeobox 1, glucose transporter 2 and glucokinase, were observed to be upregulated after mangiferin treatment. Taken together, it was indicated that mangiferin treatment significantly induced ß­cell proliferation and inhibited ß­cell apoptosis by regulating cell cycle checkpoint proteins. Furthermore, mangiferin was also demonstrated to regulate genes associated with insulin secretion. Collectively these, results suggest the therapeutic potential of mangiferin in the treatment of diabetes in aged individuals.

Mutual regulation of the Hippo/Wnt/LPA/TGF­ß signaling pathways and their roles in glaucoma (Review).

Glaucoma is the leading cause of irreversible blindness worldwide and there is no effective treatment thus far. The trabecular meshwork has been identified as the major pathological area involved. Certain signaling pathways in the trabecular meshwork, including the Wnt, lysophosphatidic acid and transforming growth factor­ß pathways, have been identified as novel therapeutic targets in glaucoma treatment. Meanwhile, it has been reported that key proteins in these pathways, particularly the primary transcription regulator Yes­associated protein (YAP) and transcriptional co­activator with PDZ­binding motif (TAZ), exhibit interactions with the Hippo pathway. The Hippo pathway, which was first identified in Drosophila, has drawn great focus with regard to various aspects of studies in recent years. One role of the Hippo pathway in the regulation of organ size was indicated by more recent evidence. Defining the relevant physiological function of the Hippo pathway has proven to be extremely complicated. Studies have ascribed a role for the Hippo pathway in an overwhelming number of processes, including cell proliferation, cell death and cell differentiation. Therefore, the present review aimed to unravel the roles of YAP and TAZ in the Hippo pathway and the pathogenesis of glaucoma. Furthermore, a new and creative study for the treatment of glaucoma is provided.

Notoginsenoside R1 attenuates glucose-induced podocyte injury via the inhibition of apoptosis and the activation of autophagy through the PI3K/Akt/mTOR signaling pathway.

Injury to terminally differentiated podocytes contributes ignificantly to proteinuria and glomerulosclerosis. The aim of this study was to examine the protective effects of notoginsenoside R1 (NR1) on the maintenance of podocyte number and foot process architecture via the inhibition of apoptosis, the induction of autophagy and the maintenance pf podocyte biology in target cells. The effects of NR1 on conditionally immortalized human podocytes under high glucose conditions were evaluated by determining the percentage apoptosis, the percentage autophagy and the expression levels of slit diaphragm proteins. Our results revealed that NR1 protected the podocytes against high glucose-induced injury by decreasing apoptosis, increasing autophagy and by promoting cytoskeletal recovery. The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was further investigated in order to elucidate the mechanisms responsible for the protective effects of NR1 on podocytes. Our data indicated that treatment with NR increased the phosphorylation levels of PI3K, Akt and mTOR, leading to the activation of the PI3K/Akt/mTOR signaling pathway in podocytes. To the best of our knowledge, this is the first in vitro study to demonstrate that NR1 protects podocytes by activating the PI3K/Akt/mTOR pathway.

Notoginsenoside R1 ameliorates podocyte injury in rats with diabetic nephropathy by activating the PI3K/Akt signaling pathway.

The present study was designed to examine the protective effect of notoginsenoside R1 (NR1) on podocytes in a rat model of streptozotocin (STZ)­induced diabetic nephropathy (DN), and to explore the mechanism responsible for NR1-induced renal protection. Diabetes was induced by a single injection of STZ, and NR1 was administered daily at a dose of 5 mg/kg (low dose), 10 mg/kg (medium) and 20 mg/kg (high) for 16 weeks in Sprague-Dawley rats. Blood glucose levels, body weight and proteinuria were measured every 4 weeks, starting on the day that the rats received NR1. Furthermore, on the day of sacrifice, blood, urine and kidneys were collected in order to assess renal function according to general parameters. Pathological staining was performed to evaluate the renal protective effect of NR1, and the expression of the key slit diaphragm proteins, namely neprhin, podocin and desmin, were evaluated. In addition, the serum levels of inflammatory cytokines [tumor necrosis factor-α (TNF-α), tumor growth factor-ß1 (TGF-ß1), interleukin (IL)-1 and IL-6] as well as an anti-inflammatory cytokine (IL-10) were assessed, and the apoptosis of podocytes was quantified. Finally, the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and the involvement of nuclear factor-κB (NF-κB) inactivation was further analyzed. In this study, NR1 improved renal function by ameliorating histological alterations, increasing the expression of nephrin and podocin, decreasing the expression of desmin, and inhibiting both the inflammatory response as well as the apoptosis of podocytes. Furthermore, NR1 treatment increased the phosphorylation of both PI3K (p85) and Akt, indicating that activation of the PI3K/Akt signaling pathway was involved. Moreover, NR1 treatment decreased the phosphorylation of NF-κB (p65), suggesting the downregulation of NF-κB. This is the first study to the best of our knowledge, to clearly demonstrate that NR1 treatment ameliorates podocyte injury by inhibiting both inflammation and apoptosis through the PI3K/Akt signaling pathway.

Characteristics of α-Gal epitope, anti-Gal antibody, α1,3 galactosyltransferase and its clinical exploitation (Review).

The α-Gal epitope (Galα1,3Galα1,4GlcNAc­R) is ubiquitously presented in non-primate mammals, marsupials and New World Monkeys, but it is absent in humans, apes and Old World monkeys. However, the anti-Gal antibody (~1% of immunoglobulins) is naturally generated in human, and is found as the immunoglobulin G (IgG), IgM and IgA isotypes. Owing to the specific binding of the anti­Gal antibody with the α­Gal epitope, humans have a distinct anti­α­gal reactivity, which is responsible for hyperacute rejection of organs transplanted from α­gal donors. In addition, the α1,3 galactosyltransferases (α1,3GT) can catalyze the synthesis of the α­Gal epitope. Therefore, the α1,3GT gene, which encodes the α1,3GT, is developed profoundly. The distributions of the α­Gal epitope and anti­Gal antibody, and the activation of α1,3GT, reveal that the enzyme of α1,3GT in ancestral primates is ineffective. Comparison of the nucleotide sequence of the human α1,3­GT pseudogene to the corresponding different species sequence, and according to the evolutionary tree of different species, the results of evolutionary inactivation of the α1,3GT gene in ancestral primates attribute to the mutations under a stronger selective pressure. However, on the basis of the structure, the mechanism and the specificity of the α­Gal epitope and anti­Gal antibody, they can be applied to clinical exploitation. Knocking out the α1,3GT gene will eliminate the xenoantigen, Gal(α1,3)Gal, so that the transplantation of α1,3GT gene knockout pig organ into human becomes a potential clinically acceptable treatment for solving the problem of organ shortage. By contrast, the α­Gal epitope expressed through the application of chemical, biochemical and genetic engineering can be exploited for the clinical use. Targeting anti­Gal­mediated autologous tumor vaccines, which express α­Gal epitope to antigen­presenting cells, would increase their immunogenicity and elicit an immune response, which will be potent enough to eradicate the residual tumor cells. For tumor vaccines, the way of increasing immunogenicity of certain viral vaccines, including flu vaccines and human immunodeficiency virus vaccines, can also be used in the elderly. Recently, α­Gal epitope nanoparticles have been applied to accelerate wound healing and further directions on regeneration of internally injured tissues.