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Abstract It is essential to elucidate the molecular mechanisms underlying liver transplant tolerance and rejection. In cases of mouse liver transplantation between inbred strains, immunological rejection of the allograft is reduced with spontaneous apoptosis without immunosuppressive drugs, which differs from the actual clinical result. This may be because inbred strains are genetically homogeneous and less heterogeneous than others. We exploited outbred CD1 mice, which show highly heterogeneous genotypes among individuals, to search for biomarkers related to immune responses and to construct a model for predicting the outcome of liver allografting. Of the 36 mice examined, 18 died within 3 weeks after transplantation, while the others survived for more than 6 weeks. Whole-exome sequencing of the 36 donors revealed more than 9 million variants relative to the C57BL/6 J reference. We selected 6517 single-nucleotide and indel variants and performed machine learning to determine whether or not we could predict the prognosis of each genotype. Models were built by both deep learning with a one-dimensional convolutional neural network and linear classification and evaluated by leave-one-out cross-validation. Given that one short-lived mouse died early in an accident, the models perfectly predicted the outcome of all individuals, suggesting the importance of genotype collection. In addition, linear classification models provided a list of loci potentially responsible for these responses. The present methods as well as results is likely to be applicable to liver transplantation in humans.

Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers but are frequently deficient or dysfunctional in patients with hepatocellular carcinoma (HCC). In the present study, we explored a novel therapy for HCC using NK cells derived from donor liver graft perfusate. These liver-derived NK cells, named LMNC-NK cells, are more abundant in liver mononuclear cells (LMNCs) than in peripheral blood mononuclear cells (PBMCs) from the same donor. We developed a method to expand LMNC-NK cells by 33.8±54.4-fold, enhancing their cytotoxic properties and cytokine production, including granzyme B, CD107a, TNF-α, and IFN-γ. These cells also showed an increased expression of cytotoxicity receptors. An RNA-seq analysis revealed considerable differences in gene expression between LMNC-NK and PBMC-NK cells, with 453 genes upregulated and 449 downregulated in LMNC-NK cells. These genes are involved in the mitogen-activated protein kinase cascade and cell differentiation, explaining the increased activity of LMNC-NK cells. Quantitative reverse transcription polymerase chain reaction confirmed the significant upregulation of TLR6, KIT, MMP14, IRF8, TCF7, FCERIG, LEF1, NLRp3, and IL16 in LMNC-NK cells. LMNC-NK cells effectively eliminated HepG-2-Luc cells in vitro, and in an orthotopic murine model of HCC, they exhibited a potent anti-tumor effect, outperforming PBMC-NK cells. The expression of the activation marker CD69+ in LMNC-NK cells was also significantly higher among tumor-infiltrating lymphocytes compared to PBMC-NK cells. Our research suggests that the adoptive transfer of LMNC-NK cells could be a promising treatment for HCC, offering a novel and effective source of NK cells with superior cytotoxic functions.

BackgroundKidney transplantation is considered the most effective treatment for end-stage renal failure. Recent studies have shown that the significance of the immune microenvironment after kidney transplantation in determining prognosis of patients. Therefore, this study aimed to conduct a bibliometric analysis to provide an overview of the knowledge structure and research trends regarding the immune microenvironment and survival in kidney transplantation.MethodsOur search included relevant publications from 2013 to 2023 retrieved from the Web of Science core repository and finally included 865 articles. To perform the bibliometric analysis, we utilized tools such as VOSviewer, CiteSpace, and the R package “bibliometrix”. The analysis focused on various aspects, including country, author, year, topic, reference, and keyword clustering.ResultsBased on the inclusion criteria, a total of 865 articles were found, with a trend of steady increase. China and the United States were the countries with the most publications. Nanjing Medical University was the most productive institution. High-frequency keywords were clustered into 6 areas, including kidney transplantation, transforming growth factor β, macrophage, antibody-mediated rejection, necrosis factor alpha, and dysfunction. Antibody mediated rejection (2019-2023) was the main area of research in recent years.ConclusionThis groundbreaking bibliometric study comprehensively summarizes the research trends and advances related to the immune microenvironment and survival after kidney transplantation. It identifies recent frontiers of research and highlights promising directions for future studies, potentially offering fresh perspectives to scholars in the field.

Abstract Hydrogen has been reported to act as an antioxidant, anti-apoptosis and anti-inflammatory agent. Coral calcium carried hydrogen (G2-SUISO) is a safer and more convenient form of hydrogen agent than others. The mechanism underlying the hepatoprotective effects of G2-SUISO using an elderly non-alcoholic steatohepatitis (NASH) rat model was investigated. Two days after fasting, six-month-old elderly male F344/NSlc rats were given a choline deficient high carbohydrate fat-free (CDHCFF) diet from day 0 to day 3 as CDHCFF control group, and then switched to a normal diet from days 4 to 7 with or without 300 mg/kg G2-SUISO. Rats in each group were finally being sacrificed on day 3 or day 7. In the CDHCFF diet group, G2-SUISO decreased the liver weight-to-body weight ratio, the serum AST, ALT, total cholesterol levels, inflammatory infiltration, pro-inflammatory cytokine expression and lipid droplets with inhibiting lipogenic pathways by reducing sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase and fatty acid synthase gene expression compared with the CDHCFF diet alone. G2-SUISO had beneficial effects of anti-apoptosis as well the down-regulation of pro-apoptotic molecules including NF-κB, caspase-3, caspase-9 and Bax. These findings suggest that G2-SUISO treatment exerts a significant hepatoprotective effect against steatosis, inflammation and apoptosis in elderly NASH rats.

Abstract Background Ferroptosis, which is characterized by lipid peroxidation and iron accumulation, is closely associated with the pathogenesis of acute renal injury (AKI). Cyanidin-3-glucoside (C3G), a typical flavonoid that has anti-inflammatory and antioxidant effects on ischemia‒reperfusion (I/R) injury, can induce AMP-activated protein kinase (AMPK) activation. This study aimed to show that C3G exerts nephroprotective effects against I/R-AKI related ferroptosis by regulating the AMPK pathway. Methods Hypoxia/reoxygenation (H/R)-induced HK-2 cells and I/R-AKI mice were treated with C3G with or without inhibiting AMPK. The level of intracellular free iron, the expression of the ferroptosis-related proteins acyl-CoA synthetase long chain family member 4 (ACSL4) and glutathione peroxidase 4 (GPX4), and the levels of the lipid peroxidation markers 4-hydroxynonenal (4-HNE), lipid reactive oxygen species (ROS) and malondialdehyde (MDA) were examined. Results We observed the inhibitory effect of C3G on ferroptosis in vitro and in vivo, which was characterized by the reversion of excessive intracellular free iron accumulation, a decrease in 4-HNE, lipid ROS, MDA levels and ACSL4 expression, and an increase in GPX4 expression and glutathione (GSH) levels. Notably, the inhibition of AMPK by CC significantly abrogated the nephroprotective effect of C3G on I/R-AKI models in vivo and in vitro. Conclusion Our results provide new insight into the nephroprotective effect of C3G on acute I/R-AKI by inhibiting ferroptosis by activating the AMPK pathway.

Summary: The liver has long been deemed a tolerogenic organ. We employed high-dimensional mass cytometry and immunohistochemistry to depict the temporal and spatial dynamics of immune cells in the spleen and liver in a murine model of spontaneous liver allograft acceptance. We depicted the immune landscape of spontaneous liver tolerance throughout the rejection and acceptance stages after liver transplantation and highlighted several points of importance. Of note, the CD4+/CD8+ T cell ratio remained low, even in the tolerance phase. Furthermore, a PhenoGraph clustering analysis revealed that exhausted CD8+ T cells were the most dominant metacluster in graft-infiltrating lymphocytes (GILs), which highly expressed the costimulatory molecule CD86. The temporal and spatial dynamics of immune cells revealed by high-dimensional analyses enable a fine-grained analysis of GIL subsets, contribute to new insights for the discovery of immunological mechanisms of liver tolerance, and provide potential ways to achieve clinical operational tolerance after liver transplantation.

BackgroundLynch syndrome (LS)-associated glioblastoma (GBM) is rare in clinical practice, and simultaneous occurrence with cutaneous porokeratosis is even rarer. In this study, we analyzed the clinicopathological and genetic characteristics of LS-associated GBMs and concurrent porokeratosis, as well as evaluated the tumor immune microenvironment (TIME) of LS-associated GBMs.MethodsImmunohistochemical staining was used to confirm the histopathological diagnosis, assess MMR and PD-1/PD-L1 status, and identify immune cell subsets. FISH was used to detect amplification of EGFR and PDGFRA, and deletion of 1p/19q and CDKN2A. Targeted NGS assay analyzed somatic variants, MSI, and TMB status, while whole-exome sequencing and Sanger sequencing were carried out to analyze the germline mutations.ResultsIn the LS family, three members (I:1, II:1 and II:4) were affected by GBM. GBMs with loss of MSH2 and MSH6 expression displayed giant and multinucleated bizarre cells, along with mutations in ARID1A, TP53, ATM, and NF1 genes. All GBMs had TMB-H but not MSI-H. CD8+ T cells and CD163+ macrophages were abundant in each GBM tissue. The primary and recurrent GBMs of II:1 showed mesenchymal characteristics with high PD-L1 expression. The family members harbored a novel heterozygous germline mutation in MSH2 and FDPS genes, confirming the diagnosis of LS and disseminated superficial actinic porokeratosis.ConclusionLS-associated GBM exhibits heterogeneity in clinicopathologic and molecular genetic features, as well as a suppressive TIME. The presence of MMR deficiency and TMB-H may serve as predictive factors for the response to immune checkpoint inhibitor therapy in GBMs. The identification of LS-associated GBM can provide significant benefits to both patients and their family members, including accurate diagnosis, genetic counseling, and appropriate screening or surveillance protocols. Our study serves as a reminder to clinicians and pathologists to consider the possibility of concurrent genetic syndromes in individuals or families.

Mosquitoes are a formidable reservoir of viruses and important vectors of zoonotic pathogens. Blood-fed mosquitoes have been utilized to determine host infection status, overcoming the difficulties associated with sampling from human and animal populations. Comprehensive surveillance of potential pathogens at the interface of humans, animals, and the environment is currently an accredited method to provide an early warning of emerging or re-emerging infectious diseases and to proactively respond to them. Herein we performed comprehensive sampling of mosquitoes from seven habitats (residential areas, hospital, airplane, harbor, zoo, domestic sheds, and forest park) across five cities in Guangdong Province, China. Our aim was to characterize the viral communities and blood feeding patterns at the human-animal-environment interface and analyze the potential risk of cross-species transmission using meta-transcriptomic sequencing. 1898 female adult mosquitoes were collected, including 1062 Aedes and 836 Culex mosquitoes, of which approximately 12% (n = 226) were satiated with blood. Consequently, 101 putative viruses were identified, which included DNA and RNA viruses, and positive-stranded RNA viruses (+ssRNA) were the most abundant. According to viral diversity analysis, the composition of the viral structure was highly dependent on host species, and Culex mosquitoes showed richer viral diversity than Aedes mosquitoes. Although the virome of mosquitoes from different sampling habitats showed an overlap of 39.6%, multiple viruses were specific to certain habitats, particularly at the human-animal interface. Blood meal analysis found four mammals and one bird bloodmeal source, including humans, dogs, cats, poultry, and rats. Further, the blood feeding patterns of mosquitoes were found to be habitat dependent, and mosquitoes at the human-animal interface and from forests had a wider choice of hosts, including humans, domesticated animals, and wildlife, which in turn considerably increases the risk of spillover of potential zoonotic pathogens. To summarize, we are the first to investigate the virome of mosquitoes from multiple interfaces based on the One Health concept. The characteristics of viral community and blood feeding patterns of mosquitoes at the human-animal-environment interface were determined. Our findings should support surveillance activities to identify known and potential pathogens that are pathogenic to vertebrates.

Dendritic cells (DCs) can initiate both naïve and memory T cell activation, as the most potent antigen-presenting cells. For efficient anti-tumor immunity, it is essential to enhance the anti-tumoral activity of tumor-associated DCs (TADCs) or to potently restrain TADCs so that they remain immuno-stimulating cells. Combined phospholipids (cPLs) adjuvant may act through the activation of DCs. This study demonstrated the potential mechanism of tumor growth inhibition of cPLs adjuvant, and confirmed that cPLs adjuvant could induce the maturation and activation (upregulation of MHC-II, CD80, CD40, IL-1β, IL-12, IL-6 expression) of BMDCs in vitro. Then we isolated tumor infiltrating lymphocytes (TILs) from solid tumor and analyzed the phenotype and cytokines of TILs. The examination of the TILs revealed that cPLs adjuvant upregulated the expression of co-stimulatory molecules (MHC-II, CD86), phosphatidylserine (PS) receptor (TIM-4) on TADCs and enhanced the cytotoxic effect (CD107a), as well as pro-inflammatory cytokine production (IFN-γ, TNF-α, IL-2) by the tumor-resident T cells. Taken together, cPLs adjuvant may be an immune-potentiating adjuvant for cancer immunotherapy. This reagent may lead to the development of new approaches in DC-targeted cancer immunotherapy.

Backgroud and study aims: Esophageal stricture is a serious adverse event occurring after circular endoscopic submucosal dissection (ESD) involving the whole esophagus. However, there is still a lack of effectively preventive methods. The main purpose of this study is to evaluate the efficacy of application of acellularized dermis matrix (ADM) for the prevention of post-ESD esophageal stricture. The main objective of this study was to evaluate the use of decellularized dermal matrix (ADM) in the prevention of post-esophageal ESD strictures. Patients and methods: A pilot, single-center, prospective study was conducted. The study enrolled seven patients who had high-risks with extended resection of developing post-ESD esophageal stricture. After undergoing ESD, we attached different size of ADM patches to the mucosal defects using titanium clips then fixed with a metal mesh stent. The stent covered with metal mesh was removed at the median time of 27 days after the endoscopic procedure. Follow-up and repeated outpatient endoscopic screening were performed at appropriate scheduled times. Results: The average longitudinal diameter of the resected specimens was 58.3 mm (range 38–90 mm). There were three patients developing strictures postoperatively at a mean time of 87 days (range 42–140). The median number of postoperative endoscopic balloon dilatation (EBD) in patients with stenosis was 2 (range 2–9). There were no deaths during a median follow-up period of 6 moths (range 1–12). Conclusions: This study was performed to assess the efficacy and safe method of relieving the severity of esophageal stricture after ESD through transplantation of ADM.

Abstract CD4+CD25+ regulatory T cells (Tregs), a subpopulation of naturally CD4+ T cells that characteristically express transcription factor Forkhead box P3 (FOXP3), play a pivotal role in the maintenance of immune homeostasis and the prevention of autoimmunity. With the development of biological technology, the understanding of plasticity and stability of Tregs has been further developed. Recent studies have suggested that human Tregs are functionally and phenotypically diverse. The functions and mechanisms of different phenotypes of Tregs in different disease settings, such as tumor microenvironment, autoimmune diseases, and transplantation, have gradually become hot spots of immunology research that arouse extensive attention. Among the complex functions, CD4+CD25+FOXP3+ Tregs possess a potent immunosuppressive capacity and can produce various cytokines, such as IL‐2, IL‐10, and TGF‐β, to regulate immune homeostasis. They can alleviate the progression of diseases by resisting inflammatory immune responses, whereas promoting the poor prognosis of diseases by helping cells evade immune surveillance or suppressing effector T cells activity. Therefore, methods for targeting Tregs to regulate their functions in the immune microenvironment, such as depleting them to strengthen tumor immunity or expanding them to treat immunological diseases, need to be developed. Here, we discuss that different subpopulations of Tregs are essential for the development of immunotherapeutic strategies involving Tregs in human diseases.

The fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), is an important agricultural pest with a strong migratory ability. While the species is native to the Americas, it has recently invaded China, ravaging crops in many provinces (autonomous regions, municipalities). Its long-distance migration, which is critical to understand for pest management programs, has been well studied in its native region. In other regions however, the moth's migration patterns have not yet been characterized. Here, the migratory behavior of FAW populations in China was studied on an isolated island located in the center of the Bohai Strait from spring to autumn 2019, the year in which FAW first reached this region, by using searchlight trapping, stable carbon isotopes and trajectory simulation. The main results were summarized as follows: (i) The number of FAW moths caught by searchlight trapping provided direct evidence that the species migrated across the Bohai Sea. (ii) Species identification was confirmed by both morphology and molecular methods, and only the “corn-strain” that preferentially infests maize and sorghum was found in the collections. (iii) Stable carbon isotope measurements showed that up to 95.07% of captured moths displayed the C4 isotope signature, thus providing additional evidence that this species is a migrant as there are no major C4 plants at the trapping site. (iv) Backward trajectory analysis indicated that the pest now threatens to expand its range into the agriculturally important region of Northeast China, and thus authorities in this region should be vigilant to the threat posed by this species. Together, these findings add to our knowledge concerning the occurrence of FAW in northern China, and will help us to develop sustainable and effective monitoring, forecasting, and pest management strategies.

Background: Streptococcus suis (SS) is a major swine pathogen and a serious zoonotic pathogen causing septicemia and meningitis in piglets and humans. Using an immunoproteomic approach, we previously brought evidence that ornithine carbamoytransferase (OCT) may represent a vaccine candidate to protect against S. suis biofilm-related and acute infections. Method: In this study, the gene encoding OCT was cloned into the expression vector pET-28a and the recombinant protein was expressed in Escherichia coli BL21. The immunogenicity and protective efficacy of the SS OCT was further investigated in a mouse model. Results: The protein was found to be expressed in vivo and elicited high antibody titers following SS infections in mice. An animal challenge experiment with SS showed that 62.5% of mice immunized with the OCT protein were protected. Using an in vitro competitive adherence inhibition assay of adherence, evidence was obtained that OCT could significantly reduce the number of SS cells adhered to porcine kidney PK-15 cells. The bacterial levels recovered in mice of the OCT immunized group were significantly decreased in some organs, compared with the control group. Conclusion: In summary, our results suggest that the recombinant SS OCT protein, which is involved in bacterial adherence, may efficiently stimulate an immune response conferring protection against SS infections. It may therefore be considered as a potential vaccine candidate, although further studies are necessary to evaluate their use in swine. Keywords: Strepcotoccus suis, Ornithine carbamoyltransferase, Adherence, Vaccine

A series of freezing experiments were carried out on the Qinghai silty clay using a one-dimensional soil column particle image velocimetry (PIV) system. The effects of the initial water content and temperature gradient on the frost heave of the soil column were analysed. The experimental results show that the total volumetric water content after the Qinghai silty clay freezing is much greater than the initial water content before freezing, and the total volumetric water content at the segregated layer reaches the maximum value. The average unfrozen water content firstly increases sharply with time and then decreases slowly. The obvious horizontal layered segregation cracks filled with unsaturated needle-columnar ice lenses have occurred above the soil column frozen fringe. The thickness of segregated ice is about 43% of the total frost heave. There is a small frost heave in the frozen fringe and a small compression deformation amount in the unfrozen zone. Under the same temperature gradient conditions, the soil column with the low initial water content has a smaller frozen depth, larger total volumetric water content, more water intake, smaller segregated ice thickness, and less total frost heave amount than that of the soil column with the high initial water content. Under the same initial water content conditions, the soil column with a large temperature gradient has a greater frozen depth, consistent total volumetric water content, larger segregated ice thickness, and greater total frost heave amount than that of the soil column with a small temperature gradient.

BackgroundOridonin (Ori), the main bioactive ingredient of the natural anti-inflammatory herb Rabdosia rubescens, could be a covalent inhibitor of the NLRP3 inflammasome. Solid organ transplantation provides a life-saving optional therapy for patients with end-stage organ dysfunction. The long-term survival of solid organ transplantation remains restricted because of the possibility of rejection and the toxicity, infection, cardiovascular disease, and malignancy related to immunosuppressive (IS) drugs. However, the pathogenic mechanisms involved remain unclear. The ideal IS drugs to prevent allograft rejection have not been identified. Here, we investigated whether Ori could prolong the in vivo survival of completely mismatched cardiac allografts.MethodsThe cardiac transplantation models were conducted among three groups of mice from C57BL/6NCrSlc (B6/N) or C3H/HeNSlc (C3H) to C3H: the syngeneic and the allogeneic group, whose recipients were treated with vehicle of Ori, and the Ori treatment group, in which the recipients were transplanted hearts from MHC-I mismatched donors and treated with different dosages of Ori from post-operative day (POD) 0 to 7. Then, we investigated the effect of Ori on bone marrow-derived dendritic cell (BMDC) and allogeneic mixed lymphocyte reaction in vitro.ResultsOri with 3, 10, and 15 mg/kg Ori could prolong the survival (MST = 22.8, 49.2, and 65.3 days, respectively). We found that infiltrating CD8+ T cells and macrophages were decreased, and regulatory T cells (Tregs) were expanded in allografts on POD7. The mRNA level of IL-1β and IFN-γ of allografts was downregulated. Mechanistically, Ori-treated BMDCs suppressed T-cell proliferation and IFN-γ+CD4+ T-cell differentiation, along with the expansion of Tregs and IL-10+CD4+ T cells. Ori inhibited NOD, LRR-, and pyrin domain-containing protein 3 (NLRP3) expression; attenuated NF-κB and IκBα phosphorylation in LPS-activated BMDCs; downregulated NLRP3, Caspase-1, IL-1β, IL-18, and IFN-γ; and upregulated IL-10 expression.ConclusionsOur findings highlight the potential of Ori as a novel and natural IS agent to improve transplant tolerance. Ori could exert IS activity through decreasing IL-1β and IL-18 production and Th1 differentiation and proliferation and expanding Tregs via inhibiting the NF-κB/NLRP3 signaling pathway.

Both acute and chronic antibody-mediated allograft rejection (AMR), which are directly mediated by B cells, remain difficult to treat. Long-lived plasma cells (LLPCs) in bone marrow (BM) play a crucial role in the production of the antibodies that induce AMR. However, LLPCs survive through a T cell-independent mechanism and resist conventional immunosuppressive therapy. Desensitization therapy is therefore performed, although it is accompanied by severe side effects and the pathological condition may be at an irreversible stage when these antibodies, which induce AMR development, are detected in the serum. In other words, AMR control requires the development of a diagnostic method that predicts its onset before LLPC differentiation and enables therapeutic intervention and the establishment of humoral immune monitoring methods providing more detailed information, including individual differences in the susceptibility to immunosuppressive agents and the pathological conditions. In this study, we reviewed recent studies related to the direct or indirect involvement of immunocompetent cells in the differentiation of naïve-B cells into LLPCs, the limitations of conventional methods, and the possible development of novel control methods in the context of AMR. This information will significantly contribute to the development of clinical applications for AMR and improve the prognosis of patients who undergo organ transplantation.

A black yeast, Aureobasidium pullulans, extracellularly produces β-(1,3), (1,6)-D-glucan (β-glucan) under certain conditions. The β-glucan is known to be an immunomodulatory agent, and β-glucan enriched A. pullulans cultured fluid (AP-CF) is used in supplements to maintain human health. Concanavalin A (ConA) is a lectin, and when injected it is known to cause T cell mediated autoimmune hepatitis in mice. The present study investigated the effects of oral administration of AP-CF on ConA injection in mice. The results demonstrated that increases in serum alanine transaminase (ALT) levels after ConA injection were significantly suppressed in an AP-CF administered group of mice. To understand the mechanism of the ALT lowering effects of AP-CF, we used Foxp3 (forkhead box P3) knock-in mice which express the green fluorescent protein (GFP) in Foxp3 induced cells, and the effects of AP-CF on the regulatory T cell (Treg) populations were investigated. The results show that the basal level of Foxp3+ Treg populations in peripheral blood lymphocytes, liver infiltrating lymphocytes, and splenocytes was decreased after 7 days of administration of AP-CF. These findings suggest that oral administration of AP-CF suppresses the basal level of inflammation, and that it may be postulated to be involved in the ALT lowering effects of AP-CF.

Cyclosporin A (CsA) is a common immunosuppressant, but its use is limited as it can cause chronic kidney injury. Oxidative stress and apoptosis play a key role in CsA‐induced nephrotoxicity. This study investigated the protective effect of 5‐aminolevulinic acid and iron (5‐ALA/SFC) on CsA‐induced injury in murine proximal tubular epithelial cells (mProx24). 5‐ALA/SFC significantly inhibited apoptosis in CsA‐treated mProx24 cells with increases in heme oxygenase (HO)‐1, nuclear factor E2‐related factor 2 (Nrf2), and p38, and Erk‐1/2 phosphorylation. Moreover, 5‐ALA/SFC suppressed production of reactive oxygen species in CsA‐exposed cells and inhibition of HO‐1 suppressed the protective effects of 5‐ALA/SFC. In summary, 5‐ALA/SFC may have potential for development into a treatment for the anti‐nephrotoxic/apoptotic effects of CsA.

Abstract Background Liver transplantation (LT) is considered the standard treatment for end-stage liver disease, but ideal donors remain in limited supply, resulting in an unavoidable increase in the need to use grafts from marginal donors. The attenuation of ischemia-reperfusion injury (IRI) in such marginal donors is therefore crucial for reducing the possibility of the primary non-function of grafts and graft loss. Some reports have found that molecular-hydrogen showed antioxidant and anti-inflammatory effects in preventing IRI in some non-hepatic transplant models. Therefore, we investigated whether or not molecular-hydrogen could attenuate IRI in LT model rats. Methods We used a hydrogen-rich water bath to dissolve hydrogen into solution and graft tissues and performed isogenic and orthotopic LT in Lewis rats with University of Wisconsin (UW) solution. Blood and tissue samples were collected 6 h after the reperfusion. Hepatic enzymes in serum were measured. Pathological findings including the expressions of cytokines and heme oxygenase (HO)-1 in liver tissues were evaluated. Results The concentration of hydrogen inside the graft tissues increased depending on the storage time, plateauing after 1 h. Serum liver enzyme levels were significantly lower and the histology score of liver damage markedly attenuated in the group given grafts preserved in hydrogen-rich UW solution than in the control group. The hydrogen-rich UW solution group also showed less oxidative damage and hepatocyte apoptosis than the control group, and the expression of proinflammatory cytokines tended to be lower while the protein levels of HO-1 were significantly increased (n = 3–12 per group, P

Methods for suppressing the host immune system over the long term and improving transplantation tolerance remain a primary issue in organ transplantation. Cell therapy is an emerging therapeutic strategy for immunomodulation after transplantation. Mesenchymal stem cells (MSCs) are adult multipotent stem cells with wide differentiation potential and immunosuppressive properties, which are mostly used in regenerative medicine and immunomodulation. In addition, emerging research suggests that MSC-derived exosomes have the same therapeutic effects as MSCs in many diseases, while avoiding many of the risks associated with cell transplantation. Their unique immunomodulatory properties are particularly important in the immune system-overactive graft environment. In this paper, we review the effects of MSC-derived exosomes in the immune regulation mechanism after organ transplantation and graft-versus-host disease (GvHD) from various perspectives, including immunosuppression, influencing factors, anti-inflammatory properties, mediation of tissue repair and regeneration, and the induction of immune tolerance. At present, the great potential of MSC-derived exosomes in immunotherapy has attracted a great deal of attention. Furthermore, we discuss the latest insights on MSC-derived exosomes in organ transplantation and GvHD, especially its commercial production concepts, which aim to provide new strategies for improving the prognosis of organ transplantation patients.

Allograft rejection has been an obstacle for the long-term survival of patients. CD70, a tumor necrosis factor (TNF) family member critically expressed on antigen-presenting cells and strongly but transiently up-regulated during lymphocyte activation, represents an important co-stimulatory molecule that induces effective T cell responses. We used a mouse heterotopic cardiac transplantation model to evaluate the effects of monotherapy with the antibody targeting mouse CD70 (FR70) on transplantation tolerance and its immunoregulatory activity. FR70-treated C3H recipient mice permanently accepted B6 fully mismatched cardiac allografts. Consistent with the graft survival, the infiltration of CD8+ T cells in the graft was reduced, dendritic cells were differentiated into a tolerogenic status, and the number of regulatory T cells was elevated both in the graft and the recipient’s spleen. In addition, naïve C3H given an adoptive transfer of spleen cells from the primary recipients with FR70 treatment accepted a heart graft from a matching B6 donor but not third-party BALB/c mice. Our findings show that treatment with FR70 induced regulatory cells and inhibited cytotoxic T cell proliferation, which led to long-term acceptance of mouse cardiac allografts. These findings highlight the potential role of anti-CD70 antibodies as a clinically effective treatment for allograft rejection.

Current organ transplantation therapy is life-saving but accompanied by well-recognized side effects due to post-transplantation systematic immunosuppressive treatment. Dendritic cells (DCs) are central instigators and regulators of transplantation immunity and are responsible for balancing allograft rejection and tolerance. They are derived from monocyte-macrophage DC progenitors originating in the bone marrow and are classified into different subsets based on their developmental, phenotypical, and functional criteria. Functionally, DCs instigate allograft immunity by presenting donor antigens to alloreactive T cells via direct, indirect, and semidirect recognition pathways and provide essential signaling for alloreactive T cell activation via costimulatory molecules and pro-inflammatory cytokines. Regulatory DCs (DCregs) are characterized by a relatively low expression of major histocompatibility complex, costimulatory molecules, and altered cytokine production and exert their regulatory function through T cell anergy, T cell deletion, and regulatory T cell induction. In rodent transplantation studies, DCreg-based therapy, by in situ targeting or infusion of ex vivo generated DCregs, exhibits promising potential as a natural, well-tolerated, organ-specific therapeutic strategy for promoting lasting organ-specific transplantation tolerance. Recent early-phase studies of DCregs have begun to examine the safety and efficacy of DCreg-induced allograft tolerance in living-donor renal or liver transplantations. The present review summarizes the basic characteristics, function, and translation of DCregs in transplantation tolerance induction.

Liver transplantation is the ideal treatment approach for a variety of end-stage liver diseases. However, life-long, systemic immunosuppressive treatment after transplantation is required to prevent rejection and graft loss, which is associated with severe side effects, although liver allograft is considered more tolerogenic. Therefore, understanding the mechanism underlying the unique immunologically privileged liver organ is valuable for transplantation management and autoimmune disease treatment. The unique hepatic acinus anatomy and a complex cellular network constitute the immunosuppressive hepatic microenvironment, which are responsible for the tolerogenic properties of the liver. The hepatic microenvironment contains a variety of hepatic-resident immobile non-professional antigen-presenting cells, including hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells, that are insufficient to optimally prime T cells locally and lead to the removal of alloreactive T cells due to the low expression of major histocompatibility complex (MHC) molecules, costimulatory molecules and proinflammatory cytokines but a rather high expression of coinhibitory molecules and anti-inflammatory cytokines. Hepatic dendritic cells (DCs) are generally immature and less immunogenic than splenic DCs and are also ineffective in priming naïve allogeneic T cells via the direct recognition pathway in recipient secondary lymphoid organs. Although natural killer cells and natural killer T cells are reportedly associated with liver tolerance, their roles in liver transplantation are multifaceted and need to be further clarified. Under these circumstances, T cells are prone to clonal deletion, clonal anergy and exhaustion, eventually leading to tolerance. Other proposed liver tolerance mechanisms, such as soluble donor MHC class I molecules, passenger leukocytes theory and a high-load antigen effect, have also been addressed. We herein comprehensively review the current evidence implicating the tolerogenic properties of diverse liver cells in liver transplantation tolerance.

IgA nephropathy (IgAN) is the most prevalent primary glomerulonephritis worldwide, with diverse clinical manifestations characterized by recurrent gross hematuria or microscopic hematuria, and pathological changes featuring poorly O-galactosylated IgA1 deposition in the glomerular mesangium. Pathogenesis has always been the focus of IgAN studies. After 50 years of research, most scholars agree that IgAN is a group of clinicopathological syndromes with certain common immunopathological characteristics, and multiple mechanisms are involved in its pathogenesis, including immunology, genetics, and environmental or nutritional factors. However, the precise pathogenetic mechanisms have not been fully determined. One hypothesis about the pathogenesis of IgAN suggests that immunological factors are engaged in all aspects of IgAN development and play a critical role. A variety of immune cells (e.g., dendritic cells, NK cells, macrophages, T-lymphocyte subsets, and B-lymphocytes, etc.) and molecules (e.g., IgA receptors, Toll-like receptors, complements, etc.) in innate and adaptive immunity are involved in the pathogenesis of IgAN. Moreover, the abnormality of mucosal immune regulation is the core of IgAN immunopathogenesis. The roles of tonsil immunity or intestinal mucosal immunity, which have received more attention in recent years, are supported by mounting evidence. In this review, we will explore the latest research insights on the role of immune modulation in the pathogenesis of IgAN. With a better understanding of immunopathogenesis of IgAN, emerging therapies will soon become realized.

Abstract Fatty liver has lower tolerance against ischemia-reperfusion (I/R) injury in liver operations, including liver transplantation. Seeking to ameliorate liver injury following I/R in fatty liver, we examined the protective effect of hydrogen (H2) saline on I/R liver injury in a methionine and choline-deficient plus high fat (MCDHF) diet-induced fatty liver mouse model. Saline containing 7 ppm H2 was administrated during the process of I/R. Livers were obtained and analyzed. Primary hepatocytes and Kupffer cells (KCs) were obtained from fatty liver and subjected to hypoxia/reoxygenation. Apoptosis-related proteins and components of the signaling pathway were analyzed after treatment with hydrogen gas. The MCDHF I/R group showed higher levels of AST and ALT in serum, TUNEL-positive apoptotic cells, F4/80 immunopositive cells, mRNA levels of inflammatory cytokines, constituents of the signaling pathway, pro-apoptotic molecules in liver, and KCs and/or primary hepatocytes, compared to the control group. In contrast, H2 treatment significantly suppressed the signs of I/R injury in fatty liver. Moreover, the expression of Bcl-2, HO-1, and Sirt1 in liver, KCs, and hepatocytes by hydrogen gas were increased, whereas caspase activation, Bax, and acetylation of p53 were suppressed by hydrogen gas. These results demonstrated that H2 treatment ameliorated I/R liver injury in a fatty liver model by reducing hepatocyte apoptosis, inhibiting macrophage activation and inflammatory cytokines, and inducing HO-1 and Sirt1 expression. Taken togather, treatment with H2 saline may have a protective effect and safe therapeutic activity during I/R events, such as in liver transplantation with fatty liver.

Myeloid-derived suppressor cells (MDSCs) are markedly increased in cancer patients and tumor-bearing mice and promote tumor growth and survival by inhibiting host innate and adaptive immunity. In this study, we generated and characterized MDSCs from murine-induced pluripotent stem cells (iPSCs). The iPSCs were co-cultured with OP9 cells, stimulated with GM-CSF, and became morphologically heterologous under co-culturing with hepatic stellate cells. Allogeneic and OVA-specific antigen stimulation demonstrated that iPS-MDSCs have a T-cell regulatory function. Furthermore, a popliteal lymph node assay and autoimmune hepatitis model showed that iPS-MDSCs also regulate immune responsiveness in vivo and have a therapeutic effect against hepatitis. Taken together, our results demonstrated a method of generating functional MDSCs from iPSCs and highlighted the potential of iPS-MDSCs as a key cell therapy resource for transplantation and autoimmune diseases. Keywords: Myeloid-derived suppressor cells, Induced pluripotent stem cells, T cell response

Systemic sclerosis (SSc) is a connective tissue disease that results in fibrosis in multiple organs. Various animal models for this disease have been developed, both genetic and induced. One of the induced models, sclerodermatous graft‐versus‐host disease (scl‐GvHD), exhibits the main characteristics of SSc, but involves lethal γ‐irradiation of recipients. We sought to develop a modified scl‐GvHD model. Spleen cells from B10.D2 donor mice were transplanted into immunodeficient Rag‐2 recipients on the BALB/c genetic background. Tissue fibrosis was analyzed at 3 and 9 weeks after transplantation. In addition to serum levels of anti‐Scl‐70 autoantibody and cytokines, tissue inflammation, fibrosis, expression of collagen‐I and α‐smooth muscle actin (α‐SMA), infiltration of leukocytes, mRNA expression of transforming growth factor (TGF)‐β, collagen‐I, α‐SMA, tumor necrosis factor (TNF)‐α, and interleukin (IL)‐6, the classical signal pathway of TGF‐β, Smad‐3, and p‐Smad‐3 expression in tissue were analyzed. Skin thickening and increased collagen synthesis, as well as the manifestation of tissue fibrosis, could be detected in skin, kidney, and lung of modified scl‐GvHD mouse model. Increased serum levels of anti‐Scl‐70 autoantibody, IL‐10, and TGF‐β could be detected. Increased CD4+ T cells and F4/80+ macrophage infiltration were found in skin, kidney, and lung. Gene expression of collagen‐I, TGF‐β, α‐SMA, TNF‐α, and IL‐6 was increased in tissue of the scl‐GvHD model. Moreover, TGF‐β expression and Smad‐3 phosphorylation were detected in skin, kidney, and lung of scl‐GvHD mice. Our data show that spleen cells from B10.D2 donor mice transplanted into immunodeficient Rag‐2 recipients could induce typical fibrosis not only of the skin and kidney but also of lung, which was missing from previous scl‐GvHD models. Thus, the modified scl‐GvHD model might be a promising model to explore the immunologic mechanisms of SSc and may be useful for investigation of new therapies for systemic sclerosis.

Regulatory dendritic cell (DCregs)-based immunotherapy is a potential therapeutic tool for transplant rejection. We generated DCregs from murine induced pluripotent stem cells (iPSCs), which could remain in a “stable immature stage” even under strong stimulation. Harnessing this characteristic, we hypothesized that iPS-DCregs worked as a negative vaccine to generate regulatory T cells (Tregs), and induced donor-specific allograft acceptance. We immunized naive CBA (H-2Kk) mice with B6 (H-2Kb) iPS-DCregs and found that Tregs (CD4+CD25+FOXP3+) significantly increased in CBA splenocytes. Moreover, immunized CBA recipients permanently accepted B6 cardiac grafts in a donor-specific pattern. We demonstrated mechanistically that donor-type iPS-DCregs triggered transforming growth factor β1 secretion, under which the donor-antigen peptides directed naive CD4+ T cells to differentiate into donor-specific FOXP3+ Tregs instead of into effector T cells in vivo. These findings highlight the potential of iPS-DCregs as a key cell therapy resource in clinical transplantation.

Worldwide morbidity and mortality from acute myocardial infarction (AMI) and related heart failure remain high. While effective early reperfusion of the criminal coronary artery after a confirmed AMI is the typical treatment at present, collateral myocardial ischemia-reperfusion injury (MIRI) and pertinent cardioprotection are still challenging to address and have inadequately understood mechanisms. Therefore, unveiling the related novel molecular targets and networks participating in triggering and resisting the pathobiology of MIRI is a promising and valuable frontier. The present study specifically focuses on the recent MIRI advances that are supported by sophisticated bio-methodology in order to bring the poorly understood interrelationship among pro- and anti-MIRI participant molecules up to date, as well as to identify findings that may facilitate the further investigation of novel targets.

Antibody-mediated allograft rejection (AMR) hinders patient prognosis after organ transplantation. Current studies concerning AMR have mainly focused on the diagnostic value of immunoglobulin G (IgG)-type donor-specific antihuman leukocyte antigen antibodies (DSAs), primarily because of their antigen specificity, whereas the clinical significance of immunoglobulin M (IgM)-type DSAs has not been thoroughly investigated in the context of organ transplantation because of their nonspecificity against antigens. Although consensus regarding the clinical significance and role of IgM antibodies is not clear, as discussed in this review, recent findings strongly suggest that they also have a huge potential in novel diagnostic as well as therapeutic application for the prevention of AMR. Most serum IgM antibodies are known to comprise natural antibodies with low affinity toward antigens, and this is derived from B-1 cells (innate B cells). However, some of the serum IgM-type antibodies reportedly also produced by B-2 cells (conventional B cells). The latter are known to have a high affinity for donor-specific antigens. In this review, we initially discuss how IgM-type antibodies of different origins participate in the pathology of various diseases, directly or through cell surface receptors, complement activation, or cytokine production. Then, we discuss the clinical applicability of B-1 and B-2 cell-derived IgM-type antibodies for controlling AMR with reference to the involvement of IgM antibodies in various pathological conditions.

The CD98 heavy chain (CD98hc) regulates virus-induced cell fusion and monocyte fusion, and is involved in amino acid transportation. Here, we examined the role that CD98hc plays in the formation of osteoclasts using CD98hcflox/floxLysM-cre peritoneal macrophages (CD98hc-defect macrophages). Peritoneal macrophages were stimulated with co-cultured with osteoblasts in the presence of 1,25(OH)2 vitamin D3, and thereafter stained with tartrate-resistant acid phosphatase staining solution. The multinucleated osteoclast formation was severely impaired in the peritoneal macrophages isolated from the CD98hc-defect mice compared with those from wild-type mice. CD98hc mediates integrin signaling and amino acid transport through the CD98 light chain (CD98lc). In integrin signaling, suppression of the M-CSF-RANKL-induced phosphorylation of ERK, Akt, JNK and p130Cas were observed at the triggering phase in the CD98h-defect peritoneal macrophages. Moreover, we showed that the general control non-derepressible (GCN) pathway, which was activated by amino acid starvation, was induced by the CD98hc-defect peritoneal macrophages stimulated with RANKL. These results indicate that CD98 plays two important roles in osteoclast formation through integrin signaling and amino acid transport.

One-way mixed lymphocyte reaction (MLR) is a classic tool to measure how T cells react to external stimuli. However, MLR is an in vitro reaction system, which shows different response intensity compared with in vivo trails sometimes due to the lack of cytokines, tissue matrix and other immune response associated factors. The following popliteal lymph node assay (PLNA) protocol is designed to test the T cells antigen-specific reaction in vivo by using ovalbumin (OVA) specific reacted transgenic mouse OT-1 and OT-2.

Steatosis has a low tolerance against ischemia-reperfusion injury (IRI). To prevent IRI in the steatotic liver, we attempted to elucidate the protective effect of astaxanthin (ASTX) in the steatotic liver model by giving mice a methionine and choline-deficient high fat (MCDHF) diet. Levels of lipid peroxidation and apoptosis, the expression of inflammatory cytokines and heme oxygenase (HO)-1, in the liver were assessed. Reactive oxygen species (ROS), inflammatory cytokines, apoptosis-related proteins and members of the signaling pathway were also examined in isolated Kupffer cells and/or hepatocytes from the steatotic liver. ASTX decreased serum ALT and AST levels, the amount of TUNEL, F4/80, or 4HNE-positive cells and the mRNA levels of inflammatory cytokines in MCDHF mice by IRI. Moreover, HO-1 and HIF-1α, phosphorylation of Akt and mTOR expressions were increased by ASTX. The inflammatory cytokines produced by Kupffer, which were subjected to hypoxia and reoxygenation (HR), were inhibited by ASTX. Expressions of Bcl-2, HO-1 and Nrf2 in hepatocytes by HR were increased, whereas Caspases activation, Bax and phosphorylation of ERK, MAPK, and JNK were suppressed by ASTX. Pretreatment with ASTX has a protective effect and is a safe therapeutic treatment for IRI, including for liver transplantation of the steatotic liver.

Background and Aim. The connection between gene polymorphisms of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and primary biliary cholangitis (PBC) is still vague and blurred. The purpose of this study is to precisely estimate the association of the polymorphisms of CTLA4 with the risk of PBC by using a meta-analysis. Methods. PubMed and the Chinese National Knowledge Infrastructure (CNKI) database were used to search correlative literatures, and the documents which were about the relationships between the polymorphisms of CTLA4 (rs231775, rs231725, rs3087243, and rs5742909) and PBC were collected as of June 2016. The strength of correlation based on odds ratios (ORs) and its 95% confidence intervals (95%CIs) was computed by STATA. Results. Generally, in rs231775, a significant risk was found in G allele, the value of OR was 1.32, and its 95%CI was 1.19 to 1.47. The same situation was found in A allele of rs231725, the value of OR was 1.33, and its 95%CI was 1.22 to 1.45. As genotypic level, different genotypic models were also found to have obvious relevance with PBC in rs231775 and rs231725. No obvious connections were found in other SNPs. Conclusion. This study indicated that the polymorphisms of rs231775 and rs231725 would be the risk factors of PBC.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that consists of myeloid progenitor cells and immature myeloid cells. They have been identified as a cell population that may affect the activation of CD4+ and CD8+ T-cells to regulate the immune response negatively, which makes them attractive targets for the treatment of transplantation and autoimmune diseases. Several studies have suggested the potential suppressive effect of MDSCs on allo- and autoimmune responses. Conversely, MDSCs have also been found at various stages of differentiation, accumulating during pathological situations, not only during tumor development but also in a variety of inflammatory immune responses, bone marrow transplantation, and some autoimmune diseases. These findings appear to be contradictory. In this review, we summarize the roles of MDSCs in different transplantation and autoimmune diseases models as well as the potential to target these cells for therapeutic benefit.

Hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of morbidity and mortality in neonates. Because of high concentrations of sensitive immature cells, metal-catalyzed free radicals, non-saturated fatty acids, and low concentrations of antioxidant enzymes, the brain requires high levels of oxygen supply and is, thus, extremely sensitive to hypoxia. Strong evidence indicates that oxidative stress plays an important role in pathogenesis and progression. Following hypoxia and ischemia, reactive oxygen species (ROS) production rapidly increases and overwhelms antioxidant defenses. A large excess of ROS will directly modify or degenerate cellular macromolecules, such as membranes, proteins, lipids, and DNA, and lead to a cascading inflammatory response, and protease secretion. These derivatives are involved in a complex interplay of multiple pathways (e.g., inflammation, apoptosis, autophagy, and necrosis) which finally lead to brain injury. In this review, we highlight the molecular mechanism for oxidative stress in HIE, summarize current research on therapeutic strategies utilized in combating oxidative stress, and try to explore novel potential clinical approaches.

During aortic surgery, interruption of spinal cord blood flow might cause spinal cord ischemia-reperfusion injury (IRI). The incidence of spinal cord IRI after aortic surgery is up to 28%, and patients with spinal cord IRI might suffer from postoperative paraplegia or paraparesis. Spinal cord IRI includes two phases. The immediate spinal cord injury is related to acute ischemia. And the delayed spinal cord injury involves both ischemic cellular death and reperfusion injury. Inflammation is a subsequent event of spinal cord ischemia and possibly a major contributor to spinal cord IRI. However, the development of inflammatory mediators is incompletely demonstrated. And treatments available for inflammation in spinal cord IRI are insufficient. Improved understanding about spinal cord IRI and the development of inflammatory cells and cytokines in this process will provide novel therapeutic strategies for spinal cord IRI. Inflammatory cytokines (e.g., TNF-α and IL-1) may play an important role in spinal cord IRI. For treatment of several intractable autoimmune diseases (e.g., rheumatoid arthritis), where inflammatory cytokines are involved in disease progression, anti-inflammatory cytokine antagonist is now available. Hence, there is great potential of anti-inflammatory cytokine antagonist for therapeutic use of spinal cord IRI. We here review the mediators and several possibilities of treatment in spinal cord IRI.

Osteonecrosis of the talus (ONT) may severely affect the function of the ankle joint. Most orthopedists believe that ONT should be treated at an early stage, but a concise and effective surgical treatment is lacking. In this study, porous titanium alloy rods were prepared and implanted into the tali of sheep with early-stage ONT (IM group). The curative effect of the rods was compared to treatment by core decompression (DC group). No significant differences in bone reconstruction were observed between the two groups at 1 month after intervention. After 3 months, the macroscopic view of gross specimens of the IM group showed ordinary contours, but the specimens of the DC group showed obvious partial bone defects and cartilage degeneration. Quantitative analysis of the reconstructed trabeculae by micro-CT and histological study suggested that the curative effect of the IM group was superior to that of the DC group at 3 months after intervention. These favorable short-term results of the implantation of porous titanium alloy rods into the tali of sheep with early-stage ONT may provide insight into an innovative surgical treatment for ONT.

Bone marrow-derived mesenchymal stem cells (MSCs) could exert a potent immunosuppressive effect, and therefore may have a therapeutic potential in T-cell-dependent pathologies. In the present study, we aimed to determine whether MSCs could be used to control graft-versus-host disease (GvHD), a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). MSCs were isolated from Lewis rat bone morrow and then cultured in 10% FBS DMEM at 37°C for 4 weeks. The enriched conventional MSCs and macrophages were purified by auto-MACS. Cloned MSCs were obtained by cloning using the limiting dilution method and expanded up to more than 6 months. The identity of MSCs was confirmed by their typical spindle-shaped morphology and immunophenotypic criteria, based on the absence of expression of CD45 and CD11b/c molecules. Both types of MSCs were also tested for their ability to differentiate into adipocytes. We showed that MSCs, like macrophages, exhibit immunomodulatory properties capable of inhibiting T-cell proliferation stimulated by alloantigens, anti-CD3e/CD28 mAbs, and ConA in a dose-dependent manner in vitro. After performing adoptive transfer, MSCs suppressed systemic Lewis to (Lewis × DA)F1 rat GvHD. In contrast to the immunosuppressive activities of conventional MSCs, the cloned MSCs enhanced T-cell proliferation in vitro and yielded no clinical benefit in regard to the incidence or severity of GvHD. Therefore, these rat models have shown intriguing differences in the suppression effects of lymphocyte proliferation and GvHD prevention between short-term cultured conventional MSCs and cloned MSCs.

It is currently said that CO has anti-inflammatory and antiapoptosis effects and it has attracted attention as a medical gas. We used CO for rat hearts and conducted a preservation experiment. We isolated rat hearts, placed them into a specially made chamber, filled the chamber with a gas mixture of PCO (4,000 hPa) and PO 2 (3,000 hPa), and preserved the hearts in a refrigerator at 4°C for 48 h. We then performed a heterotrophic transplantation on the neck of each recipient rat and resuscitated the preserved hearts. We herein report our findings.

Naturally occurring CD4 + CD25 + Treg cells (nTregs) can be exploited to establish an immunologic tolerance to non-self-antigens. The in vivo administration of a single superagonistic CD28-specific monoclonal antibody (supCD28mAb) to naive rat preferentially expanded the nTregs, which induced a potent inhibition of lethality of the graft-versus-host (GvH) diseases. The appearance of increased Foxp3 molecules was accompanied with a polarization towards a Th2 cytokine profile with a decreased production of IFN-γ and increased production of IL-4 and IL-10 in the serum of the antibody-treated rat. The peripheral Foxp3 nTregs are decreased in acute GvHD, while supCD28mAb administration showed that nTregs were preferentially proliferating in vivo, thus resulting in the significant prevention of the GvH disease. Furthermore, antigen-specific nTregs could suppress conventional T-cell proliferation stimulated with alloantigen in vitro. Taken together, our findings demonstrate that the potent regulatory functions of the Tregs for the treatment of GvHD are antigen specific. These data also provide evidence that GvHD is associated with decrease of Tregs in the periphery of the host. The determination of the Foxp3 Tregs can be a helpful tool to discriminate GvHD severity and lethality after allogeneic stem cell transplantation.

Regulatory lymphocytes play a pivotal role in preventing organ-specific autoimmune disease and in induction and maintenance of tolerance in various experimental transplantation models. The enhancement of the number and activity of peripheral CD4 + CD25 + Treg cells is an obvious goal for the treatment of autoimmunity and for the suppression of alloreactions. The present study demonstrates that naturally occurring CD4 + CD25 + Treg (nTreg) cells preferentially proliferate to a fourfold increase within 3 days in response to the administration of a single superagonistic CD28-specific monoclonal antibody (supCD28 mAb). The appearance of increased Foxp3 molecules was accompanied with polarization toward a Th2 cytokine profile with decreased production of IFN-γ and increased production of IL-4 and IL-10 in the expanded Treg subset. Adoptive transfer of supCD28 mAb-expanded cells in a graft-versus-host disease (GvHD) model induced a potent inhibition of lethality. These results suggest that this therapeutic effect is mediated by the in vivo expansion of nTreg cells. Taken together, these data demonstrate that supCD28-mAb may target nTreg cells in vivo and maintain and enhance their potent regulatory functions for the treatment GvHD.

Cell-mediated therapy for mucopolysaccharidosis type VII (MPSVII) was studied using monkey amniotic epithelial cells (mAEC). The cells were transduced with a recombinant adenovirus expressing human β-glucuronidase (GUSB), and cells overexpressing GUSB were generated. The cells expressed 2000-fold higher activities than the endogenous GUSB activities of nontransduced mAEC, demonstrating that mAEC were successfully transduced with adenoviral vectors. These cells also secreted high levels of GUSB. To clarify the cross-correction of GUSB secreted from mAEC, the conditioned medium containing high levels of GUSB was added into the medium for culturing human or murine fibroblasts established from an MPSVII patient or a mouse model of the disease. Dramatic increases in GUSB activities were observed in both fibroblasts. We then transplanted the cells transduced with an adenovirus expressing LacZ into the caudate-putamen of monkey brain. Survival and distribution of the transplanted cells 1 month after the treatment were evaluated. Histochemical analysis showed that LacZ-positive cells were widely distributed in the brain, suggesting that the transplanted cells had migrated and were distributed even at regions far from the implantation site. These findings suggest that local intracerebral engraftment of genetically engineered amniotic epithelial cells is favorable for the treatment of lysosome storage disorders, whose pathological abnormalities are not restricted to specific regions of the brain.

It is hoped that amniotic epithelial cells can be useful in cell-mediated gene therapy. We report here an experimental cell transplantation model of amniotic cells in rats. There is an anatomical difference between human and rodent embryos. We established a method to isolate amniotic cells that are equivalent to human amniotic epithelial cells. An amniotic membrane distinct from the yolk sac was carefully collected and teased in saline containing deoxyribonuclease and hyaluronidase, followed by collagenase digestion. The cell yield was approximately 106 cells per pregnant female (105 cells per fetus), roughly in proportion to the age of fetus used, and 60% of the isolated cells were attached to the dish under culture conditions. Telomerase activity was higher in the cells isolated from fetuses in the middle stage (day 13.5 to 15.5) than in the late stage (day 17.5 to 21.5). Adherent cells exhibited two to three times more cell division, resulting in a ninefold increase in the number of cells. Immunohistochemical analysis revealed that approximately half of the adherent cells were albumin positive and formed clusters. The senescent cells survived for 2 months without apparent morphological changes. The adherent cells were able to be stored in liquid nitrogen and had a viability of 70% when thawed. Gene transduction with adenovirus vector was highly effective for rat amniotic cells. Transplantation of lacZ transfected amniotic cells into syngeneic rat liver resulted in the integration of the transplanted cells in the liver structure and the cells survived for at least 30 days.

Hepatocyte transplantation has been proposed as a potential therapeutic method to treat irreversible liver failure and inherited hepatic disorders, although transplanted cells do not easily reconstruct the liver tissue under intact conditions. This study was aimed at modulating the recipient liver conditions to promote repopulation of the liver after hepatocyte transplantation. Hepatocytes isolated from male MRL-lpr/lpr (lpr) mice with a mutation of Fas antigen were transplanted in a number of 1 × 106 cells in female MRL-+/+ (wildtype mice) by intrasplenic injection. An agonistic anti-Fas antibody (0.15 mg/kg) was administered intravenously 24 h after cell transplantation. We also administrated the antibody at 0.3 mg/kg 1 week after grafting and at 0.6 mg/kg 2 weeks after transplantation. The liver specimens were taken at different time intervals for histological examination. The reconstructed male lpr hepatocytes in the female wild-type mice were determined by a real-time quantitative PCR assay using the primers and probe for the sry gene. The pathologic findings of the recipient livers after treatment with anti-Fas antibody revealed a large number of apoptotic hepatocytes. The grafted lpr hepatocytes were observed to reconstruct as much as 6.9% of the recipient liver in the anti-Fas antibody-treated group 3 months after transplantation. In contrast, we observed the transplanted cells at lower than 0.1% in the nontreated livers. These findings demonstrated that repeated induction of apoptosis in recipient hepatocytes shifts the environment of the liver to a regenerative condition. This method may be useful to promote the reconstruction of transplanted hepatocytes in a recipient liver.

Glutamine synthetase (GS) is involved in an accessory pathway of ammonia removal in mammals. To develop a bioartificial liver with a human cell line, GS gene was transfected into HepG2 cells, which had no ammonia removal activity. After culturing in the presence of methionine sulfoximine (MSX), a GS inhibitor, we obtained a MSX-resistant HepG2 subline (GS-HepG2), which had amplified GS gene; ammonia removal activity was estimated to be 1/7 of that of rat primary culture hepatocytes. The cells were cultured in a circulatory flow bioreactor for 109 days, while they multiplied from 5 × 10 7 to 4 × 10 9 cells. Three days after inoculation, the ammonia level of the culture medium was lowered to a level maintained thereafter, suggesting that using recombinant cell lines for bioartificial livers enables long-term repeated treatment for hepatic failure patient. Judging from the rate of decrease in the amount of the added ammonia, the ammonia removal capability of 4 × 10 9 GS-HepG2 cells was almost equivalent to 5 × 10 8 porcine hepatocytes inoculated into the circulatory flow bioreactor. Apart from their ammonia removal activity, GS-HepG2 cells eliminated human tumor necrosis factor-α (TNF-α). Cytokine removal therefore promises to be another useful property of bioreactor cells.

For successful gene therapy in hepatic enzyme deficiencies, it is essential to use promoters that can maintain strong transcriptional activity for the long term in the liver. Using Gunn rats, a model animal for Crigler-Najjar syndrome type I, the long-term transcriptional function of the CAG promoter (a combination of chicken β-actin promoter and cytomegalovirus immediate-early enhancer) was evaluated in the rat liver. We constructed a plasmid pCAGGHUGT, containing expression cassettes of human bilirubin UDP-glucurono-syltransferase (BUGT) and hygromycin phosphotransferase, under the control of the CAG promoter and murine phosphoglycerate kinase promoter, respectively. Conditionally immortalized Gunn rat hepatocytes (IGRH), which had been established using mutant SV40 large T antigen ( TS T), were transfected with pCAGGHUGT. A stably transfected clone IGRHUGT, expressing a high level of BUGT, was obtained after selection with hygromycin. At 33°C, the cells doubled in number in approximately 72 h; however, at 37°C, cell proliferation stopped, indicating that the characteristic of temperature-dependent proliferation was retained in this clone. Ten million cells were injected into the spleen of syngeneic Gunn rats five times at 10-day intervals. Serum bilirubin levels were reduced by 45–50% at 70 days after the first transplantation and remained so throughout the duration of the study (120 days). These results suggested that the CAG promoter was able to maintain strong transcriptional activity in rat liver for at least 120 days.

Cell therapy with human amniotic epithelial (HAE) cells was developed as an alternative method for enzyme replacement therapy in congenital lysosomal storage disorders, but only limited therapeutic efficacy has been reported. A major drawback is insufficient production and secretion of lysosomal enzymes from HAE cells. In this study, we infected HAE cells with an E1-deleted adenoviral vector expressing human β-glucuronidase (GUSB), and generated cells overexpressing GUSB by a hundred times as much as endogenous GUSB in untreated HAE cells. GUSB secreted from the gene-transferred HAE cells were efficiently transported to murine fibroblasts with endocytosis mediated by mannose-6-phosphate receptors. The cells were administered into the spleen of the mice with the lysosomal storage disease mucopolysaccharidosis type VII (B6/MPSVII). Approximately 10–15% of the normal GUSB activity was detected in both liver and spleen 7 days after the cell administration. Histopathological examination showed that lysosomal enlargement in tissue macrophages in the liver and the spleen had disappeared by day 14. These results suggest that transplantation of the HAE cells transduced with adenoviral vectors can be employed for the treatment of congenital lysosomal storage disorders.