BACKGROUND:Xenotransplantation of porcine cells, tissues or organs offers a promising strategy to address the critical shortage of human donor organs. However, cross-species pathogen transmission, instant blood-mediated inflammatory reaction (IBMIR)-related liver injury, chronic over-immunosuppression, and long-term safety remain major challenges in porcine islet xenotransplantation. In this study, we performed a clinical trial aimed to evaluate the biosafety of neonatal porcine islet xenotransplantation in patients with type 1 diabetes. METHODS:Ten recipients were assigned to two groups receiving non-encapsulated neonatal islet cell clusters via the jugular-hepatic-portal vein at mean doses of 6354 ± 835 IEQ/kg and 11 600 ± 1100 IEQ/kg, respectively. Donor pigs originated from a highly inbred, PERV-C-negative colony reared in designated pathogen-free (DPF) conditions, with rigorous pathogen and PERV screening. Immunosuppression included mycophenolate mofetil, tacrolimus, belatacept, and autologous regulatory T-cell therapy, combined with tocilizumab. Continuous low-dose heparin was infused via a portal vein catheter was used for 7 days as anticoagulation therapy. Recipients and spouses were monitored for over 5 years, with 7 followed for more than 10 years. RESULTS:No PERV transmission or transplant-related infections were detected in any recipient or contact during long-term follow-up. The procedure was safe and well-tolerated, with only transient liver function and coagulation changes and mild adverse events. Both groups showed reduced exogenous insulin requirements and markedly lower hypoglycemia incidence, with better glucose control in the higher-dose group. CONCLUSION:This study demonstrates that DPF PERV-C-free neonatal porcine islet xenotransplantation is biologically safe and partially effective, supporting its value as a reliable donor source and foundational platform for advancing clinical islet xenotransplantation. TRIAL REGISTRATION:ClinicalTrials.gov identifier: NCT03162237.

Background Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related deaths worldwide, especially in advanced stages where limited treatment options result in poor prognosis. The immunosuppressive tumor immune microenvironment (TIME), characterized by low immune cell infiltration and exhaustion, limits immunotherapy efficacy. To address this, our study investigates the role of C-C motif chemokine ligand 3 (CCL3) in modulating the HCC TIME.Methods We analyzed CCL3 expression in human HCC samples from The Cancer Genome Atlas database, focusing on its correlation with inflammatory gene signatures and immune cell infiltration. High-dimensional single-cell RNA sequencing (scRNA-seq), flow cytometry, and multiplex immunofluorescence were used to investigate CCL3’s effects on macrophage function and T cell activation. The biological impact of CCL3 on macrophages was assessed using co-culture systems, confocal imaging, metabolite detection, and inhibition assays. Preclinical HCC models and ex vivo tumor fragment assays further explored how CCL3 modulates immune responses and enhances immune checkpoint blockade efficacy.Results Our study shows that CCL3 is suppressed in the tumor microenvironment and positively correlates with immune infiltration and inflammatory responses. Targeted liver delivery of rAAV-Ccl3 reprograms the immune microenvironment in HCC, promoting immune cell recruitment and tertiary lymphoid structure formation, thus suppressing tumor growth via immune engagement. Through scRNA-seq, flow cytometry, and multiplex immunofluorescence, we found that CCL3 enhances macrophage antigen uptake and activates cytotoxic T cells. In vivo and in vitro experiments confirmed that CCL3 facilitates T cell infiltration and upregulates MHC II expression on macrophages, enhancing antigen presentation. The CCL3-CCR5 pathway also boosts macrophage metabolism, increasing lysosomal activity and antigen uptake, thereby strengthening adaptive immune responses and increasing sensitivity to immune checkpoint blockade therapies in preclinical models.Conclusions This study highlights the pivotal role of CCL3 in reshaping the TIME and enhancing antitumor immunity in HCC. By promoting immune cell recruitment and enhancing antigen presentation, CCL3 demonstrates significant potential to improve the efficacy of immunotherapy, particularly in combination with immune checkpoint inhibitors. Targeting CCL3 may help to overcome the immunosuppressive TIME in HCC and improve patient outcomes.

Hepatocellular carcinoma (HCC) resists immunotherapy due to its immunosuppressive microenvironment. Sarcoma homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) inhibits T cell receptor signaling, and its pharmacological inhibition is limited by poor selectivity and membrane permeability. Here, we generated CRISPR-edited SHP-1-knockout (KO) CD8+ T cells to enhance adoptive therapy against HCC. Single-cell RNA sequencing of HCC patient T cells revealed elevated SHP-1 in exhausted subsets. SHP-1-KO T cells exhibited increased effector memory T cells (TEM) proportions and enhanced IFN-γ/Granzyme B/perforin secretion, improving cytotoxicity against HCC lines. In humanized PDX models, SHP-1-KO T cells demonstrated superior tumor-killing activity. Transcriptomics identified upregulated lipid metabolism pathways, with HMGCR as a hub gene. Combining SHP-1-KO T cells with simvastatin (HMGCR inhibitor) synergistically amplified anti-HCC efficacy. This study proposes a dual strategy combining SHP-1-targeted cell therapy and metabolic modulation to overcome immunotherapy resistance, offering a translatable approach for HCC treatment.

Background Hepatocellular carcinoma (HCC) represents one of the most significant causes of mortality due to cancer-related deaths. It has been previously reported that the TGF-beta signaling pathway may be associated with tumor progression. However, the relationship between TGF-beta signaling pathway and HCC remains to be further elucidated. The objective of our research was to investigate the impact of TGF-beta signaling pathway on HCC progression as well as the potential regulatory mechanism involved.Methods We conducted a series of bioinformatics analyses to screen and filter the most relevant hub genes associated with HCC. E. coli was utilized to express recombinant protein, and the Ni-NTA column was employed for purification of the target protein. Liquid liquid phase separation (LLPS) of protein in vitro, and fluorescent recovery after photobleaching (FRAP) were utilized to verify whether the target proteins had the ability to drive force LLPS. Western blot and quantitative real-time polymerase chain reaction (qPCR) were utilized to assess gene expression levels. Transcription factor binding sites of DNA were identified by chromatin immunoprecipitation (CHIP) qPCR. Flow cytometry was employed to examine cell apoptosis. Knockdown of target genes was achieved through shRNA. Cell Counting Kit-8 (CCK-8), colony formation assays, and nude mice tumor transplantation were utilized to test cell proliferation ability in vitro and in vivo.Results We found that Smad2/3/4 complex could regulate tyrosine aminotransferase (TAT) expression, and this regulation could relate to LLPS. CHIP qPCR results showed that the key targeted DNA binding site of Smad2/3/4 complex in TAT promoter region is -1032 to -1182. In addition. CCK-8, colony formation, and nude mice tumor transplantation assays showed that Smad2/3/4 complex could repress cell proliferation through TAT. Flow cytometry assay results showed that Smad2/3/4 complex could increase the apoptosis of hepatoma cells. Western blot results showed that Smad2/3/4 complex would active caspase-9 through TAT, which uncovered the mechanism of Smad2/3/4 complex inducing hepatoma cell apoptosis.Conclusion This study proved that Smad2/3/4 complex could undergo LLPS to active TAT transcription, then active caspase-9 to induce hepatoma cell apoptosis in inhibiting HCC progress. The research further elucidate the relationship between TGF-beta signaling pathway and HCC, which contributes to discover the mechanism of HCC development.

Dysregulation of splicing factor expression plays a crucial role in the progression of hepatocellular carcinoma (HCC). Our research found that the expression level of splicing factor ZMAT2 was increased in HCC, promoting the proliferation of HCC cells. RNAseq data indicated that the absence of ZMAT2 induced skipping exon of mRNA, while RIPseq data further revealed the mRNA binding motifs of ZMAT2. A comprehensive analysis of RNAseq and RIPseq data indicateed that ZMAT2 played a crucial role in the maturation process of TRIM28 mRNA. Knocking down of ZMAT2 led to the deletion of 25 bases in exon 11 of TRIM28, ultimately resulting in nonsense-mediated decay (NMD). Our data revealed that ZMAT2 could regulate TRIM28 to reduce the accumulation of ROS in HCC cells, thereby promoting their proliferation. Our research also discovered that ZMAT2 was capable of undergoing phase separation, resulting in the formation of liquid droplet condensates within HCC cells. Additionally, it was found that ZMAT2 was able to form protein-nucleic acid condensates with TRIM28 mRNA. In summary, this study is the first to reveal that ZMAT2 and TRIM28 mRNA form protein-nucleic acid condensates, thereby regulating the splicing of TRIM28 mRNA. The increased expression of ZMAT2 in HCC leads to upregulated TRIM28 expression and reduced ROS accumulation, ultimately accelerating the proliferation of HCC cells.

Introduction: Islet xenotransplantation has emerged as a prospective β-cell replacement therapy for insulin-deficient diabetes. Pigs can be genetically modified to protect islet xenografts from immune challenges, including an intense innate response triggered by intraportal delivery, the immediate blood-mediated inflammatory response (IBMIR). The rhesus macaque is one of the most widely used NHP models for islet xenotransplantation. Therefore, we used transgenic porcine islets to treat diabetic rhesus monkeys to verify its effectiveness. Methods: GGTA1P gene knockout and humanized CD55 and CD59 gene insertion were performed on Xeno-1 pigs by CRISPR/Cas9 technology, and the successful insertion of the two gene sequences was confirmed by gene sequencing. Diabetes was induced by i.v. injection of streptozotocin. Rhesus macaques were considered truly diabetic if they had persistent hyperglycemia requiring exogenous insulin administration and were unresponsive to IVGTT and AST. The islets of gene-modified adult pig (over 2 years old) were extracted by enzyme perfusion and continuous density gradient centrifugation. Porcine islets were transplanted into two diabetic monkeys by mesenteric venipuncture. One monkey received 10,000 islet equivalents (IE)/kg and the other 20,000 IE/kg. Graft function was monitored by measuring blood glucose and porcine C-peptide. Results: The first diabetic monkey returned to normal levels of blood glucose 110 days after transplantation. Before the transplantation, the average blood glucose was about 18mmol/L, and the insulin dosage was about 12 IU/day. 110 days after the transplant, the average blood glucose was about 8mmol/L, and the insulin dosage was about 3 IU/day. The amount of insulin is reduced by about 75%. Postoperative D-dimer increased slightly, reached the peak on the 3rd day, and returned to normal on the 5th day. The C-peptide test result was 42.5 pmol/L 3 months after the operation. Blood glucose in the second diabetic monkey returned to normal levels 30 days after transplantation. The average blood glucose was about 17 mmol/L and the insulin dosage was about 12 IU/day before the transplantation. 30 days after transplantation, the average blood glucose was about 7 mmol/L, and the insulin dosage was about 2.5 IU/day. Insulin doses were reduced by approximately 79%. There was no significant fluctuation of D-dimer after operation. Conclusion: GTKO-CD55-CD59-HT adult porcine islets can effectively treat diabetic monkeys. Compared with before transplantation, the blood glucose of the two diabetic monkeys returned to normal after transplantation, the blood glucose fluctuation was small, the average insulin consumption decreased by 77%, and the islet transplantation dosage was positively correlated with the transplantation effect. The blood coagulation indexes after transplantation were basically normal, indicating that there was no postoperative thrombosis. the National Key Research and Development Program (Grant No. 2019YFA0110703).

Objective: Ulcerative colitis(UC) is a complex disease characterized by chronic inflammation of the colon. The incidence and prevalence of UC are on the rise, and it has become a global disease.At present, The existing UC treatment drugs have defects in effectiveness and safety, thus optimizing drug delivery system has become a key point to improving the treatment efficiency. Spermidine (SPD) has strong anti-inflammatory effects and regulate oxidative stress. In this study, an overexpressing spermidine synthase (SRM) engineered cells (SEC) was established to form a new drug delivery system that can automatically produce spermidine in vivo. We used sodium alginate bio-semipermeable membrane to wrap SEC to prepare engineered cell microcapsules (SECM) which can prevent the SEC from being attacked by the recipient immune system. The efficacy of the spermidine automatic drug delivery system was explored by in vitro study and the UC mouse model. Methods: (1) We used lentivirus system and HEK293T cell line to construct SEC cell line. qRT-PCR was used to detect SRM overexpression efficiency. ELISA was performed to detect spermidine secretion of SEC; (2) The viability of microencapsulated cell was explored by AO/EB staining. ELISA was used to detect the secretion of spermidine. Flow CBA was used to detect the expression of spermidine-related pro-inflammatory cytokines; (3) SECM was implanted into the peritoneal cavity of ulcerative colitis mice model, CCK8 and trypan blue staining were used to detect the cell viability of SECM after nine days. (4) Before and after the SECM implantation, body weight, stool shape, blood in the stool and other indicators were monitored for disease activity index (DAI) scoring. (5) The treatment efficacy of SECM was evaluated by DAI, inflammation-associated histological scores, changes of colon length and spleen weight. Results: (1) The RT-PCR results showed that SRM mRNA expression in SEC was significantly higher than control group. The spermidine secreted by SEC at 48h was 1.65 times of the control group, and at 72h was 1.49 times, respectively. (2) AO/EB staining showed that the viability of the cells packed in 4×106/mL density was the best. The spermidine secreted by SECM was 1.59 times of the negative control group. The spermidine secreted by SECM and SEC was the same. The CBA results showed that the secretion of TNF-α, IL-6 and IFN-γ of SECM was significantly lower than control group. (3) The SECM and control group have the same survival rate by CCK8 test and trypan blue staining. SECM could significantly improve clinical symptoms and other pathological manifestations. And finally reduce DAI and inflammation-associated histological score. Conclusion: (1) Successfully established SEC formed a new SPD automatic drug delivery system; (2) sodium alginate microcapsules can effectively protect SEC; (3) SPD automatic drug delivery system was an efficacy way for DSS-induced UC in mice. the National Key Research and Development Program (Grant No. 2019YFA0110703). The Science and Technology Innovation Foundation of Hunan Province (Grant No. 2020SK53614). The National Natural Science Foundation of China (Grant Nos. 82272102 and 81971721).

INTRODUCTION:PTEN often mutates in tumors, and its manipulation is suggested to be used in the development of preclinical tools in cancer research. This study aims to explore the biological impact of gene expression related to PTEN mutations and to develop a prognostic classification model based on the heterogeneity of PTEN expression, and to explore its sensitivity as an indicator of prognosis and molecular and biologic features in hepatocellular carcinoma (HCC).MATERIAL AND METHODS:RNA-seq data and mutation data of the LIHC cohort sample downloaded from The Cancer Genome Atlas (TCGA). The HCC samples were grouped according to the mean expression of PTEN, and the tumor microenvironment (TME) was evaluated by ESTIMATE and ssGSEA. The prognostic classification model related to PTEN were constructed by COX and LASSO regression analysis of differentially expressed genes (DEGs) between PTEN-high and -low expressed group.RESULTS:The expression of PTEN was affected by copy number variation (CNV) and negatively correlated with immune score, IFNγ score and immune cell infiltration. 1281 DEGs were detected between PTEN-high and PTEN-low expressed group, 8 of the DEGs were finally filtered for developing a prognosis classification model. This model showed better prognostic value than other clinicopathological parameters, and the prediction accuracy of prognosis and ICB treatment for immunotherapy cohorts was better than that of TIDE model.CONCLUSIONS:This study demonstrated the effect of CNV on PTEN expression and the negative immune correlation of PTEN, and constructed a classification model related to the expression of PTEN, which was of guiding significance for evaluating prognostic results of HCC patients and ICB treatment response of cancer immunotherapy cohorts.

Clinically, xenotransplantation often leads to T-cell-mediated graft rejection. Immunosuppressive agents including polyclonal regulatory T cells (poly-Tregs) promote global immunosuppression, resulting in serious infections and malignancies in patients. Xenoantigen-expanded Tregs (xeno-Tregs) have become a promising immune therapy strategy to protect xenografts with fewer side effects. In this study, we aimed to identify an efficient and stable subset of xeno-Tregs. We enriched CD27 + xeno-Tregs using cell sorting and evaluated their suppressive functions and stability in vitro via mixed lymphocyte reaction (MLR), real-time polymerase chain reaction, inflammatory induction assay, and Western blotting. A STAT5 inhibitor was used to investigate the relationship between the function and stability of CD27 + xeno-Tregs and the JAK3–STAT5 signaling pathway. A humanized xenotransplanted mouse model was used to evaluate the function of CD27 + xeno-Tregs in vivo . Our results show that CD27 + xeno-Tregs express higher levels of Foxp3, cytotoxic T-lymphocyte antigen-4 (CTLA4), and Helios and lower levels of interleukin-17 (IL-17) than their CD27 − counterparts. In addition, CD27 + xeno-Tregs showed enhanced suppressive function in xeno-MLR at ratios of 1:4 and 1:16 of Tregs:responder cells. Under inflammatory conditions, a lower percentage of CD27 + xeno-Tregs secretes IL-17 and interferon-γ (IFN-γ). CD27 + xeno-Tregs demonstrated an upregulated JAK3–STAT5 pathway compared with that of CD27 − xeno-Tregs and showed decreased Foxp3, Helios, and CTLA4 expression after addition of STAT5 inhibitor. Mice that received porcine skin grafts showed a normal tissue phenotype and less leukocyte infiltration after reconstitution with CD27 + xeno-Tregs. Taken together, these data indicate that CD27 + xeno-Tregs may suppress immune responses in a xenoantigen-specific manner, which might be related to the activation of the JAK3–STAT5 signaling pathway.

FOXP3+ regulatory T cells (Tregs) are central to maintaining peripheral tolerance and immune homeostasis. They have the potential to be developed as a cellular therapy to treat various clinical ailments such as autoimmune disorders, inflammatory diseases and to improve transplantation outcomes. However, a major question remains whether Tregs can persist and exert their function effectively in a disease state, where a broad spectrum of inflammatory mediators could inactivate Tregs. In this study, we investigated the potential of mesenchymal stem cell (MSC)-derived exosomes to promote and sustain Tregs function. MSC-conditioned media (MSC-CM) cultured Tregs were more suppressive in both polyclonal and allogeneic responses and were resistant to inflammatory stimulation in vitro compared with the controls. A similar enhancement of Treg function was also observed by culturing Tregs with MSC-derived exosomes alone. The enhanced suppressive activity and stability of Treg cultured in MSC-CM was reduced when exosomes were depleted from MSC-CM. We identified that MSC-derived exosomes could upregulate the expression of LC3(II/I), phosphorylate Jak3 and Stat5 to promote Treg survival, and regulate FOXP3 expression in Tregs. Overall, our study demonstrates that MSC-derived exosomes are capable of enhancing Hucb-Tregs function and stability by activating autophagy and Stat5 signalling pathways. Our findings provide a strong rationale for utilizing MSC-derived exosomes as an effective strategy to enhance Treg function, and improve the overall Tregs-based cell therapy landscape.

Background: The clinical application of xenotransplantation often leads to T cell-mediated graft rejection. Immunosuppressive agents including polyclonal regulatory T cells (poly-Tregs) promote global immunosuppression, resulting in serious infection and malignancy in patients. Xenoantigen-expanded Tregs (xeno-Tregs) has become a promising immune therapy strategy that can protect xenografts with less side effects. In this study, we aimed to uncover a more efficient and stabler species of xeno-Tregs. Methods: We enriched the CD27+ xeno-Tregs by cell sorting and evaluated their suppressive functions and stability in vitro via mixed lymphocyte reaction (MLR), RT-PCR, an inflammatory induction assay and western blotting. A humanized xenotransplanted mouse model was used to evaluate the function of CD27+ xeno-Tregs in vivo. Results: Our results showed that CD27+ xeno-Tregs expressed higher levels of Foxp3, CTLA-4, and Helios and lower IL-17 than their CD27- counterparts. In addition, CD27+ xeno-Tregs showed significantly enhanced potency in suppressing the proliferation of xenoantigen-specific responder T cells at ratios of 1:4 and 1:16. Under inflammatory conditions, CD27+ xeno-Tregs displayed lower levels of IL-17 and IFN-gamma and less converted to IL17+ cells. Mice received porcine grafts showed normal tissue phenotype and less leukocyte infiltration after the injection of CD27+ xeno-Tregs. Conclusion: Taken together, these data indicated that CD27+ xeno-Tregs could suppress immune responses in a xenoantigen specific manner, effectively protecting xenografts from tissue damage. Grant number: 81971721.

The role of Regulatory T cells (Tregs) in tolerance induction post-transplantation is well-established, but Tregs adoptive transfer alone without combined immunosuppressants have failed so far in achieving clinical outcomes. Here we applied a set of well-designed criteria to test the influence of commonly used immunosuppressants (belatacept, tacrolimus, and mycophenolate) on cord blood-derived Tregs (CB-Tregs). Our study shows that while none of these immunosuppressants modulated the stability and expression of homing molecules by CB-Tregs, belatacept met all other selective criteria, shown by its ability to enhance CB-Tregs-mediated in vitro suppression of the allogeneic response without affecting their viability, proliferation, mitochondrial metabolism and expression of functional markers. In contrast, treatment with tacrolimus or mycophenolate led to reduced expression of functional molecule GITR in CB-Tregs, impaired their viability, proliferation and mitochondrial metabolism. These findings indicate that belatacept could be considered as a candidate in Tregs-based clinical immunomodulation regimens to induce transplant tolerance.

Background: Peripheral blood derived Treg can be cultured and trained as xenoantigen specific Treg which is a promising way for xenotransplantation. However an abnormal quantity and/or quality of regulatory T cells were reported in some autoimmune diseases such as T1D. Thus using an alternative source and selecting Treg-friendly drugs may supply a efficacy immunosuppressive regimen for islet xenotransplantation. This study aims to give experimental evidence regarding the influence of current using immunosuppressive drugs on xenoantigen-stimulated cord blood derived Treg. Methods: Human cord blood derived CD4+CD25+CD127low Treg were cultured with IL-2, rapamycin and irradiated porcine PBMC as xenoantigen stimulation for 2 weeks. The effects of major ISDs as tacrolimus (TAC), mycophenolate (MPA) and new ISD beletacept (BEL) and the mixture of three ISDs (ISDs mix) on xenospecific Treg were test to analysis the cell viability, phenotype, proliferation, function, stability and metabolism. Results: After two weeks induction, xenoantigen stimulated cord blood derived-Tregs (Xn-Treg) have enhanced suppressive capacity in xeno MLR and higher expression of chemokine receptor CCR4, CCR6, CXCR3 and CCR7 compared to polyclonal Treg (Pc-Treg). Further the expression of function molecular CD39, CD73, ICOS, GITR, CTLA-4 and IDO was also upregulated compared to Pc-Treg which confirmed the function of Xn-Treg. Mycophenolate have a negative impact on viability and proliferative capacity of Xn-Treg in a dose-dependent manner. however, BEL and TAC did not affect the viability and proliferative capacity of xenospecific Treg severely. No matter Treg were treated with ISD alone or the mixture of ISDs, the immunosuppressive treatments have no effect on the expression of Treg functional molecules but affected chemokine receptors. From the OCR assay, MPA and ISDs mix significantly increased the maximal respiratory capacity and mitochondrial mass of Xn-Treg compared to the control group. While TAC and BEL did not have any change. For xeno-MLR assays, TAC and MPA did not have any negative effect on the suppressive capacity of Xn-Treg but rather BEL strengthened it at the ratio of responder:Treg 64:1. The results of CBA assay showed when activated CD25-responder cells co-cultured with Treg and ISDs, BEL and Treg have synergetic effect to reduction of IL6, TNF-α, IL17A and IFN-γ which explained why BEL could strengthen the suppressive ability of Treg. Conclusion: Our results suggested xenoantigen-stimulated UCB-Treg show enhanced suppressive capacity in the pig-human MLR and different immune modulatory drugs have different influence on Xn-Treg. We speculate that a low dose combination of ISDs would be better able to support Treg while adequately preventing rejection while minimizing toxicity.

Background: Xenotransplantation using porcine cells, tissues or organs is a promising method to alleviate the shortage of donated cadaveric human organs. Cross-species transmission of an infectious agent from the pig graft to the recipient, hepatic impact caused by an instant blood-mediated reaction, chronic over-immunosuppression and long-term potential infectious risk are the main concerns regarding xenotransplantation. Therefore, we designed a clinical trial to evaluate a biosafety system based on the principle of Changsha Communique for porcine islet xenotransplantation. Methods: Based on current WHO guidelines and the existing pathogen list for designated pathogen-free (DPF) animals, we designed our updated DPF pathogen screening list. We had selectively inbred a Xiang pig herd for 12 years and confirmed their PERV-C free status and, most recently, their DPF status (Xeno-1). We here in report a clinical trial of ten adult patients (9 M:1 F) with type 1 diabetes who received DPF porcine islet xenotransplantation via the portal vein. Clinically accepted immunosuppressant protocol, tacrolimus, MMF, beletacept and autologous Tregs, were used for the recipients. The recipients were observed for follow-up biosafety evaluations such as infectious signs, microbiological detection, liver function, immunocytes and cytokines as the time schedule post-Tx. The recipients and their spouse were also examined the transmission of porcine endogenous retroviruses (PERV) and the micro-chimerism using PCR and cross-species infection monitoring. Results: PERV-C negative DPF donor piglets were certified in China by CFDA. All recipients and their spouse were minimal 1 year monitoring and up to 5 years. No cross-species pathogen infections and no PERV transmission were observed, and no severe transplant-related side effects were observed. Mild liver function damage was found post transplantation, but recover to normal within 28 days. We did not negatively alter the cytokine and immune responses in patients at 12 months’ post-islet xenotransplantation monitoring. The metabolic outcome improved, and hypoglycemic episodes decreased in all recipients. The exogenous insulin requirement decreased by an average of 45%, and the HbA1c decreased by 22.5% in recipients who received 11,400 ± 1828 IEQ/kg (n=4) at one year post transplantation. Conclusion: This first-in-the-world pilot clinical trial for xenotransplantation biosafety system evaluation of fresh neonatal porcine islets demonstrates that validated DPF PERV-C-free pigs are safe from biohazard risks and transmission and are a valuable donor source of tissue for xenotransplantation. Our data demonstrate that this therapy is safe and efficacious, and these findings provide an important basis for future xenotransplantation studies. This study was supported by the National Grant Program on Key Infectious Disease (2018ZX10301101-003), National Natural Science Foundation of China (Grant No. NSFC 81671752, 31671324, 31970176; Grant No. 2018SK1020). J.C is supported by CAS Pioneer Hundred Talents Program.

Tumor targeting delivery of SPM functionalized micelles via PTS binding and their endocytosis and pH-triggered endo/lysosome drug release for anti-cancer therapy.

Objective To explore the homing of DiR labeled regulatory T cells ( Tregs) in human-ized heterologous liver tissue transplantation mouse model. Methods The fluorescence intensities of Tregs labeled with different concentrations of DiR dye and different incubation times were measured, and the cell viability was measured by 3-( 4, 5-dimethylthiazol-2-yl )-5-( 3-carboxymethoxyphenyl )-2-( 4-sulfophenyl )-2H-tetrazolium ( MTS) assay to determine the optimal incubation time and dye concentration. The effect of DiR dye on the function and phenotype of Tregs was verified by flow cytometry. The xenogeneic liver tissue transplantation mouse model was constructed and the immune system was reconstituted. Small animal fluores-cence imaging was performed at different time points after infusion of Tregs. Immunohistochemistry analysis was used to analyze immune reconstitution and lymphocyte distribution in vivo. One-way analysis of variance and Dunnett-t test were used to analyze the data. Results With the increase of DiR concentration and incu-bation time, the fluorescence intensity of Tregs increased and gradually weakened after reaching the peak at 3 d. The cell viability of the 5. 00 μg/ml and 20. 00 μg/ml groups was significantly lower than that of the control group (culture medium) at various time points (F=120.142-182.025, t=9.969-19.329, all P<0. 05) . After incubation for 30 min and 60 min, the activity of Tregs was also significantly lower than that of the control group (F=21.826-301.968, t=6.897-40.016, all P<0.05). Tregs were finally co-incubated with DiR dye at a concentration of 2.50 μg/ml for 5 min, which was used further in vivo experiments. The flow cytometry showed that DiR dye did not affect the phenotype or the function of Tregs. The small animal fluorescence imaging showed that Tregs could locate in the graft area of mouse model. Immunohistochemical analysis showed that Tregs could improve lymphocyte infiltration induced by immune reconstitution. Conclu-sion After labeling Tregs with DiR dye, the distribution of Tregs can be directly observed by fluorescence imaging, which is a promising imaging method for Tregs tracer.

Uniform hollow spheres of Yb and Er codoped Gd2O2SO4 (noted as Gd2O2SO4:Yb,Er) have been developed as novel bifunctional contrast agents for efficient upconversion optical and magnetic resonance imaging (MRI) for the first time. Gd-containing organic precursory spheres were first obtained by a facile hydrothermal process. Owing to the innate hydrophilic nature of the precursory spheres, surface modification with a layer of stable and biocompatible silica could be readily achieved through the Stober sol gel method and subsequent calcination. The morphology and thickness of the silica shell can be tailored by adjusting the reaction time. Compared with Gd2O2SO4:Yb,Er hollow spheres without silica coating, well-dispersed Gd2O2SO4:Yb,Er@SiO2 double-shell hollow spheres could generate intense upconversion fluorescence, and showed a significant contrast enhancement of T1-weighted MM both in vitro and in vivo. These gadolinium oxysulfate-based hollow spheres are thus regarded as a new type of potential bimodal optical-MRI contrast agents.