In vivo induction of regulatory T cells by anti-CD45RB antibody causes transferable tolerance to discordant human xenogenic islets.

BACKGROUND: The treatment of diabetes by islet cell transplantation has become an accepted therapy, with transplantation of xenogeneic islet cells an attractive alternative to the problem. Previous studies in mice have demonstrated that anti-CD45RB induce immune tolerance in human pancreatic islet cells. The current study was to define the mechanism of action of anti-CD45RB induced nonspecific immune tolerance to heteroantigens. METHODS: A total of 1500 IEQ human islets were transplanted to diabetic B6µMT-/- mice, B6 mice, and µMT-/- diabetic mice undergoing thymectomy. These mice were treated short-term with doses of anti-CD45RB. CD4+Foxp3+Tregs were detected in the blood, peripheral lymphatic organs by flow cytometry, and immunohistochemistry. In addition, anti-CD25 mAb was administered to tolerant human islet cells B6µMT-/-mice. Mice then were transplanted with other human islet cells and received CD4+CD25+Tregs isolated from tolerant human islets mice to observe islet destruction. RESULTS: Anti-CD45RB treatment-induced tolerance to islets in both immunocompetent and B-cell-deficient mice (µMT-/- mice) by processes that were dependent on CD25+ Tregs, but not B cells. Anti-CD45RB treatment increased the number of CD4+Foxp3+Tregs cells. Anti-CD45RB treatment-induced immune tolerance that was antigen nonspecific, with Tregs playing an important role. Anti-CD45RB treatment-induced tolerance generated Tregs that could be transferred to another individual to manifest nonspecific immune tolerance. CONCLUSION: The results of the experiment suggest that anti-CD45RB induced tolerance to human islet xenografts is mediated by the proliferation of Tregs. These tolerogenic Tregs can be transferred to other mice and induce nonspecific immune tolerance.

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

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

An immunosufficient murine model for the study of human islets.

For the sake of therapy of diabetes, it is critical to understand human beta cell function in detail in health and disease. Current studies of human beta cell physiology in vivo are mostly limited to immunodeficient mouse models, which possess significant technical limitations. This study aimed to create a new model for the study of human islets through induction of transplant tolerance in immunosufficient mice. B6 diabetic mice were transplanted with human islets and treated with anti-CD45RB. To assess whether anti-CD45RB-induced transplant tolerance requires B cells, B6 recipients received additional anti-CD20 or B6µMT-/- mice were used. For some anti-CD45RB-treated B6µMT-/- mice, additional anti-CD25 mAb was applied at the early or late stage post-transplant. Immunohistology was performed to show the Foxp3 cells in grafted anti-CD45RB/anti-CD20-treated Foxp3-GFP B6 mice. The results showed that anti-CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice, however 100% of xenografts were accepted in mice treated simultaneously with anti-CD20, and 88.9% of xenografts accepted in anti-CD45RB-treated µMT-/- mice. These µMT-/- mice accepted the islets from another human donor but rejected the islets from baboon. Additional administration of anti-CD25 mAb at the time of transplantation resulted in 100% rejection, whereas 40% of grafts were rejected while the antibody was administrated at days 60 post-transplant. Immunohistologic examination showed Foxp3+ cells accumulated around grafts. We conclude that induction of tolerance to human islets in an immunosufficient mouse model could be generated by targeting murine CD45RB and CD20. This new system will facilitate study of human islets and accelerate the dissection of the critical mechanisms underlying islet health in human disease.

Clinical outcome of autologous hematopoietic stem cell infusion via hepatic artery or portal vein in patients with end-stage liver diseases.

OBJECTIVE: To investigate the efficacy of hematopoietic stem cell (HSC) transplantation via the hepatic artery vs. the portal vein for end-stage liver disease (ESLD). METHODS: Patients with hepatic decompensation were prospectively recruited from September 2010 to September 2012 to receive HSC transplantation via the hepatic artery or the portal vein. Liver function was examined at 3, 6, and 12 months after transplantation. Liver biopsy Results were analyzed using the Knodell score. RESULTS: Eighty patients (58 males and 22 females) were enrolled in the study. The Child-Pugh score was grade B in 69 cases, and grade C in the remaining 11 cases. HSC transplantation was performed via the portal vein in 36 patients and via the hepatic artery in 44 patients. ALT levels decreased while serum albumin levels increased significantly in both groups at 6 and 12 months after HSC transplantation (P<0.05 compared with pre-transplantation levels). Total bilirubin levels decreased significantly in both groups at 3, 6, and 12 months after HSC transplantation (P<0.05 compared with pre-transplantation levels). Additionally, prothrombin time decreased in both groups at 12 months after HSC transplantation (P<0.05 compared with pre-transplantation level). There were no significant differences in ALT, total bilirubin and prothrombin time between the two groups either before or after transplantation. Moreover, Knodell score decreased significantly at 6 and 12 months. Histological examination showed that liver cell edema, degeneration, necrosis, and inflammation were significantly relieved at 3, 6, and 12 months after transplantation. The incidence of portal vein thrombosis, upper gastrointestinal bleeding, and hepatic encephalopathy were 1.25%, 3.75%, and 2.5% respectively. The one-year survival rate was 100%. CONCLUSIONS: Autologous HSC transplantation improves liver function and histology in ESLD patients. The administration route of HSC has no significant impact on the efficacy of transplantation.

[Autologous peripheral blood CD34+ stem cells transplanted into 100 patients with advanced cirrhosis].

OBJECTIVE: To investigate whether transplantation of autologous peripheral blood CD34+ stem cells is a viable approach for treating patients with advanced cirrhosis,which is currently hindered by a shortage in liver donors. METHODS: A total of 100 patients with advanced cirrhosis and who had failed to respond to conservative therapy were recruited for transplantation of autologous peripheral blood CD34+ stem cells.The success of transplantation was investigated 6-and 12-months later by measuring markers of liver biosynthesis function (coagulation,albumin level,indocyanine green clearance,Child-Pugh score) and assessing pathological changes (Knodell score) and morphologic changes in the liver tissue.Complications were also recorded during follow-up. RESULTS: The 1-year cumulative survival rate was 100%. Fifty-two patients with massive ascites showed gradual reduction and disappearance of the ascites.Four patients experienced upper gastrointestinal bleeding and three patients developed with hepatic encephalopathy (I-II degree) at 3 months post-transplantation.All patients showed significantly improved liver biosynthesis function,liver elasticity and Knodell score after transplantation (P less than 0.05). CONCLUSION: Autologous peripheral blood CD34+ stem cell transplantation is a safe and effective treatment for advanced cirrhosis,and has high cost-benefit since it improves liver function,liver histology,and quality of life.

SEMA4D/VEGF surface enhances endothelialization by diminished-glycolysis-mediated M2-like macrophage polarization.

Cardiovascular disease remains the leading cause of death and morbidity worldwide. Inflammatory responses after percutaneous coronary intervention led to neoathrosclerosis and in-stent restenosis and thus increase the risk of adverse clinical outcomes. In this work, a metabolism reshaped surface is engineered, which combines the decreased glycolysis promoting, M2-like macrophage polarization, and rapid endothelialization property. Anionic heparin plays as a linker and mediates cationic SEMA4D and VEGF to graft electronically onto PLL surfaces. The system composed by anticoagulant heparin, immunoregulatory SEMA4D and angiogenic VEGF endows the scaffold with significant inhibition of platelets, fibrinogen and anti-thrombogenic properties, also noteworthy immunometabolism reprogram, anti-inflammation M2-like polarization and finally leading to rapid endothelializaiton performances. Our research indicates that the immunometabolism method can accurately reflect the immune state of modified surfaces. It is envisioned immunometabolism study will open an avenue to the surface engineering of vascular implants for better clinical outcomes.

LncRNA H19 secreted by umbilical cord blood mesenchymal stem cells through microRNA-29a-3p/FOS axis for central sensitization of pain in advanced osteoarthritis.

OBJECTIVE: To explore the molecular mechanism of umbilical cord blood mesenchymal stem cells (UCBMSCs) in the treatment of advanced osteoarthritis pain. METHODS: Normal healthy rats were selected to establish advanced osteoarthritis (OA) model, and the rats were randomly divided into control group, intravenous group, intracavitary group and intrathecal group. The intravenous group received intravenous injection of UCBMSCs, intracavitary group received intra-articular injection of UCBMSCs, and intrathecal group received subarachnoid injection of UCBMSCs. The pain behavior and serum pro-inflammatory factor levels were evaluated before and after treatment. microRNA-29a-3p and FOS mRNA in spinal dorsal horn was detected using qPCR, the phosphorylation of c-fos protein and NR1, NR2B, ERK and PKCg was detected using Western blot, and the level of LncRNA H19 was detected using qPCR. RESULTS: LncRNA H19 was enriched in the exosomes of UCBMSCs. microRNA-29a-3p was the target gene of LncRNA H19, while FOS was the downstream target of microRNA-29a-3p. Pain and inflammation of rats in the intrathecal group improved best, and the phosphorylation levels of c-fos and NR1, NR2B, ERK and PKCg in the spinal dorsal horn of the intrathecal group decreased. LncRNA H19 regulated the central sensitization of astrocytes through microRNA-29a-3p/FOS axis. CONCLUSION: Intrathecal injection of umbilical cord blood mesenchymal stem cells can improve the pain and central sensitization of advanced osteoarthritis through LncRNA H19/microRNA-29a-3p/FOS axis.

A novel prevascularized tissue-engineered chamber as a site for allogeneic and xenogeneic islet transplantation to establish a bioartificial pancreas.

Although sites for clinical or experimental islet transplantation are well established, pancreatic islet survival and function in these locations remain unsatisfactory. A possible factor that might account for this outcome is local hypoxia caused by the limited blood supply. Here, we modified a prevascularized tissue-engineered chamber (TEC) that facilitated the viability and function of the seeded islets in vivo by providing a microvascular network prior to transplantation. TECs were created, filled with Growth Factor-Matrigel™ (Matrigel™) and then implanted into the groins of mice with streptozotocin-induced diabetes. The degree of microvascularization in each TECs was analyzed by histology, real-time PCR, and Western blotting. Three hundred syngeneic islets were seeded into each chamber on days 0, 14, and 28 post-chamber implantation, and 300, 200, or 100 syngeneic islets were seeded into additional chambers on day 28 post-implantation, respectively. Furthermore, allogeneic or xenogeneic islet transplantation is a potential solution for organ shortage. The feasibility of TECs as transplantation sites for islet allografts or xenografts and treatment with anti-CD45RB and/or anti-CD40L (MR-1) was therefore explored. A highly developed microvascularized network was established in each TEC on day 28 post-implantation. Normalization of blood glucose levels in diabetic mice was negatively correlated with the duration of prevascularization and the number of seeded syngeneic islets. Combined treatment with anti-CD45RB and MR-1 resulted in long-term survival of the grafts following allotransplantation (5/5, 100%) and xenotransplantation (16/20, 80%). Flow cytometry demonstrated that the frequency of CD4+Foxp3-Treg and CD4+IL-4+-Th2 cells increased significantly after tolerogenic xenograft transplantation, while the number of CD4+IFN-γ-Th1 cells decreased. These findings demonstrate that highly developed microvascularized constructs can facilitate the survival of transplanted islets in a TECs, implying its potential application as artificial pancreas in the future.

Quantitative Analysis of HER2 Amplification by Droplet Digital PCR in the Follow-Up of Gastric Cancer Patients Being Treated with Trastuzumab after Surgery.

BACKGROUND: Circulating tumor DNA (ctDNA) derived from tumors is a promising biomarker for monitoring tumor status and evaluating therapeutic effects and prognosis. We studied the plasma human epidermal growth factor receptor 2 (HER2) amplification in gastric cancer (GC) patients by droplet digital PCR (ddPCR) during therapy with trastuzumab. METHODS: A total of 12 patients were recruited after surgery. All patients received FOLFOX chemotherapy combined with trastuzumab as a treatment regimen. During the 12 months of the follow-up period, using elongation factor Tu GTP binding domain containing 2 (EFTUD2) as a reference gene, plasma HER2 to EFTUD2 ratios (the HER2 ratio) were determined for each patient every 2 months by ddPCR. RESULTS: The concordance rate of HER2 amplification examined in plasma and formalin-fixed paraffin-embedded (FFPE) samples with ddPCR was 81.4%, with a sensitivity of 76.5% and a specificity of 83.8%. Plasma HER2 ratios were correlated with the primary tumor size (p < 0.01). A significant decrease in the plasma HER2 ratio was found after two months of treatment (p < 0.0001). Nine patients experienced partial response, and three patients had stable disease. Seven patients had progressive disease (PD) during follow-up, and four of them had died. The median progression-free survival (PFS) was 9.8 months. For each patient who developed PD, the plasma HER2 ratio was approximately 2.3-4.1 times higher than the cut-off value at the time of PD, which was the highest during the whole follow-up period. CONCLUSION: Longitudinal monitoring for the plasma HER2 ratio by ddPCR in the clinical courses of GC patients holds great promise for use as an indicator of tumor progression and treatment efficacy.

Inhibition of transplantation tolerance by immune senescence is reversed by endocrine modulation.

The senescent immune system responds poorly to new stimuli; thymic involution, accumulation of memory cells against other specificities, and general refractoriness to antigen signaling all may contribute to poor resistance to infection. These same changes may pose a significant clinical barrier to organ transplantation, as transplantation tolerance requires thymic participation and integrated, tolerance-promoting responses to novel antigens. We found that after the age of 12 months, mice became resistant to the tolerance-inducing capacity of the monoclonal antibody therapy anti-CD45RB. This resistance to tolerance to cardiac allografts could be overcome by surgical castration of male mice, a procedure that led to thymic regeneration and long-term graft acceptance. The potential for clinical translation of this endocrine-immune interplay was confirmed by the ability of Lupron Depot injections, which temporarily disrupt gonadal function, to restore tolerance in aged mice. Furthermore, we demonstrated that the restoration of tolerance after surgical or chemical castration depended on thymic production of regulatory T cells (T(regs)); thymectomy or T(reg) depletion abrogated tolerance restoration. The aging of the immune system ("immune senescence") is a significant barrier to immune tolerance, but this barrier can be overcome by targeting sex steroid production with commonly used clinical therapeutics.