Hitomi Nishinakamura

HMGB1-Mediated Early Loss of Transplanted Islets Is Prevented by Anti-IL-6R Antibody in Mice.

Objectives The limited success in achieving insulin independence of patients with type 1 diabetes mellitus after islet transplantation from a single donor, mainly due to early loss of transplanted islets, hampers clinical application of islet transplantation. Previously, we have shown in mice that the early loss of transplanted islets in the liver, the site of islet transplantation, is caused by innate immune rejection triggered by high-mobility group box 1 (HMGB1) protein released from transplanted islets. We herein determined whether the HMGB1-mediated early loss of transplanted mouse islets is prevented by anti–interleukin-6 receptor (IL-6R) antibody. Methods The effect of anti–IL-6R antibody on amelioration of hyperglycemia in streptozocin-induced diabetic mice receiving 200 islets into the liver from a single donor was evaluated in association with HMGB1-stimulated interferon-&ggr; production of hepatic mononuclear cells. Results Hyperglycemia of diabetic mice receiving 200 syngeneic islets was ameliorated with down-regulation of interferon-&ggr; production of hepatic natural killer T cells and neutrophils when anti–IL-6R was administered at the time of transplantation. This beneficial effect was also seen in allografts when alloimmune rejection was prevented by anti-CD4 antibody. Conclusions These findings demonstrate that anti–IL-6R antibody prevented the early loss of intrahepatic islet grafts with inhibiting HMGB1-induced immune activation after islet transplantation.

Vascular endothelial growth factor-C derived from CD11b+ cells induces therapeutic improvements in a murine model of hind limb ischemia

OBJECTIVE The use of bone marrow cells (BMCs) in therapeutic angiogenesis has been studied extensively. However, the critical paracrine effects of this treatment are still unclear. Therefore, we studied autotransfusable cells that produce vascular endothelial growth factor (VEGF), especially VEGF-C. METHODS Male C57BL/6 mice with hind limb ischemia were administered intramuscular injections of phosphate-buffered saline as controls, or unsorted BMCs, sorted CD11b(+), or CD11b(-) cells from BMCs, and recombinant VEGF-C. To evaluate the treatments, perfusion was measured by laser Doppler scanning performed on days 0, 1, 3, 7, 14, 21, and 28. A functional assay was performed in parallel, with mice traversing an enclosed walkway. Capillary density was determined by directly counting vessels stained positive with von Willebrand factor at individual time points. Lymphangiogenesis was assessed by LYVE-1 positive cells. RESULTS Postischemic recovery of hind limb perfusion significantly improved in BMC, CD11b(+), and VEGF-C treatment groups compared with the control groups, as assessed by laser Doppler scanning. On early operative days 1 and 3, the blood flow recovery ratio was higher in the CD11b(+)-treated group compared with BMC and VEGF-C treatment groups. In the functional assay, the VEGF-C group dramatically recovered compared with the control group. The capillary/myofiber ratio in the thigh muscle and number of LYVE-1 positive cells was higher in the CD11b(+) and VEGF-C groups than in controls. Furthermore, expression of VEGF-A, VEGF-C, and VEGF receptor messenger ribonucleic acid and protein was observed in CD11b(+) cells. CONCLUSIONS The VEGF-C derived from CD11b(+) cells play a critical role in angiogenesis and lymphangiogenesis in a murine model of hind limb ischemia. Consequently, treatment with self-CD11b(+) cells accelerated recovery from ischemia and may be a promising therapeutic strategy for peripheral arterial disease patients.

Pretreatment of donor islets with the Na(+) /Ca(2+) exchanger inhibitor improves the efficiency of islet transplantation.

Pancreatic islet transplantation is an attractive therapy for the treatment of insulin‐dependent diabetes mellitus. However, the low efficiency of this procedure necessitating sequential transplantations of islets with the use of 2–3 donors for a single recipient, mainly due to the early loss of transplanted islets, hampers its clinical application. Previously, we have shown in mice that a large amount of HMGB1 is released from islets soon after their transplantation and that this triggers innate immune rejection with activation of DC, NKT cells and neutrophils to produce IFN‐γ, ultimately leading to the early loss of transplanted islets. Thus, HMGB1 release plays an initial pivotal role in this process; however, its mechanism remains unclear. Here we demonstrate that release of HMGB1 from transplanted islets is due to hypoxic damage resulting from Ca2+ influx into β cells through the Na+/Ca2+ exchanger (NCX). Moreover, the hypoxia‐induced β cell damage was prevented by pretreatment with an NCX‐specific inhibitor prior to transplantation, resulting in protection and long‐term survival of transplanted mouse and human islets when grafted into mice. These findings suggest a novel strategy with potentially great impact to improve the efficiency of islet transplantation in clinical settings by targeting donor islets rather than recipients.

Prevention of high-mobility group box 1-mediated early loss of transplanted mouse islets in the liver by antithrombin III.

Background The low efficiency of pancreatic islet transplantation mainly because of the early loss of transplanted islets hampers its clinical application. Previously, we have shown in mice that the early loss of transplanted islets in the liver is caused by innate immune rejection in concert with dendritic cells, natural killer T cells, and neutrophils to produce interferon (IFN)-&ggr;, which is triggered by high-mobility group box 1 (HMGB1) released from transplanted islets. We herein determined whether the HMGB1-mediated early loss of transplanted mouse islets is prevented by antithrombin (ATIII). Methods The effect of ATIII on in vitro and in vivo HMGB1-stimulated IFN-&ggr; production of hepatic mononuclear cells was examined. Then, the effect of ATIII on amelioration of hyperglycemia in streptozotocin-induced diabetic mice receiving 200 syngeneic islets from a single donor was determined. Results In vitro and in vivo IFN-&ggr; production of mononuclear cells in the liver of mice in response to HMGB1 was suppressed by ATIII. Hyperglycemia of streptozotocin-induced diabetic mice receiving 200 syngeneic islets into the liver from a single donor was ameliorated with down-regulation of IFN-&ggr; production of natural killer T cells and neutrophils in the liver when ATIII but not vehicle was administered once at the time of islet transplantation. The favorable effect of ATIII was similarly achieved in mice receiving islet allografts when rejection was prevented with anti-CD4 antibody treatment. Conclusions These findings demonstrate that ATIII prevents HMGB1-mediated early loss of transplanted islets caused by innate immune rejection, suggesting a potential application of ATIII to improve efficiency of clinical islet transplantation.

GM-CSF Treated F4/80+ BMCs Improve Murine Hind Limb Ischemia Similar to M-CSF Differentiated Macrophages

Novel cell therapy is required to treat critical limb ischemia (CLI) as many current approaches require repeated aspiration of bone marrow cells (BMCs). The use of cultured BMCs can reduce the total number of injections required and were shown to induce therapeutic angiogenesis in a murine model of hind limb ischemia. Blood flow recovery was significantly improved in mice treated with granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent BMCs that secreted inflammatory cytokines. Angiogenesis, lymphangiogenesis, and blood flow recovery ratio were significantly higher in the GM-CSF-cultured F4/80+ macrophage (GM-Mø)-treated group compared with controls. Furthermore, Foxp3+ cell numbers and tissue IL-10 concentrations were significantly increased compared with controls. There was no significant difference in blood flow recovery between GM-Mø and M-CSF-cultured F4/80+ macrophages (M-Mø). Thus, GM-Mø were associated with improved blood flow in hind limb ischemia similar to M-Mø. The selective methods of culturing and treating GM-Mø cells similar to M-Mø cells could be used clinically to help resolve the large number of cells required for BMC treatment of CLI. This study demonstrates a novel cell therapy for CLI that can be used in conjunction with conventional therapy including percutaneous intervention and surgical bypass.

The Spleen Is an Ideal Site for Inducing Transplanted Islet Graft Expansion in Mice

Alternative islet transplantation sites have the potential to reduce the marginal number of islets required to ameliorate hyperglycemia in recipients with diabetes. Previously, we reported that T cell leukemia homeobox 1 (Tlx1)+ stem cells in the spleen effectively regenerated into insulin-producing cells in the pancreas of non-obese diabetic mice with end-stage disease. Thus, we investigated the spleen as a potential alternative islet transplantation site. Streptozotocin-induced diabetic C57BL/6 mice received syngeneic islets into the portal vein (PV), beneath the kidney capsule (KC), or into the spleen (SP). The marginal number of islets by PV, KC, or SP was 200, 100, and 50, respectively. Some plasma inflammatory cytokine levels in the SP group were significantly lower than those of the PV group after receiving a marginal number of islets, indicating reduced inflammation in the SP group. Insulin contents were increased 280 days after islet transplantation compared with those immediately following transplantation (p<0.05). Additionally, Tlx1-related genes, including Rrm2b and Pla2g2d, were up-regulated, which indicates that islet grafts expanded in the spleen. The spleen is an ideal candidate for an alternative islet transplantation site because of the resulting reduced inflammation and expansion of the islet graft.

Islet-derived damage-associated molecular pattern molecule contributes to immune responses following microencapsulated neonatal porcine islet xenotransplantation in mice.

Clinical allogeneic islet transplantation has become an attractive procedure for type 1 diabetes mellitus treatment. However, there is a severe shortage of human donors. Microencapsulated neonatal porcine islet (NPI) xenotransplantation may be an alternative transplantation procedure. Currently, the efficacy of microencapsulated NPI xenotransplantation into the peritoneal cavity is limited because of early non‐function resulting from inflammation, which is a serious hindrance to promoting this procedure as a standard therapy. Previously, we have demonstrated that high‐mobility group box 1 (HMGB1), a damage‐associated molecular pattern (DAMP) molecule, was released from transplanted islets and triggered inflammatory reactions leading to early loss of intrahepatic syngeneic islet grafts in mice. In this study, we hypothesized that the inflammatory reaction in the peritoneal cavity following the transplantation of microencapsulated NPIs is more severe than that of empty capsules. Additionally, we predicted that HMGB1 released from transplanted microencapsulated NPIs triggers further inflammatory reactions in mice. Finally, we hypothesized that microencapsulated NPI xenotransplantation efficacy would be improved by treatment‐targeting inflammatory reactions in a mouse model.

Inhibitory Effect of Thrombomodulin on HMGB1-Stimulated IFN-γ Production of Hepatic NKT and Gr-1+ Cells, Facilitating to Prevent Early Loss of Transplanted Islets in the Liver of Mice: 1512

Currently, insulin independence in patients with IDDM has been hardly achieved after pancreatic islet transplantation (tx) from a single donor mainly due to early loss of transplanted islets. Previously, we have shown in mice that pancreatic islets contain abundant HMGB1, released into circulation and triggering NKT cell-dependent IFN-γ production of Gr-1+ cells (neutrophils) in the liver receiving islets (JCI 2010) which is an essential component of early loss of transplanted islets (JEM 2005). In the present study, we hypothesize that the beneficial effect of thrombomodulin (TM) on engraftments of islets in the liver might be mediated through inhibition of HMGB1-NKT-Gr-1+cell pathways since TM has been reported to produce sequestration of HMGB1 (JCI 2005). Hyperglycemia of STZ-diabetic mice (C57BL/6) receiving 200 syngenic islets from a single donor into the liver via the portal vein was ameliorated when TM was administered IV for 3 times (0, 12 and 24hrs, 200μg/ injection/mouse, n=5), while those of mice (n=5) treated with saline did not. Morphologically, intact islet grafts with well granulated β cells were seen in the liver of normoglycemic recipients treated with TM, while in contrast, degenerated islets with de-granulated β cells were seen in hyperglycemic control mice. IPGTT (1g/kg glucose) at 60 days after tx revealed that the glucose tolerance of TM-treated mice receiving 200 islets (n=5) was superior to that of normoglycemic mice receiving 400 islets without TM treatment (n=5). FACS analysis showed that IFN-γ production of NKT cells and Gr-1+ cells accumulated in the liver of mice receiving islets and treated with saline was up-regulated at 6 hours after tx as reported previously, while in contrast, that in mice receiving 200 islets and treated with TM was down-regulated with reduction in number of infiltrating Gr-1+ cells. IFN-γ production of NKT cells and Gr-1+ cells accumulated in the liver of mice at 2 hours after the IV injection of HMGB1 (100 μg/injection/mouse) without islet transplantation was up-regulated, while in marked contrast, that in mice treated with TM (500μg, iv) prior to the HMGB1 injection was down-regulated with reduced number of infiltrated Gr-1+ cells. These findings indicate that TM prevents the early loss of transplanted islets in the liver of mice through inhibition of stimulatory effects of HMGB1. Importantly, recombinant TM has already been used in clinics with great impact on its efficacy for the treatment of sepsis with disseminated intravascular coagulation in Japan. Thus, the safety issue regarding the clinical use of TM has been cleared and it seems ready to apply this to clinical islet transplantation to improve the efficiency of intraportal islet transplantation. 1290

Master Regulator for Splenic Development, Tlx1 Contributes the Engraftment of the Transplanted Islets in the Spleen

Introduction Clinical islet transplantation (Tx) is a useful treatment for the severe diabetes mellitus. The liver has been recognized as an ideal transplant site for islet Tx due to the similarity of native insulin flow, however it has a disadvantage in poor transplant efficacy due to the active innate immune system. To establish an alternative transplant site, we have focused on the spleen, a component of portal circulation as same as pancreas and liver, and splenocytes promote immune tolerance which might prevent graft rejection. And we previously reported that Tlx1+ stem cells in the spleen regenerated into insulin-producing cells in the pancreas. This study aimed to show the effectiveness of intrasplenic islet Tx. Materials and Methods We tried to clarify the marginal numbers of islets for achieving normoglycemia using diabetic C57BL/6 mice, which underwent the transplantation of 25, 50, 100 or 200 islets equivalents (IEQs) into the liver (L), beneath the left kidney capsule (KC), or into the spleen (SP). Intraperitoneal glucose tolerance tests (IGTT) were performed at 50 days after Tx, and a splenectomy was done at 60 or 160 days. Plasma MCP-1, G-CSF and HMGB-1 levels at 6 hours after Tx was also measured using ELISA or MAGPIX systems. Next, we transplanted 25 IEQs into the spleen to figure out the minimum dose of islets under controlling insulin with transplantation of 100 IEQs beneath KC (SP25+KC100 or SP25 alone group). To assess the graft function in the spleen, the nephrectomy was performed at 240 days after Tx and the splenectomy was done at 290 days. Additionally, microarray was performed to detect the gene regulation associated with improving islet engraftment. Results and Discussions The marginal islets Tx number in L, KC, or SP or 200, 100, or 50 IEQs, respectively. The pattern of glucose change in IGTT of the mice transplanted 50 IEQs into the SP (SP50) was comparable to that of naïve control mice. All the SP50 mice became hyperglycemia after splenectomy. All the plasma MCP-1, G-CSF and HMGB-1 levels in the SP50 mice were significantly lower (p < 0.05) than those of the mice received 200 IEQs into the L (43.4±11.1 pg/mL, vs. 77.0±40.2 pg/mL, 4,970.7±1,176.1 pg/mL vs. 7,862.6±1,023.4 pg/mL, 12.4±1.2 ng/mL vs. 27.7±6.6 ng/mL, respectively) indicating that reduction of inflammation was seen in the SP group. All SP25+KC100 mice (n = 11) became normoglycemia, and the eight of them remained normoglycemia even after nephrectomy. All of them became hyperglycemia after splenectomy that meant the islet graft in the spleen kept normoglycemia. Additionally, Tlx1-related genes including Rrm2b and Pla2g2d, which encode a transcription factor essential for the spleen development, were up-regulated following islet engraftment in the SP. Figure. No caption available. Figure. No caption available. Conclusion The spleen is an ideal transplant site for the islet to reduce inflammation and support the engraftment of islets.

Pretreatment of donor islets with papain improves allograft survival without systemic immunosuppression in mice

ABSTRACT Although current immunosuppression protocols improve the efficacy of clinical allogenic islet transplantation, T cell-mediated allorejection remains unresolved, and major histocompatibility complexes (MHCs) play a crucial role in this process. Papain, a cysteine protease, has the unique ability to cleave the extracellular domain of the MHC class I structure. We hypothesized that pretreatment of donor islets with papain would diminish the expression of MHC class I on islets, reducing allograft immunogenicity and contributing to prolongation of islet allograft survival. BALB/c islets pretreated with papain were transplanted into C57BL/6J mice as an acute allorejection model. Treatment with 1 mg/mL papain significantly prolonged islet allograft survival. In vitro, to determine the inhibitory effect on T cell-mediated alloreactions, we performed lymphocyte proliferation assays and mixed lymphocyte reactions. Host T cell activation against allogenic islet cells was remarkably suppressed by pretreatment of donor islet cells with 10 mg/mL papain. Flow cytometric analysis was also performed to investigate the effect of papain treatment on the expression of MHC class I on islets. One or 10 mg/mL papain treatment reduced MHC class I expression on the islet cell surface. Pretreatment of donor islets with papain suppresses MHC class I-mediated allograft rejection in mice and contributes to prolongation of islet allograft survival without administration of systemic immunosuppressants. These results suggest that pretreatment of human donor islets with papain may reduce the immunogenicity of the donor islets and minimize the dosage of systemic immunosuppressants required in a clinical setting.