Costimulation blockade and its possible future use in clinical transplantation

The nonimmune effects of currently used immunosuppressive drugs result in a high incidence of late graft loss due to nephrotoxicity and death of patients. As an immune-specific alternative to conventional immunosuppressants, new biotechnology tools can be used to block the costimulation signals of T-cell activation. Many experimental studies--particularly preclinical studies in nonhuman primates--have focused on blocking the 'classical' B7/CD28 and CD40/CD40L pathways, which are critical in primary T-cell activation. Here, we review the limitations, the recent advances and the first large-scale clinical application of the CTLA4-Ig fusion protein to block the B7/CD28 costimulation pathway. We also focus on new B7/CD28 and tumor necrosis factor (TNF)/TNF-R family costimulatory molecules that can deliver positive or negative costimulation signals regulating the alloimmune response. Strategies that use single agents to block costimulation have often proved to be insufficient. Given the diversity of the different costimulation molecules, future strategies for human transplantation may involve the simultaneous blockade of several selected pathways or the simultaneous use of conventional immunosuppressants.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

Transplantation tolerance: lessons from experimental rodent models

Immunological tolerance or functional unresponsiveness to a transplant is arguably the only approach that is likely to provide long-term graft survival without the problems associated with life-long global immunosuppression. Over the past 50 years, rodent models have become an invaluable tool for elucidating the mechanisms of tolerance to alloantigens. Importantly, rodent models can be adapted to ensure that they reflect more accurately the immune status of human transplant recipients. More recently, the development of genetically modified mice has enabled specific insights into the cellular and molecular mechanisms that play a key role in both the induction and maintenance of tolerance to be obtained and more complex questions to be addressed. This review highlights strategies designed to induce alloantigen specific immunological unresponsiveness leading to transplantation tolerance that have been developed through the use of experimental models.     

Conventional Immunosuppression is Compatible with Costimulation Blockade-Based, Mixed Chimerism Tolerance Induction

T-cell costimulatory blockade has emerged as an effective strategy to prevent allograft rejection in experimental models. We and others have reported that the beneficial effects of costimulation blockade can be negated when combined with certain immunosuppressants. The current study evaluates the compatibility of various immunosuppressive agents in a costimulation blockade-based, mixed chimerism tolerance protocol. The addition of conventional agents, including calcineurin inhibitors, did not interfere with tolerance induction. All mice developed multilineage macrochimerism and accepted donor allografts. Analysis of specific T-cell receptor utilization demonstrated selective deletion of donor-reactive T cells. Challenge with donor and third-party allografts confirmed donor-specific tolerance. Clinical introduction of costimulation blockade-based strategies will likely incorporate currently approved immunosuppressive agents. While it has been reported that certain conventional agents are detrimental to costimulation blockade-based strategies, our results suggest that these agents could safely be combined in clinical trials when used as part of a nonmyelosuppressive, mixed chimerism-based tolerance strategy.     

Multiple Combination Therapies Involving Blockade of ICOS/B7RP-1 Costimulation Facilitate Long-Term Islet Allograft Survival

In recent years a series of novel costimulatory molecules have been identified, including inducible costimulator (ICOS). In a fully major histocompatibility complex (MHC)-mismatched mouse model of islet transplantation, we demonstrate that while monotherapy with CTLA4-Ig, CD40 ligand monoclonal antibody (CD40L mAb) or rapamycin each improves islet allograft survival, graft rejection eventually develops. Immunohistologic analysis of rejected grafts revealed increased ICOS expression, suggesting a role for this costimulatory molecule as an alternate pathway for T-cell activation. The combination of a blocking anti-ICOS mAb with each of the above therapies resulted in significantly improved islet allograft survival, confirming the importance of ICOS signaling in islet allograft rejection. Mechanistic studies conducted in mice treated with anti-ICOS mAb and rapamycin demonstrated a lack of donor-specific immunological tolerance and an absence of regulatory T-cell activity. However, a dramatic effect was seen on acute anti-donor responses whereby anti-ICOS mAb and rapamycin significantly reduced the initial expansion and function of alloreactive T cells. These data demonstrate that blockade of the ICOS/B7RP-1 pathway has potential therapeutic benefit given its role in enhancing islet allograft survival and regulating acute alloresponses in vivo.     

Transient Depletion of Dividing T Lymphocytes in Mice Induces the Emergence of Regulatory T Cells and Dominant Tolerance to Islet Allografts

We previously showed that transient depletion of dividing T cells at the time of an allogeneic transplantation induces long-term tolerance to the allograft. Here we investigated the role of homeostatic perturbation and regulatory T cells (Treg) in such tolerance. Transient depletion of dividing T cells was induced at the time of an allogeneic pancreatic islets graft, by administration of ganciclovir for 14 days, into diabetic transgenic mice expressing a thymidine kinase (TK) conditional suicide gene in T cells. Allograft tolerance was obtained in 63% of treated mice. It was not due to global immunosuppression, permanent deletion or anergy of donor-alloantigens specific T cells but to a dominant tolerance process since lymphocytes from tolerant mice could transfer tolerance to naïve allografted recipients. The transient depletion of dividing T cells induces a 2- to 3-fold increase in the proportion of CD4⁺CD25⁺Foxp3⁺ Treg, within 3 weeks that persisted only in allograft-bearing mice but not in nongrafted mice. Tolerance with similar increased proportion of Treg cells was also obtained after a cytostatic hydroxyurea treatment in normal mice. Thus, the transient depletion of dividing T cells represents a novel means of immuno-intervention based on disturbance of T-cell homeostasis and subsequent increase in Treg proportion.     

Combined ''En Bloc'' Liver and Pancreas Transplantation in Patients with Liver Disease and Type 1 Diabetes Mellitus

Liver disease alters the glucose metabolism and may cause diabetes, but this condition is potentially reversible with liver transplantation (LTx). Type 1 diabetes mellitus may be coincidentally present in a LTx candidate and immunosuppressive drugs will aggravate diabetes and make its management more difficult for posttransplant. In addition, diabetes negatively influences outcome after LTx. Therefore, the question arises as to why not transplanting the pancreas in addition to the liver in selected patients suffering from both liver disease and Type 1 diabetes. We report two cases of en bloc combined liver and pancreatic transplantation, a technique originally described a decade ago in the treatment of upper abdominal malignancies but rarely used for the treatment of combined liver disease and Type 1 diabetes. Both recipients are currently liver disease-free and insulin-free more than 2 and 4 years posttransplant, respectively. Surgical, medical and immunological aspects of combined liver-pancreas transplantation are discussed in the light of the existing relevant literature.     

Islet Transplantation for Brittle Type 1 Diabetes: The UIC Protocol

This prospective phase 1/2 trial investigated the safety and reproducibility of allogeneic islet transplantation (Tx) in type I diabetic (T1DM) patients and tested a strategy to achieve insulin-independence with lower islet mass. Ten C-peptide negative T1DM subjects with hypoglycemic unawareness received 1–3 intraportal allogeneic islet Tx and were followed for 15 months. Four subjects (Group 1) received the Edmonton immunosuppression regimen (daclizumab, sirolimus, tacrolimus). Six subjects (Group 2) received the University of Illinois protocol (etanercept, exenatide and the Edmonton regimen). All subjects became insulin- independent. Group 1 received a mean total number of islets (EIN) of 1460   080 ± 418   330 in 2 (n = 2) or 3 (n = 2) Tx, whereas Group 2 became insulin- independent after 1 Tx (537   495 ± 190   968 EIN, p = 0.028). All Group 1 subjects remained insulin free through the follow-up. Two Group 2 subjects resumed insulin: one after immunosuppression reduction during an infectious complication, the other with exenatide intolerance. HbA1c reached normal range in both groups (6.5 ± 0.6 at baseline to 5.6 ± 0.5 after 2–3 Tx in Group 1 vs. 7.8 ± 1.1 to 5.8 ± 0.3 after 1 Tx in Group 2). HYPO scores markedly decreased in both groups. Combined treatment of etanercept and exenatide improves islet graft function and facilitates achievement of insulin-independence with less islets .     

Allograft Acceptance Despite Differential Strain-Specific Induction of TGF-β/IL-10-Mediated Immunoregulation

We examined the immune approaches that C57BI/6 and BALB/c mice take when treated to accept cardiac allografts. C57BI/6 mice accept DBA/2 cardiac allografts when treated with gallium nitrate (GN) or anti-CD40L mAb (MR1). These allograft acceptor mice fail to mount donor-reactive delayed type hypersensitivity (DTH) responses, and develop a donor-induced immunoregulatory mechanism that inhibits DTH responses. In contrast, BALB/c mice accept C57BI/6 cardiac allografts when treated with MR1 but not with GN. These allograft acceptor mice display modest donor-reactive DTH responses, and do not develop donor-induced immune regulation of DTH responses. Real-time PCR analysis of rejecting graft tissues demonstrated no strain-related skewing in the production of cytokines mRNAs. In related studies, C57BI/6 recipients of cytokine and alloantigen educated syngeneic peritoneal exudate cells (PECs) failed to mount DTH responses to the alloantigens unless neutralizing antibodies to transforming growth factor-beta (TGF-p were present at the DTH site demonstrating regulation of cell-mediated alloimmune responses. In contrast, BALB/c recipients of cytokine-and alloantigen-educated PECs expressed strong DTH responses to alloantigens demonstrating a lack of regulated alloimmunity. In conclusion, C57BI/6 mice respond to immunosuppression by accepting cardiac allografts and generating TGF-beta-related regulation of donor-reactive T cell responses, unlike BALB/c mice that do not generate these regulatory responses yet still can accept cardiac allografts.     

Posttransplant Diabetes Mellitus After Pancreas Transplantation

Some patients do not achieve normoglycemia after an otherwise successful pancreas transplant. The aim of this study was to define the incidence and risk factors for the development of persistent diabetes mellitus after pancreas transplantation. We studied the outcomes of 144 pancreas transplants performed at our institution between January 2001 and December 2005. Diabetes mellitus was defined as the persistent need for pharmacologic treatment of diabetes mellitus despite evidence of allograft function. Data are expressed as median (25–75% inter-quartile range). Median follow-up was 39 months (IQR 26–55 months). During the follow-up period, 28 patients (19%) developed diabetes mellitus with a functioning allograft. Factors predicting hyperglycemia included: pretransplant insulin dose, BMI and acute rejection episodes (p < 0.0001, p = 0.0002 and p < 0.02, respectively). The median pretransplant hemoglobin A1c for patients developing diabetes was 8.3% (IQR 7.0–9.4%) compared to 6.2% (IQR 5.8–7.4%) at 2 years after transplant (p = 0.0069). In conclusion, persistent diabetes mellitus can occur despite the presence of a functioning pancreas allograft and is due to increased pretransplant BMI, high pretransplant insulin requirements and episodes of acute rejection.     

Repaglinide in the Management of New-Onset Diabetes Mellitus After Renal Transplantation

The purpose of this study was to investigate the use of the short-acting insulin secretion drug repaglinide in new-onset diabetes mellitus (NODM) after renal transplantation. Twenty-three Caucasian patients with NODM after renal transplantation were selected to receive repaglinide therapy and were followed for at least 6 months. A control group treated with rosiglitazone was chosen for comparison. Successful repaglinide treatment was defined as a significant improvement of blood glucose concentrations and HbA1c <7% in the absence of glucosuria and without the need for the addition of further anti-diabetic agents. After 6 months of treatment with repaglinide, 14 of the 23 patients were successfully treated. Mean HbA1c decreased from 7.6 +/- 0.6% to 5.8 +/- 0.6% in 14 patients treated successfully. In nine patients, hyperglycemia persisted, and they were switched to insulin treatment (HbA1c 8.5 +/- 2.9% at the beginning to 7.4 +/- 2.2%). Mean serum creatinine levels, cyclosporine A and tacrolimus blood levels did not change significantly following institution of repaglinide therapy. The rate of successful treatment and the degree of HbA1c decrease were similar compared to rosiglitazone-treated control patients. The data from our observational study indicate that repaglinide can be an effective treatment option in Caucasian patients with NODM after renal transplantation.     

Influence of Sirolimus on Cyclosporine-Induced Pancreas Islet Dysfunction in Rats

This study was performed to investigate the effect of sirolimus (SRL) on cyclosporine (CsA)-induced pancreatic islet dysfunction in rats. Three separate studies were performed. First, diabetogenic effect of SRL was evaluated with three different doses (0.15, 0.3 and 0.6 mg/kg). Second, rats were treated with SRL (0.3 mg/kg) with or without CsA (15 mg/kg) for 4 weeks. Third, rats were treated with CsA for 4 weeks, and then switched to SRL for 4 weeks. The effect of SRL on CsA-induced pancreatic islet dysfunction was evaluated by an intraperitoneal glucose tolerance test, plasma insulin concentration, HbA1c level, HOMA-R index, immunohistochemistry of insulin and pancreatic beta islet cell mass. The SRL treatment increased blood glucose concentration in a dose-dependent manner. The combined treatment with SRL and CsA increased blood glucose concentration, Hemoglobin A1c (HbA1c) level, HOMA-R [fasting insulin (mU/mL) x fasting glucose (mmol/L)]/22.5] index and decreased plasma insulin concentration, immunoreactivity of insulin and pancreatic beta islet cell mass compared with rats treated with CsA. CsA withdrawal for 4 weeks improved pancreatic beta-cell function and structure. However, conversion from CsA to SRL further increased blood glucose levels compared with the rats converted from vehicle to SRL. The results of our study demonstrate that SRL is diabetogenic and aggravates CsA-induced pancreatic islet dysfunction.     

Morphometric Studies of Pituitary Glands and Testes in Rats with Streptozotocin-Induced Diabetes

Morphometrische Untersuchungen von Hypophysen und Hoden bei Ratten mit Streptozotocin-induziertem Diabetes Beim Tiermodell für Diabetes mellitus sind Hodenveränderungen und gestörte Fruchtbarkeit seit vielen Jahren bekannt. Doch hat sich bei diabetischem Hypogonadisms das Interesse erst in letzter Zeit auf das hypothalamo-hypophysäre System konzentriert. Bei Streptozotocin-diabetischen Ratten beschrieben wir kürzlich morphologische und immunohistochemische Veränderungen im Nucleus arcuatus und in der Eminentia mediana, welche von Hypophysen- und Hodenveränderungen begleitet waren. In der vorliegenden morphometrischen Arbeit werden die zeitliche Entwicklung von hypophysären und testikulären Läsionen untersucht und mögliche Korrelationen zwischen klinischen Werten und morphologischen Veränderungen geprüft. Bei Wistarratten wurden nach 4, 8 und 12 Monaten Streptozotocin-Diabetes Blutglukose, Körper- und Hodengewicht, Querschnittsfläche der Hodentubuli und Zahl der LH-Gonadotrophen in der Hypophyse untersucht. Bei den Diabetikern war das Hodengewicht nach 4 und 12 Monaten deutlich erniedrigt, nach 8 und 12 Monaten auch die Tubulusfläche signifikant kleiner. Bei 20–25% der Tiere traten die Hodenveränderungen früh auf (nach 4 Monaten oder früher), Tubulusflächen waren stark verkleinert, mit einem (sub-) totalen Block der Spermatogenese. Bei den übrigen Tieren erschienen die Veränderungen geringgradiger und traten später auf (nach 8–12 Monaten). Die Leydigzellen waren vermindert, verkleinert und oft degeneriert; die LH-Gonadotrophen stark vermehrt. Dabei handelte es sich vorwiegend um kleine atypische Zellen. Zwischen Körper- und Hodengewicht bestand eine positive, zwischen Tubulusflächen und LH-Gonadotrophen eine negative Korrelation. Diese Befunde bestätigen die pathogenetische Bedeutung der Läsionen im hypothalamo-hypophysären System bei diabetischem Hypogonadismus, wie er bei Streptozotocin-diabetischen Ratten auftritt. Zudem lassen die Befunde einen zweiphasigen Mechanismus dieser diabetischen testikulären Veränderungen vermuten.     

Combined Pancreatic Islet and Kidney Transplantation in a Child With Unstable Type 1 Diabetes and End‐Stage Renal Disease

Islet transplantation after successful kidney transplantation is a recognized treatment for adults with diabetes and end‐stage renal disease (ESRD), but has not been considered an option in the pediatric population. To our knowledge, we report the first combined islet and kidney transplant in a child. The patient was born with bilateral renal hypoplasia and was diagnosed with type 1 diabetes mellitus at age 13 months. He had erratic glycemic control and hypoglycemia unawareness. At 6 years of age, the child safely underwent simultaneous islet and live donor kidney transplantation. Although function of the islet graft was transient, the combined transplant provided significant benefits in terms of glucose control and overall growth and development. Such an approach represents a viable treatment option for pediatric patients with ESRD and unstable diabetes.     

胃袖带切除术对GK大鼠2型糖尿病的影响及其机理

目的研究胃袖带切除术（sleevegastrectomy,SG）对GK大鼠2型糖尿病（type2diabetesmellitus,T2DM）的治疗作用及其可能机理。方法将13只12周龄的GK大鼠随机分为2组：SG组7只和假手术组（SO组）6只,分别行SG术和假手术。于术前及术后1、4、10和26周测量2组大鼠的体质量、24h进食量、空腹血糖值、血清胰高血糖素样肽-1（glucagon-likepeptide-1,GLP-1）和血清生长激素释放肽（Ghrelin）浓度;于术后10周检测2组大鼠的粪便能量含量,并进行口服葡萄糖耐量实验（OGTT）和胰岛素耐受性实验（ITT）。结果①体质量：各时点2组大鼠的体质量比较差异均无统计学意义（P〉0．05）;与术前比较,术后1周2组大鼠的体质量均降低（P〈0．01）,术后10和26周的体质量均增加（P〈0．01）。②24h进食量：与SO组比较,术后4和10周SG组大鼠的24h进食量均较低（P〈0。05）。与术前比较,SG组大鼠术后1、4及10周的进食量均较低（P〈0．05）,SO组大鼠术后1周的进食量低于术前（P〈0．05）。③空腹血糖值：与SO组比较,术后各时点SG组大鼠的空腹血糖值均较低（P〈0．01）。与术前比较,SG组大鼠术后各时点的空腹血糖值均较低（P〈0．01）,而SO组大鼠仅术后1周明显低于术前∽〈0．OD。④血清GLP-1水平：与SO组比较,术后4、10及26周SG组大鼠的血清GLP．1水平均较高（P〈0．05）。与术前比较,术后4、10及26周SG组大鼠的血清GLP-1水平较高（P〈0．05）,而术后SO组大鼠的血清GLP．1水平无明显变化（P〉0．05）。⑤血清Ghrelin水平：与SO组比较,术后各时点SG组大鼠的血清Ghrelin水平均较低（P〈0．01）。与术前比较,术后各时点SG组大鼠的血清Ghrelin水平均较低（P〈0．001）,而SO组大鼠的血清Ghrelin水平无明显变化（P〉0．05）。⑥曲线下面积（AUC）：SG组大鼠的AUC（OGTT和ITT）均较SO组低（P〈0．01）。结论SG术可以明显降低GK大鼠的空腹血糖值,改善葡萄糖耐量及增强胰岛素敏感性,该作用可能是GLP．1、Grelin等多种胃肠道激素共同作用的结果。SG术可能是潜在的非肥胖型T2DM的治疗方法。     

Current advances in using tolerogenic dendritic cells as a therapeutic alternative in the treatment of type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β-cells of the pancreatic islets by autoreactive T cells, leading to high blood glucose levels and severe long-term complications. The typical treatment indicated in T1D is exogenous insulin administration, which controls glucose levels; however, it does not stop the autoimmune process. Various strategies have been implemented aimed at stopping β-cell destruction, such as cellular therapy. Dendritic cells (DCs) as an alternative in cellular therapy have gained great interest for autoimmune disease therapy due to their plasticity to acquire immunoregulatory properties both in vivo and in vitro, performing functions such as anti-inflammatory cytokine secretion and suppression of autoreactive lymphocytes, which are dependent of their tolerogenic phenotype, displayed by features such as semimature phenotype, low surface expression of stimulatory molecules to prime T cells, as well as the elevated expression of inhibitory markers. DCs may be obtained and propagated easily in optimal amounts from peripheral blood or bone marrow precursors, such as monocytes or hematopoietic stem cells, respectively; therefore, various protocols have been established for tolerogenic (tol)DCs manufacturing for therapeutic research in the treatment of T1D. In this review, we address the current advances in the use of tolDCs for T1D therapy, encompassing protocols for their manufacturing, the data obtained from preclinical studies carried out, and the status of clinical research evaluating the safety, feasibility, and effectiveness of tolDCs.