Allogeneic Islet Transplantation Research Articles

Long Term Outcomes of Allogeneic Islet Transplantation: The Collaborative Islet Transplant Registry (CITR) 1999-2010

Introduction: CITR (www.citregistry.org) operates to expedite progress and promote safety in islet/beta cell transplantation (ITx) by collecting, analyzing, and communicating current data on all clinical ITx performed in North America and JDRF-funded sites in Europe and Australia. Methods: From 1999-2010, 27 CITR sites registered 659 recipients of one or more allogeneic ITx consenting to share their data with CITR: 276 in 1999-2003, 193 in 2004-2006 and 190 in 2007-2010; 559 were islet transplant alone (ITA) and 100 were islet after or simultaneous islet-kidney (IAK/SIK); 7% came from the Clinical Islet Transplant Consortium (www.citisletstudy.org, 2008-2010); 63% were from North American sites and 37% from European and Australian JDRF sites, representing 81% of all allogeneic ITx conducted at these sites. Results: Rates of insulin independence (≥14 days, Figure 1) post last infusion increased from 29% at 3-years in the early (1999-2003) to 41% in the most recent era (2007-2010), p=0.02). Factors most predictive of a superior outcome included age ≥35 years (p< 0.01), insulin requirement < 0.41U/kg-day (p< 0.01), total IEQs infused >600 (p=0.01), 2 but not ≥3 infusions (p< 0.01), induction with T-cell depletion plus TNF-α inhibition (p=0.02) and maintenance that includes calcineurin inhibitors and IMPDH inhibitors (p=0.02). These factors also predicted longer duration of basal C-peptide≥0.3 ng/mL secretion, as did culturing the islets (p< 0.001), and donor given insulin (p=0.02) (Figure 2). Similar improvements over the decade were seen in HbA1c levels, fasting blood glucose levels, which stabilize dramatically following ITx, and resolution of severe hypoglycemic events. Death rates have remained low and stable across the eras. A total of 29 neoplasms occurred over 1,800 person-years of follow-up: 72% were benign. Of 12 (1.8%) patients developing non-skin cancers, 6 recovered completely, 5 did not recover and 1 died (lung). CKD-eGFR improved somewhat over the decade. The incidence of life-threatening events declined significantly (p=0.002), as did overall incidence of any adverse event in the first year of ITx (p=0.02)[Figure 1][Figure 2]Conclusions: The efficacy and risk profiles of islet transplantation showed steady improvement over 1999-2010.

Induction of Tolerance to Human Allogeneic Islet Transplants in Humanized NOD/SCID Mice

Introduction: A recombinant soluble form of the inhibitory receptor ILT3 (ILT3Fc) was tested as an immunosuppressive treatment in a pancreatic islet transplantation model in humanized NOD/SCID mice. We sought to determine its efficacy in blocking allo-reactions against transplanted pancreatic islets and to determine its mechanism of action. Methods: Diabetes was induced in NOD/SCID mice in by streptozotocin injection. Mice were then transplanted with human pancreatic islets (1500 IEQ). Upon restoration of euglycemia, the mice were ‘humanized’ with allogeneic peripheral blood mononuclear cells. Treatment with ILT3Fc, control human IgG, or saline was initiated immediately, at day 15 or day 20 after humanization. Rejection was diagnosed when two glycemic readings >350 mg/dl were recorded. Histological studies of the tolerated and rejected grafts were performed. CD4 and CD8 T-cells from the spleen were assayed for suppressor activity, cytokine production, and costimulatory phenotypic profiles. Results: Although human T-cell engraftment was similar in all groups, ILT3Fc-treated mice tolerated the islets for the entire period of observation, while untreated mice rejected the graft within 7 weeks (P < 0.0001). Initiation of treatment at day 15 reversed rejection and rescued 70% of grafted mice, however, initiation at day 20 resulted in loss of all grafts. ILT3Fc treatment suppressed Th1 and Th2 cytokine production and costimulatory molecules. Human CD8+ T cells (but not CD4+ T cells) isolated from tolerant mice exhibited suppressor activity inhibiting Th alloreactivity. Histochemical studies showed a striking difference between the insulin production and T cell infiltration of human pancreatic islets from tolerant, ILT3Fc-treated mice and non-ILT3Fc treated controls. Conclusion: ILT3-Fc is an effective immunoregulatory agent that suppressed or reversed islet allograft rejection in this humanized mouse model by induction of allospecific CD8 T suppressor cells.

Acute Systemic Profiles of Inflammation and Coagulation Following Autologous Islet Transplantation

Introduction: Prevention of early graft loss is a major issue in improving clinical outcomes of allogeneic as well as autologous islet transplantation. The instant blood mediated inflammatory reaction (IBMIR) has been well investigated in allogeneic islet transplant setting; however, inflammatory or coagulatory responses in autologous islet transplantation (AIT) have not been clearly documented. We focused on systemic inflammatory and coagulatory responses in early phase of clinical AIT. Methods: A total of ten consecutive patients with refractory chronic pancreatitis who underwent AIT at our institute were included in this study. Serum C-peptide, inflammatory and coagulation factors were measured at the following time points: prior, middle and end of islet infusion as well as 15 min, 1 hour, 3 hours, 6 hours and 24 hours after the IAT. Serum C-peptide response during islet infusion was considered as a surrogate maker of early islet destruction and the patients with the early C-peptide response over or less than 1000 pmol/L were grouped as high- or low-damage groups, respectively. Peak values of cytokine and coagulation factors within 24 hours after AIT were compared between the two groups using Mann-Whitney U test non-parametrically. Results: No significant differences were found in patient characteristics and transplant condition except for body mass index (p=0.04, Table 1)[Table 1. Patient and transplant characteristics]Tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, interferon γ-induced protein (IP)-10/CXCL10, monocyte chemotactic protein (MCP)-1/CCL2, interferon (IFN)-γ, thrombin-antithrombin complex (TAT), complement 3a (C3a) were significantly elevated within 24 hours after AIT compared to the prior infusion levels (p with nonparametric matched Wilcoxon signed-rank test < 0.01). Among these factors, significant differences were found in TNF-α and C3a levels between the two group (p < 0.05) while IP-10 had marginally significant difference (p = 0.06).[Fig. 1. Elevated inflammatory/coagulatory factors]Conclusions: Systemic inflammatory and coagulatory responses were observed in in early phase of AIT for the patients with chronic pancreatitis. Greater islet graft damage may be associated with higher release of inflammatory/coagulatory factors, suggesting that inhibition of these factors could enhance islet engraftment in AIT. The impact of IBMIR should be validated in large scale clinical studies on AIT.

Regulatory T Cell Therapy Combined with Donor-Specific T Cell Deletion Promotes Long-Term Survival in Mouse Model of Allogeneic Islet Transplantation: Preconditioning Requirements and Mechanisms of Action

Regulatory T cell (Treg) prevention of transplant rejection requires drastic change of the balance of donor antigen-reactive Tregs to conventional T (Tconv) cells at the time of transplantation. To accomplish this, we preconditioned recipient mice using donor-specific transfusion (DST) followed by cyclophosphamide (Cy) treatment to reduce donor-reactive T cells before Treg infusion. This DST+Cy approach deleted 70-80% of donor-reactive T cells, but failed to prolong islet allograft survival. Infusion of 5 × 106 Tregs with direct donor reactivity led to long-term survival of BALB/c islets in 70% of the DST+Cy conditioned C57BL/6 recipients. Equal numbers of polyclonal Tregs significantly prolonged graft survival, but 90% of the recipients rejected their grafts by 100 days. However, increasing the numbers of polyclonal Tregs to 25 × 106 was able to confer long-term graft protection in 90% of the recipients. Mice receiving Treg therapy showed marked reduction of CD8+ T cells, but not CD4+ T cells, and concomitant increase of Tregs in the grafts during the first 14 days after transplantation. Mice with long-term protected grafts harbored donor-reactive T cells in the periphery and but had minimal T cells without detectable therapeutic Tregs within the grafts. Majority of the leukocytes present in the protected grafts were F4/80+CD11c-macrophages. However, the original islet grafts remained to be protected while the recipients were challenged with and rejected skin grafts from the same donor strain. Therapeutic Tregs altered the balance as well as the size of lymphocytic infiltrates early after transplantation likely through altering local inflammatory milieu, and conferred long-term graft protection locally in the grafts via infectious tolerance. Together, these results demonstrate a clinically viable approach to harness the tolerogenic property of Tregs for inducing long-term allograft survival and suggest Treg-mediated protection and biomarkers for Treg-induced tolerance reside locally in the grafts.

Protection of Human Islets from Instant Blood-Mediated Inflammatory Reaction by NF-κB Blockade

Introduction: Instant blood-mediated inflammatory reaction (IBMIR) is known to cause the loss of a significant mass of transplanted islets. Major characteristics of IBMIR include activation of complement and coagulation cascades and infiltration of innate immune cells into the islets. We provide evidence using an in vitro model that blockade of transcription factor NF-κB results in significant inhibition of this inflammatory reaction. Methods: Purified human islets were mixed with heparinized autologous or ABO-matched allogeneic blood in an in vitro tube model. The in vitro model was validated by measurement of pO2, pH and glucose in the blood+islet mix which showed that the mixing of islets and blood retained normal physiological conditions up to 6 h. Low-molecular weight dextran sulfate (LMWDS) alone and in combination with Withaferin A (WA)or resveratrol (known NF-kB inhibitors) were added to the blood-islet mix. Islet viability was measured using Hoechst 33341/propidium iodide staining followed by image analysis. Plasma samples and islet clots were retrieved at regular time points to analyze cytokines and cellular infiltration respectively. Panel of pro-inflammatory cytokines including IFN-γ, IL-6, and IL-8 were measured using Luminex assay. Results: Islet viability increased by 26% over control in the presence of LMWDS; however, addition of WA or resveratrol significantly improved islet viability over control (107-112%; p< 0.02). Exposure of purified islets to both autologous and allogeneic blood increased production of inflammatory cytokines with significant release at 6 hour. Mixing of allogeneic blood invoked a stronger cytokine response (>10 fold) when compared to autologous combination. Presence of WA or resveratrol significantly inhibited release of IFN-γ (4.6±0.1 vs. 2.0±1.0 pg/mL; p< 0.04), IL-6 (6444±196 vs. 712±127 pg/mL; p< 0.001), IL-8 (6368±309 vs. 4257±84 pg/mL; p< 0.005) in allogeneic combination. Immunohistochemistry of islets showed significant reduction in infiltration of neutrophils in both allogeneic (8.2±2.9 vs. 0.5±0.7 cells/islet; p< 0.005) and autologous samples (8.1±2.7 vs. 1.7±0.8 cells/islet; p< 0.001). Conclusion: The results showed that combination of LMWDS and NF-κB blockers offered remarkable protection of islets from IBMIR during the initial phase of the exposure of islets to blood. In summary, control of NF-kB activation could be a key strategy to minimize damage to both autologous and allogeneic islet transplants due to IBMIR.

A Novel Monoclonal Antibody to CD40 Prolongs Islet Allograft Survival

The importance of CD40/CD154 costimulatory pathway blockade in immunosuppression strategies is well-documented. Efforts are currently focused on monoclonal antibodies specific for CD40 because of thromboembolic complications associated with monoclonal antibodies directed towards CD154. Here we present the rational development and characterization of a novel antagonistic monoclonal antibody to CD40. Rhesus macaques were treated with the recombinant anti-CD40 mAb, 2C10, or vehicle before immunization with keyhole limpet hemocyanin (KLH). Treatment with 2C10 successfully inhibited T cell-dependent antibody responses to KLH without significant peripheral B cell depletion. Subsequently, MHC-mismatched macaques underwent intraportal allogeneic islet transplantation and received basiliximab and sirolimus with or without 2C10. Islet graft survival was significantly prolonged in recipients receiving 2C10 (graft survival time 304, 296, 265, 163 days) compared to recipients receiving basiliximab and sirolimus alone (graft survival time 8, 8, 10 days). The survival advantage conferred by treatment with 2C10 provides further evidence for the importance of blockade of the CD40/CD154 pathway in preventing alloimmune responses. 2C10 is a particularly attractive candidate for translation given its favorable clinical profile.

SUITO Index for Evaluation of Clinical Islet Transplantation

The major endpoints for clinical islet transplantation for type 1 diabetes are insulin independence and reduction of hypoglycemic episodes. Both endpoints are influenced by patients' and physicians' preferences regarding the use of exogenous insulin. Therefore, development of an objective endpoint for assessing clinical islet transplantation is desirable. HOMA-beta score is useful in assessing functional β-cell mass. However, this score uses blood insulin levels that are influenced by exogenous insulin injection and therefore is not suitable for patients who receive exogenous insulin. For assessing functional β-cell mass for type 1 diabetic patients after islet transplantation, we created the Secretory Unit of Islet Transplant Objects (SUITO) index using fasting C-peptide and fasting glucose. The formula of the SUITO index is fasting C-peptide (ng/ml)/[fasting blood glucose − 63 (mg/dl)] × 1500. We demonstrated that, within 1 month of islet transplantation, an average SUITO index of >26 was an excellent predictor of achieving insulin independence. In addition, daily SUITO index scores correlated with a reduction of insulin dose and adversely correlated with blood glucose levels during an intravenous glucose tolerance test. Other important endpoints, reduction of hypoglycemic episodes and quality of life, also correlated with the SUITO index. Thus, the SUITO index is excellent for assessing important endpoints (insulin independence, reduction of hypoglycemia, improved quality of life) after allogeneic islet transplantation.

Islet Cell Transplantation in Type 1 Diabetes: An Analysis of Efficacy Outcomes and Considerations for Trial Designs

To estimate treatment effect size and other parameters required for planning the designs and analyses of future phase 3 islet transplant trials, we analyzed key clinical and laboratory outcomes of 347 allogeneic islet transplant recipients, using data from the Collaborative Islet Transplant Registry (CITR). At 1 year, approximately 59% of all transplant recipients were free of severe hypoglycemic events and maintained hemoglobin A1c (HbA1c) level of ≤ 6.5%. The Kaplan-Meier (KM) survival analyses showed that 69%, 54% and 44% of these 1-year responders maintained this composite endpoint at 2, 3 and 4 years, respectively. Ninety-one percent of all recipients were free of severe hypoglycemic episodes at 1 year. Furthermore, the KM survival estimates showed that 91%, 85% and 80% of these subjects maintained this clinical benefit at 2, 3 and 4 years, respectively. These results can be very useful in developing framework for study designs, sample size estimates, and statistical analysis plans for future pivotal trials of islet cell transplantation in type 1 diabetes.

Regenerative Medicine Approach as an Alternative Treatment to Islet Transplantation

Islet transplantation is considered to be one of the most promising treatment for type I diabetes mellitus (TID). Development of the Edmonton protocol opened the possibility of insulin independence for the patients with TID. However, there is the problem of the donor shortage. Herein we have discussed recent approaches to overcome the problem. It is neccessary to develop a new cellular source for donor islets and to achieve a high engraftment rate. One advantage in TID therapy is that allogeneic islet transplantation is allowed to avoid autoimmunity. That opens broad candidates for the beta-cell source. To achieve a high engraftment rate, is several attempts have sought to develop an appropriate site for transplantation and to modify beta-cells for long-term survival. It is also important to achieve early onset of blood perfusion after transplantation by prevascularization of the islets in vitro. These multiple approaches will bring a milestone in diabetes therapy.

Rescue of a Pancreatic Islet Graft After Steroid Therapy

Rescue of a Pancreatic Islet Graft After Steroid Therapy

Mesenchymal stem cells differentially mediate regulatory T cells and conventional effector T cells to protect fully allogeneic islet grafts in mice

Limited information is available on the cellular interactions between regulatory T (T(reg)) cells and mesenchymal stem cells (MSCs). In particular, a direct effect of MSCs on the survival and proliferation of T(reg) cells has not been demonstrated. We investigated the effects of MSCs on effector T (T(eff)) cells and T(reg) cells, and the molecular mechanisms involved in the distinct regulation of these two cell populations by MSCs in vivo and in vitro. We show that MSCs are capable of selectively suppressing T(eff) cells and fostering the generation of T(reg) cells. T(eff) cells, but not T(reg) cells, fail to respond to IL-2 and undergo profound apoptosis in the presence of MSCs. The differential regulations of these two T cell subsets by MSCs are associated with their distinct expressions of CD25, with MSCs specifically reducing the expression of CD25 on T(eff) and sparing T(reg) cells intact. In vivo, the administration of MSCs significantly delays the rejection of allogeneic islet grafts in adaptive transferred recipients by favouring the induction of T(reg) cells. In this model, MSCs inhibit the proliferation and development of alloreactive T(eff) but potently enhance the induction of T(reg) cells. We demonstrate that MSCs are capable of regulating T(eff) and T(reg) cells differentially in vitro. MSCs inhibit T(eff) cells by inducing apoptosis and impairing the proliferative response to IL-2 in T(eff) cells, but favour the survival and expansion of T(reg) cells. This result is further demonstrated in mice that have undergone allogeneic islet transplantation, in which MSCs suppress alloreactive T(eff) cells while favouring the induction of T(reg) cells, thus protecting the islet allografts from rejection.

Tolerance-Inducing Strategies in Islet Transplantation

Allogeneic islet transplantation is a promising approach for restoring normoglycemia in type 1 diabetic patients. Current use of immunosuppressive therapies for management of islet transplant recipients can be counterintuitive to islet function and can lead to complications in the long term. The induction of donor-specific tolerance eliminates the dependency on immunosuppression and allows recipients to retain responses to foreign antigens. The mechanisms by which tolerance is achieved involve the deletion of donor-reactive T cells, induction of T-cell anergy, immune deviation, and generation of regulatory T cells. This review will outline the various methods used for inducing donor-specific tolerance in islet transplantation and will highlight the previously unforeseen potential of tissue stromal cells in promoting islet engraftment.

Macro- or microencapsulation of pig islets to cure type 1 diabetes

Although allogeneic islet transplantation can successfully cure type 1 diabetes, it has limited applicability. For example, organs are in short supply; several human pancreas donors are often needed to treat one diabetic recipient; the intrahepatic site may not be the most appropriate site for islet implantation; and immunosuppressive regimens, which are associated with side effects, are often required to prolong survival of the islet graft. An alternative source of insulin-producing cells would therefore be of major interest. Pigs represent a possible alternative source of beta cells. Grafting of pig islets may appear difficult because of the immunologic species barrier, but pig islets have been shown to function in primates for at least 6 mo with clinically incompatible immunosuppression. Therefore, a bioartificial pancreas made of encapsulated pig islets may resolve issues associated with islet allotransplantation. Although several groups have shown that encapsulated pig islets are functional in small-animal models, less is known about the use of bioartificial pancreases in large-animal models. In this review, we summarize current knowledge of encapsulated pig islets, to determine obstacles to implantation in humans and possible solutions to overcome these obstacles.

Autologous islet cell transplantation to prevent surgical diabetes

Autologous islet transplantation (AIT) is performed to prevent surgical diabetes after total or semi-total pancreatectomy for the treatment of chronic pancreatitis with severe abdominal pain. In addition, AIT is used in cases of benign pancreatic tumors and pancreatic trauma. It has been shown that AIT results in better outcomes in terms of glycemic control compared with allogeneic islet transplantation. The reasons for the favorable outcomes of AIT are thought to be: (i) patients have no autoimmune diseases; (ii) the transplanted islets do not suffer allogeneic rejection; (iii) diabetogenic antirejection drugs are not required; (iv) pancreata do not undergo a cytokine storm as a result of periods of brain death; (v) the period of cold preservation of retrieved pancreata is short; (vi) the isolated islets are immediately transplanted without culture; and (vii) pancreata with pancreatitis may contain more progenitor cells. Further research into AIT would help improve the results of allogeneic islet transplantation. Conversely, the technical difficulties associated with islet isolation appear to be the largest hurdle for AIT; therefore, remote center islet isolation may prove to be key in the promotion of this treatment.

Alterations of the Female Reproductive System in Islet Recipient Receiving Immunosuppression

Pancreatic islet allotransplantation is an option for patients with unstable type 1 diabetes mellitus (T1DM). Major improvements in islet isolation techniques and the implementation of steroid-free immunosuppressive regimens can maintain insulin independence in the majority of T1DM for at least 1 year after transplantation. Recent studies have emphasized the impact of sirolimus on female reproductive tract. In this communication we report on the alterations of the female reproductive tract in 18 chronically immunosuppressed patients with T1DM following allogenic islet transplantation. Previous research has shown development of ovarian cysts in islet transplant patients receiving sirolimus. We extensively reevaluated this and other possible side effects on the female reproductive system. These side effects have been underestimated, although they are significant, requiring surgical or intensive medical treatment. Pre- and posttransplant gynecological evaluation should be performed to address the development of complications secondary to sirolimus in order to intervene sooner with alternative therapies.

Usefulness of the Secretory Unit of Islet Transplant Objects (SUITO) Index for Evaluation of Clinical Autologous Islet Transplantation

Background Assessing the engrafted islet mass is important in evaluating the efficacy of islet transplantation. We previously demonstrated that the average secretory unit of islet transplant objects (SUITO) index within 1 month of allogeneic islet transplantation was an excellent predictor of insulin independence. However, the usefulness of the SUITO index for evaluating autologous islet transplantation has not been explored. The purpose of the present study was to assess the relationship between the SUITO index and clinical outcomes after total pancreatectomy followed by autologous islet transplantation. Methods We performed 27 total pancreatectomies followed by autologous islet transplantation from October 2006 to January 2011. Cases were divided into an insulin-independent group (IIG; n = 12) and an insulin-dependent group (lDG; n = 15). The SUITO index was calculated by the formula [fasting C-peptide (ng/mL)/fasting glucose (mg/dL) −63] × 1,500. The average SUITO index within the first month of transplantation except for days 0, 1, and 2, maximum SUITO index, and most recent SUITO index were calculated in each case, and values were compared between the IIG and the IDG. Results The average SUITO index within 1 month was significantly higher in the IIG than in the IDG (24.6 ± 3.4 vs 14.9 ± 2.0, respectively; P < .02). The maximum SUITO indices were 45.7 ± 7.7 in the IIG and 30.1 ± 8.1 in the IDG (not significant), and the recent SUITO indices were 36.9 ± 6.7 in the IIG and 22.8 ± 6.1 in the IDG (not significant). Conclusions The average SUITO index within 1 month was an excellent predictor of insulin independence after total pancreatectomy followed by autologous islet transplantation.

Pig islet for xenotransplantation in human: structural and physiological compatibility for human clinical application

Allogeneic islet transplantation has proven difficult because organ shortages are recurrent, several pancreas donors are often needed to treat one diabetic recipient, and the intrahepatic site of islet implantation may not be the most appropriate site. Thus, another source of insulin-producing cells would be beneficial; and pigs represent a possible and viable source for obtaining such cells. Although the use of pig islet grafts appears to be difficult because of the species barrier, recent reports demonstrated that pig islet xenotransplantation can overcome the immunological barrier following strong immunosuppression and function successfully in primates for at least 6 months. Before becoming clinically applicable, however, pig islet xenotransplantation must still overcome the structural and physiological incompatibility between pig donor and human recipient. Researchers agree that it is necessary to produce more preclinical data in the pig-to-primate model before any pig-to-human transplantation of islets can be considered. Therefore, in this review, we provide a summary of the present state of knowledge about pig and human islet compatibility.

A comparison of islet autotransplantation with allotransplantation and factors elevating acute portal pressure in clinical islet transplantation

Acute portal pressure rise is occasionally observed during intraportal islet infusion, especially in islet autotransplantation (IAT) where tissue purification is rarely applied. In this paper we investigate factors associated with acute portal pressure rise, a known risk factor for portal vein thrombosis. Retrospective data was collected on 15 islet autotransplant and 122 allogeneic islet transplant subjects. Non-purified pancreatic cells were transplanted in islet autotransplants, and purified islet cells were transplanted in allogeneic transplants. Portal pressure was documented throughout the islet infusion. The total numbers of transplanted islets were significantly smaller in autotransplants than allografts, although the packed cell volume in autotransplants was larger. Autoislet infusion, with a larger packed cell volume, caused higher transient portal venous pressures than allogeneic islet transplant. Univariate analysis and multivariate linear regression revealed that packed cell volume and the number of transplanted cells were significant risk factors for acute portal pressure rise in both autotransplants and allogeneic transplants. Non-purified IAT has a higher risk for acute portal pressure rise than allogeneic islet transplantation, and the rise is associated with the packed cell volume and the number of transplanted cells. Minimization of packed cell volume and cautious monitoring of portal pressure are important to avoid potential complications of portal hypertension.

α-1-Antitrypsin Gene Delivery Reduces Inflammation, Increases T-Regulatory Cell Population Size and Prevents Islet Allograft Rejection

Antiinflammatory clinical-grade, plasma-derived human α-1 antitrypsin (hAAT) protects islets from allorejection as well as from autoimmune destruction. hAAT also interferes with disease progression in experimental autoimmune encephalomyelitis (EAE) and in collagen-induced arthritis (CIA) mouse models. hAAT increases IL-1 receptor antagonist expression in human mononuclear cells and T-regulatory (Treg) cell population size in animal models. Clinical-grade hAAT contains plasma impurities, multiple hAAT isoforms and various states of inactive hAAT. We thus wished to establish islet-protective activities and effect on Treg cells of plasmid-derived circulating hAAT in whole animals. Islet function was assessed in mice that received allogeneic islet transplants after mice were given hydrodynamic tail-vein injection with pEF-hAAT, a previously described Epstein-Barr virus (EBV) plasmid construct containing the EBV nuclear antigen 1 (EBNA1) and the family of repeat EBNA1 binding site components (designated "EF") alongside the hAAT gene. Sera collected from hAAT-expressing mice were added to lipopolysaccharide (LPS)-stimulated macrophages to assess macrophage responsiveness. Also, maturation of peritoneal cells from hAAT-expressing mice was evaluated. hAAT-expressing mice accepted islet allografts (n = 11), whereas phosphate-buffered saline-injected animals (n = 11), as well as mice treated with truncated-hAAT-plasmid (n = 6) and untreated animals (n = 20) rapidly rejected islet allografts. In hAAT-expressing animals, local Treg cells were abundant at graft sites, and the IL-1 receptor antagonist was elevated in grafts and circulation. Sera from hAAT-expressing mice, but not control mice, inhibited macrophage responses. Finally, peritoneal cells from hAAT-expressing mice exhibited a semimature phenotype. We conclude that plasmid-derived circulating hAAT protects islet allografts from acute rejection, and human plasma impurities are unrelated to islet protection. Future studies may use this in vivo approach to examine the structure-function characteristics of the protective activities of AAT by manipulation of the hAAT plasmid.

Mesenchymal stem cells as a gene delivery vehicle for successful islet transplantation.

To evaluate the efficacy of human bone marrow-derived mesenchymal stem cells (hBMSCs) as gene delivery vehicles to simultaneously express human hepatocyte growth factor (HGF) and interleukin 1 receptor antagonist (IL-1Ra) to improve the outcome of islet transplantation. Morphology and islet-binding affinity of hBMSCs were checked by microscope. The expression of target genes and endogenous genes was determined by ELISA. Protection of islets by hBMSCs was evaluated in vitro by Calcein-AM/Propidium Iodide staining and in vivo by allogeneic islet transplantation study. Function and revascularization of islets was evaluated by immune fluorescence study. Non-donor-specific hBMSCs showed strong binding affinity to human islets and protected viability and function. Transduction of hBMSCs with adenovirus encoding human HGF and human IL-1Ra (Adv-hHGF-hIL-1Ra) prior to co-culturing with islets further protected from apoptotic cell death, helped maintain 3D structures and morphology, and enhanced insulin secretion. Transplantation of human islets reconstituted with Adv-hHGF-hIL-1Ra transduced hBMSCs under the kidney capsule of streptozotocin-induced diabetic non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice reversed diabetes by reducing blood glucose levels to ≤ 200 mg/dL for up to 15 weeks and reduced the number of islets required to achieving normoglycemia. Blood glucose levels of mice transplanted with islets alone reversed to ≥ 500 mg/dL 4 weeks post-transplantation. Results indentified hBMSCs as effective gene delivery vehicles to improve the outcome of islet transplantation.