[Show abstract][Hide abstract] ABSTRACT: Non-human primates (NHPs) are important preclinical models for pancreatic islet transplantation (PIT) because of their close phylogenetic and immunological relationship with humans. However, low availability of NHP tissue, long learning curves and prohibitive expenses constrain the consistency of isolated NHP islets for PIT studies. To advance preclinical studies, we attempted to identify key variables that consistently influence the quantity and quality of NHP islets.
Seventy-two consecutive pancreatic islet isolations from rhesus macaques were reviewed retrospectively. A scaled down, semi-automated islet isolation method was used, and monkeys with streptozotocin-induced diabetes, weighing 3-7 kg, served as recipients for allotransplantation. We analysed the effects of 22 independent variables grouped as donor factors, surgical factors and isolation technique factors. Islet yields, success of isolation and transplantation results were used as quantitative and qualitative outcomes.
In the multivariate analysis, variables that significantly affected islet yield were the type of monkey, pancreas preservation, enzyme lot and volume of enzyme delivered. The variables associated with successful isolation were the enzyme lot and volume delivered. The transplant result was correlated with pancreas preservation, enzyme lot, endotoxin levels and COBE collection method.
Islet quantity and quality are highly variable between isolations. The data reviewed suggest that future NHP isolations should use bilayer preservation, infuse more than 80 ml of Liberase into the pancreas, collect non-fractioned tissue from the COBE, and strictly monitor for infection.
[Show abstract][Hide abstract] ABSTRACT: Natural killer T cells (NKT) possess dual functions of innate and adaptive immune systems, controlling viral infections and regulating autoimmune diseases. Non-human primates (NHP) are penultimate models for advancing therapeutic immunoregulatory strategies for translational application in humans, though, little is known about NHP NKT cells. Here we characterized rhesus macaque NKT cells ex vivo.
The frequency, phenotype and intracellular cytokine production of V alpha 24+ 6B11+ invariant NKT (iNKT) cells were analyzed by multi-color flow cytometry. V alpha 24J alpha Q mRNA expression was analyzed by real-time RT-PCR.
The frequencies of peripheral blood (PB) and spleen V alpha 24+ 6B11+ iNKT cells were not significantly different. The iNKT cell subset in spleen was significantly increased for CD4+ CD8+ and CD3+ CD56+ co-expression as well as intracellular interleukin-4 production, which was rarely observed in circulating PB.
Spleen iNKT cells in rhesus macaques are Th2 biased and display phenotypically and functionally distinct profiles from their PB counterpart.
Journal of Medical Primatology 03/2008; 37(1):1-11. · 1.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanisms mediating T-cell depletion plus 15-deoxyspergualin (DSG)-induced prolonged allograft survival or tolerance are uncertain. The purpose of this study is to evaluate the role of IL-4 and IL-10 in prolonged allograft survival induced by T-cell depletion plus DSG. MHC mismatched skin allograft transplantation was performed, using wild-type and three separate knockout (i.e., IL-4-/-, Stat6-/-, or IL-1-/ -) mice as recipients. Induction therapy consisted of T-cell depletion and or brief course of DSG. The data demonstrate that monotherapy with T-cell-depleting mAbs or DSG prolonged skin allograft survival, compared to controls, in wild-type Balb/c recipients [median survival time (MST) = 25 and 21 vs. 10 days, p < 0.007]. T-cell depletion plus DSG further augmented skin allograft survival in wild-type animals relative to monotherapy (MST = 35 days vs. 25 and 21 days, p < 0.006 vs. mAbs or DSG only), and was equally effective in IL-4-/- and Stat6-/- recipients. In contrast, combined therapy was no better than monotherapy in IL-10-/- animals (p > 0.05). Furthermore, skin allograft survival after combined therapy was shorter in IL-10-/- versus wild-type recipients (MST 20 and 41 days, respectively, p < 0.001). IL-4-mediated signaling through Stat6 is dispensable for prolonged allograft survival induced by T-cell depletion plus DSG. In contrast, IL-10 appears to be important for prolonged allograft survival induced by combined therapy in this model.
[Show abstract][Hide abstract] ABSTRACT: The powerful anti-inflammatory and immunosuppressive activities of IL-10 make it attractive for supplemental therapy in translational tolerance induction protocols. This is bolstered by reports of IL-10-mediated inhibition of innate immunity, association of human stem cell and nonhuman primate (NHP) islet allograft tolerance with elevated serum IL-10, and evidence that systemic IL-10 therapy enhanced pig islets survival in mice. IL-10 has not been examined as adjunctive immunosuppression in NHP. To enable such studies, we cloned and expressed rhesus macaque (RM) IL-10 fused to a mutated hinge region of human IgG1 Fc to generate IL-10/Fc(ala-ala). RM IL-10/Fc(ala-ala) was purified to approximately 98% homogeneity by affinity chromatography and shown to be endotoxin-free (<0.008 EU/microg protein). The biological activity of IL-10/Fc(ala-ala) was demonstrated by (1) costimulation of the mouse mast cell line, MC/9 proliferation in a dose-dependent fashion, (2) suppression of LPS-induced septic shock in mice and (3) abrogation of LPS-induced secretion of proinflammatory cytokines/chemokines in vitro and in vivo in NHP. Notably, RM IL-10/Fc(ala-ala) had significantly greater potency than human IL-10/Fc(ala-ala) and exhibited a circulating half-life of approximately 14 days. The availability of this reagent will facilitate definitive studies to determine whether supplemental therapy with RM IL-10/Fc(ala-ala) can influence tolerance outcomes in NHP.
[Show abstract][Hide abstract] ABSTRACT: Fibrin glue has proven to be a good delivery system for cell transplantation but the factors that influence the fibrin-cell relationships are not well understood. The purpose of this study was to assess the effect of different concentrations of fibrin glue components (thrombin and fibrinogen) on the function of pancreatic islets.
Islets were isolated from rat pancreata and combined with 6 different fibrin glue formulations. Each islet sample was incubated sequentially with RPMI containing low and high glucose, and culture supernatants were harvested for insulin determination using enzyme-linked immunosorbent assay (ELISA).
The control group (no fibrin glue) and group 3 (with thrombin 50 U/mL and fibrinogen 10 mg/mL) had the highest insulin secretion in response to glucose stimulation. These were followed by groups 5 and 4 with 2.6 and 1.8 stimulation indexes, respectively. Group 2 (with thrombin 50 U/mL and fibrinogen 5 mg/mL) and group 6 (commercial kit with thrombin 250 U/mL and fibrinogen 75-115 mg/mL) had the lowest insulin response after glucose stimulation.
This study demonstrates that different fibrin glue formulations significantly impact pancreatic islets function. In the future, when using fibrin glue as a carrier for pancreatic islet transplantation, lower concentrations of fibrinogen and thrombin are recommended to obtain more viable and functional grafts.
[Show abstract][Hide abstract] ABSTRACT: CD4(+)CD25(+) regulatory T cells (Tregs) play an important role in allograft and self-tolerance and thus have potential therapeutic application in transplantation, autoimmunity, and allergy. Although nonhuman primate (NHP) provide the most accepted preclinical models for translational studies in allograft tolerance and infectious diseases, CD4(+)CD25(+) Tregs have been rarely studied in NHP. The low frequencies of Tregs in peripheral blood will likely necessitate ex vivo expansion to enable Tregs adaptive immune therapy in NHP and humans. Tregs were isolated by magnetic and flow sorting and then stimulated weekly with antirhesus CD3 clone FN18 and antihuman CD28-coated Dynal beads plus 100 U/ml rhIL-2. Under these conditions, the Tregs were expanded 300- to 2000-fold in 4 weeks. Expanded CD4(+)CD25(+) Tregs expressed high to moderate levels of FOXP3 as well as CD95, CD62L, CD69, and CCR7 surface antigens. Expanded rhesus Tregs were anergic and suppressed the proliferation of autologous peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion, and the suppression was partially reversed by anti-transforming growth factor (TGF)-beta1 neutralizing antibody (Ab). These results demonstrate that rhesus macaque suppressive regulatory CD4(+)CD25(+)FOXP3(+) Tregs can be efficiently expanded in vitro under rhesus-specific stimulation, which enables preclinical testing of Treg therapy in the NHP model.
Human Immunology 07/2007; 68(6):478-90. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pancreatic islet grafts are difficult to manipulate and implant in the recipient site mainly because they are formed by a group of cells suspended in a solution. This physical property determines various characteristics that are unique for pancreatic islet transplantation. The purpose of this study was to evaluate the role of fibrin glue as a delivery method for islet transplantation.
C3H mouse islets were syngeneically transplanted into streptozotocin-diabetic recipients using fibrin glue in a subcutaneous pocket (Group 1) and using liquid islets injected under the kidney capsule (Group 2). Blood glucose levels were measured during 4 weeks of follow-up and compared against normal (Group 3) and diabetic levels (Group 4).
No statistical differences were observed between the normal, kidney capsule, and fibrin glue groups. Only the diabetic group had a statistical difference when compared with the normal control group (P < .01). At the beginning, levels in Group 1 (fibrin glue) were higher than in Group 2 (kidney capsule), but turned into similar values after time and no statistical differences were observed between them during follow-up.
Islet/fibrin glue grafts placed in a subcutaneous pocket obtained the same results as liquid grafts placed under the kidney capsule, proving to be an adequate delivery method for islet transplantation and solving some of the engraftment problems we find with liquid grafts.
[Show abstract][Hide abstract] ABSTRACT: Enzymatic digestion of the pancreas is a fundamental step in islet isolation and there are many ways to administer the enzyme during procurement. The aim of this study was to evaluate the influence of different methods of Liberase delivery during pancreas harvest on the quality and quantity of islets.
Depending on the type of Liberase delivery, 4 groups were created. Group 1 was intraductal, Group 2 was interstitial, Group 3 was intragallbladder, and Group 4 was no infusion of enzyme. After injection, the pancreata were harvested, digested in Liberase solution, mechanically disrupted, and purified using discontinuous gradient centrifugation. After 24-hour culture, the number, purity, and viability of the isolated islets were determined.
Intraductal injection of the enzyme yielded statistically significantly more islets per mouse when compared with interstitial, intragallbladder, and no injection administration. Although there was a trend toward better islet purity and viability for Group 1, this was not statistically significant.
Intraductal administration is the best enzyme delivery method for pancreatic islet isolation. The pancreatic ducts are the most anatomic and physiological way to transport the enzyme uniformly inside the pancreas, determining an adequate digestion and better islet quantity and quality when compared with other delivery methods.
[Show abstract][Hide abstract] ABSTRACT: Regulatory T cells (Tregs) are implicated in immune tolerance and are variably dependent on IL-10 for in vivo function. Brief peritransplant treatment of multiple nonhuman primates (NHP) with anti-CD3 immunotoxin and deoxyspergualin has induced stable (5-10 years) rejection-free tolerance to MHC-mismatched allografts, which associated with sustained elevations in serum IL-10. In this study, we demonstrate that resting and activated PBMC from long-term tolerant NHP recipients are biased to secrete high levels of IL-10, compared with normal NHP PBMC. Although IL-10-producing CD4+ Tregs (type 1 regulatory cells (TR1)/IL-10 Tregs) were undetectable (<0.5%) in normal rhesus monkeys, 7.5 +/- 1.7% of circulating CD4+ T cells of tolerant rhesus recipients expressed IL-10. In addition to this >15-fold increase in Tr1/IL-10 Tregs, the tolerant monkeys exhibited a nearly 3-fold increase in CD4+CD25+ Tregs, 8.1 +/- 3.0% of CD4 T cells vs 2.8 +/- 1.4% in normal cohorts (p < 0.02). The frequency of CD4+CD25+IL-10+ cells was elevated 5-fold in tolerant vs normal NHP (1.8 +/- 0.9% vs 0.4 +/- 0.2%). Rhesus CD4+CD25+ Tregs exhibited a memory phenotype, and expressed high levels of Foxp3 and CTLA-4 compared with CD4+CD25- T cells. Also, NHP CD4+CD25+ Tregs proliferated poorly after activation and suppressed proliferation of CD4+CD25- effector T cells, exhibiting regulatory properties similar to rodent and human CD4+CD25+ Tregs. Of note, depletion of CD4+CD25+ Tregs restored indirect pathway antidonor responses in tolerant NHP. Our study demonstrates an expanded presence of Treg populations in tolerant NHP recipients, suggesting that these adaptations may be involved in maintenance of stable tolerance.
The Journal of Immunology 01/2006; 175(12):8060-8. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peritransplant treatment with anti-CD3 immunotoxin plus deoxyspergualin induces tolerance to kidney allografts in most rhesus macaque recipients. Tolerant recipients maintain normal function for years without evidence of chronic rejection. Indirect alloantigen presentation is implicated in chronic rejection. Accordingly, we determined if anti-CD3 immunotoxin plus deoxyspergualin induced rejection-free tolerance associates with suppression of anti-donor indirect pathway responses. Tolerant recipients exhibited an early decrease in direct anti-donor responses with recovery to baseline levels by 3 years posttransplantation. In contrast, tolerant monkeys were unresponsive to donor antigens presented by the indirect pathway. Recipients that rejected their allografts retained vigorous direct and indirect anti-donor responses. Therefore, following temporary donor-specific hyporesponsiveness, direct responses recover in tolerant recipients >1.5 years after transplantation. However, tolerant recipients tested at 1.9-4 years posttransplant are specifically unresponsive to donor antigens presented by the indirect pathway. Thus, the rejection-free state of tolerant recipients may depend on mechanisms regulating indirect pathway responsiveness.
[Show abstract][Hide abstract] ABSTRACT: CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed on various cell types in addition to hematopoietic cells. Recent studies show that CD40 expression is related to several carcinomas, although its role in cancer pathobiology is unknown. In this study, we demonstrate the expression of CD40 on several ovarian carcinoma cell lines and the ability of CD40 to mediate targeted adenoviral infection in vitro.
CD40 expression on ovarian cancer cell lines and clinical patient samples was examined by reverse transcription-PCR and flow cytometry. To study the utilization of CD40 for gene delivery, we precomplexed a luciferase coding adenovirus (Ad), Ad5luc1, with a CD40-targeting molecule (CAR/G28).
According to our studies, all of the examined ovarian cancer cell lines are expressing CD40. In addition, mRNA for CD40 was detected in every primary tumor sample, suggesting that CD40 is also expressed in vivo. Compared with nontargeted Ad, gene transfer was increased up to 40-fold in CD40+ cells when CD40-targeted Ad was used. Supporting the relation of targeted system to CD40, increasing the amount of targeting fusion protein results in dose response. Furthermore, blockade of CD40 receptors on cell surface decreases the infectability of CD40+ cells with CD40-targeted virus, indicating the specificity of the targeting system for CD40.
These results suggest that CD40 is present in ovarian cancer cells and can be used for targeted gene delivery in a CAR-independent manner, circumventing the problem of the low expression levels of CAR in various cancer cells.
Clinical Cancer Research 03/2003; 9(2):619-24. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brief treatment of rhesus macaques with immunotoxin plus 15-deoxyspergualin has yielded exceptional numbers (54%) of stable tolerant kidney allograft recipients, surviving over 6 years without rejection or immunosuppression. An early increase in IL-10 and reduction in IFNgamma distinguished recipients that subsequently became tolerant. Furthermore, analysis suggested that this immune switch was programmed within hours of transplantation. Administering deoxyspergualin within 5 h of surgery gave a higher incidence of tolerance (76%) compared to administration >5 h before or after surgery (11%, P<0.01). Deoxyspergualin inhibits nuclear translocation of activated NF-kappaB through heat shock proteins. Lymph node biopsies from tolerant recipients showed significant reductions in cytoplasmic expression of Hsp70 and RelB and almost complete inhibition of nuclear translocation of both. The early timing effect of deoxyspergualin suggests a crucial limitation to induction of stable tolerance is activation of Hsp-dependent innate responses to damage by ischemia-reperfusion. This was supported by studies in murine kidney reperfusion injury, where deoxyspergualin given 5 h before reperfusion protected renal function and reduced levels of IL-6 and IL-12. The narrow timing window for initiating deoxyspergualin treatment suggests the innate immune system is poised to defeat allograft tolerance induction, so effective blockade of NF-kappaB-mediated innate immunity must be in place early, to enable development of a tolerogenic environment.
[Show abstract][Hide abstract] ABSTRACT: The tolerogenic activity of allogeneic bone marrow cells (BMCs) associates with functional inactivation of alloreactive T cells and has been attributed to a veto effect. Studies in mice and rhesus monkeys indicated that the CD8alpha molecule expressed on a subpopulation of allogeneic BMCs is necessary to induce signal transduction within the BMCs to increase veto effector molecules such as transforming growth factor (TGF)-beta1. In vitro activation of alloreactive cytotoxic T-lymphocyte precursor enhances their susceptibility to veto-mediated functional inactivation by specific alloantigen-bearing BMCs. Accordingly, we examined a hypothesis that mature rhesus monkey (Rh) monocyte-derived dendritic cells (MDDCs) modified by gene transfer to over-express active TGF-beta1 might mediate veto activity without the need to express CD8alpha.
Rh MDDCs were modified by recombinant adenovirus (Ad) transduction and characterized by phenotype and functional studies.
Rh MDDC transduction with Ad vectors using conventional methods was remarkably inefficient. However, a single-chain anti-CD40/soluble Coxsackie and adenovirus receptor-fusion protein (G28/sCAR) permitted high-efficiency transduction of Rh MDDCs by retargeting Ad to Rh MDDC CD40. Mature Rh MDDCs that were transduced to overexpress active TGF-beta1 (AdTGF-beta1 Rh MDDC) significantly suppressed alloimmune responses in [ H]thymidine uptake mixed leukocyte reaction assays. We showed by the carboxyfluorescein succinimidyl ester dilution method that allogeneic mature AdTGF-beta1 Rh MDDCs inhibited proliferation of CD4 and CD8 responder T cells. Notably, AdTGF-beta1 Rh MDDC abrogated alloimmune responses induced by control AdGFP Rh MDDC in an antigen-specific manner.
These results suggest that nonhuman primate mature MDDCs can be genetically engineered to function as alloantigen-specific cellular immunosuppressants, an approach that has potential to facilitate induction of allograft tolerance in vivo.