Islet Recipients Research Articles

Amyloid and Transplanted Islets

TO THE EDITOR: In their letter to the editor, Westermark and colleagues (Aug. 28 issue)1 report on their identification of amyloid in 43% of intrahepatically transplanted islets on postmortem examination of a recipient with type 1 diabetes. Amyloid is composed of amylin (islet amyloid polypeptide [IAPP]) that is cosecreted from the beta cell with insulin but normally is inhibited from forming amyloid by appropriate proportions of insulin and other factors in the beta cell.2,3 Proportions of insulin and amylin within the beta cell are best estimated in vivo following secretion after acute stimulation.4 To determine whether insulin and amylin are secreted in appropriate proportions after human islet transplantation, we measured plasma concentrations of both hormones before and after arginine stimulation under fasting and hyperglycemic-clamp conditions in four insulin-independent islet recipients and matched controls; the characteristics and C-peptide data of the subjects were reported previously.5 Insulin responses to arginine were relatively more reduced during the hyperglycemic clamps than were amylin responses, resulting in markedly lower ratios between insulin and amylin (Fig. 1). These data indicate that during hyperglycemia, intrahepatically transplanted islets secrete disproportionately more amylin than normal, suggesting that hyperglycemia in the islet recipient may have contributed to the observed amyloid deposition. Figure 1 Plasma Insulin, Amylin, and Molar Ratios between Insulin and Amylin in Four Insulin-Independent Recipients of Islet Transplantation and Four Control Subjects without Diabetes

Effect of Glucagon-Like Peptide-1 on β- and α-Cell Function in Isolated Islet and Whole Pancreas Transplant Recipients

Glucose-dependent insulin secretion is often impaired after islet transplantation where reduced beta-cell secretory capacity indicates a low functional beta-cell mass. We sought to determine whether glucagon-like peptide-1 (GLP-1) enhanced glucose-dependent insulin secretion and glucagon suppression in islet recipients, and whether GLP-1 effects were dependent on functional beta-cell mass by simultaneously studying recipients of whole pancreas transplants. The study was performed in a clinical and translational research center. Five intraportal islet and six portally drained pancreas transplant recipients participated in the study. Subjects underwent glucose-potentiated arginine testing with GLP-1 (1.5 pmol . kg(-1) . min(-1)) or placebo infused on alternate randomized occasions, with 5 g arginine injected under basal and hyperglycemic clamp conditions. Basal glucose was lower with increases in insulin and decreases in glucagon during GLP-1 vs. placebo in both groups. During the hyperglycemic clamp, a significantly greater glucose infusion rate was required with GLP-1 vs. placebo in both groups (P < 0.05), an effect more pronounced in the pancreas vs. islet group (P < 0.01). The increased glucose infusion rate was associated with significant increases in second-phase insulin secretion in both groups (P < 0.05) that also tended to be greater in the pancreas vs. islet group (P = 0.08), whereas glucagon was equivalently suppressed by the hyperglycemic clamp during GLP-1 and placebo infusions in both groups. The GLP-1-induced increase in second-phase insulin correlated with the beta-cell secretory capacity (P < 0.001). The proinsulin secretory ratio (PISR) during glucose-potentiated arginine was significantly greater with GLP-1 vs. placebo infusion in both groups (P < 0.05). GLP-1 induced enhancement of glucose-dependent insulin secretion, but not glucagon suppression, in islet and pancreas transplant recipients, an effect dependent on the functional beta-cell mass that may be associated with depletion of mature beta-cell secretory granules.

C-Jun N-terminal kinase 1 is deleterious to the function and survival of murine pancreatic islets

Inhibition of c-jun N-terminal kinase (JNK) favours pancreatic islet function and survival. Since two JNK isoforms are present in the pancreas (JNK1 and JNK2), we addressed their specific roles in experimental islet transplantation. C57BL/6J (wild-type [WT]), Jnk1 (also known as Mapk8)(-/-) and Jnk2 (also known as Mapk9)(-/-) mice were used as donor/recipients in a syngeneic islet transplantation model. Islet cell composition, function, viability, production of cytokines and of vascular endothelial growth factor (VEGF) were also studied in vitro. Jnk1 ( -/- ) islets secreted more insulin in response to glucose and were more resistant to cytokine-induced cell death compared with WT and Jnk2 (-/-) islets (p < 0.01). Cytokines reduced VEGF production in WT and Jnk2 (-/-) but not Jnk1 ( -/- ) islets; VEGF blockade restored Jnk1 ( -/- ) islet susceptibility to cytokine-induced cell death. Transplantation of Jnk1 ( -/- ) or WT islets into WT recipients made diabetic had similar outcomes. However, Jnk1 ( -/- ) recipients of WT islets had shorter time to diabetes reversal (17 vs 55 days in WT, p = 0.033), while none of the Jnk2 (-/-) recipients had diabetes reversal (0% vs 71% in WT, p = 0.0003). Co-culture of WT islets with macrophages from each strain revealed a discordant cytokine production. We have shown a deleterious effect of JNK2 deficiency on islet graft outcome, most likely related to JNK1 activation, suggesting that specific JNK1 blockade may be superior to general JNK inhibition, particularly when administered to transplant recipients.

Factors influencing the regulation of cytokine balance during islet transplantation in mice

Many experimental islet studies compare the effect of allogeneic transplantations with either syngeneically transplanted or sham-operated animals. Presently we examined multiple “control” treatments to be able to distinguish effects by the operating procedures themselves versus reactions induced by islet graft rejection. Herein, we have studied untreated, sham-operated, syngeneically or allogeneically (C57BL/6) islet transplanted BALB/c mice, and subsequently examined cytokine production (TNFα, IFNγ, IL-4, IL-10, IL-17 and TGF-β) in vitro by RT-PCR and ELISA in spleen cells and transplants. To investigate if the strain of the recipient mice influences cytokine production we also performed allogeneic islet transplantations in the reverse direction. So-called control treatments such as sham operations and syngeneic transplantations had a distinct effect on cytokines in spleen cells, possibly induced by surgery and/or anaesthesia. This seems to decrease the regulatory T cells, thereby leading to increased cytokine expression. Furthermore, spleen cells from surgically manipulated animals seem to have a decreased responsive capacity to con A stimulation in culture. Cytokine generation, FoxP3 mRNA expression and COX-2 mRNA expression in the two investigated mouse strains were sometimes altered in opposite directions by the treatments. In conclusion, the genetic background of both the islet donor and recipient has a major impact on both the magnitude and skewing of a cytokine response. Moreover, factors not directly related to allorejection influences systemic cytokine production in connection to islet transplantation.

Differential Susceptibility of Allogeneic Targets to Indirect CD4 Immunity Generates Split Tolerance

CD4 T cells frequently help to activate CD8 T and B cells that effect transplant rejection. However, CD4 T cells alone can reject transplants, either directly or indirectly. The relative effectiveness of indirect CD4 immunity in rejecting different types of allogeneic grafts is unknown. To address this, we used a TCR transgenic mouse model in which indirect CD4 alloimmunity alone can be studied. We challenged transgenic recipients with hematopoietic cells and shortly thereafter skin transplants that could only be rejected indirectly, and observed Ag-specific indirect donor B cell and skin rejection, but not T cell elimination, reflecting a state of split tolerance. Deficiency of indirect CD4 alloimmunity in donor T cell rejection was also apparent when acute indirect rejection of donor islets occurred despite generation and maintenance of mixed T cell chimerism, due to migration of the few passenger T cells into recipient circulation. Although passenger lymphocytes delayed indirect islet rejection, they enhanced rejection by a full repertoire capable of both direct and indirect reactivity. Interestingly, the persistence of chimerism was associated with the eventual development of tolerance, as demonstrated by acceptance of donor skin grafts given late to hematopoietic cell recipients, and hyporesponsiveness of transgenic T cells from islet recipients in vitro. Mechanistically, tolerance was recessive and associated with progressive down-regulation of CD4. Collectively, our data indicate that indirect CD4 immunity is not equally destructive toward different types of allogeneic grafts, the deficiency of which generates split tolerance. The futility of these responses can convert immunity into tolerance.

Current challenges in islet transplantation

Allogeneic islet transplantation is becoming a treatment option for patients with unstable type 1 diabetes mellitus (T1DM). Around 80% of the islet recipients achieve insulin independence after one or two islet infusions under "Edmonton-like" immunosuppressive protocol, but only 10% will remain insulin independent over the long term. Islet transplantation leads to glucose stabilization, and severe hypoglycemia is prevented even in patients back on insulin injections. Thus, islet transplantation has achieved the proposed targets in patients with unstable T1DM: normalizing blood glucose and hemoglobin A(1c), preventing severe hypoglycemic episodes, and improving quality of life. The current aims of islet transplantation programs are to maintain the success achieved and to overcome remaining obstacles and limitations.

Retraction

We review a novel strategy for tolerance induction developed in rhesus macaques and termed STEALTH. We summarize the evolution of the STEALTH model, the results of successful trials in inducing long-term, stable transplant tolerance in rhesus kidney and diabetic islet recipients and discuss information related to the mechanism by which durable tolerance is induced. STEALTH tolerance is induced by a 3-day treatment course of CD3epsilon immunotoxin (IT) combined with a 14-day treatment with deoxyspergualin (DSG). IT causes profound depletion of sessile lymph node T cells as well as the more accessible circulating T cells. DSG, an inhibitor of HSC 70-mediated NF-kappaB nuclear translocation, arrests maturation of myeloid dendritic cells, blocks production of proinflammatory cytokines induced by IT administration, and promotes systemic production of Th2 type cytokines that persist indefinitely. Such Th2 cytokine deviation has not been reported in NHP transplant recipients. These studies provide proof of principle in a preclinical model that prevention of both acute and chronic allograft rejection, for at least 2.2-4.9 years of follow-up, can be achieved in NHP in the absence of chronic immunosuppressive drugs or other interventions. This strategy for inducing NHP tolerance is discussed in relation to current tolerance paradigms.

Reconstructing the Pancreas: Restoration of Normoglycemia, Exocrine Function, and Islet Innervation by Islet Transplantation to the Pancreas

Impaired function in transplanted islets may be ascribed in part to disturbed reinnervation. The objectives of this study were to determine whether islet transplantation to the pancreas in the presence of nerve growth factor (NGF) would restore islet innervation and endocrine and exocrine pancreatic function. Streptozotocin-diabetic Lewis rats received 800 syngeneic islets beneath the pancreatic capsule in the presence or absence of NGF (20 ng/d for 14 days). Fasting blood glucose was measured for 3 months. The pancreata were isolated and perfused in situ. Pancreatic juice was collected for amylase determination. The sympathetic trunks were isolated and stimulated electrically. The tissues were immunostained for nerve markers. All islet recipients remained euglycemic (4.2 ± 0.6 mmol/L glucose). Ductal amylase concentrations were restored to near normal levels in contrast to diabetic controls (normal rat 98 ± 8 U/L, islet transplant 78.4 ± 9 U/L, diabetic control 14.5 ± 8 U/L). NGF enhanced the innervation of transplanted islets in contrast to control islet transplants. Sympathetic adrenergic innervation was significantly increased by NGF (tyrosine hydroxylase [ P < .001] and neuropeptide Y [ P < .05]). No differences in parasympathetic innervation were observed (vesicular acetylcholine transporter). Electrical stimulation of the sympathetic trunks in the presence of 4 μmol/L phentolamine and 5 μmol/L atropine resulted in increased insulin secretion in NGF-treated islet transplants (164%) compared with control transplants (30%). The combination of growth factors and the pancreatic site may allow the use of fewer islets than conventional islet transplant sites and promote more normal transplanted islet function by the enhancement of islet reinnervation.

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

Activation of c-jun N-terminal kinase (JNK) has been described in islet isolation and engraftment, making JNK a key target in islet transplantation. The objective of this study was to investigate if JNK inhibition with a cell-permeable TAT peptide inhibitor (L-JNKI) protects functional beta cell mass in human islets and affects AKT and its substrates in islet cells. The effect of L-JNKI (10 micromol/l) on islet count, mitochondrial membrane potential, glucose-stimulated insulin release and phosphorylation of both AKT and its substrates, as well as on reversal of diabetes in immunodeficient diabetic Nu/Nu mice was studied. In vitro, L-JNKI reduced the islet loss in culture and protected from cell death caused by acute cytokine exposure. In vivo, treatment of freshly isolated human islets and diabetic Nu/Nu mice recipients of such islets resulted in improved functional beta cell mass. We showed that L-JNKI activates AKT and downregulates glycogen synthase kinase-3 beta (GSK-3B) in human islets exposed to cytokines, while other AKT substrates were unaffected, suggesting that a specific AKT/GSK-3B regulation by L-JNKI may represent one of its mechanisms of cytoprotection. In conclusion, we have demonstrated that targeting JNK in human pancreatic islets results in improved functional beta cell mass and in the regulation of AKT/GSK3B activity.

Acute Insulin Responses to Glucose and Arginine as Predictors of β-Cell Secretory Capacity in Human Islet Transplantation

Islet transplantation for type 1 diabetes can enable the achievement of near-normal glycemic control without severe hypoglycemic episodes. How much an islet (beta-cell) graft may be contributing to glycemic control can be quantified by stimulatory tests of insulin (or C-peptide) secretion. Glucose-potentiation of arginine-induced insulin secretion provides a measure of functional beta-cell mass, the beta-cell secretory capacity, as either AIR(pot) or AIR(max), but requires conduct of a hyperglycemic clamp. We sought to determine whether acute insulin responses to intravenous glucose (AIR(glu)) or arginine (AIR(arg)) could predict beta-cell secretory capacity in islet recipients. AIR(arg) was a better predictor of both AIR(pot) and AIR(max) (n=10, r2=0.98, P<0.0001 and n=7, r2=0.97, P<0.0001) than was AIR(glu) (n=9, r2=0.78, P=0.002 and n=6, r2=0.76, P=0.02). Also, the measures of beta-cell secretory capacity were highly correlated (n=7, r2=0.98, P<0.0001). These results support the use of AIR(arg) as a surrogate indicator of beta-cell secretory capacity in islet transplantation.

Metabolic aspects of pig-to-monkey (Macaca fascicularis) islet transplantation: implications for translation into clinical practice

Attempts to use an alternative source of islets to restore glucose homeostasis in diabetic patients require preclinical islet xenotransplantation models to be tested. These models raise questions about metabolic compatibility between species and the most appropriate metabolic parameters to be used to monitor graft function. The present study investigated and compared relevant gluco-metabolic parameters in pigs, monkeys and the pig-to-monkey islet transplantation model to gain insight into the potential clinical outcome of pig-to-human islet transplantation. Basal and IVGTT-stimulated blood glucose, C-peptide, insulin and glucagon levels were assessed in non-diabetic pigs and monkeys. The same parameters were used to evaluate the performance of porcine islet xenografts in diabetic monkeys. Non-diabetic cynomolgus monkeys showed lower levels of fasting and stimulated blood glucose but higher levels of C-peptide and insulin than non-diabetic pigs. The reported levels in humans lie between those of monkeys and pigs, and differences in metabolic parameters between pigs and humans appear to be smaller than those between pigs and cynomolgus monkeys. The transplantation data indicated that the degree of graft function (evaluated by the measurement of C-peptide levels) necessary to normalise blood glucose in the recipient was determined by the recipient levels rather than by the donor levels. The differences between donor and recipient species may affect the transplantation outcome and need to be considered when assessing graft function in xenotransplantation models. Given the differences between monkeys and humans as potential recipients of pig islets, it should be easier to reach glucose homeostasis in pig-to-human than in pig-to-non-human primate islet xenotransplantation.

Quality of Life After Islet Transplant: Impact of the Number of Islet Infusions and Metabolic Outcome

The health-related quality of life (HRQL; Health Utilities Index Mark 2) and the fear of hypoglycemia (Hypoglycemia Fear Survey) were assessed after islet transplant; the impact of a single islet infusion and of the metabolic outcome were determined. A control group included 166 patients with type 1 diabetes. Islet transplant had no impact on overall HRQL. Prior to transplant, islet recipients had more fear of hypoglycemia than controls (P<0.000001), but this improved up to 36 months after transplant (P<0.00001, pretransplant vs. each time point). With a single islet infusion, this fear improved substantially (P<0.00001), but improved further with subsequent islet infusions (P</=0.01). Fear of hypoglycemia correlated with the occurrence of hypoglycemia (r=0.47, P=0.01), and even more so with blood glucose stability (r=0.56, P=0.0007) and insulin requirement (r=0.69, P=0.000002).

Delayed functional maturation of neonatal porcine islets in recipients under strict glycemic control

The aim of this study was to compare the functional maturation of neonatal porcine islet (NPI) grafts exposed to long-term hyperglycemia with those implanted under euglycemic conditions. mice Neonatal porcine islets were transplanted under the left renal capsule of diabetic SCID mice (group H), or in diabetic SCID mice who were also implanted with 500 BALB/c islets under the right renal capsule (group N). On day 42, the right kidneys were removed in both groups. No animals in group H achieved euglycemia within 3 weeks after transplantation. Thus, these mice were exposed to long-term hyperglycemia. Mice in group N became euglycemic immediately after transplantation, however after removal of BALB/c grafts on day 42 they exhibited significantly higher blood glucose levels than in group H and showed glucose intolerance after glucose administration. Cellular insulin content of NPI grafts harvested on day 58 or 72 was significantly lower in group N mice compared to group H. These results suggest that tight control of glycemia reduces the functional maturation of NPI grafts.

Encapsulation of Porcine Islets Permits Extended Culture Time and Insulin Independence in Spontaneously Diabetic BB Rats

The ability to culture porcine islets for extended times allows for both their functional assessment and the assurance of their microbiological safety prior to transplantation. We have previously shown that agarose-encapsulated porcine islets can be cultured for at least 24 weeks. In the current study, porcine islet agarose macrobeads cultured for up to 67 weeks were assessed for their ability to restore normoglycemia, respond to an intraperitoneal glucose challenge, maintain spontaneously diabetic BB rats free of insulin therapy for more than 6 months, and for their biocompatibility. Porcine islets were encapsulated in agarose macrobeads and subjected to weekly static perifusion assays for the assessment of insulin production. After in vitro culture for either 9, 40, or 67 weeks, 56-60 macrobeads were transplanted to each spontaneously diabetic BB rat. Transplanted rats were monitored daily for blood glucose levels. Glucose tolerance tests and assessments for porcine C-peptide were conducted at various intervals throughout the study. Normoglycemia (100-200 mg/dl) was initially restored in all islet transplanted rats. Moderate hyperglycemia (200-400 mg/dl) developed at around 30 days posttransplantation and continued throughout the study period of 201-202 days. Importantly, all rats that received encapsulated porcine islets continued to gain weight and were free of exogenous insulin therapy for the entire study. Porcine C-peptide (0.2-0.9 ng/ml) was detected in the serum of islet recipients throughout the study period. No differences were detected between recipient animals receiving islet macrobeads of various ages. These results demonstrate that the encapsulation of porcine islets in agarose macrobeads allows for extended culture periods and is an appropriate strategy for functional and microbiological assessment prior to clinical use.

Healthy Twin Birth After Autologous Islet Transplantation in a Pancreatectomized Patient Due to a Benign Tumor

Objective Autologous islet transplantation has been reported to show favorable outcomes on glucose metabolism. The objective of this study was to describe successful delivery of twins in an islet recipient who had undergone distal pancreatectomy. Patient A 35-year-old woman who underwent distal pancreatectomy owing to a solid pseudopapillary neoplasm received an autologous islet transplantation (140,000 islet equivalents). After 2.5 years, she unexpectedly became pregnant. Cesarean section was performed at 35 weeks delivering male twins without complications. Plasma glucose and insulin levels, insulinogenic index, and hemoglobin A1c were measured from the preoperative to the postpartum state as the main outcome. Results The patient showed impaired glucose tolerance before pancreatectomy, but improved to a normal glucose tolerance after transplantation, maintaining euglycemia until pregnancy. Because her fasting glucose levels were within the normal range during pregnancy, fasting insulin represented insulin resistance. Her fasting insulin levels abruptly increased in the third trimester of pregnancy, but returned after delivery. Insulinogenic index increased over 1 year after transplantation, but gradually decreased thereafter. During pregnancy, it increased again, but could not compensate for the insulin resistance. Therefore, gestational diabetes mellitus developed: glucose homeostasis recovered to normal after delivery. Conclusions The current report suggested a successful pregnancy after autologous islet transplantation that did not itself permanently deteriorate graft function.

Inhalation of glutamic acid decarboxylase 65-derived peptides can protect against recurrent autoimmune but not alloimmune responses in the non-obese diabetic mouse

Systemic administration of islet-derived antigens has been shown to protect against diabetes in the non-obese diabetic (NOD) mouse by the induction of antigen-specific regulatory T cells. Bystander regulation to related and unrelated islet-derived antigens (intramolecular and intermolecular recognition) in this context is recognized. We tested if intranasal administration of glutamic acid decarboxylase 65 (GAD 65)-derived peptides could protect against both autoimmune and, through bystander regulation, alloimmune responses in a NOD mouse model. Spontaneously diabetic female NOD mice underwent islet transplantation from either C57Bl/6 or NOD islet donors. Islet recipients were treated with intranasal GAD 65-derived peptides or control (ovalbumin) peptide pre- and post-transplantation. In-vitro analysis of the effect of inhalation was defined using lymph node proliferation assays and supernatant analysis for cytokines. GAD 65-derived peptide inhalation resulted in significant protection against recurrent autoimmune disease, with the generation of an interleukin (IL)-10-producing immune phenotype in a syngeneic islet transplant model. This phenotype, however, was not robust enough to protect against alloimmune responses. Inhalation of GAD-derived peptides induces an immunoregulatory response that protects against recurrent autoimmune, but not alloimmune responses in the NOD mouse.

Immature Syngeneic Dendritic Cells Potentiate Tolerance to Pancreatic Islet Allografts Depleted of Donor Dendritic Cells in Microgravity Culture Condition

Previously we showed that pancreatic islets cultured for seven days in rotating bioreactors survived for >100 days in allogeneic recipients without immunosuppression. This survival coincided with almost complete elimination of "passenger" donor dendritic cells (DCs). Herein, we examined the necessity of DCs in the generation of CD4+ CD25+ T regulatory (Treg) cells. Allogeneic fresh islets or islets cultured for three days in bioreactors were transplanted to streptozotocin-induced diabetic Balb/c(stat4 -/-) as well as signal transducers and activators of transcription (Stat)4-deficient Balb/c(stat6 -/-) or Balb/c(stat4 -/-) mice. Some Balb/c recipients of fresh islet allografts were also treated with a tolerogenic protocol of anti-CD40 Ligand MR1 mAb and CTLA4Ig. Islet allografts cultured for three days in bioreactors survived >100 days in all Balb/c(stat4 -/-) recipients and in 56% of Balb/c(stat6 -/-) recipients, but in none of the Balb/c recipients; the same recipients rejected fresh islet allografts. Purified T cells from long-term surviving Balb/c(stat4 -/-) recipients failed to transfer tolerance to SCID recipients of donor-type fresh islet allografts. In contrast, MR1/CTLA4Ig therapy induced tolerance to fresh islet allografts and their T cells adoptively transferred tolerance. When Balb/c or Balb/c(stat4 -/-) recipients of bioreactor-cultured islets were injected intravenously with immature syngeneic DCs, they became tolerant and developed potent alloantigen-specific CD4+ CD25+ Treg cells expressing Foxp3. Allogeneic islets depleted of donor DCs by culture in bioreactors have almost twofold better acceptance in Balb/c(stat4 -/-) than in Balb/c(stat6 -/-) mice, but lack Treg cells. Additional injection of host immature DCs improves tolerance in Balb/c and Balb/c(stat4 -/-) recipients by inducing potent CD4+ CD25+ Treg cells.

Measurement of anti‐CD154 monoclonal antibody in primate sera by competitive inhibition ELISA

Anti-human CD154 monoclonal antibody (mAb)-based regimens have been demonstrated to prevent T cell-dependent elicited antibody response in baboon recipients of pig hematopoietic progenitor cells, organs and islets. Monitoring of anti-CD154 mAb in serum is important to ensure maintenance of adequate levels and for adjusting dosage of the anti-CD154 mAb. We describe a method for measuring the level in primate sera. The anti-CD154 mAb level in primate serum was measured with a competitive inhibition enzyme linked immunosorbent assay in which the extent of inhibition of binding by anti-CD154 mAb conjugated to horseradish peroxidase (anti-CD154-HRP) to soluble CD154 was used to determine the serum level. Briefly, a 96-well maxisorb plate coated with soluble human CD154, and blocked with bovine serum albumin, was loaded with graded doses of anti-CD154 mAb or primate sera containing anti-CD154 mAb. Both were mixed with a known dosage of anti-CD154-HRP before loading. Bound anti-CD154-HRP was detected by color developed using 3,3',5,5' tetramethyl-benzidine as substrate. Absorbance was measured in a Synergy HT Multi-Detection Microplate Reader at a wavelength of 450 nm. Data analysis was carried out using BioTek's KC4 Data Analysis Software. The standard curve was generated from the wells loaded with the mixture of anti-CD154 mAb and anti-CD154-HRP. The assay has been used successfully to measure anti-CD154 mAb levels in the serum of both baboons and monkeys.

Remission of Digestive Insufficiency by Islet Transplantation to the Pancreas

Pancreatic digestive insufficiency is a common problem in both Type 1 and Type 2 diabetes and remains a serious consequence of diabetes in developing countries. The problem is not corrected by supportive therapies including exogenous insulin injections. It is our hypothesis that digestive insufficiency may be corrected or diminished by the transplantation of islets to the pancreas, thereby supplying islet hormones directly to acinar tissue analogous to the normal pancreas. Diabetic rats received 1000 syngeneic islets and dogs received 7600 autologous islets per kilogram as a transplant to the pancreas. Blood glucose and amylase concentrations were normalized in islet recipients in contrast with controls receiving no islets or islets transplanted to the renal capsule. These results suggest that diabetic digestive insufficiency may be corrected by intrapancreatic islet transplantation.

Beneficial Effects of Simultaneous Treatment With 15-deoxyspergualin and Monoclonal Antibodies to CD45RB and CD154 on Murine Islet Transplantation Recipients

Treatment of transplant recipients with either 15-deoxyspergualin (DSG) or monoclonal antibodies (mAbs) to T-cell proteins CD45RB and CD154 (a two-signal blockade) has been shown to prolong islet graft survival. Therefore, we investigated the combined effect of DSG, anti-CD45RB, and anti-CD154 in murine islet model. Chemically induced diabetic C57BL/6 mice underwent allografting with islets from BALB/c mice or xenografting with rat islets. After transplantation, they were treated with either DSG, the two-signal blockade, or both (the triple treatment). The tolerogenic effects of the posttransplant treatments were measured with an intraperitoneal glucose tolerance test (IPGTT), immunohistology, enzyme-linked immunosorbent assays, and flow cytometry. Blood glucose profiles measured after glucose challenges were improved in all islet recipients. Enhancement of xenograft survival in triple-treated groups was not statistically significant (P = 0.08), compared to graft survival in group received only the two-signal blockade. However, 15 days after transplantation, xenografts in the triple-treated group showed a significant decrease in the proportion of CD4, CD8, and CD4CD45RB T-cells, and in the expression of interleukin-10 and interferon-gamma, relative to grafts in the other treatment groups. In addition, reduced infiltration of the xenografts by CD3 T-cells was observed in groups that had received either the two-signal blockade or the triple treatment. With long-term (>248 days) xenografts, only those in the triple-treated group were free of inflammatory infiltrates. These grafts also exhibited larger islet clusters and contained more insulin- and glucagon-positive cells, relative to grafts in the other treatment groups. Triple treatment has a beneficial effect in murine islet xenotransplantation.