Prevention of nonimmunologic loss of transplanted islets in monkeys.

The nonimmunologic loss of islets in the pre-, peri-, and early post-islet transplant periods is profound. To determine the potential role that transplantation of only a marginal mass of functioning beta cells may play in triggering late nonimmunologic graft loss, we studied the effect of treatment with alpha-1-antitrypsin (AAT) in the autologous cynomolgus islet transplant model. A marginal mass of autologous islets, that is islets prepared from 70% to 80% of the pancreas, was transplanted at 1600-4100 IEQ/kg into subtotal pancreatectomized, streptozotocin-treated and insulin-deficient diabetic hosts. In this marginal mass islet transplant model, islet function is insidiously lost over time and diabetes recurs in all untreated monkeys by 180 days posttransplantation. Short-term treatment with AAT, an acute phase reactant, in the peri-transplant period serves to terminate inflammation through effects upon expression of TGFß, NFκB and AKT and favorably altering expression of cell death and survival pathways, as detected by a system biology approach and histology. These effects enabled functional expansion of the islet mass in transplanted hosts such that graft function improves rather than deteriorating over time.

Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term triple therapy.

Preclinical studies in nonhuman primates (NHP) are particularly useful to evaluate the safety and efficacy of new therapeutic proteins developed for use in clinical transplantation. We hypothesized that a treatment that selectively destroys activated cytopathic donor reactive T cells while sparing resting and immunoregulatory T cells in a mouse model might also produce long-term drug-free engraftment and tolerance without the hazards of lymphopenia in the challenging nonhuman primate islet allograft model. Short-term treatment with a regimen consisting of rapamycin, and IL-2.Ig plus mutant antagonist-type IL-15.Ig cytolytic fusion proteins (triple therapy) posttransplantation results in prolonged, drug-free engraftment of cynomolgus islet allografts. Moreover slow progressive loss of islet function in some recipients was not associated with obvious pathologic evidence of rejection.

Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term anti-CD154-based therapy: nonimmunologic graft failure?

Conventional drug therapy and several anti-CD154 mAb-based regimens were tested in the nonhuman primate (NHP) islet allograft model and found to be inadequate because islets were lost to rejection. Short-term therapy with an optimized donor-specific transfusion (DST) + rapamycin (RPM) + anti-CD154 mAb regimen enables immunosuppression drug-free islet allograft function for months following cessation of therapy in the NHP islet allograft model. After a substantial period of drug-free graft function, these allografts slowly and progressively lost function. Pathologic studies failed to identify islet allograft rejection as a destructive islet invasive lymphocytic infiltration of the allograft was not detected. To evaluate the mechanism, immunologic versus nonimmunologic, of the late islet allograft loss in hosts receiving the optimized therapeutic regimen, we performed experiments with islet autografts and studied islet function in NHPs with partial pancreatectomy. The results in both experiments utilizing autologous islet allografts and partially pancreatectomized hosts reinforce the view that the presence of a marginal islet mass leads to slowly progressive nonimmunological islet loss. Long-term clinically successful islet cell transplantation cannot be realized in the absence of parallel improvements in tolerizing regimens and in the preparation of adequate numbers of islets.

Effects of streptozotocin on autoimmune diabetes in NOD mice.

Non-obese diabetic (NOD) mice develop autoimmunity that destroys their native beta cells causing diabetes. Their autoimmunity will also destroy syngeneic transplanted islets and transfer both autoimmunity and diabetes via spleen cells to non-diabetic mice. In this report, we studied the effects of streptozotocin (STZ) on the autoimmune diabetes in NOD mice. We transplanted NOD.SCID islets into three groups of NOD mice: (1) spontaneously diabetic NOD mice (NOD-sp.); (2) prediabetic NOD mice made diabetic by streptozotocin (NOD-stz); and (3) diabetic NOD mice also treated with streptozotocin (NOD-sp./stz). In the first group, the transplants were rejected within 3 weeks. In the second and third groups, the transplants survived indefinitely. Alloxan, a drug similar to streptozotocin, did not have the same effect as streptozotocin. The ability of streptozotocin to prevent diabetes in young NOD mice was reversed by anti-CD8 antibody treatment but not by anti-CD4 treatment. Streptozotocin also made spleen cells from diabetic NOD mice less effective transferring diabetes. These results indicate that streptozotocin treatment both prevents and reverses the islet destructive autoimmunity in NOD mice. We postulate that the effects of streptozotocin treatment may be mediated in part by regulatory T cells.

The effect of low versus high dose of streptozotocin in cynomolgus monkeys (Macaca fascilularis).

Streptozotocin (STZ) is often used to induce diabetes in animal models. However, morbidity associated with STZ and its ability to induce diabetes vary with different dosages among different animal species, including nonhuman primates. To find an optimal dose of STZ that would cause diabetes with minimal toxicity, we compared low and high doses of STZ. Male cynomolgus monkeys (3-6 years old) were given a single dose of 100 mg/kg (high dose, 4 animals) or 55 mg/kg (low dose, 20 animals) of STZ. Blood glucose levels, intravenous glucose tolerance test (IVGTT), pancreatic biopsies, liver function tests (LFTs), liver biopsies, kidney function tests, and kidney biopsies were performed periodically. Animals from both groups developed diabetes within 24 h after administration of STZ. Serum C-peptide levels in both groups decreased from 2 to 8 ng/mL before STZ to between 0.01 and 0.6 ng/mL after STZ. Animals with the high dose of STZ developed transient vomiting within minutes after injection. During the first week after STZ injection, high-dose animals developed elevated LFTs, BUN and creatinine. In contrast, low-dose animals had normal liver and kidney function tests. Histological analysis showed that animals given the high dose of STZ developed marked steatosis of the liver and tubular injury in the kidneys, whereas animals given the low dose of STZ had normal-looking liver and kidney histology. The pancreatic islets in both groups were indistinguishable by immunoperoxidase staining for insulin, and showed either no insulin-positive cells or rare insulin-positive cells. Glucagon staining was normal. Over time, low-dose diabetic monkeys remained persistently hyperglycemic with negligible C-peptide stimulation by intravenous glucose. We conclude that low-dose STZ at 55 mg/mL successfully induces diabetes in cynomolgus monkeys with minimal liver and kidney toxicity.