Dana Elias

β-cell function in new-onset type 1 diabetes and immunomodulation with a heat-shock protein peptide (DiaPep277): a randomised, double-blind, phase II trial

Summary Background Type 1 diabetes results from autoimmune destruction of insulin-producing pancreaticcells. The 60 kDa heat-shock protein (hsp60) is one of the known target self antigens. An immunomodulatory peptide from hsp60, p277, arrested � -cell destruction and maintained insulin production in newly diabetic NOD mice. We did a randomised, double-blind, phase II study of peptide treatment in patients with newly diagnosed (<6 months) type 1 diabetes.

Peptide therapy for diabetes in NOD mice

NOD mice spontaneously develop autoimmune diabetes that mimics insulin-dependent diabetes mellitus (IDDM) in man. A peptide of the 60 kDa heat shock protein (hsp60), designated p277, can serve as a target for diabetogenic T-cell clones, and diabetes was prevented by using the p277 peptide to turn off anti-p277 immunity early in life. We report that the p277 peptide, administered once, can arrest the autoimmune process even after it is far advanced. Successful therapy was associated with down-regulation of the autoimmune process and regression of islet inflammation. Thus the immune system is responsive to manipulation by a specific signal even in the face of a virulent, full-blown autoimmune process.

Hsp60 Peptide Therapy of NOD Mouse Diabetes Induces a Th2 Cytokine Burst and Downregulates Autoimmunity to Various β-Cell Antigens

A peptide of the human 60-kDa heat-shock protein (hsp60), designated p277, was found to be useful as a therapeutic agent to arrest the autoimmune process responsible for diabetes in nonobese diabetic (NOD) mice. The effectiveness of peptide treatment was associated with the induction of peptide-specific antibodies of the IgG1 but not of the IgG2a isotype, suggesting the possibility that a Th2-type response may have been induced. We now report that the effectiveness of p277 treatment is associated with the transient activation of anti-p277 splenic T-cells that produce the Th2 cytokines interleukin-4 (IL-4) and IL-10. The Th2 response to p277 was associated with reduced Thl-type autoimmunity to hsp60 and to two other target antigens associated with diabetes: GAD and insulin. The Th2 shift appeared to be relatively specific; spontaneous T-cell reactivity to a bacterial antigen peptide remained in the Th1 mode in the p277-treated mice. Moreover, treatment with the bacterial peptide did not induce a change in cytokine profile, and it did not affect progression of the disease. Thus, effective peptide treatment of the diabetogenic process associated with the induction of antibodies may be explained by selective and transient activation of Th2 autoimmune reactivity.

Treatment of Autoimmune Diabetes and Insulitis in NOD Mice With Heat Shock Protein 60 Peptide p277

We recently showed that a peptide of the Mr 60,000 heat shock protein molecule, designated peptide p277, is a target of T-cells in autoimmune diabetes in NOD mice. Indeed, the p277 peptide could be used as a therapeutic agent to arrest the autoimmune process even after it was faradvanced. The present study was done to document the effects of p277 therapy on inflammation of the islets and on T-cell responsiveness to p277. Groups of female NOD mice of various ages up to 17 weeks were treated with a single inoculation of p277 given before or after the onset of overt hyperglycemia. We now report that fragments of p277 can affect diabetes but that optimal therapy requires the whole peptide. The positive response to p277 was dependent on administration of a threshold dose of peptide. Therapy was accompanied by the regression of intra-islet inflammation and the reappearance of histologically normal islets. Successful peptide therapy was associated with downregulation of T-cell immunity to p277. Adoptive transfer experiments demonstrated that the spleen cells of p277-treated mice were no longer diabetogenic and also could suppress the diabetogenic potential of cotransferred spleen cells of untreated female NOD mice. These results indicate that specific treatment of diabetes with a defined peptide can reprogram the autoimmune response.

Treatment of new‐onset type 1 diabetes with peptide DiaPep277® is safe and associated with preserved beta‐cell function: extension of a randomized, double‐blind, phase II trial

Treatment with DiaPep277®, a peptide derived from HSP60, has been shown to preserve beta‐cell function in non‐obese diabetic mouse (NOD) mice and in a trial with newly diagnosed human patients with type 1 diabetes treated over a 10‐month period. This article extends the clinical trial observations to a total of 20 months of treatment to determine the safety and the effects of repeated doses of DiaPep277 on endogenous insulin secretion, metabolic control, and exogenous insulin requirements.

Autoimmune Diabetes Induced by the β-cell Toxin STZ: Immunity to the 60-kDa Heat Shock Protein and to Insulin

Administered at a suitably low dose, the toxin streptozotocin (STZ) can trigger an autoimmune process leading to destruction of the β-cells of the pancreatic islets. In this study, we examined specific immunological reactions in mice before and during the development of STZ-induced autoimmune diabetes. We now report that the development of spontaneous autoantibodies to insulin can serve as a marker of susceptibility to a low dose of STZ. Susceptible male mice of the C57BL/KsJ strain manifested such anti-insulin antibodies, and resistant female mice did not. Administration of a low dose of STZ (five daily doses each of 30 mg/kg) induced transient hyperglycemia ∼20–30 days later, which temporarily remitted but was followed by intractable diabetes ∼2.5 months later. The diabetogenic process triggered by the low dose of STZ was associated with an increase in the level of anti-insulin antibodies bearing the Dana and Micha (DM) idiotype, later followed by the appearance of anti-idiotypic antibodies that peaked before the onset of diabetes. Antibodies and T-cells reactive to hsp60 (heat shock protein) were triggered by the low-dose STZ administration and persisted throughout the period that preceded clinical diabetes. T-cells reactive to the p277 peptide of hsp60 were also observed. Finally, active immunization to hsp60 caused transient hyperglycemia by itself and also aggravated the hyperglycemia induced by low-dose STZ. Thus, autoantibodies to insulin can indicate susceptibility to a toxic trigger of diabetes, and a low dose of a toxin can activate the insulin and hsp60 autoimmunity that has been detected previously in the spontaneous autoimmune diabetes of NOD strain mice.

The hsp60 Peptide p277 Arrests the Autoimmune Diabetes Induced by the Toxin Streptozotocin

The development of autoimmune diabetes in the NOD strain of mice (H-2g7) is marked by the presence of T-cells reactive to the p277 peptide of the 60-kDa heat shock protein (hsp60). We have found that the p277 peptide can be used as a therapeutic vaccine to arrest NOD diabetes. Recently, we found that T-cell autoimmunity to p277 also develops spontaneously in C57BL/KsJ mice (H-2d) during the induction of autoimmune diabetes by a very low dose of the β-cell toxin streptozotocin (STZ). We now report the inhibition of STZ toxin-induced autoimmune diabetes by p277 peptide therapy. Administration of two doses each of 100 µg of peptide p277 in mineral oil given 1 week after toxin induction and 85 days later was most effective. The effect of p277 on STZ toxin-induced diabetes was marked by a shift in p277 autoimmunity from a T-cell proliferative response to the production of anti-p277 antibodies. The anti-p277 antibodies were predominantly of the IgGl and IgG2b isotypes, known to be regulated by Th2 type cytokines; IgG2a antibody, known to be dependent on interferon (IFN)-γ, was induced to a much lesser degree. Peptide p277 therapy was specific: treatment of the mice with an immunogenic peptide from the sequence of another antigen, GADp34, failed to prevent the development of diabetes. The GADp34 peptide induced lower titers of specific antibodies, and the antibodies were predominantly of the IgG2a class. Thus, p277 peptide therapy, marked by the induction of Th2-type antibodies, can be effective in toxin-induced autoimmune diabetes.

The pathogenicity of islet-infiltrating lymphocytes in the non-obese diabetic (NOD) mouse

The aim of the present study was to investigate the pathogenic properties of islet‐infiltrating lymphocytes related to the severity of the autoimmune destruction of islet β‐cells in the NOD mouse. We analysed the development of insulin‐dependent diabetes mellitus (IDDM) produced by adoptive transfer of islet lymphocytes from NOD into NOD.scid mice. Here we show that the transfer was most effective when both CD4+ and CD8+ T cells were present in the infiltrate, but CD4+ T cells alone were sufficient to cause the disease. Islet lymphocytes from both females and males transferred diabetes effectively, but the severity of IDDM was higher when female islet lymphocytes were used. Unexpectedly, the sensitivity of male islets to β‐cell damage was greater than that of female islets. Treatment of NOD females with a peptide of heat shock protein (hsp)60, p277, known to protect NOD mice from IDDM, reduced the pathogenicity of the islet lymphocytes. In contrast, administration of cyclophosphamide to males, a treatment that accelerates the disease, rendered the islet lymphocytes more pathogenic. More severe disease in the recipient NOD.scid mice was associated with more interferon‐gamma (IFN‐γ)‐secreting islet T cells of the NOD donor. The disease induced by islet lymphocytes was strongly inhibited by co‐transfer of spleen cells from prediabetic mice, emphasizing the regulatory role of peripheral lymphocytes. Thus, the cellular characteristics of the islet infiltrate and the pathogenicity of the cells are subject to complex regulation.

A quantitative model of autoimmune disease and T-cell vaccination: does more mean less?

According to a simple mathematical model, the activated effector T cells that cause an autoimmune disorder can also cure the disease if administered in large doses. This prediction has been tested in the nonobese diabetic (NOD) mouse model and demonstrates that administration of intermediate doses of a diabetogenic T-cell clone caused early hyperglycemia, whereas a higher dose cured the disease. As discussed here by Lee Segel and colleagues, the proposed application of T-cell vaccination to treat clinical disease obliges immunologists to consider the quantitative complexities of regulation.

Treatment of Recent-Onset Type 1 Diabetic Patients With DiaPep277: Results of a Double-Blind, Placebo-Controlled, Randomized Phase 3 Trial

OBJECTIVE To evaluate safety and efficacy of DiaPep277 in preserving β-cell function in type 1 diabetic patients. RESEARCH DESIGN AND METHODS DIA-AID 1 is a multinational, phase 3, balanced-randomized, double-blind, placebo-controlled, parallel-group clinical study. Newly diagnosed patients (N = 457, aged 16–45 years) were randomized to subcutaneous injections of DiaPep277 or placebo quarterly for 2 years. The primary efficacy end point was the change from baseline in the area under the glucagon-stimulated C-peptide curve. Secondary end points were the change from baseline in mixed-meal stimulated C-peptide secretion and in fasting C-peptide and achieving target HbA1c ≤7% (≤53 mmol/mol). Partial remission (target HbA1c on insulin ≤0.5 units/kg/day) and hypoglycemic event rate were exploratory end points. RESULTS DiaPep277 was safe and well tolerated. Significant preservation of C-peptide secretion was observed in the DiaPep277-treated group compared with the placebo (relative treatment effects of 23.4%, P = 0.037, and 29.2%, P = 0.011, in the modified intent-to-treat [mITT] and per-protocol [PP] populations, respectively). The mixed-meal stimulation failed to distinguish between the groups. There was a trend toward efficacy in fasting C-peptide levels, though not statistically significant. Significantly more DiaPep277-treated than placebo-treated patients maintained target HbA1c (mITT 56% versus 44%, P = 0.03; PP 60% versus 45%, P = 0.0082) and entered partial remission (mITT 38% versus 29%, P = 0.08; PP 42% versus 30%, P = 0.035). DiaPep277 treatment reduced the relative hypoglycemic event risk (mITT by 20%; PP by 28%). CONCLUSIONS DiaPep277 safely contributes to preservation of β-cell function and to improved glycemic control in patients with type 1 diabetes.