Hiroaki Moriyama

Insulin autoimmunity: prediction/precipitation/prevention type 1A diabetes

Type 1 diabetes of both the NOD mouse and man is associated with autoimmunity directed against insulin which is the only beta cell specific autoantigen identified to date. One can use autoantibodies to insulin to predict diabetes, use insulin peptides to create insulin autoantibodies, insulitis and diabetes, and use insulin or its peptides in animal models to prevent diabetes. An expanding set of resources are now available for the development and testing in man of therapies to prevent type 1 diabetes, and a number of trials utilizing insulin peptides are now underway.

Induction of Insulin Autoantibodies and Protection from Diabetes with Subcutaneous Insulin B: 9-23 Peptide without Adjuvant

Abstract: Insulin B chain peptide B: 9‐23 given with incomplete Freunds adjuvant (IFA) subcutaneously to NOD and BALB/c mice induces insulin autoantibodies (IAA). We also found that subcutaneous administration of the peptide without adjuvant induced IAA in normal BALB/c mice. The autoantibodies react with intact insulin and cannot be absorbed by the B: 9‐23 peptide. With the induction of IAA by the self‐peptide without adjuvant, we hypothesized that the peptide given subcutaneously without adjuvant would prevent the development of diabetes mellitus in NOD mice. The peptide B: 9‐23, when given in standard doses of 100 μg and low doses of 10 μg, protected female NOD mice versus unvaccinated controls from diabetes. Presently, NOD mice vaccinated with the standard dose and the low dose have a 44% and 60% survival, respectively, at 26 weeks compared to controls with a 10% diabetes‐free survival at 22 weeks (n= 10 for each group, P < 0.001 for both vaccine doses). As expected, the level of IAA expressed was significantly higher for the vaccinated mice versus the control group. We conclude that insulin B chain peptide B: 9‐23 can confer protection from diabetes in NOD mice even when administered subcutaneously without adjuvant.

Nondepleting anti-CD4 monoclonal antibody prevents diabetes and blocks induction of insulin autoantibodies following insulin peptide B:9-23 immunization in the NOD mouse.

INTRODUCTIONnInsulin peptide B:9-23 is a major autoantigen in type 1 diabetes that induces insulin autoantibodies and prevents diabetes in the NOD. However, immunization with peptide without adjuvant may be insufficient to reverse disease or induce long-term tolerance. Furthermore, recent experience has demonstrated the potential dangers of disease exacerbation or anaphylaxis with peptide immunotherapy.nnnMETHODSnCombination therapy of B:9-23 with a nondepleting anti-CD4 monoclonal antibody (YTS 177.9) was studied in female NOD mice from 4 through 6 weeks of age. Injections of either B:9-23 in saline, YTS 177.9 antibody, or both peptide and antibody were given to mice.nnnRESULTSnBy 52 weeks follow-up, 40% of B:9-23-treated, 100% of YTS177.9-treated, and 70% of B:9-23 and YTS177.9 combination-treated mice remained diabetes-free. IAA, both spontaneous and induced by B:9-23, was almost completely suppressed in mice receiving YTS 177.9. In addition to suppression of IAA expression, anti-B:9-23 peptide antibodies are also suppressed in mice receiving B:9-23 with YTS 177.9, compared to B:9-23 alone.nnnCONCLUSIONnA brief course of the nondepleting anti-CD4 monoclonal antibody (YTS 177.9) in NOD mice confers long-term protection from diabetes and insulitis and profoundly blocks spontaneous and B:9-23 peptide-induced insulin autoantibodies.

Establishing Insulin 1 and Insulin 2 Knockout Congenic Strains on NOD Genetic Background

Abstract: As insulin is a major autoantigen in autoimmune diabetes and because the insulin gene region locus in humans has been linked to diabetes risk, we have bred insulin gene knockouts onto the NOD mouse. Mice differ from humans in terms that they express two nonallelic genes of insulin. Insulin 2 is the murine homologue of the human insulin gene and is located on mouse chromosome 7. Insulin 1 is thought to have evolved by a gene duplication event, lacks the second intron of the insulin 2 gene, and is located on mouse chromosome 19. The differential thymic expression of the insulin gene may be important for central tolerance induction. Here, we present the initial establishment of congenic knockouts and characterization of the congenic intervals corresponding to insulin 1 and insulin 2 knockout genes on mouse chromosome 19 and 7, respectively.

Animal Models of Insulin-Dependent Diabetes

Animal models have contributed enormously to study in the field of type 1 diabetes. Perhaps the most intensively studied model is the nonobese diabetic (NOD) mouse, which develops an autoimmune-mediated spontaneous diabetes associated with the development of insulin autoantibodies and insulitis. Accurate measurement of antiislet autoantibodies by radioassay and detection of antigen-specific T cells using major histocompatibility complex tetramers are possible. Various strategies have been developed in preventing diabetes in animal models; a peptide-induced model of type 1 diabetes has been described. Finally, the development of peptide vaccines is hampered by the risk of anaphylaxis in both mouse and humans. In this chapter, methods and strategies to measure antiinsulin autoantibodies, to detect antigen-specific T cells by tetramer analysis, and to prevent diabetes using peptide vaccines are discussed. Along with these topics, a protocol of peptide-induced diabetes and peptide vaccine-induced anaphylaxis are described, serving as a reminder of the potential dangers that could exist in human trials. In summary, animal models have become necessary in the study of type 1 diabetes and provide researchers important tools to conduct studies that could not otherwise be performed in humans.

Establishment of Native Insulin‐Negative NOD Mice and the Methodology to Distinguish Specific Insulin Knockout Genotypes and a B:16 Alanine Preproinsulin Transgene

Abstract: We hypothesize that NOD mice without native insulin, but with an altered insulin B:9‐23 sequence, will be completely protected from diabetes/insulitis if insulin B:9‐23 is an essential T cell epitope. To investigate this hypothesis, we have established initial insulin 1‐ and 2‐negative NOD mice with a transgene directing production of preproinsulin with alanine at position B:16 rather than the native tyrosine of both insulin 1 and insulin 2. Sets of primers for PCR‐based assays have been created and validated. They are able to distinguish the presence or absence of the insulin gene knockouts and of both native insulin 1 and insulin 2 (and thus distinguish heterozygous versus homozygous knockouts), as well as the presence of the altered insulin transgene, B:16 alanine preproinsulin. Four B:16 alanine transgenic founders were produced directly in NOD mice and, by intercrossing, initial live native insulin‐negative B:16 alanine transgenic mice have been generated.

Genetics and environmental factors in endocrine/organ-specific autoimmunity: have there been any major advances?

The field of endocrine/organ-specific autoimmunity is the beneficiary of a wealth of new knowledge and techniques in genetics and immunology, with the promise of much more to come in the current decade (www.barbaradaviscenter.org, Immunology of Type 1 Diabetes web book). Major advances can perhaps best be discussed in the setting of the central questions that are being addressed for almost all autoimmune disorders (Table 1). For specific diseases or disease subsets (as genetic heterogeneity is recognized) we now have a few clear answers in man or animal models, and we will address what we believe are important advances. New concepts arise from major advances and we will emphasize the discovery of autoimmune genes with characteristic disease syndromes, the dissection of diseases influenced by multiple genes within the major histocompatibility complex (MHC), the molecular characterization of class II HLA molecules and the development of technologies to define T cell receptors, the realization that many “autoantigens” recognized by either T cells or antibodies are expressed within the thymus and many are post-transcription-

Comparative study of oral versus subcutaneous B:9-23 insulin peptide in Balb/c mice as an experimental model for autoimmune diabetes.

Abstract: Insulin peptide B:9–23 (amino acids 9 to 23 of the B chain) can induce immune targeting of insulin and islets in normal Balb/c mice. The insulin autoantibodies induced react with insulin and not the immunizing peptide. Oral administration of insulin as well as subcutaneous insulin can sensitize to insulin.

Differential immune induction with subcutaneous versus oral administration of a diabetogenic insulin peptide in the NOD mouse.

Abstract: The B chain insulin peptide 9 to 23 (B:9–23) is a dominant T cell epitope of the NOD mouse. Given in oral form with multiple different vehicles, it did not alter expression of insulin autoantibodies in contrast to subcutaneous administration.