Translating Data from Animal Models into Methods for Preventing Human Autoimmune Diabetes Mellitus: Caveat Emptor and Primum non Nocere

Clinical Immunology Vol. 100, No. 2, August, pp. 134 –143, 2001 doi:10.1006/clim.2001.5075, available online at http://www.idealibrary.com on

SHORT ANALYTICAL REVIEW Translating Data from Animal Models into Methods for Preventing Human Autoimmune Diabetes Mellitus: Caveat Emptor and Primum non Nocere Dale L. Greiner, 1 Aldo A. Rossini, and John P. Mordes Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655

Type 1 diabetes in humans is a serious autoimmune disorder of children that is still poorly understood, unpreventable, and irreversible. Study of its animal models, notably the NOD mouse and BB rat, has generated a wealth of information concerning genetics and immunopathogenesis, but that information has still not altered the way in which we treat children with diabetes. In this review we attempt to identify the most promising avenues of continuing research in these models and the most important issues that must be faced by the designers of human therapies based on the animal dataset. © 2001 Academic Press THE LONG WAIT: CAUSE AND PREVENTION OF TYPE 1 DIABETES

Insulin deficiency in humans with type 1 diabetes results from the selective destruction of pancreatic ␤ cells, a process found by von Meyenberg to be associated with lymphocytic inflammation of the islets or “insulitis” (1). His 1940 observation led to the hypothesis that type 1 diabetes is an autoimmune disease. In the 1960s Gepts substantiated that concept in a study of 22 young persons with juvenile diabetes who had died within 6 months of onset; 15 had insulitis (2). The first animal model of the disorder may have been that of Renold, Soeldner, and Steinke, who reported in 1964 that insulitis could be induced in cattle and other species by immunization with insulin in adjuvant (3). In 1968 encephalomyocarditis virus was shown to cause a diabetic syndrome resembling autoimmune juvenile diabetes (4). In 1976 it was reported that treatment of mice with multiple small doses of streptozotocin caused similar pathology (5). Studies of some of these syndromes have continued for more than 3 de1 To whom correspondence and reprint requests should be addressed at Diabetes Division, Biotech 2, 373 Plantation St., Worcester, MA 01605-2377. Fax: 508-856-4093. E-mail: dale.greiner@ umassmed.edu.

1521-6616/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

cades (6, 7). The BB rat was discovered in the mid1970s (8) and the nonobese diabetic (NOD) mouse soon thereafter (9). Unlike previous models, the BB rat and NOD mouse developed both insulitis and diabetes spontaneously. Embraced as models for studying what was then called insulin-dependent diabetes mellitus, both animals engendered widespread interest. Medline lists ⬎900 BB rat and ⬎1500 NOD mouse publications through 2000. In 1985 the advent of molecular engineering gave us the first of even more advanced model systems based on transgenic and knockout technology (10). The hope, expressed often in these publications, was that the models would reveal the causes of autoimmune diabetes and then lead to therapies for children. Early in the new millennium, the human disorder has been renamed type 1 diabetes, but the promise of the animal model research has yet to be fulfilled. By any name, human autoimmune diabetes mellitus remains incompletely understood, unpreventable, difficult to treat, and difficult to cure by transplantation (11). Here we review the promise and problems inherent in the animal models, and we explore some of the reasons the translation of preventive strategies from bench to beside has proven so arduous. The use of animals to perfect new methods of transplantation as a cure for type 1 diabetes is reviewed elsewhere (12). AUTOIMMUNE DIABETES MELLITUS IN CHILDREN

Phenotype Diabetes mellitus is a group of disorders characterized simply by hyperglycemia. Though diagnostic criteria continue to evolve (13), most cases are currently classified as either type 1, insulin-dependent diabetes mellitus, or type 2 non-insulin-dependent diabetes mellitus. Type 1 diabetes accounts for ⬃10% of all cases and most often presents as rapid onset polyuria, polydipsia, polyphagia, and weight loss in children and

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young adults. All cardinal features of the disease can be traced to an absolute deficiency of insulin, which leads to hyperglycemia, release of free fatty acids from adipose tissue, and hepatic overproduction of ketones. Untreated individuals succumb to ketoacidosis, dehydration, and electrolyte disturbances (14). Treatment with exogenous insulin controls the disorder and good control reduces the frequency of complications. Immunopathogenesis A substantial clinical dataset supports the autoimmune hypothesis of type 1 diabetes. Persons affected often suffer from other intercurrent autoimmune disorders. These include Addison’s disease, vitiligo, celiac disease (11), and lymphocytic thyroiditis (15). Additional data were obtained when a pancreas was transplanted from a nondiabetic donor to a discordant, diabetic monozygotic twin. Despite the absence of transplantation barriers, inflammation of the transplanted islets and ␤ cell destruction developed, suggesting recurrence of a tissue-specific autoimmune process (16). Type 1 diabetes has also occurred when bone marrow was transplanted from type 1 diabetic donors to nondiabetic HLA-identical (17) and allogeneic (18) recipients. The presence of diabetes-associated humoral immune markers in patients with type 1 diabetes also supports the autoimmunity hypothesis (11). Autoantibodies include those directed against islet cell antigens (ICA), insulin, glutamic acid decarboxylase (GAD), and the tyrosine phosphatase IA-2, among others (11). The presence of various combinations of these autoantibodies is used to predict disease in children who are still euglycemic (19). Genetics Human type 1 diabetes is familial, but its mode of inheritance is non-Mendelian (20 –22). Consistent with the autoimmunity hypothesis, the disease is most clearly associated with permissive HLA haplotypes; the MHC-associated susceptibility locus is designated IDDM1 (21). Another susceptibility locus, designated IDDM2 is located in the 5⬘ VNTR of the insulin gene (23). The results of genomewide searches for additional human IDDM genes have confirmed the large effect associated with IDDM1 (24 –27) and identified multiple other but different chromosomal regions linked to diabetes. Concordance for type 1 diabetes among identical twins is only 30 –50%, however (28), suggesting that dietary and/or environmental modifiers influence expression of the disease (29). Leading candidate environmental agents include diet (30) and viral infections (6).

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Prevention and Cure Confidence in the autoimmunity hypothesis of type 1 diabetes eventually led to a trial of cyclosporine immunosuppression in diabetic children; it was found to prolong endogenous insulin production (31). Unfortunately its toxicity was unacceptably high, but trials involving newer agents are planned or under way (see www.jdrf.org and www.clinicaltrials.gov). These clinical trials involve strategies based on immunosuppression, immunomodulation, and oral tolerance. To date, however, there are no interventions documented to be both safe and efficacious that will prevent or reverse type 1 diabetes. The only available curative treatment for established type 1 diabetes mellitus is the combination of a pancreas or islet graft and lifelong systemic immunosuppression (32, 33). Animal Models of Type 1 Diabetes The ethical and technical impediments to studying diseases in human subjects are obvious, and the restrictions being placed on such research are stringent. Reliance on animal “models” is in part a recognition of the primacy of patient safety—primum non nocere, first do no harm. Unlike children, diabetic animals can be bred to study and manipulate inheritance. They can be made diabetic, biopsied, and autopsied. Their genome can be altered or fixed by inbreeding. Therapies to prevent or reverse the disease can readily be tested. Efforts to understand the causes and pathophysiology of type 1 diabetes-like syndromes have involved animal models with diabetes that is spontaneous, induced by environmental perturbation, and induced by genetic manipulation (transgenes and knockouts) (7). Essentially all translational research (i.e., research directed at prediction and prevention) in type I diabetes is currently based on the BB rat and NOD mouse, and this review focuses on them. In each, the observation of insulitis succeeded by selective ␤ cell destruction and finally ketoacidosis propelled broad programs of research. Few facets of the immune systems of these rodents have escaped study. The study of cyclosporin in children (31) was engendered after documentation of efficacy in animals (34, 35). Diabetes-Prone and Diabetes-Resistant BB Rats Clinical and immunological phenotype. Inbred diabetes-prone BBDP rats develop spontaneous T-cell-dependent, ketosis-prone autoimmune diabetes in the context of pancreatic insulitis and autoantibodies, but the rats are severely T cell lymphopenic (36). In the reference BBDP/Wor colony at BRM, Inc. (Worcester, MA, www.biomere.com), ⬎90% of adolescent animals of both sexes develop insulitis and diabetes. Diabetes-

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resistant BBDR rats are phenotypically normal and disease-free in clean environments, but they are susceptible to induction of type 1 diabetes (v.i.). The peripheral lymphopenia in BBDP rats represents primarily a lack of T cells that express ART2 (formerly termed RT6) (37). ART2 is a marker of T cells with regulatory capability. Transfusions of histocompatible ART2 ⫹ cells prevent spontaneous diabetes in BBDP rats (38), and only ART2 ⫺ cells adoptively transfer the disease to naı¨ve recipients (39). BBDR rats circulate normal numbers of peripheral ART2 ⫹ T cells and become diabetic only after environmental perturbation (36). Diabetogenic perturbants include (1) infection with Kilham rat virus (KRV), (2) the interferon inducer poly(I:C), and (3) lower doses of either of these agents in combination with a depleting anti-ART2 monoclonal antibody (mAb) (36). Characteristics and key features of BB rats have recently been reviewed in detail (36). Genetics. Genetic analysis indicates that diabetes in BB rats requires the presence of at least one MHC class II RT1 u allele (40). The MHC susceptibility locus is designated iddm2. Interestingly, susceptibility to induced autoimmune diabetes appears in many rat strains that express the RT1 u haplotype (41). In BBDP rats, a second locus designated lyp/iddm1 has been identified. The lyp gene is responsible for lymphopenia (42), which is permissive to the expression of spontaneous hyperglycemia (42). Additional susceptibility loci in BB rats have been mapped to chromosomes 4 and 13 using environmental perturbation to induce diabetes in backcross animals (43, 44). The chromosome 4 locus, iddm4, is linked to, but not identical to, lyp and contributes strongly to diabetes susceptibility in both BBDP and BBDR rats. The iddm4 gene is located in a region containing other major autoimmunity loci in the rat (44). Prevention and cure. Most systemic immunosuppressive agents prevent diabetes in BBDP rats. As noted above, a preventive strategy unique to the model is transfusion of ART2 ⫹ T cells to overcome the effects of lymphopenia (38). The disease can also be prevented by intrathymic transplantation of islets (45). Oral tolerance is ineffective (46). Certain diets reduce the prevalence of the disease, but do not prevent it (36). Cure has been achieved with islet transplantation plus either immunosuppression (47) or costimulatory blockade (48). NOD Mice Clinical and immunological phenotype. Makino reported this mouse model in 1980 (9). NOD mice develop insulitis when 4 to 5 weeks old. In the colony at The

Jackson Laboratory (Bar Harbor, ME, www.jax.org), ⬃90% of female and ⬃60% of male NOD/Lt mice typically become diabetic by 12 months of age. Ketoacidosis in the affected animals is mild. Diabetic animals can survive for weeks without exogenous insulin. By the time clinical hyperglycemia occurs, frank insulitis is always present (49). Characteristics and key features of NOD mice have been reviewed elsewhere (7, 49, 50). As in humans and BB rats, additional lines of evidence suggest that NOD mouse diabetes is an autoimmune disorder. Autoantibodies are present. Splenocytes, CD4 ⫹ and CD8 ⫹ lymphocytes, and various lymphocyte clones from adult NOD mice can adoptively transfer disease to appropriate recipients (49). Conversely, a broad array of interventions prevent the disease (50). Genetics. Analyses of NOD mice (51) reveal at least 18 loci on 11 different chromosomes that associate with diabetes or insulitis. Analogous to the human IDDM1 and rat iddm2 loci, idd1 in the mouse is a major susceptibility locus that has been assigned to an MHC class-II-associated region. Also available are diabetessusceptible and -resistant congenic NOD mice (51). Prevention and cure. There are reports of more than 125 interventions that delay, ameliorate, or prevent diabetes in the NOD mouse (49, 50). These include many reagents that cause immunosuppression and a wide variety of immunomodulatory agents, such as nicotinamide, cytokines, cytokine inhibitors, BCG, and LPS. In addition, some interventions that have no documented effect on human immunity (elevated temperature, reduced caloric intake) also seem to be effective (7, 50). NOD mouse diabetes can be cured by islet transplantation and immunosuppression, but not peripheral tolerance-based islet transplantation. Animal Models and Translational Type I Diabetes Research The sections above summarize only a fraction of the data that have been generated using only two animal models of type 1 diabetes. They point out clearly that we can identify genetic loci and pathophysiological pathways relevant to type 1 diabetes-like syndromes in animals. They point out that we can use that information to prevent and cure animal diabetes with remarkable success. But they also point out other key issues. First, we have not yet identified in any species the non-MHC genes or sets of genes whose actions will predictably lead to autoimmunity. Second, we have not precisely defined the exact immunological pathways that cause autoimmune diabetes in any species. Finally, we have not yet been able to translate the wealth of animal data into therapies for children that meet “gold standard” criteria of safety and efficacy.

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This may seem like a poor return for 30 years of investment in animal research. Indeed, it has been suggested that human type 1 diabetes stems from the chance concatenation of any of a host of genetic and environmental factors that act through any of many pathways that we may never fully understand (52). By extension it can be argued that studying “distant cousin” processes in rodents is futile. In the following sections, we address this issue of futility specifically with respect to the promise of genetic and environmental analyses in animals. Genetics Like all humans, children with type 1 diabetes are outbred. Few if any inbred pedigrees exist. But all our animal models of spontaneous diabetes have been inbred for dozens of generations. The process of inbreeding has given us a stable scientific framework on which to design studies and identify genes. Inbreeding, however, is inherently artificial and aleatory. It is artificial because no humans are comparably inbred. It is aleatory in that most of the genes that become fixed are fixed by chance. Inbreeding is a process of selecting for a target phenotype, in this case hyperglycemia. But endless rounds of selection also fix countless other genes in a purely chance fashion. The result is, in the case of the NOD mouse, a robust disease phenotype on an immunological framework riddled with defects. NOD mice lack the MHC class II I-E locus, C5a (and hence lack hemolytic complement), have defective NK cell activity, display defective NK T cell function, have defects in APC activity, and exhibit T cell hyperplasia (49). In addition, they are deaf, a phenotype that would seem to be the oddest happenstance, were it not for the fact that certain odd mitochondrial– diabetes syndromes in humans are also associated with deafness (53). Given so many immunological defects, it should not be surprising that multiple interventions that perturb that system prevent diabetes in these animals (50). The BBDP rat owes its discovery to a chance mutation at the lyp locus leading to severe T cell lymphopenia. Upon inbreeding, that defect cosegregated with the diabetes phenotype and was fixed. Spontaneous diabetes in BB rats requires lymphopenia (36), but of course no children with diabetes are lymphopenic. But which defects in mice and rats are counterparts of the human defects? The lesson of these animal data is not that analyses of rats and mice are futile. Rather, we need to be very cautious if not frankly skeptical of interventions that are successful in only one inbred rodent model system that has an idiosyncratic and fragile immune system. Using the data from inbred and congenic rodents with spontaneous diabetes, many candidate genes have been identified, and it appears clear that polymor-

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phisms in genes encoding cytokines or their regulatory proteins are likely to contribute to disease susceptibility. It is also clear, however, that more than one set of genes, interacting with the environment, can lead to type 1 diabetes. Candidate loci in the human, NOD mouse, and BB rat other than the MHC are not syntenic and do not appear to be common to the three species. So what will genetic analyses reveal? What can an inbred rodent really tell us about disease in outbred human populations? We believe that the key point to make is that these model systems will reveal at least one road to autoimmunity, and that is one more than we have available now in children. The genetics are complex but the data coming from inbred and congenic animals will inevitably identify pathways that underlie physiology. Artificial Environments, Diet, and Infection Animal models of spontaneous type 1 diabetes are inbred to facilitate genetic analysis and kept pathogen free to minimize confounding variables in metabolic and immunological analyses. Initially housed conventionally, both BB rats and NOD mice were moved first to specific pathogen-free facilities and then to viral antibody-free facilities. In both cases, as their environment became cleaner, the frequency of diabetes rose (36, 49). BB rats are so “clean” that lymph nodes can be exceedingly difficult to find. In contrast, the T cell hyperplasia gives NOD mice generous lymphoid tissues despite their immunological naı¨vete´. Humans, of course, are not only outbred but also immunized to microbial and viral pathogens from birth, the result of naturally occurring infection and pediatric immunizations that begin during the first year of life. But just as inbreeding will facilitate our identification of the genes that predispose to diabetes, the environmental control affords us the opportunity to test at least generically the hypothesis that diet, toxins, or infection are important in a defined state of genetic predisposition. In the end, the rodents are unlikely to prove informative as to which, if any, viruses abet human type 1 diabetes expression, but they will inform us as to the process by which such events can occur. Diet. The relevance of diet, particularly protein that is derived from cow’s milk, in the pathogenesis of human type 1 diabetes is controversial (54, 55). In part, this line of research is driven by the finding that changes in food, particularly its protein content, influence the onset of diabetes in NOD mice (56, 57) and BB rats (58, 59). These observations argue that complex natural ingredients in standard human and rodent diets can be diabetogenic accelerators. The specific issue of cow’s milk proteins in animal and human studies is addressed below.

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Infectious agents. Certain viruses influence the expression of insulin-dependent diabetes in NOD mice, BB rats, and humans. In humans, the temporal relationship of infection to diabetes is well documented, but the exact role of the virus in pathogenesis is unknown (6). Implicated viruses include Coxsackie B4 and perhaps other enteroviruses, rubella, and mumps. Compared with mice raised in conventional animal facilities, gnotobiotic NOD mice become diabetic more often and at a younger age (60). Viruses that decrease the incidence of diabetes in NOD mice include mouse hepatitis virus, Sendai, lymphocytic choriomeningitis virus (LCMV), vaccinia, lactate dehydrogenase virus, and Pichinde (61). As in the NOD mouse, LCMV infection decreases the frequency of type 1 diabetes in BBDP rats. This constellation of findings in NOD mice and BBDP rats suggests that neither of these models is likely to be a good candidate for understanding the role of viral infection in the induction of autoimmune diabetes. In contrast, BBDR rats housed in VAF facilities remain free of spontaneous diabetes, but BBDR rats infected with KRV can become diabetic. Naturally occurring infection, transmitted by close contact, typically induces diabetes in ⬃1–2% of animals (62); direct injection of KRV renders ⬃30% of animals diabetic (63). Disease induction does not involve infection of the pancreatic ␤ cells (64), and several other viruses, notably SDA, H1, and Sendai, appear not to be diabetogenic in BBDR rats. Interpretation of the data holds that KRV triggers diabetes by altering the balance between autoreactive T cells and regulatory T cells (65). Understanding in detail this inbred, viral antibody-free rodent system may provide important generic information on the mechanism by which viral infection can induce autoimmunity.

Immunosuppression The classic method for preventing type 1 diabetes is the use of cyclosporine to induce generalized immunosuppression. This approach was shown to be effective in preventing and ameliorating diabetes in both the BB rat (34, 35) and the NOD mouse (66). In these trials, primary prevention was almost uniformly successful, therapy had to be given only for a matter of weeks, and no toxicities were reported. In human clinical trials, cyclosporine was documented clearly to ameliorate type 1 diabetes and preserve insulin secretory capability when given promptly after onset (67), but disease usually recurred when therapy was stopped, and longterm results were disappointing (68). Therapy was sometimes complicated by drug-induced nephrotoxicity (69, 70), and long-term follow up of some study subjects 7 years after discontinuing the drug suggests that they may be at increased risk for late onset nephropathy (71). Looking to the future, there is little reason to doubt that newer immunosuppressive agents would also be effective in preventing and possibly reversing diabetes. Many have already been documented to be effective in diabetes prevention in animal models (7). The experience with cyclosporine very clearly illustrates, however, that efficacy in rodents often outstrips that in humans, that the duration of treatment required may vary across species, and that toxicities may appear unexpectedly and long after the completion of a trial. The crucial contemporary issue is to use the animal models to identify immunosuppressive agents that possess a better benefit to risk ratio, i.e., have lower toxicity profiles demonstrated first in nonhuman primates and in human organ transplantation. For the moment, no agents with such properties are available, and no large-scale trials of generalized immunosuppression for type 1 diabetes in children are in progress.

PREVENTION AND CURE: HOW BEST TO MOVE INTO THE CLINIC

Parenteral Insulin We hope that our arguments make it clear that it would be premature to call off the pursuit of answers in animal models before we understand at least the NOD mouse or the BB rat in detail. On the other hand, it is not premature to recognize that our lack of insight into cause at the present time should give us pause in the design of “translational” experimentation in humans. How have the data obtained in animal models of diabetes been used to design current and proposed clinical trials for the prevention and cure of type 1 diabetes? What are the basic criteria currently being used to justify advancement of therapies to the clinic? Below are a few specific examples to illustrate how the animal dataset has been used to generate translational research.

Vaccination trials designed as antigen-specific therapy for type 1 diabetes are under way, and therapies with altered peptide ligands have been proposed. Although based on efficacy in animal models, we believe that such trials must be entertained with caution. Injections of insulin at an early age prevent diabetes in NOD mice (72), and the effect requires only modest doses of insulin. These observations, suggestive of the induction of tolerance, led to the generation of suggestive preliminary data in six humans (73), and subsequently the very large scale human Diabetes Prevention Trial of Type 1 Diabetes or DPT-1 was initiated (74). Interestingly, parenteral insulin also prevents diabetes in BBDP rats (75) and BBDR rats treated to

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induce the disease (76). The BB rat data suggest that the intervention requires some degree of ␤ cell involution, as relatively substantial doses of insulin are required (36). If insulin injections induce tolerance (as suggested by the mouse data), the intervention may well be appropriate for humans, but if ␤ cell rest is required (as suggested by the rat data), then auguries for the human trials may be less favorable. In theory, the administration of a parenteral autoantigen as a tolerizing agent is not free of the risk of disease induction. Vaccination with adjuvant is sometimes discussed, but we would point out that the available animal data suggest that adjuvant plus islet antigen does have the capability of inducing insulitis (3). Oral Insulin and GAD65 Oral tolerance induction by feeding peptides has been shown to prevent several autoimmune diseases, including diabetes in NOD mice (77). Feeding oral intact insulin (78) to NOD mice prevents diabetes very effectively, as does mucosal administration of insulin ␤ chain peptide (79) and the putative autoantigen GAD65 (80). Based on this dataset, one arm of the DPT has been designed to test the efficacy of oral insulin in preventing diabetes: enrollment in this trial is continuing. One preliminary report was not encouraging (81), but the DPT and other trials are still ongoing. Interestingly, oral insulin in BB rats appears not to prevent diabetes (46), and there are data to suggest that the combined oral administration of insulin and adjuvant could exacerbate rather than tolerize in this model (82).

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trials in children at risk are in progress despite the interspecies discordance. The treatment appears to be safe (89), but the data available at this time suggest that the magnitude of any therapeutic effect may be small (90, 91). Another immunomodulatory approach that has recently been translated from mouse to human trials is treatment with anti-CD3 mAb. This reagent prevents and may reverse diabetes in NOD mice (92, 93). Therapy using the human-specific reagent hOKT3␥1 (alaala) induces transient depletion of T cells, and the drug is currently being tested in patients with new onset of type 1 diabetes. The long-term effects of this transient depletion are unknown. It has not been tested in the BB rat or animal models of type 1 diabetes other than the NOD mouse. Diet Antibodies reactive against a peptide sequence contained within bovine serum albumin (BSA) occur in humans, BB rats, and NOD mice, and they cross-react with an islet antigen designated p69 (94). Together with suggestive epidemiological data, these findings have given rise to the controversial theory that dietary cow’s milk may contribute to the pathogenesis of type 1 diabetes in susceptible individuals (95). Removal of dietary bovine proteins does prevent diabetes in NOD mice (96) but not BB rats (97). A large-scale trial of dietary BSA restriction in children at risk of type 1 diabetes is under way in Finland (98) despite skepticism on the part of some researchers (99). Data on prophylactic efficacy are not yet available. EXECUTING TRANSLATIONAL RESEARCH

Immunomodulation Adjuvants like BCG and CFA in NOD mice (83, 84) and CFA in BB rats (85) prevent diabetes. The data suggest that nonspecific immunization can prevent autoimmune diabetes; polarization has been proposed as the underlying mechanism, but definitive proof is lacking. Trials of BCG to prevent diabetes have been implemented in children, but to date neither harm nor benefit has been reported (86, 87). Although benefit has not been shown for this agent, it is worth noting that the risk to participants was small. Large numbers of people worldwide are safely immunized with BCG to prevent tuberculosis, and the margin of safety in the diabetes trial was presumably large. Nicotinamide is a precursor for nicotine adenine dinucleotide (NAD) synthesis and an inhibitor of poly(ADP ribose) synthetase, which is known to protect ␤ cells from certain toxins and the effects of nitric oxide. Nicotinamide prevents type 1 diabetes in NOD mice (88). It is not effective in the BB rat (36). Prevention

When we last reviewed the issue of translational research for preventing type 1 diabetes (100), we emphasized the need to consider the caveats that apply to such research—caveat emptor, let the buyer beware. Six years later, the caveats are clearly still important. The only intervention to date that is clearly efficacious in preventing or at least retarding the development of type 1 diabetes is immunosuppression. This is not surprising, as there is relatively uniform agreement that type 1 diabetes in humans and spontaneous type 1 diabetes-like syndromes in animal models are autoimmune disorders. These agents are also effective in many other models of autoimmunity, and they require no knowledge of specific autoantigens or specific immunological pathways. In contrast, interventions to prevent diabetes based on specific processes in animal models like the NOD mouse have generated inconclusive results. How then do we move observations made in animal models to the clinic? Given (1) the animal models that

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are currently available, (2) the inchoate status of our knowledge of the pathophysiology of type 1 diabetes, and (3) the translational “track record” just discussed, we would like to suggest that new trials be undertaken cautiously. The obvious first step remains to prove that a therapy is effective in NOD mice or BB rats. However, given the obvious differences among the mouse, rat, and human syndromes described above, we propose that the second step should be an attempt to determine if the therapy works in more than one animal model of diabetes. If a therapy is based on the NOD mouse, it might, depending on the hypothesis being considered, be tested in BB rats, mice treated with streptozotocin, or a transgenic mouse. Third, we propose that safety be addressed independently of the issues of efficacy not only in rodents but also larger animals, such as nonhuman primates. Nonhuman primate models of autoimmune diabetes are unavailable, but they are still appropriate to use to analyze safety issues before proceeding to the clinic. We also suggest that explicit consideration be given not only to the hypothesis that underlies the expectation of efficacy (i.e., vaccination will tolerize) but also the caveats (i.e., immunization could induce autoimmunity or anaphylaxis). Fourth, when possible, trials in humans should be initiated in cases where the risk to benefit ratio is appropriate. For example, hematopoietic stem cell therapy protocols that might be used for treatment of autoimmunity and autoimmune diabetes (101) should first be evaluated in patients with malignancy or advanced autoimmunity who have exhausted other therapeutic options. Fifth, in the case of generic, “non-antigen-specific” therapeutics, notably immunosuppression, we propose that they be shown to be efficacious in other models of cell-mediated autoimmunity. Any therapy that fundamentally alters the immune response in a non-antigenspecific fashion should work in many T-cell-mediated autoimmune diseases, not just in type 1 diabetes. Efficacy across model systems would strengthen the case for application in human diabetes. Finally, issues of acute and long-term consequences of the therapy need to be considered as the dose and duration of therapy are scaled up following phase I trials for safety. As noted above, there may be longterm consequences of transient cyclosporine therapy for children who participated in early trials (71). Risk benefit assessments are seldom made in the design of animal studies but cannot be accorded too much importance in the design of translational research. In summary, we believe that the animal models of diabetes still hold immense promise for the discovery of genes and pathways that will eventually lead us to a better understanding of diabetes. But in addition to the

new language of genomics and bioinformatics that is informing our efforts to discover causes, a few ancient words should continue to inform our translational research: caveat emptor and primum non nocere. ACKNOWLEDGMENTS This work was supported in part by Grants DK49106, DK25306, and DK36024 from the National Institutes of Health, by Program Project Grants AI42669 and DK53006 (jointly funded by NIH and the Juvenile Diabetes Research Foundation International), and by an institutional Diabetes and Endocrinology Research Center Grant from the National Institutes of Health (DK32520). REFERENCES ¨ ber ‘Insulitis’ bei Diabetes. Schweiz. 1. von Meyenburg, J. V., U Med. Wochenschr. 24, 554 –557, 1940. 2. LeCompte, P. M., and Gepts, W., The pathology of juvenile diabetes. In “The Diabetic Pancreas” (B. W. Volk and K. F. Wellmann, Eds.), pp. 325–363, Plenum, New York, 1977. 3. Klo¨ppel, G., “Insulin” induced insulitis. In “Immunological Aspects of Diabetes Mellitus. International Symposium, Steno Memorial Hospital, Denmark” (O. Ortved Andersen, T. Deckert, and J. Nerup, Eds.), pp. 107–121, Acta Endocrinologica, Copenhagen, 1976. 4. Craighead, J. E., and McLane, M. F., Diabetes mellitus: Induction in mice by encephalo-myocarditis virus. Science 162, 913– 914, 1968. 5. Like, A. A., and Rossini, A. A., Streptozotocin-induced pancreatic insulitis: New model of diabetes mellitus. Science 193, 415– 417, 1976. 6. Yoon, J. W., and Jun, H.-S., Role of viruses in the pathogenesis of type 1 diabetes mellitus. In “Diabetes Mellitus: A Fundamental and Clinical Text” (D. LeRoith, S. I. Taylor, and J. M. Olefsky, Eds.), pp. 419 – 429, Lippincott Williams & Wilkins, Philadelphia, 2000. 7. Mordes, J. P., Greiner, D. L., and Rossini, A. A., Animal models of autoimmune diabetes mellitus. In “Diabetes Mellitus: A Fundamental and Clinical Test” (D. LeRoith, S. I. Taylor, and J. M. Olefsky, Eds.), pp. 430 – 441, Lippincott Williams & Wilkins, Philadelphia, 2000. 8. Nakhooda, A. F., Like, A. A., Chappel, C. I., Murray, F. T., and Marliss, E. B., The spontaneously diabetic Wistar rat. Metabolic and morphologic studies. Diabetes 26, 100 –112, 1977. 9. Makino, S., Kunimoto, K., Munaoko, Y., Mizushima, Y., Katagiri, K., and Tochino, Y., Breeding of a non-obese diabetic strain of mice. Exp. Anim. 29, 1–13, 1980. 10. Hanahan, D., Heritable formation of pancreatic ␤-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 315, 115–122, 1985. 11. Park, Y., and Eisenbarth, G. S., The natural history of autoimmunity in type 1A diabetes mellitus. In “Diabetes Mellitus: A Fundamental and Clinical Text” (D. LeRoith, S. I. Taylor, and J. M. Olefsky, Eds.), pp. 347–362, Lippincott Williams & Wilkins, Philadelphia, 2000. 12. Rossini, A. A., Greiner, D. L., and Mordes, J. P., Induction of immunological tolerance for transplantation. Physiol. Rev. 79, 99 –141, 1999. 13. Gabir, M. M., Hanson, R. L., Dabelea, D., Imperatore, G., Roumain, J., Bennett, P. H., and Knowler, W. C., The 1997 American Diabetes Association and 1999 World Health Organization

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