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Åke Lernmark, MD, Ph.D., and his colleagues atHagedorn Research Laboratory in Denmark, firstreported 64K proteins in humans in 1982.Steinunn Baekkeskov, Ph.D., played a primary role indetecting the 64K protein in humans and later worked incollaboration with Yale researchers to identify the proteinas GAD.Mark Atkinsson, Ph.D., and colleagues at the Universityof Florida in Gainesville, concluded in 1990 that 64Kautoantibodies might be the earliest and best marker fordiabetes yet identified.techniques for detecting membrane proteins while studyingthe role of antigens in African sleeping sickness. Usingnewly developed and very sensitive tests such as the“immunoprecipitation technique”, Drs. Baekkeskov,Lernmark and colleagues at the Hagedorn found 80-90percent of blood serum samples from children withnewly diagnosed diabetes had antibodies to an unidentifiedICSA with molecular weight of approximately 64,000daltons-or 64 kilodaltons. They dubbed the mystery protein64K, and reported their findings in 1982 in Nature.In 1984, they reported that 64K antibodies were oftenfound in diabetic BB rats (a commonly used animalmodel for human diabetes). One intriguing aspect of thisfinding was that the antibodies appeared 40 to 70 daysbefore the onset of diabetes. This suggested that the 64Kprotein was an early and major target of the immunesystem. But did this same predictive effect hold true forhumans as well?They next looked at blood serum samples of 14 peoplewho later developed diabetes. Eleven of them had antibodiesto the 64K protein - for up to seven years before theybegan to show symptoms of diabetes. This suggested that64K antibodies might be an early warning sign for theeventual development of diabetes.In the summer of 1986 Dr. Atkinson who had beenstudying environmental influences on diabetes in rats atthe University of Florida in Gainesville, traveled toHagedorn as a visiting scientist. There Baekkeskov taughthim the immunoprecipitation technique for detecting64K antibodies. Armed with this new skill, he returned tothe University of Florida, where he and Dr. NoelMaclaren, detected the 64K antibodies in nonobese diabetic(NOD) mice, a newly bred type of animal model fordiabetes. Finding the 64K antibodies in animals helpedconfirm that the 64K antigen was an important feature indiabetes.Drs. Atkinson, Maclaren, and colleagues also studiedfirst-degree relatives of people with diabetes, who were athigher risk for developing diabetes themselves. In 1990, inthe British Journal The Lancet, they reported finding 64Kautoantibodies in 23 of 28 people who developed diabetesup to seven years later. Based on these findings and thoseof the Hagedorn researchers they concluded that 64Kmight be the earliest and best marker for diabetes yetidentified and might be especially useful for predictingdiabetes.As diabetes researchers became increasingly aware ofhow important this protein was, they set out to analyzeits chemical characteristics to compare with known proteins.In 1988, Drs. Solimena, Camilli, and their colleagues atYale University reported in The New England Journal ofMedicine that they had found the target antigen in patientswith an autoimmune disorder called Stiff-ManSyndrome (“SMS”). SMS is a rare disorder of the centralnervous system in which a person’s muscles become moreand more rigid, and painful spasms occur. Patients withthe disorder were found to have antibodies to an enzymecalled Glutamic acid Decarboxylase, or GAD.GAD is responsible for converting the amino acid glutamateinto a protein called GABA, which the brain cells useto communicate. It turned out that many patients withpage 8 dmccad june 2003
GAD antibodies had diabetes – even the ones who neverdeveloped SMS. Moreover, they found that antibodies fromthe serum of SMS patients targeted the beta cells, and thesewere not any of the previously known antibodies.This led to an historic collaboration between Dr.Baekkeskov, now at the University of California, SanFrancisco, and the Yale research team. Dr. Camilli sentBaekkeskov blood serum samples from SMS patients (containingGAD antibodies), and she sent him blood serumsamples from diabetes patients (containing 64K antibodies).Then they used various methods to demonstrate thatthe proteins were completely identical. The end result ofthis meeting of the minds was their landmark paper inNature identifying the 64K protein as the enzyme GAD.The discovery set off a flurry of interest in GAD by diabetesresearchers.Send in the ClonesDr. Kaufman, who had been studying the role of GAD inneurological diseases such as epilepsy, became interested indiabetes one day in 1990 when his car broke down. Whilewaiting for it to be fixed, he went to the library, wherehe stumbled on the article by Dr. Atkinson and colleaguesin The Lancet describing the 64K antibodies as predictorsof diabetes.According to Kaufman, the landmark paper identifying64K as GAD had yet to appear in Nature, but he was ableto put the two together for himself: He knew about the roleof GAD in SMS and the fact that it was found in beta cells,and he also knew that that molecular weight of GAD wasapproximately 64kD. He joined forces with the researchteam at the University of Florida, as well as with his formermentor at the UCLA Dr. Tobin, who was researching therole of GAD in Huntington’s disease and epilepsy. Therewere known to be slightly different molecules of GADfound in the brain – one weighing 67kD (GAD67) and asmaller one weighing 65kD (GAD65). As a graduate studentworking with Tobin, Kaufman had worked out the geneticsequence for GAD67, and another graduate student Dr.Erlander, had done the same for GAD65. Thanks in part tothis work, Dr. Tobin’s laboratory was now able to makeboth forms of GAD in quantity using genetically alteredbacteria.Spurred by the news that 64K was GAD, other researchgroups cloned GAD. In 1988, Dr. Lernmark had moved tothe University of Washington in Seattle with some membersof his former Hagedorn research team. One of theseDr. Allan Karlsen, cloned the gene for human isletGAD65. One of Lernmark’s former graduate student’s, Dr.Birgitte Michelsen, cloned GAD67. According to Dr.Lernmark, cloning GAD enabled researchers to makerecombinant GAD in quantities they had never dreamedAllan Tobin, Ph.D., and Daniel L. Kaufman, Ph.D., at the University of California at Los Angeles published one of two1993 papers in “Nature” confirming that GAD is the first known target of the T cell attack in diabetes.of. Cloning GAD65 also enabled researchers to determinewhich molecule was the alter ego of 64K.The researchers in Tobin’s lab sought help from aUCLA diabetes expert, Dr. Michael Clare-Salzler, tostudy GAD’s role in diabetes. Using blood serum samplesfrom diabetic patients with newly diagnosed diabetes providedby Dr. Clare-Salzler, the researchers showed that96% of patients with newly diagnosed diabetes had antibodiesto one or both forms of GAD. Furthermore, itappeared that 64K was GAD65, the smaller of the twomolecules that had been identified.Dr. Erlander also used a computer search that uncoveredstructural similarities between both forms of GADand proteins in the Coxsackie virus, a virus known toinfect many people who eventually developed diabetes.“That moment was probably the most exciting moment inmy whole scientific life!” Dr. Tobin recalled. This findingsupported a theory known as “molecular mimcry”, whichsuggested that infection by the Coxsackie virus might setoff the immune reaction in diabetes. According to the“true identity”pageAs diabetes researchers became increasinglyaware of how important thismystery protein was, they were eager to discover itsdmccad june 20039
Page 4 and 5: forewordResearch Scientists through
Page 6 and 7: The Story ofGADRobert Dinsmoor1975R
Page 10 and 11: theory, the immune system mistakenl
Page 12 and 13: GAD Back to the Future…Åke Lernm
Page 14 and 15: 100GAD in GraphsGAD65 DNA vaccinati
Page 16 and 17: In Nature, Anything that CanHappen
Page 18 and 19: References1. Baekkeskov, S., et al,
Page 20 and 21: GAD in GraphsIncidence of diabetes
Page 22 and 23: References1. Quinn A, et al,MHC cla
Page 24 and 25: References1. Tisch, R., et al,Induc
Page 26 and 27: Vaccination with GAD PlasmidSuppres
Page 28 and 29: GADGAD in GraphsGAD ELISPOT outperf
Page 30 and 31: GAD in GraphsA1004 wks of age% Inci
Page 32 and 33: Mark Atkinson, Ph.D.,is an American
Page 34 and 35: GAD in GraphsIL-4 (pg/ml)2501007550
Page 36 and 37: References1. Kobayashi T, et al,Isl
Page 38 and 39: References1. A.Falorni, et al,Radio
Page 40 and 41: References1. Chattopadhyay, S., et
Page 42 and 43: Diamyd’s Commercial Development o
Page 44: T cell GAD65For use of GAD in immun

Check GAD antibody positivity with the Diamyd anti-GAD RIA plate*

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