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

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GAD in GraphsA1004 wks of age% Incidence of diabetes% Incidence of diabetes80604020B10001480604018 22 2610 wks of age(n=12)PlasmidsGAD65GAD65-GMCSFcontrol(n=8)(n=13)Vaccination with GAD plasmid DNAprevents diabetes in NOD mice(n=11)PlasmidsGAD65GAD65-GMCSFcontrol20(n=8)(n=7)016 14 20 24 28 32 36Balasa et al, 2001. Vaccination with GAD plasmidDNA protects mice from spontaneousautoimmune diabetes and B7/CD28 costimulationcircumvents that protectionClin Immunol 99:241-252• Mice vaccinated at both 4-5 (A)and 10-11 weeks of age (B) wereprotected from developing diabetesIs GAD All There Is?TBart Roephe discovery of GAD65 as oneof the first islet autoantigens inthe pathogenesis of type 1 diabeteswas a breakthrough thatenabled study of specific autoimmuneresponses. I worked asa postdoc in the laboratory of Steinunn Baekkeskovin San Francisco at the time that the identity ofthe 64,000 kDa target of autoantibodies was unraveledas GAD65. My aim as a T cell immunologistwas to determine T cell autoreactivity to this proteinin humans, and to study whether immunizationof genetically predisposed mice with GAD65led to the induction of insulitis or diabetes. Theresults in these pioneer studies to check for T cellautoimmunity were unexpected since I foundvery few differences between patients and controlsubjects, although in both groups significantresponses were detectable. With few exceptions,this finding turned out to be reproducible bymany colleagues all over the world during the folllowingdecade. My second aim to induce diabetesby immunization with GAD65 was in vain:although very strong immune responses could beinduced in various strains of mice, none of thesedeveloped insulitis or diabetes. The latter observationproved useful for the application of usingGAD65 as an immune modulator rather than asan inducer of pathogenesis. Currently there is consensusthat, immunization with GAD65 delays orprevents diabetes in mice, rather than inducing oraccelerating the disease.For the next step as T cell immunologists, weintroduced GAD65 as a candidate target autoantigenin our immune monitoring of Type 1 diabetespatients receiving islet allografts. Indeed, chronicrecurrent loss of islet allograft function wasaccompanied with increment in T cell autoreactivityto GAD65, even in the absence of increases inantibody titers, while this reactivity was neverobserved in patients successfully transplanted withislets. This clinical trial proved that longitudinalstudies on cellular immune responses to GAD65were informative to determine the clinical fate ofbeta-cells in Type 1 diabetes patients. It proved oftremendous importance to test GAD65 of highpurity to avoid reactivity to contaminants in thepreparation of recombinant GAD65. This was theprime result from the first international workshopon T cell reactivity to islet autoantigens. Eversince, I have used GAD65 produced and purifiedby diamyd as golden standard, since this materialwas of reproducibly high quality and void of toxicor mitogenic contaminants. A second satisfactoryencounter with GAD65 came from studies in arare autoimmune disorder called Stiff-ManSyndrome (SMS) that shares GAD65 with Type 1diabetes as major target autoantigen. One-third ofSMS patients develop Type 1 diabetes. We studieda case of SMS without Type 1 diabetes to understandwhy SMS patients often do not developType 1 diabetes despite phenomenal autoimmunityto this neuroendocrine autoantigen GAD65, inorder to develop immunotherapy for Type 1 diabetesin patients that lost tolerance to this protein.Indeed, the GAD65 reactivity measured in thispage 30 dmccad june 2003
“Indeed, chronic recurrent loss of islet allograftfunction was accompanied with incrementin T cell autoreactivity to GAD65, even”in theabsence of increases in antibody titersnon-diabetic SMS patient was characterized as nonoreven anti-inflammatory by its IL-10 producingnature. Unexpectedly, this SMS patient did not elicitprimary proliferative responses to GAD65.However, when she developed Type 1 diabetesfour years later, strong primary responses weredetectable, while the only cytokine produced bythese T cells was the proinflammatory cytokineinterferon-γ. At that time, all symptoms of SMShad resolved. This observation demonstrated thatthe nature of cellular autoimmunity to GAD65 isan important parameter to distinguish healthy subjectsfrom Type 1 diabetics from SMS patients.Moreover, this was a clinical demonstration thatan anti-inflammatory cytokine profile was favorablefor diabetes-prone subjects against developmentof Type 1 diabetes.Thirteen years after the discovery of GAD65 astarget autoantigen in Type 1 diabetes mellitusthrough its recognition by autoantibodies, we arestill left with many open issues. The disease specificityof immune responses to GAD65 is unclear.Autoantibodies against GAD65 are rarely found innon-diabetic subjects, but the majority of subjectswho do have such antibodies will remain healthy,while others suffer from different autoimmunediseases. With regard to T cell responses toGAD65, the consensus is that there are only minimaldifferences in T cell proliferation to GAD65in Type 1 diabetes patients versus controls. Thedifference most likely lies in the quality of theimmune response, and how GAD65 autoimmunityis regulated. The big question now, however, iswhether islet autoantigens such as GAD65 can beused as an immunotherapeutic to divert autoimmmunityfrom destruction to regulation.It should also be considered to assess whethercombinations of immunotherapies includingGAD65 as an immune modifying agent are effectiveto suppress disease activity. In preliminary invitro experiments we have observed that stimulationof GAD65 specific autoreactive T cell clonesisolated from a prediabetic subject is affected bythe new generation of humanized monoclonalantibodies against CD3, and only upon stimulationwith GAD65.Bart Roep is AssociateProfessor in Medicine,in particular theimmunology andimmunogenetics ofType 1 diabetes, at theLeiden UniversityMedical Center, Leiden,The Netherlands. Roep pioneered studies onthe role of T cells in the pathogenesis ofType 1 diabetes, demonstrating that autoreactiveT cells play a key role in beta celldestruction in humans. Roep is currentlyinvolved in the design and evaluation of newimmunotherapies to prevent beta-cell autoimmunity.dmccad june 2003page 31
Page 4 and 5: forewordResearch Scientists through
Page 6 and 7: The Story ofGADRobert Dinsmoor1975R
Page 8 and 9: Åke Lernmark, MD, Ph.D., and his c
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 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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