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

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In Nature, Anything that CanHappen Does HappenDaniel Kaufman, Ph.D.,is a Professor, activewithin the departmentof Molecular andMedical Pharmacologyat the UCLA School ofMedicine in Los Angeles.In a paper in November1993, Kaufman demonstrated that the administrationof GAD to mice that would otherwisedevelop insulin-dependent diabetes preventedthe outbreak of this disorder.Kaufman was also a member of the groupassociated with Allan Tobin, which was thefirst to submit a patent application for thefull cDNA code for GAD, the application thatDiamyd Medical licenses. Kaufman’s sphereof interest is focused on GAD and its relationto diabetes.IDan Kaufmandid not want to clone GAD. It was themid-1980’s, and I was first year graduatestudent of Allan Tobin at UCLA.New cloning technologies had just beendeveloped that could allow neurobiologiststo clone rare brain mRNAs. Allanand I thought that some of the most importantgenes in brain development and disease would beneurotransmitter receptors and neurotransmittersynthesizing proteins. I wanted to use antibodiesto screen brain cDNA expression libraries forGABA receptors, or use serum from multiple sclerosispatients to identify the protein targets oftheir autoimmune response. Allan, however, knewthat there was a good antibody available againstGAD (developed by Oertel) and instructed me togo after GAD.I constructed and screened a brain cDNAexpression library and quickly isolated one immunoreactiveclone. In those days, there was greatcontention about the molecular weight of GAD,so I tested the putative GAD clone for its GADenzymatic activity. Luckily, the one immunoreactiveclone I had isolated encoded an enzymaticallyactive protein when expressed in E. coli. Ourreport describing the cloning of GAD (Science,1986) showed that expression libraries could bescreened for functional activity, led to a greater ofunderstanding GAD biochemistry, and providedneuroscientists with a cDNA probe for studyingGAD and GABA in brain development and disease.My discussions with a new graduate student inthe Tobin lab, Mark Erlander, led us to suspect thatanother GAD gene existed, despite our failure todetect another gene by low stringency screening.Using degenerate PCR techniques, Mark was thefirst to isolate the second GAD gene, which encodeda 65 kD protein (GAD65). The clone that I had isolatedwas renamed GAD67, based on its molecularweight.Using recombinantly produced GAD67 protein,I next generated a GAD67-specific antibody. Thisantibody has been widely used by neuroscientiststo study GABAergic neurons. Using this antibodytogether with a GAD65-specific antibody, I showedfor the first time that while GAD67 is distributedthroughout the neuron, GAD65 is localizedin the nerve axon terminals. While most ofGAD67 is saturated with its cofactor, less thanhalf of GAD65 exists as active holoenzyme. Wesuggested that the modulation of GAD65apo/holoenzyme levels in nerve terminals couldcouple GABA production to neuronal activity (J.Neurochem, 1991).My change in course to study Type I diabetes(T1D) resulted from my car breaking down. I wasthen a postdoc at the Salk Institute in San Diego.After working in the lab and finding that my carwouldn’t start, I decided to pass time in the library.I picked up the latest issue of Lancet, and it fellopen to a report entitled “64,000 Mr autoantibodiesas predictors of insulin-dependent diabetes” byMark Atkinson and colleagues. A then littleknown factlet came to mind that besides the brain,GAD was also expressed in the cells that madeinsulin. From my work in the Tobin lab, I knewthat there were two forms of GAD, both of whichcould be candidates for this 64K autoantigen.I learned that the 64K autoantigen was a longsought after ß-cell antigen described by SteinunnBaekkeskov and Åke Lernmark. In addition,Michele Solimena and Pietro De Camilli had justreported an association between GAD autoimmunityand Stiffman syndrome. However, they alsoreported that they did not detect GAD autoimmmunityin T1D patients, which in retrospect, wasmissed only because the GAD antibodies in T1Dpatients could not recognize the denatured GADepitopes in their assay system. Despite this reportedlack of association, I decided that it wasworth a try to test whether T1D sera could recognizerecombinant GAD67 or GAD65. This wouldbe an easy experiment using the recombinantGADs available in the Tobin lab. I was again fortunate,since at this time, the existence of twoGADs was only known, and their cDNAs onlyavailable, in the Tobin lab.I got permission from Allan to do an immunoprecipitationexperiment in his lab over the nextweekend. I got T1D sera from Michael Clare-Salzler, and later, from Mark Atkinson and NoelMaclaren. Using the recombinant GADs, I quicklypage 16 dmccad june 2003
found that sera from T1D patients contained GADautoantibodies that were primarily directed againstGAD65. Using recombinantly produced fragmentsof GAD65, I was able to map some of the autoantibodyepitope recognition patterns. These were thefirst T1D diagnostics based on using recombinantGADs. I had also read about an epidemiologicalassociation between Coxsackie virus and T1D.When we compared Mark Erlander’s GAD65 DNAsequence with other sequences in the DNA sequencedata banks, we found a large sequence similaritywith a Coxsackie virus. This was a very provocativefinding, which could explain how ß-cell autoimmunitywas started, or augmented.We submitted a manuscript with our findingsto Science essentially at the same time asSteinunn Baekkeskov and colleagues submittedtheir findings to Nature. Unfortunately, a reviewerinsisted that we provide further proof ofGAD-Coxsackie virus molecular mimicry beforeit could be published. Consequently, we were notable to publish our work until after that ofBaekkeskov and colleagues. While very disappointingat the time, this may have been in a way fortuitous– I had just become a new AssistantProfessor at UCLA, and it prompted me to goafter the next major question – what was the roleof GAD in T1D?As T1D is thought to be T cell, and not autoantibodymediated, it was crucial to understand Tcell responses to ß-cell antigens. I was again extremelyfortunate to have Jide Tian join my newlab, who was highly knowledgeable and skilled incellular immunology. Using the NOD mousemodel of T1D, Jide found that a Th1-type responsefirst arose to GAD65, and then spread intraandinter-molecularly to other ß-cell antigens. Jidetreated very young NOD mice with GAD65 orother ß-cell antigens in a way that inactivatedreactive T cells. The mice that were tolerized toGAD65 had no autoimmune responses or insulitis,while those tolerized to other antigens did displayß-cell autoreactivity (Nature, 1993). Thus,the early inactivation of GAD65 reactive T cellscould circumvent the development of autoimmunity,qualifying GAD65 as a key antigen in theinduction of murine T1D.We next examined what could be done to inhibitan autoimmune process after it had alreadystarted. Jide showed that injecting GAD65 in anadjuvant that induced Th2 responses to GAD65could greatly inhibit the progression of the autoimmuneprocess in NOD mice that already had“These and subsequent experiments suggested that the greaterprotective effect of GAD65 was due to its greater abilityto induce Th2 responses. It is these experiments that”led to the GAD65vaccine that Diamyd is testing in clinical trialsan established autoimmune process. Moreover,GAD65 vaccination could also protect transplantedislets in diabetic mice, significantly betterthan the other ß-cell autoantigens (Nature-Medicine, 1996). These and subsequent experimentssuggested that the greater protective effectof GAD65 was due to its greater ability to induceTh2 responses. It is these experiments that led tothe GAD65 vaccine that diamyd is testing in clinicaltrails.We also began to examine the possibility of Tcell cross-reactivity between GAD and Coxsackievirus. Jide showed that T cell cross-reactivity didoccur, both at the peptide level and at the wholeprotein level between GAD65 and the Coxsackievirus protein – but only in mice with the diabetessusceptibly MHC II allele, and not in mice withother MHC II alleles (J. Exp. Med, 1994). Thejury is still out as to whether the epidemiologicalassociation of Coxsackie virus with T1D is due tothe ability of Coxsackie virus to infect islet cells,or due to T cell cross-reactivity with GAD. As itis generally found that in nature, anything thatcan happen does happen, it would not be surprisingto find that both mechanisms can occur, andin some cases, initiate or augment ß-cell autoimmmunity.It has been most gratifying to see our work atthe bench contribute to new diagnostics andpotential treatments for diabetes, as well as neurologicaldiseases. Our work has been highly dependenton the support of Allan Tobin, as well ascontributions by Jide Tian, Michael Clare-Salzler,Mark Atkinson and Paul Lehmann – who havealso had the patience of saints in teaching meimmunology.dmccad june 2003page 17
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 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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