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

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References1. Baekkeskov, S., et al,Autoantibodies in newly diagnosed diabetic childrenimmunoprecipitate specific human pancreatic islet cellproteins.Nature 298, 167-169 (1982).2. Baekkeskov, S., et al,Antibodies to a Mr 64,000 human islet cell antigen precedethe clinical onset of insulin-dependent diabetes. J.Clin. Invest. 79, 926-934 (1987).3. Baekkeskov, S., et al,Revelation of specificity of 64k autoantibodies in IDDMserums by high-resolution 2D-gel electrophoresis.Unambiguous identification of 64k target antigen.Diabetes 38, 1133-1141 (1989).4. Christgau, S., et al,Pancreatic ß-cells express two autoantigenic forms of glutamicacid decarboxylase, a 65kDa hydrophilic form anda 64kDa amphiphilic form which can be both membrane-boundand soluble.J. Biol. Chem. 266, 21257-21264 (1991).5. Baekkeskov, S., et al,Identification of the 64k autoantigen in insulin-dependentdiabetes as the GABA-synthesizing enzyme glutamicacid decarboxylase.Nature 347, 151-156 (1990).6. Kim, J., et al,Higher autoantibody levels and recognition of a linearN-terminal epitope in the autoantigen GAD65 distinguishstiff-man syndrome from insulin dependent diabetes.J. Exp. Med. 180, 595-606 (1994).7. Kanaani, J., et al,A combination of three distinct trafficking signals mediatesaxonal targeting and presynaptic clustering of GAD65.J. Cell Biol. 158, 1229-1238. (2002).8. 8. Kash, S. F., et al,Increased anxiety and altered responses to anxiolytics inmice deficient in the 65 kDa isoform of glutamic aciddecarboxylase.Proc. Natl. Acad. Sci. 96, 1698-1703 (1999).9. Shi, Y., et al,Increased expression of glutamic acid decarboxylase andGABA in pancreatic b-cells impairs glucose-stimulatedinsulin secretion at a step proximal to membrane depolarization.Am. J. Physiol. Endocrinol. Metab. 279, 684-694, (2000).10. Schwartz, H., et al,High resolution epitope mapping and structural modelingof human glutamic acid decarboxylase 65.J. Mol. Biol. 287, 983-999 (1999).11. Jaume, J. C., et al,Suppressive effect of GAD65-specific autoimmune Blymphocytes on processing of T cell determinants locatedwithin the antibody epitope.J. Immunology 169, 665-672, (2002).Contributions tothe GAD65 FieldISteinunn Baekkeskovn the 1960’’s and 1970’s several investigatorsdiscovered evidence to show thatinsulin dependent diabetes mellitus(Type 1 diabetes or T1D) is an autoimmmunedisease characterized by circulatingautoantibodies to the β cell in humanpancreatic islets and infiltration of lymphocytesinto the islets. However, the target antigen of thisautoimmunity was completely unknown, when Ijoined the Hagedorn Laboratory in Copenhagen inlate 1979 to work with Åke Lernmark. Usingexpertise I had gained in studying target antigensin trypanosomes, I developed a method that alloweda very sensitive detection of human β cellproteins that bound to circulating autoantibodiesin the blood of patients. To our surprise, we discoveredthat autoantibodies in the blood from 8 outof 10 diabetic children bound to the same protein,a 64kD antigen from human islets while none of10 healthy children recognized this protein. Thiswas the very first indication that there was a specifictarget antigen in the autoimmune response inT1D (1). We then showed that the autoantibodiesto the 64kD protein can be present many yearsbefore clinical onset of T1D (2), suggesting thatthese autoantibodies can be used as a sensitivemarker to identify individuals at risk of developingT1D. Also, in all autoimmune diseases the targetantigen is of critical importance because of itspotential to be used to prevent the autoimmunedisease. Having established my own laboratoryfirst at the Hagedorn Research Laboratory andlater at the University of California, San Francisco,we developed amethod for partiallypurifyingthe 64kD protein(2-3). Wecharacterizedimportantbiochemicaland biophysicalparameters ofthe antigen (2-4) in a painstakingeffort thatculminated inits identificationas theSteinunn Baekkeskov,Ph.D., is a Professor atthe University ofCalifornia. Baekkeskov´scurrent research projectsfocus on four mainareas: i) structure,function, and cell biologyof GAD65, IA-2 and glima 38; ii) characterizationof disease specific B-cell epitopesin GAD65, and IA2, and the temporalpattern of their recognition during early andlate phases of §-cell destruction; iii) characterizationof autoimmune T cell epitopes inGAD65 and IA2, and how they can be used toinduce apoptosis in autoimmune T cells duringthe preclinical phase to prevent disease; iv)and mechanisms of transplantation tolerance,and development of methods to preventallo- as well as autoimmune destruction ofislet cell transplants.smaller isoform of the GABA-synthesizing enzymeglutamic acid decarboxylase or GAD65 (5). Theidentification of a component of the 64kD autoantigenas GAD65 transformed the field ofImmunology of Diabetes, because it became possibleto use recombinant protein for developmentof autoantibody assays and for studies of autoimmunemechanisms in the NOD mouse. A fewyears later, a second component of the 64kD autoantigen,left behind by our purification method,was identified as the protein IA-2 by Michaelpage 18 dmccad june 2003
GAD65 moleculeChristie, my first postdoctoral fellow, in his ownlab. Together, GAD65 and IA-2, which are expresssedin both ß cells and neurons, are recognized by80-90% of T1D patients. In neurons, GAD65 isalso a target of autoimmunity in a rare neurologicaldisorder Stiff-man syndrome (6).Following the discovery of GAD65 as a major targetautoantigen in T1D (5), my lab has focused onunderstanding the structure, cell biology, and functionof this molecule, and how it is recognized bythe immune system. The subcellular trafficking ofGAD65 is the parameter that most clearly distinguishesit from the highly homologous GAD isoform,GAD67, and is dependent on unique trafficking signalsin the N-terminal region (7 and refs therein).By knocking-out GAD65 in the mouse, we showedthat GABA generated by GAD65 is involved infine tuning of inhibitory neurotransmission inresponse to a variety of environmental stimuli (8and refs therein), while overexpression of the proteinin β cells revealed a role of GABA in negativeregulation of first phase insulin secretion (9). Finemapping of the autoimmune epitopes recognizedby GAD65 autoantibodies in human patients revealedthat they target almost the entire surface of themolecule (10 and refs therein) and led to the firstThe figure is printed with permission from Academic Press (Journal of Molecular Biology, Vol.287,No 5, April 16, 1999, pp. 983-999, Baekkeskov S, Schwartz HL).3D model of the GAD65 dimer (10). This structuralinformation enabled us to show how the epitopespecificity of autoimmune B cells, a critical playerin GAD65 presentation to T cells and developmentof T1D, influences the autoimmune T cellepitope repertoire in the protein (11).dmccad june 2003 page 19
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 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

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