Добірка наукової літератури з теми "Human GAD65"

Two isoforms of the glutamate decarboxylase (GAD) enzyme exist, GAD65 and GAD67, which are associated with type 1 diabetes (T1D) and stiff-person syndrome (SPS), respectively. Interestingly, it has been reported that T1D patients seldom develop SPS, whereas patients with SPS occasionally develop T1D. In addition, coxsackievirus B4 (CVB4) has previously been proposed to be involved in the onset of T1D through molecular mimicry. On this basis, we aimed to examine antibody cross-reactivity between a specific region of GAD65 and GAD67, which has high sequence homology to the nonstructural P2C protein of CVB4 to determine potential correlations at antibody level. Monoclonal peptide antibodies generated in mice specific for a region with high similarity in all three proteins were screened for reactivity along with human sera in immunoassays. In total, six antibodies were generated. Two of the antibodies reacted to both GAD isoforms. However, none of the antibodies were cross-reactive to CVB, suggesting that antibody cross-reactivity between GAD65 and CVB, and GAD67 and CVB may not contribute to the onset of T1D and SPS, respectively.

Glutamic acid decarboxylase GAD65 autoantibodies (GADA) are an established marker for autoimmune diabetes. Recently, the autoantigen GAD65 itself was proposed as biomarker of beta-cell loss for prediction of autoimmune diabetes and graft rejection after islet transplantation. Therefore, the GAD65 content in pancreatic islets of different species and its serum degradation kinetics were examined in this study using a sensitive immunoassay. GAD65 was found in quantities of 78 (human), 43.7 (LEW.1A rat) and 37.4 (BB/OK rat) ng per 1,000 islets, respectively, but not in mouse islets. Thein vitrohalf-life of porcine GAD65 and human recombinant GAD65 ranged from 1.27 to 2.35 hours at 37°C in human serum, plasma and blood, and was unaffected by presence of GAD65 autoantibodies. After injecting 2,000 ng recombinant human GAD65 into LEW.1A rats, thein vivohalf-life was 2.77 hours. GAD65 was undetectable after 24 hours in these animals, and for up to 48 hours following diabetes induction by streptozotocin in LEW.1A rats. Estimated from these data, at least 13 islets in rat and 1,875 in human must be simultaneously destroyed to detect GAD65 in circulation. These results should be taken into consideration in further studies aimed at examining the diagnostic relevance of GAD65.

The functional role of glutamate decarboxylase (GAD) and its product GABA in pancreatic islets has remained elusive. Mouse β-cells express the larger isoform GAD67, whereas human islets express only the smaller isoform GAD65. We have generated two lines of transgenic mice expressing human GAD65 in pancreatic β-cells (RIP7-hGAD65, Lines 1 and 2) to study the effect that GABA generated by this isoform has on islet cell function. The ascending order of hGAD65 expression and/or activity in β-cells was Line 1 heterozygotes < Line 2 heterozygotes < Line 1 homozygotes. Line 1 heterozygotes have normal glucose tolerance, whereas Line 1 homozygotes and Line 2 heterozygotes exhibit impaired glucose tolerance and inhibition of insulin secretion in vivo in response to glucose. In addition, fasting levels of blood glucose are elevated and insulin is decreased in Line 1 homozygotes. Pancreas perfusion experiments suggest that GABA generated by GAD65 may function as a negative regulator of first-phase insulin secretion in response to glucose by affecting a step proximal to or at the KATP +channel.

GAD65 autoantibodies (GAD65Ab) are important markers for type 1 (insulin-dependent) diabetes mellitus. Although most patients have GAD65Ab at the time of clinical diagnosis, there are also GAD65Ab-positive individuals in the population at low risk of developing type 1 diabetes. The aim of this study was to test the hypothesis that the GAD65Ab reactivity to GAD65 cloned from human, mouse, and rat in newly diagnosed type 1 diabetic patients differ from antibody-positive healthy individuals. Sera from 254 new-onset 0- to 34-yr-old type 1 diabetic patients and 270 controls were assayed for their reactivity to human, mouse, and rat GAD65. Among the type 1 diabetic patients there was a significant better binding of human GAD65 compared to either mouse (P = 0.03) or rat GAD65 (P = 0.0005). The preference for human GAD65 increased with increasing age at onset (P = 0.0002). This differentiation was not observed in 88 GAD65Ab-positive control subjects. Our data indicate that recognition of epitopes by GAD65Ab in type 1 diabetes is different from that in nontype 1 diabetes, GAD65Ab-positive individuals.

The use of glutamic acid decarboxylase (GAD65) from bovine brain has been studied to obtain basic knowledge and diagnosis and prediction of Type 1 Diabetes Mellitus (DM) patients. The importance of GAD65 in DM diagnosis based on its patogenesis. One of the autoimmune marker that can be used to detect beta-pancreas destruction in Diabetes Type I is the antibody to glutamic acid decarboxylase (GAD65). Most of the pre-diabetic patients indicate the reactive autoantibody to GAD65. For early detection of anti-GAD65 in the serum of the patient, human recombinat GAD65 has been succeed to be used. However this is not economical, therefore, it is necessary to find the alternative source of cheaper GAD65. The aim of this research is to develop an early detection kit of Type 1 DM based on antibody-GAD65, since the longest patient suffering from DM has higher probability to be complicated, especially for uncured patients. The anti- GAD65 antibodies induced by anti-GAD65 synthetized and labelled by alkaline phosphatase can be used as reagent detection early DM patients. The ten patients of DM as samples (positive of anti-GAD65) and five rats of DM were positive with western blott technique using reagents as result of this research. It can be concluded, GAD65 enzyme isolated from bovine brain induced anti-GAD65 production and have possibilities to be packaged in a diagnostic kit for patient pre DM.

Abstract Glutamic acid decarboxylase 65 (GAD65) is one of the major autoantigens in type 1 diabetes. We investigated whether there is variation in the processing of GAD65 epitopes between individuals with similar HLA backgrounds and whether the processing characteristics of certain immunogenic epitopes are different in distinct APC subpopulations. Using DR401-restricted T cell hybridomas specific for two immunogenic GAD65 epitopes (115–127 and 274–286), we demonstrate an epitope-specific presentation pattern in human B-lymphoblastoid cell lines (B-LCL). When pulsed with the GAD protein, some DRB1*0401-positive B-LCL, which presented GAD65 274–286 epitope efficiently, were unable to present the GAD65 115–127 epitope. However, all B-LCL presented synthetic peptides corresponding to either GAD epitope. In addition, when pulsed with human serum albumin, all cell lines gave equal stimulation of a DR4-restricted human serum albumin-specific T hybridoma. GAD65-transfected cell lines displayed the same presentation phenotype, showing that lack of the presentation of the 115–127 epitope was not due to inefficient uptake of the protein. Blood mononuclear adherent cells, B cells, or dendritic cells derived from the same individual displayed the same presentation pattern as observed in B cell lines, suggesting that the defect most likely is genetically determined. Therefore, individual differences in Ag processing may result in the presentation of distinct set of peptides derived from an autoantigen such as GAD65. This may be an important mechanism for the deviation of the immune response either into a regulatory pathway or into an inflammatory autoimmune reactivity.

Riassunto Il diabete insulino-dipendente di tipo 1 (T1D), il quale colpisce lo 0.03-0.04% della popolazione, è causato dalla distruzione autoimmune delle cellule beta del pancreas deputate alla produzione di insulina, rendendo necessaria una terapia sostitutiva continua con questo ormone (Gepts, 1965). Questo forma di diabete è considerata una malattia cronica con un forte impatto sociale, a causa della comparsa di complicazioni legate al progredire della malattia ed alla giovane età dei pazienti essendo la più diffusa malattia cronica in bambini e ragazzi al di sotto dei 14 anni di età. Attualmente non esiste una cura della malattia ed il trattamento con insulina rappresenta semplicemente una terapia sostitutiva continua. L’induzione della tolleranza mediante l’utilizzo di auto antigeni è considerata una strategia utile per la prevenzione o il rallentamento della progressione delle malattie autoimmuni (Harrison, 2005). In particolare, la GAD65 è considerata un possibile vaccino per il diabete di tipo 1 e, di conseguenza, sono stati effettuati diversi studi di immunoterapia antigene-specifica (ASI) utilizzando questo auto antigene per testare la sua efficacia nell’induzione della tolleranza. I risultati ottenuti nel modello animale, rappresentato dai topi NOD (Non-Obese Diabetic mouse), dimostrano la possibilità di fornire un trattamento preventivo contro il diabete mediante la somministrazione di GAD65. Recentemente, un’azienda svedese, “Diamyd Medical”, ha condotto gli studi clinici di Fase II e III in uomo. Nel primo caso, è stato dimostrato che due iniezioni subcutanee da 20 µg ciascuna del farmaco in esame (GAD-alum) può revocare il progredire del T1D in pazienti di età compresa fra 10 e 18 anni in cui la malattia è insorta da breve tempo e fornire protezione contro di essa (Ludvigsson J. et al., 2008), mentre le prove cliniche di Fase III sono fallite. Sono stati proposti ulteriori studi clinici per testare la possibilità di intervento a tempi diversi rispetto all’insorgenza e sviluppo della malattia, tipi diversi di terapia e nuove modi di somministrazione dell’autoantigene. Le attuali proposte includono terapie preventive in individui ad alto rischio (attualmente in Fase II di sperimentazione clinica), terapie di combinazione che sfruttano l’associazione dell’immunoterapia non antigene-specifica (NASI) mediante un immunosoppressore, con l’immunoterapia antigene-specifica (ASI), utilizzando per esempio la GAD65, e ASI con auto antigeni multipli (Lernmark and Larsson, 2011; Larsson and Lernmark, 2011). Inoltre, la modalità di somministrazione necessita di ulteriori studi, quali, per esempio, l’induzione di tolleranza orale sfruttando la GAD65, come è stato fatto mediante la somministrazione orale dell’insulina. Viste le prove cliniche in corso o previste in futuro con l’obiettivo di testare diverse formulazioni del vaccino basate su auto antigeni del T1D, attualmente, la produzione di GAD65 umana è un punto cruciale per la pianificazione delle strategie future di prevenzione della malattia. Fino ad ora, la GAD65 umana (hGAD65) è stata ottenuta da diversi sistemi omologi ed eterologi, ma, le attuali piattaforme di produzione sono troppo costose ed incapaci di fornire quantità di auto antigene sufficienti a soddisfare la richiesta per i trattamenti di immunoterapia. Le due forme della GAD65 umana, hGAD65 e hGAD65mut, sono state, quindi, espresse nel sistema eterologo Escherichia coli per stabilire se entrambe le molecole vengono accumulate come proteine insolubili, come precedentemente già osservato nel caso della hGAD65 (Mauch L. et al., 1993), e se la produzione di hGAD65mut risulta maggiore di quella della forma nativa dell’enzima, come precedentemente descritto in piante di Nicotiana tabacum var. Sr1 (Avesani L. et al., 2010). E stato dimostrato che entrambe le forme della GAD65 umana sono accumulate in corpi di inclusione insolubili e vengono solubilizzate solo in presenza di concentrazioni denaturanti di urea. Inoltre, è stato provato che anche nel sistema batterico la hGAD65mut è prodotta in quantità maggiori rispetto alla hGAD65. I sistemi di produzione vegetali possono offrire vantaggi dal punto di vista economico e della produzione su larga scala di proteine ampiamente utilizzate in ambito terapeutico (Ma J.C.K. et al., 2005 a, b; Barasan and Rodriguez-Cerezo, 2008). Di conseguenza, il principale obiettivo del progetto di ricerca è la valutazione dell’idoneità del sistema vegetale per la produzione della GAD65 umana. La GAD65 umana è stata precedentemente espressa in piante di tabacco transgeniche, ma i livelli di produzione si sono rivelati deludenti raggiungendo valori massimi pari allo 0.25% delle proteine solubili totali (Total Soluble Proteins, TSP) (Porceddu A. et al., 1999; Ma S. et al., 2004; Wang et al., 2008; Avesani L. et al., 2003). Recentemente, una forma mutata e cataliticamente inattiva della GAD65 umana, hGAD65mut, è stata espressa in piante transgeniche di Nicotiana tabacum var. Sr1. Le piante più alte esprimenti hanno accumulato livelli di proteina ricombinante dieci volte maggiori (2.2% TSP) rispetto a quelli ottenuti nelle piante più alte esprimenti trasformate con la hGAD65 (Avesani L. et al., 2010). Tale sistema di produzione vegetale necessita di essere ulteriormente caratterizzato in modo da poter verificare l’ipotesi secondo cui le proprietà catalitiche della forma nativa della GAD65 umana possono contribuire ai bassi livelli di produzione. In studi precedenti, infatti, è stato dimostrato l’assenza di attività enzimatica in vitro per la forma mutata della GAD65. Di conseguenza, si è effettuato un saggio enzimatico in vivo con il quale è stata confermata l’assenza di attività enzimatica della stessa molecola anche nel sistema eterologo vegetale. Un secondo sistema vegetale è stato testato per la produzione di entrambe le forme della GAD65 umana. Nicotiana tabacum var. Maryland mammoth è una varietà di tabacco che produce una maggiore quantità di biomassa della più diffusa Sr1 e, quindi, potrebbe essere un eccellente candidato per la produzione di proteine ricombinanti ampiamente utilizzate in ambito terapeutico. Per questo, entrambe le forme della GAD65 umana, hGAD65 e hGAD65mut, sono state espresse in piante di tabacco transgeniche della varietà Maryland mammoth, dimostrando che, anche in tale sistema vegetale, la produzione di hGAD65mut è maggiore di quella della forma nativa dell’enzima, come precedentemente descritto in piante di tabacco transgeniche della varietà Sr1 (Avesani L. et al., 2010). L’1% delle proteine solubili totali è generalmente il livello di espressione minimo richiesto per considerare l’estrazione di proteine ad uso farmaceutico derivate da pianta economicamente vantaggiosa (Ma J.C.K. et al., 2003). Visto che tale valore è stato raggiunto e superato già nella prima generazione di piante di Nicotiana tabacum var. Sr1, si è proceduto alla messa a punto del downstream processing della proteina ricombinante dal sistema vegetale. I risultati ottenuti durante il processo di selezione della popolazione di piante esprimenti la hGAD65mut in modo stabile ed omogeneo e di studio e valutazione del protocollo di estrazione e purificazione della proteina dal tessuto fogliare sono riportati e discussi.
Abstract Type 1 insulin-dependent diabetes (T1D) which afflicts 0.03-0.04% of population is caused by autoimmune destruction of insulin-secreting beta cells, leading to an insulin deficiency (Gepts, 1965). It is considered a chronic disease with a strong social impact because of high prevalence of late-onset complications and the young age of affected patients being the most frequent chronic disease in children younger than 14-years old. Until now there are no possibilities to cure it and insulin treatment is only a life-long replacement therapy. Tolerance induction through autoantigen administration is one of the strategies useful to prevent or to slow down autoimmune diseases (Harrison, 2005). In particular, for T1D GAD65 has been evaluated as a good candidate vaccine and different Antigen-Specific Immunotherapy (ASI) studies using this autoantigen have been done to test its efficacy in tolerance induction. Results obtained in the non-obese diabetic (NOD) mouse models indicate the potential of GAD65 administration to provide a preventive treatment for diabetes. Recently, Diamyd Medical, a Swedish company, has conducted phase II and phase III clinical trials in humans: in the first case it was demonstrated that two subcutaneous injections of 20µg of alum-formulated GAD65 can reverse the progress of recent-onset T1D in 10 to 18-years-old patients and give protection against it (Ludvigsson et al., 2008), while phase III trials failed. New clinical studies have been proposed including new challenges in timing, different types of therapies and new administration routes. Current proposals include preventive therapies in high-risk individuals (current Phase II trial), combination therapies exploiting the combination of Non-Antigen Specific Immunotherapy (NASI) through an immunosoppressor, together with Antigen-Specific Immunotherapy (ASI), using for example GAD65, and ASI using multiple autoantigens (Lernmark and Larsson, 2011; Larsson and Lernmark, 2011). In addition the route of administration needs further studies, such as oral tolerance induction through GAD65, as it has been done using oral insulin. At present, human GAD65 production is a central point for planning future T1D prevention strategies because of the undergoing and future trials using different vaccine preparations based on T1D autoantigens. Until now human GAD65 (hGAD65) has been obtained from different homologous and heterologous platforms. However, actual production platforms are too expensive and unable to provide sufficient quantity of this autoantigen to meet demand for immunotherapy treatments. Both forms of human GAD65, hGAD65 and hGAD65mut, are expressed in E.coli heterologous system to sort out if they are both accumulated as insoluble proteins, as previously described for human GAD65 (Mauch L. et al., 1993), and if hGAD65mut yield is higher than that of the wild type form of the enzyme, as previously reported in Nicotiana tabacum var. Sr1 plants (Avesani L. et al., 2010). They demonstrated to be both accumulated as insoluble inclusion bodies and were solubilized by the use of denaturing concentration of urea. Western and radioimmunoassay analyses demonstrated that hGAD65mut accumulated at higher levels than hGAD65. Plant-based systems may offer advantages in terms of economy and scalability for the large-scale production of therapeutic proteins in high demand (Ma J.C.K. et al., 2005 a, b; Barasan and Rodriguez-Cerezo, 2008). Thus, the principal aim of the PhD project is the evaluation of plant-based platform feasibility for human GAD65 production. Human GAD65 has previously been expressed in transgenic tobacco plants but yields were disappointing (maximum 0.25% of total soluble protein, TSP) (Porceddu et al., 1999; Ma S. et al., 2004; Wang et al., 2008; Avesani L. et al., 2003). In a recent study, a mutated catalytically-inactive form of human GAD65 (hGAD65mut) was expressed in transgenic Nicotiana tabacum var. Sr1 plants. hGAD65mut-highest expressing plants accumulated 10-fold (2.2% TSP) higher levels of recombinant protein than hGAD65-highest expressing plants (Avesani L. et al., 2010). This plant platform production system needs to be characterized thoroughly in order to verify the hypothesis by which the catalytic properties of native hGAD65 could contribute to its poor yields. Since in previous studies it has been demonstrated in vitro the lack of the enzymatic activity for hGAD65mut, an enzymatic assay in vivo is performed in order to demonstrated the absence of enzymatic activity of the mutated form of GAD65 also in the heterologous plant-based system. Results of the assay are discussed. An additional plant-based platform is tested for the production of both forms of human GAD65. In fact, being Nicotiana tabacum var. Maryland mammoth a higher leaf biomass producing variety than the most widely used one, Sr1, it can be, potentially, an excellent candidate for the production of recombinant pharmaceutical proteins with a large demand. Both forms of human GAD65, hGAD65 and hGAD65mut, are expressed in this plant-based platform, demonstrating that also in this system hGAD65mut yield is higher than that of the wild type form of the enzyme, as previously already described in Nicotiana tabacum var. Sr1 plants (Avesani L. et al., 2010). 1% TSP yield is usually regarded as the minimum required to make the extraction of a plant-derived pharmaceutical protein economically feasible (Ma J.K.C. et al., 2003). Since this threshold was exceeded in the first generation of hGAD65mut-transgenic Nicotiana tabacum var. Sr1 plants, a protocol for downstream processing of the recombinant protein from plant systems was investigated. Results obtained during the set up of the extraction and purification protocol are discussed.