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Characteristics of GADA in Type 1 Diabetes following Immunomodulation with GAD65
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type 1 diabetes (T1D) is a serious autoimmune disease which increases worldwide and affects children at a young age, but there still is no cure available. Clinical intervention trials in recent onset T1D patients are therefore very important, since even a modest preservation of β-cell function has proven to reduce end-organ complications. Glutamic acid decarboxylase 65 (GAD65) is one of the major antigens in T1D, to which autoantibodies (GADA) are formed. Immunomodulation with aluminum-formulated GAD65 (GAD-alum) has been considered both in the prevention and intervention of T1D. In a phase II trial using GADalum we showed clinical benefits in C-peptide preservation, but unfortunately a following larger European phase III trial failed to reach primary end-point. The general aim of this thesis was to study the characteristics and phenotypes of GADA following immunomodulation with GAD-alum in T1D patients during a phase II and III trial.

In the phase II trial, a transient increase of the GADA IgG3 and IgG4 subclasses, and a decrease in IgG1 was detected as part of the treatment-induced GADA levels after 2 GADalum doses, a result interpreted to be T helper (Th) 2-associated. This Th2-associated immune response was also observed, in parallel to increased GADA levels, during the following phase III trial including a larger group of patients. However, enhanced Th2-like IgG subclass distribution, reflected as increased IgG4 frequency, was in contrast only observed in the group treated with 4 doses of GAD-alum. In addition, the GADA fold-change was associated with in vitro GAD65-stimulated cytokine secretion, but only in patients receiving 2 GAD-alum doses. Furthermore, a 4-year follow-up of the phase II trial showed that the effect of GAD-alum treatment was long-lasting as GADA titers remained elevated. Even though the phase III trial did not reach primary end-point, and was closed after 15 months, preservation of β-cell function was observed in the small sub-group of Swedish patients receiving 2 GAD-alum doses that completed the 30 months trial-period. During the trials, concerns were raised whether the elevated GADA titers might induce Stiff person syndrome (SPS), a disease affecting the nervous system, but in vitro analysis of GADA phenotypes showed that the GAD65-enzyme activity and GADA epitope distribution differed from that detected in SPS patients.

Continued research to clarify how immunomodulation with autoantigens affects immune responses and also to identify which patients are suitable for treatment, is crucial for optimizing future T1D intervention- and prevention trials.

Place, publisher, year, pages
Linköping: Linköping University Electronic Press, 2012. 83 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1337
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84842 (URN)978-91-7519-774-6 (ISBN)oai:DiVA.org:liu-84842 (OAI)diva2:562438 (DiVA)
Public defence
2012-11-15, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from2012-10-24 Created:2012-10-24 Last updated:2012-10-30Bibliographically approved
List of papers
1. GAD-alum treatment in patients with type 1 diabetes and the subsequent effect on GADA IgG subclass distribution, GAD(65) enzyme activity and humoral response
Open this publication in new window or tab >>GAD-alum treatment in patients with type 1 diabetes and the subsequent effect on GADA IgG subclass distribution, GAD(65) enzyme activity and humoral response
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2010 (English)In: Clinical Immunology, ISSN 1521-6616, E-ISSN 1521-7035, Vol. 137, no 1, 31-40Article in journal (Refereed) Published
Abstract [en]

We have previously shown that two injections of 20 mu g GAD-alum to recent onset type 1 diabetic children induced GADA levels in parallel to preservation of insulin secretion. Here we investigated if boosted GADA induced changes in IgG1, 2, 3 and 4 subclass distributions or affected GAD(65) enzyme activity. We further studied the specific effect of GAD-alum through analyses of IA-2A, tetanus toxoid and total IgE antibodies. Serum from children receiving GAD alum or placebo was collected pre-treatment and after 3, 9, 15 and 21 months. At 3 months a reduced percentage of IgG1 and increased IgG3/IgG4 were detected in GAD-alum treated. Further, IA-2A, IgE and tetanus toxoid antibodies, as well as GAD(65) enzyme activity, were unaffected confirming the specific effect of treatment. In the GAD-alum group, higher pretreatment GADA were associated to more pronounced C-peptide preservation. The induced IgG3/IgG4 and reduced IgG1 suggest a Th2 deviation of the immune response.

Publisher, range
Elsevier Science B.V., Amsterdam, 2010
Keyword
Immunotherapy, GAD(65), GAD-alum, GADA, Type 1 diabetes, T1D, IgG, IgG subclass
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-60511 (URN)10.1016/j.clim.2010.06.001 (DOI)000282204900005 ()
Note
<p>Original Publication: Mikael Chéramy, Camilla Skoglund, Ingela Johansson, Johnny Ludvigsson, Christiane S Hampe and Rosaura Casas, GAD-alum treatment in patients with type 1 diabetes and the subsequent effect on GADA IgG subclass distribution, GAD(65) enzyme activity and humoral response, 2010, CLINICAL IMMUNOLOGY, (137), 1, 31-40. http://dx.doi.org/10.1016/j.clim.2010.06.001 Copyright: Elsevier Science B.V., Amsterdam http://www.elsevier.com/</p>Available from2010-10-15 Created:2010-10-15 Last updated:2013-09-12Bibliographically approved
2. Characteristics of in-vitro phenotypes of glutamic acid decarboxylase 65 autoantibodies in high-titre individuals
Open this publication in new window or tab >>Characteristics of in-vitro phenotypes of glutamic acid decarboxylase 65 autoantibodies in high-titre individuals
2013 (English)In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 171, no 3, 247-254Article in journal (Refereed) Published
Abstract [en]

Previous studies have indicated phenotypical differences in glutamic acid decarboxylase 65 autoantibodies (GADA) found in type 1 diabetes (T1D) patients, individuals at risk of developing T1D and stiff-person syndrome (SPS) patients. In a Phase II trial using aluminium-formulated GAD65 (GAD-alum) as an immunomodulator in T1D, several patients responded with high GADA titres after treatment, raising concerns as to whether GAD-alum could induce GADA with SPS-associated phenotypes. This study aimed to analyse GADA levels, immunoglobulin (Ig)G1–4 subclass frequencies, b78- and b96·11-defined epitope distribution and GAD65 enzyme activity in sera from four cohorts with very high GADA titres: T1D patients (n = 7), GAD-alum-treated T1D patients (n = 9), T1D high-risk individuals (n = 6) and SPS patients (n = 12). SPS patients showed significantly higher GADA levels and inhibited the in-vitro GAD65 enzyme activity more strongly compared to the other groups. A higher binding frequency to the b78-defined epitope was found in the SPS group compared to T1D and GAD-alum individuals, whereas no differences were detected for the b96·11-defined epitope. GADA IgG1–4 subclass levels did not differ between the groups, but SPS patients had higher IgG2 and lower IgG4 distribution more frequently. In conclusion, the in-vitro GADA phenotypes from SPS patients differed from the T1D- and high-risk groups, and GAD-alum treatment did not induce SPS-associated phenotypes. However, occasional overlap between the groups exists, and caution is indicated when drawing conclusions to health or disease status.

Publisher, range
John Wiley & Sons, 2013
Keyword
GAD65 immunotheraphy; GADA; stiff-person syndrome; type 1 diabetes
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84840 (URN)10.1111/cei.12026 (DOI)000314655100003 ()
Note
<p>The title of the article in manuscript form was "<em>Characteristics of GAD<sub>65</sub> autoantibodies (GADA) in high titer individuals</em>".</p>Available from2012-10-24 Created:2012-10-24 Last updated:2013-03-14Bibliographically approved
3. Long-Lasting Immune Responses 4 Years after GAD-Alum Treatment in Children with Type 1 Diabetes
Open this publication in new window or tab >>Long-Lasting Immune Responses 4 Years after GAD-Alum Treatment in Children with Type 1 Diabetes
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2011 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 6, no 12Article in journal (Refereed) Published
Abstract [en]

A phase II clinical trial with glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) has shown efficacy in preserving residual insulin secretion in children and adolescents with recent-onset type 1 diabetes (T1D). We have performed a 4-year follow-up study of 59 of the original 70 patients to investigate long-term cellular and humoral immune responses after GAD-alum-treatment. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with GAD(65). Frequencies of naive, central and effector memory CD4+ and CD8+ T cells were measured, together with cytokine secretion, proliferation, gene expression and serum GAD(65) autoantibody (GADA) levels. We here show that GAD-alum-treated patients display increased memory T-cell frequencies and prompt T-cell activation upon in vitro stimulation with GAD(65), but not with control antigens, compared with placebo subjects. GAD(65)-induced T-cell activation was accompanied by secretion of T helper (Th) 1, Th2 and T regulatory cytokines and by induction of T-cell inhibitory pathways. Moreover, post-treatment serum GADA titres remained persistently increased in the GAD-alum arm, but did not inhibit GAD(65) enzymatic activity. In conclusion, memory T- and B-cell responses persist 4 years after GAD-alum-treatment. In parallel to a GAD(65)-induced T-cell activation, our results show induction of T-cell inhibitory pathways important for regulating the GAD(65) immunity.

Publisher, range
Public Library of Science, 2011
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-74156 (URN)10.1371/journal.pone.0029008 (DOI)000298366600057 ()
Note
Funding Agencies|Swedish Research Council|K2008-55x-20652-01-3|Swedish Child Diabetes Foundation (Barndiabetesfonden)||Medical Research Council of Southeast Sweden||Juvenile Diabetes Research Foundation (JDRF)|1-2008-106|Ile-de-France CODDIM||Inserm Avenir Program||Available from2012-01-20 Created:2012-01-20 Last updated:2012-10-24
4. GAD65 autoantibody (GADA) responses in Type 1 diabetes patients participating in a phase III GAD-alum intervention trial
Open this publication in new window or tab >>GAD65 autoantibody (GADA) responses in Type 1 diabetes patients participating in a phase III GAD-alum intervention trial
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Glutamic acid decarboxylase 65 kDa isoform (GAD65) is a major autoantigen in type 1 diabetes (T1D). Although aluminum-formulated GAD65 (GAD-alum) induced preservation of residual insulin secretion in a previous clinical phase II trial, recent phase II and III trials failed to reach their primary end-points. The European phase III trial was therefore closed after 15 months, and the entire study period was completed only for a minority of the patients. This study aimed to characterize GAD65 autoantibodies (GADA) and Tyrosine phosphatase IA-2 autoantibody (IA-2A) levels, GADA IgG1-4 subclass distribution, B-cell frequencies/phenotypes and cytokine secretion. We also assessed whether GAD-alum preserved β-cell function in the small subgroup of Swedish patients who completed the 30 months visit. Serum samples and peripheral blood mononuclear cells (PBMC) were collected at baseline and after 1, 3, 9, 15 and 21 months from the 148 Swedish subjects included in the trial, and also at 30 months from the 45 patients who reached the final visit. Patients were randomly assigned to; i) 4 doses of GAD-alum (4D), ii) 2 doses of GAD-alum followed by two doses of placebo (2D), or iii) 4 doses of placebo.

GADA titers were induced both in the 4D and 2D group compared to placebo, and 4D patients also displayed a higher GADA fold-change after receiving the  two additional injections compared to the 2D group. The 4D group switched to a higher frequency of GADA IgG4, associated to a Th2 type response at 9 months, whereas an association between GADA fold-change and GAD65-induced in vitro cytokine secretion was observed in the 2D group. These findings suggest that the humoral response, induced by the 2D treatment,  seems to be associated with a GAD65-specific cellular response, while 4D induces a distinct humoral response. Even though GADA titers were elevated, no changes in B-cell frequencies or phenotype were observed in any group. IA-2A levels declined at a similar rate in all groups during the trial.The subgroup of patients who completed the 30 month visit receiving 2 doses of GAD-alum had less decline of both fasting and stimulated C-peptide after 30 months compared to placebo. These results support the concept of GAD-alum treatment, but no specific immune markers have been identified.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84841 (URN)
Available from2012-10-24 Created:2012-10-24 Last updated:2012-10-24Bibliographically approved

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