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The Importance of Cell-Mediated Immunity for the Development of Type 1 Diabetes
Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background Type I (insulin-dependent) diabetes mellitus is an autoimmune disease characterised by infiltration of T-lymphocytes in the islets of Langerhans. In particular, activated Th1-like lymphocytes secreting IFN-γ are suggested to contribute to the inflammatory process and the destruction of ß-cells, whereas Th2-like cells producing IL-4 might even be protective. Environmental factors (diet, viruses, stress etc.) and autoantigens, e.g. Glutamic Acid Decarboxylase (GAD65) and insulin, are suggested to initiate the autoimmune process resulting in type I diabetes.

Aim To estimate the immunological balance of Th1/Th2-like lymphocytes, spontaneously and after stimulation with antigens, in high-risk first degree relatives of type 1 diabetic children and in children with newly diagnosed type 1 diabetes.

Materials and methods Peripheral blood mononuclear cells (PBMC) from healthy high-risk first-degree relatives (ICA ≥ 20) and newly diagnosed type 1 diabetic children were examined and compared with the response seen in PBMC from healthy controls matched for age and HLA-type (DR3 and/or DR4).

Expression of IFN-γ and IL-4 mRNA was determined by RT-PCR or real-time RTPCR and IFN-γ and IL-4 by ELISPOT or ELISA, spontaneously and after stimulation with GAD65 , insulin, bovine serum albumin (BSA), the ABBOS-peptide and ß-lactoglobulin (ßLG). Cytokine expression and secretion was compared to the production of diabetes-associated autoantibodies and to the secretion of endogenous insulin.

Results The epitope of GAD65, that mimics the Coxsackie B virus, caused increased IFN-γ mRNA expression in activated Th1-like lymphocytes from newly diagnosed diabetic children. This suggests that GAD65 might be involved in the development of type I diabetes. On the contrary, cow's milk proteins caused increased IFN-γ and IL- 4 mRNA expression in activated Th1- and Th2-like lymphocytes from both diabetic and healthy children. This does not support the hypothesis that cow's milk antigens are important for the development of type 1 diabetes.

Overwhelming secretion of IFN-γ was observed in high-risk first-degree relatives of type 1 diabetic children. High-risk individuals still have the ability to change a Th1-like immune deviation into a more protective Th2-like response in the presence of GAD65 and insulin.

Conclusions GAD65, but not cow's milk proteins, causes a Th1-like deviation in type 1 diabetic children. High-risk individuals are capable to deviate a Th1-like immune system into a more protective Th2-like response in the presence of autoantigens. These results can be useful in future therapeutic approaches.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2000. , 130 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 644
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28590Local ID: 13744ISBN: 91-7219-745-5 (print)OAI: oai:DiVA.org:liu-28590DiVA: diva2:249401
Public defence
2000-10-20, Berzeliussalen, Hälsouniversitet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-08-14Bibliographically approved
List of papers
1. Determination of mRNA expression for IFN-γ and IL-4 in lymphocytes from children with IDDM by RT-PCR technique
Open this publication in new window or tab >>Determination of mRNA expression for IFN-γ and IL-4 in lymphocytes from children with IDDM by RT-PCR technique
1998 (English)In: Diabetes Research and Clinical Practice, ISSN 0168-8227, E-ISSN 1872-8227, Vol. 40, no 1, 21-30 p.Article in journal (Refereed) Published
Abstract [en]

Insulin-dependent diabetes mellitus (IDDM) is characterized by infiltration of T-lymphocytes in the islets of Langerhans. Antigens are presented to Th-lymphocytes which can be divided into Th1- and Th2-lymphocytes, producing interferon-γ (IFN-γ) and interleukin-4 (IL-4) respectively. The aim of our study was to determine the messenger-RNA (mRNA) for these cytokines by RT-PCR in antigen-stimulated lymphocytes from children with newly diagnosed IDDM. The expression of mRNA for IL-4, and to a lesser degree IFN-γ, is increased in lymphocytes stimulated with tetanus toxoid (TT). Loss of activity after freezing and thawing could be compensated for, by increased amplification, while the use of EDTA or sodium heparin in the blood samples did not influence the results. In a pilot application, the lymphocytes from children with newly diagnosed IDDM were stimulated with a peptide of glutamic acid decarboxylase (GAD) (a.a. 247–279) known to have a similar aminoacid sequence as the Coxsackie B virus (a.a. 32–47). Increased IFN-γ mRNA could be seen in two out of four children, whereas IL-4 showed a less pronounced mRNA expression. No increased mRNA expression for IFN-γ and IL-4 could be seen in healthy HLA-matched controls. Further studies are needed to confirm whether increased IFN-γ mRNA in Th1-like lymphocytes stimulated with this specific GAD-peptide play a role in the cell-mediated immune response seen in children early after the onset of IDDM.

Place, publisher, year, edition, pages
Elsevier, 1998
Keyword
IDDM, Th-lymphocytes, IFN-γ, IL-4, mRNA, Glutamic acid decarboxylase
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79850 (URN)10.1016/S0168-8227(98)00014-X (DOI)9699087 (PubMedID)
Available from: 2012-08-14 Created: 2012-08-14 Last updated: 2017-12-07Bibliographically approved
2. Peptide from glutamic acid decarboxylase similar to coxsackie B virus stimulates IFN- γ mRNA expression in Th1-like lymphocytes from children with recent-onset insulin-dependent diabetes mellitus
Open this publication in new window or tab >>Peptide from glutamic acid decarboxylase similar to coxsackie B virus stimulates IFN- γ mRNA expression in Th1-like lymphocytes from children with recent-onset insulin-dependent diabetes mellitus
1998 (English)In: Acta Diabetologica, ISSN 0940-5429, E-ISSN 1432-5233, Vol. 35, no 3, 137-144 p.Article in journal (Refereed) Published
Abstract [en]

At the clinical onset of insulin-dependent diabetes mellitus (type 1 diabetes), inflammation within the pancreatic islets of Langerhans causes insulitis. CD4+ or Th-lymphocytes will be activated after stimulation resulting in interferon-gamma (IFN-γ) production by Th1-like lymphocytes and/or interleukin-4 (IL-4) secretion from Th2-like lymphocytes. The antigens responsible for this activation are unknown, but studies have suggested glutamic acid decarboxylase (GAD) to be a possible candidate. One peptide from this enzyme (amino acid 247–279) with a similar amino acid sequence to coxsackie B virus may cause lymphocyte proliferation in diabetic patients. In this study we have shown that this peptide activates Th1-like lymphocytes which produce increased amounts of IFN-γ mRNA, but seldom mRNA for IL-4. Lymphocytes from healthy HLA-matched controls (DR3/4) did not respond with an upregulated mRNA expression for these cytokines when stimulated by the GAD-peptide (P<0.05). A low or absent expression of IFN-γ mRNA was significantly correlated to a high fasting C-peptide at 3 months' duration (P<0.05). In conclusion, we suggest that GAD65 is involved in the development of type 1 diabetes and that the Th1-response may play a role in the destruction of β cells.

Keyword
Th-lymphocytes, IFN-γ, mRNA, GAD65, coxsackie B virus
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79851 (URN)10.1007/s005920050118 (DOI)9840449 (PubMedID)
Available from: 2012-08-14 Created: 2012-08-14 Last updated: 2017-12-07Bibliographically approved
3. The ABBOS-peptide from bovine serum albumin causes an IFN-γ and IL-4 mRNA response in lymphocytes from children with recent onset of type 1 diabetes
Open this publication in new window or tab >>The ABBOS-peptide from bovine serum albumin causes an IFN-γ and IL-4 mRNA response in lymphocytes from children with recent onset of type 1 diabetes
2000 (English)In: Diabetes Research and Clinical Practice, ISSN 0168-8227, E-ISSN 1872-8227, Vol. 47, no 3, 199-207 p.Article in journal (Refereed) Published
Abstract [en]

The ABBOS-peptide from bovine serum albumin (BSA) in cow’s milk has been suggested to initiate the autoimmune process against the β-cells leading to type 1 diabetes. The aim of this study was to elucidate if the ABBOS-peptide is a possible trigger of type 1 diabetes. The cytokines IL-4 and IFN-γ were determined at the level of transcription as mRNA in lymphocytes, stimulated with the ABBOS-peptide. Sixteen children with newly diagnosed type 1 diabetes were compared with 10 healthy controls matched for the diabetes associated HLA-type DR3/4. Antibodies to bovine serum albumin (BSA), insulin antibodies (IA), and antibodies against islet cells (ICA) were determined, as well as serum C-peptide. Increased mRNA expression for IFN-γ and/or IL-4 could be observed in lymphocytes from 13/16 children with recent onset of diabetes after in vitro stimulation with the ABBOS-peptide. Low expression of IFN-γ mRNA was associated with high secretion of C-peptide, whereas a positive relationship could be observed between expression of IL-4 mRNA and insulin antibodies. Expression of IFN-γ and/or IL-4 mRNA was also detected in lymphocytes from 6/10 healthy controls. ABBOS may have a role as a reactive epitope in the upregulation of the autoimmune process against the β-cells but ABBOS does not seem to cause any specific Th1 response. An increased mRNA expression could also be seen in lymphocytes from healthy controls. Thus, the ABBOS-peptide might just cause or reflect an unspecific immune activity.

Keyword
ABBOS, IFN-γ, IL-4, Type 1 diabetes, Th-lymphocytes
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25986 (URN)10.1016/S0168-8227(99)00127-8 (DOI)10438 (Local ID)10438 (Archive number)10438 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
4. Cows' milk proteins cause similar Th1- and Th2-like immune response in diabetic and healthy children
Open this publication in new window or tab >>Cows' milk proteins cause similar Th1- and Th2-like immune response in diabetic and healthy children
2001 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 44, no 9, 1140-1147 p.Article in journal (Refereed) Published
Abstract [en]

Aims/hypothesis:   Cows' milk proteins have been proposed to play a part in the pathogenesis of Type I (insulin-dependent) diabetes mellitus but both epidemiological and immunological studies have given conflicting results. Thus we aimed to study the immunological response to cows' milk proteins among diabetic and healthy children, focusing on the balance of Th1- and Th2-like lymphocytes.

Methods:   Peripheral blood mononuclear cells from 30 Type I diabetic children (4 to 18 years old) were examined and compared with peripheral blood mononuclear cells from 18 healthy age-matched control children (7 to 15 years old). Expression of IFN-γ and IL-4 mRNA were detected by realtime RT-PCR and as protein by ELISA after stimulation with BSA, the ABBOS-peptide (a. a. 152–169) and β-lactoglobulin (βLG) from cows' milk and ovalbumin from hens' egg. Phytohaemagglutinin and keyhole limpet haemocyanin were used as positive and negative controls, respectively.

Results:   Bovine serum albumin caused a weak Th2-like response in Type I diabetic children, whereas BSA antibodies decreased with age only among healthy children. Otherwise, cows' milk proteins (BSA, ABBOS and βLG) caused increased expression for IFN-γ and IL-4 mRNA in diabetic and healthy children. βLG caused the strongest immunological response, which decreased with age only among diabetic children. However, ovalbumin from egg caused a similar activation of the immune system and the immune response was similar in both diabetic and healthy children.

Conclusion/interpretation:   Proteins from cows' milk caused an equal Th1- and Th2-like immune response in diabetic and healthy children. Thus, our results do not support the hypothesis that cows' milk antigens are important for the immune process associated with Type I diabetes.

Keyword
Th-lymphocytes, IFN-γ, IL-4, bovine serum albumin, ABBOS, β-lactoglobulin, real-time RT-PCR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25928 (URN)10.1007/s001250100611 (DOI)10370 (Local ID)10370 (Archive number)10370 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
5. Th1-like dominance in high-risk first-degree relatives of Type I diabetic patients
Open this publication in new window or tab >>Th1-like dominance in high-risk first-degree relatives of Type I diabetic patients
2000 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 43, no 6, 742-749 p.Article in journal (Refereed) Published
Abstract [en]

Aims/hypothesis. The humoral part of the immune system, including autoantibodies, is known to predict manifest Type I (insulin-dependent) diabetes mellitus in first-degree relatives but the cell-mediated immune process preceding the manifest disease still is not known. The aim of this investigation was to estimate the immunological balance of Th-like lymphocytes (Th1/Th2) in high-risk first-degree relatives of Type I diabetic children.

Methods. Peripheral blood mononuclear cells (PBMC) from 21 healthy high-risk first-degree relatives (ICA ≥ 20) were examined and compared with the response seen in PBMC from children with newly diagnosed Type I diabetes and healthy control subjects of similar age, sex and HLA-type. Expression of interleukin-4 (IL-4) and interferon-γ (IFN-γ) mRNA were determined by RT-PCR and as protein by ELISPOT after stimulation with specific epitopes of GAD65 (a. a. 247–279, 509–528, 524–543), bovine serum albumin, the ABBOS peptide (a. a. 152–169) and insulin.

Results. High-risk relatives had a high ratio of IFN-γ:IL-4 compared with both diabetic children (p = 0.0005) and healthy control subjects (p = 0.004). Production of IFN-γ seen in high-risk relatives was negatively correlated to production of GADA (r = –0.44, p = 0.05). The high concentration of IFN-γ from high-risk relatives, decreased after stimulation with peptides of GAD65, the ABBOS peptide, BSA and insulin. Increased secretion of IL-4 was observed after stimulation with two peptides of GAD65 (a. a. 509–528 and 524–543), the ABBOS peptide and insulin.

Conclusion/interpretation. Overwhelming production of IFN-γ seen in peripheral blood mononuclear cells from high-risk first-degree relatives of children with Type I diabetes suggests a Th1-like immune deviation in the prediabetic phase.

Keyword
Type I diabetes, Th-lymphocyte, high-risk relatives, IFN-γ, IL-4, GAD65, ABBOS, BSA, insulin, GADA.
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25985 (URN)10.1007/s001250051372 (DOI)10437 (Local ID)10437 (Archive number)10437 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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