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Determination of mRNA expression for IFN-γ and IL-4 in lymphocytes from children with IDDM by RT-PCR technique
Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
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. Vol. 40, no 1, 21-30 p.
Keyword [en]
IDDM, Th-lymphocytes, IFN-γ, IL-4, mRNA, Glutamic acid decarboxylase
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-79850DOI: 10.1016/S0168-8227(98)00014-XPubMedID: 9699087OAI: oai:DiVA.org:liu-79850DiVA: diva2:544403
Available from: 2012-08-14 Created: 2012-08-14 Last updated: 2017-12-07Bibliographically approved
In thesis
1. The Importance of Cell-Mediated Immunity for the Development of Type 1 Diabetes
Open this publication in new window or tab >>The Importance of Cell-Mediated Immunity for the Development of Type 1 Diabetes
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:nbn:se:liu:diva-28590 (URN)13744 (Local ID)91-7219-745-5 (ISBN)13744 (Archive number)13744 (OAI)
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

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