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  • 1.
    Axelsson, Stina
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics .
    Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics .
    Hedman, Maria
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Ludvigsson, Johnny
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Casas, Rosaura
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics .
    Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children2008In: Cryobiology, ISSN 0011-2240, E-ISSN 1090-2392, Vol. 57, no 3, p. 201-208Article in journal (Refereed)
    Abstract [en]

    Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD(65) or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-gamma and TNF-alpha) and chemokines (IP-10, MCP-1, MIP-1 alpha, MIP-1 beta and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-beta mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-gamma and MCP-1, and mRNA expression of FOXP3 and TGF-beta, was detected after cryopreservation. Stimulation with GAD65 induced higher levels of IL-6, IFN-gamma, TNF-alpha and MIP-1 alpha, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCPA and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.

  • 2. Bohmova, K
    et al.
    Hladikova, Z
    Cerny, M
    Flajsmanova, K
    Vrabelova, Z
    Skramlikova, T
    Spalova, I
    Cerna, M
    Chudoba, D
    Pithova, P
    Stadlerova, G
    Bartaskova, D
    Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Stechova, K
    Cord blood cytokine profile detection in neonates with T1D parents - Monitoring of cellular auto-reactivity using protein microarray2007In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 66, no 5, p. 563-571Article in journal (Refereed)
    Abstract [en]

    Type 1 diabetes (T1D) is a great medical challenge and its incidence rises rapidly. T lymphocytes and their cytokine production are supposed to play a major role in T1D development. So far, there is no potent tool to recognize the early signs of cellular auto-reactivity which leads to β-cell damage. The naïve immune system of the newborn (not yet influenced by external factors) can be used as an important model for T1D pathogenesis studies. Cord blood samples of 22 healthy neonates born at term to a diabetic parent (T1DR) and 15 newborns with no family history of any autoimmune disease (controls) were collected. Determination of 23 cytokines was performed before and after the stimulation with diabetogenic autoantigens using protein microarray. We observed lower basal production of all detected cytokines in the T1DR group - granulocyte/macrophage colony-stimulating factor (GM-CSF) (P = 0.025), growth regulated protein (GRO) (P = 0.002), GRO-α (P = 0.027), interleukin (IL)-1-α (P = 0.051), IL-3 (P = 0.008), IL-7 (P = 0.027), IL-8 (P = 0.042), monocyte chemoattractant proteins (MCP)-3 (P = 0.022), monokine-induced by IFN-γ (MIG) (P = 0.034) and regulated upon activation normal T-cell express sequence (RANTES) (P = 0.004). Exclusively lower post-stimulative levels of G-CSF (P = 0.030) and GRO-α (P = 0.04) were observed in controls in comparison with the basal levels. A significant post-stimulative decrease in G-CSF (P = 0.030) and MCP-2 (P = 0.009) levels was observed in controls in comparison with T1DR neonates. We also observed the interesting impact of the risky genotype on the protein microarray results. Protein microarray seems to be a useful tool to characterize a risk pattern of the immune response for T1D also in newborns. © 2007 The Authors.

  • 3.
    Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Type 1 diabetes - probably a T-helper-1 associated disease, but when? Before, at or after the clinical onset of disease?2007In: Curr. Res. in Immunology, Vol. 1, p. 159-170Article in journal (Refereed)
  • 4.
    Faresjö, Maria
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Ernerudh, Jan
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Immunology and Transfusion Medicine.
    Berlin, Gösta
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Transfusion Medicine. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Immunology and Transfusion Medicine.
    Garcia, Jorge
    Ludvigsson, Johnny
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    The immunological effect of photopheresis in children with newly diagnosed type 1 diabetes2005In: Pediatric Research, ISSN 0031-3998, E-ISSN 1530-0447, Vol. 58, no 3, p. 459-466Article in journal (Refereed)
    Abstract [en]

    Photopheresis has been claimed to have immune-modulating effects, but the mechanisms of action are unknown. This study investigated the immune effect of photopheresis in children with type 1 diabetes, with a focus on the balance of Th1- and Th2-like cytokines. Ten children with newly diagnosed type 1 diabetes (10-17 y) were treated with five double treatments of photopheresis and 10 children matched for disease, age, and gender were given placebo tablets and sham pheresis. Expression of IFN-γ and IL-4 mRNA was determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and secretion of IFN-γ, IL-10, and IL-13 in cell-culture supernatants by ELISA after stimulation with glutamic acid decarboxylase (GAD65) (a.a. 247-279), the ABBOS peptide (a.a. 152-169), insulin, phytohemagglutinin (PHA), and keyhole limpet hemocyanin (KLH). Photopheresis changed antigen-stimulated immune balance in line with a Th2-like shift. Thus, the ratio of IFN-γ/IL-4 mRNA expression after in vitro stimulation with a peptide of the autoantigen GAD 65 was reduced after treatment in the photopheresis group. The IFN-γ/IL-4 mRNA expression ratio after in vitro stimulation with insulin was also lower in children treated with photopheresis compared with the placebo group. Photopheresis has an immune-modulating effect in children with type 1 diabetes, causing a Th2-like deviation. Copyright © 2005 International Pediatric Research Foundation, Inc.

  • 5.
    Faresjö, Maria
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Ernerudh, Jan
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Immunology and Transfusion Medicine.
    Ludvigsson, Johnny
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Cytokine profile in children during the first 3 months after the diagnosis of type 1 diabetes2004In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 59, no 5, p. 517-526Article in journal (Refereed)
    Abstract [en]

    Type 1 diabetes is an autoimmune disease with an inflammatory process directed against the β cells in pancreas. This investigation aimed at studying the immune response during the first 3 months after the diagnosis of type 1 diabetes, with focus on the balance of T-helper 1 (Th1)- and Th2-like cytokines, produced spontaneously and in response to relevant autoantigens. Peripheral blood mononuclear cells (PBMCs) were collected from type 1 diabetic children (10-17 years) at 5, 20, 35 and 90 days after diagnosis. Expression of interferon-γ (IFN-γ) and interleukin-4 (IL-4) mRNA were detected by real-time reverse transcriptase polymerase chain reaction and IFN-γ, IL-10 and IL-13 by enzyme-linked immunosorbent assay in cell supernatant after stimulation with a glutamic acid decarboxylase 65 (GAD65)-peptide [amino acid (a.a.) 247-279], insulin, the ABBOS-peptide (a.a. 152-169), phytohaemagglutinin and keyhole limpet haemocyanin. Spontaneous and antigen-induced expression and secretion of cytokines were low at the diagnosis of type 1 diabetes. During the first month, after diagnosis, the GAD 65-peptide caused an increased ratio of IFN-γ/IL-4 mRNA expression (P < 0.05) and increased secretion of IFN-γ (P = 0.07). Expression of IFN-γ mRNA did also increase from stimulation with insulin (P < 0.05), even though cytokine secretion remained low. Thus, duration after diagnosis as well as metabolic state should be carefully considered both in studies of the pathogenesis of type 1 diabetes and in immune intervention studies at onset.

  • 6.
    Faresjö, Maria
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Ludvigsson, Johnny
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Diminished Th1-like response to autoantigens in children with a high risk of developing type 1 diabetes2005In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 61, no 2, p. 173-179Article in journal (Refereed)
    Abstract [en]

    The exact role of T-helper (Th) cells that precede the clinical manifestation of type 1 diabetes remains unclear. The aim of this investigation was to study the Th1- and Th2-like profile in children and adults with high risk of developing the disease. Peripheral blood mononuclear cells were collected from high-risk children and adults and from healthy individuals matched for age and gender. Using the sensitive enzyme-linked immunospot (ELISPOT) technique to divide Th1- from Th2-like lymphocytes, secretion of interferon-γ (IFN-γ) and interleukin-4 was analysed from lymphocytes spontaneously and after in vitro stimulation with different antigens, based on present paradigms regarding the pathogenesis of type 1 diabetes. Compared to the response observed in healthy individuals, we found that individuals with a high risk of developing type 1 diabetes, especially children, responded with less IFN-γ secretion to the three autoantigens glutamic acid decarboxylase 65 (GAD 65), insulin and tyrosinphosphatase (IA-2). Thus, a diminished Th1-like response by in vitro autoantigen stimulation was observed in especially children with a high risk of developing type 1 diabetes. Reduced Th1/Th2 response was related to signs of β cell exhaustion.

  • 7.
    Hedman Hjorth, Maria
    et al.
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Axelsson, Stina
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Ludvigsson, Johnny
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Casas, Rosaura
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Impaired CD4+ and CD8+ T cell phenotype and reduced chemokine secretion in recent-onset type 1 diabetic children2008In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 153, no 3, p. 360-368Article in journal (Refereed)
    Abstract [en]

    Although the role of the T cell-mediated autoimmune reaction in type 1 diabetes (T1D) is conclusive, studies including data from human circulating CD4+ and CD8+ lymphocytes subsets during the disease onset and posterior development are scarce. Further, chemokines and chemokine receptors are key players in the migration of pathogenic T cells into the islets of NOD mice developing T1D, but few studies have investigated these markers in human T1D patients. We studied the expression of T helper 1 (Th1) and Th2 associated chemokine receptors, and the two isoforms of CD45 leukocyte antigen on CD4+ and CD8+ lymphocytes from T1D and healthy children, as well as the secretion of chemokines in cell supernatants in peripheral blood mononuclear cells. Our results showed increased expression of CCR7 and CD45RA, and reduced CD45RO on CD8+ cells among recent-onset T1D patients. The percentages of CD4+ cells expressing CXC chemokine receptor 3 (CXCR3), CXCR6 and CCR5, and the secretion of interferon-γ-induced protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1a and MIP-1β was lower among diabetics. Low expression of Th1-associated receptors and secretion of chemokines, together with an increased amount of CD8+ cells expressing CD45RA and CCR7 in T1D patients therefore might represent suboptimal Th function in T1D, leading to impaired T cytotoxic (Tc) responses or alternatively reflect a selective recruitment of Th1 cells into the pancreas.

  • 8.
    Hedman Hjorth, Maria
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Ludvigsson, Johnny
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Nicotinamide reduces high secretion of IFN-γ in high-risk relatives even though it does not prevent type 1 diabetes2006In: Journal of Interferon and Cytokine Research, ISSN 1079-9907, E-ISSN 1557-7465, Vol. 26, no 4, p. 207-213Article in journal (Refereed)
    Abstract [en]

    Type 1 diabetes (T1D) is an autoimmune disease suggested to be of a T helper (Th)1-like origin. This study aimed to investigate the Th1-like and Th2-like profile in high-risk individuals during the prediabetic phase and the immunologic effect of treatment with nicotinamide. High-risk first-degree relatives of T1D patients participating in the European Nicotinamide Diabetes Intervention Trial (ENDIT) were treated with either nicotinamide or placebo. Peripheral blood mononuclear cells (PBMC) were obtained during the prediabetic phase and close to the onset of manifest T1D and from nondiabetic high-risk individuals. Using the sensitive enzyme-linked immunospot (ELISPOT) technique to distinguish Th1-like from Th2-like lymphocytes, secretion of interferon-γ (IFN-γ) and interleukin-4 (IL-4) was analyzed from PBMCs spontaneously and after in vitro stimulation with the diabetes-associated autoantigens, glutamic acid decarboxylase 65 (GAD65, protein and peptide, aa 247-279), recombinant tyrosine phosphatase (IA-2), and heat shock protein (HSP, aa 437-460). High-risk individuals showed high spontaneous as well as autoantigen-induced IFN-γ secretion. Secretion of IFN-γ and the IFN-γ/IL-4 ratio, induced by autoantigens, decreased in individuals developing T1D (p < 0.05), whereas nondiabetic individuals showed an increased IL-4 response (p < 0.05). Thus, a Th1-dominated cytokine profile observed in high-risk individuals inclined toward a diagnosis of diabetes. Nicotinamide caused decreased spontaneous (p = 0.05) and in vitro autoantigen-induced IFN-γ secretion (p < 0.05) and may play a role in immune regulation, even though it has not been shown to prevent T1D. © Mary Ann Liebert, Inc.

  • 9.
    Hjorth, Maria
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Axelsson, Stina
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Ryden, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Faresjo, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Ludvigsson, Johnny
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Casas, Rosaura
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    GAD-alum treatment induces GAD(65)-specific CD4(+)CD25(high)FOXP3(+) cells in type 1 diabetic patients2011In: CLINICAL IMMUNOLOGY, ISSN 1521-6616, Vol. 138, no 1, p. 117-126Article in journal (Refereed)
    Abstract [en]

    Type 1 diabetes results from autoimmune destruction of insulin producing pancreatic beta-cells. We have shown that treatment with alum-formulated glutamic acid decarboxylase 65 (GAD-alum) preserved residual insulin secretion and induced antigen-specific responses in children with recent onset type 1 diabetes. The aim of this study was to further investigate the immunomodulatory effect of GAD-alum, focusing on CD4(+)CD25(high) cells and their association to cytokine secretion. Samples obtained 21 and 30 months after the initial injection of GAD-alum or placebo were included in the present study. GAD(65)-stimulation enhanced the percentage of CD4(+)CD25(high)FOXP3(+) cells, but reduced the percentage of CD4(+)CD25(+) cells, in samples from the GAD-alum treated group. Further, the GAD(65)-induced secretion of IL-5, -10, and -13 correlated with the expression of CD4(+)CD25(high)FOXP3(+) cells, but inversely with CD4(+)CD25(+) cells. These new data suggest that GAD-alum treatment induced GAD(65)-specific T cells with regulatory features.

  • 10.
    Jonson, Carl-Oscar
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Hedman, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Casas, Rosaura
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Ilonen, Jorma
    JDRF Center for Prevention of Type 1 Diabetes in Finland and Department of Virology, University of Turku, Turku, Finland .
    Ludvigsson, Johnny
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Vaarala, Outi
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    CTLA-4 Polymorfism, Type 1 Diabetes-risk Human Leukocyte Antigen-genotypes, insulin gene polymorphism and Regulatory T-cell Marker Expression in 5-year-old children2006In: Clinical & Experimental Immunology, ISSN 0009-9104, Vol. 145, no 1, p. 48-55Article in journal (Refereed)
    Abstract [en]

    Regulatory T cells (Treg) are involved in the maintenance of peripheral tolerance by suppression of autoreactive lymphocytes that have avoided thymic depletion. The defective function of Treg cells has recently attracted attention in autoimmune diseases such as type 1 diabetes (T1D), rheumatoid arthritis and multiple sclerosis. Susceptibility to these diseases is associated with specific human leucocyte antigen (HLA) class II and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) gene polymorphisms. This study aimed to investigate the relationship between HLA class II and CTLA +49 A/G polymorphisms associated with susceptibility to T1D and the number and characteristics of Treg cells in children. Samples from 47 5-year-old children who participated in the All Babies in South-east Sweden (ABIS) follow-up study were grouped according to the presence of the T1D risk-associated HLA genotype (DQA1*0501–DQB1*0201, DQA1*0301–DQB1*0302) or neutral HLA genotypes. Lower percentages of CD4+ T cells (= 0·03) and CD4+ CD25high cells (= 0·06) expressing intracellular CTLA-4 were detected in samples from children with CTLA-4 +49GG compared to children with the +49AA genotype. Similarly, lower percentages of CD4+ (= 0·002) and CD4+ CD25high (= 0·002) cells expressing CTLA-4 were observed in children positive for HLA DQA1*0501–DQB1*0201 and DQA1*0301–DQB1*0302 (= 0·04 for CD4+ and P = 0·02 for CD4+ CD25high) risk haplotypes when compared to children without these alleles. The percentage of CD25high cells among CD4+ cells was correlated inversely with CTLA-4 mRNA expression in PBMC (r = –0·56, P = 0·03). Decreased levels of CTLA-4 in CD4+ and CD4+ CD25high cells in individuals with CTLA-4 and HLA class II alleles associated with T1D may contribute to the initiation and/or progression of autoimmune response.

  • 11.
    Jonson, Carl-Oscar
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Lernmark, Åke
    Department of Medicine, University of Washington, Seattle, WA, USA.
    Ludvigsson, Johnny
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Rutledge, Elizabeth A.
    Department of Medicine, University of Washington, Seattle, WA, USA.
    Hinkkanen, Ari
    Department of Biochemistry and Pharmacy, Åbo Akademi, Turku, Finland.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    The importance of CTLA-4 polymorphism and Human leukocyte antigen genotype for the induction of diabetes-associated cytokine response in healthy school children2007In: Pediatric Diabetes, ISSN 1399-543X, Vol. 8, no 4, p. 185-192Article in journal (Refereed)
    Abstract [en]

    Background: Type 1 diabetes (T1D) is an autoimmune disease associated with the destruction of pancreatic β cells and genetically linked to human leukocyte antigen (HLA) class II DR3-DQ2 and DR4-DQ8 haplotypes. The +49A/G polymorphism of the immunoregulatory cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene is also associated with T1D. Genetic and environmental risk factors precede the onset of T1D, which is characterized by a T helper 1 cell-dominating cytokine response to diabetes-related autoantigens.

    Aim: To investigate immunological differences between healthy children with and without CTLA-4 +49A/G and HLA genetic susceptibility for T1D.

    Study design: Young, 7–15 years of age, healthy subjects (n = 58) were investigated to test whether CTLA-4 +49A/G genotype was associated with enzyme-linked immunospot assay T-cell responses to T1D-related autoantigens. Because T1D is primarily HLA-DQ associated, we stratified the healthy subjects by HLA genotypes associated with the disease.

    Results: Peptide of heat shock protein 60 induced a higher interferon-γ (IFN-γ) response in subjects with risk-associated CTLA-4 polymorphism (GG genotype) (p = 0.02) while glutamic acid decarboxylase 65-induced interleukin-4 (IL-4) secretion was lower in GG genotype subjects (p = 0.02).

    Conclusion: The increased IFN-γ response and lower IL-4 response toward diabetes-related autoantigens shown in CTLA-4 +49 GG risk subjects show a possible mechanism for the association between CTLA-4 and T1D.

  • 12.
    Jonson, Carl-Oscar
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Pihl, Mikael
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Nyholm, Caroline
    CRC, Lund University, Department of Clinical Sciences, Malmö, Sweden.
    Cilio, Corrado M
    CRC, Lund University, Department of Clinical Sciences, Malmö, Sweden.
    Ludvigsson, Johnny
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Regulatory T-cell associated activity in Photopheresis-induced Immune tolerance in Recent Onset Type 1 Diabetes Children2008In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 153, no 2, p. 174-181Article in journal (Refereed)
    Abstract [en]

    Extracorporeal photochemotherapy (ECP) has demonstrated immunological effects. The proposed cytotoxic lymphocyte antigen 4 (CTLA-4) involvement, together with forkhead box P3 (FoxP3) and transforming growth factor (TGF)-β are associated with regulatory T cell activity. The aim of the study was to evaluate the regulatory T cell-associated effect of ECP in recent onset type 1 diabetic (T1D) children. Children (n = 20) with T1D received photopheresis 8-methoxypsoralen + ECP or placebo + shampheresis. Peripheral blood mononuclear cells (PBMC) collected pretreatment (day 1) and post-treatment (day 90) were stimulated with phytohaemagglutinin (PHA) and T1D-associated glutamic acid decarboxylase 65 (GAD65) peptide a.a. 247–279. CTLA-4, sCTLA-4, FoxP3 and TGF-β mRNA transcription was quantified. Photopheresis-treated individuals' relative mRNA expression was generally maintained during the course of the study. Placebo individuals increased in spontaneous CTLA-4 mRNA (P < 0·05) but decreased in expression after stimulation with GAD65-peptide (P < 0·05) and PHA (P < 0·05). Spontaneous TGF-β (P < 0·05) increased whereas PHA- (P < 0·01) and GAD65-peptide (P < 0·01)-induced TGF-β expression decreased in the placebo group, whereas it was maintained in the treated group. Without intervention, expression of CTLA-4 and TGF-β, stimulated with PHA and GAD65 peptide, decreased with time, with a parallel reduction of GAD65-peptide and PHA-stimulated TGF-β expression. These parameters were counteracted by ECP. In conclusion, our results indicate that ECP maintains regulatory T cell-associated activity in recent-onset T1D.

  • 13.
    Karlsson Faresjö, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Vaarala, Outi
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Thuswaldner, Sophie
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Ilonen, Jorma
    JDRF Center for Prevention of Type 1 Diabetes in Finland and the Department of Virology, University of Turku, Turku, Finland.
    Hinkkanen, Ari
    Åbo Akademi, Turku University, Turku, Finland.
    Ludvigsson, Johnny
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Diminished IFN-γ response to diabetes-associated autoantigens in children at diagnosis and during follow up of type 1 diabetes2006In: Diabetes/Metabolism Research Reviews, ISSN 1520-7552, E-ISSN 1520-7560, Vol. 22, no 6, p. 462-470Article in journal (Refereed)
    Abstract [en]

    Background

    Imbalance of T-helper (Th)1- and Th2-like cytokines has been associated with type 1 diabetes. We therefore studied the immune deviation in antigen-specific T cells from diagnosis onwards in type 1 diabetic children.

    Methods

    Peripheral blood mononuclear cells (PBMC) were collected from 15 children after 4 days, 3 months and 18 months of being diagnosed with type 1 diabetes, from 15 healthy children matched by age and gender to the type 1 diabetic children and from 14 children with and 35 children without HLA-risk genes. Secretion of interferon-γ (IFN-γ) and interleukin-4 (IL-4) was detected by ELISPOT after stimulation with glutamic acid decarboxylase (GAD65, protein and aa 247–279), recombinant tyrosinphosphatase (IA-2), insulin, ovalbumin and phytohaemagglutinin (PHA).

    Results

    Secretion of IFN-γ in PBMC stimulated with GAD65 (p < 0.05), the GAD65-peptide (p < 0.01), IA-2 (p < 0.01), and insulin (p < 0.01) was lower in diabetic children at diagnosis than in healthy children. Stimulation of PBMC with GAD65 and IA-2 decreased the secretion of IFN-γ in children with HLA-risk genotype. Spontaneous and antigen-induced IFN-γ secretion increased significantly after diagnosis of the disease, but did not exceed the levels observed in healthy children. Fasting C-peptide levels at diagnosis correlated with insulin-induced IFN-γ (R = 0.52; p = 0.05) and negatively with spontaneous IL-4 secretion (R = −0.62; p < 0.05).

    Conclusion

    A diminished IFN-γ secretion and the association of fasting C-peptide levels with cytokine response in children with type 1 diabetes suggest that factors related to β-cell function in type 1 diabetes may modify T-cell function. Thus, the T-cell responses detected at or after diagnosis may not reflect the pathogenic process leading to type 1 diabetes.

  • 14.
    Karlsson, Maria G. E.
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Garcia, Jorge
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Ludvigsson, Johnny
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Cows' milk proteins cause similar Th1- and Th2-like immune response in diabetic and healthy children2001In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 44, no 9, p. 1140-1147Article in journal (Refereed)
    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.

  • 15.
    Karlsson, Maria G. E.
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Ludvigsson, Johnny
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    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 diabetes2000In: Diabetes Research and Clinical Practice, ISSN 0168-8227, E-ISSN 1872-8227, Vol. 47, no 3, p. 199-207Article in journal (Refereed)
    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.

  • 16.
    Karlsson, Maria G. E.
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Sederholm Lawesson, S.
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Ludvigsson, Johnny
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Th1-like dominance in high-risk first-degree relatives of Type I diabetic patients2000In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 43, no 6, p. 742-749Article in journal (Refereed)
    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.

  • 17.
    Kivling, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Nilsson, Lennart
    Linköping University, Department of Clinical and Experimental Medicine, Allergy Centre. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Allergy Centre UHL.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    How and when to pick up the best signals from markers associated with T-regulatory cells?2009In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 345, no 1-2, p. 29-39Article in journal (Refereed)
    Abstract [en]

    Regulatory T (Treg) cells are important tools with the purpose to control and regulate the immune system. These cells use FOXP3, TGF-beta, CTLA-4 and sCTLA-4 to regulate other T-cells. Since cryopreservation of peripheral blood mononuclear cells (PBMC) is a convenient way to handle samples, we investigated whether these cells will change their mRNA expression of Treg associated markers, as well as secretion of TGF-beta1, after cryopreservation. Additionally, we aimed to investigate the mRNA expression after two different time intervals of in vitro antigen stimulation. PBMC from healthy adults were stimulated either fresh (48/96 h) or after cryopreservation (48 h), with PHA, tetanus toxoid, beta-lactoglobulin, ovalbumin or in culture medium exclusively (spontaneous). The mRNA expression of FOXP3, TGF-beta, CTLA-4 and sCTLA-4 were studied with multiplex real-time RT-PCR and TGF-beta1 with ELISA. Cryopreserved PBMC were appropriate for detection of the Treg associated markers FOXP3, TGF-beta, CTLA-4 and sCTLA-4 expressed spontaneously. Also antigen-induced mRNA expression of CTLA-4, sCTLA-4 and TGF-beta and secreted TGF-beta1, with the exception of FOXP3, preserved a stable transcription activity after cryopreservation. Further, a stimulation period of 48 h in general revealed the highest mRNA expression of the markers studied. In conclusion, cryopreserved cells are in general suitable for studying Treg associated markers.

  • 18.
    Kivling, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Nilsson, Lennart
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Fälth-Magnusson, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Sollvander, Sofia
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Johanson, Calle
    Ryhov Jonkoping City Hospital.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Diverse FOXP3 Expression in Children with Type 1 Diabetes and Celiac Disease2008In: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 1150, p. 273-277Article in journal (Refereed)
    Abstract [en]

    Imbalance between different types of T lymphocytes, such as T helper (Th) and regulatory T cells (Tregs), has been reported to play a part in the pathogenesis behind such autoimmune diseases as type 1 diabetes (T1D) and celiac disease (CD). Defects in Tregs are proposed to at least partly explain the imbalance of Th cells found in children with immunologic diseases. Peripheral blood mononuclear cells from 24 children with T1D and/or CD, and reference children (that is, those without any of these diseases) were stimulated with disease-associated antigens (insulin, gluten, transglutaminase [tTG]), and phytohemagglutinin (PHA). The mRNA expression of the Treg-associated marker FOXP3 was analyzed with multiplex real-time RT-PCR. Children with T1D showed both a low spontaneous (P < 0.05) and PHA-induced (P < 0.01) expression of FOXP3 mRNA compared to children with CD. Children with T1D also had a low PHA-induced FOXP3 mRNA expression compared to the group of children diagnosed with both T1D and CD (P < 0.05). Spontaneous (P < 0.05) and PHA-induced (P < 0.05) FOXP3 mRNA expression was high in children with CD compared to reference children. In contrast, stimulation with insulin tended to induce high FOXP3 mRNA expression in T1D children compared to reference children (P = 0.057). In conclusion, children with only T1D generally showed a lower FOXP3 mRNA expression than did children with CD, or with T1D in combination with CD, which suggests impaired regulation of the immune system in children with T1D.

  • 19.
    Kivling, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Nilsson, Lennart
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Åkesson, Karin
    Clinic of Paediatrics, Ryhov County Hospital, Jönköping, Sweden 4.
    Fälth-Magnusson, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Diverging immune responses when allergy, type 1 diabetes and celiac disease coexistManuscript (preprint) (Other academic)
    Abstract [en]

    An imbalance between different immune cells, among them T-cells and inflammatory cells, has been proposed to be part of the disease process in type 1 diabetes (T1D), celiac disease and allergy. T-cells and inflammatory cells exert their actions through cytokines and chemokines, and the secretion of those can be used to describe the cell milieu during an immune response.

    This study included seventy-two children, diagnosed with T1D, celiac disease, allergy, or a combination of two of these diseases and compared to reference children. The study aimed to evaluate the secretion of 27 different cytokines and chemokines in cell culture supernatant after in vitro stimulation with disease-associated antigens (birch, gluten, insulin) detected by Luminex technique.

    Combination of allergy with either T1D or celiac disease gave diverging results. Children with combination of T1D and allergy showed an increased secretion of several cytokines (IL-2, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12, IL-15, IL-17 and CCL11), in comparison to almost all groups from birch stimulation. In contrast, when allergy was combined with celiac disease, the spontaneous secretion of IL-1β, IL-5, IL-6, IL-9, IL-10, IL-12 and CCL3 was decreased compared to children with T1D or allergy, as well as children with celiac disease alone, children with combination of T1D and allergy and reference children.

    In conclusion, our results shed some light on the immune responses in children with common immunological diseases. Our study indicates diverging immune responses when allergy, type 1 diabetes and celiac disease coexist.

  • 20.
    Kivling, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Åkesson, Karin
    Clinic of Paediatrics, Ryhov County Hospital, Jönköping, Sweden 4.
    Nilsson, Lennart
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Combined type 1 diabetes and celiac disease in children give raise to a pronounced Treg populationManuscript (preprint) (Other academic)
    Abstract [en]

    Regulatory T-cells (Treg) suppress the immune response in order to avoid harmful effects. A wide range of highly expressed markers are associated to Treg, among them are CD4, CD25, CD39 and forkhead box p3 (FOXP3). Others are expressed in a low number on Tregs, for example CD45RA and CD127. Type 1 diabetes (T1D) and celiac disease are both autoimmune diseases, caused by an unwanted immune response, and Treg cells have been associated to both diseases. As T1D and celiac disease share same risk genes, patients have the risk of developing the other disease subsequently. Treg cells have been implicated to be associated with development of T1D combined with celiac disease.

    This pilot study aimed to investigate the expression of Treg associated markers in both CD4+CD25+ and CD4+CD25high cells by flow cytometry. In order to evaluate the involvement of Treg cells, CD39, CD45RA, CD127 and FOXP3 were studied in children with combination of T1D and celiac disease, in comparison to children with either T1D or celiac disease, and healthy children.

    Our data point out that children with combination of T1D and celiac disease have higher expression of FOXP3 as well as CD39, together with a decreased expression of both CD127 and CD45RA, in comparison to children with exclusively T1D or celiac disease. Even though none of the groups differed from the reference group, our data indicates that children with combination of T1D and celiac disease have a more pronounced Treg population, both in frequency and MFI, compared to children with either T1D or celiac disease.

  • 21.
    Ludvigsson, Johnny
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics .
    Hjorth, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics .
    Axelsson, Stina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics .
    Chéramy, Mikael
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics .
    Pihl, Mikael
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics .
    Vaarala, Outi
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics .
    Forsander, Gun
    Ivarsson, Sten
    Johansson, Calle
    Lindh, Agne
    Nilsson, NO
    Åman, Jan
    Örtqvist, Eva
    Zerhouni, Peter
    Casas, Rosaura
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Pediatrics .
    GAD treatment and insulin secretion in recent-onset type 1 diabetes2008In: New England Journal of Medicine, ISSN 0028-4793, E-ISSN 1533-4406, Vol. 359, no 18, p. 1909-1920Article in journal (Refereed)
    Abstract [en]

    Background: The 65-kD isoform of glutamic acid decarboxylase (GAD) is a major autoantigen in patients with type 1 diabetes mellitus. This trial assessed the ability of alum-formulated GAD (GAD-alum) to reverse recent-onset type 1 diabetes in patients 10 to 18 years of age. Methods: We randomly assigned 70 patients with type 1 diabetes who had fasting C-peptide levels above 0.1 nmol per liter (0.3 ng per milliliter) and GAD autoantibodies, recruited within 18 months after receiving the diagnosis of diabetes, to receive subcutaneous injections of 20 μg of GAD-alum (35 patients) or placebo (alum alone, 35 patients) on study days 1 and 30. At day 1 and months 3, 9, 15, 21, and 30, patients underwent a mixed-meal tolerance test to stimulate residual insulin secretion (measured as the C-peptide level). The effect of GAD-alum on the immune system was also studied. Results: Insulin secretion gradually decreased in both study groups. The study treatment had no significant effect on change in fasting C-peptide level after 15 months (the primary end point). Fasting C-peptide levels declined from baseline levels significantly less over 30 months in the GAD-alum group than in the placebo group (-0.21 vs. -0.27 nmol per liter [-0.62 vs. -0.81 ng per milliliter], P = 0.045), as did stimulated secretion measured as the area under the curve (-0.72 vs. -1.02 nmol per liter per 2 hours [-2.20 vs. -3.08 ng per milliliter per 2 hours], P = 0.04). No protective effect was seen in patients treated 6 months or more after receiving the diagnosis. Adverse events appeared to be mild and similar in frequency between the two groups. The GAD-alum treatment induced a GAD-specific immune response. Conclusions: GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent-onset type 1 diabetes, although it did not change the insulin requirement. (ClinicalTrials.gov number, NCT00435981.) Copyright © 2008 Massachusetts Medical Society. All rights reserved.

  • 22.
    Michalek, J
    et al.
    Cell Immunotherapy Center, Masaryk University Brno, Czech Republic.
    Vrabelova, Z
    Dept of Pediatrics, Masaryk University Brno, Czech Republic.
    Hrotekova, Z
    Dept of Pediatrics, Masaryk University Brno, Czech Republic.
    Kyr, M
    Dept of Pediatrics, Masaryk University Brno, Czech Republic.
    Kolouskova, S
    Dept of Pediatrics, University Hospital Motol Prague, Czech Republic.
    Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Stechova, K
    Dept of Pediatrics, University Hostpial Motol Prague, Czech Republic.
    Immune regulatory T cells in siblings of children suffering from type 1 diabetes mellitus2006In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 64, no 5, p. 531-535Article in journal (Refereed)
    Abstract [en]

    Patients with type 1 diabetes are suffering from defects in immune regulatory cells. Their siblings may be at increased risk of type 1 diabetes especially if they are carriers of certain human leucocyte antigen (HLA) alleles. In a prospective non-randomized study, we intended to evaluate 31 healthy siblings of paediatric patients with type 1 diabetes and explore immune regulatory populations of CD4+CD25+ T cells and natural killer (NK) T cells. Tested siblings of type 1 diabetes patients were stratified according to the HLA-associated risk of possible diabetes development. Immune regulatory function of CD4+CD25+ T cells was tested in vitro. Significant differences in CD4+CD25+ but not in NK T cells have been identified. Siblings of type 1 diabetes patients carrying high risk HLA alleles (DQA1*05, DQB1*0201, DQB1*0302) had significantly lower number of immune regulatory CD4+CD25+ T cells than the age-matched healthy controls or siblings carrying low-risk HLA alleles (DQB1*0301, DQB1*0603, DQB1*0602). Regulatory function of CD4+CD25+ T cells demonstrated a dose-escalation effect. In siblings of type 1 diabetes patients, the defect in immune regulatory CD4+CD25+ T cells exists in association with genetic HLA-linked risk for type 1 diabetes. © 2006 The Authors.

  • 23.
    Nilsson, Hans
    et al.
    Linköping University, Department for Studies of Social Change and Culture, Centre for Local History. Linköping University, Faculty of Arts and Sciences.
    Faresjö, Tomas
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Twin Cities Research Group: en växande forskningsplattform i Jan Sundins anda2008In: Se människan : demografi, rätt och hälsa: en vänbok till Jan Sundin / [ed] Annika Sandén, Linköping: Linköpings universitet , 2008, p. 195-202Chapter in book (Other academic)
    Abstract [en]

    An interdisciplinary research group entitled "Twincities Research Group" has been initiated at Linkoping University. The term twin cities refer to the Swedish cities Linkoping and Norrkoping, neighbors that are nearly equal in size. These two cities, located within a distance of only 40 km, are governed by the same county council and consequently have the same health care structure. However, public health is remarkably different in these twin cities. The comparison of public health in these two cities during the development from the industrial to the post-industrial era has a design similar to classical experiments with a control and an experiment group, since the social history and the socio-economic structures of the cities are radically different. Through an interdisciplinary research design including historical, epidemiological and clinical competence we have a unique opportunity to increase our understanding of how the social environment may affect public health.

  • 24.
    Nilsson, Lennart
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Kivling, Anna
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Jalmelid, M
    Fälth-Magnusson, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Combinations of common chronic paediatric diseases deviate the immune response in diverging directions2006In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 146, no 3, p. 433-442Article in journal (Refereed)
    Abstract [en]

    The cytokine pattern of T lymphocytes has not been characterized in children with combinations of paediatric immunological disorders. We describe cytokine secretion in children with type 1 diabetes, coeliac disease and allergy and combinations of two of these diseases after stimulation with 'disease-specific' antigens. Peripheral blood mononuclear cells (PBMC) were collected from 68 children with type 1 diabetes, allergy or coeliac disease, two of these diseases in combination or none of these diseases. Using the enzyme-linked immunospot (ELISPOT) technique, interferon (IFN)-γ and interleukin (IL)-4 were analysed from fresh PBMC spontaneously and after in vitro stimulation with antigens associated with one or more of these diseases (insulin, gluten, birch and cat extract, β-lactoglobulin, ovalbumin and phytohaemagglutinin) in order to divide T helper (Th)1- from Th2-like lymphocytes. Stimulation with birch and cat extract caused increased IL-4 secretion in allergic children. A low IFN-γ response to insulin was found in type 1 diabetic children, whereas allergic children responded to insulin by increased IL-4 secretion. Children suffering from both type 1 diabetes (Th1-prone) and allergy (Th2-prone) reacted distinctly to general mitogen stimulation. Children suffering from two Th1-dominated diseases (type 1 diabetes and coeliac disease) showed hardly any response to either food or inhalation allergens. Our results indicate an important interplay between common immunological diseases in children. The combination of two Th1-deviated diseases is associated with a suppressed immune response, whereas a combination of Th1- and Th2-dominated diseases appears to increase the general immune response. © 2006 The Author(s).

  • 25.
    Rydén, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Bolmeson, Caroline
    Cellular Autoimmunity Unit, Department of Clinical Sciences, Lund University, Malmö, Sweden.
    Jonson, Carl-Oscar
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Cilio, Corrado M.
    Cellular Autoimmunity Unit, Department of Clinical Sciences, Lund University, Malmö, Sweden.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Low expression and secretion of circulating soluble CTLA-4 in peripheral blood mononuclear cells and sera from Type 1 diabetic children2012In: Diabetes/Metabolism Research Reviews, ISSN 1520-7552, E-ISSN 1520-7560, Vol. 28, no 1, p. 84-96Article in journal (Refereed)
    Abstract [en]

    High levels of soluble cytotoxic T-lymphocyte antigen 4 (soluble CTLA-4), an alternative splice form of the regulatory T-cell (Treg) associated CTLA-4 gene, have been associated with type 1 diabetes (T1D) and other autoimmune diseases, such as Grave's disease and myasthenia gravis. At the same time, studies have shown soluble CTLA-4 to inhibit T-cell activation through B7 binding. This study aimed to investigate the role of soluble CTLA-4 in relation to full length CTLA-4 and other Treg associated markers in T1D children and in individuals with high or low risk of developing the disease.

    T1D children were studied at four days, one and two years after diagnosis in comparison to individuals with high or low risk of developing the disease. Isolated peripheral blood mononuclear cells (PBMC) were stimulated with the T1D-associated glutamic acid decarboxylase 65 (GAD65) and phytohaemagglutinin (PHA). Subsequently, soluble CTLA-4, full length CTLA-4, FOXP3 and TGF-β mRNA transcription were quantified and protein concentrations of soluble CTLA-4 were measured in culture supernatant and sera.

    Low protein concentrations of circulating soluble CTLA-4 and a positive correlation between soluble CTLA-4 mRNA and protein were seen in T1D, in parallel to a negative relation in healthy subjects. Further, low mitogen-induced soluble CTLA-4 was accompanied by low C-peptide, together indicating an inverse relation of soluble CTLA-4 between health and disease. Moreover, low mitogen-induced soluble CTLA-4 mRNA and low TGF-β mRNA expression were seen in high-risk individuals, indicating an alteration in activation and downregulating immune mechanisms already during the pre-diabetic phase.

  • 26.
    Rydén, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Altered immune profile from pre-diabetes to manifestation of type 1 diabetes2013In: Diabetes Research and Clinical Practice, ISSN 0168-8227, E-ISSN 1872-8227, Vol. 100, no 1, p. 74-84Article in journal (Refereed)
    Abstract [en]

    Background: While the mechanisms leading to beta-cell destruction and clinical onset of T1D are still unclear, the composition of the immune profile is probably important for the outcome of immune activity. The aim of this study was to investigate the composition and possible changes of the immunological profile, spontaneously and following stimulation with the autoantigens GAD(65), and HSP60, at high-risk and T1D onset and further to 8 months post diagnosis. Methods: Fifteen first-degree relatives of T1D patients with a high risk of developing the disease within five years, 25 children approximately four days and 8 months after diagnosis of T1D and 16 healthy children were included in the study. Cytokines (IL-1 beta, -6, -7, -10, -13, -17, IFN-gamma and TNF-alpha) and chemokines (CCL2, -3, -4, -5 and CXCL10) associated with Th1, Th2, Tr1 and inflammatory cells were detected in cell culture supernatants by Luminex-technique, and markers associated with regulatory T-cells (FOXP3, CTLA-4 and TGF-beta) by real-time RT-PCR. Results: High-risk individuals differed in immunity from that seen in healthy and T1D children. High-risk individuals had a low TNF-alpha response and fewer responders from mitogen exposure as well as low spontaneous secretions of IL-13 compared to healthy children. High-risk individuals that later developed T1D, had a lower FOXP3 and CTLA-4 mRNA expression, following stimulation with GAD(65), in combination with higher secretion of the pro-inflammatory chemokine CCL4. Conclusion: Changes in immunity seen in individuals with high risk of developing T1D points to alterations/actions in the immune system already early in the pre-diabetic phase.

  • 27.
    Rydén, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Efficient expansion of cryopreserved CD4+CD25+CD127lo/- cells in Type 1 diabetes2011In: Results in Immunology, ISSN 2211-2839, Vol. 1, no 1, p. 36-44Article in journal (Refereed)
    Abstract [en]

    Increased attention has been drawn to the important role played by regulatory T-cells (Treg) in immune homoeostasis. However, the small numbers of Tregs make them elusive to study. We investigated the cryostability of Tregs and whether they can be expanded from cryopreserved peripheral blood mononuclear cells (PBMCs). Further, to elucidate if there is a difference in ex-vivo frequency or in vitro expansion of Tregs among T1D children (n=9), high-risk (n=7) and healthy (n=10) individuals, Tregs defined as CD4+CD25+CD127lo/− were isolated from cryopreserved PBMCs.

    Cryopreserved PBMCs maintained a stable expression of Treg-markers. Tregs were efficiently expanded in vitro from all donors and Tregs from T1D children acquired higher FOXP3 expression compared to healthy subjects. T1D children had a significantly lower percentage of Tregs among CD4+ T-cells and also lower Treg to CD4+CD25 cell ratios compared to healthy individuals.

  • 28.
    Rydén, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Stechova, Katerina
    Charles University Prague.
    Durilova, Marianna
    Charles University Prague.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Switch from a dominant Th1-associated immune profile during the pre-diabetic phase in favour of a temporary increase of a Th3-associated and inflammatory immune profile at the onset of type 1 diabetes2009In: Diabetes/Metabolism Research Reviews, ISSN 1520-7552, E-ISSN 1520-7560, Vol. 25, no 4, p. 335-343Article in journal (Refereed)
    Abstract [en]

    Background: Type 1 diabetes (T1D) is all autoimmune disease dominated by loss of self-tolerance resulting in depletion of the beta-cells. This Study aims to confirm previous observations of a dominant T-helper (Th)1-like profile during the period close to onset of disease. Further, to follow the immune response from onset to 2 years duration, the Study focused on spontaneous as well as autoantigen-induced immune profile.

    Methods: Peripheral blood mononuclear cells were collected 4 days and 1 and 2 years after diagnosis of T1D children, from healthy children carrying the human leukocyte antigen-risk genes and from high-risk children (ICA andgt;= 20 IJDF units). Peripheral blood mononuclear cells were stimulated with glutamic acid decarboxylase (GAD(65)) and phytohaemagglutinin (PHA). Cytokines and chemokines were detected in cell-culture supernatants by protein microarray (naive T-cells; interleukin (IL)-7, Th1; interferon-gamma, turnout necrosis factor-beta, Th2; IL-5, Th3; transforming growth factor-beta, T-regulatory cell type 1; IL-10 and inflammatory cytokines; tumour necrosis factor-alpha, IL-6 and chemokines; monocyte chemoattractant protein-1, monokine upregulated by IFN-gamma) in relation to clinical outcome (C-peptide).

    Results: High-risk children showed a dominant Th1-associated profile with high spontaneous and GAD(65)-induced secretion. The mitogen PHA instead induced a Th2-associated response exclusively in high-risk children. In contrast, newly diagnosed T1D children showed a pronounced Th3-associated cytokine profile as well as a burst of inflammatory cytokines and chemokines secreted both spontaneously and by GAD(65) and PHA stimulation. The immune response to GAD(65) and PHA, however, diminished with duration of disease.

    Conclusion: A dominant Thl-associated immune profile was observed during the pre-diabetic phase. This Th1 dominance, however, diminished in favour of a temporary increase in a Th3-associated and inflammatory immune profile at the onset of disease.

  • 29. Schloot, N
    et al.
    Meierhoff, G
    Karlsson Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Ott, Patrick
    Putnam, A
    Lehman, P
    Gottlieb, P
    Roep, BO
    Peakman, M
    Tree, T
    Comparison of cytokine ELISpot assay formats for the detection of islet antigen autoreactive T cells: Report of the third immunology of diabetes society T-cell workshop2003In: Journal of Autoimmunity, ISSN 0896-8411, E-ISSN 1095-9157, Vol. 21, no 4, p. 365-376Article in journal (Refereed)
    Abstract [en]

    The identification of sensitive assay formats capable of distinguishing islet autoreactive T cells directly ex vivo in blood is a major goal in type 1 diabetes research. Recently, much interest has been shown in the cytokine enzyme linked immunospot assay (CK ELISpot), an assay potentially capable of fulfilling these difficult criteria. To address the utility of this assay in detecting autoreactive T cells, a 'wet' workshop was organized using the same fresh blood sample and coded antigens. Five different laboratories participated, using three distinct CK ELISpot assay formats. Samples from two subjects were pre-tested for responses to sub-optimal concentrations of tetanus toxoid, representing a low frequency recall response, and peptides from diabetes associated autoantigens GAD65, IA-2 and HSP60. All participants measured interferon-? production and combinations of interleukins-4, -5, -10 and -13. In the workshop 4 of 5 laboratories detected low frequency recall responses in both subjects and 3 of 5 detected at least one of the autoreactive peptide responses concordant with pre-testing. Significant assay format related differences in sensitivity and signal-to-noise ratio were observed. The results demonstrate the potential for detection of low-level autoreactive T cell responses and identify assay characteristics that will be useful for studies in type 1 diabetes. ⌐ 2003 Elsevier Ltd. All rights reserved.

  • 30.
    Stechova, K.
    et al.
    2nd Medical Faculty of Charles University.
    Spalova, I.
    2nd Medical Faculty of Charles University.
    Durilova, M.
    2nd Medical Faculty of Charles University.
    Bartaskova, D.
    2nd Medical Faculty of Charles University.
    Cerny, M.
    2nd Medical Faculty of Charles University.
    Cerna, M.
    Institute for the Care of Mother and Child, Prague, Czech Republic.
    Pithova, P.
    2nd Medical Faculty of Charles University.
    Chudoba, D.
    2nd Medical Faculty of Charles University.
    Stavikova, V.
    2nd Medical Faculty of Charles University.
    Ulmannova, T.
    2nd Medical Faculty of Charles University.
    Faresjo, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Influence of Maternal Hyperglycaemia on Cord Blood Mononuclear Cells in Response to Diabetes-associated Autoantigens2009In: SCANDINAVIAN JOURNAL OF IMMUNOLOGY, ISSN 0300-9475, Vol. 70, no 2, p. 149-158Article in journal (Refereed)
    Abstract [en]

    Perfect maternal diabetes compensation is crucial for the outcome of the baby. However, little is known how hyperglycaemia influences the specific immune response. Furthermore, babies of type 1 diabetes (T1D) mothers have less risk of development T1D than babies with a T1D father. This study aimed to analyze the effect of maternal hyperglycaemia on newborns with focus on the response to diabetes-associated autoantigens. Populations: (1) Newborns of T1D mothers split into groups according to maternal diabetes compensation during the 3rd trimester: perfect (n = 15) or acceptable (n = 25) compensation. (2) newborns with T1D father (n = 12) (3) newborns with a mother treated for either gestational or type 2 diabetes (n = 10) (4) control newborns (n = 25). Spontaneous as well as diabetes-associated autoantigen-stimulated production of 23 cytokines and chemokines were tested using protein microarray. In addition, the influence of glucose on cytokine and chemokine responsiveness was analyzed in vitro. The study groups differed in their spontaneous as well as stimulated cytokine and chemokine spectra. A prominent Th1 response (high IFN-gamma) from autoantigen stimulation was observed especially in babies of T1D fathers (P = 0.001) and also in mothers with perfect diabetes compensation during the 3rd trimester (P = 0.016) in comparison with control newborns. By contrast, cord blood mononuclear cells cultivated in vitro in high glucose concentration decreased the diabetogenic stimulated Th1 cytokine response. Maternal sweet as well as autoimmune environment may both lead to lower occurrence of T1D within their offspring. Further studies will reveal the exact immunological mechanism of this observation.

  • 31. Stechova, Katerina
    et al.
    Bohmova, Kristyna
    Vrabelova, Zuzana
    Sepa, Anneli
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Stadlerova, Gabriela
    Zacharovova, Klara
    Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    High T-helper-1 cytokines but low T-helper-3 cytokines, inflammatory cytokines and chemokines in children with high risk of developing type 1 diabetes2007In: Diabetes/Metabolism Research Reviews, ISSN 1520-7552, E-ISSN 1520-7560, Vol. 23, no 6, p. 462-471Article in journal (Refereed)
    Abstract [en]

    Background. Type 1 diabetes (T1D) is suggested to be of T-helper (Th)1-like origin. However, recent reports indicate a diminished interferon (IFN)-γ secretion at the onset of the disease. We hypothesize that there is a discrepancy in subsets of Th-cells between children with a high risk of developing T1D, children newly diagnosed with T1D and healthy children. Methods. Peripheral blood mononuclear cells (PBMC) were collected from children at high risk for T1D (islet cells antibodies [ICA] ≥ 20 IJDF-U), those newly diagnosed and healthy children carrying the HLA-risk gene DQB1*0302 or DQB1*0201 and DQA1*0501. Th1- (IFN-γ, tumour necrosis factor [TNF]-β, interleukin [IL]-2), Th2- (IL-4,-5,-13), Th3- (transforming growth factor [TGF-β], IL-10) and inflammatory associated cytokines (TNF-α, IL-1α,-6) and chemokines (monocyte chemoattractant protein [MCP]-1,-2,-3, Monokine unregulated by IFN-γ [MIG], Regulated on Activation, Normal T-cell Expressed and Secreted [RANTES], IL-7,-8,-15) were detected in cell-culture supernatants of PBMC, stimulated with glutamic acid decarboxylase 65 (GAD65) and phytohaemagglutinin (PHA), by protein micro array and enzyme linked immunospot (ELISPOT) technique. Results. The Th1 cytokines IFN-γ and TNF-β, secreted both spontaneously and by GAD65- and mitogen stimulation, were seen to a higher extent in high-risk children than in children newly diagnosed with T1D. In contrast, TNF-α and IL-6, classified as inflammatory cytokines, the chemokines RANTES, MCP-1 and IL-7 as well as the Th3 cytokines TGF-β and IL-10 were elevated in T1D children compared to high-risk children. Conclusion. High Th-1 cytokines were observed in children with high risk of developing TID, whereas in children newly diagnosed with T1D Th3 cytokines, inflammatory cytokines and chemokines were increased. Thus, an inverse relation between Th1-like cells and markers of inflammation was shown between children with high risk and those newly diagnosed with T1D. Copyright © 2007 John Wiley & Sons, Ltd.

  • 32.
    Stechova, Katerina
    et al.
    University Hospital Motol.
    Halbhuber, Zbynek
    Central European Biosyst, Prague.
    Hubackova, Miluse
    Charles University of Prague.
    Kayserova, Jana
    Charles University of Prague.
    Petruzelkova, Lenka
    Charles University of Prague.
    Vcelakova, Jana
    Charles University of Prague.
    Kolouskova, Stanislava
    Charles University of Prague.
    Ulmannova, Tereza
    Charles University of Prague.
    Faresjö, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Neuwirth, Ales
    Institute Molecular Genet AS CR.
    Spisek, Radek
    Charles University of Prague.
    Sediva, Anna
    Charles University of Prague.
    Filipp, Dominik
    Institute Molecular Genet AS CR.
    Sumnik, Zdenek
    Charles University of Prague.
    Case report: type 1 diabetes in monozygotic quadruplets2012In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 20, no 4, p. 457-462Article in journal (Refereed)
    Abstract [en]

    Type 1 diabetes (T1D) is an autoimmune disease characterized by the lack of insulin due to an autoimmune destruction of pancreatic beta cells. Here, we report a unique case of a family with naturally conceived quadruplets in which T1D was diagnosed in two quadruplets simultaneously. At the same time, the third quadruplet was diagnosed with the pre-diabetic stage. Remarkably, all four quadruplets were positive for anti-islet cell antibodies, GAD65 and IA-A2. Monozygotic status of the quadruplets was confirmed by testing 14 different short tandem repeat polymorphisms. Serological examination confirmed that all quadruplets and their father suffered from a recent enteroviral infection of EV68-71 serotype. To assess the nature of the molecular pathological processes contributing to the development of diabetes, immunocompetent cells isolated from all family members were characterized by gene expression arrays, immune-cell enumerations and cytokine-production assays. The microarray data provided evidence that viral infection, and IL-27 and IL-9 cytokine signalling contributed to the onset of T1D in two of the quadruplets. The propensity of stimulated immunocompetent cells from non-diabetic members of the family to secrete high level of IFN-alpha further corroborates this conclusion. The number of T regulatory cells as well as plasmacytoid and/or myeloid dendritic cells was found diminished in all family members. Thus, this unique family is a prime example for the support of the so-called fertile-field hypothesis proposing that genetic predisposition to anti-islet autoimmunity is fertilized and precipitated by a viral infection leading to a fully blown T1D.

  • 33.
    Stechova, Katerina
    et al.
    Dept of Pediatrics Prague.
    Kolouskova, Stanislava
    Dept of Pediatrics Prague.
    Sumnik, Zdenek
    Dept of Pediatrics Prague.
    Cinek, Ondrej
    Dept of Pediatrics Prague.
    Kverka, Milosav
    The Inst of Microbiology Prague.
    Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Chudoba, Daniel
    Dept of Biology and Genetics Prague.
    Dovolilova, Eva
    Diabetes Centrum Prague.
    Pechova, Marta
    Dept of Biochemistry Prague.
    Vrabelova, Zuzana
    Dept of Pediatrics Prague.
    Böhmova, Kristyna
    Dept of Pediatrics Prague.
    Janecek, Lukas
    Dept of Pediatrics Prague.
    Saudek, Frantisek
    Diabetes Centrum Prague.
    Vavrinec, Jan
    Dept of Pediatrics Prague.
    Anti-GAD65 reactive peripheral blood mononuclear cells in the pathogenesis of cystic fibrosis related diabetes mellitus2005In: Autoimmunity, ISSN 0891-6934, E-ISSN 1607-842X, Vol. 38, no 4, p. 319-323Article in journal (Refereed)
    Abstract [en]

    Objective: A role of autoreactive T cells for type 1 diabetes pathogenesis is considered crucial. In our pilot study we addressed if autoreactive mononuclear cells are present also in peripheral blood of patients with other specific forms of diabetes as cystic fibrosis related diabetes (CFRD). Methods: Cellular immune responses to a known β-cell autoantigen (GAD65 and GAD65 derived peptides) were analysed by ELISPOT (IFN-γ) and by protein microarray analysis in four patients suffering from CFRD, in four cystic fibrosis (CF) patients without diabetes, in eight type 1 diabetes patients (without CF) and in four healthy controls. Results: Response to the autoantigen GAD65 (protein and peptides) was observed in 7/8 patients suffering from CF and in all type 1 diabetes patients. Post-stimulation production of Th1 cytokines (IFN-γ, TNF-β) was observed in 2/4 CFRD, 1/4 CF patients and in 7/8 type 1 diabetes patients. All these patients carry prodiabetogenic HLA-DQ genotype. Th2- and Th3 type of cytokine pattern was observed in 2/4 CF patients. Production of IL-8 was observed in the third CFRD as well as in the third CF patient and in 1/8 type 1 diabetes patient and borderline production of this chemokine was also observed in 2/4 healthy controls. No reaction was observed in the other 2/4 healthy controls and in the fourth CFRD patient who carried a strongly protective genotype and did not produce autoantibodies. The most potent peptide of GAD65 was amino acids 509-528. Conclusions: We consider our observations as a sign of a reaction directed against the self-antigen GAD65 that are closely connected to type 1 diabetes. In CF patients who do not develop diabetes autoreactive mechanisms are very probably efficiently suppressed by immune self-tolerance mechanisms. CFRD patients are a heterogenic group. To disclose those who may display features of autoimmune diabetes could have an impact for their therapy and prognosis. © 2005 Taylor & Francis Group Ltd.

  • 34. Vrabelova, Z
    et al.
    Kolouskova, S
    Böhmova, K
    Faresjö, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Pediatrics.
    Sumnik, Z
    Pechova, M
    Kverka, M
    Chudoba, D
    Zacharovova, K
    Stadlerova, G
    Pithova, P
    Hladikova, M
    Stechova, K
    Protein microarray analysis as a tool for monitoring cellular autoreactivity in type 1 diabetes patients and their relatives2007In: Pediatric Diabetes, ISSN 1399-543X, E-ISSN 1399-5448, Vol. 8, no 5, p. 252-260Article in journal (Refereed)
    Abstract [en]

    Background: Autoreactive T cells have a crucial role in type 1 diabetes (T1D) pathogenesis.: Objectives: The aim of our study was to monitor the in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs) after stimulation with diabetogenic autoantigens. Subjects: Ten T1D patients (tested at the time of diagnosis and 6 and 12 months later), 10 first-degree relatives of the T1D patients, and 10 controls underwent the study. Methods: PBMCs were stimulated with glutamic acid decarboxylase 65 (GAD65) amino acids (a.a.) 247-279, 509-528, and 524-543, proinsulin a.a. 9-23, and tyrosine phosphatase (islet antigen-2)/R2 a.a. 853-872. Interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-γ, tumor necrosis factor β, transforming growth factor β1, and granulocyte colony-stimulating factor (GCSF) were analyzed by protein microarray. Results: Differences in cytokine(s) poststimulatory and mainly in basal production were observed in all groups. The most prominent findings were in controls, the higher basal levels of IL-2, IL-4, IL-5, IL-13, and GCSF were observed when compared with relatives (p < 0.05, for all). After stimulation in controls, there was a significant decrease in IL-2, IL-13, GCSF, and IFN-γ (p < 0.05, for all). The group of relatives was the most variable in poststimulatory production. A strong correlation between cytokines production was found but groups differed in this aspect. Conclusion: By multiplex analysis, it may be possible, for example, to define the risk immunological response pattern among relatives or to monitor the immune response in patients on immune modulation therapy. © 2007 The Authors Journal compilation © 2007 Blackwell Munksgaard.

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