Zuzana Vrabelová

High T‐helper‐1 cytokines but low T‐helper‐3 cytokines, inflammatory cytokines and chemokines in children with high risk of developing type 1 diabetes

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.

Immune regulatory T cells in siblings of children suffering from type 1 diabetes mellitus

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.

Anti-GAD65 reactive peripheral blood mononuclear cells in the pathogenesis of cystic fibrosis related diabetes mellitus

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.

Protein microarray analysis as a tool for monitoring cellular autoreactivity in type 1 diabetes patients and their relatives

Background:  Autoreactive T cells have a crucial role in type 1 diabetes (T1D) pathogenesis.

Cord Blood Cytokine Profile Detection in Neonates with T1D Parents – Monitoring of Cellular Auto‐reactivity Using Protein Microarray

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.