BACKGROUND: Autoimmune insulitis leading to insulin dependent diabetes mellitus (IDDM, Type 1 Diabetes) is accompanied by autoantibodies as its invaluable markers. The aim of the study was to determine the frequency of autoantibodies against GAD65, IA2 and insulin in Czech diabetic children at the disease onset. METHODS AND RESULTS: Sera of 105 newly diagnosed children with IDDM drawn within 24 hours after the first insulin dose were investigated for anti-GAD65, anti-IA2 and insulin autoantibodies (IAA) using RIA methods. The cut-off normal levels were determined as the 99th percentile of 105 non-diabetic children. At given 99% specificity, the sensitivity was 71% for anti-GAD65, 73% for anti-IA2, and 46% for IAA. 29% diabetic children were positive for all three autoantibodies, 25% had anti-GAD65 and anti-IA2 (IAA negative), 5.7% anti-GAD65 and IAA (anti-IA2 negative), 7.6% anti-IA2 and IAA (anti-GAD65 negative). As the only positive autoantibody, anti-GAD65 was found in 12%, anti-IA2 in 11%, and IAA in 3.8% children. In 5.7% children, none of the investigated autoantibodies was positive. Diabetic children diagnosed before the age of 5 years had significantly higher prevalence of IAA than the older ones. CONCLUSIONS: We have determined normal levels in healthy children, and prevalence at childhood IDDM onset of autoantibodies against three main molecular-defined autoantigens.

• MeSH
• autoprotilátky krev   MeSH
• diabetes mellitus 1. typu imunologie   MeSH
• dítě MeSH
• glutamát dekarboxyláza imunologie   MeSH
• inzulin imunologie   MeSH
• izoenzymy imunologie   MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• předškolní dítě MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• předškolní dítě MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoprotilátky MeSH
• glutamát dekarboxyláza MeSH
• glutamate decarboxylase 2 MeSH Prohlížeč
• ICA512 autoantibody MeSH Prohlížeč
• inzulin MeSH
• izoenzymy MeSH

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 beta-cell autoantigen (GAD65 and GAD65 derived peptides) were analysed by ELISPOT (IFN-gamma) 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-gamma, TNF-beta) 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.

• MeSH
• autoprotilátky krev   imunologie   MeSH
• čipová analýza proteinů MeSH
• cystická fibróza komplikace   imunologie   MeSH
• diabetes mellitus etiologie   imunologie   MeSH
• dítě MeSH
• dospělí MeSH
• glutamát dekarboxyláza imunologie   MeSH
• HLA-DQ antigeny imunologie   MeSH
• interferon gama krev   MeSH
• interleukin-8 krev   MeSH
• izoenzymy imunologie   MeSH
• leukocyty mononukleární imunologie   MeSH
• lidé MeSH
• lymfotoxin-alfa krev   MeSH
• mladiství MeSH
• pilotní projekty MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoprotilátky MeSH
• glutamát dekarboxyláza MeSH
• glutamate decarboxylase 2 MeSH Prohlížeč
• HLA-DQ antigeny MeSH
• interferon gama MeSH
• interleukin-8 MeSH
• izoenzymy MeSH
• lymfotoxin-alfa MeSH

BACKGROUND: Individuals initially diagnosed with type 2 diabetes (T2D) might exhibit positivity for diabetes-associated autoantibodies (DAA +). We investigated the prevalence of DAA positivity in a group of individuals with T2D who were referred to a tertiary diabetes centre within a pre-specified period of time. We aimed to identify characteristics linked with DAA positivity by comparing DAA + individuals with their DAA-negative counterparts. METHODS: This was a cross-sectional study into which all T2D patients referred to the National Institute of Endocrinology and Diabetology, Ľubochňa, Slovakia, between 1 January and 30 June 2016 were included. Data on > 70 participants' characteristics, including antibodies against glutamic acid decarboxylase (anti-GAD65), insulinoma-associated antigen IA-2 (IA-2A) and insulin (IAA), were collected. RESULTS: Six hundred and ninety-two individuals (387, 55.6% female) with a median (range) age of 62 (24-83) years, HbA1c of 8.9 (5.0-15.7)% [74 (31-148 mmol/mol)] and diabetes duration of 13.0 (0-42) years were analysed. One hundred and forty-five (145/692, 21.0%) tested positive for at least one DAA; 136/692 (19.7%) were positive for anti-GAD65, 21/692 (3.0%) were positive for IA-2A and 9/692 (1.3%) were positive for IAA. Only 84.9% of the DAA + individuals aged > 30 years at the time of diabetes diagnosis met the current diagnostic criteria for latent autoimmune diabetes of adults (LADA). DAA + differed from DAA - individuals in multiple characteristics, including the incidence of hypoglycaemia. CONCLUSION: Several pathological processes linked with distinct types of diabetes can develop in parallel, including insulin resistance and autoimmune insulitis. In this single-centre cross-sectional study from Slovakia, we report a higher than previously published prevalence of DAA positivity in a group of individuals with a formal diagnosis of T2D.

• Klíčová slova
• Antibody against glutamic acid decarboxylase (anti-GAD65), Antibody against insulin (IAA), Antibody against insulinoma-associated antigen IA-2 (IA-2A), Diabetes-associated autoantibodies (DAA), Latent autoimmune diabetes of adults (LADA), Type 2 diabetes mellitus (T2D),
• Publikační typ
• časopisecké články MeSH

Type 1 diabetes (T1D) is an autoimmune disorder characterised by the immune-mediated destruction of insulin-producing pancreatic beta cells. The inflammatory process appears to be primarily mediated by pro-inflammatory Th1 lymphocytes, while the role Th17 cells in T1D is currently being investigated. T1D is characterised by the presence of autoantigen-specific autoantibodies. This study was conducted using patients with confirmed T1D and healthy control subjects. We examined the effect of the patient's autoantibody profile on peripheral blood mononuclear cell (PBMC) cytokine production following stimulation with the major diabetogenic autoantigens GAD65 and IA2. IFN-gamma and IL17 production was detected by ELISPOT and the ratio of basic cellular populations in PBMCs was measured by flow cytometry. We demonstrated a significant interaction between the patient's autoantibody profile and mode of stimulation. This suggests that autoantigen stimulation has a different effect on different groups of patients depending on their autoantibody profile. An increased production of IL17 was found in patients with high IA2 autoantibodies compared to patients with low levels of autoantibodies and healthy controls regardless of the mode of stimulation. The titre of IA2 autoantibodies positively correlates with the proportion of Tc lymphocytes and negatively correlates with the proportion of Th lymphocytes. Our results show that a patient's autoantibody profile reflects the type of cellular immune responses. It seems that the high titre of IA2 autoantibodies is related to increased production of IL17 and an increased proportion of Tc lymphocytes. This finding may be useful in designing immunointervention studies to prevent T1D.

• Klíčová slova
• Autoantibodies, GAD65, IA2, IL17, Type 1 diabetes,
• MeSH
• autoprotilátky krev   imunologie   MeSH
• diabetes mellitus 1. typu krev   imunologie   MeSH
• dítě MeSH
• glutamát dekarboxyláza imunologie   MeSH
• interferon gama biosyntéza   krev   MeSH
• interleukin-17 biosyntéza   krev   MeSH
• leukocyty mononukleární metabolismus   MeSH
• lidé MeSH
• předškolní dítě MeSH
• průtoková cytometrie MeSH
• regulační T-lymfocyty imunologie   MeSH
• T-lymfocyty pomocné-indukující imunologie   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoprotilátky MeSH
• glutamát dekarboxyláza MeSH
• glutamate decarboxylase 2 MeSH Prohlížeč
• interferon gama MeSH
• interleukin-17 MeSH

AIMS: Residual beta cell function in type 1 diabetes (T1D) is associated with lower risk of complications. Autoantigen therapy with GAD-alum (Diamyd) given in 3 intralymphatic injections with oral vitamin D has shown promising results in persons with T1D carrying the human leukocyte antigen (HLA) DR3-DQ2 haplotype in the phase 2b trial DIAGNODE-2. We aimed to explore the efficacy of intralymphatic GAD-alum on blood glucose recorded by continuous glucose monitoring (CGM). METHODS: DIAGNODE-2 (NCT03345004) was a multicenter, randomized, placebo-controlled, double-blind trial of 109 recent-onset T1D patients aged 12 to 24 years with GAD65 antibodies and fasting C-peptide > 0.12 nmol/L, which randomized patients to 3 intralymphatic injections of 4 μg GAD-alum and oral vitamin D, or placebo. We report results for exploratory endpoints assessed by 14-day CGM at months 0, 6, and 15. Treatment arms were compared by mixed-effects models for repeated measures adjusting for baseline values. RESULTS: We included 98 patients with CGM recordings of sufficient quality (DR3-DQ2-positive patients: 27 GAD-alum-treated and 15 placebo-treated). In DR3-DQ2-positive patients, percent of time in range (TIR, 3.9-10 mmol/L) declined less between baseline and month 15 in GAD-alum-treated compared with placebo-treated patients (-5.1% and -16.7%, respectively; P = 0.0075), with reduced time > 13.9 mmol/L (P = 0.0036), and significant benefits on the glucose management indicator (P = 0.0025). No differences were detected for hypoglycemia. GAD-alum compared to placebo lowered the increase in glycemic variability (standard deviation) observed in both groups (P = 0.0219). Change in C-peptide was correlated with the change in TIR. CONCLUSIONS: Intralymphatic GAD-alum improves glycemic control in recently diagnosed T1D patients carrying HLA DR3-DQ2.

• Klíčová slova
• C-peptide, Diamyd, GAD-alum, GAD65, HLA DR3-DQ2, HbA1c, antigen-specific immune therapy, continuous glucose monitoring, type 1 diabetes,
• MeSH
• C-peptid MeSH
• diabetes mellitus 1. typu * MeSH
• dítě MeSH
• glutamát dekarboxyláza MeSH
• HLA-DR3 antigen MeSH
• kamencové sloučeniny MeSH
• krevní glukóza MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• regulace glykemie MeSH
• selfmonitoring glykemie MeSH
• vitamin D terapeutické užití   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH
• Názvy látek
• aluminum sulfate MeSH Prohlížeč
• C-peptid MeSH
• glutamát dekarboxyláza MeSH
• HLA-DR3 antigen MeSH
• kamencové sloučeniny MeSH
• krevní glukóza MeSH
• vitamin D MeSH

OBJECTIVE: Refractory epilepsy may have an underlying autoimmune etiology. Our aim was to assess the prevalence of neural autoantibodies in a multicenter national prospective cohort of patients with drug-resistant epilepsy undergoing epilepsy surgery utilizing comprehensive clinical, serologic, and histopathological analyses. METHODS: We prospectively recruited patients undergoing epilepsy surgery for refractory focal epilepsy not caused by a brain tumor from epilepsy surgery centers in the Czech Republic. Perioperatively, we collected cerebrospinal fluid (CSF) and/or serum samples and performed comprehensive commercial and in-house assays for neural autoantibodies. Clinical data were obtained from the patients' medical records, and histopathological analysis of resected brain tissue was performed. RESULTS: Seventy-six patients were included, mostly magnetic resonance imaging (MRI)-lesional cases (74%). Mean time from diagnosis to surgery was 21 ± 13 years. Only one patient (1.3%) had antibodies in the CSF and serum (antibodies against glutamic acid decarboxylase 65) in relevant titers; histology revealed focal cortical dysplasia (FCD) III (FCD associated with hippocampal sclerosis [HS]). Five patients' samples displayed CSF-restricted oligoclonal bands (OCBs; 6.6%): three cases with FCD (one with FCD II and two with FCD I), one with HS, and one with negative histology. Importantly, eight patients (one of them with CSF-restricted OCBs) had findings on antibody testing in individual serum and/or CSF tests that could not be confirmed by complementary tests and were thus classified as nonspecific, yet could have been considered specific without confirmatory testing. Of these, two had FCD, two gliosis, and four HS. No inflammatory changes or lymphocyte cuffing was observed histopathologically in any of the 76 patients. SIGNIFICANCE: Neural autoantibodies are a rare finding in perioperatively collected serum and CSF of our cohort of mostly MRI-lesional epilepsy surgery patients. Confirmatory testing is essential to avoid overinterpretation of autoantibody-positive findings.

• Klíčová slova
• CSF, GAD65, epilepsy, epilepsy surgery, neural antibodies,
• MeSH
• autoprotilátky MeSH
• epilepsie * epidemiologie   chirurgie   komplikace   MeSH
• lidé MeSH
• magnetická rezonanční tomografie MeSH
• malformace mozkové kůry * komplikace   MeSH
• prevalence MeSH
• prospektivní studie MeSH
• refrakterní epilepsie * diagnostické zobrazování   chirurgie   komplikace   MeSH
• retrospektivní studie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• Názvy látek
• autoprotilátky MeSH

UNLABELLED: Tolerogenic DCs (tolDCs) are being researched as a promising intervention strategy also in autoimmune diseases including type 1 diabetes (T1D). T1D is a T-cell-mediated, organ-specific disease with several well-defined and rather specific autoantigens, i.e., proinsulin, insulin, glutamic acid decarboxylase 65 (GAD65), that have been used in animal as well as human intervention trials in attempts to achieve a more efficient, specific immunotherapy. In this study, we have tested tolerogenic DCs for their effectiveness to prevent adoptive transfer of diabetes by diabetogenic splenocytes into non-obese diabetes (NOD)-severe combined immunodeficiency (NOD-SCID) recipients. While i.p. application of tolDCs prepared from bone marrow of prediabetic NOD mice by vitamin D2 and dexamethasone significantly reduced diabetes transfer into the NOD-SCID females, this effect was completely abolished when tolDCs were loaded with the mouse recombinant GAD65, but also with a control protein-ovalbumin (OVA). The effect was not dependent on the presence of serum in the tolDC culture. Similar results were observed in NOD mice. Removal of possible bystander antigen-presenting cells within the diabetogenic splenocytes by negative magnetic sorting of T cells did not alter this surprising effect. Tolerogenic DCs loaded with an immunodominant mouse GAD65 peptide also displayed diminished diabetes-preventive effect. Tolerogenic DCs were characterized by surface maturation markers (CD40, CD80, CD86, MHC II) and the lipopolysaccharide stability test. Data from alloreactive T cell proliferation and cytokine induction assays (IFN-γ) did not reveal the differences observed in the diabetes incidence. Migration of tolDCs, tolDCs-GAD65 and tolDCs-OVA to spleen, mesenteric- and pancreatic lymph nodes displayed similar, mucosal pattern with highest accumulation in pancreatic lymph nodes present up to 9 days after the i.p. APPLICATION: These data document that mechanisms by which tolDCs operate in vivo require much better understanding for improving efficacy of this promising cell therapy, especially in the presence of an antigen, e.g., GAD65.

• Klíčová slova
• autoantigen, cell therapy, dendritic cells, glutamic acid decarboxylase 65, non-obese diabetes mice, non-obese diabetes-severe combined immunodeficiency mouse, tolerogenic, type 1 diabetes,
• MeSH
• autoantigeny imunologie   MeSH
• dendritické buňky imunologie   MeSH
• diabetes mellitus 1. typu imunologie   MeSH
• glutamát dekarboxyláza imunologie   MeSH
• imunologická tolerance imunologie   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši inbrední NOD MeSH
• myši SCID MeSH
• myši MeSH
• převzatá imunita MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoantigeny MeSH
• glutamát dekarboxyláza MeSH
• glutamate decarboxylase 2 MeSH Prohlížeč

Tolerogenic dendritic cells (tolDCs) may offer an interesting intervention strategy to re-establish Ag-specific tolerance in autoimmune diseases, including type 1 diabetes (T1D). T1D results from selective destruction of insulin-producing β cells leading to hyperglycemia that, in turn, specifically affects a patient's immune system. In this study, we prepared monocyte-derived tolDCs modulated by dexamethasone and vitamin D2 from 31 T1D patients with optimal glycemic control and 60 T1D patients with suboptimal glycemic control and assessed their tolerogenic properties in correlation with metabolic state of patients. tolDCs differentiated from both groups of patients acquired a regulatory phenotype and an anti-inflammatory profile. Interestingly, tolDCs from well-controlled patients expressed higher levels of inhibitory molecules IL-T3 and PD-L1. Additionally, glutamic acid decarboxylase (GAD)65-loaded tolDCs from well-controlled patients decreased significantly primary Th1/Th17 responses, induced stable GAD65-specific T cell hyporesponsiveness, and suppressed markedly control DC-induced GAD65-specific T cell activation compared with poorly controlled patients. The ability of tolDCs from poorly controlled patients to induce durable GAD65-specific T cell hyporesponsiveness was reversed once the control of glycemia improved. In both groups of patients, tolDCs were able to induce regulatory T cells from autologous naive CD4+ T cells. However, regulatory T cells from well-controlled patients had better suppressive abilities. The functionality of tolDCs was confirmed in the adoptive transfer model of NOD-SCID mice where tolDCs delayed diabetes onset. These results suggest that metabolic control of T1D affects the functional characteristics of tolDCs and subsequent effector T cell responses. Metabolic control may be relevant for refining inclusion criteria of clinical trials in the settings of T1D.

• MeSH
• aktivace lymfocytů imunologie   MeSH
• dendritické buňky imunologie   MeSH
• diabetes mellitus 1. typu imunologie   MeSH
• imunologická tolerance imunologie   MeSH
• lidé MeSH
• myši inbrední NOD MeSH
• myši SCID MeSH
• myši MeSH
• převzatá imunita MeSH
• průtoková cytometrie MeSH
• regulační T-lymfocyty imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Changes in the levels of gamma-aminobutyric acid (GABA) are known to occur in different parts of the brain during aging. In our study we attempted to define the effect that aging has on glutamate decarboxylase (GAD), the key enzyme in the synthesis of GABA, in the central parts of the auditory system. Age-related changes in GAD65 and GAD67 levels were investigated using immunohistochemistry and Western blotting in the inferior colliculus (IC), the auditory cortex (AC) and the visual cortex in Long-Evans rats. The results show that aging is associated with a decrease in the numbers of GAD65- and 67-immunoreactive neurons and the optical density of their somas in both the IC and AC. Western blot analysis revealed a pronounced age-related decline in the levels of GAD65 and 67 proteins in both the IC and AC. For comparison, in the visual cortex the decrease in both proteins was less pronounced than in the IC and AC. A similar pattern of age-related changes was found in Fischer 344 rats, a strain that manifests a rapid loss of hearing function with aging. The observed age-related decline in the levels of GAD65 and 67 may contribute significantly to the deterioration of hearing function that accompanies aging in mammals, including man.

• MeSH
• analýza rozptylu MeSH
• colliculus inferior metabolismus   MeSH
• druhová specificita MeSH
• GABA metabolismus   fyziologie   MeSH
• glutamát dekarboxyláza metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• potkani inbrední F344 MeSH
• potkani Long-Evans MeSH
• presbyakuze metabolismus   MeSH
• protein - isoformy analýza   metabolismus   MeSH
• sluchové korové centrum metabolismus   MeSH
• stárnutí metabolismus   MeSH
• western blotting metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• GABA MeSH
• glutamát dekarboxyláza MeSH
• protein - isoformy MeSH

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

• Klíčová slova
• GAD65 and VGAT, long-lasting pain reversal, neuropathic pain, precision spinal vector delivery, subpial injection technique, subpial non-human primates, subpial pig, targeted AAV9 delivery,
• MeSH
• buňky zadních rohů míšních MeSH
• mícha MeSH
• myši MeSH
• neuralgie * etiologie   terapie   MeSH
• nociceptory * MeSH
• prasata MeSH
• technika přenosu genů MeSH
• zadní rohy míšní MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH