Processing in the endoplasmic reticulum generates an epitope on the insulin A chain that stimulates diabetogenic CD8 T cell responses.

RIP-B7.1 mice express the costimulator molecule B7.1 (CD80) on pancreatic beta cells and are a well-established model for studying de novo induction of diabetogenic CD8 T cells. Immunization of RIP-B7.1 mice with preproinsulin (ppins)-encoding plasmid DNA efficiently induces experimental autoimmune diabetes (EAD). EAD is associated with an influx of CD8 T cells specific for the K(b)/A(12-21) epitope into the pancreatic islets and the subsequent destruction of beta cells. In this study, we used this model to investigate how ppins-derived Ags are expressed and processed to prime diabetogenic, K(b)/A(12-21)-specific CD8 T cells. Targeting the K(b)/A(12-21) epitope, the insulin A chain, or the ppins to the endoplasmic reticulum (ER) (but not to the cytosol and/or nucleus) efficiently elicited K(b)/A(12-21)-specific CD8 T cell responses. The K(b)/A(12-21) epitope represents the COOH terminus of the ppins molecule and, hence, did not require COOH-terminal processing before binding its restriction element in the ER. However, K(b)/A(12-21)-specific CD8 T cells were also induced by COOH-terminally extended ppins-specific polypeptides expressed in the ER, indicating that the epitope position at the COOH terminus is less important for its diabetogenicity than is targeting the Ag to the ER. The K(b)/A(12-21) epitope had a low avidity for K(b) molecules. When epitopes of unrelated Ags were coprimed at the same site of Ag delivery, "strong" K(b)-restricted (but not D(b)-restricted) CD8 T cell responses led to the suppression of K(b)/A(12-21)-specific CD8 T cell priming and reduced EAD. Thus, direct expression and processing of the "weak" K(b)/A(12-21) epitope in the ER favor priming of autoreactive CD8 T cells.

Allelic variants in the PHTF1-PTPN22, C12orf30 and CD226 regions as candidate susceptibility factors for the type 1 diabetes in the Estonian population.

BACKGROUND: Type 1 diabetes is a multifactorial disease with a strong genetic component. The aim of the study was to assess the impact of single nucleotide polymorphisms (SNPs) in several genes as susceptible markers in the risk of type 1 diabetes in the Estonian population. METHODS: The rs6679677 (1p13), rs17696736 (12q24) and rs763361 (18q22) were genotyped in a total of 230 controls and 154 type 1 diabetes patients of Estonian origin. RESULTS: The rs6679677 A (OR = 2.13, 95%CI = 1.48-3.08, p = 0.00001), rs17696736 G (OR = 1.53, 95%CI = 1.14-2.04, p = 0.0046) and rs763361 T (OR = 1.48, 95%CI = 1.11-1.98, p = 0.0084) alleles were associated with risk of type 1 diabetes. CONCLUSIONS: The current study supports the rs6679677 (PHTF1-PTPN22), rs17696736 (C12orf30) and rs763361 (CD226) SNPs as susceptibility factors for type 1 diabetes outside the major histocompatibility region (MHC) region. The full study had 80% or above to detect an odds ratio of 1.8 under the assumption of an additive model at type 1 error rate, alpha = 0.05.

Antigenic proteins of Lactobacillus acidophilus that are recognised by serum IgG antibodies in children with type 1 diabetes and coeliac disease.

Immune responses to lactobacilli have been so far insufficiently investigated in patients with autoimmune diseases. We used whole-cell lysate of an indigenous Lactobacillus acidophilus strain isolated from an Estonian child to study serum IgG antibodies in children groups with type 1 diabetes [insulin dependent diabetes mellitus (IDDM)] (n = 21, age 4-18 yr) and with acute coeliac disease (CD) (n = 20, age 0.6-15 yr) and to compare the results with the controls (n = 24, age 2-17 yr). We found that our developed 1-D immunoblot assay readily enables to reveal antibodies against 28 L. acidophilus antigenic proteins in patients' and controls' sera. As verified by immunoproteomics analysis with 2-D and LC ESI-MS/MS the antigens of L. acidophilus were mainly common cytoplasmic proteins GroEL (HSP60), enolase, transcription factor EF-Ts and EF-Tu. However, in addition we identified formyl-CoA transferase being target for antibodies in every tested IDDM patients' serum. We have characterized for the first time the antigenic profile of L. acidophilus whole-cell lysate using sera from children with IDDM, CD, and controls. The different prevalence of reactions against tested antigens in patients and controls sera may indicate significant differences in immune system and commensal bacteria cross-talk in these groups.

Lack of disease specificity limits the usefulness of in vitro costimulation in HIV- and HCV-infected patients.

Measurements of antigen-specific T cell responses in chronic diseases are limited by low frequencies of antigen-specific cells in the peripheral blood. Therefore, attempts have been made to add costimulatory molecules such as anti-CD28 or IL-7/IL-15 to ELISPOT assays to increase sensitivity. While this approach has been successful under certain circumstances, results are often inconsistent. To date, there are no comprehensive studies directly comparing the in vitro effects of multiple costimulatory molecules in different disease settings. Therefore, in the present study we tested the effects of IL-7/IL-15, IFN-alpha, anti-ICOS, and anti-CD28 on antigen-specific T cell responses in patients infected with HCV or HIV versus healthy individuals. Our data show that none of the aforementioned molecules could significantly increase ELISPOT sensitivity, neither in HCV nor in HIV. Moreover, all of them caused false-positive responses to HCV and HIV antigens in healthy individuals. Our results question the broad use of in vitro costimulation.

Demonstration of natural autoantibodies against the neurofilament protein alpha-internexin in sera of patients with endocrine autoimmunity and healthy individuals.

Serum anti-pituitary antibodies (APAs) to cytosolic antigens have been found in association with autoimmune hypophysitis, idiopathic hypopituitarism, and other autoimmune endocrinopathies. Here, an immunoblot method was used to search for serum autoantibody (AAb) reactivities against pituitary antigens, including nuclear and cytoskeletal proteins, in six patients with idiopathic hypopituitarism, 60 patients with type 1 diabetes, nine patients with autoimmune polyglandular syndrome (APS) type 1, and in 74 healthy controls. Frequent patient serum IgG reactivity was observed against a 60 kDa human pituitary antigen, and the cross-reactive 62 kDa protein from rat brain was identified as alpha-internexin (alpha-INX) by proteomic methods. IgG and IgM AAbs to this neuron-specific type IV intermediate filament (IF) protein were found in most sera of patients with endocrine autoimmunity as well as healthy subjects with no significant differences in frequencies between the groups, but the levels of IgM alpha-INX AAbs were higher in patients with hypopituitarism as compared to healthy controls (P = 0.032, Mann-Whitney U-test). These findings suggest that alpha-INX AAbs are not specifically related to autoimmune endocrine diseases and most probably are a part of the natural AAb repertoire. This is the first demonstration of alpha-INX AAbs as one of the predominant neuronal IF AAbs in human sera.

Preproinsulin-specific CD8+ T cells secrete IFNgamma in human type 1 diabetes.

In animal models autoreactive CD8(+) T cells are crucial in the development of type 1 diabetes (T1D); however, their role in human T1D is still not known. To address the role of CD81 T cells we performed a pilot study by investigating CD8(+) T cell-mediated cytokine secretion after in vitro stimulation with 94 preproinsulin (PPI) peptides. We were able to show that CD8(+) T cells contribute to a strong IFNgamma reactivity against PPI in human T1D. Further investigations defining epitope specificity, cytokine secretion, and cytotoxic capacity are important to clarify their role in T1D development.

Experimental autoimmune diabetes: a new tool to study mechanisms and consequences of insulin-specific autoimmunity.

(Prepro)insulin is considered a central antigenic determinant in diabetic autoimmunity. Insulin has been used to modify diabetes development in NOD mice and prediabetic individuals. We have recently shown that (prepro)insulin can adversely promote diabetes development in murine type 1 diabetes. Based on these findings we have developed experimental autoimmune diabetes (EAD), a new mouse model characterized by (1) CD4(+)/CD8(+) insulitis, induced by (2) (prepro)insulin DNA vaccination, leading to (3) beta cell damage and insulin deficiency in (4) RIP-B7.1 transgenic mice (H-2(b)). EAD develops rapidly in 60-95% of mice after intramuscular, but not intradermal ("gene gun"), vaccination; and DNA plasmids expressing insulin or the insulin analogues glargine, aspart, and lispro are equally potent to induce EAD. Similar to NOD mice, diabetes is adoptively transferred into syngeneic recipients by spleen cell transplantation in a dose-dependent fashion. We have devised a two-stage concept of EAD in which T cell activation and expansion is driven by in vivo autoantigen expression, followed by islet damage that requires beta cell expression of costimulatory B7.1 for disease manifestation. Taken together, EAD is a novel, genetically defined animal model of type 1 diabetes suitable to analyze mechanisms and consequences of insulin-specific T cell autoimmunity.

CC-chemokine receptor CCR5-del32 mutation as a modifying pathogenetic factor in type I diabetes.

The purpose of this study was to determine the CCR5-del32 allele frequency in type I (insulin-dependent) and type II (noninsulin-dependent) diabetes patients, and to test whether and how this mutation is associated with both types of diabetes. Thirty-eight type I diabetes and 111 type II diabetes patients' genotyping was performed by polymerase chain reaction assaying, and amplified products were digested with restriction enzyme EcoRI. The results were analyzed using statistical methods. No statistical differences were found in CCR5-del32 allele frequencies in types I and II diabetes patients compared with the control group of native Estonians. However, an association exists between CCR5 gene polymorphism and the clinical course of type I diabetes. In the case of wild-type CCR5, the disease starts at an earlier age. In type II diabetes, there was a difference between genotypes in morbidity to concomitant diseases, being higher in the CCR5 wild-type genotype.

Gender differences in serum high-molecular-weight adiponectin levels in metabolic syndrome.

OBJECTIVE: The objective of this study was to estimate gender-specific associations between metabolic syndrome (MS) and high-molecular-weight (HMW) adiponectin in an Estonian adult population. METHODS: Plasma HMW adiponectin was measured in 458 subjects (191 men) who participated in a population-based cross-sectional multicenter study (n=495) on the prevalence of metabolic disorders in Estonia. MS was defined according to National Cholesterol Education Program Adult Treatment Panel III criteria. RESULTS: Median HMW adiponectin levels (µg/ml) were significantly lower among all subjects with MS compared with subjects without MS: 2.1 vs 2.8 in men (P=0.002) and 3.1 vs 5.1 in women (P<0.001). In a fully adjusted, logistic regression model containing HMW adiponectin, homeostasis model assessment of insulin resistance (HOMA-IR), BMI, and age, HMW adiponectin was significantly associated with MS only in women. Comparison of HMW adiponectin and HOMA-IR as markers for MS indicated that HOMA-IR predicted MS better than did HMW adiponectin in both genders. However, after adjusting for age and BMI, HOMA-IR was a significantly better predictor only in men. HMW adiponectin and HOMA-IR predicted the presence of MS at the same level in women. Areas under the receiver operating characteristic curves for HMW adiponectin and HOMA-IR were 0.833 vs 0.88 in men (P=0.02) and 0.897 vs 0.907 in women (P=0.5). CONCLUSIONS: These data suggest that the association between low HMW adiponectin levels and presence of MS might be stronger in women compared with men.

Metabolic syndrome in estonia: prevalence and associations with insulin resistance.

Recently, it has been suggested that metabolic syndrome should be considered a premorbid condition in younger individuals. We evaluated the prevalence of metabolic syndrome in Estonia and compared the characteristic profiles between morbid metabolic syndrome (previously established diabetes, hypertension, or dyslipidaemia) and premorbid metabolic syndrome subgroups. Our study was a cross-sectional, population-based sample of the general population in Estonia aged 20-74 years (n = 495). Metabolic syndrome was diagnosed by National Cholesterol Education Program Adult Treatment Panel III criteria. Insulin resistance was estimated using the homeostasis model assessment (HOMA-IR). The crude and weighted prevalence of metabolic syndrome was 27.9% and 25.9%, respectively. Despite being significantly younger, the premorbid subgroup showed similar levels of insulin resistance as the morbid subgroup (mean HOMA-IR ± SD 2.73 ± 1.8 versus 2.97 ± 2.1, P = 0.5). The most important attribute of metabolic syndrome is insulin resistance, which already characterises metabolic syndrome in the early stages of its metabolic abnormalities.

Deficiency in B7-H1 (PD-L1)/PD-1 coinhibition triggers pancreatic beta-cell destruction by insulin-specific, murine CD8 T-cells.

OBJECTIVE: RIP-B7.1 mice expressing the costimulator molecule B7.1 (CD80) on pancreatic beta-cells are a well established model to characterize preproinsulin-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD). Different immunization strategies could prime preproinsulin-specific CD8 T-cells in wild-type C57BL/6 (B6) mice, but did not induce diabetes. We tested whether altering the B7-H1 (PD-L1) coinhibition on pancreatic beta-cells can reveal a diabetogenic potential of preproinsulin-specific CD8 T-cells. RESEARCH DESIGN AND METHODS: DNA-based immunization and adoptive T-cell transfers were used to characterize the induction of preproinsulin-specific CD8 T-cell responses and EAD in RIP-B7.1, B6, B7-H1(-/-), PD-1(-/-) or bone marrow chimeric mice. RESULTS: Preproinsulin-specific CD8 T-cells primed in B6 mice revealed their diabetogenic potential after adoptive transfer into congenic RIP-B7.1 hosts. Furthermore, preproinsulin-specific CD8 T-cells primed in anti-B7-H1 antibody-treated B6 mice, or primed in B7-H1(-/-) or PD-1(-/-) mice induced EAD. Immunization of bone marrow chimeric mice showed that deficiency of either B7-H.1 in pancreatic beta-cells or of PD-1 in autoreactive CD8 T-cells induced EAD. CONCLUSIONS: An imbalance between costimulator (B7.1) and coinhibitor (B7-H1) signals on pancreatic beta-cells can trigger pancreatic beta-cell-destruction by preproinsulin-specific CD8 T-cells. Hence, regulation of the susceptibility of the beta-cells for a preproinsulin-specific CD8 T-cell attack can allow or suppress EAD.

Higher FoxP3 mRNA expression in peripheral blood mononuclear cells of GAD65 or IA-2 autoantibody-positive compared with autoantibody-negative persons.

The role of regulatory T cells (Tregs) in type 1 diabetes has been studied extensively. The most prevalent way to define Tregs has been by their surface expression of CD4 and CD25. As currently the transcription factor FoxP3 and the low expression of CD127 are regarded to be the most specific markers of Tregs, we analysed the number of Tregs defined by these molecules in peripheral blood mononuclear cells of diabetic patients and healthy controls. The gene expression of transforming growth factor beta and two isoforms of FoxP3 was measured as well. There were no significant differences between diabetic patients and healthy controls regarding the number of Tregs, or the expression of FoxP3 isoforms and TGFbeta in peripheral blood mononuclear cells. However, we found significantly higher expression of both full-length and Delta2FoxP3 in study subjects, positive for either GAD65 or IA-2 autoantibodies. The ratio of the expression of different isoforms was not changed. This study shows the possible role of FoxP3 in the development of tissue characteristic humoral immunity in type 1 diabetes.

The diabetogenic, insulin-specific CD8 T cell response primed in the experimental autoimmune diabetes model in RIP-B7.1 mice.

Type 1 diabetes mellitus can result from the specific destruction of pancreatic beta cells by autoreactive T cells. As shown here, experimental autoimmune diabetes (EAD) is efficiently induced in RIP-B7.1 mice by preproinsulin (ppins)-encoding DNA vaccines. EAD develops in RIP-B7.1 mice within 3-4 wk after a single immunization with ppins-encoding plasmid DNA. RIP-B7.1 mice develop insulitis, insulin deficiency and hyperglycemia after vaccination with plasmids encoding murine ppins-I or murine ppins-II or human hu-ppins. EAD induction critically depends on CD8 T cells and is independent of CD4 T cells. To be diabetogenic, ppins-specific CD8 T cells had to express IFN-gamma. Neither expression of perforin nor signaling through the type I IFN receptor is an essential component of this pathogenic CD8 T cell phenotype. Using plasmids encoding truncated ppins variants, we show that EAD is only induced by DNA vaccines encoding the insulin A-chain. Diabetogenic CD8 T cells specifically recognize the Kb-restricted A12-21 epitope of the insulin A-chain. The RIP-B7.1 model hence represents an attractive model for the characterization of cellular and molecular events involved in the CD8 T cell-mediated immune pathogenesis of diabetes.

Lactase non-persistence and milk consumption in Estonia.

AIM: To define the frequency of the C/T-13910 variant associated with lactase persistence/non-persistence trait and to analyze the milk consumption of lactase non-persistent subjects in Estonia. METHODS: We genotyped 355 Estonians by polymerase chain reaction and direct sequencing. Milk consumption was analyzed by a questionnaire, specially developed to analyze milk consumption and abdominal complaints. RESULTS: The frequency of the genotype of the C/C-13910 (lactase non-persistence) was found to be 24.8% in native Estonians. No other single nucleotide polymorphisms covering the region of 400 bp adjacent to the C/T-13910 variant were found. Lactase non-persistence subjects were found to consume less milk than lactase persistence subjects. CONCLUSION: The frequency of lactase non-persistence defined by the C/C-13910 genotype confirms the results of the previous studies based on indirect methods of determining hypolactasia. Milk consumption of lactase non-persistence subjects is consistent with previously reported figures of adult-type hypolactasia in Estonia. However, lactase non-persistence does not prevent the intake of milk in many adults.