Jochen Seissler

Titre and combination of ICA and autoantibodies to glutamic acid decarboxylase discriminate two clinically distinct types of latent autoimmune diabetes in adults (LADA)

Aims/hypothesis: This study aimed to define the immunological parameters which could be used to identify patients with the distinct metabolic features of adult latent autoimmune diabetes. Methods: Sera of 312 patients with short-term diabetes (duration < 5 years) over 35 years of age at diagnosis were screened for ICA, GAD- and IA2-Ab by antibody assays validated in workshops. The antibody status was correlated with age, BMI, residual beta-cell function, measured by fasting C-peptide, onset of diabetes-related complications and markers of the metabolic syndrome (hypertension and hyperlipidaemia). Results: A total of 51 antibody positive patients were identified. These patients had lower fasting C-peptide and less neuropathy and hypertension compared with matched antibody-negative patients. However, only patients with two or more antibodies had reduced residual beta-cell function compared with antibody-negative or single antibody-positive (ICA or GAD-Ab only) patients. Patients with two or more antibodies were also leaner and had diabetes-related complications or hypertension less frequently than single antibody-positive or antibody negative-patients. IA2 antibody status did not substantially contribute to the diagnosis or differentiation of LADA patients. Conclusion/interpretation: We concluded that the combination of ICA and GAD antibodies and high titre of GAD antibodies are characteristic of patients with insulin deficiency with the clinical features of Type I (insulin-dependent) diabetes mellitus (LADA-type 1). Single antibody positivity and low titre antibodies are markers for LADA-type 2 associated with the clinical and metabolic phenotype of Type II (non-insulin-dependent) diabetes patients. [Diabetologia (2001) 44: 1005–1010]

Autoantibodies from patients with coeliac disease recognize distinct functional domains of the autoantigen tissue transglutaminase

The enzyme tissue transglutaminase (tTG) has been recently identified to represent a highly sensitive and specific target of autoantibodies in coeliac disease. To characterize autoantigenic epitopes, we generated novel tTG deletion mutants by polymerase chain reaction, produced radiolabelled fragments by in vitro transcription/translation, immunoprecipitated the mutants using sera from patients with coeliac disease, and related the binding data with putative structural and functional domains of human tTG. We show that tTG antibody positive sera display a heterogeneous autoantibody response covering distinct regions of the molecule. The N‐terminal and C‐terminal third of tTG, comprising amino acid (aa) 1–281 and aa 473–687, harbour the dominant epitopes (67·4% and 69·4% positive), whereas the catalytic region is of minor antigenicity (22·5% positive). Autoantibodies directed to one, two and three domains were observed in 36·7%, 28·6% and 22·4% of patients, respectively. Comparative analysis revealed the presence of strictly conformational epitopes which were dependent on the N‐terminus (aa 1–12) or the intact β‐barrel domains in the C‐terminus (aa 473–497, aa 649–687). In conclusion, we here demonstrate for the first time that the humoral autoimmunity is directed against distinct functional tTG domains. The spectrum of autoantibodies indicates that the native folded protein may be the target of autoantibodies.

Calcitonin-specific antitumor immunity in medullary thyroid carcinoma following dendritic cell vaccination.

Abstract. In this study, we investigated the immune response following immunotherapy with calcitonin-pulsed dendritic cells (DC) in 7 patients with metastasized medullary thyroid carcinoma. After immunization with 1–5×106 autologous DC, significant calcitonin-specific T cell proliferation was detectable in 3 patients. Measurement of cytokine release from T lymphocytes demonstrated high post-treatment interferon-γ (IFN-γ) secretion after stimulation with calcitonin in 5 patients, one of whom experienced significant tumor regression. In contrast, antigen-specific interleukin-4 (IL-4) production was only slightly increased in 4 patients. All 7 patients developed a strong delayed-type hypersensitivity (DTH) skin reaction, which was confirmed to be mediated by infiltrating CD4+ T-helper cells and CD8+ cytotoxic T cells in all 3 patients who underwent skin biopsy. This is the first study to show that a polypeptide hormone can be used to develop a DC vaccination strategy for the immunotherapy of highly malignant endocrine cancers.

Response of human monocyte-derived dendritic cells to immunostimulatory DNA.

Activated dendritic cells (DC) are of key importance for the initiation of primary immune responses and represent promising tools for immunotherapies in humans. Since DNA containing CpG motifs have been described as potent immunostimulatory (IS) adjuvants for murine DC, we here studied maturation and stimulation of functional activity in human monocyte‐derived DC (MODC) in response to several immunostimulatory oligodeoxynucleotides (IS‐ODN) and plasmid DNA (IS‐PL). We show that exposure of MODC to IS‐PL, but not IS‐ODN, induced a dose‐dependent strong up‐regulation of HLA class II and co‐stimulatory molecules (CD80, CD86), similar to that observed after treatment with TNF‐α. Functional activity was assessed by the detection of increased secretions of IL‐6 and IL‐12(p75) following treatment with IS‐PL. In addition, IS‐PL‐stimulated MODC acquired a high T cell‐stimulatory capacity. T cells stimulated by tetanus toxoid‐pulsed, IS‐PL‐matured MODC were significantly more frequently IFN‐γ positive (25.2±2.7%) as compared to TNF‐α‐treated MODC (15.4±1.4 %), indicating a strong activation of Th1 lymphocytes. In conclusion, we demonstrate that human MODC are activated by IS‐PL but not IS‐ODN previously used as adjuvants in animal models. The Th1‐like immune response observed after stimulation with IS‐PL‐treated DC suggests that preincubation of human MODC with IS‐PL or coimmunization with IS‐PL may represent an useful approach to generate strongly activated human MODC for several therapeutic applications such as DC‐based tumor immunotherapy.

Mapping of novel autoreactive epitopes of the diabetes-associated autoantigen IA-2.

IA‐2, a member of the tyrosine phosphatase family, has been identified as a dominant autoantigen in type 1 diabetes. To define humoral IA‐2 epitopes, we generated a panel of IA‐2 deletion mutants and chimeric proteins using the highly homologous tyrosine phosphatase‐like protein IA‐2β. Analysis of autoantibody reactivity in 111 IA‐2 antibody positive sera from patients with type 1 diabetes revealed that humoral epitopes cluster to several domains of the intracytoplasmic part of IA‐2 [IA‐2ic, amino acid (aa) 604–979]. Immunodominant epitopes were found in the first N‐terminal 73 amino acids (56% positive), in the middle domain residing between residues 699–874 (45% positive) and the C‐terminus depending on the presence of aa 931–979 (at least 37% positive). Competition experiments with overlapping peptides revealed that autoantibody binding towards the N‐terminus was dependent on residues 621–628. In the C‐terminal domain, two novel conformation‐dependent epitopes were identified. The first epitope requires the presence of the C‐terminal part of IA‐2 (aa 933–979) and an IA‐2‐specific region between residues 771–932. Reactivity against the second epitope was dependent on intact C‐terminal domains as well as residues in the middle (aa 887–932) and N‐terminal regions (aa 604–771) which are conserved in IA‐2 and IA‐2β. We here defined novel autoantigenic determinants in the N‐terminus of IA‐2 and characterized conformational epitopes residing in the C‐terminal region or spanning from C‐terminal residues to the N‐terminal domain of IA‐2ic. The identification of dominant target regions of diabetes‐specific autoantibodies may help to elucidate the molecular mechanisms involved in the autoimmunity towards IA‐2.

Vasoregulatory peptides pro-endothelin-1 and pro-adrenomedullin are associated with metabolic syndrome in the population-based KORA F4 study.

BACKGROUND Metabolic alterations and endothelial dysfunction are interrelated processes in type 2 diabetes (T2D) and metabolic syndrome (MetS) that often develop in parallel. We assessed the association of vasoactive precursor peptides (VPPs) with MetS and T2D. DESIGN AND METHODS Plasma levels of C-terminal pro-endothelin-1 (CT-proET-1) and midregional pro-adrenomedullin (MR-proADM) were measured by novel sensitive assays in 1590 participants of the population-based KORA F4 study. The association of the VPPs with T2D, MetS defined by IDF criteria, the components of MetS, and insulin resistance (IR) was assessed in logistic regression models. RESULTS Elevated levels of CT-proET-1 and MR-proADM were associated with T2D, MetS, and IR in age- and sex-adjusted models. After adjustment for age, sex, former vascular complications, lifestyle factors, high-sensitive C-reactive protein, and serum creatinine, significant associations with MetS were found for MR-proADM (OR=5.94, 95% CI 3.78-9.33) and CT-proET-1 (OR=5.18, 95% CI 3.48-7.71) (top quartile vs bottom quartile). CT-proET-1 and MR-proADM were strongly associated with all components of MetS as defined by IDF criteria. After multivariable adjustment, association of CT-proET-1 and MR-proADM with pathological glucose tolerance and T2D disappeared and a borderline association with IR was found only for CT-proET-1 (OR=1.34, 95% CI 0.96-1.87). CONCLUSIONS We here demonstrate for the first time that plasma levels of both MR-proADM and CT-proET-1 levels are related to MetS and its components, thus suggesting that they possibly have a role as a surrogate biomarker for the disease and its complications.

Dendritic cell vaccination: new hope for the treatment of metastasized endocrine malignancies

Dendritic cells (DCs) are antigen-presenting cells that are involved in the induction of primary immune responses. The unique ability of DCs to activate naive and memory CD4+ and CD8+ T cells suggests that they could be used for the induction of a specific antitumour immunity. In the past few years, several in vitro and in vivo studies in rodents and humans have demonstrated that immunizations with DCs pulsed with tumour antigens result in protective immunity and rejection of established tumours in various malignancies. Here, we focus on recent results of how DCs regulate immune responses that are important for generating antitumour cytotoxic T cells, and summarize clinical vaccination trials for the treatment of endocrine and nonendocrine carcinomas. Preliminary results suggest that DC vaccines might be novel tools for antitumour immunotherapies to treat chemotherapy-resistant and radioresistant endocrine cancers, such as metastasized medullary thyroid carcinomas and other neuroendocrine carcinomas.

Autoantigen-specific protection of non-obese diabetic mice from cyclophosphamide-accelerated diabetes by vaccination with dendritic cells

Aims/hypothesisDendritic cells (DCs) are professional antigen presenting cells involved in the initiation of primary immune responses and the preservation of peripheral tolerance. The aim of this study was to develop a DC vaccine for autoantigen-specific prevention of autoimmune diabetes.MethodsSplenocytes from diabetes-prone NOD mice were cultured in conditioned media to obtain a homogeneous DC sub-population for vaccination experiments. These cells were used to modulate autoimmune responses in NOD mice after synchronization of diabetes with cyclophosphamide. After immunisation with insulin-pulsed DCs the incidence of diabetes, the insulitis grade and the cytokine production was examined.ResultsThe long-term culture of splenocytes resulted in the generation of a cell line, termed NOD-DC1, which have a phenotype of myeloid DCs (CD11c, CD11b, DEC-205), express MHC class II and co-stimulatory molecules (CD40, CD80, CD86). The NOD-DC1 cells have preserved functional activity shown by the detection of a high antigen uptake capacity, the induction of a mixed lymphocyte reaction and stimuli-dependent IL-6 and TNF-α secretion. Vaccination with insulin-pulsed NOD-DC1 cells results in an antigen-specific prevention of diabetes. This was mediated by a reduction of the severity of insulitis and a decrease of T helper 1 effector cells.Conclusion/interpretationWe describe the generation of a DC line which confers protection from diabetes in an antigen-specific way. Our data shows that autoantigen-loaded DCs can induce strong immunoregulatory effects supporting the hypothesis that DCs are promising candidates to develop novel vaccines for the prevention of autoimmune diabetes.

Preliminary evidence that an endogenous retroviral long‐terminal repeat (LTR13) at the HLA‐DQB1 gene locus confers susceptibility to Addison's disease

OBJECTIVE Addisons disease is associated with particular haplotypes of the human leucocyte antigen (HLA) region [DQA1*0501‐DQB1*0201 (DQ2) and DQA1*0301‐DQB1*0302 (DQ8)]. This locus harbours several human endogenous retroviral (HERV) long‐terminal repeats (LTR). LTRs within the HLA region have been shown to confer additional susceptibility to type 1 diabetes and rheumatoid arthritis.

Are we ready to predict and prevent endocrine/organ specific autoimmune diseases?

Endocrine autoimmune-mediated disorders (EADs) are characterised by an infiltration of the target organ with mononuclear cells such as macrophages, dendritic cells and lymphocytes. According to the predominant pathogenic mechanisms that contribute to the disease development, EADs can be roughly divided into celland humoral-mediated diseases. Cell-mediated disorders include among others, Hashimotos’s thyroiditis (HT), autoimune Addison’s disease (AD) and type 1 (insulin-dependent) diabetes (T1D) [42, 127, 145]. For each of these disorders, cellular immunity is associated with progressive tissue destruction, either via direct cytotoxicity by autoreactive CD8+ T cells or by indirect cytotoxic mechanisms through several components of the infiltrating cells (proinflammatory cytokines IL-1β, IFN-γ, TNF-α, free radicals) [96, 115]. In autoimmune diabetes both CD4+ and CD8+ T cells specific to β-cell antigens were sufficient to transfer diabetes in the non-obese diabetic (NOD) mouse model [40, 165], suggesting that autoantibodies play a minor role in the pathogenesis of βcell destruction. Indeed, for human T1D we observed the case of a boy who developed autoreactive T cells and T1D despite the presence of severe hereditary B lymphocyte deficiency [97]. In contrast to T1D, in Graves’disease (GD) autoantibodies can induce pathology by stimulating the function of the thyroid-stimulating hormone (TSH) receptor resulting in diffuse thyroid hyperplasia and hyperthyroidism [98]. Epidemiological studies have shown that genetic as well as environmental factors trigger the development of autoimmune diseases. It is believed that various susceptibility genes are necessary for the induction and potentiation of the autoimmune process, but the genetic susceptibility alone would not be sufficient for disease expression. The 30–50% concordance rate in monozygotic twins [92] documents that various environmental factors, such as virus infections and several dietary components, may have a major influence on the appearance and quality of the autoimmune re-