Lessons from mouse models of Graves' disease.

Graves' disease (GD) is an autoimmune condition with the appearance of anti-TSH receptor (TSHR) autoantibodies in the serum. The consequence is the development of hyperthyroidism in most of the patients. In addition, in the most severe cases, patients can develop orbitopathy (GO), achropachy and dermopathy. The central role of the TSHR for the disease pathology has been well accepted. Therefore immunization against the TSHR is pivotal for the creation of in vivo models for the disease. However, TSHR is well preserved among the species and therefore the immune system is highly tolerant. Many differing attempts have been performed to break tolerance and to create a proper animal model in the last decades. The most successful have been achieved by introducing the human TSHR extracellular domain into the body, either by injection of plasmid or adenoviruses. Currently available models develop the whole spectrum of Graves' disease-autoimmune thyroid disease and orbitopathy and are suitable to study disease pathogenesis and to perform treatment studies. In recent publications new immunomodulatory therapies have been assessed and also diseaseprevention by inducing tolerance using small cyclic peptides from the antigenic region of the extracellular subunit of the TSHR.

Genetic immunization with mouse thyrotrophin hormone receptor plasmid breaks self-tolerance for a murine model of autoimmune thyroid disease and Graves' orbitopathy.

Experimental models of Graves' hyperthyroid disease accompanied by Graves' orbitopathy (GO) can be induced efficiently in susceptible inbred strains of mice by immunization by electroporation of heterologous human TSH receptor (TSHR) A-subunit plasmid. In this study, we report on the development of a bona fide murine model of autoimmune Graves' disease induced with homologous mouse TSHR A-subunit plasmid. Autoimmune thyroid disease in the self-antigen model was accompanied by GO and characterized by histopathology of hyperplastic glands with large thyroid follicular cells. Examination of orbital tissues showed significant inflammation in extra-ocular muscle with accumulation of T cells and macrophages together with substantial deposition of adipose tissue. Notably, increased levels of brown adipose tissue were present in the orbital tissue of animals undergoing experimental GO. Further analysis of inflammatory loci by 19 F-magnetic resonance imaging showed inflammation to be confined to orbital muscle and optic nerve, but orbital fat showed no difference in inflammatory signs in comparison to control ß-Gal-immunized animals. Pathogenic antibodies induced to mouse TSHR were specific for the self-antigen, with minimal cross-reactivity to human TSHR. Moreover, compared to other self-antigen models of murine Graves' disease induced in TSHR knock-out mice, the repertoire of autoantibodies to mouse TSHR generated following the breakdown of thymic self-tolerance is different to those that arise when tolerance is not breached immunologically, as in the knock-out models. Overall, we show that mouse TSHR A-subunit plasmid immunization by electroporation overcomes tolerance to self-antigen to provide a faithful model of Graves' disease and GO.

Modeling Graves' Orbitopathy in Experimental Graves' Disease.

Graves' orbitopathy (GO), also known as thyroid eye disease is an inflammatory disease of the orbital tissue of the eye that arises as a consequence of autoimmune thyroid disease. The central feature of the disease is the production of antibodies to the thyrotropin hormone receptor (TSHR) that modulate the function of the receptor leading to autoimmune hyperthyroidism and GO. Over the years, all viable preclinical models of Graves' disease have been incomplete and singularly failed to progress in the treatment of orbital complications. A new mouse model of GO based upon immunogenic presentation of human TSHR A-subunit plasmid by close field electroporation is shown to lead to induction of prolonged functional antibodies to TSHR resulting in chronic disease with subsequent progression to GO. The stable preclinical GO model exhibited pathologies reminiscent of human disease characterized by orbital remodeling by inflammation and adipogenesis. Inflammatory lesions characterized by CD3+ T cells and macrophages were localized in the orbital muscle tissue. This was accompanied by extensive adipogenesis of orbital fat in some immune animals. Surprisingly, other signs of orbital involvement were reminiscent of eyelid inflammation involving chemosis, with dilated and congested orbital blood vessels. More recently, the model is replicated in the author's independent laboratories. The pre-clinical model will provide the basis to study the pathogenic and regulatory roles of immune T and B cells and their subpopulations to understand the initiation, pathophysiology, and progression of GO.

Autoantibody epitopes to the smaller isoform of glutamate decarboxylase do not differ in Swedish and Japanese type 1 diabetes patients and may be associated with high-risk human leucocyte antigen class II alleles.

Type 1 diabetes (T1D) is an autoimmune disease with a strong human leucocyte antigen (HLA) class II association. Depending on geographic locations, the disease-associated HLA class II alleles vary. We evaluated the beta cell-specific autoimmunity reflected in autoantibodies directed to the smaller isoform of glutamate decarboxylase (GAD65) in Japanese and Swedish T1D patients. GAD65Ab epitope specificities were assessed using GAD65-specific recombinant Fab. GAD65Ab epitope specificities did not differ between Swedish and Japanese patients. Only recognition of the MICA-4-defined middle epitope was significantly stronger in the Japanese T1D patient group compared to the Swedish T1D patients (P = 0.001). Binding to the b96.11-defined middle epitope was substantial in both groups and showed significant associations with high-risk HLA class II haplotypes. In the Japanese T1D group the association was with haplotype DRB1*0802-DQB1*0302 (P = 0.0008), while in the Swedish T1D patients binding to the b96.11-defined epitope as associated with the presence of high-risk HLA genotypes DR3-DQB1*0201 and/or DR4-DQB1*0302 (P = 0.02). A significant association between reduction in binding in the presence of recombinant Fab (rFab) DPD and high-risk allele DQB1*0201 was found (P = 0.008) in the Swedish T1D patients only. We hypothesize that epitope-specific autoantibodies effect the peptide presentation on HLA class II molecules by modulating antigen uptake and processing. Molecular modelling of the high-risk HLA class II molecules will be necessary to test whether these different molecules present similar peptide-binding specificities.

Dynamic changes of GAD65 autoantibody epitope specificities in individuals at risk of developing type 1 diabetes.

AIMS/HYPOTHESIS: Progression to type 1 diabetes is associated with intramolecular epitope spreading to disease-specific antibody epitopes located in the middle region of glutamic acid decarboxylase 65 (GAD65). METHODS: The relationship between intramolecular epitope spreading of autoantibodies specific to GAD65 in relation to the risk of developing type 1 diabetes was tested in 22 high-risk individuals and 38 low-risk individuals. We determined the conformational epitopes in this longitudinal study by means of competition experiments using recombinant Fab of four GAD65-specific monoclonal antibodies. RESULTS: Sera from high-risk children in the preclinical stage recognise a specific combination of GAD65 antibody epitopes located in the middle and the C-terminus of GAD65. High risk of progressing to disease is associated with the emergence of antibodies specific for conformational epitopes at the N-terminus and the middle region. Binding to already established antibody epitopes located in the middle and at the N-terminus increases and shows a significant relation (p=0.005) with HLA, which confers risk of developing diabetes. CONCLUSIONS/INTERPRETATION: In type 1 diabetes, GAD65 antibodies are initially generated against the middle and C-terminal regions of GAD65. In genetically predisposed subjects the autoimmune response may then undergo intramolecular epitope spreading towards epitopes on the N-terminus and further epitopes located in the middle. These findings clearly demonstrate that the GAD65 autoantibody response in the preclinical stage of type 1 diabetes is dynamic and related to the HLA genotypes that confer risk of diabetes. GAD65-specific Fab should prove useful in predicting progression from islet autoimmunity to clinical onset of type 1 diabetes.

Multiplicity of the antibody response to GAD65 in Type I diabetes.

Type I diabetes (TID) is an autoimmune disease characterized in part by the presence of autoantibodies directed against glutamic acid decarboxylase 65 (GAD65), among other pancreatic islet antigens. We investigated the independent epitope specificities of these GAD65 antibodies (GAD65Ab) and their combinations in the sera of new onset TID patients and first-degree relatives positive for GAD65Ab. For our analysis, we used four GAD65-specific recombinant Fabs (rFabs) that recognize different conformational determinants of GAD65 located throughout the molecule, including the N-terminal, the middle and the C-terminal regions. We used these epitope-specific rFabs in competition assays to determine the binding specificity of the autoantibodies found in patient sera. Among the 61 sera from newly diagnosed GAD65Ab-positive TID patients GAD65 binding was competed for 23 sera by all four rFabs, 29 by at least two rFabs, and in nine sera were displaced by one or no rFab. In contrast, none of the 24 sera from GAD65Ab-positive first-degree relatives of TID patients were displaced by all four rFabs. When using all four rFabs simultaneously to compete with GAD65Ab binding, binding of sera from TID patients was reduced by an average of 70%. A significantly weaker competition was observed when evaluating sera of GAD65Ab-positive first-degree relatives (P < 0.0001).

Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice.

Murine experimental autoimmune thyroiditis (EAT), characterized by thyroid destruction after immunization with thyroglobulin (Tg), has long been a useful model of organ-specific autoimmune disease. More recently, porcine thyroid peroxidase (pTPO) has also been shown to induce thyroiditis, but these results have not been confirmed. When (C57BL/6 x CBA)F(1) mice, recently shown to be susceptible to mouse TPO-induced EAT, were immunized with plasmid DNA to human TPO (hTPO) and cytokines IL-12 or GM-CSF, significant antibody (Ab) titres were generated, but minimal thyroiditis was detected in one mouse only from the TPO + GM-CSF immunized group. However, after TPO DNA immunization of HLA-DR3 transgenic class II-deficient NOD mice, thyroiditis was present in 23% of mice injected with TPO + IL-12 or GM-CSF. We also used another marker for assessing the closeness of the model to human thyroid autoimmunity by examining the epitope profile of the anti-TPO Abs to immunodominant determinants on TPO. Remarkably, the majority of the anti-TPO Abs was directed to immunodominant regions A and B, demonstrating the close replication of the model to human autoimmunity. TPO protein immunizations of HLA-DR3 transgenic mice with recombinant hTPO did not result in thyroiditis, nor did immunization of other mice expressing HLA class II transgenes HLA-DR4 or HLA-DQ8, with differential susceptibility to Tg-induced EAT. Moreover, our efforts to duplicate exactly the experimental procedures used with pTPO also failed to induce thyroiditis. The success of hTPO plasmid DNA immunization of DR3(+) mice, similar to our reports on Tg-induced thyroiditis and thyrotropin receptor DNA-induced Graves' hyperthyroidism, underscores the importance of DR3 genes for all three major thyroid antigens, and provides another humanized model to study autoimmune thyroid disease.

Induction of hyperthyroidism in mice by intradermal immunization with DNA encoding the thyrotropin receptor.

Intramuscular injection with plasmid DNA encoding the human thyrotropin receptor (TSHR) has been known to elicit symptoms of Graves' disease (GD) in outbred but not inbred mice. In this study, we have examined, firstly, whether intradermal (i.d.) injection of TSHR DNA can induce hyperthyroidism in BALB/c mice and, secondly, whether coinjection of TSHR- and cytokine-producing plasmids can influence the outcome of disease. Animals were i.d. challenged at 0, 3 and 6 weeks with TSHR DNA and the immune response was assessed at the end of the 8th or 10th week. In two experiments, a total of 10 (67%) of 15 mice developed TSHR-specific antibodies as assessed by flow cytometry. Of these, 4 (27%) mice had elevated thyroxine (TT4) levels and goitrous thyroids with activated follicular epithelial cells but no evidence of lymphocytic infiltration. At 10 weeks, thyroid-stimulating antibodies (TSAb) were detected in two out of the four hyperthyroid animals. Interestingly, in mice that received a coinjection of TSHR- and IL-2- or IL-4-producing plasmids, there was no production of TSAbs and no evidence of hyperthyroidism. On the other hand, coinjection of DNA plasmids encoding TSHR and IL-12 did not significantly enhance GD development since two out of seven animals became thyrotoxic, but had no goitre. These results demonstrate that i.d. delivery of human TSHR DNA can break tolerance and elicit GD in inbred mice. The data do not support the notion that TSAb production is Th2-dependent in murine GD but they also suggest that codelivery of TSHR and Th1-promoting IL-12 genes may not be sufficient to enhance disease incidence and/or severity in this model.

Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis.

Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549-563), P14 (aa 599-617) and P18 (aa 210-225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75%. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81.5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model.

Graves' hyperthyroidism and thyroiditis in HLA-DRB1*0301 (DR3) transgenic mice after immunization with thyrotropin receptor DNA.

Familial and twin studies in Caucasians have established that the MHC class II allele HLA-DRB1*0301 (DR3) is a strong susceptibility gene in Graves' hyperthyroid disease (GD). To determine if a DR3 transgene could help establish an animal model for GD, we expressed DR3 molecules in class II-knockout NOD mice (H2Ag7-). DR3+g7- mice were given cardiotoxin prior to immunization on weeks 0, 3 and 6 with plasmid DNA encoding human thyrotropin receptor (TSHR). Two groups of mice were also coimmunized with plasmid DNA for IL-4 or GM-CSF. Serial bleeds on weeks 8, 11 and 14 showed that approximately 20% of mice produced thyroid-stimulating antibodies (Abs), and approximately 25% had elevated T4 levels. In particular, a subset displayed both signs of hyperthyroidism, resulting in approximately 30% with some aspect of GD syndrome. Additional mice had thyroid-stimulating blocking Abs and/or TSH-binding inhibitory immunoglobulins, while most mice showed strong labelling of TSHR+ cells by flow cytometry. Interestingly, lymphocytic infiltration with thyroid damage and Abs to mouse thyroglobulin were also noted. Vector controls were uniformly negative. Thus, DR3 transgenic mice can serve as a model for GD, similar to our earlier reports that this allele is permissive for the Hashimoto's thyroiditis model induced with human thyroglobulin.

Modulation of antigen presentation by autoreactive B cell clones specific for GAD65 from a type I diabetic patient.

We used a GAD65-specific human B-T cell line cognate system in vitro to investigate the modulation of GAD65 presentation by autoantibody, assessed in a proliferation assay. Generally, if the T cell determinant overlaps or resides within the antibody epitope, effects of presentation are blunted while if they are distant can lead to potent presentation. For three different autoreactive B-T cell line cognate pairs, the modulation of GAD65 presentation followed the mode of overlapping or distant epitopes with resultant potent or undetectable presentation. However, other cognate pairs elicited variability in this pattern of presentation. Notably, one B cell line, DPC, whose antibody epitope did not overlap with the T cell determinants, was consistently poor in presenting GAD65. Using the fluorescent dye Alexa Fluor 647 conjugated to GAD65 to study receptor-mediated antigen endocytosis showed that all the antigen-specific B cell clones were efficient in intracellular accumulation of the antigen. Additionally, multicolour immunofluorescence microscopy showed that the internalized GAD65/surface IgG complexes were rapidly targeted to a perinuclear compartment in all GAD-specific B cell clones. This analysis also demonstrated that HLA-DM expression was reduced strongly in DPC compared to the stimulatory B cell clones. Thus the capability of antigen-specific B cells to capture and present antigen to human T cell lines is dependent on the spatial relationship of B and T cell epitopes as well other factors which contribute to the efficiency of presentation.

Role of complex asparagine-linked oligosaccharides in the expression of a functional thyrotropin receptor.

To evaluate the functional role of complex asparagine-linked oligosaccharides of the human thyrotropin receptor (TSHR), a Chinese hamster ovary cell line (JP09) and a K562 cell line (K562-TSHR) expressing this receptor were treated with deoxymannojirimycin (dMM), a mannosidase I inhibitor. dMM blocks the formation of complex-type structures and leads to the formation of high-mannose-type structures. Treatment of cells with dMM led to a decrease in the number of thyrotropin (TSH)-binding sites at the cell surface. Detection of the TSHR at the cell surface using a monoclonal antibody directed against the A subunit showed that this decrease was not due to a decrease in the number of TSHRs expressed at the cell surface. However the recognition of TSHR by a monoclonal antibody directed against the C peptide was greatly decreased. On immunoblotting, after deglycosylation using peptide N-glycanase F, the A subunit was visualized as a doublet (36 and 41 kDa). In control cells the species of higher molecular mass was more abundant whereas after dMM treatment the species of lower molecular mass became more abundant. This difference in molecular mass between the two peptides is compatible with the removal of the C peptide. In conclusion, the results show that inhibition of complex-type structure formation leads to (i) an incapacity for TSHR to bind TSH, without affecting its intracellular transport and (ii) an increase of TSHR susceptibility to proteases that remove the C peptide. We then hypothesized that removal of the C peptide could contribute to the formation of a non-functional TSHR.

Human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface.

The enzyme, thyroid peroxidase (TPO), is a dominant antigen in thyroid autoimmune diseases. Autoantibodies recognised two major dominant conformational epitopes termed A and B. The epitopes have been defined by mAbs, but the amino acid residues which constitute these determinants remain unknown. Using a model of TPO, built from the structure of myeloperoxidase (MPO), we have synthesised peptides corresponding to exposed loops and generated rabbit antibodies to the peptides. Antisera to peptide sequence 599-617 (peptide 14) representing a highly protrusive loop on the TPO, showed the highest inhibition in 65 sera from patients positive with anti-TPO antibodies. The inhibition was by 15-80% (mean 41%), and no other antibody showed any inhibition. Binding of hFabs to the B determinant on TPO was inhibited by anti-peptide 14 antibodies more then 85%, but not Fabs to the A determinant. In conclusion, the peptide 14 defines a sequence taking part in building up the B major conformational epitope. None of generated anti-peptide antibodies alone inhibited the binding of human Fabs to the A epitope, however a combination of four anti-peptide antibodies (P1, P12, P14 and P18) inhibits Fabs binding to the A determinant by more then 60% and autoantibodies binding from 65% to 94%. Combination of antibodies reacting with peptides outside the surface defined by those four antipeptide antibodies did not give any inhibition of Fabs to TPO. The inhibition of Fabs and auto Abs to TPO by this combination of anti-peptide Abs is the result of steric hindrance as none of these Abs individually inhibited auto Abs' or Fabs' binding to TPO. The four peptides define an area on the enzyme surface where the A and B major conformational epitopes are localised.

Two amino acids in glutamic acid decarboxylase act in concert for maintenance of conformational determinants recognised by Type I diabetic autoantibodies.

AIMS/HYPOTHESIS: Glutamic acid decarboxylase 65 is a major autoantigen in Type I (insulin-dependent) diabetes mellitus, autoimmune polyendocrine syndrome and stiff-man syndrome. These disorders are characterised by the presence of multiple autoantibodies to the autoantigen which can be distinguished in a variety of different ways. We have investigated the role of single amino-acid mutations in glutamic acid decarboxylase 65 in distinguishing the binding of serum antibodies and a variety of patient-derived human IgG monoclonal antibodies directed to different determinants of the autoantigen. METHODS: We identified a mutant of glutamic acid decarboxylase 65 that contained four single amino-acid mutations from the wild-type molecule. The role of these mutations was investigated by site-directed mutagenesis. We investigated the binding of patient-derived serum antibodies to glutamic acid decarboxylase 65 to a number of single and double amino-acid mutants using immunoprecipitation with labelled, recombinant antigen. To overcome the heterogeneity of different anti-glutamic acid decarboxylase 65 antibodies present in a patient's serum, the binding of a panel of eleven patient-derived human monoclonal antibodies recognising different determinants on the autoantigen was also studied. RESULTS: Two replacements in glutamic acid decarboxylase 65 at Asn247Ser and Leu574Pro were identified that preferentially influence the anti-glutamic acid decarboxylase 65 serum antibodies of Type I diabetic patients, without statistically significantly effecting those recognised in other disorders. Single or double amino-acid replacements Asn247Ser and Leu574Pro in the autoantigen showed differential affects on expression of epitopes recognised by the human monoclonals. The double replacement of Asn247Ser and Leu574Pro in glutamic acid decarboxylase 65 resulted in the loss of binding of all eleven human monoclonal antibodies, irrespective of their epitope recognition. In contrast, single replacement of Leu574Pro statistically significantly reduced the binding of some carboxyl terminal-directed antibodies such as MICA 1, MICA 3 and DP-A without influencing the binding of other monoclonals. Replacement of Asn247Ser did not, however, influence the binding of any patients serum or human monoclonal antibodies. CONCLUSION/INTERPRETATION: Two distantly spaced amino acids, Asn247 and Leu574 in glutamic acid decarboxylase 65 were identified that act in concert to greatly influence the conformational structure of the autoantigen and statistically significantly influence the binding of antibodies present in Type I diabetic sera. The single or double amino-acid mutants can be used to distinguish some anti-glutamic acid decarboxylase-65 autoantibodies and could prove useful in distinguishing Type I diabetic from autoimmune polyendocrine syndrome and stiff-man syndrome patients' sera as well as to study changes in antibody patterns during disease progression.

Evidence for recognition of novel islet T cell antigens by granule-specific T cell lines from new onset type 1 diabetic patients.

Type 1 diabetes is a T cell-mediated autoimmune disease where a number of islet beta-cell target autoantigens have been characterized on the basis of reactivity with autoantibodies. Nevertheless, there remains uncertainty of the nature of another group of autoantigens associated with the secretory granule fraction of islet beta-cells that appear to be targeted predominantly by autoreactive T cells. We have previously characterized CD4+, HLA-DR-restricted T cell lines from new onset type 1 diabetic patients that are specific for the secretory granule fraction of rat tumour insulinoma, RIN. The T cell line from the first patient, HS, proliferates in response to crude microsomal membranes prepared from a recently established, pure human islet beta-cell line NES2Y. In addition, the HS line also responds to secretory granule fractions prepared from a murine tumour insulinoma grown in RIP-Tag mice, showing the recognition of species-conserved antigen(s) in beta-cells. Using partially matched antigen-presenting cells, the HS T cells and another line derived from a second patient, MR, were shown to be restricted by disease-associated DRB1*0101 and DRB1*0404 alleles, respectively. Neither the HS or MR T cell lines proliferate in response to a large panel of candidate islet cell antigens, including insulin, proinsulin, glutamic acid decarboxylase, the protein tyrosine phosphatase IA-2/phogrin, imogen-38, ICA69 or hsp60. Our data provide compelling evidence of the presence of a group of antigens associated with the secretory granule fraction of islet beta-cells recognized by the T cell lines, whose definition may contribute to our knowledge of disease induction as well as to diagnosis.

Identification of an immunodominant region recognized by human autoantibodies in a three-dimensional model of thyroid peroxidase.

Autoimmune thyroid diseases (AITD) are characterized by the presence of autoantibodies to thyroid peroxidase (TPO). This response is dominated by autoantibodies to two conformational determinants, termed A and B, that have been defined by monoclonal antibodies but whose structures and location within TPO are unknown. We have modeled the three-dimensional structure of the extracellular region of TPO, raised antisera to prominent surface structures, and identified an epitope that we show to be a critical part of the B determinant. Antibodies to this epitope inhibit the binding to TPO of human autoantibodies in virtually all serum samples from 65 patients with AITD that were tested. This first description of a model of the three-dimensional structure and location of a major autoantigenic determinant within the TPO molecule may provide structural clues for identifying causative agents or developing novel therapeutic strategies.

Comparative analysis of epitope recognition of glutamic acid decarboxylase (GAD) by autoantibodies from different autoimmune disorders.

Autoantibodies to GAD, an important marker of the autoimmune process in type I or insulin-dependent diabetes mellitus (IDDM), are also found in non-diabetic individuals with autoimmune polyendocrine syndrome type 1 (APS1), APS2, and stiff man syndrome (SMS). Most IDDM sera contain two distinct GAD antibody specificities, one of which targets an epitope region in the middle-third of GAD65 (IDDM-E1; amino acids 221-359) and one of which targets the carboxy-third of GAD65 (IDDM-E2; amino acids 453-569). Using 11 chimeric GAD65/GAD67 proteins to maintain conformation-dependent epitopes of GAD65, we compared the humoral repertoire of IgG antibodies from an individual with APS2-like disease (b35, b78, and b96) and MoAbs from an IDDM patient (MICA-2, MICA-3, and MICA-4). Neither the APS2 IgG antibodies nor the IDDM MoAbs bind the amino-terminal third of GAD65, but instead target the carboxy-terminal two-thirds of GAD65. Amino acids 270-359 (IDDM-E1) are targeted by one APS2 IgG antibody and MICA-4, while two other APS2 IgG antibodies, MICA-2 and MICA-3, target amino acids 443-585 (IDDM-E2). Using GAD65/67 chimera that span the IDDM-E2 region, we found that MICA-2 binds amino acids 514-528 of GAD65, but two APS2 IgG antibodies require this region and amino acids 529-570. In contrast, the binding of MICA-3 requires two discontinuous amino acid segments of GAD65 (452-513 and 528-569), but not amino acids 514-528. These results indicate that there are both similarities and differences in the humoral response to GAD65 in APS2 and IDDM.

Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease.

Graves' disease (GD) is characterized by the presence of autoantibodies against the TSH-receptor (TSH-R) which are pathogenic and, upon binding to the receptor, trigger intracellular signal transduction. The autoantibodies are oligoclonal and as they are responsible for disease activity, their characterization would lead to a better understanding of the development of GD. Attempts to isolate anti-TSH-R antibodies from patients have proved to be difficult due to the exceedingly low serum levels due to rarity of these B cells, together with difficulties in obtaining purified TSH-R capable of interacting with patients autoantibodies. We employed phage antibody display technology and performed selection with a previously characterized semisynthetic antibody library on the purified extracellular ectodomain of the TSH-R. We report the isolation of six different anti-TSH-R monoclonal phage antibodies (moPhabs) from this library. All the moPhabs recognized TSH-R and its recombinant fragments by Western blotting, but failed to recognize the native TSH-R by flow cytometry. Consequently, the moPhabs did not lead to TSH-R activation. As these were the first moPhabs to TSH-R, they were analysed in terms of nucleotide and amino acid sequence and epitope specificity on the receptor. The moPhabs used immunoglobulin VH1 and VH3 germ line genes, all associated with Vlambda3 genes. Interestingly, the CDR3 regions of all moPhabs were remarkably similar, though not identical. In light of the common CDR3 usage, the epitopes recognized on TSH-R appeared to be restricted to amino acids residues 405-411 and 357-364. In summary, our results show that semisynthetic libraries may be limited in isolating human monoclonal antibodies that resemble pathogenic antithyrotropin receptor autoantibodies present in patients with GD. It is likely that until preparations of purified TSH-R that can be recognized by patients autoantibodies become available, similar to the recently described glycosylphosphatidylinositol (GPI) anchored TSH-R ectodomain, monoclonal antibodies from phage antibody display to TSH-R will be limited for isolating the rare, pathogenic antibodies of GD.

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.