Nanoparticles Bearing TSH Receptor Protein and a Tolerogenic Molecule Do Not Induce Immune Tolerance but Exacerbate Thyroid Autoimmunity in hTSHR/NOD.H2h4 Mice.

Transgenic NOD.H2h4 mice that express the human (h) TSHR A-subunit in the thyroid gland spontaneously develop pathogenic TSHR autoantibodies resembling those in patients with Graves disease. Nanoparticles coupled to recombinant hTSHR A-subunit protein and a tolerogenic molecule (ligand for the endogenous aryl-hydrocarbon receptor; ITE) were injected i.p. four times at weekly intervals into hTSHR/NOD.H2h4 mice with the goal of blocking TSHR Ab development. Unexpectedly, in transgenic mice, injecting TSHR A-subunit-ITE nanoparticles (not ITE-nanoparticles or buffer) accelerated and enhanced the development of pathogenic TSHR Abs measured by inhibition of TSH binding to the TSHR. Nonpathogenic TSHR Abs (ELISA) were enhanced in transgenics and induced in wild-type littermates. Serendipitously, these findings have important implications for disease pathogenesis: development of Graves TSHR Abs is limited by the availability of A-subunit protein, which is shed from membrane bound TSHR, expressed at low levels in the thyroid. The enhanced TSHR Ab response following injected TSHR A-subunit protein-nanoparticles is reminiscent of the transient increase in pathogenic TSHR Abs following the release of thyroid autoantigens after radio-iodine therapy in Graves patients. However, in the hTSHR/NOD.H2h4 model, enhancement is specific for TSHR Abs, with Abs to thyroglobulin and thyroid peroxidase remaining unchanged. In conclusion, despite the inclusion of a tolerogenic molecule, injected nanoparticles coated with TSHR A-subunit protein enhanced and accelerated development of pathogenic TSHR Abs in hTSHR/NOD. NOD.H2h4 These findings emphasize the need for sufficient TSHR A-subunit protein to activate the immune system and the generation of stimulatory TSHR Abs in genetically predisposed individuals.

Critical Differences between Induced and Spontaneous Mouse Models of Graves' Disease with Implications for Antigen-Specific Immunotherapy in Humans.

Graves' hyperthyroidism, a common autoimmune disease caused by pathogenic autoantibodies to the thyrotropin (TSH) receptor (TSHR), can be treated but not cured. This single autoantigenic target makes Graves' disease a prime candidate for Ag-specific immunotherapy. Previously, in an induced mouse model, injecting TSHR A-subunit protein attenuated hyperthyroidism by diverting pathogenic TSHR Abs to a nonfunctional variety. In this study, we explored the possibility of a similar diversion in a mouse model that spontaneously develops pathogenic TSHR autoantibodies, NOD.H2h4 mice with the human (h) TSHR (hTSHR) A-subunit transgene expressed in the thyroid and (shown in this article) the thymus. We hypothesized that such diversion would occur after injection of "inactive" hTSHR A-subunit protein recognized only by nonpathogenic (not pathogenic) TSHR Abs. Surprisingly, rather than attenuating the pre-existing pathogenic TSHR level, in TSHR/NOD.H2h4 mice inactive hTSHR Ag injected without adjuvant enhanced the levels of pathogenic TSH-binding inhibition and thyroid-stimulating Abs, as well as nonpathogenic Abs detected by ELISA. This effect was TSHR specific because spontaneously occurring autoantibodies to thyroglobulin and thyroid peroxidase were unaffected. As controls, nontransgenic NOD.H2h4 mice similarly injected with inactive hTSHR A-subunit protein unexpectedly developed TSHR Abs, but only of the nonpathogenic variety detected by ELISA. Our observations highlight critical differences between induced and spontaneous mouse models of Graves' disease with implications for potential immunotherapy in humans. In hTSHR/NOD.H2h4 mice with ongoing disease, injecting inactive hTSHR A-subunit protein fails to divert the autoantibody response to a nonpathogenic form. Indeed, such therapy is likely to enhance pathogenic Ab production and exacerbate Graves' disease in humans.

A unique mouse strain that develops spontaneous, iodine-accelerated, pathogenic antibodies to the human thyrotrophin receptor.

Abs that stimulate the thyrotropin receptor (TSHR), the cause of Graves' hyperthyroidism, only develop in humans. TSHR Abs can be induced in mice by immunization, but studying pathogenesis and therapeutic intervention requires a model without immunization. Spontaneous, iodine-accelerated, thyroid autoimmunity develops in NOD.H2(h4) mice associated with thyroglobulin and thyroid-peroxidase, but not TSHR, Abs. We hypothesized that transferring the human TSHR A-subunit to NOD.H2(h4) mice would result in loss of tolerance to this protein. BALB/c human TSHR A-subunit mice were bred to NOD.H2(h4) mice, and transgenic offspring were repeatedly backcrossed to NOD.H2(h4) mice. All offspring developed Abs to thyroglobulin and thyroid-peroxidase. However, only TSHR-transgenic NOD.H2(h4) mice (TSHR/NOD.H2(h4)) developed pathogenic TSHR Abs as detected using clinical Graves' disease assays. As in humans, TSHR/NOD.H2(h4) female mice were more prone than male mice to developing pathogenic TSHR Abs. Fortunately, in view of the confounding effect of excess thyroid hormone on immune responses, spontaneously arising pathogenic human TSHR Abs cross-react poorly with the mouse TSHR and do not cause thyrotoxicosis. In summary, the TSHR/NOD.H2(h4) mouse strain develops spontaneous, iodine-accelerated, pathogenic TSHR Abs in female mice, providing a unique model to investigate disease pathogenesis and test novel TSHR Ag-specific immunotherapies aimed at curing Graves' disease in humans.

Detection of COVID-19 by quantitative analysis of carbonyl compounds in exhaled breath.

COVID-19 has caused a worldwide pandemic, creating an urgent need for early detection methods. Breath analysis has shown great potential as a non-invasive and rapid means for COVID-19 detection. The objective of this study is to detect patients infected with SARS-CoV-2 and even the possibility to screen between different SARS-CoV-2 variants by analysis of carbonyl compounds in breath. Carbonyl compounds in exhaled breath are metabolites related to inflammation and oxidative stress induced by diseases. This study included a cohort of COVID-19 positive and negative subjects confirmed by reverse transcription polymerase chain reaction between March and December 2021. Carbonyl compounds in exhaled breath were captured using a microfabricated silicon microreactor and analyzed by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). A total of 321 subjects were enrolled in this study. Of these, 141 (85 males, 60.3%) (mean ± SD age: 52 ± 15 years) were COVID-19 (55 during the alpha wave and 86 during the delta wave) positive and 180 (90 males, 50%) (mean ± SD age: 45 ± 15 years) were negative. Panels of a total of 34 ketones and aldehydes in all breath samples were identified for detection of COVID-19 positive patients. Logistic regression models indicated high accuracy/sensitivity/specificity for alpha wave (98.4%/96.4%/100%), for delta wave (88.3%/93.0%/84.6%) and for all COVID-19 positive patients (94.7%/90.1%/98.3%). The results indicate that COVID-19 positive patients can be detected by analysis of carbonyl compounds in exhaled breath. The technology for analysis of carbonyl compounds in exhaled breath has great potential for rapid screening and detection of COVID-19 and for other infectious respiratory diseases in future pandemics.

A feasibility study on exhaled breath analysis using UV spectroscopy to detect COVID-19.

A 23-subject feasibility study is reported to assess how UV absorbance measurements on exhaled breath samples collected from silicon microreactors can be used to detect COVID-19. The silicon microreactor technology chemoselectively preconcentrates exhaled carbonyl volatile organic compounds and subsequent methanol elution provides samples for analysis. The underlying scientific rationale that viral infection will induce an increase in exhaled carbonyls appears to be supported by the results of the feasibility study. The data indicate statistically significant differences in measured UV absorbance values between healthy and symptomatic COVID-19 positive subjects in the wavelength range from 235 nm to 305 nm. Factors such as subject age were noted as potential confounding variables.

The NOD Mouse Beyond Autoimmune Diabetes.

Autoimmune diabetes arises spontaneously in Non-Obese Diabetic (NOD) mice, and the pathophysiology of this disease shares many similarities with human type 1 diabetes. Since its generation in 1980, the NOD mouse, derived from the Cataract Shinogi strain, has represented the gold standard of spontaneous disease models, allowing to investigate autoimmune diabetes disease progression and susceptibility traits, as well as to test a wide array of potential treatments and therapies. Beyond autoimmune diabetes, NOD mice also exhibit polyautoimmunity, presenting with a low incidence of autoimmune thyroiditis and Sjögren's syndrome. Genetic manipulation of the NOD strain has led to the generation of new mouse models facilitating the study of these and other autoimmune pathologies. For instance, following deletion of specific genes or via insertion of resistance alleles at genetic loci, NOD mice can become fully resistant to autoimmune diabetes; yet the newly generated diabetes-resistant NOD strains often show a high incidence of other autoimmune diseases. This suggests that the NOD genetic background is highly autoimmune-prone and that genetic manipulations can shift the autoimmune response from the pancreas to other organs. Overall, multiple NOD variant strains have become invaluable tools for understanding the pathophysiology of and for dissecting the genetic susceptibility of organ-specific autoimmune diseases. An interesting commonality to all autoimmune diseases developing in variant strains of the NOD mice is the presence of autoantibodies. This review will present the NOD mouse as a model for studying autoimmune diseases beyond autoimmune diabetes.

Insulin-like Growth Factor 1 and Prolactin Levels in Chimpanzees (Pan troglodytes) Across the Lifespan.

As human and chimpanzee genomes show high homology for IGF1 and PRL, we analyzed the sera of 367 healthy chimpanzees obtained during routine physical examinations in a single colony and measured chimpanzee insulin-like growth factor (IGF)-1 and prolactin (PRL) levels across the lifespan using standard human immunoassays. Assuming chimpanzee IGF-1 levels peak during puberty as in humans, we randomly defined puberty as the age at which most IGF-1 levels were equal to or above the 90th percentile for each sex (males, ages ≥7.00 but <9.20 years; females, ≥5.00 but <8.00 years). IGF-1 levels steadily increased at a similar rate in juvenile males and females and peaked in puberty, strongly correlating with age, then slowly decreased faster in adult males than in adult females. As a group, males had a higher mean IGF-1 level than did females, but comparison by age category showed similar mean IGF-1 levels in males and females. PRL levels increased with age in females more than in males and levels were twice as high in females than in males. One pubertal male reported to have short stature had lower IGF-1 and weight compared with other males in the age group, confirming suspected growth hormone deficiency; a second male of normal height but low IGF-1 may have had delayed puberty. Overall, results show that differences in IGF-1 levels over the lifespan in this cohort of chimpanzees largely mimic those seen in humans, while patterns of PRL changes are less similar.

A Mouse Thyrotropin Receptor A-Subunit Transgene Expressed in Thyroiditis-Prone Mice May Provide Insight into Why Graves' Disease Only Occurs in Humans.

Background: Graves' disease, caused by autoantibodies that activate the thyrotropin (TSH) receptor (TSHR), has only been reported in humans. Thyroiditis-prone NOD.H2h4 mice develop autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO) but not to the TSHR. Evidence supports the importance of the shed TSHR A-subunit in the initiation and/or amplification of the autoimmune response to the holoreceptor. Cells expressing the gene for the isolated A-subunit secrete A-subunit protein, a surrogate for holoreceptor A-subunit shedding. NOD.H2h4 mice with the human TSHR A-subunit targeted to the thyroid (a "self" antigen in such transgenic (Tgic) animals), unlike their wild-type (wt) siblings, spontaneously develop pathogenic TSHR antibodies to the human-TSH holoreceptor. These autoantibodies do not recognize the endogenous mouse-TSH holoreceptor and do not cause hyperthyroidism. Methods: We have now generated NOD.H2h4 mice with the mouse-TSHR A-subunit transgene targeted to the thyroid. Tgic mice and wt littermates were compared for intrathyroidal expression of the mouse A-subunit. Sera from six-month-old mice were tested for the presence of autoantibodies to Tg and TPO as well as for pathogenic TSHR antibodies (TSH binding inhibition, bioassay for thyroid stimulating antibodies) and nonpathogenic TSHR antibodies (ELISA). Results: Expression of the mouse TSHR A-subunit transgene in the thyroid was confirmed by real-time polymerase chain reaction in the Tgics and had no effect on the spontaneous development of autoantibodies to Tg or TPO. However, unlike the same NOD.H2h4 strain with the human-TSHR A-subunit target to the thyroid, mice expressing intrathyroidal mouse-TSHR A subunit failed to develop either pathogenic or nonpathogenic TSHR antibodies. The mouse TSHR A-subunit differs from the human TSHR A-subunit in terms of its amino acid sequence and has one less glycosylation site than the human TSHR A-subunit. Conclusions: Multiple genetic and environmental factors contribute to the pathogenesis of Graves' disease. The present study suggests that the TSHR A-subunit structure (possibly including posttranslational modification such as glycosylation) may explain, in part, why Graves' disease only develops in humans.

Shared and unique susceptibility genes in a mouse model of Graves' disease determined in BXH and CXB recombinant inbred mice.

Susceptibility genes for TSH receptor (TSHR) antibodies and hyperthyroidism can be probed in recombinant inbred (RI) mice immunized with adenovirus expressing the TSHR A-subunit. The RI set of CXB strains, derived from susceptible BALB/c and resistant C57BL/6 (B6) mice, were studied previously. High-resolution genetic maps are also available for RI BXH strains, derived from B6 and C3H/He parents. We found that C3H/He mice develop TSHR antibodies, and some animals become hyperthyroid after A-subunit immunization. In contrast, the responses of the F1 progeny of C3H/He x B6 mice, as well as most BXH RI strains, are dominated by the B6 resistance to hyperthyroidism. As in the CXB set, linkage analysis of BXH strains implicates different chromosomes (Chr) or loci in the susceptibility to induced TSHR antibodies vs. hyperthyroidism. Importantly, BXH and CXB mice share genetic loci controlling the generation of TSHR antibodies (Chr 17, major histocompatibility complex region, and Chr X) and development of hyperthyroidism (Chr 1 and 3). Moreover, some chromosomal linkages are unique to either BXH or CXB strains. An interesting candidate gene linked to thyroid-stimulating antibody generation in BXH mice is the Ig heavy chain locus, suggesting a role for particular germline region genes as precursors for these antibodies. In conclusion, our findings reinforce the importance of major histocompatibility complex region genes in controlling the generation of TSHR antibodies measured by TSH binding inhibition. Moreover, these data emphasize the value of RI strains to dissect the genetic basis for induced TSHR antibodies vs. their effects on thyroid function in Graves' disease.

Aberrant Iodine Autoregulation Induces Hypothyroidism in a Mouse Strain in the Absence of Thyroid Autoimmunity.

We investigated factors underlying the varying effects of a high dietary iodide intake on serum T4 levels in a wide spectrum of mouse strains, including thyroiditis-susceptible NOD.H2h4, NOD.H2k, and NOD mice, as well as other strains (BALB/c, C57BL/6, NOD.Lc7, and B10.A4R) not previously investigated. Mice were maintained for up to 8 months on control or iodide-supplemented water (NaI 0.05%). On iodized water, serum T4 was reduced in BALB/c (males and females) in association with colloid goiters but was not significantly changed in mice that developed thyroiditis, namely NOD.H2h4 (males and females) or male NOD.H2k mice. Neither goiters nor decreased T4 developed in C57BL/6, NOD, NOD.Lc7, or B10.A4R female mice. In further studies, we focused on males in the BALB/c and NOD.H2h4 strains that demonstrated a large divergence in the T4 response to excess iodide. Excess iodide ingestion increased serum TSH levels to the same extent in both strains, yet thyroidal sodium iodide symporter (NIS) messenger RNA (mRNA) levels (quantitative polymerase chain reaction) revealed greatly divergent responses. NOD.H2h4 mice that remained euthyroid displayed a physiological NIS iodine autoregulatory response, whereas NIS mRNA was inappropriately elevated in BALB/c mice that became hypothyroid. Thus, autoimmune thyroiditis-prone NOD.H2h4 mice adapted normally to a high iodide intake, presumably by escape from the Wolff-Chaikoff block. In contrast, BALB/c mice that did not spontaneously develop thyroiditis failed to escape from this block and became hypothyroid. These data in mice may provide insight into the mechanism by which iodide-induced hypothyroidism occurs in some humans without an underlying thyroid disorder.

Thyroid Hemiagenesis in a Thyroiditis Prone Mouse Strain.

BACKGROUND: Thyroid hemiagenesis, a rare congenital condition detected by ultrasound screening of the neck, is usually not manifested clinically in humans. This condition has been reported in mice with hypothyroidism associated with induced deficiency in paired box 8 and NK2 homeobox 1, sonic hedgehog, or T-box 1. Unexpectedly, we observed thyroid hemiagenesis in NOD.H2h4 mice, an unusual strain that spontaneously develops iodide enhanced thyroid autoimmunity but remains euthyroid. OBJECTIVES AND METHODS: First, to compare mice with thyroid hemiagenesis versus bilobed littermates for serum T4, autoantibodies to thyroglobulin (ELISA) and thyroid peroxidase (TPO; flow cytometry with eukaryotic cells expressing mouse TPO), gross anatomy, and thyroid histology; second, to estimate the percentage of mice with thyroid hemiagenesis in the NOD.H2h4 mice we have studied over 6 years. RESULTS: Thyroid hemiagenesis was observed in 3 of 1,025 NOD.H2h4 mice (2 females, 1 male; 0.3$). Two instances of hemiagenesis were in wild-type females and one in a transgenic male expressing the human TSHR A-subunit in the thyroid. Two mice had very large unilobed glands, as in some human cases with this condition. Thyroid lymphocytic infiltration, serum T4, and the levels of thyroid autoantibodies were similar in mice with thyroid hemiagenesis and bilobed littermates. CONCLUSIONS: Unlike hypothyroidism associated with hemiagenesis in transcription factor knockout mice, hemiagenesis in euthyroid NOD.H2h4 mice occurs spontaneously and is phenotypically similar to that occasionally observed in humans.

The link between Graves' disease and Hashimoto's thyroiditis: a role for regulatory T cells.

Hyperthyroidism in Graves' disease is caused by thyroid-stimulating autoantibodies to the TSH receptor (TSHR), whereas hypothyroidism in Hashimoto's thyroiditis is associated with thyroid peroxidase and thyroglobulin autoantibodies. In some Graves' patients, thyroiditis becomes sufficiently extensive to cure the hyperthyroidism with resultant hypothyroidism. Factors determining the balance between these two diseases, the commonest organ-specific autoimmune diseases affecting humans, are unknown. Serendipitous findings in transgenic BALB/c mice, with the human TSHR A-subunit targeted to the thyroid, shed light on this relationship. Of three transgenic lines, two expressed high levels and one expressed low intrathyroidal A-subunit levels (Hi- and Lo-transgenics, respectively). Transgenics and wild-type littermates were depleted of T regulatory cells (Treg) using antibodies to CD25 (CD4(+) T cells) or CD122 (CD8(+) T cells) before TSHR-adenovirus immunization. Regardless of Treg depletion, high-expressor transgenics remained tolerant to A-subunit-adenovirus immunization (no TSHR antibodies and no hyperthyroidism). Tolerance was broken in low-transgenics, although TSHR antibody levels were lower than in wild-type littermates and no mice became hyperthyroid. Treg depletion before immunization did not significantly alter the TSHR antibody response. However, Treg depletion (particularly CD25) induced thyroid lymphocytic infiltrates in Lo-transgenics with transient or permanent hypothyroidism (low T(4), elevated TSH). Neither thyroid lymphocytic infiltration nor hypothyroidism developed in similarly treated wild-type littermates. Remarkably, lymphocytic infiltration was associated with intermolecular spreading of the TSHR antibody response to other self thyroid antigens, murine thyroid peroxidase and thyroglobulin. These data suggest a role for Treg in the natural progression of hyperthyroid Graves' disease to Hashimoto's thyroiditis and hypothyroidism in humans.

Variable Effects of Dietary Selenium in Mice That Spontaneously Develop a Spectrum of Thyroid Autoantibodies.

Selenium (Se) is a critical element in thyroid function, and variable dietary Se intake influences immunity. Consequently, dietary Se could influence development of thyroid autoimmunity and provide an adjunct to treat autoimmune thyroid dysfunction. Nonobese diabetic (NOD).H2h4 mice spontaneously develop autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO). This mouse strain expressing a human thyroid-stimulating hormone receptor (TSHR) A-subunit transgene in the thyroid also develops pathogenic TSHR autoantibodies. In this report, we investigated whether dietary Se influences these immune processes. Male and female wild-type and transgenic NOD.H2h4 mice were maintained on normal-, low-, or high-Se (0.1, 0, or 1.0 mg/kg) rodent diets. After 4 months, Se serum levels were extremely low or significantly increased on 0 or 1.0 mg/kg Se, respectively. Varying Se intake affected Tg antibody (TgAb) levels after 2 (but not 4) months; conversely, TPO antibody (TPOAb) levels were altered by dietary Se after 4 (but not 2) months. These data correspond to the earlier development of TgAb than TPOAb in NOD.H2h4 mice. In males, TgAb levels were enhanced by high Se and in females by low Se intake. Se intake had no effect on pathogenic TSHR autoantibodies in TSHR transgenic NOD.H2h4 females. In conclusion, in susceptible NOD.H2h4 mice, we found no evidence that a higher dietary Se intake ameliorates thyroid autoimmunity by reducing autoantibodies to Tg, TPO, or the TSHR. Instead, our finding that low dietary Se potentiates the development of autoantibodies to Tg and TPO in females is consistent with reports in humans of an increased prevalence of autoimmune thyroiditis in low-Se regions.

Genes Outside the Major Histocompatibility Complex Locus Are Linked to the Development of Thyroid Autoantibodies and Thyroiditis in NOD.H2h4 Mice.

Thyroiditis and autoantibodies to thyroglobulin (TgAb) and thyroid peroxidase (TPOAb) develop spontaneously in NOD.H2h4 mice, a phenotype enhanced by dietary iodine. NOD.H2h4 mice were derived by introducing the major histocompatibility class (MHC) molecule I-Ak from B10.A(4R) mice to nonobese diabetic (NOD) mice. Apart from I-Ak, the genes responsible for the NOD.H2h4 phenotype are unknown. Extending serendipitous observations from crossing BALB/c to NOD.H2h4 mice, thyroid autoimmunity was investigated in both genders of the F1, F2, and the second-generation backcross of F1 to NOD.H2h4 (N2). Medium-density linkage analysis was performed on thyroid autoimmunity traits in F2 and N2 progeny. TgAb develop before TPOAb and were measured after 8 and 16 weeks of iodide exposure; TPOAb and thyroiditis were studied at 16 weeks. TgAb, TPOAb, and thyroiditis, absent in BALB/c and F1 mice, developed in most NOD.H2h4 and in more N2 than F2 progeny. No linkages were observed in F2 progeny, probably because of the small number of autoantibody-positive mice. In N2 progeny (equal numbers of males and females), a chromosome 17 locus is linked to thyroiditis and TgAb and is suggestively linked to TPOAb. This locus includes MHC region genes from B10.A(4R) mice (such as I-Ak and Tnf, the latter involved in thyrocyte apoptosis) and genes from NOD mice such as Satb1, which most likely plays a role in immune tolerance. In conclusion, MHC and non-MHC genes, encoded within the chromosome 17 locus from both B10.A(4R) and NOD strains, are most likely responsible for the Hashimoto disease-like phenotype of NOD.H2h4 mice.

Probing the genetic basis for thyrotropin receptor antibodies and hyperthyroidism in immunized CXB recombinant inbred mice.

Immunization with adenovirus encoding the TSH receptor (TSHR) or its A-subunit induces Graves' hyperthyroidism in BALB/c and BALB/c x C57BL/6 offspring but not C57BL/6 mice. High-resolution genetic maps are available for 13 recombinant inbred CXB strains generated from BALB/c x C57BL/6 progeny by repeated brother x sister matings to establish fully inbred lines. CXB strains were studied before and after A-subunit adenovirus immunization for TSHR antibodies (TBI, inhibition of TSH binding), serum T4, and thyroid histology. All strains developed TBI activity (at variable levels), six strains became hyperthyroid, and one was overtly thyrotoxic. No low TBI responders became hyperthyroid, but high TBI did not predict hyperthyroidism. Preimmunization T4 levels varied in different CXB strains and was unrelated to subsequent T4 elevation. Linkage analysis indicated that different chromosomes were involved in generating TSHR antibodies and serum T4 before and after immunization. TBI activity was linked in part with major histocompatibility (MHC) genes on chromosome 17 (Chr 17) but induced Graves' disease involved non-MHC genes (Chr 19 and 10). The Chr 10 locus is close to the Trhde gene that encodes TSH-releasing hormone degrading enzyme. Expression of Trhde is controlled by thyroid hormones and linkage with a thyroid function-related gene is intriguing. Our data, the first genome scan in murine Graves' disease, provides insight into the role of MHC and non-MHC genes in human and murine Graves' disease. Finally, our study demonstrates the potential of recombinant inbred mice for discriminating between immune-response genes and thyroid function susceptibility genes in Graves' disease.

Blockade of costimulation between T cells and antigen-presenting cells: an approach to suppress murine Graves' disease induced using thyrotropin receptor-expressing adenovirus.

OBJECTIVE: Immune responses require costimulatory interactions between molecules on antigen-presenting cells and T cells: CD40 binding to CD40 ligand and B7 binding to CD28. Graves' hyperthyroidism is induced in BALB/c mice by immunization with thyrotropin receptor (TSHR) A-subunit adenovirus (Ad-A-subunit). We attempted to modulate Ad-A-subunit-induced Graves' disease using adenoviruses expressing costimulation "decoys": CD40-IgG-Fc (CD40-Ig) to block CD40:CD40-ligand interactions and CTLA4-Fc (CTLA4-Ig) to prevent B7:CD28 binding. OUTCOME: Unexpectedly, coimmunizing mice with Ad-A-subunit and excess control adenovirus (1:10 Ad-A-subunit:Ad-control) reduced TSHR antibody levels (thyrotropin binding inhibition [TBI]). Furthermore, only 15% of mice developed hyperthyroidism versus 75% using the same Ad-A-subunit dose (10(8) particles) without Ad-control. This effect was related to the dose of control adenovirus but not to the adenovirus insert, the timing or immunization site. Increasing the Ad-subunit dose (10(9) particles) and decreasing the control adenovirus dose (10:1 Ad-A-subunit:Ad-control) induced high TBI levels and 80% of mice were hyperthyroid. Coimmunization with Ad-CD40-Ig (but not Ad-CTLA4-Ig) reduced the incidence of hyperthyroidism to 40%. CONCLUSIONS: Using appropriate controls and adenovirus ratios, our data suggest the importance of CD40:CD40-ligand interactions for inducing Graves' hyperthyroidism by Ad-A-subunit. Furthermore, our observations emphasize the potential pitfalls of non-specific inhibition by coimmunization with two adenovirus species.

Dissociation between iodide-induced thyroiditis and antibody-mediated hyperthyroidism in NOD.H-2h4 mice.

NOD.H-2h4 mice are genetically predisposed to thyroid autoimmunity and spontaneously develop thyroglobulin autoantibodies (TgAb) and thyroiditis. Iodide administration enhances TgAb levels and the incidence and severity of thyroiditis. Using these mice, we investigated the interactions between TSH receptor (TSHR) antibodies induced by vaccination and spontaneous or iodide-enhanced thyroid autoimmunity (thyroiditis and TgAb). Mice were immunized with adenovirus expressing the TSHR A-subunit (or control adenovirus). Thyroid antibodies, histology, and serum thyroxine levels were compared in animals on a regular diet or on a high-iodide diet (0.05% NaI-supplemented water). Thyroiditis severity and TgAb levels were enhanced by iodide administration and were independent of the type of adenovirus used for immunization. In contrast, TSHR antibodies, measured by TSH-binding inhibition, thyroid-stimulating activity, and TSH-blocking activity, were induced in the majority of animals immunized with TSHR (but not control) adenovirus and were unaffected by dietary iodide. The NOD.2h4 strain of mice was less susceptible than BALB/c or BALB/k mice to TSHR adenovirus-induced hyperthyroidism. Nevertheless, hyperthyroidism developed in approximately one third of TSHR adenovirus-injected NOD.2h4 mice. This hyperthyroidism was suppressed by a high-iodide diet, probably by a nonimmune mechanism. The fact that inducing an immune response to the TSHR had no effect on thyroiditis raises the possibility that the TSHR may not be the target involved in the variable thyroiditis component in some humans with Graves' disease.

Relationship between thyroid peroxidase T cell epitope restriction and antibody recognition of the autoantibody immunodominant region in human leukocyte antigen DR3 transgenic mice.

We investigated the relationship between thyroid peroxidase (TPO) antibody and T lymphocyte epitopes in TPO-adenovirus (TPO-Ad) immunized BALB/c mice and mice transgenic for the human class II molecule DR3 associated with human thyroid autoimmunity. TPO autoantibodies are largely restricted to an immunodominant region (IDR). BALB/c mice immunized with fewer (10(7) vs. 10(9)) TPO-Ad particles developed TPO antibodies with lower titers that displayed greater restriction to the IDR. However, as with higher-dose TPO-Ad immunization, T cell epitopes (assessed by splenocyte interferon-gamma response to TPO in vitro) were highly diverse and variable in different animals. In contrast, DR3 mice immunized the higher TPO-Ad dose (10(9) particles) had high TPO antibody levels that showed relative focus on the IDR. Moreover, T cell epitopes recognized by splenocytes from DR3 mice showed greater restriction than BALB/c mice. Antibody affinities for TPO were higher in DR3 than in BALB/c mice. The present study indicates that weak TPO-Ad immunization of BALB/c mice (with consequent low TPO antibody titers) is required for enhanced IDR focus yet is not associated with T cell epitopic restriction. Humanized DR3 transgenic mice, despite stronger TPO-Ad immunization, develop higher titer TPO antibodies that do focus on the autoantibody IDR with T cells that recognize a more limited range of TPO peptides. These data suggest a relationship between major histocompatibility complex class II molecules and the development of antibodies to the IDR, a feature of human thyroid autoimmunity.

Evidence that TSH Receptor A-Subunit Multimers, Not Monomers, Drive Antibody Affinity Maturation in Graves' Disease.

CONTEXT: The TSH receptor (TSHR) A-subunit shed from the cell surface contributes to the induction and/or affinity maturation of pathogenic TSHR autoantibodies in Graves' disease. OBJECTIVE: This study aimed to determine whether the quaternary structure (multimerization) of shed A-subunits influences pathogenic TSHR autoantibody generation. DESIGN: The isolated TSHR A-subunit generated by transfected mammalian cells exists in two forms; one (active) is recognized only by Graves' TSHR autoantibodies, the second (inactive) is recognized only by mouse monoclonal antibody (mAb) 3BD10. Recent evidence suggests that both Graves' TSHR autoantibodies and mAb 3BD10 recognize the A-subunit monomer. Therefore, if the A-subunit monomer is an immunogen, Graves' sera should have antibodies to both active and inactive A-subunits. Conversely, restriction of TSHR autoantibodies to active A-subunits would be evidence of a role for shed A-subunit multimers, not monomers, in the pathogenesis of Graves' disease. Therefore, we tested a panel of Graves' sera for their relative recognition of active and inactive A-subunits. RESULTS: Of 34 sera from unselected Graves' patients, 28 were unequivocally positive in a clinical TSH binding inhibition assay. None of the latter sera, as well as 8/9 sera from control individuals, recognized inactive A-subunits on ELISA. In contrast to Graves' sera, antibodies induced in mice, not by shedding from the TSHR holoreceptor, but by immunization with adenovirus expressing the free human A-subunit, were directed to both the active and inactive A-subunit forms. CONCLUSIONS: The present study supports the concept that pathogenic TSHR autoantibody affinity maturation in Graves' disease is driven by A-subunit multimers, not monomers.

"Hijacking" the thyrotropin receptor: A chimeric receptor-lysosome associated membrane protein enhances deoxyribonucleic acid vaccination and induces Graves' hyperthyroidism.

Naked DNA vaccination with the TSH receptor (TSHR) does not, in most studies, induce TSHR antibodies and never induces hyperthyroidism in BALB/c mice. Proteins expressed endogenously by vaccination are preferentially presented by major histocompatibility complex class I, but optimal T cell help for antibody production requires lysosomal processing and major histocompatibility complex class II presentation. To divert protein expression to lysosomes, we constructed a plasmid with the TSHR ectodomain spliced between the signal peptide and transmembrane-intracellular region of lysosome-associated membrane protein (LAMP)-1, a lysosome-associated membrane protein. BALB/c mice pretreated with cardiotoxin were primed intramuscularly using this LAMP-TSHR chimera and boosted twice with DNA encoding wild-type TSHR, TSHR A-subunit, or LAMP-TSHR. With each protocol, spleen cells responded to TSHR antigen by secreting interferon-gamma, and 60% or more mice had TSHR antibodies detectable by ELISA. TSH binding inhibitory activity was present in seven, four, and two of 10 mice boosted with TSHR A-subunit, LAMP-TSHR, or wild-type TSHR, respectively. Importantly, six of 30 mice had elevated T4 levels and goiter (5 of 6 with detectable thyroid-stimulating antibodies). Injecting LAMP-TSHR intradermally without cardiotoxin pretreatment induced TSHR antibodies detectable by ELISA but not by TSH binding inhibitory activity, and none became hyperthyroid. These findings are consistent with a role for cardiotoxin-recruited macrophages in which (unlike in fibroblasts) LAMP-TSHR can be expressed intracellularly and on the cell surface. In conclusion, hijacking the TSHR to lysosomes enhances T cell responses and TSHR antibody generation and induces Graves'-like hyperthyroidism in BALB/c mice by intramuscular naked DNA vaccination.