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.

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.

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.

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.

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.

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.

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.

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.

Immunoglobulin heavy chain variable region and major histocompatibility region genes are linked to induced graves' disease in females from two very large families of recombinant inbred mice.

Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans.

Sex, genetics, and the control of thyroxine and thyrotropin in mice.

BACKGROUND: Previously, we studied the genetic basis for variability in total thyroxine (TT4) as part of investigating induced Graves' hyperthyroidism in panels of genetically diverse recombinant inbred (RI) mice. Because Graves' disease occurs predominantly in women, we used female mice. A limitation of this approach is that thyrotropin (TSH) is undetectable by some assays in most female mice. METHOD: Variation in levels of serum TSH, TT4, and free thyroxine (FT4) was measured in males from three related RI families (CXB, BXH, and AXBXA) followed by quantitative genetic analysis and mapping of these traits. RESULTS: In general, TSH levels were higher in males than females. FT4 levels were also higher in males than in females, but TT4 sex differences were absent or inconsistent. Chromosomal linkage was only observed for TSH in BXH males and for FT4 in AXBXA males. Different chromosomes were linked to TT4 in males of the three RI sets. The most striking finding came from genetic linkages in males versus our previous data for females. TT4 was linked to the same chromosomal loci in CXB males and females. In contrast, TT4, FT4, and TSH were linked to different "sex-specific chromosomes" in AXBXA and BXH families. CONCLUSIONS: In three RI mouse families, TSH and FT4 were significantly higher in males than females. Linkage analysis revealed chromosomal overlap for TT4 in males and females for one RI set but striking sex differences for TT4, FT4, and TSH linkage in two RI sets. Our findings provide a cautionary note: genetic linkage analysis of thyroid hormones traits in mice should be studied separately in males and females.

Thyroid autoantibodies are rare in nonhuman great apes and hypothyroidism cannot be attributed to thyroid autoimmunity.

The great apes include, in addition to Homo, the genera Pongo (orangutans), Gorilla (gorillas), and Pan, the latter comprising two species, P. troglodytes (chimpanzees) and P. paniscus (bonobos). Adult-onset hypothyroidism was previously reported in 4 individual nonhuman great apes. However, there is scarce information on normal serum thyroid hormone levels and virtually no data for thyroid autoantibodies in these animals. Therefore, we examined thyroid hormone levels and TSH in all nonhuman great ape genera including adults, adolescents, and infants. Because hypothyroidism in humans is commonly the end result of thyroid autoimmunity, we also tested healthy and hypothyroid nonhuman great apes for antibodies to thyroglobulin (Tg), thyroid peroxidase (TPO), and the TSH receptor (TSHR). We established a thyroid hormone and TSH database in orangutans, gorillas, chimpanzees, and bonobos (447 individuals). The most striking differences are the greatly reduced free-T4 and free-T3 levels in orangutans and gorillas vs chimpanzees and bonobos, and conversely, elevated TSH levels in gorillas vs Pan species. Antibodies to Tg and TPO were detected in only 2.6% of adult animals vs approximately 10% in humans. No animals with Tg, TPO, or TSHR antibodies exhibited thyroid dysfunction. Conversely, hypothyroid nonhuman great apes lacked thyroid autoantibodies. Moreover, thyroid histology in necropsy tissues was similar in euthyroid and hypothyroid individuals, and lymphocytic infiltration was absent in 2 hypothyroid animals. In conclusion, free T4 and free T3 are lower in orangutans and gorillas vs chimpanzees and bonobos, the closest living human relatives. Moreover, thyroid autoantibodies are rare and hypothyroidism is unrelated to thyroid autoimmunity in nonhuman great apes.

Genetic linkages for thyroxine released in response to thyrotropin stimulation in three sets of recombinant inbred mice provide evidence for shared and novel genes controlling thyroid function.

BACKGROUND: Graves' hyperthyroidism is induced by immunizing mice with adenovirus expressing the human thyrotropin (TSH)-receptor. Using families of recombinant-inbred mice, we previously discovered that genetic susceptibility to induced thyroid-stimulating antibodies and hyperthyroidism are linked to loci on different chromosomes, indicating a fundamental genetic difference in thyroid sensitivity to ligand stimulation. An approach to assess thyroid sensitivity involves challenging genetically diverse lines of mice with TSH and measuring the genotype/strain-specific increase in serum thyroxine (T4). METHODS: We investigated genetic susceptibility and genetic control of T4 stimulation by 10 mU bovine TSH in female mice of the CXB, BXH, and AXB/BXA strain families, all previously studied for induced Graves' hyperthyroidism. RESULTS: Before TSH injection, T4 levels must be suppressed by inhibiting endogenous TSH secretion. Three daily intraperitoneal L-triiodothyronine injections efficiently suppressed serum T4 in females of 50 of 51 recombinant inbred strains. T4 stimulation by TSH was more strongly linked in CXB and BXH sets, derived from parental strains with divergent T4 stimulation, than in AXB/BXA strains generated from parents with similar TSH-induced responses. Genetic loci linked to the acute TSH-induced T4 response (hours) were not the same as those linked to induced hyperthyroidism (which develops over months). CONCLUSIONS: Genetic susceptibility for thyroid sensitivity to TSH stimulation was distinct for three families of inbred mouse lines. These observations parallel the human situation with multiple genetic loci contributing to the same trait and different loci associated with the same trait in different ethnic groups. Of the genetic loci highlighted in mice, three overlap with, or are located up or downstream, of human TSH-controlling genes. Other studies show that human disease genes can be identified through cross-species gene mapping of evolutionary conserved processes. Consequently, our findings suggest that novel thyroid function genes may yet be revealed in humans.

Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the TSHR) displaying strong self-tolerance.

Role of self-tolerance and chronic stimulation in the long-term persistence of adenovirus-induced thyrotropin receptor antibodies in wild-type and transgenic mice.

BACKGROUND: Graves'-like disease, reflected by thyrotropin receptor (TSHR) antibodies and hyperthyroidism in some mouse strains, can be induced by immunization with adenovirus-expressing DNA for the human TSHR or its A-subunit. The conventional approach involves two or three adenovirus injections at 3-week intervals and euthanasia 10 weeks after the first injection. To investigate TSHR antibody persistence in mice with differing degrees of self-tolerance to the TSHR A-subunit, we studied the effect of delaying euthanasia until 20 weeks after the initial immunization. METHODS: Wild-type (WT) mice and transgenic (tg) mice expressing low intrathyroidal levels of the human TSHR A-subunit were immunized with A-subunit-adenovirus on two occasions; a second group of mice was immunized on three occasions. Sera obtained 4, 10, and 20 weeks (euthanasia) after the initial immunization were tested for thyrotropin (TSH) binding inhibition (TBI), antibody binding to TSHR A-subunit protein-coated enzyme-linked immunosorbent assay (ELISA) plates, and thyroid stimulating antibody activity (TSAb; cyclic adenosine monophosphate [cAMP] generation). Serum thyroxine (T4) and thyroid histology were studied at euthanasia. RESULTS: THE majority of WT mice retained high TSHR antibody levels measured by TBI or ELISA at euthanasia but only about 50% were TSAb positive. Low-expressor tgs exhibited self-tolerance, with fewer mice positive by TBI or ELISA and antibody levels were lower than in WT littermates. In WT mice, antibody persistence was similar after two or three immunizations; for tgs, only mice immunized three times had detectable TSAb at 20 weeks. Unlike our previous observations of hyperthyroidism in WT mice examined 4 or 10 weeks after immunization, all mice were euthyroid at 20 weeks. CONCLUSIONS: Our findings for induced TSHR antibodies in mice, similar to data for human thyroid autoantibodies, indicate that the parameters that contribute to the concentration of the antibody and thereby play a critical role in long-term persistence of TSHR antibodies are the degree of self-tolerance to the TSHR and chronic stimulation.

Long term persistence of thyrotropin receptor antibodies in wild-type and transgenic mice in a Graves' disease model.

Background: Graves'-like disease, reflected by TSHR antibodies and hyperthyroidism in some mouse strains, can be induced by immunization with adenovirus expressing DNA for the human thyrotropin receptor (TSHR) or it's A-subunit. The conventional approach involves two or three adenovirus injections at three-weekly intervals and euthanasia 10 weeks after the first injection. In order to investigate TSHR antibody persistence in mice with differing degrees of self-tolerance to the TSHR A-subunit, we studied the effect of delaying euthanasia until 20 weeks after the initial immunization. Methods: Wild-type mice and transgenic mice expressing low intrathyroidal levels of the human TSHR A-subunit were immunized with A-subunit-adenovirus on two occasions; a second group of mice was immunized on three occasions. Sera obtained 4, 10 and 20 weeks (euthanasia) after the initial immunization were tested for TSH binding inhibition (TBI), antibody binding to TSHR A-subunit protein-coated ELISA plates and thyroid stimulating antibody activity (TSAb; cAMP generation). Serum thyroxine and thyroid histology were studied at euthanasia. Results: The majority of wild-type mice retained high TSHR antibody levels measured by TBI or ELISA at euthanasia but only about 50% were TSAb positive. Low expressor transgenics exhibited self-tolerance, with fewer mice positive by TBI or ELISA and antibody levels were lower than in wild-type littermates. In wild-type mice, antibody persistence was similar after two or three immunizations; for transgenics, only mice immunized three times had detectable TSAb at 20 weeks. Unlike our previous observations of hyperthyroidism in wild-type mice examined 4 or 10 weeks after immunization, all mice were euthyroid at 20 weeks. Conclusions: Our findings for induced TSHR antibodies in mice, similar to data for human thyroid autoantibodies, indicate that the parameters that contribute to the concentration of the antibody and thereby play a critical role in long term persistence of TSHR antibodies are the degree of self-tolerance to the TSHR and chronic stimulation.