Proximity ligation assay (PLA) is a methodology that permits detection of protein-protein closeness, that is, proteins that are within 40 nanometers of each other, in cells or tissues at endogenous protein levels or after exogenous overexpression. It detects the protein(s) with high sensitivity and specificity because it employs a DNA hybridization step followed by DNA amplification. PLA has been used successfully with many types of proteins. In this methods paper, we will describe the workings of PLA and provide examples of its use to study TSH/IGF-1 receptor crosstalk in Graves\' orbital fibroblasts (GOFs) and TSH receptor homodimerization in primary cultures of human thyrocytes.

We aimed to investigate the relationship between the effects excessive of fluoride on thyroid health in children and the moderating role of thyroid stimulating hormone receptor (TSHR) or protein tyrosine phosphatase nonreceptor-22 (PTPN22) gene polymorphisms. Four hundred thirteen children (141 with dental fluorosis and 198 boys) were enrolled from both historical endemic and non-endemic areas of fluorosis in Tianjin, China. The fluoride exposure levels, thyroid health indicators, and TSHR (rs2268458) and PTPN22 (rs3765598) polymorphisms were examined. Multiple logistic models were applied to evaluate the relationship between dental fluorosis and thyroid abnormalities. Children over 9 year old with dental fluorosis have lower FT4 and TGAb levels and thyroid volume and higher TPOAb levels (all P < 0.05). In overall participants, children with dental fluorosis were more likely to have thyroid antibody single positive issues (adjusted P = 0.039) and less likely to have a goiter according to age or body surface area (age or BSA) (adjusted P = 0.003); In the TSHR (rs2268458) SNP = CC/CT or PTPN22 (rs3765598) SNP = CC subgroup, dental fluorosis may cause thyroid antibody single positive (adjusted P = 0.036; adjusted P = 0.002); in the TSHR (rs2268458) SNP = TT or PTPN22 (rs3765598) SNP = CC subgroup, dental fluorosis may protect children from goiter (age or BSA) (adjusted P = 0.018; adjusted P = 0.013). Excessive fluoride may induce thyroid antibody single positive and reduce goiter in children. Heterogeneity exists in the relationship between excessive fluoride and thyroid antibody single positive or goiter issues across children carrying different TSHR (rs2268458) or PTPN22 (rs3765598) genotypes.

BACKGROUND: Urinary albumin excretion (UAE) is a risk factor for cardiovascular diseases, metabolic syndrome, chronic kidney disease, etc. Only a few genome-wide association studies (GWAS) for UAE have been conducted in the European population, but not in the Asian population. Here we conducted GWAS and identified several candidate genes harboring single nucleotide polymorphisms (SNPs) responsible for UAE in the Japanese population.
METHODS: We conducted GWAS for UAE in 7805 individuals of Asian ancestry from health-survey data collected by Tohoku Medical Megabank Organization (ToMMo) and Iwate Tohoku Medical Megabank Organization (IMM). The SNP genotype data were obtained with a SNP microarray. After imputation using a haplotype panel consisting of 2000 genome sequencing, 4,962,728 SNP markers were used for the GWAS.
RESULTS: Eighteen SNPs at 14 loci (GRM7, EXOC1/NMU, LPA, STEAP1B/RAPGEF5, SEMA3D, PRKAG2, TRIQK, SERTM1, TPT1-AS1, OR5AU1, TSHR, FMN1/RYR3, COPRS, and BRD1) were associated with UAE in the Japanese individuals. A locus with particularly strong associations was observed on TSHR, chromosome 14 [rs116622332 (p = 3.99 × 10-10)].
CONCLUSIONS: In this study, we successfully identified UAE-associated variant loci in the Japanese population. Further study is required to confirm this association.

Graves\' hyperthyroidism can be treated but not cured. Antigen-specific immunotherapy would accomplish this goal, for which purpose an animal model is an invaluable tool. Two types of animal models are available. First, pathogenic TSHR antibodies (TSHRAb) can be induced by injecting mice with fibroblasts co-expressing the human TSHR (hTSHR) and MHC class II, or in mammals using plasmid or adenovirus vectors encoding the hTSHR or its A-subunit. Second, a mouse model that spontaneously develops pathogenic TSHRAb resembling those in human disease was recently described. This outcome was accomplished by transgenic intrathyroidal expression of the hTSHR A-subunit in NOD.H2h4 mice that are genetically predisposed to develop thyroiditis but, without the transgene, do not generate TSHRAb. Recently, novel approaches to antigen-specific immunotherapy have been tested, primarily in the induced model, by injecting TSHR A-subunit protein or cyclic TSHR peptides. T-cell tolerance has also been induced in \"humanized\" HLA-DR3 mice by injecting synthetic peptides predicted in silico to mimic naturally processed TSHR T-cell epitopes. Indeed, a phase 1 study based on the latter approach has been conducted in humans. In the spontaneous model (hTSHR/NOD.H2h mice), injection of soluble or nanoparticle-bearing hTSHR A-subunits had the unwanted effect of exacerbating pathogenic TSHRAb levels. A promising avenue for tolerance induction, successful in other conditions and yet to be tested with the TSHR, involves encapsulating the antigen. In conclusion, these studies provide insight into the potential outcome of immunotherapeutic approaches and emphasize the importance of a spontaneous model to test future novel, antigen-specific immunotherapies for Graves\' disease.