Surprisingly, the levels were higher at the younger age (Fig. protein which is usually shed from membrane bound TSHR, expressed at low levels in the thyroid. The enhanced TSHR antibody response following injected TSHR A-subunit protein-nanoparticles is usually reminiscent of the transient increase in pathogenic TSHR antibodies following the release of thyroid autoantigens after radio-iodine therapy in Graves patients. However, in the hTSHR/ NOD.model, enhancement is specific for TSHR antibodies, with antibodies to thyroglobulin and thyroid peroxidase remaining unchanged. In conclusion, despite the inclusion of a Tmem34 tolerogenic molecule, 2′-Deoxycytidine hydrochloride injected nanoparticles coated with TSHR A-subunit protein enhanced and accelerated development of pathogenic TSHR antibodies in hTSHR/NOD. NOD.mice that express the human (h) TSHR A-subunit in the thyroid and the thymus and consequently, unlike their wild-type littermates, spontaneously develop pathogenic stimulating TSHR antibodies, as in Graves patients (11). Like their non-transgenic counterparts, hTSHR/NOD.mice also spontaneously develop autoantibodies to the other two major thyroid autoantigens, thyroglobulin (Tg) (12C14) and thyroid peroxidase(TPO)(15). These transgenic mice provide the tools to test the outcome of injecting nanoparticles coated with hTSHR A-subunit 2′-Deoxycytidine hydrochloride protein plus ITE to specifically block the development of pathogenic TSHR antibodies. Unlike with insulin and myelin basic protein, this approach exacerbated, rather than ameliorated the autoimmune response. This obtaining provides insight into a limiting factor critical for the spontaneous development of TSAb. METHODS Reagents: Recombinant human (mice (originally from your Jackson Laboratory, Bar Harbor, ME) and transgenic TSHR/NOD.mice (11), that express low levels 2′-Deoxycytidine hydrochloride of the human TSHR A-subunit in the thyroid and thymus, were bred at Cedars-Sinai Medical Center. [Sperm from hTSHR/NOD. have been frozen by MMRRC; Strain Name: NOD.Cg-Tg(TG-TSHR)51.9Smcl/Mmmh; Stock Number: 037586]. The two types of nanoparticles, 200 l of np-ITE-TSHR or np-ITE, and phosphate buffered saline as an additional control, were injected intraperitoneally (ip) four occasions at weekly intervals starting in 7-week aged mice, following the approach used by Quintana and colleagues for NOD mice. (5). (Fig. 1A). From the age of 8 weeks, mice were provided with drinking water containing sodium iodide (NaI, 0.05%). Blood was drawn from your tail vein after 8 weeks on iodide (mice aged 16 weeks). Mice were euthanized to obtain blood and thyroid tissue after 16 weeks on iodide (mice aged 24 weeks). Open in a separate window Physique 1. Panel A, Protocol for intra-peritoneal (ip) injection of TSHR transgenic (Tgic) and wild-type (WT) NOD.mice with 2′-Deoxycytidine hydrochloride buffer, nanoparticles (np) coated with ITE, or np coated with both ITE and hTSHR A-subunit protein. In this and subsequent figures, the upward arrows show the times of injection and the downward arrows the time blood samples taken. As mentioned (Methods), 29 TSHR Tgic (10 males, 19 females) and 36 WT mice (15 males and 21 females) were analyzed (total 65 mice). More females than males were investigated because high levels of TSH in males on iodized water give spurious positives in TBI assays (observe below) which precludes measuring pathogenic TSHR antibodies in this sex (11). Panel B, Non-pathogenic (ELISA) TSHR antibody levels (Mean + SE) are enhanced in transgenic hTSHR/NOD.and wild-type (WT) NOD.mice injected ip with np coated with ITE-TSHR A- subunit compared with mice injected with buffer or np coated with ITE only. Non-pathogenic TSHR antibodies develop similarly in males and females; therefore, data for males and females are combined. The numbers of mice are indicated in parentheses. * significantly higher in transgenics injected with np-ITE-TSHR than with buffer or np-ITE (p<0.05, ANOVA). Panel C, TgAb levels for the mice analyzed in Panel B are not significantly different in hTSHR/NOD.or in WT 2'-Deoxycytidine hydrochloride mice injected with buffer, np-ITE, or np-ITE-TSHR A subunit. TgAb were measured by ELISA and data reported as the OD 490 nm (mean +SE). TgAb levels do not differ between males and females on iodide (12C14, 19) and therefore data are shown for males and females combined. The numbers of mice are.