Successful switch to cisplatin-based chemotherapy in a patient with lung cancer who developed a carboplatin-induced hypersensitivity reaction.

INTRODUCTION: Platinum-based chemotherapy is the mainstay of first-line therapy for advanced-stage non-small cell lung cancer (NSCLC). Although carboplatin-induced hypersensitivity reactions (HSRs) commonly occur following multiple cycles of therapy, they are rarely observed during the first cycle of the treatment. CASE REPORT: Here, we report the case of a 70-year-old man with advanced-stage NSCLC who developed HSR possibly caused by carboplatin during the first cycle of induction with platinum-doublet chemotherapy plus pembrolizumab. The patient presented with bronchial obstruction due to a centrally located tumor. No driver mutations were detected, and the programmed death-ligand 1 expression ranged from 1% to 24%. Consequently, the patient was treated with pembrolizumab combined with carboplatin and paclitaxel. However, immediately after the start of carboplatin, the blood pressure and oxygen levels of the patient dropped and he began exhibiting an altered level of consciousness. These findings indicated carboplatin-induced anaphylaxis. Hypotension and oxygen desaturation improved following carboplatin discontinuation and normal saline administration. MANAGEMENT AND OUTCOME: The basophil activation test for both carboplatin and cisplatin was negative. Thus, the risk of anaphylaxis owing to both drugs was ruled out, and carboplatin was believed to have induced grade 3 HSR. Subsequently, carboplatin-based chemotherapy was switched to cisplatin-based chemotherapy. HSR was not observed during the four treatment cycles with pembrolizumab, cisplatin, and pemetrexed, and best response was partial response. DISCUSSION: Cisplatin-based chemotherapy could be used as an alternate treatment in patients with NSCLC who develop severe carboplatin-induced HSR.

Exacerbation of autoimmune thyroiditis by a single low dose of whole-body irradiation in non-obese diabetic-H2h4 mice.

PURPOSE: To evaluate how irradiation affects thyroid autoimmunity in mouse models of Hashimoto's thyroiditis and Graves' hyperthyroidism. MATERIALS AND METHODS: Non-obese diabetic (NOD)-H2(h4) mice spontaneously develop anti-thyroglobulin (Tg) antibodies and thyroiditis when supplied with sodium iodine (NaI) in the drinking water. BALB/c mice develop anti-thyrotropin receptor (TSHR) antibodies and hyperthyroidism following immunization with adenovirus expressing TSHR (Ad-TSHR). Mice were irradiated as follows: A single whole-body irradiation with 0.05, 0.5 or 3 Gy one week before or after the beginning of NaI or immunization with Ad-TSHR, fractionated whole-body irradiations with 0.05 Gy twice a week or 0.5 Gy once a week from one week before NaI or Ad-TSHR immunization, or a single regional irradiation to the thyroid gland with 0.5 Gy one week before NaI. The effect of a single irradiation with 0.05, 0.5 or 3 Gy on splenocytes was also evaluated. RESULTS: A single whole-body irradiation with 0.5 Gy one week before NaI exacerbated thyroiditis and increased anti-Tg antibody titers in NOD-H2(h4) mice. In contrast, any irradiation protocols employed did not affect incidence of hyperthyroidism or anti-TSHR antibody titers in BALB/c mice. High-dose irradiation increased the relative ratios of effector T cells to regulatory T cells (an indication of enhanced immune status) but kills most of T cells. CONCLUSIONS: These results indicate that a single whole-body low-dose irradiation with 0.5 Gy exacerbates thyroiditis in NOD-H2(h4) mice, data consistent with some clinical evidence for increased incidence of thyroid autoimmunity by environmental irradiation.

CD8+CD122+ T cells, a newly identified regulatory T subset, negatively regulate Graves' hyperthyroidism in a murine model.

Graves' disease is a thyroid-specific autoimmune disease mediated by stimulatory autoantibodies against the TSH receptor (TSHR). We have previously shown in our mouse model with adenovirus expressing the TSHR that antibody mediated depletion of CD4(+)CD25(+) regulatory T cells (Tregs) enhances incidence and severity of hyperthyroidism in resistant and susceptible mouse strains, respectively. These data indicate that balance between effector T cells and Tregs is critical for disease development. This study was designed to evaluate the role played by another recently identified type of Treg, CD8(+)CD122(+) T cells, in our mouse model to delineate the significance of different types of Tregs in Graves' disease. Flow cytometry analysis showed that CD4(+)CD25(+) and CD8(+)CD122(+) T cells are distinct cell types, and anti-CD122 antibody effectively and selectively depleted CD8(+)CD122(+) T cells. As for CD4(+)CD25(+) Treg, CD8(+)CD122(+) T cell depletion increased the incidence of hyperthyroidism both in resistant and susceptible mice. Of interest, intrathyroidal lymphocytic infiltration was observed in some CD8(+)CD122(+) T cell-depleted, hyperthyroid resistant mice. These results indicate that in addition to CD4(+)CD25(+) T cells, CD8(+)CD122(+) T cells also play a crucial role in disease susceptibility in mouse Graves' disease. Thus, different types of Tregs appear to be involved in tolerance to a self-antigen, the TSHR.

Lack of effect of methimazole on dendritic cell (DC) function and DC-induced Graves' hyperthyroidism in mice.

In addition to the biochemical inhibition of thyroid hormone synthesis, antithyroid drugs including methimazole (MMI) may have immunosuppressive effect through inhibition of major histocompatibility complex (MHC) class I and II expressions on non-professional (thyrocytes) and professional (macrophages and B cells) antigen presenting cells (APCs). Dendritic cells (DCs) are another professional APCs and very likely play the most important role in the primary immune response. Therefore, we focused in this study on evaluating the effect of MMI on DC function in mice. Bone marrow cells cultured with granulocyte macrophage colony stimulating factor and interleukin (IL)-4 expressed high levels of CD11c and moderate levels of MHC class II, both of which are widely used markers for DCs. In vitro incubation of this DC-containing cell population with 10(- 6)-10(- 4) M MMI for 2 days did not change basal- and maturation signal (adenoviral infection and lipopolysaccharide)-induced levels of the cell surface marker expressions such as MHC class I and II, CD86, CD40 and DEC205, and of proinflammatory cytokine IL-6 release. Further we found that treatment of the DC-containing cell population with MMI did not influence the incidence of Graves' hyperthyroidism and anti-thyrotropin receptor (TSHR) antibody titers in a mouse Graves' model we have recently established with DCs infected with adenovirus expressing the TSHR A subunit. Although we cannot completely exclude immunosuppressive effect of MMI on other immune cells, our data indicate that DCs do not appear to be the primary target for the immunosuppressive effect of MMI.

Distinct responses of two hepatocellular carcinoma cell lines of a similar origin to immunotherapies targeting regulatory or effector T cells.

Balance between effector T cells (Teff) and regulatory T cells (Treg) appears to be very crucial for effective anti-tumor immunotherapy. The therapeutic efficacies of enhancement of Teff and suppression of Treg were compared between two murine hepatoma cell lines of a similar origin, MH129 and MH134. Enhancement of Teff was achieved by infection of tumor cells with adenovirus expressing glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), and suppression of Treg, by depletion of CD4+CD25+ naturally occurring Treg by administration of anti-CD25 monoclonal antibody (PC61) or low-dose cyclophosphamide. Our data show that MH129 cells were susceptible to Treg depletion but resistant to GITR expression, and vice versa for MH134 cells. Thus, in MH129 cells, injection of PC61 prior to or after tumor cell inoculation completely or partially, respectively, eradicated tumor growth. Low-dose cyclophosphamide administered after tumor cell inoculation also delayed tumor growth. However, GITR expression either in vitro or in vivo exhibited little effect. In contrast, in MH134 cells, PC61 induced partial tumor growth delay only when injected prior to tumor cell inoculation, and low-dose cyclophosphamide showed no effect, but GITR, particularly when administered in vitro, inhibited tumor growth. An additive effect of PC61 and GITR was observed only in MH134 cells. The ratios of peripheral CD4+CD25+ to CD4+ T cells remained unaltered during the experimental course in both tumor models. From these results we speculate that this different sensitivity may be due to a difference in relative induction levels of Teff versus Treg, not due to different immunogenicity or different kinetics of peripheral Treg, between the two tumor models. Future studies identifying antigen(s) or epitope(s) specific for Teff and Treg in these tumor cell lines are necessary as analysis of the immune response to such antigen(s) or epitope(s) may in general help predict the relative efficacy of different immunotherapies against distinct tumors.

Regulation of Graves' hyperthyroidism with naturally occurring CD4+CD25+ regulatory T cells in a mouse model.

Graves' hyperthyroidism can be efficiently induced in susceptible mouse strains by repeated immunization with recombinant adenovirus coding the TSH receptor (TSHR). This study was designed to evaluate the role(s) played by naturally occurring CD4(+)CD25(+) regulatory T cells in the development of Graves' hyperthyroidism in resistant C57BL/6 and susceptible BALB/c mice. Depletion of CD4(+)CD25(+) T cells rendered some C57BL/6 mice susceptible to induction of hyperthyroidism. Thus, hyperthyroidism developed in 30% of the CD4(+)CD25(+) T cell-depleted C57BL/6 mice immunized with adenovirus expressing the TSHR A-subunit (AdTSHR289) vs. 0% of those immunized with AdTSHR289 alone. This immunological manipulation also enhanced disease severity in susceptible BALB/c mice, as reflected by a significant increase in mean T(4) levels by CD4(+)CD25(+) T cell depletion. The immunoenhancing effect of CD4(+)CD25(+) T cell depletion appears to be attributable to an increase in thyroid-stimulating antibody production and/or a decrease in thyroid-blocking antibody synthesis, but not immune deviation to either T helper 1 or 2 cells. Interestingly, unlike BALB/c mice, some hyperthyroid C57BL/6 mice showed some intrathyroidal lymphocytic infiltration with follicular destruction. These results indicate that CD4(+)CD25(+) T cells play a role in disease susceptibility and severity in adenovirus-TSHR-induced Graves' hyperthyroidism. Overall, the imbalance between effector and regulatory T cells appears to be crucial in the pathogenesis of Graves' disease.

Different mechanisms for anti-tumor effects of low- and high-dose cyclophosphamide.

It is known that, besides its direct cytotoxic effect as an alkylating chemotherapeutic agent, cyclophosphamide also has immuno-modulatory effects, such as depletion of CD4+CD25+ regulatory T cells. However, its optimal concentration has not yet been fully elucidated. Therefore, we first compared the effects of different doses of cyclophosphamide on T cell subsets including CD4+CD25+ T cells in mice. Cyclophosphamide (20 mg/kg) decreased the numbers of splenocytes, CD4+ and CD8+ T cells by approximately 50%, while a decline in CD4+CD25+ T cell number was more profound, leading to the remarkably lower ratios of CD4+CD25+ T cells to CD4+ T cells. In contrast, 200 mg/kg cyclophosphamide severely decreased the numbers of all the T cell subsets by > 90% although the decreased ratios of CD4+CD25+ T cells to CD4+ T cells were still observed. Next, low-dose cyclophosphamide significantly inhibited in vivo growth of murine hepatoma MH129 tumor in immuno-competent but not immuno-deficient mice. This anti-tumor effect was abolished by CD4+CD25+ T cell repletion. In contrast, high-dose cyclophosphamide exhibited similar anti-tumor effects in both mice. In addition, contrary to antibody-mediated CD4+CD25+ T cell depletion, administration of low-dose cyclophosphamide after tumor inoculation was more efficacious than the prior administration. Our data show that low-dose cyclophosphamide selectively depletes CD4+CD25+ T cells, leading to enhanced anti-tumor effects against pre-existing tumors, while the anti-tumor effect of high-dose cyclophosphamide is solely attributed to its direct cytotoxicity. These findings appear to be highly crucial in a clinical setting of combined chemotherapy and immunotherapy for cancer treatment.

Vaccination with dendritic cells pulsed with apoptotic cells elicits effective antitumor immunity in murine hepatoma models.

Dendritic cell (DC)-based vaccine is a developing strategy to treat cancer including hepatoma. We evaluated the antitumor efficacy of vaccination with DCs pulsed with apoptotic cells, as compared to vaccination with DCs pulsed with cell lysates, in murine hepatoma models. Murine hepatoma cells, Hepa1-6, MH134 and BNL1ME.A.7R.1, and their syngeneic mice, C57BL/6, C3H/HeN and BALB/c, respectively, were used in the study. Protective and therapeutic antitumor effects of vaccination with bone marrow-derived DCs pulsed with irradiation or sulindac-induced apoptotic cells or cell lysates were analyzed. Immature DCs efficiently phagocytosed apoptotic cells and increased expression of CD86, a cell surface maturation marker. Vaccination with apoptotic cell-pulsed, but not cell lysate-pulsed, DCs promoted significant protective immunity against parental hepatoma in vivo. Spleen cells from mice vaccinated with apoptotic cell-pulsed DCs showed higher cytolytic activity and contained higher number of IFN-gamma producing cells against parental hepatoma cells than those from mice vaccinated with cell lysate-pulsed DCs in vitro. Polyriboinosinic polyribocytidylic acid [poly (I:C)], double strand RNA, further enhanced CD86 expression and the therapeutic efficacy of vaccination with DCs pulsed with apoptotic cells for pre-established hepatoma. These results suggest that vaccination with DCs pulsed with apoptotic cells and treated with poly (I:C) appears to be a promising approach as a new therapeutic means for hepatoma.

Genetic polymorphisms of OCT-1 confer susceptibility to severe progression of primary biliary cirrhosis in Japanese patients.

BACKGROUND: To identify the genetic factors involved in the pathogenesis of primary biliary cirrhosis (PBC), we focused on the organic cation transporter 1 (OCT1/SLC22A1), which is closely associated with phosphatidylcholine synthesis in hepatocytes. METHODS: We selected four (rs683369, rs2282143, rs622342 and rs1443844) OCT-1 single nucleotide polymorphisms (SNPs), and genotyped these SNPs using the TaqMan probe method in 275 Japanese PBC patients and 194 gender-matched, healthy volunteers as controls. RESULTS: The Chi-square test revealed that the rs683369 variant allele (G) was associated with insusceptibility to PBC development [P = 0.009, odds ratio (OR) 0.60, 95 % confidence interval (CI) 0.40-0.88] in an allele model, and that the rs683369 variant allele (G) was associated with jaundice-type progression in a minor allele dominant genotype model (P = 0.032, OR 3.10, 95 % CI 1.05-9.14). The OCT-1 rs2282143 variant (T) and rs622342 variant (C) were also associated with jaundice-type progression in a minor allele recessive genotype model (P = 0.0002, OR 10.58, 95 % CI 2.36-47.54, and P = 0.006, OR 7.84, 95 % CI 1.39-44.36, respectively). Furthermore, the association of OCT-1 rs683369 and rs622342 with susceptibility to jaundice-type progression was confirmed by a replication study with a distinct set of PBC patients who underwent liver transplantation. CONCLUSIONS: The present study is the first report on the association of OCT-1 genetic polymorphisms with the overall development and jaundice-type progression of PBC.

Distinct role of T helper Type 17 immune response for Graves' hyperthyroidism in mice with different genetic backgrounds.

T helper type 17 (Th17) cells, a newly identified effector T-cell subset, have recently been shown to play a role in numerous autoimmune diseases, including iodine-induced autoimmune thyroiditis in non-obese diabetic (NOD)-H2(h4) mice, which had previously been thought Th1-dominant. We here studied the role of Th17 in Graves' hyperthyroidism, another thyroid-specific autoimmune disease, in a mouse model. Two genetically distinct BALB/c and NOD-H2(h4) strains with intact or disrupted IL-17 genes (IL-17(+/+) or IL-17(-/-)) were immunized with adenovirus (Ad) expressing the thyrotropin receptor (TSHR) A-subunit (Ad-TSHR289). Both IL-17(+/+) and IL-17(-/-) mice developed anti-TSHR antibodies and hyperthyroidism at equally high frequencies on the BALB/c genetic background. In contrast, some IL-17(+/+), but none of IL-17(-/-), mice became hyperthyroid on the NOD-H2(h4) genetic background, indicating the crucial role of IL-17 for development of Graves' hyperthyroidism in non-susceptible NOD-H2(h4), but not in susceptible BALB/c mice. In the T-cell recall assay, splenocytes and lymphocytes from the draining lymph nodes from either mouse strains, irrespective of IL-17 gene status, produced IFN-γ and IL-10 but not other cytokines including IL-17 in response to TSHR antigen. Thus, the functional significance of Th17 may not necessarily be predictable from cytokine expression patterns in splenocytes or inflammatory lesions. In conclusion, this is, to our knowledge, the first report showing that the role of Th17 cells for the pathogenesis of a certain autoimmune disease depends on the mouse genetic backgrounds.

Fibroblast-mediated in vivo and in vitro growth promotion of tumorigenic rat thyroid carcinoma cells but not normal Fisher rat thyroid follicular cells.

BACKGROUND: It is known that genetic abnormalities in oncogenes and/or tumor suppressor genes promote carcinogenesis. Numerous recent articles, however, have demonstrated that epithelial-stromal interaction also plays a critical role for initiation and progression of carcinoma cells. Furthermore, ionizing radiation induces alterations in the tissue microenvironments that promote carcinogenesis. There is little or no information on epithelial-stromal interaction in thyroid carcinoma cells. The objective of this study was to determine if epithelial-stromal interaction influenced the growth of thyroid carcinoma cells in vivo and in vitro and to determine if radiation had added or interacting effects. METHODS: Normal Fisher rat thyroid follicular cells (FRTL5 cells) and tumorigenic rat thyroid carcinoma cells (FRTL-Tc cells) derived from FRTL5 cells were employed. The cells were injected into thyroids or subcutaneously into left flanks of rats alone or in combination with skin-derived fibroblasts. In groups of rats, fibroblasts were irradiated with 0.1 or 4 Gy x-ray 3 days before inoculation. In vitro growth of FRTL-Tc and FRTL-5 cells were evaluated using the fibroblast-conditioned medium and in a co-culture system with fibroblasts. RESULTS: The in vivo experiments demonstrated that FRTL-Tc cells injected intrathyroidally grew faster than those injected subcutaneously, and that admixed fibroblasts enhanced growth of subcutaneous FRTL-Tc tumors, indicating that the intrathyroidal milieu, particularly in the presence of fibroblasts, confer growth-promoting advantage to thyroid carcinoma cells. This in vivo growth-promoting effect of fibroblasts on FRTL-Tc cells was duplicated in the in vitro experiments using the fibroblast-conditioned medium. Thus, our data demonstrate that this effect is mediated by soluble factor(s), is reversible, and is comparable to that of 10% fetal bovine serum. However, normal FRTL5 cells did not respond to the fibroblast-conditioned medium. Furthermore, high- and low-dose irradiation enhanced and suppressed, respectively, the in vivo fibroblast-mediated growth promotion. This effect was, however, not observed in the in vitro experiment with conditioned medium or even that allowing cell-cell contact. CONCLUSIONS: The intrathyroidal stromal microenvironments, particularly fibroblasts, appear to enhance the growth of thyroid carcinomas through soluble factor(s), which is modulated differently by high- and low-dose irradiation. To our knowledge this is the first study to show epithelial-stromal interaction in thyroid carcinoma.

Induction of late-onset spontaneous autoimmune thyroiditis by a single low-dose irradiation in thyroiditis-prone non-obese diabetic-H2h4 mice.

The previous data regarding the effect of irradiation on thyroid autoimmunity are controversial. We have recently reported the exacerbation of autoimmune thyroiditis by a single low dose (0.5 Gy) of whole body irradiation in thyroiditis-prone non-obese diabetic (NOD)-H2(h4) mice treated with iodine for 8 weeks. However, it is uncertain in that report whether the results obtained by the provision of iodine in a relatively short period of time (8 weeks) accurately reflects the long-term consequences of low-dose irradiation on thyroid autoimmunity. Therefore, we repeated these experiments with mice that were monitored after irradiation without iodine treatment for up to 15 months. We found that a single low-dose (0.5 Gy) irradiation increased the incidence and severity of thyroiditis and the incidence and titers of anti-thyroglobulin autoantibodies at 15 months of age. The numbers of splenocytes and percentages of various lymphocyte subsets were not affected by irradiation. Thus, we conclude that low-dose irradiation also exacerbates late-onset spontaneous thyroiditis in NOD-H2(h4) mice; one plausible explanation for this may be the acceleration of immunological aging by irradiation.

Expression of immunoregulatory molecules by thyrocytes protects nonobese diabetic-H2h4 mice from developing autoimmune thyroiditis.

One approach to prevent tissue destruction by autoimmune attack in organ-specific autoimmune diseases is to protect the target tissue from autoimmune reaction, regardless of its persistent activity. To provide proof-of-principle for the feasibility of this approach, the immunoregulatory molecules, TNF-related apoptosis-inducing ligand (TRAIL) and indoleamine 2, 3-dioxygenase, were expressed in the thyroid glands using adenovirus vector in nonobese diabetic-H2(h4) mice that spontaneously develop thyroiditis. Mice were anesthetized, and the thyroid glands were exposed by neck dissection, followed by in situ infection with adenovirus vector (5 x 10(10) particles per mouse) twice or thrice, starting 1 d or 4 wk before mice were supplied with sodium iodine (NaI) water. After 8 wk NaI provision, the extent of thyroiditis, serum titers of antithyroglobulin antibodies, and cytokine expression in the spleen were examined. In situ infection of adenovirus expressing TRAIL or indoleamine 2, 3-dioxygenase, but not green fluorescent protein, significantly suppressed thyroiditis scores. However, antithyroglobulin antibody titers and expression levels of cytokines (interferon-gamma and IL-4) in the spleen remained unaltered. Importantly, adenovirus infection 4 wk after NaI provision was also effective at suppressing thyroiditis. The suppressive effect of TRAIL appears to be mediated at least partly by accumulation of CD4(+)Foxp3(+) regulatory T cells into the thyroid glands. Thus, localized expression of immunoregulatory molecules efficiently protected the thyroid glands from autoimmune attack without changing the systemic autoimmunity in nonobese diabetic-H2(h4) mice. This kind of immunological intervention, although it does not suppress autoimmune reactivity, may have a potential for treating organ-specific autoimmune diseases.

T helper type 17 immune response plays an indispensable role for development of iodine-induced autoimmune thyroiditis in nonobese diabetic-H2h4 mice.

T helper type 1(Th1)/Th2 paradigm has been expanded by discovery of a novel effector T cell (T(eff)) subset, Th17 cells, which produce a proinflammatory cytokine IL-17. Th17 cells have recently been shown to play a major role in numerous autoimmune diseases that had previously been thought to be Th1-dominant diseases. We here studied the significance of Th17 cells in iodine-induced autoimmune thyroiditis in nonobese diabetic-H2(h4) mice, a mouse model of Hashimoto's thyroiditis in humans, which spontaneously develop antithyroglobulin autoantibodies and intrathyroidal lymphocyte infiltration when supplied with iodine in the drinking water. We observed increased numbers of Th1 and Th17 cells in spleen and accumulation of both types of T(eff) in the thyroid glands of iodine-fed wild-type mice, indicating that Th17 cells as well as Th1 cells constitute thyroid lesions. Furthermore, the incidence and severity of intrathyroidal lymphocyte infiltration, and the titers of antithyroglobulin autoantibodies were markedly reduced in iodine-treated IL-17(-/-) mice as compared with wild-type mice. Of interest, IL-17(+/-) mice showed an intermediate phenotype. Therefore, the present study, together with a previous report demonstrating the importance of Th1, not Th2, immune response for developing thyroiditis using mice deficient for interferon-gamma or IL-4, clearly indicates that both Th1 and Th17 cells are critical T(eff) subsets for the pathogenesis of spontaneous autoimmune thyroiditis in nonobese diabetic-H2(h4) mice.

CD4+CD25+ naturally occurring regulatory T cells and not lymphopenia play a role in the pathogenesis of iodide-induced autoimmune thyroiditis in NOD-H2h4 mice.

NOD-H2(h4) mice, which express I-A(k) on the NOD background, spontaneously develop autoimmune thyroiditis, a model of Hashimoto thyroiditis in humans, by adding iodide in the drinking water. Parental NOD mice have previously been shown to have intrinsic numerical abnormalities in peripheral lymphocytes and lymphocyte subpopulations such as CD4(+)CD25(+) naturally occurring regulatory T cells (Treg). Therefore we first investigated whether the similar abnormalities exist in NOD-H2(h4) mice. We observed that, compared with other non-autoimmune disease prone BALB/c and C57BL/6 mice, NOD-H2(h4) mice have lower numbers of splenocytes, CD3(+)T, CD4(+)T and CD8(+)T cells but the ratios of Treg to CD4(+)T cells were comparable. Increasing the numbers of peripheral lymphocytes by Complete Freund's Adjuvant immunization or splenocyte transfer did not affect development of thyroiditis, indicating that lymphopenia does not play a critical role in the pathogenesis of thyroiditis. We next examined the significance of Treg by depleting this lymphocyte subset with anti-CD25 antibody. Treg depletion, performed 4days before the administration of NaI water for 8 weeks, significantly exacerbated thyroiditis (p<0.01). Anti-thyroglobulin antibody titers also increased by Treg depletion (p<0.01) without changing the IgG2b to IgG1 ratios. In addition, expression levels of mRNA for IFN-gamma and IL-4 were enhanced in parallel. However, T(4) levels were similar between antibody-treated and untreated groups. Additional anti-CD25 administration at 3 weekly intervals did not influence these results. These data, together with previous studies on other mouse models of inducible thyroiditis and Graves' disease, indicate the role played by Treg in keeping anti-thyroid autoimmune reaction in check in experimental autoimmune thyroid diseases.

Adenovirus encoding the thyrotropin receptor A-subunit improves the efficacy of dendritic cell-induced Graves' hyperthyroidism in mice.

Stimulating the immune system by in vivo expression of the thyrotropin receptor (TSHR) is an efficient means to induce Graves' disease experimentally. For example, BALB/c mice injected with dendritic cells (DCs) infected with adenovirus encoding the full-length TSHR (AdTSHR) develop hyperthyroidism, albeit at a low incidence (36%). Recent observations suggest that the shed TSHR A-subunit, rather than the full-length receptor, is the autoantigen responsible for initiating/enhancing immune responses leading to thyroid stimulating antibodies (TSAb) and hyperthyroidism. Therefore, we attempted to improve the efficacy of the DC-based approach for Graves' disease using adenovirus encoding the TSHR A-subunit (AdTSHR289). Three injections of DCs infected with AdTSHR289 induced hyperthyroidism in 70% of BALB/c mice, approximately twice the disease induction rate with AdTSHR. TSAb activity was detected in most hyperthyroid mice, whereas virtually all immunized mice developed antibodies that inhibit [125I]TSH binding to the TSHR or recognize linear or conformational epitopes on the TSHR. TSHR antibodies were of IgG1 and IgG2a, indicating mixed T-helper type 1 (Th1)/Th2 immune responses. In conclusion, immunization with DC infected with adenovirus expressing the TSHR A-subunit is a highly efficient protocol to induce Graves' hyperthyroidism in BALB/c mice. This improved model will permit studies of the pathogenic role and therapeutic potential of DCs in Graves' hyperthyroidism.