The Essential Role of Circulating Thyroglobulin in Maintaining Dominance of Natural Regulatory T Cell Function to Prevent Autoimmune Thyroiditis.

Several key findings from the late 1960s to mid-1970s regarding thyroid hormone metabolism and circulating thyroglobulin composition converged with studies pertaining to the role of T lymphocytes in autoimmune thyroiditis. These studies cemented the foundation for subsequent investigations into the existence and antigenic specificity of thymus-derived natural regulatory T cells (nTregs). These nTregs prevented the development of autoimmune thyroiditis, despite the ever-present genetic predisposition, autoantigen (thyroglobulin), and thyroglobulin-reactive T cells. Guided by the hypothalamus-pituitary-thyroid axis as a fixed set-point regulator in thyroid hormone metabolism, we used a murine model and compared at key junctures the capacity of circulating thyroglobulin level (raised by thyroid-stimulating hormone or exogenous thyroglobulin administration) to strengthen self-tolerance and resist autoimmune thyroiditis. The findings clearly demonstrated an essential role for raised circulating thyroglobulin levels in maintaining the dominance of nTreg function and inhibiting thyroid autoimmunity. Subsequent identification of thyroglobulin-specific nTregs as CD4(+)CD25(+)Foxp3(+) in the early 2000s enabled the examination of probable mechanisms of nTreg function. We observed that whenever nTreg function was perturbed by immunotherapeutic measures, opportunistic autoimmune disorders invariably surfaced. This review highlights the step-wise progression of applying insights from endocrinologic and immunologic studies to advance our understanding of the clonal balance between natural regulatory and autoreactive T cells. Moreover, we focus on how tilting the balance in favor of maintaining peripheral tolerance could be achieved. Thus, murine autoimmune thyroiditis has served as a unique model capable of closely simulating natural physiologic conditions.

Experimental autoimmune thyroiditis. In vitro cytotoxic effects of T lymphocytes on thyroid monolayers.

Effector mechanisms in experimental autoimmune thyroiditis (EAT) were studied in vitro by establishing a cytotoxicity system with thyroid target cells. Lymph node cells (LNC) from popliteal and inguinal lymph nodes were obtained from CBA/J mice (8-10 wk old) 12-18 d after immunization with 120 micrograms mouse thyroglobulin (MTg) in complete Freund's adjuvant (0.2 ml to both hind footpads and thighs) and were cultured with MTg (10-50 micrograms/ml). On day 5 of culture, viable LNC were added to labeled thyroid monolayers and their cytoxicity was assayed after 16 h. Functional thyroid target cells, as reflected by MTg production for up to 9 d, were prepared by adding 1 mM dibutyryl adenosine 3',5'-cyclic monophosphate and 60 microU thyroid-stimulating hormone/ml to the culture medium. On days 5-7, confluent monolayers were labeled with 111In and used as targets. Specific 111In-release ranged from 56 to 85%. The cytotoxic response is MTg specific and H-2 restricted. Pretreatment of thyroid target cells with rabbit antiserum to MTg completely inhibited cytotoxicity. Pretreatment with mouse antiserum to either Kk or Dk products resulted in approximately 50% inhibition, whereas the combined use of both antisera led to total inhibition. No cytotoxicity was observed when control BALB/c thyroid cultures were the target cells. The kinetics of the expansion of Thy-1+ cytotoxic cells by in vitro exposure to MTg were then studied. The cytotoxic response required 5 d to develop and was abolished by treating LNC on day 4 with monoclonal antibody to Lyt-1.1, but not to Lyt-2.1, plus complement. In contrast, by day 5, cytotoxicity was abrogated by similar treatment with antiserum to Lyt-2.1, but not to Lyt-1.1. We conclude that cytotoxic cells derived from MTg-immunized mice are Lyt-2-bearing cells but require the presence of Lyt-1-bearing cells for their generation and/or differentiation.

[Hyperglycemia caused by mutation of GCK gene in 10 patients analysis of clinical and mutation characteristics].

Objective: To explore the gene mutation characteristics and detailed clinical presentations of hyperglycemia caused by GCK mutations in 10 patients. Methods: The clinical and follow-up data of 10 patients with hyperglycemia caused by mutation of GCK gene were reviewed. The patients were ascertained between January 1, 2014 and August 31, 2018 at the Department of Pediatrics, the First Affiliated Hospital of Zhejiang University and Ningbo Women & Children's Hospital. Clinical data were collected, including age, gender, main complaint, family history, fasting blood glucose, fasting blood insulin, 2-hour blood glucose, 2-hour blood insulin after oral glucose tolerance test, glycosylated hemoglobin, anti-glutamic acid decarboxylase antibody and body mass index. Mutations of GCK gene were detected by Sanger sequencing or high-throughput sequencing of diabetes-related genes in the patients and their family members. Results: There were ten patients, 8 of them were male, 2 were female.The ages at diagnosis varied between 4.7 to 12.3 years. The patients usually did not have obvious clinical symptoms of diabetes mellitus. Most of them were unexpectedly found to have hyperglycemia and with impaired glucose metabolism in three consecutive generations. The fasting blood glucose of patients was 6.8-7.7 mmol/L, 2-hour postprandial blood glucose was 7.8-11.6 mmol/L. Fasting blood insulin was 0.5-8.5 mU/L, glucose tolerance test results showed that 2 h postprondial blood insulin was 1.3-55.4 mU/L. The level of glycosylated hemoglobin was 6.1%-6.8%. Anti-glutamic acid decarboxylase antibody was negative in all patients. The GCK mutations identified in patients and one of their parents were located at exon5 (4 cases), exon9 (2 cases), exon2 (1 case), exon4 (1 case), exon6 (1 case) and exon7 (1 case). Conclusions: Most of the hyperglycemia patients caused by GCK mutations did not have typical clinical symptoms of diabetes. The fasting blood glucose was slightly elevated. Abnormal glucose tolerance test results were found in all 10 patients. Three consecutive generations of family had impaired glucose metabolism. GCK mutations located at exon 5 were common in 10 cases. There was no correlation between type of mutations and plasma glucose levels in domestic and international researches. When fasting glucose was found abnormal in clinic, a complete family history should be taken and the GCK gene should be sequenced to confirm the diagnosis in time.

Prospective study of T cell receptor utilization following the induction of murine thyroiditis.

Murine experimental autoimmune thyroiditis (EAT) is a well established model of autoimmune disease initiated by immunization with thyroglobulin. We have previously analyzed the T cell receptor (TcR) V gene families used by the intrathyroidal lymphocytic infiltrate in CBA/J mice with well established thyroiditis EAT and have implicated T cells expressing the mTcR V beta 13 gene family. We have now proceeded to examine the time course of mTcR V gene family use following immunization with mTg. We used a radiolabelled RT-PCR technique with oligonucleotides detecting 17 mouse TcR V beta gene families to examine the heterogeneity of the amplified V-D-J (CDR3) fragments. As previously, the TcR V beta 13 amplifications showed the expression of two similar homogeneous CDR3 sizes consistent with two clonally expanded T cell populations. However, such T cell clonal expansion was observed to peak at day 25 and by 90 days had markedly diminished despite the continuing presence of extensive histologic infiltration. An additional immunization with mTg at 63 days failed to maintain the mTcR V beta 13 clonal presence. Further confirmation of these observations was obtained by direct analysis of intrathyroidal T cells rescued from mice with EAT. Such intrathyroidal T cells, 25 days after mTg, demonstrated a marked increase in mTcR V13 expressing T cells to 9.4% compared to 2% of T cells in peripheral blood. It appeared, therefore, that in EAT the accumulation of V13 expressing T cells was a transient phenomenon which peaked at 25 days after immunization. The persistence of an intrathyroidal infiltration indicated that such T cells must have been accompanied by the accumulation and recruitment of additional selected bystander T cells. Such non-specific T cells may also have an integral role in the progression of autoimmune thyroiditis.

Depletion of CD4+CD25+ regulatory T cells exacerbates sodium iodide-induced experimental autoimmune thyroiditis in human leucocyte antigen DR3 (DRB1*0301) transgenic class II-knock-out non-obese diabetic mice.

Both genetic and environmental factors contribute to autoimmune disease development. Previously, we evaluated genetic factors in a humanized mouse model of Hashimoto's thyroiditis (HT) by immunizing human leucocyte antigen DR3 (HLA-DR3) and HLA-DQ8 transgenic class II-knock-out non-obese diabetic (NOD) mice. DR3+ mice were susceptible to experimental autoimmune thyroiditis (EAT) induction by both mouse thyroglobulin (mTg) and human (h) Tg, while DQ8+ mice were weakly susceptible only to hTg. As one environmental factor associated with HT and tested in non-transgenic models is increased sodium iodide (NaI) intake, we examined the susceptibility of DR3+ and/or DQ8+ mice to NaI-induced disease. Mice were treated for 8 weeks with NaI in the drinking water. At 0 x 05% NaI, 23% of DR3+, 0% of DQ8+ and 20% of DR3+DQ8+ mice had thyroid destruction. No spleen cell proliferation to mTg was observed. Most mice had undetectable anti-mTg antibodies, but those with low antibody levels usually had thyroiditis. At 0.3% NaI, a higher percentage of DR3+ and DR3+DQ8+ mice developed destructive thyroiditis, but it was not statistically significant. However, when DR3+ mice had been depleted of CD4+CD25+ regulatory T cells prior to NaI treatment, destructive thyroiditis (68%) and serum anti-mTg antibodies were exacerbated further. The presence of DQ8 molecules does not alter the susceptibility of DR3+DQ8+ mice to NaI-induced thyroiditis, similar to earlier findings with mTg-induced EAT. Susceptibility of DR3+ mice to NaI-induced EAT, in both the presence and absence of regulatory T cells, demonstrates the usefulness of HLA class II transgenic mice in evaluating the roles of environmental factors and immune dysregulation in autoimmune thyroid disease.

Mitogen-stimulated glutaraldehyde-fixed spleen cells: ability to stimulate in the mixed lymphocyte reaction and generate effector cells in cell-mediated lympholysis.

Mouse spleen cells treated with glutaralde lose their stimulating ability in the MLR. If the spleen cells are first converted to a blastogenic state by lipopolysaccharide and subsequently fixed with glutaraldehyde, their stimulating capacity is maintained.

Studies on interference with tolerance induction in T cells.

The mechanism of T-cell tolerance to a thymus-dependent antigen was examined, using the adjuvant polyadenylic-polyuridylic acid (poly A:U). In adoptive transfer experiments, thymus cells obtained from donor mice 2 days after treatment with a tolerogenic dose of bovine gamma-globulin (sBGG) did not cooperate with bone marrow (BM) cells in irradiated recipients challenged with aggregated BGG (aBGG). In contrast, thymus cells from donors given sBGG plus poly A:U retained their helper activity, as assayed by hemagglutination and rosette formation of spleen cells. The effect of poly A:U in preventing tolerance induction was also demonstrable in the cortisone-resistant population, in that thymus cells from cortisone-treated donors that had received sBGG and poly A:U retained their helper function. The presence of suppressor cells and the effect of poly A:U on their stimulation were also examined. sBGG-treated thymus cells suppressed the response of BGG-primed spleen cells in lethally irradiated mice, whereas thymus cells from donors treated with sBGG and poly A:U were not suppressive. These observations show that poly A:U prevents tolerance induction and the development of suppressor activity in T cells.

In vitro tolerance induction to a thymus-dependent antigen with BALB/c and C57BL/6 lymph node cells: effect of tolerogen concentration and duration of exposure.

Further studies on tolerance induction in vitro to bovine gamma globulin (BGG) in nonadherent BALB/c lymph node cells showed that it was dependent upon both the dose of tolerogen and the time of exposure. Nearly complete tolerance was achieved at a dose of 1.0 mg/ml and an incubation time of 12-18 hours. When C57BL/6 strain was used for comparison with the BALB/c strain because of its relative ease to become tolerant after in vivo injection of tolerogen, the rate of tolerance induction of its nonadherent lymph node cells was not different from that of BALB/c cells. Thus, in the absence of other host factors, the acquisition of tolerance by lymphocytes is gradual and may reflect the time required for a cell to reach a susceptible phase of the cell cycle and/or the activation of suppressor cells.

The role of T cells expressing TcR V beta 13 in autoimmune thyroiditis induced by transfer of mouse thyroglobulin-activated lymphocytes: identification of two common CDR3 motifs.

The transfer of lymphocytes from mouse thyroglobulin (mTg)-immunized CBA/J (H-2k) mice following in vitro activation with mTg initiates experimental autoimmune thyroiditis (EAT) in syngeneic recipients. We have analyzed the T cell receptor (TcR) V gene families used by the intrathyroidal lymphocytic infiltrate of such mice. Using a radiolabeled RT-PCR technique with oligonucleotides detecting 17 mouse TcR V beta gene families to examine the heterogeneity of the amplified V-D-J (CDR3) fragments, we demonstrated that only the TcR V beta 13 amplifications consistently showed two similar homogeneous CDR3 sizes consistent with two clonally expanded T cell populations. Sequencing of the homogeneous RT-PCR products from these V beta 13++ populations confirmed the presence of clonal expanded T cells and identified two recurrent CDR3 motifs LTGKDTQ and LGEQ present in six of the seven samples. Both these motifs had been found as contributors to the T cell population in our previous studies of CBA/J mouse thyroiditis induced by active immunization with heterologous human (h) Tg. These data suggest that the autoepitope recognized was shared between hTg and mTg. It appears, therefore, that in transfer thyroiditis the intrathyroidal T cell clonal proliferation follows the homing of V beta 13 antigen-specific T cells which have been expanded by a brief (3 day) in vitro activation to mTg and utilize two distinct CDR3 motifs. CDR3 size heterogeneity in many of the other expressed V gene families also suggested the accumulation and recruitment of selected bystander T cells responding to additional but limited Tg or other self epitopes, perhaps on the basis of CDR3 shape rather than sequence. Such T cells may also have integral roles in the development of autoimmune thyroiditis.

Suppressive effects of soluble histocompatibility antigens on the in vitro generation of cytotoxic T cells to D-end alloantigens.

Murine histocompatibility antigens were solubilized from the spleens and lungs of C57BL/6 (H-2b) animals with hypertonic salt (3 M KC1). Aggregate-free soluble antigens were incubated with nonadherent lymph node cells from BALB/c (H-2d) mice for 18 hr prior to their use as responder cells in the mixed-lymphocyte reaction (MLR). It was found that the generation of cytotoxic cells was suppressed while the proliferative response was not affected. The observed suppression was not due to a shift in the kinetics of the generation of cytotoxicity as determined throughout a 10-day culture period. The suppression was specific in that the response in MLR to unrelated H-2f stimulator cells and the subsequent generation of cytotoxic cells were unchanged. Using various H-2 recombinant strains as target cells in the assay of cell-mediated lympholysis, suppression of cytotoxicity was observed when the D end, but not the K end, was shared with the C57BL/6 strain from which the antigens were derived.

Resistance to experimental autoimmune thyroiditis: L3T4+ cells as mediators of both thyroglobulin-activated and TSH-induced suppression.

Mechanisms suppressive to induction of murine experimental autoimmune thyroiditis (EAT) can be activated by pretreatment with tolerogenic doses of mouse thyroglobulin (MTg) or prior TSH infusion to raise circulatory MTg levels. MTg-activated suppressor T cells (Ts), shown earlier to be Thy-1+ and probably I-J+, were further characterized by in vivo administration of paired rat monoclonal antibodies to distinct epitopes on the L3T4 or Lyt-2 molecule, either on the day of, or subsequent to, initiation of the tolerogenic regimes. The cells required at the time of MTg pretreatment were L3T4+, Lyt-2- and low anti-L3T4 doses had no effect on their activation. The cells that mediated the strong MTg-induced resistance following pretreatment were also L3T4+; their suppressor function could only be abrogated by depletion of L3T4+, but not Lyt-2+, cells. Injection of cyclophosphamide (20-100 mg/kg) either prior to EAT induction or after Ts activation did not affect the severity of disease. Similarly, the suppressor state evoked by TSH infusion could only be abrogated by anti-L3T4 treatment. These findings indicate that both MTg-activated and TSH-induced suppression are mediated by L3T4+ cells. We hypothesize that MTg-specific Ts are present in normal, EAT-susceptible mice in low numbers to contribute to the maintenance of self-tolerance and that they are stimulated by increased levels of circulatory MTg to expand/differentiate and mediate the marked resistance to EAT induction.

Characterization of resistance to murine experimental autoimmune thyroiditis: duration and afferent action of thyroglobulin- and TSH-induced suppression.

Genetically susceptible mice develop experimental autoimmune thyroiditis (EAT) after immunization with mouse thyroglobulin (MTg). Earlier studies have shown that resistance to EAT induction can be activated by two regimens, pretreatment with deaggregated MTg (dMTg) or with thyroid-stimulating hormone (TSH). With both protocols, suppression is linked to a > or = 2-3 day increase in circulatory MTg level and is mediated by CD4+ suppressor T cells (Ts). To assess the duration of suppression, CBA (H-2k) mice were injected with dMTg or infused with TSH via an osmotic pump for 3-4 days and then challenged with MTg + adjuvant at intervals up to 73 days for dMTg-pretreated mice or up to 94 days for TSH-pretreated mice. Suppression was measurable for at least 73 days after injected dMTg. TSH-induced suppression was also long-lasting; resistance was strong 38 days after TSH infusion and was still measurable on Day 66. The Thy-1+, CD4+ Ts which transfer MTg-induced suppression were further characterized by treatment with I-J antibodies plus complement prior to transfer. This treatment abolished the transfer of suppression which acts in the afferent phase to interfere with EAT induction. The capacity of Ts to suppress the efferent phase of EAT was assessed in vitro and in vivo. Cells from dMTg-pretreated mice did not block the in vitro proliferative response of MTg-primed cells to MTg, nor did these cells, when left intact in situ, reduce the severity of disease produced by the adoptive transfer of thyroiditogenic cells. Similarly, TSH-induced suppression was ineffective in preventing adoptively transferred EAT. Since suppression, which correlates with a temporary increase of circulatory MTg, occurs only at the afferent phase of active immunization, these findings lend support to our earlier hypotheses that circulatory MTg serves a physiologic role in maintaining normal self-tolerance by sustaining low levels of Ts activation and that additional rise above baseline increases and prolongs resistance to EAT induction.

The effect of hot pressing on the physical properties of glass reinforced hydroxyapatite.

Hydroxyapatite (HA), being of physiological importance, can be developed synthetically for implant application. A number of avenues have been explored in order to improve the physical and biological properties of a variety of hydroxyapatite composites. However, the fact remains, hydroxyapatite lacks the mechanical properties needed to sustain high loads. This study investigates the advantages of hot pressing on the physical properties of HA and glass reinforced HA (GR-HA). The results show a significant enhancement in the mechanical properties of GR-HA composites compared to HA e.g. flexural bending strength values were given at 91.75 and 88.87 M Nm(-2) for GR-HA (CP15F) and GR-HA (CP20F) respectively, compared to 78.9 M Nm(-2) for HA. The results for other properties such as elastic modulus, fracture toughness, Vicker's hardness, density and porosity also demonstrate the benefit of adding phosphate based glasses as a sintering aid. This is supported by XRD analysis, highlighting the presence of a secondary phase (beta-TCP) in GR-HA systems and the positive effect it has on the physical properties. It must be brought to attention that densification of hot pressed HA and GR-HA composites is reached at a lower temperature compared to a previous study on the same materials that have undergone pressureless sintering.

Syngeneic thyroglobulin is immunogenic in good responder mice.

Mouse thyroglobulin (MTg) or thyroid extract (TE) was given repeatedly to good responder C3H/Anf (H-2k) and poor responder BALB/c (H-2d) mice in the absence of adjuvant. Anti-MTg antibodies reached high levels in good responder mice given high doses of thyroid antigen. To eliminate stimulation by alloantigenic determinants and reduce the chance of denaturation. TE from syngeneic mice was prepared freshly each week and injected into good and poor responder strains. Again, significant antibody titers were observed in good responder mice. The antibody was specific for MTg since (a) it was not inhibited by extracts of other organs and (b) it reacted strongly with the closely related rat Tg and weakly with Tg from other species. Histology revealed mononuclear cell infiltration of the thyroid of good responder, but not of poor responder mice, regardless of the strain used to provide the thyroid antigen. The data demonstrate the presence of both T and B cell populations reactive with this self antigen and their stimulation by repeated high doses of antigen, without the aid of adjuvant, to override the regulatory controls that normally prevent autoimmune responses.