Immunological abnormality of peripheral blood B cells in patients with autoimmune thyroid disease

We investigated the response to immunoglobulin G-secreting cells (ISC) by peripheral blood mononuclear cells (PB-MNC) and purified B cells following stimulation with Staphylococcus aureus Cowan 1 (SAC) or with B cell stimulatory factor 2 (interleukin 6: IL-6), using the reverse hemolytic plaque assay in an attempt to clarify the immunological functions of peripheral blood B cells in patients with autoimmune thyroid disease (AITD). ISC response by PB-MNC following stimulation with SAC was significantly decreased in patients in the hyperthyroid state of Graves' disease and Hashimoto's thyroiditis as compared with that of normal controls. The difference in SAC-response was not significant between patients with euthyroid state of Graves' disease and normal controls. ISC response by PB-MNC following stimulation with SAC exhibited a reciprocal relationship to TRAb in patients with Graves' disease. Using purified B cells, some spontaneous ISC response without SAC stimulation was observed in patients in the hyperthyroid state of Graves' disease and Hashimoto's thyroiditis. This spontaneous ISC response was further enhanced by IL-6. These results suggest that in organ-specific autoimmune diseases such as AITD, immunological abnormalities exist in B cells and some B cells are nonspecifically activated in the immunologically active state.     

Development of an animal model of autoimmune thyroid eye disease

In previous studies we have transferred thyroiditis to naive BALB/c and NOD mice with human thyrotropin (TSH) receptor (TSHR)-primed splenocytes. Because the TSHR has been implicated in the pathogenesis of thyroid eye disease (TED) we have examined the orbits of recipients of TSHR-primed T cells, generated using a TSHR fusion protein or by genetic immunization. In the NOD mice, 25 of 26 animals treated with TSHR-primed T cells developed thyroiditis with considerable follicular destruction, numerous activated and CD8+ T cells, and immunoreactivity for IFN-gamma. Thyroxine levels were reduced. Thyroiditis was not induced in controls. None of the NOD animals developed any orbital pathology. Thirty-five BALB/c mice received TSHR-primed spleen cells. Thyroiditis was induced in 60-100% and comprised activated T cells, B cells, and immunoreactivity for IL-4 and IL-10. Autoantibodies to the receptor were induced, including TSH binding inhibiting Igs. A total of 17 of 25 BALB/c orbits displayed changes consisting of accumulation of adipose tissue, edema caused by periodic acid Schiff-positive material, dissociation of the muscle fibers, the presence of TSHR immunoreactivity, and infiltration by lymphocytes and mast cells. No orbital changes or thyroiditis were observed in control BALB/c mice. We have induced orbital pathology having many parallels with human TED, only in BALB/c mice, suggesting that a Th2 autoimmune response to the TSHR may be a prerequisite for the development of TED.     

Antigen presentation in human autoimmune thyroid disease

Monocyte/macrophage function in patients with autoimmune thyroiditis was investigated by their presentation of two distinct antigens; sheep red blood cells (SRBC) and human thyroglobulin (hTg) using in vitro systems designed for antibody induction. Purified peripheral blood monocyte/macrophages were primed by prefeeding with antigen for 60 min at 37 degrees C, washed, and co-cultured with autologous lymphocytes under a variety of incubation conditions. The most successful system employed 5% monocyte/macrophages with autologous T-B cells in the presence of the mitogen Staphylococcus aureus and B-cell differentiating factors. Under such conditions the anti-SRBC plaque-forming cell (PFC) response was amplified equally (approximately 10-fold) by SRBC-fed monocyte/macrophages in normal controls and patients with autoimmune thyroiditis rendered euthyroid with thyroxine replacement. hTg-fed monocyte/macrophages induced a 4-fold increase in anti-hTg PFC in selected patients with autoimmune thyroiditis examined under similar conditions (mean 36 +/- 3 PFC per 10(6) T-B cells). These data indicated that antigen processing by monocyte/macrophages was normal in patients with autoimmune thyroid disease.     

Gene expression signatures for autoimmune disease in peripheral blood mononuclear cells

The relatively new technology of DNA microarrays offers the possibility to probe the human genome for clues to the pathogenesis and treatment of human disease. While early studies using this approach were largely in oncology, many new reports are emerging in other fields including infectious diseases and pharmacology, and applications in autoimmunity have been recently reported by our group and others. Some of these investigations have examined animal models of autoimmune disease, but a number of human studies have also been carried out. Of special interest are those that have used peripheral blood samples because, unlike tissue biopsies, these are readily available from all subjects. Using this approach, patterns of gene expression can be detected that distinguish patients with autoimmune conditions from normal subjects. Furthermore, the genes that are identified provide clues to possible pathogenetic mechanisms and are likely to be useful in developing tests to establish diagnostic categories and predict therapeutic responses.     

Activation, proliferation, and differentiation of circulating B cells in autoimmune thyroid disease

Several studies of thyroid autoantibody production in vitro have been reported with the use of pokeweed mitogen, but the conclusions that have resulted regarding the immunoregulation of B cell function in thyroid disease are difficult to interpret due to the relatively nonphysiologic nature of pokeweed mitogen stimulation. We have therefore examined the responses of circulating B cells in Graves' disease and Hashimoto's thyroiditis by using a combination of lymphokines and other stimuli that act at various stages of the B cell cycle. In patients with autoimmune thyroid disease, nonspecific B cell proliferation and differentiation into IgG-secreting cells were both normal. However, a previously unsuspected heterogeneity among patients was found in their ability to produce autoantibodies in vitro. B cells of certain patients produced maximal autoantibody in response to pokeweed mitogen, some in response to Staphylococcus aureus Cowan strain I, and some in response to the lymphokines contained in the supernatants of stimulated T cell cultures. There was no correlation between serum autoantibody levels and those achieved in vitro. Attempts to stimulate antibody production by autoantigen (thyroglobulin) were unsuccessful, even when B cells were cultured with purified autologous OKT4+ T cells to avoid potential suppressor effects in the OKT8+ population. However, OKT4+ T cells enhanced pokeweed mitogen-driven autoantibody production. Our results show that several different functional stages of B cells exist in the circulation of patients with autoimmune thyroid disease, and that circulating B cells from such patients do not manifest a uniform response to B cell stimulators. This is presumably the result of differences in migration of circulating B cells and in their level of activation at the major sites of autoantibody production, such as the thyroid gland itself. In the light of these findings, caution is required in interpreting the results obtained from studies of circulating B cells as a means of elucidating the pathophysiology of autoimmune thyroid disease.     

Fetal cells in the maternal blood

Summary In an attempt to stimulate fetal cells in the maternal blood to mitotic division, peripheral blood lymphocytes were cultured from ten primiparous women and six multiparous women. In the case of the ten primiparous women, PWM was used to stimulate lymphocytes in 3- and 7-day cultures made at the 16th, 20th, 24th, and 28th week of gestation. Altogether, 10565 mitoses were analyzed after quinacrine staining of cells from five mothers who each subsequently gave birth to a male infant, and not a single XY mitosis was found.In the case of the multiparous women, lymphocyte cultures, with PHA or LPS as mitogen and MLC, were initiated between the 13th and 20th week of pregnancy. Four of the mothers were pregnant with a male child, and two with a female child. From cultures of each of the four mothers expecting a boy, a total of 9721 mitoses were analyzed after quinacrine staining, and not a single XY mitosis was found. However, one XY cell was found in the culture from one of the two women who delivered a girl. The XY mitosis probably originated from a pregnancy 8 months earlier which terminated in a male infant.In an attempt to culture and obtain good chromosome preparations from small numbers of cells, it was shown that a good mitotic response and good chromosome preparations could be obtained from as few as 6000 lymphocytes.     

Age-related prevalence of platelet-associated immunoglobulins G in nonthrombocytopenic patients with autoimmune thyroid disease and autoimmune polyglandular syndrome

OBJECTIVE: The prevalence of platelet-associated IgG (paIgG) in nonthrombocytopenic patients with autoimmune thyroid disease (AITD) alone or associated with autoimmune polyglandular syndrome (APS) has been studied. SUBJECTS: A total of 164 individuals were enrolled in this study: 81 patients with AITD alone, 33 patients with APS, and 50 healthy controls. RESULTS: The presence of paIgG was recorded in 41 of 81 patients with AITD (51%) as compared with 2 of 50 control subjects (4%, p < 0.0001). The prevalence of paIgG in patients with APS was higher even when compared with patients with AITD alone (25/33, 76%; p = 0.02). The presence of paIgG was not related to the functional thyroid parameters. The prevalence of paIgG was higher in the older than in the younger patients (75 vs. 47%, p = 0.0037). CONCLUSIONS: The results indicate that the prevalence of paIgG in patients with AITD is higher than previously thought, namely in elderly patients and in patients with APS, and not related to the thyroid function.     

Zn concentration in thyroid tissue and whole blood of women with different diseases of thyroid

The Zn concentration in thyroid tissue and whole blood of patients with Graves’ disease, thyroid cancer, and nodular goiter disease was determined using the total-reflection X-ray fluorescence method. The dependence of obtained concentrations on the clinical stage of the examined disease, histopathological grading, and kind of analyzed material (thyroid tissue and blood) was studied. The determined concentration of Zn was the lowest in the thyroid tissue of patients with thyroid cancer (23.1 μg/g) and it was the highest in the case of Graves’ disease (41.7 μg/g), whereas in the blood samples, the reverse results were found (7.1 μg/g and 4.8 μg/g, respectively). The physical basis of the method used, the experimental setup, and the procedure of sample preparation are described.     

Peptides of human thyroglobulin reactive with sera of patients with autoimmune thyroid disease

Autoantibodies to thyroglobulin (Tg) are a prominent feature of the two autoimmune thyroid diseases, chronic lymphocytic (Hashimoto's) thyroiditis and Graves' disease. Similar autoantibodies are found in the serum of many normal individuals without evidence of thyroid disease. Previous studies have indicated that patients with autoimmune thyroid disease recognize epitopes of Tg which are not usually recognized by normal individuals. The goal of this investigation was to identify peptide fragments of Tg bearing these disease-associated epitopes. For this purpose, we utilized a panel of mAbs that bind to different epitopes of the Tg molecule. One of these mAbs (137C1) reacted with an epitope that was also recognized by the sera of patients with autoimmune thyroiditis. In the present study, we show that two peptides (15 and 23 kDa) that reacted with mAb 137C1 are located in different parts of the Tg molecule. Each peptide inhibited the binding of mAb 137C1 to the other peptide and to the intact Tg, indicating that the same epitope was represented on the two peptides. Loops and helices of the secondary structure of the two peptides might be involved in the conformational epitope recognized by mAb 137C1. A striking finding of this study is that two apparently unrelated fragments of the Tg molecule bind to the same mAb. These findings may have important ramifications with regard to epitope spread and the progression of the autoimmune response to disease.     

Radiosensitivity of peripheral blood lymphocytes in autoimmune disease

The proliferation of peripheral blood lymphocytes, cultured with Con A, can be inhibited by ionizing radiation. Lymphocytes from patients with conditions associated with autoimmunity, such as rheumatoid arthritis, systemic lupus erythematosus and polymyositis, are more radiosensitive than those from healthy volunteers or patients with conditions not associated with autoimmunity. The nuclear material isolated from the lymphocytes of patients with autoimmune diseases is, on average, lighter in density than the nuclear material from most healthy controls. This difference in density is not related to increased sensitivity to ionizing radiation but the degree of post-irradiation change in density (lightening) is proportional to the initial density, i.e. more dense nuclear material always shows a greater upward shift after radiation. The recovery of preirradiation density of nuclear material, 1 h after radiation exposure, taken as an indication of DNA repair, correlates with the radiosensitivity of lymphocyte proliferation (Con A response); failure to return to pre-irradiation density being associated with increased sensitivity of proliferative response. These results require extension but, taken with previously reported studies of the effects of DNA methylating agents, support the idea that DNA damage and its defective repair could be important in the aetio-pathogenesis of autoimmune disease.     

Dendritic cells under investigation in autoimmune disease

Autoimmune disorders play an increasing role in public health, especially in light of the fact of the growing aged population, which primarily develop such diseases. A clear understanding of the mechanisms leading to the development of autoimmune responses and finally to autoimmune disease does not exist. Autoimmunity is characterized by the presence of autoantibodies and/or autoreactive T cells and the corresponding organ manifestation. Following the discovery of autoreactive T cells found in the periphery of mice and humans, the old immunological concept that autoreactive T cells are completely deleted in the thymus during evolution has been revised in recent years. Although antigen-presenting cells and particularly dendritic cells are known to play an important role in the regulation of immune responses and the activation of T cells, recent evidence suggests that the role of dendritic cells in the development of autoimmunity has been underestimated previously. This article aims to give a general overview on the basic immunological principles involved and gives a short review of the current literature on the functional relevance of dendritic cells in various human and murine autoimmune disorders.     

Methodology for detecting trace amounts of microchimeric DNA from peripheral murine white blood cells by real-time PCR

Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We report methodology using primers for a portion of the H2-k^b murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000 μM primer, a 20-fold increase in the median manufacturer’s recommended concentration, the assay could be optimized to detect 34 pg of C57BL/6J DNA in a background of 2.5 μg of carrier BALB/cJ DNA (1/100,000). These conditions resulted in a detection limit half as sensitive as that found when no carrier DNA was present. Published: April 7, 2003     

基于16S rDNA测序的自身免疫性甲状腺疾病患者肠道菌群研究

探讨Graves病患者、桥本甲状腺炎患者及健康人的肠道菌群结构、多样性和丰度差异。

选取2020年8月至2020年12月于湖北省中医院甲状腺疾病诊疗中心就诊的新诊断且未经治疗的Graves病患者（GD组）、桥本甲状腺炎患者（HT组）各15例以及15例年龄、性别相匹配的健康受试者（健康对照组）。采集参与者粪便样本,采用16S rDNA高通量测序法分析肠道菌群,比较各组对象肠道菌群物种组成及丰度差异。

与健康对照组相比,GD组患者肠道菌群Sobs指数、Ace指数、Chao指数偏低,Coverage指数偏高（均P＜0.05）。GD组与HT组以及HT组与健康对照组比较,其肠道菌群Sobs指数、Ace指数、Chao指数、Coverage指数差异均无统计学意义（均P＞0.05）。3组对象肠道优势菌门为拟杆菌门、厚壁菌门、变形菌门、梭杆菌门。瘤胃球菌属、罗斯伯里菌属、罗姆布茨菌属、链球菌属等在3组对象中差异均有统计学意义（均P＜0.05）,其中GD组患者肠道乳杆菌目、链球菌属、链球菌科的相对丰度较高;HT组患者肠道瘤胃球菌属、消化链球菌科、消化链球菌—蒂氏菌目、罗姆布茨菌、梭菌科、严格厌氧梭状芽胞杆菌1、梭菌目的相对丰度较高;而罗斯伯里菌属、光冈菌属在健康对照组中丰度较高。GD组患者肠道中严格厌氧梭状芽胞杆菌1、Odoribacter、Phocea丰度低于HT组,而Streptococcus的丰度高于HT组（均P＜0.05）。

Graves病患者、桥本甲状腺炎患者的肠道菌群与健康人群有显著差异,促进脂肪累积与能量吸收的菌群在Graves病患者中降低,而条件致病菌在自身免疫性甲状腺疾病患者中丰度增加,这些菌种变化可能通过破坏肠道稳态,干预免疫调节,促进炎症反应介导自身免疫性甲状腺疾病的发病。严格厌氧梭状芽胞杆菌1、Odoribacter、Phocea、链球菌属可能是Graves病和桥本甲状腺炎的关键差异菌属,但还需要进一步的研究以明确这些菌株介导疾病的机制。

     

Encephalopathy associated with autoimmune thyroid disease in patients with Graves' disease: clinical manifestations,follow-up,and outcomes

Background  

The encephalopathy associated with autoimmune thyroid disease (EAATD) is characterized by neurological/psychiatric symptoms, high levels of anti-thyroid antibodies, increased cerebrospinal fluid protein concentration, non-specific electroencephalogram abnormalities, and responsiveness to the corticosteroid treatment in patients with an autoimmune thyroid disease. Almost all EAATD patients are affected by Hashimoto's thyroiditis (HT), although fourteen EAATD patients with Graves' disease (GD) have been also reported.     

Experimental autoimmune orchitis: in vitro induction of an autoimmune disease.

Experimental autoimmune orchitis (EAO) can be induced in vitro. Normal lymph node lymphocytes cultured with autologous dissociated testis cells form rosette-like aggregates and later undergo blast transformation and proliferation. These stimulated lymphocytes cause in vivo EAO lesions, when injectd into syngeneic recipients. Moreover, their autoimmune reactivity can be monitored by an in vitro cytostasis assay. Density gradient analysis of the early lymphocyte-testis cultures reveals that the autoimmune reactive lymphocytes are enriched in the rosette populations. It therefore appears that testicular self-antigens are recognized by clonally preformed autologous lymphocytes.     

Increment in the Ta1+ cells in the peripheral blood and thyroid tissue of patients with Graves' disease

The present study utilized the anti-Ta1 mAb to characterize the cell surface phenotypes of peripheral blood and intrathyroidal lymphocytes in patients with Graves' disease. We found an increase in PBL bearing the Ta1 Ag in untreated patients. The euthyroid patients in remission, induced by antithyroidal drugs, radioisotope therapy, and subtotal thyroidectomy, had lower percentages of Ta1+ cells than did untreated patients. An increased percentage of Ta1+ cells in untreated patients was found in both CD4+ cells and CD8+ cells. The ratio of CD4+Ta1+ cells to CD8+Ta1+ cells in untreated patients was significantly higher than that of normal subjects. There was a positive correlation between the percentage of Ta1+ cells and the level of anti-TSH receptor antibody. In this prospective study, the proportion of Ta1+ cells was decreased in parallel with the reduction in anti-TSH receptor antibody and free T3 levels. In the chronically treated patients, the proportion of Ta1+ cells in the thyroid tissue was, yet similar to that in the peripheral blood, markedly increased in comparison to that of normal subjects. In contrast to Ta1+ cells, the thyroid tissue had a significantly higher percentage of HLA-DR+ T cells than did the paired peripheral blood. The proliferative responses of the Ta1+ cell-enriched population isolated from untreated patients toward thyroglobulin and microsomal Ag were markedly higher than those in a Ta1+ cell-depleted population, but both populations were able to respond equally to PHA. These results suggest that the Ta1+ cells may include Ag-triggered memory cells that are reactive with thyroid-specific Ag. Furthermore, monitoring such cells may provide an objective measure of abnormal immunologic activity.     

Insulin-dependent diabetes, an autoimmune disease

In type I (insulin-dependent) diabetes evidence for an autoimmune process is now fully established. This is also true for a similar disease observed in the NOD mouse and the BB rat. In addition to circulating antipancreatic antibodies, we demonstrated T-lymphocyte mediated cellular immunity in both these diabetic animals and in the human. Immunological abnormalities precede the development of diabetes and may be responsible for beta cell alteration. Evidence for this interpretation appears stronger for cell-mediated than for humoral immunity. However, full demonstration and understanding of the relationship between anti-beta cell immunity and beta cell alteration still raise many unresolved problems.