Autoantibodies against Forssman glycolipids in Graves'' disease and Hashimoto''s thyroiditis.

Sera from patients with Graves' disease and Hashimoto's thyroiditis have been shown to react with the Forssman glycolipid antigen (Gb5) using the techniques of high performance thin-layer chromatography (HPTLC) immunostaining and ELISA. Human monoclonal antibodies (MoAbs) have been prepared by fusion of human myeloma with peripheral lymphocytes from patients with Graves' disease. A MoAb, TRMo-4, reacted strongly and specifically with Gb5. These results suggest that anti-Forssman antibody may be involved in the pathogenesis of these autoimmune diseases. The detection of anti-Forssman glycolipid antibody may provide a useful means for clinical diagnosis and therapy.     

Cytotoxic lymphocytes in Hashimoto''s thyroiditis

The ability of peripheral lymphocytes from patients with Hashimoto's disease to destroy target cells coated with thyroid-related-autoantigens has been demonstrated. Direct cytotoxicity was demonstrable against mouse mastocytoma target cells coated with human thyroglobulin or microsomal antigen from thyrotoxic thyroid glands. No correlation was noted between circulating antibody and the ability of these cells to be cytotoxic.     

Clonal analysis of T lymphocytes infiltrating the thyroid gland in Hashimoto's thyroiditis

T cells isolated from thyroid tissue and peripheral blood of 2 patients with Hashimoto's thyroiditis were studied by a high cloning efficiency microculture technique. Clonal efficiencies of 37 and 24% were obtained from thyroid-derived T cell cultures, while 40 and 90% efficiencies resulted from peripheral-blood-derived cultures. A prevalence of T4-/T8+ T cell clones were found in thyroid infiltrates. The functional analysis of the clones demonstrated significantly higher proportions of clones with cytolytic activity in a lectin-dependent assay in thyroid-derived microcultures, as compared to peripheral blood-derived ones. The proportion of clones displaying natural-killer-like activity was increased in 1 patient only. Cytolytic activity was displayed not only by all T4-/T8+, but also by several T4+/T8- intrathyroid clones. Remarkable proportions of cytolytic clones were also able to release interleukin-2 upon phytohemagglutinin stimulation. Finally, the proportion of T cell clones able to release gamma-interferon following mitogen stimulation was significantly higher in thyroid- vs. peripheral-blood-derived microcultures. These results provide further data about the possible pathogenetical role of both regulatory and effector T lymphocytes in human autoimmune thyroiditis.     

桥本甲状腺炎伴甲状腺癌临床病理分析

目的　探讨桥本甲状腺炎 (Hashimoto’sthyroiditis,HT)与甲状腺癌的关系。 方法　对 80例HT和其中 14例并发甲状腺癌的HE片进行形态学观察 ,部分病例进行PCNA、p5 3免疫组化标记。结果　HT及甲状腺癌旁组织有滤泡上皮细胞增生、乳头状增生、不典型增生至癌变移行 (过渡 )的形态 ,其PCNA表达逐渐增强 (P <0 0 1) ,p5 3在少数不典型增生及癌变区呈低度阳性表达 (P <0 0 5 )。结论　HT与甲状腺癌发生关系密切 ,HT中甲状腺滤泡上皮增殖活跃 ,易于癌变。HT是一种具有恶变潜能的病变 ,应引起高度重视。     

In vitro production of interferon-gamma by peripheral blood from patients with Graves'' disease, Hashimoto''s thyroiditis and rheumatoid arthritis.

The production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells (PBMC), CD4 cells, or CD8 cells in response to interleukin-2 (IL-2) stimulation has been studied; the samples were obtained from 12 healthy control subjects, 19 patients with Graves' disease (10 hyperthyroid and nine euthyroid), 13 patients with Hashimoto's thyroiditis (four hypothyroid and nine euthyroid), and 15 patients with rheumatoid arthritis (11 active and four inactive). A dose of IL-2 (25 U/ml) was utilized to induce IFN-gamma by PBMC from all four groups. The incremental increase in IFN-gamma values (with IL-2 stimulation minus without stimulation) was significantly less in PBMC from patients with Graves' disease, Hashimoto's thyroiditis, and rheumatoid arthritis than that in PBMC from control subjects. The values from PBMC in patients with Graves' disease in a euthyroid state were below normal but greater than those from patients with Graves' disease in a hyperthyroid state. The incremental increase in IFN-gamma values from Graves' disease PBMC correlated with the serum TSH values (r = 0.622, P less than 0.01), but not with thyroid autoantibodies (anti-thyroid microsomal antibodies, anti-thyroid microsomal antibodies, nor TSH-binding inhibitory immunoglobulin activities). The incremental increase in IFN-gamma from PBMC from both control subjects and Graves' disease was correlated with that from CD4 cells (r = 0.711, P less than 0.01), but not with that from CD8 cells. The production of IFN-gamma in response to IL-2 from PBMC in Graves' disease correlated inversely with thyroid function, appearing to reflect the very effect of hyperthyroidism in this process. The precise explanation of these phenomena remains unclear. The decreased response of IFN-gamma to IL-2 stimulation by PBMC from patients with Graves' disease, Hashimoto's thyroiditis, and rheumatoid arthritis seems to be a non-specific phenomenon occurring in both organ specific autoimmune disease and systemic autoimmune disease. It may be due to a down-regulation in autoimmune disease of CD4 cells in response to IL-2, a decreased level of IL-2 cellular receptors or a decreased receptor affinity, associated increased soluble IL-2 receptors, or a defect of the intra-CD4 cellular IL-2 signal to produce or release IFN-gamma in the conditions studied.     

Intrathyroidal HLA-DR expression and T lymphocyte phenotypes in Graves'' thyrotoxicosis, Hashimoto''s thyroiditis and nodular colloid goitre.

Thyroid surgical biopsies from 21 individuals were examined by a double immunoenzymatic technique with respect to HLA-DR expression and lymphocytic infiltration. HLA-DR positive thyrocytes were observed in two examined Hashimoto goitres and in nine of 11 specimens from patients with Graves' disease. HLA-DR positive thyrocytes were localized to areas harbouring infiltrating lymphocytes, whereas regions with no lymphocytes only rarely expressed HLA-DR antigens. In two specimens of nodular goitre HLA-DR positive thyrocytes were observed in the vicinity of lymphocytic infiltration. Tissues from another three nodular goitres, from one follicular adenoma and from two normal individuals contained no HLA-DR positive thyrocytes and no or only a few lymphocytes. The lymphocytic infiltrates were dominated by cells with the Leu 3a helper/inducer phenotype irrespective of underlying disease, although most pronounced in Hashimoto's thyroiditis. The results indicate that HLA-DR antigens is expressed on thyrocytes in thyroid disorder. The extent of expression correlated with lymphocytic infiltration, which suggests that the two findings are related and of importance for the development of thyroid autoimmunity.     

Subpopulations of thyroid autoantibody secreting lymphocytes in Graves'' and Hashimoto thyroid glands.

Lymphocytes isolated from Graves' and Hashimoto thyroid tissue by enzymatic (dispase) digestion or mechanical disaggregation were markedly different in terms of their ability to synthesize thyroid autoantibodies in culture. Dispase digestion, followed by removal of thyroid follicular cells, gave a lymphocyte population with a high T:B cell ratio (6:1). However, the ability of these cell suspensions to synthesize microsomal (Mic) and thyroglobulin (Tg) antibodies spontaneously was significantly increased compared with lymphoid suspensions isolated by mechanical means. Spontaneous synthesis of thyroid autoantibodies was not markedly enhanced in cell suspensions prepared from patients' lymph node tissue by digestion compared with mechanical disaggregation. Further, Mic and Tg antibody production by thyroid lymphocytes prepared using dispase was inhibited by pokeweed mitogen (PWM) whereas in most cases suspensions prepared from the same tissues by mechanical dispersion synthesized low or undetectable levels of autoantibodies whether PWM was present or absent. Digestion of tissue debris remaining after mechanical removal of lymphocytes gave suspensions which had an increased proportion of suppressor/cytotoxic T cells compared with suspensions produced mechanically or by digestion alone; however, in terms of spontaneous autoantibody synthesis and PWM induced inhibition, these suspensions were similar to these obtained by digestion alone. It would therefore seem that enzymatic digestion of thyroid tissue resulted in the isolation of a lymphoid population which was different from that extracted by mechanical disaggregation. The digestion process appears to permit the recovery of lymphocytes closely associated with thyroid follicular cells and our studies suggest that it is this population which makes the major contribution to autoantibody synthesis.     

Immunohistological characterisation of tumour infiltrating lymphocytes in melanocytic skin lesions

BACKGROUND: Although the presence of tumour infiltrating lymphocytes (TIL) is a constant feature in melanomas, their immunophenotypic characterisation is still incomplete. We hypothesise that the transition from normal skin to benign naevi (BN) to melanocytic dysplastic naevi (MDN) to radial growth phase cutaneous malignant melanoma (RGP-CMM) to vertical growth phase cutaneous malignant melanoma (VGP-CMM) is associated with alterations in TIL. This study attempted to test this hypothesis and to characterise TIL in the melanocytic skin lesions. METHODS: In total, 74 lesions (12 BN, 12 MDN, 13 RGP-CMM, 26 VGP-CMM, and 11 metastatic melanomas) were examined using immunoperoxidase staining methods and antibodies targeting leukocyte common antigen (LCA+), T (CD3+) and B (CD20+) lymphocytes, and resting cytotoxic T cells (TIA-1+). RESULTS: Histologically, the transitions from normal skin to BN to MDN to RGP-CMM to VGP-CMM was associated with a gradual increase in the numbers of TIL (total, parenchymal, stromal, perivascular, and epidermal TIL, as well as TIL at the base of the lesions). The numbers of TIL were higher at the stroma than at the parenchyma. Similarly, immunostaining revealed that these transitions were associated with a gradual increase in the staining values (staining intensity, percentage of positive cells, and immunoreactivity score) for LCA+, CD20+, CD3+, and TIA-1+cells. The number of CD3+ cells was higher than that of CD20+ cells. All these differences between the normal skin and the lesional ones reached statistical significance (p<0.01). The majority of CD3+ cells were TIA-1+ T cells with cytotoxic potential. Compared with primary melanomas, there was a decrease in TIL in metastatic melanomas. CONCLUSIONS: The gradual increase in TIL during melanoma tumorigenesis may reflect increased antigenicity of the tumour cells. Although both humoral and cell mediated immunity are involved in melanomagenesis, the latter seems to have the major role. The immune profile of MDN suggests their intermediacy between BN and CMM.     

Immunohistological analysis of tumour infiltrating lymphocytes in seminoma using monoclonal antibodies

Summary Immunological characterization of tumour infiltrating lymphocytes (TIL) by immunohistological techniques was carried out in 20 cases of stage I seminoma. Routine pathological examination of these surgical specimens showed typical seminoma in 20 cases. Eighteen cases showed obvious TIL and immunohistological staining on frozen specimens was performed in 12. TIL in seminomas were predominantly T-cells but B-cells were also identified. T-cells were distributed diffusely with predominance of the CD 8+ phenotype judged semiquantitatively. In contrast to the distribution of T-cells, B-cells tended to accumulate and occasionally formed lymphoid follicles. In such follicles the phenotypic pattern of B-cell antigens was comparable with secondary lymphoid follicles in lymphoid organs. There is an immunologically complex response to seminoma by the host with a predominant infiltration of cytotoxic/ suppressor T-cells and functional maturation of B-cells.     

Spontaneous secretion of thyroid autoantibodies by cultured peripheral blood lymphocytes from patients with Hashimoto''s thyroiditis detected by micro-ELISA techniques

The spontaneous in vitro production of anti-thyroglobulin (aTg) and anti-microsomal (aM) antibodies by mononuclear cells (MNC) from patients with Hashimoto's thyroiditis (HT) was analysed by an ELISA detection system. MNC from 35 HT patients spontaneously produced detectable levels of both autoantibodies in vitro (i.e., without mitogenic or antigenic stimulation). aTg was quantified using a reference aTg IgG standard and ranged from 55 to 9,000 ng aTg. Specificity of aTg by ELISA was assessed using heterologous Tg antigen (Ag). Microsomal Ag obtained by gel filtration was far less contaminated with Tg than the ultracentrifugation pellet. Specificity of aM ELISA was assessed using insulinoma membrane as unrelated Ag and by blocking aM detection only with microsomal Ag. aM levels in the 35 supernatants ranged from 0.1 to 1.12 OD. A direct correlation was found between aM serum titres detected by haemagglutination and in vitro aM spontaneous production, but not for aTg. This lack of correlation for aTg might have biological relevance. Tg restimulation in vitro enhanced aTg production in only four out of 18 cases, of which only one was significant. This system provides a tool for studies of the immunoregulation of thyroid autoantibody formation in vitro.     

Hashimoto''s thyroiditis manifesting monoclonal lymphocytic infiltration.

Hashimoto's thyroiditis (HT) and lymphoma are sometimes difficult to distinguish between. Moreover, lymphoma sometimes develops in a thyroid gland from pre-existing HT. Open- or large-needle biopsy usually distinguishes between them; the specimen may be examined histologically and subjected to immunohistochemistry. Another possible method of examination is fine-needle aspiration biopsy (FNAB). The cells obtained may be evaluated cytologically, and subjected to flow cytometry, using various antibodies. In this study, anti-kappa and anti-lambda antibodies are especially important, as a gross predominance of kappa or lambda B lymphocytes infiltrating the thyroid is evidence for a B cell monoclone. In this study, 15 patients were selected because of their rapidly growing goitres. They all underwent FNAB. Five had cytology typical of HT, and no evidence of monoclonality on flow cytometry. They were diagnosed as HT without further histopathology. The remaining 10 patients had cytology suspected of lymphoma, or evidence of monoclonality on flow cytometry, or both. These patients underwent open- or large-needle biopsy. Only three of them were diagnosed histopathologically as lymphoma; the other seven were diagnosed histopathologically as HT, making 12 cases of HT in all. Five of these 12 cases, and one of the three cases of lymphoma showed flow cytometrical evidence of monoclonality; thus evidence of monoclonality from FNAB, while interesting, does not necessarily serve to differentiate between HT and lymphoma. Furthermore, the immunohistochemical assessment of monoclonality did not correlate with the flow cytometrical assessment. Follow-up evidence will be required to discover whether those patients with a B cell monoclone in their HT are the ones who develop a lymphoma.     

In vivo activated cytotoxic T cells in the thyroid infiltrate of patients with Hashimoto''s thyroiditis.

High proportions of T8+ cells with inverted T4/T8 ratio were found in freshly isolated thyroid lymphocytes from patients with Hashimoto's thyroiditis. In addition, about one third of thyroid infiltrating cells expressed the TAC antigen, whereas in patient peripheral blood (PB) or normal lymphocytes from PB or lymphoid organs the percentage of TAC-positive cells was consistently lower than 10%. Following negative selection with OKT4 or OKT8 monoclonal antibodies and complement, TAC+ T cells were enriched in the T8+ cell population. Thyroid infiltrating T cells from two patients underwent two different cloning procedures. In the first, single T cells were initially activated with phytohaemagglutinin (PHA) and interleukin 2 (IL-2), in the other with recombinant IL-2 (rIL-2) alone. The majority of T cell clones obtained by initial PHA-stimulation (55-65%) had the T8+ phenotype, but the frequency of T8+ clones obtained by stimulating T cells with rIL-2 alone was even higher (78 & 71%, respectively). The majority of T8+ clones elicited by PHA (35/37 & 36/38) and all the T8+ clones (36/36 & 22/22) obtained from thyroid infiltrates with initial stimulation by rIL-2 displayed cytolytic activity. Most of cytolytic T8+ clones obtained from thyroid infiltrates with both cloning procedures, displayed NK activity against human K562 and MOLT-4 target cells, but not against a NK-resistant target, such as Raji cells. These data suggest that in Hashimoto's disease a considerable proportion of thyroid infiltrating T cells are in vivo activated T8+ cytolytic T cells with NK activity, which may be of importance in determining or maintaining the tissue damage of the target gland.     

Interphase ribosomal RNA cistron staining in thyroid epithelial cells in Grave''s disease, Hashimoto''s thyroiditis and benign and malignant tumours of the thyroid gland

Aim—To evaluate the expression of ribosomal cistrons in human thyroid epithelial cells (TECs) of patients with Grave's disease, Hashimoto's thyroiditis and benign and malignant tumours of the thyroid gland.     

Peripheral blood T and B lymphocytes in patients with thyrotoxicosis and Hashimoto''s thyroiditis and in normal subjects

Lymphocytes were separated from the peripheral blood of three groups of subjects (normal controls, untreated thyrotoxicosis and confirmed Hashimoto's thyroiditis) by two separation methods: dextran sedimentation and Ficoll–Triosil gradient centrifugation. It has been shown that there is a selective loss of T lymphocytes (as measured by sheep red cell rosettes) with relative enrichment of B lymphocytes (as measured by surface immunoglobulins) in the Ficoll–Triosil-separated suspensions. This distortion of the T/B ratio was seen to a similar extent in each of the three groups of subjects. Furthermore, the mean percentage T and B lymphocytes of both patient groups were not significantly different from those of the controls when separated by the same method. Optimal E-rosette formation occurred after prolonged incubation at 4°C and in the absence of serum. Direct counts using Toluidine Blue were superior to indirect counts with unstained rosette suspensions.     

Plasmacytoma and follicular lymphoma in a case of Hashimoto''s thyroiditis

Monoclonal gammopathy (IgG, lambda) in a 37-year-old man with Hashimoto's thyroiditis, was markedly decreased after thyroidectomy. Histological examination of the thyroid showed large lymphoid follicles surrounded by a massive proliferation of plasma cells. Immunological studies revealed that interfollicular plasma cells stained monotypically for lambda chain, findings in keeping with a diagnosis of plasmacytoma of the thyroid. In addition, there was kappa monotypic staining of the lymphoid follicles and absence of tangible body macrophages, indicating the presence of a follicular lymphoma derived from a separate cell line from the plasmacytoma.     

Characterization of lymphoid cells in the thyroid of patients with Graves'' disease.

The distribution and function of lymphoid cells has been investigated in thyroid glands obtained at operation from 16 patients with Graves' disease (GD) using a peroxidase technique to enumerate total T and B lymphocytes as well as helper and suppressor T cell subsets in tissue sections. A spectrum of lymphocytic infiltration was observed and the increase from minimal numbers of immune cells in some GD thyroids to focal thyroiditis in others appeared to be due to a rise in all the lymphoid cell types analysed and was not the result of major change in any one lymphoid compartment. T cells were diffusely distributed whereas B cells tended to occur in aggregates. Small numbers of OKT6+ cells (possibly antigen presenting cells) were observed although these were less numerous than in lymphoid organs such as tonsil. Lymphoid cell suspensions prepared from the thyroid tissue of five of seven GD individuals treated pre-operatively with propranolol synthesized thyroid autoantibodies spontaneously in culture and this synthesis was decreased in the presence of pokeweed mitogen. Since the OKT8+ T cell subset has been shown to suppress immunoglobulin production by lymphocyte cultures containing mitogen, it appears that the suppressor T cells, which are readily demonstrable in GD thyroid sections, are functional. It seems unlikely, therefore, that a defect in this type of suppression is responsible for the initiation or perpetuation of the autoimmune response to thyroid antigens in GD.