Non-Hodgkin's lymphoma of the thyroid: a retrospective review of all patients diagnosed in Nottinghamshire from 1973 to 1992.

The pathological and clinical features were reviewed of all primary non-Hodgkin's lymphomas (NHL) of the thyroid gland diagnosed between 1973 and 1992 in the population (1.1 million) served by the Nottingham and North Nottinghamshire Health Authorities. Of the 43 patients with histologically proven NHL, three had low grade mucosa associated lymphoid tissue (MALT) lymphomas (Stage IEA, 2; Stage IIEA, 1), 35 had intermediate or high grade lymphomas, Stage IEA or IIEA (intermediate MALT, 2; high grade MALT, 14; B-cell diffuse centroblastic, 17; anaplastic large cell (Ki-1) of null cell type, 1; high grade unclassifiable, 1), and one had unclassifiable NHL Stage IIEA. One patient had Stage IIIEA disease (high grade MALT) and three had stage IVA disease (high grade MALT, 2; B-cell diffuse centroblastic, 1). The median age was 68 years (range 45-86) with a female: male ratio of 6:1. For the 35 patients with intermediate or high grade thyroid NHL (Stages IEA and IIEA) the 5- and 10-year cause specific survival was 60%. The 21 patients treated between 1985 and 1992 initially with chemotherapy (except stage IEA (< 5 cm diameter) had a 5-year cause specific survival of 69% (95% CI 48-90) compared with 46% (95% CI 19-73) for the 14 patients treated between 1973 and 1984 with initial radiotherapy (Chi 2 = 1.62). The survival of those patients with intermediate or high grade MALT lymphomas was not significantly greater than of those patients with B-cell diffuse centroblastic NHL.

Diagnostic approaches to parotid swellings.

Although the mainstay assessment of parotid swellings remains clinical examination, cytology, computed tomography and magnetic resonance imaging may give valuable preoperative information on both the likely histology and the location of the individual swelling. This is important for therapeutic and prognostic reasons.

Production and characterisation of a human monoclonal thyroid peroxidase autoantibody.

A human-mouse hybridoma has been produced by fusion of Hashimoto thyroid lymphocytes with the mouse myeloma line X63-Ag8.653. The cloned hybridoma secreted 2.5 micrograms per 10(6) cells per day of an IgG kappa thyroid peroxidase (TPO) autoantibody (2G4) with high affinity (2.5 x 10(9) molar-1) and specificity for human TPO. 2G4 did not react with lactoperoxidase, horseradish peroxidase or human myeloperoxidase or with porcine TPO or with human thyroglobulin. Plastic tubes coated with 2G4 bound about 50% of 125I-labelled human TPO added and the binding was inhibited by IgGs prepared from 18/18 TPO autoantibody-positive sera. This indicated that all 18 sera contained autoantibodies which recognised the same (or closely related) epitope as 2G4. Plastic tubes coated with IgGs from different TPO autoantibody-positive patient sera also bound 125I-labelled TPO but inhibition by 2G4 in this system was not complete. This suggested that the sera contained at least 2 types of TPO autoantibodies, with only one type of autoantibody reactive with the same epitope as 2G4.

Severe combined immunodeficient (SCID) mice: a model for investigating human thyroid autoantibody synthesis.

We have studied the ability of lymphocytes from the blood, thyroid and lymph nodes of patients with autoimmune thyroid disease (AITD) to produce autoantibodies to thyroglobulin (Tg) and/or thyroid peroxidase (TPO) in SCID mice. Human IgG class Tg and/or TPO antibodies were detectable in plasma from SCID mice 7 days after transfer of 15-25 x 10(6) cells/mouse and the highest levels were recorded 2-3 weeks later. In contrast, Tg and/or TPO antibodies were undetectable in recipients of lymphocytes from thyroid antibody negative controls. AITD thyroid lymphocytes produced the most antibody in recipient mice and lower levels were observed in recipients of AITD blood and lymph node lymphocytes. The amounts of Tg and/or TPO antibody detected were in accordance with the ability of thyroid and lymph node lymphocytes to secrete these autoantibodies spontaneously in culture (indicating the presence of cells activated in the patient) and with the capacity of blood lymphocytes (probably B memory cells) to secrete Tg and/or TPO antibodies in culture in response to pokeweed mitogen. Tg antibodies in plasma from SCID recipients of thyroid lymphocytes were of subclasses IgG1, IgG2 and IgG4 and the proportions closely resembled those of the donor's serum Tg antibodies. Blood lymphocytes transferred to SCID recipients were also able to produce Tg antibodies of subclasses 1, 2 and 4 but the subclass distribution varied between mice and the reason for this is not clear at present. Since SCID mice provide an environment in which B lymphocytes from patients with AITD can be activated without mitogen to secrete thyroid antibodies, this model will provide a powerful system for elucidating the mechanisms regulating the secretion of human antibodies to Tg and TPO.

Human monoclonal thyroglobulin autoantibodies of high affinity. I. Production, characterisation and interaction with murine monoclonal thyroglobulin antibodies.

Four hybridomas secreting human thyroglobulin (Tg) autoantibodies of different IgG subclasses and light chain types (IgG1 lambda, IgG1 kappa, IgG2 lambda and IgG2 kappa) were obtained by direct fusion of Hashimoto thyroid lymphocytes with the mouse myeloma X63-Ag.653. The autoantibodies were specific for human Tg and the functional affinities were high (only 2.6-3.9 log10 pM Tg required to give 50% inhibition of binding in ELISA). Using thyroid lymphocytes, 4 lines secreting Tg autoantibodies were obtained from 11 fusions compared with 1 line from 32 fusions of Epstein Barr virus infected blood lymphocytes, which emphasises the importance of using lymphocytes derived from a tissue known to be enriched in thyroid autoantibody secreting precursor B cells. These 4 human Tg autoantibodies, as well as an IgG2 lambda Tg antibody previously derived from Hashimoto blood B cells and an IgG4 kappa monoclonal Tg antibody present in a Hashimoto serum, were used in attempts to probe the interaction between human Tg autoantibodies and the Tg molecule (2 polypeptides of 330 KD). The binding to 125-I Tg by 3/7 murine monoclonal antibodies was inhibited (36-78%) by an IgG2 lambda and an IgG4 kappa human monoclonal Tg autoantibody, indicating an overlap between the epitopes recognised by these 3 murine monoclonal Tg antibodies and 2 monoclonal human Tg autoantibodies. None of the human Tg autoantibodies (or the murine monoclonal Tg antibodies) bound to Tg denatured by reduction and alkylation. Although the number of observations is limited, our study demonstrates that high affinity human monoclonal Tg autoantibodies, like polyclonal serum Tg autoantibodies, recognise non-linear B cell epitopes on conformationally intact human Tg.

Thyroid autoantigens and human T cell responses.

We investigated the ability of T cells from patients with Hashimoto's thyroiditis and with Graves' disease as well as control donors to proliferate in response to thyroid peroxidase (TPO) and thyroglobulin using (i) lymphoid cells from different lymphoid organs; (ii) unfractionated or CD8- depleted lymphoid suspensions or T cells + autologous low density cells (LDC); (iii) 200-microliters well cultures and 20-microliters hanging-drop microcultures; and (iv) intact TPO and thyroglobulin, denatured thyroglobulin and 12 synthetic peptides predicted on the basis of the amino acid sequence of TPO to be T cell epitopes. In 200-microliters well cultures, proliferative responses (assessed in terms of 3H-thymidine uptake) to intact TPO or thyroglobulin, digested thyroglobulin or synthetic TPO peptides were not significantly different in unfractionated or CD8-depleted lymphoid suspensions from blood, thyroid or lymph nodes of TPO/thyroglobulin autoantibody-positive patients, autoantibody-negative patients or control donors. In contrast, blood T cells from some high titre patients with Hashimoto's thyroiditis (but not from healthy individuals) proliferated in response to intact thyroglobulin or TPO presented by autologous LDC in hanging-drop microcultures. Heat denatured thyroglobulin (with which thyroglobulin autoantibodies do not interact) did not stimulate proliferation and this observation, together with the ability of T cells from some patients to respond to intact thyroglobulin in the absence of LDC, indicated that thyroglobulin-specific B cells may be involved in antigen presentation. As we were unable to demonstrate proliferation by blood T cells + LDC from all thyroglobulin antibody-positive patients with Hashimoto's thyroiditis, our studies suggest that the presence of sufficient precursor T cells, as well as the number and type of antigen-presenting cells, are critical for T cell proliferative responses to human TPO and thyroglobulin.

Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture.

In autoimmune thyroid disease lymphoid cells infiltrating the thyroid gland occur in conspicuous aggregates or as a diffusely distributed population invading the thyroid follicles. Consequently cytokines secreted by activated T cells or macrophages could influence neighbouring thyroid cells as well as other lymphocytes. We have investigated this possibility using recombinant cytokines. Thyroid cell survival was assessed in terms of mitochondrial dehydrogenase activity in monolayers exposed to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1 (IL-1 alpha and beta) and interleukin-2 (IL-2) in the presence or absence of thyroid-stimulating hormone (TSH). Neither TNF-alpha nor IL-2 affected thyroid cell survival, IFN-gamma was usually inhibitory and IL-1 alpha slightly enhanced cell survival in some experiments. However, the effects were small and variable and were not enhanced by potentially synergistic combinations of cytokines, longer periods of exposure, or different culture conditions. In contrast, IFN-gamma, IL-2 and TNF-alpha inhibited the ability of thyroid lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis to synthesize autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg). Comparison of lymphoid populations isolated by digestion and/or mechanical disaggregation indicated that a population of activated B cells, plasma cells and T cells, intimately associated with thyroid cells since they could only be extracted by digestion, was influenced by cytokines. Our studies suggest that in addition to its well-recognized ability to induce MHC class II antigens on thyroid cells, IFN-gamma may inhibit thyroid cell proliferation and TNF-alpha, IFN-gamma and IL-2 may down-regulate thyroid autoantibody synthesis.

Potential role of PHA in producing human monoclonal thyroid autoantibodies of different subclasses.

A human monoclonal autoantibody to thyroglobulin (Tg) of subclass IgG2 was developed by fusing a mouse myeloma with Tg antibody secreting Epstein-Barr virus (EBV)-infected B lymphocytes from a Hashimoto patient. Subsequent studies showed that EBV-infected B lymphocytes from this patient synthesized IgG2 Tg antibody while unfractionated blood lymphocytes cultured with pokeweed mitogen secreted IgG1, IgG2, and IgG4 Tg antibodies in amounts proportional to those present in the patient's serum. To investigate this discrepancy further, we cultured EBV-infected lymphocytes from blood, lymph nodes, and thyroid tissue in medium alone and with increasing concentrations of PHA. In individuals with thyroid autoantibodies predominantly of subclass IgG1, PHA enhanced the levels of total Tg antibody synthesis without affecting the IgG subclass distribution. However, in patients with serum autoantibodies of subclasses IgG1, 2, and 4, the increased levels of total Tg antibody synthesis were associated with increased amounts of thyroid autoantibodies of all of these subclasses; in some instances IgG1 and IgG4 autoantibodies were only synthesized in cultures containing PHA. These observations suggest that addition of the T-cell mitogen PHA to cultures of EBV-infected lymphocytes may ensure activation of B-cell precursors committed to synthesizing the IgG subclasses characteristic of serum antibody in the lymphocyte donor. Since Tg antibodies of subclasses IgG2 and IgG4 recognize different epitopes on Tg, the ability to produce human monoclonal antibodies of different IgG subclasses may simultaneously ensure the development of antibodies to different epitopes on the same antigen.

The thyroid microenvironment in autoimmune thyroid disease: effects of TSH and lymphokines on thyroid lymphocytes and thyroid cells.

Thyroid lymphocytes synthesize thyroid autoantibodies in close proximity to thyroid cells and consequently soluble mediators such as TSH and interleukins (IL) 1 and 2 may have unforeseen effects on lymphocytes and thyrocytes, respectively. Investigations of thyroid autoantibody synthesis by thyroid lymphocytes in vitro showed that TSH did not affect microsomal (Mic) antibody production, but thyroglobulin (Tg) antibody synthesis was decreased, probably as a result of complexing between Tg antibody and Tg secreted by small numbers of thyrocytes in the cell suspension. IL-1 and IL-2 partially mimicked the inhibitory effects on spontaneous autoantibody synthesis induced by Pokeweed mitogen (PWM) in cultures of thyroid lymphocytes. This inhibition may require a number of soluble mediators released by T cells in response to the mitogen; however, depletion studies indicated that the cell type responsible for PWM inhibition is unlikely to be a suppressor T cell and may be an NK cell. IL-1 and IL-2 had little effect on the viability of thyrocyte monolayers in an 18 h assay, but antibody dependent cells cytotoxicity (ADCC) using blood lymphocytes and thyroid autoantibody positive sera was demonstrated; further, the cytotoxicity appeared to be due to Mic antibodies. It is possible that IL-1 and/or IL-2 (as well as other cytokines) may affect thyroid cells after longer periods of exposure, either by altering them functionally or by direct damage. However, assuming that NK cells are present in sufficient numbers in the gland, ADCC could play a major role in the development of hypothyroidism in Hashimoto's disease.

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.

TSH receptor antibody synthesis by thyroid lymphocytes.

Several indirect observations have indicated that lymphocyte in the thyroid may be an important site of TSH receptor antibody synthesis in Graves' disease and we now describe an investigation of this possibility using improved lymphocyte isolation and TSH receptor antibody assay procedures. Our studies demonstrate that thyroid lymphocytes spontaneously produce TSH receptor antibody in culture. Furthermore, experiments with mitogen tend to suggest that these cells, in contrast to lymphocytes from lymph nodes draining the thyroid, are part of an active immune response to the TSH receptor.

A prospective study of thyroid ultrasound scan in the clinically solitary thyroid nodule.

Thyroid ultrasound differentiates solid from cystic lesions, solitary nodules from multinodular and diffuse enlargement, and extrathyroidal lesions. Two hundred consecutive patients with a clinically solitary thyroid nodule were investigated by ultrasound examination, thyroid function test, and thyroid auto-antibodies. Patients with confirmed solitary solid, or mixed solid and cystic nodules underwent surgery as well as those with cysts, multinodular or diffuse goitres with pressure symptoms, recurrent haemorrhage or relapsing hyperthyroidism. Comparison between the ultrasound and ultimate pathological findings in the 101 patients who underwent surgery showed that they were in agreement in 96 cases. The remaining 99 patients with cystic, multinodular or diffuse lesions have been followed up for a mean of two years. Nearly 50% of patients with a clinically solitary thyroid nodule have avoided surgery.

The effect of carbimazole on thyroid autoantibody synthesis by thyroid lymphocytes.

Thyroid autoantibody synthesis was investigated in cultures of lymphocytes isolated from several sources, including thyroid and lymph nodes from patients with hyperthyroid Graves' disease treated preoperatively with carbimazole or propranolol. The ability of thyroid lymphocytes to secrete immunoglobulins, including thyroid microsomal or thyroglobulin autoantibodies, was markedly reduced in lymphocyte suspensions obtained from patients treated with carbimazole compared with suspensions from patients treated with propranolol. This effect (which was greater in individuals treated with carbimazole for longer periods) was attributable to a significant reduction in the number of viable lymphocytes present after the 14-day culture interval. In contrast, the type of preoperative therapy had little effect on cultures of lymphocytes obtained from lymph nodes draining the thyroid. Although it is not yet clear whether carbimazole exerts its effects in vivo by direct immunosuppression or indirectly by altering the thyroid microenvironment, our observations indicate that the fall in serum levels of thyroid autoantibodies that occurs during carbimazole therapy is related to an effect of the drug on lymphocytes within the thyroid.

Thyroid autoantibody synthesis by lymphocytes from different lymphoid organs: fractionation of B cells on density gradients.

Lymphocytes from thymus, blood, lymph nodes and thyroid tissue of patients with autoimmune thyroid disease have been assessed for their ability to synthesize thyroid autoantibodies spontaneously or following stimulation by Pokeweed mitogen (PWM). Blood and thymic lymphocytes synthesized IgG and microsomal or thyroglobulin antibodies of IgG class in response to PWM (and were therefore probably B-memory cells), while thyroid lymphocytes frequently secreted autoantibodies spontaneously. Lymph node lymphocytes resembled blood lymphocytes in terms of increased production of IgG in response to PWM; however, spontaneous secretion of thyroid autoantibodies was observed in some lymph node suspensions, and the magnitude of the increment in thyroid autoantibodies synthesized in response to PWM was lower than that observed for blood lymphocytes. Fractionation of B-cell enriched populations on density gradients and subsequent incubation of the fractions with T cells and PWM demonstrated that, whereas blood B cells capable of synthesizing autoantibody were found in both medium and low density fractions, lymph node precursors of thyroid autoantibody-secreting cells were associated almost exclusively with the light fractions. The presence in lymph nodes of small numbers of low density B cells, compared with a much higher proportion of the heterogeneous population capable of secreting IgG, could account for the discrepancy between the IgG and autoantibody response to PWM. Further, it seems likely that the density difference in the autoantibody precursor population of lymph nodes and blood is related to the difference in the state of activation of B cells in these lymphoid organs.

Functional analysis of T and B cells from blood and thyroid tissue in Hashimoto's disease.

B lymphocytes from Hashimoto blood and thyroid tissue have been cultured with autologous T cells from thyroid/blood to assess their ability to synthesise IgG and thyroid autoantibody. Thyroid B cells were able to synthesize microsomal antibody spontaneously in the absence of T cells or pokeweed mitogen (PWM) and this synthesis was increased in the presence of thyroid T cells without PWM or with blood T cells with PWM. In contrast, blood B cells did not secrete thyroid autoantibody spontaneously but could be induced to do so by thyroid T cells spontaneously or by blood T cells with PWM. Despite these differences, lymphocytes from blood and thyroid tissue secreted microsomal or thyroglobulin antibodies in culture which were similar in terms of the IgG subclass distribution. It would appear, therefore, that although the state of activation of B and T cells is different in blood and thyroid tissue, the precursors of thyroid autoantibody secreting cells are the same.

Mediastinal parathyroid adenoma: a new method of localization.

201Tl-thallous chloride/99mTc sodium pertechnetate subtraction scanning is a simple and accurate means to demonstrate enlarged parathyroid glands. We report a case in which the intra-operative location of a mediastinal adenoma was greatly facilitated by using a probe radiation detector following intravenous injection of 201Tl-thallous chloride. We believe this is the first reported use of this technique in parathyroid surgery.

The influence of preoperative drug treatment on the histological appearances and in vitro 131I uptake of human hyperplastic thyroid gland.

The specimen of thyroid resected at partial thyroidectomy from 103 patients with primary thyrotoxicosis was studied with histometric and organ culture techniques. Twenty-seven patients had been prepared for operation with propranolol and seventy-six with carbimazole: all received Lugol's iodine for 10 days before operation. The resected specimen and deduced total thyroid weight was greater in the patients prepared with carbimazole. There was no absolute qualitative histopathological difference in the appearance of the glands of the two groups of patients, but histometry showed that the volume percentage of colloid and total gland colloid weight was significantly greater in the patients prepared with carbimazole: the volume percentage of epithelial cells and the total gland epithelial cell weight was similar in the two groups. The iodide concentrating capacity per g wet weight thyroid tissue or per unit volume of colloid did not differ significantly between the two groups. However, the iodide concentration capacity per unit volume of epithelial cells was significantly higher in the carbimazole prepared patients than in those prepared with propranolol.

Propranolol in the surgical management of thyrotoxicosis.

Forty-nine thyrotoxic patients prepared for partial thyroidectomy with the beta-adrenoceptor blocking drug, propranolol, and iodine are compared with 42 patients prepared with carbimazole and iodine. The age and sex distribution of the two groups were comparable, but patients with obstructive airways disease and possible cardiac insufficiency were excluded from preparation with propranolol. The mean duration of preoperative treatment with propranolol was 40 days, compared with 89 days for carbimazole. Propranolol treated patients had lower pulse rates before and after operation. The serum PB(127)I values immediately before and after operation were higher in the propranolol group than in the carbimazole group, but were the same in both groups one and four months after operation. The incidence of hypothyroidism at one year after operation was 30% in the carbimazole prepared patients and 31% in the propranolol patients. Serum calcium levels were higher in the propranolol group at the time of operation. No adverse effects from the use of propranolol and at operation the thyroid gland prepared with propranolol was firmer, less friable, more easily mobilised and less likely to bleed than the gland prepared with carbimazole. There is, consequently, less risk of damage to the parathyroid glands and recurrent laryngeal nerves. However, the basal metabolic rate remains high on propranolol therapy and very careful supervision is advised.