Modulation of Fas-mediated apoptosis of human thyroid epithelial cells by IgG from patients with Graves' disease (GD) and idiopathic myxoedema

The expression of two autoimmune thyroid diseases, GD and idiopathic myxoedema, is associated with antibodies to the thyroid-stimulating hormone (TSH) receptor. Thyroid stimulating antibodies (TSAb) in GD are TSH agonists and cause hyperthyroidism as well as goitre, whereas thyroid stimulation blocking antibodies (TSBAb) in idiopathic myxoedema are TSH antagonists and cause hypothyroidism and thyroid atrophy. We investigated the effect of antibodies to TSH receptor on Fas-mediated apoptosis of thyroid epithelial cells (thyrocytes). Human IgG was isolated from healthy donors, patients with GD and idiopathic myxoedema. Human thyrocytes were obtained from surgical specimens. Thyrocytes were cultured in the presence or absence of human IgG with or without interferon-gamma (IFN-γ) or IL-1β for a specified time. After incubation, we examined the level of cAMP in cultured supernatants and both Fas and Bcl-2 expression on thyrocytes. In addition, we examined anti-Fas-mediated apoptosis of thyrocytes. Fas expression on thyrocytes was significantly down-regulated by Graves' IgG and TSH, although idiopathic myxoedema IgG did not affect Fas expression on thyrocytes. Idiopathic myxoedema IgG abrogated the effect of TSH on both cAMP production and inhibition of Fas expression on thyrocytes. Treatment of thyrocytes with IL-1β or IFN-γ caused a marked augmentation of Fas expression on thyrocytes. The increase of Fas expression of thyrocytes induced by IL-1β or IFN-γ was significantly suppressed in the presence of TSH or Graves' IgG. Anti-Fas-induced apoptosis of thyrocytes was observed in thyrocytes treated with IL-1β or IFN-γ, but was markedly inhibited in the presence of TSH or Graves' IgG. Furthermore, idiopathic myxoedema IgG abrogated most of the inhibitory effect of TSH on Fas-mediated apoptosis of thyrocytes treated with IL-1β or IFN-γ. Bcl-2 expression of thyrocytes did not change after stimulation with TSH, Graves' IgG, idiopathic myxoedema IgG, IL-1β or IFN-γ. These results suggest that TSAb found in Graves' patients may be potentially involved in the development of goitre by inhibition of Fas-mediated apoptosis of thyrocytes. In addition, TSBAb inhibit the action of TSH and increase the sensitivity toward Fas-mediated apoptosis of thyrocytes, inducing thyroid atrophy seen in patients with idiopathic myxoedema.     

Anti-thyroid drugs and lymphocyte function: II. The in vivo effect on blastogenesis and suppressor cell activity in graves' disease

The proliferative response (PR) of T lymphocytes in PHA stimulated cultures (5 μg/ml and 0·5 μg/ml; 72 hr) was used to assess the suppressive capabilities of peripheral blood mononuclear cells (PBMC) in the thyrotoxic phase of Graves' disease and their possible modification by propylthiouracil (PTU) and methimazole (MMI) treatment. Graves' patients had 50% higher PR than controls. Treatment with PTU (n=6) for 9·5 weeks (mean) or MMI (n=6) for 18 weeks (mean) resulted in continuous decrease in PR, starting after 3 weeks and down to control values and plateau at 7-10 weeks. This decrease correlated with the decline in plasma thyroxine (T4) levels which had already dropped by 3 weeks. Grouped according to thyroid functional state PR was significantly decreased only in the euthyroid state. Suppressor cell function, expressed as suppressor removal index (PR of PBMC pre-incubated for 24 hr/PR of fresh PBMC), was significantly lower in Graves' patients compared to controls and reached above normal values under PTU treatment: 0·98±0·16, 1·39±0·09 and 1·94±0·19 (mean±s.e.m.) respectively. A direct suppressive effect of PTU in culture, observed in normal subjects, did not exist in untreated patients and evolved under MMI treatment to above normal levels. The cell-mediated PTU effect, exercised by PBMC pre-incubated with PTU on autologous cells pre-incubated in medium alone, increased under PTU treatment to above normal levels. Both this cell-mediated suppressive effect and augmented PR of pre-incubated cells were already significantly increased after 3 weeks of PTU treatment, when all patients were still thyrotoxic. We conclude therefore that PBMC of patients in the untreated, thyrotoxic phase of Graves' disease are deficient in an active cell-mediated suppressive function, a deficiency corrected—with compensatory overshoot—during anti-thyroid drug treatment.     

Serum immunoglobulin levels in thyroid disease

Serum levels of IgG, IgA and IgM were assayed by radial immunodiffusion in 261 patients with eight categories of thyroid disease. These composed eighty-three patients with a first episode of untreated active Graves' disease (toxic diffuse goitre), ten with relapsed Graves' disease, seventeen with thyrotoxicosis due to a multinodular goitre, forty-nine with Hashimoto's thyroiditis, twenty-eight with primary (non-goitrous) myxoedema, forty with non-toxic goitre, eighteen with an adenoma and sixteen with euthyroid ophthalmopathy.

Eighteen (21·7%) patients with a first episode of Graves' disease had abnormally high IgG levels whereas eight (80%) of those who had relapsed after a course of Carbimazole had high levels. Those Graves' disease patients with raised IgG levels had a significantly higher 24-hr radioiodine uptake than those with normal levels. Eight (16·3%) patients with Hashimoto's thyroiditis had abnormally high levels of IgG associated with a higher incidence of thyroglobulin autoantibodies. Very few (<6%) patients with primary myxoedema, non-toxic goitre and adenoma had abnormal levels. Euthyroid patients with ophthalmopathy had a significantly lower mean IgG level than the corresponding mean level found in the group with active Graves' disease.

However, despite the differences between groups described above, there were no significant differences of mean IgG, IgA and IgM levels in seven of the eight groups when compared with normal subjects. Only the group with relapsed Graves' disease had a significantly higher mean IgG. None of the patients studied had abnormal IgM or IgA levels.

     

Metabolism of radio-iodinated IgG in patients with abnormal serum IgG levels. I. Hypergamma-globulinaemia

Metabolic turnover studies were performed with radio-iodinated IgG in twelve patients with a serum IgG level greater than 1600 mg/100 ml (six with monoclonal gammopathy and six with a polyclonal increase in IgG associated with liver disease). The six patients with an IgG monoclonal protein comprised four multiple myeloma, one benign monoclonal gammopathy and one biclonal gammopathy presenting as Waldenström's macroglobulinaemia. The six patients with liver disease comprised two patients with cirrhosis, two with infective hepatitis and two with chronic active hepatitis. The injected IgG was either autologous normal IgG (five cases), autologous monoclonal IgG (five cases), homologous normal IgG (one case) or therapeutic intravenous HGG (two cases).

The plasma volume was increased in six patients; the plasma IgG pool in nine; and the total body IgG pool in seven. The plasma T½ was normal in one patient with monoclonal and one patient with polyclonal gammopathy but shortened in the other ten studies with mean values of 11·3 and 11·0 days in monoclonal and polyclonal gammopathy respectively. The fractional turnover rate was normal in two studies in polyclonal gammopathy and increased in the other ten with mean values of 13·6% per day in both groups of patients. The IgG synthesis rate was significantly increased in all studies except for a reduced synthesis of normal IgG in one patient with multiple myeloma. The mean synthesis rates in monoclonal and polyclonal gammopathy were respectively 6·7 and 4·1 times the mean synthesis rate in normal controls.

The pattern of increased synthesis and increased catabolism in such patients confirms published reports in some diseases and demonstrates a similar pattern in chronic active hepatitis. The findings are consistent with the `concentration-catabolism' effect.

     

The expression of the microsomal/peroxidase autoantigen in human thyroid cells is thyrotrophin-dependent.

In the present report the mechanisms responsible for the expression of the thyroid microsomal autoantigen (M-Ag) were studied in primary cultures of human thyroid cells prepared from Graves' or non-toxic goitres. The indirect immunofluorescence (IFL) technique using human sera positive for anti-microsomal antibody (anti-MAb) was employed to detect M-Ag. Studies were performed to ascertain whether M-Ag recognized by anti-MAb could be identified with thyroid peroxidase (TPO). Preabsorption experiments showed that, similarly to solubilized thyroid microsomes, purified human TPO abolished the binding of anti-MAb to thyrocytes, while no inhibition was obtained with control human tissues. The identity of M-Ag and TPO was also demonstrated using a double layer IFL technique which allowed a simultaneous staining of the antigen(s) recognized by anti-MAb and by a monoclonal anti-TPO antibody. After 5-15 days of TSH withdrawal from the culture medium the M/TPO-Ag disappeared from the surface and the cytoplasm of human thyroid cells. Readdition of TSH (0.1-100 mU/ml) to cells lacking M/TPO-Ag elicited its reappearance within 48-72 h. This effect of TSH was prevented by 10 microM cycloheximide but not by methimazole (0.1-2 mM). Two stimulators of the adenylate cyclase-cAMP system, cholera toxin and forskolin, and 8-bromo-cAMP mimicked TSH in inducing M/TPO-Ag. Thyroid stimulating antibody (TSAb) of Graves' disease also reproduced the effect of TSH on M/TPO-Ag reexpression in human thyroid cells. By contrast, epidermal growth factor, oestradiol or NaI were ineffective in inducing M/TPO-Ag. The present data indicate that: (i) the expression of M/TPO-AG in human thyroid cells is dependent on TSH stimulation, through pathways which involve cAMP production and protein synthesis, (ii) TSAb reproduces this effect of TSH; (iii) oestradiol and NaI have no direct influence on the expression of M/TPO-Ag.     

Expression of HLA-DR antigens by thyroid cells: the effect of Graves' IgG

We have investigated factors which influence HLA-DR expression on thyroid cells. While bTSH (100 mU/ml) did not enhance HLA-DR expression, it increased when brought about by IFN-gamma. Graves' IgG showed a dose-dependent (0.1-2 mg/ml) increase in DR expression and at a concentration of 2 mg/ml prolonged the time for which DR was expressed. The pathway of DR induction by Graves' IgG apparently differs from that by IFN-gamma. The humoral response in Graves' disease, by inducing DR expression, may be instrumental in propagating thyroid specific autoimmunity.     

Quantitative determination of hepatitis B antigen by means of radial immunodiffusion

The method of radial immunodiffusion for measurement of hepatitis B antigen (HB ag) levels in sera of twenty and urines of three HB ag positive patients is described. In patients suffering from acute hepatitis, the level of HB ag in serum was 0·15–2·12 mg/ml (mean = 1·08±SD 0·69 mg/ml). In patients suffering from chronic hepatitis the level of HB ag in serum was 0·36–3·61 mg/ml (mean 2·56±SD 1·13 mg/ml). The highest levels of HB ag in serum, 2·62–5·12 mg/ml (mean 3·91±SD 0·90 mg/ml) were observed in symptomless HB ag carriers.

The amounts of HB ag excreted in urine, found in two chronic hepatitis patients and one HB ag carrier, were 13·2 mg, 46 mg and 82 mg/1000 ml of urine respectively.

     

Distribution of chylomicrons and albumin in dog kidney

1. Under specified experimental conditions the distribution space of labelled chylomicrons in the kidney was 13·8 ± 0·9 ml./100 g. tissue. The assumption is supported that this provides a measure for the quantity of intravascular plasma constituents.

2. Values for red blood cells and albumin distribution spaces were 5·2 ± 0·6 and 20·2 ± 1·0 ml./100 g tissue, respectively, in the whole kidney. The ratio of tissue haematocrit over simultaneous arterial haematocrit averaged 0·56. The extravascular albumin fraction amounted to about 31·0% of the total albumin in the whole kidney.

3. A statistically significant correlation was demonstrated between osmotic urine/plasma (U/P) ratios (within the approximate limits of 0·6-1·8) and quantities of extravascular albumin in the medulla.

     

Renal contribution to thoracic duct lymph in dogs

1. The renal contribution to thoracic duct lymph was measured in seventeen anaesthetized fasting dogs by measurement of thoracic duct flow before and after renal arterial occlusion.

2. In eight experiments it was shown that thoracic duct flow and glomerular filtration rate were not significantly affected by the operation to expose the renal artery.

3. In three experiments occlusion of the renal vein, after release of arterial occlusion, resulted in a sudden increase in thoracic duct flow.

4. In animals infused with isotonic saline or dextrose (approximately 1 ml./min) the average values obtained for control thoracic duct flow, left renal flow and right renal flow were approximately 2·0, 0·7 and 0·3 ml./hr/kg body wt.: in non-infused animals these values were 1·4, 0·4 and 0·35 ml./hr/kg body wt. respectively.

5. Possible reasons for the apparently smaller lymph flow from the right than the left kidney, and the relationship between the renal contribution to thoracic duct flow and actual renal lymph flow are discussed.

6. Close correlation was found between control thoracic duct flow and body weight and between renal lymph flow and control thoracic duct flow.

     

The specific influence of carbon dioxide and carbamate compounds on the buffer power and Bohr effects in human haemoglobin solutions

1. Lymph from the lungs of lambs and sheep was found to enter both the right lymph duct and the thoracic duct. Right lymph duct flow was collected by constructing a venous sac, the venous tributaries of which were ligated but which the right lymph duct entered; thoracic duct flow was collected by cannulating the duct. Lymph from sites other than the lungs was excluded from the collections.

2. Measurements were made of the surface tension characteristics of lung extracts and of the liquid present in foetal lungs. These values were used together with gestational age and crown-rump length to designate the foetal lambs into mature and immature groups.

3. Lymph flow from the lungs averaged 0·99 ml./kg body wt./hr in immature foetal lambs, and 1·81 ml./kg/hr in mature foetal lambs before the start of ventilation. Lymph flow from the lungs of spontaneously delivered new-born lambs (mean age 51 hr) averaged 0·86 ml./kg/hr. In adult ewes right lymph duct flow averaged 0·11 ml./kg/hr and total lung lymph flow was estimated indirectly to be 0·33 ml./kg/hr. Calculated rates of protein flow in lung lymph (flow × protein concentration) were greater in foetal lambs than in adult sheep.

4. Total thoracic duct flow averaged 2·48 ml./kg/hr in immature foetal lambs, 5·30 ml./kg/hr in mature foetal lambs, 3·65 ml./kg/hr in new-born lambs, and 2·92 ml./kg/hr in adult ewes.

5. At the start of ventilation there was an increase in lymph flow from the lungs, which at 15-30 min reached a mean of 6·4 ml./kg/hr in mature lambs and 2·6 ml./kg/hr in immature lambs. At the same time the protein concentration of lymph decreased but the calculated protein flow increased.

6. The lungs of foetal lambs weighed more than the lungs of spontaneously delivered new-born lambs, and the difference could be accounted for by liquid which could be aspirated through the trachea of the foetal lamb. On ventilation of the lungs for 2 hr, without first allowing the escape of any lung liquid, lung weight measurements indicated that about 66% of the lung liquid had been taken up in mature lambs and about 50% in immature lambs.

7. It was concluded that the rate at which lymph is formed in the lungs is greater per kilogram body weight in foetal than in new-born lambs and greater in them than in ewes. The increase in lymph flow at the start of ventilation could account for the removal of about 40% of the liquid present in the lungs of the mature foetus and about 25% of the liquid in the lungs of the immature foetus.

     

Validation of autoradiography for recognition of antigen-binding lymphocytes in blood and lymphoid tissues: quantitation and specificity of binding

Antigen-binding lymphocytes were recognized by their reaction with radioiodine labelled antigens such as flagellin and haemocyanin. Counts varied according to the antigen and species studied. For flagellin, counts in human blood of antigen-binding lymphocytes (mean ± 1 SD per 1000 lymphocytes) were 19·0±3·0, and in foetal thymus 18·2±5·0 and spleen 3·5±0·5. Results depended on contact time of cells with antigen, concentration of antigen, autoradiographic exposure, presence of natural antibody and antibody levels after immunization. Antigen-binding lymphocytes in blood were not antibody-producing cells. The specificity of the antigen-binding reaction was shown by exposing lymphocytes to 0·5 μg of two antigenically distinct flagellins; there was a 67–100% increase in the counts in contrast to the 20–45% increase on doubling the dose (0·5 μg to 1 μg) of flagellin from Salmonella adelaide. Cytophilic antibody as the cause of antigen binding was excluded.

The binding of flagellin to lymphocytes was prevented by anti-human IgM and light chain antisera, but not anti-human IgG sera. The binding of labelled flagellin was prevented by unlabelled flagellin but 100 times more was needed for blood lymphocytes than thymocytes. It is inferred that thymocytes, T cells, have considerably fewer receptors than most β lymphocytes detectable in blood.

Using standardized conditions, radiolabelled antigen binding provides a reproducible, immunologically specific and flexible technique allowing study of the nature and role of antigen-binding cells and cell surface receptors.

     

Circulating complexes in leprosy studied by the platelet aggregation test. The platelet aggregation test and its relation to the rubino test and other sero-immunological parameters in 135 patients with leprosy

Sera from 135 patients with leprosy were tested by the platelet aggregation test (PAT), by the Rubino test and by other sero-immunological assays. PAT positivity (titre≥10) was 53% in the lepromatous subgroups and 5% in the tuberculoid subgroups (P<0·005). The higher PAT titres and Rubino titres clustered significantly (P<0·0005) toward the lepromatous end of the disease spectrum. A statistically significant correlation was found between the PAT and the Rubino titres (0·05>P>0·025). Removal of the effect of the disease spectrum, however, resulted in a partial correlation between the PAT and the Rubino titres that was not significant (P>0·1), suggesting different basic mechanisms for the platelet aggregation (PA) and the Rubino activity of the lepromatous sera. The correlation between the PAT titres and twenty-nine other sero-immunological parameters was calculated, and a highly significant correlation was found between the PAT and the IgG level (P<0·005) and between the PAT and the antistaphylolysin-α titre (P<0·005).

The PA activity in most lepromatous sera studied sedimented in the heavy (>19S) fractions and was inhibitable by IgM rheumatoid factor. It thus fulfilled the criteria for IgG complexes as defined in previous studies with known model Ag/Ab complexes and with sera from patients with immune complex states. The addition of an excess of soluble mycobacterial antigens affected the PA activity of some lepromatous sera, which suggests that the putative complexes were composed of mycobacterial antigens complexed with corresponding IgG antibody.

It was concluded that the PAT is a sensitive detector of IgG complexes peculiar to the lepromatous leprosy. In leprosy the discriminatory power of the PAT seems to be superior to that of other immune complex tests recently applied for the analysis of leprosy series.

     

Studies on thyroid cell surface antigens using cultured human thyroid cells.

Human thyroid cells in primary culture were used for studies of thyroid cell surface antibodies in patients with thyroid autoimmune disorders. Radioiodinated IgG preparations containing thyroid microsomal antibody (TMAb), thyroid stimulating antibody (TSAb) and/or thyroglobulin antibody (TgAb) were tested for binding to thyroid cells. Binding was observed with radioiodinated IgG from patients with Graves' disease, Hashimoto's thyroiditis and idiopathic myxoedema containing TMAb, irrespective of the presence of TSAb and TgAb, while negative results were obtained with normal IgG. A dose-dependent inhibition of binding to thyroid cells was produced by the addition of the corresponding unlabelled IgG preparations. Evidence for tissue specificity was provided by the absence of binding to human skin fibroblasts used as controls. Preabsorption with human thyroid microsomes completely abolished the binding to thyroid cells of a radioiodinated TMAb positive IgG preparation, while only incomplete removal of the reactivity to thyroid microsomes was produced by preabsorption with thyroid cells. These data suggest that some but not all microsomal antigenic determinants are expressed on the thyroid cell surface. Binding to thyroid cells was also observed with purified TgAb, indicating that thyroglobulin antigenic determinants are present on the surface of thyroid cells. No evidence of binding was obtained with a TSAb positive Graves' IgG preparation with undetectable TMAb and TgAb. Unlabelled IgG preparations containing TMAb from patients with either Hashimoto's thyroiditis or idiopathic myxoedema were shown to inhibit the binding to thyroid cells of radioiodinated TMAb positive Graves' IgG and vice versa. These data indicate that antibodies present in these thyroid autoimmune disorders share common thyroid cell surface antigens. However, the binding of radioiodinated IgG from a patient with idiopathic myxoedema was only partially inhibited by Graves' or Hashimoto's IgG, suggesting that some of the thyroid cell surface antibodies of idiopathic myxoedema may not be detectable in other thyroid autoimmune disorders.     

Intrathyroidal lymphocytes from non toxic multinodular goiter: no evidence for production of thyroid stimulating antibodies

Although an autoimmune pathogenesis for non toxic goiter has been suggested, reports concerning circulating antibodies to TSH receptor structures have been conflicting. Intra thyroid lymphocytes, capable of secreting IgG, have been shown to be involved in the pathogenesis of Graves' and Hashimoto's diseases; therefore, the ability of conditioned media obtained from intra thyroid lymphocyte culture, and of IgG purified from these media, to stimulate cAMP accumulation and [3H]-Thymidine (TdR) uptake in FRTL-5 cells was investigated. The activity of IgG produced "in vitro" was compared with that of circulating IgG. Thyroid tissue samples were obtained at surgery from 21 patients with non toxic multinodular goiter (MNG), 5 patients with active Graves' disease (GD), and from 10 normal subjects, undergoing neck surgery for non-thyroidal pathology. IgG purified from media of GD lymphocyte cultures stimulated both cAMP accumulation and [3H]-TdR in 5 out of 5 cases: all of the IgG purified from control or MNG lymphocyte culture media was not active in either assay. Circulating IgG did not affect cAMP accumulation or [3H]-TdR in any of the non toxic MNG cases: controls showed no changed at all. However, both activities represented were increased by GD IgG. Conditioned media from intra thyroid lymphocyte cultures significantly inhibited basal cAMP accumulation in 7 out of the 21 non toxic MNG samples and totally abolished the response in all GD patients. [3H]-TdR was not affected by IgG of any of the controls, but it had an inhibitory effect on 8 out of 21 non toxic MNG patients, and significantly stimulated [3H]-TdR in all GD patients. In conclusion, present data demonstrate that intra thyroid lymphocytes from non toxic MNG do not produce antibodies capable of mimicking TSH actions through the adenylate cyclase cascade. Conversely, soluble factors interacting in TSH-mediated functions of FRTL-5 cells are present in conditioned media of intra thyroid lymphocytes of GD and MNG thyroid lymphocytes of GD and MNG thyroid cultures.     

The effects of alteration of blood-volume on the concentration of circulating angiotensin in anaesthetized dogs

1. The blood-bathed organ technique was used to assay the concentration of angiotensin in the blood of anaesthetized dogs.

2. Alterations of blood volume caused inverse changes of angiotensin concentration owing to changes in the rate of generation of angiotensin which are probably due to changes of the rate of renin secretion.

3. Haemorrhage of 14-26 ml. blood/kg caused an increase of 0·25-1·5 μg/min in the rate of generation and an increase of 0·1-0·33 ng/ml. in the blood concentration of angiotensin.

4. The changes of angiotensin generation rate were not due to changes of renal arterial or venous pressure. They were abolished by blocking the renal nerves with lignocaine; they showed a consistent inverse correlation with central venous pressure but not with systemic arterial pressure.

5. It is concluded that changes of blood volume bring about changes of the rate of generation of angiotensin by a reflex mechanism the efferent limb of which involves the renal nerves. The afferent pathway remains to be elucidated, but the systemic baroceptors do not appear to be of primary importance.

6. The renin-angiotensin system is important in the homeostatic response to changes of blood volume.

     

Neutrophil‐mediated mechanisms of damage and in‐vitro protective effect of colchicine in non‐vascular Behçet’s syndrome

Behçet’s syndrome (BS) is a systemic vasculitis with several clinical manifestations. Neutrophil hyperactivation mediates vascular BS pathogenesis, via both a massive reactive oxygen species (ROS) production and neutrophil extracellular traps (NETs) release. Here, we investigated neutrophil‐mediated mechanisms of damage in non‐vascular BS manifestations and explored the in‐vitro effects of colchicine in counteracting these mechanisms. NETs and intracellular ROS production was assessed in blood samples from 80 BS patients (46 with active non‐vascular BS, 34 with inactive disease) and 80 healthy controls. Moreover, isolated neutrophils were incubated for 1 h with an oxidating agent [2,2′‐azobis (2‐amidinopropane) dihydrochloride; 250 nM] and the ability of pure colchicine pretreatment (100 ng/ml) to counteract oxidation‐induced damage was assessed. Patients with active non‐vascular BS showed remarkably increased NET levels [21.2, interquartile range (IQR) = 18.3–25.9 mU/ml] compared to patients with inactive disease (16.8, IQR = 13.3–20.2 mU/ml) and to controls (7.1, IQR = 5.1–8.7 mU/ml, p < 0.001]. Also, intracellular ROS tended to increase in active BS, although not significantly. In active non‐vascular BS, NETs correlated with neutrophil ROS production (p < 0.001) and were particularly increased in patients with active mucosal (p < 0.001), articular (p = 0.004) and gastrointestinal symptoms (p = 0.006). In isolated neutrophils, colchicine significantly reduced oxidation‐induced NET production and cell apoptosis, although not via an anti‐oxidant activity. Neutrophil‐mediated mechanisms might be directly involved in non‐vascular BS, and NETs, more than ROS, might drive the pathogenesis of mucosal, articular and intestinal manifestations. Colchicine might be effective in counteracting neutrophils‐mediated damage in BS, although further studies are needed.     

A quantitative study of the binding of ALS to various cell types

The fraction of antibody which bound to the lymphocyte surface, was determined in the [¹²⁵I]IgG preparations of rabbit-anti-mouse ALS prepared by different immunization protocols. This fraction increased with continuing immunization and was higher in sera raised by injection of lymphoid cells in Freund's complete adjuvant (5·3–9·6% of total IgG) than in sera raised by intravenous injection of lymphoid suspension (2·3–5·2% of total IgG).

8·5% of IgG from adjuvant-raised ALS bound to non-lymphoid (liver) cells as compared to 9·6% of IgG which binds to the lymphoid cells themselves.

Antibodies against these `species specific' non-lymphoid antigenic determinants were completely absorbed with lymphocyte-free liver cell suspensions. Such absorption did not substantially reduce the immunosuppressive activity of ALS.

     

The metabolism of autologous IgM and 19S rheumatoid factor in rheumatoid arthritis

The turnover of autologous preparations of radio-iodine labelled IgM and 19S rheumatoid factor was studied and compared in patients with severe rheumatoid disease. The IgM was isolated by block electrophoresis and column chromatography. 19S rheumatoid factor was isolated by a combination of euglobulin precipitation, column chromatography, and absorption onto and acid-elution from solid aggregated IgG.

Ten studies were made in seven patients, five with IgM, and five with 19S rheumatoid factor. In two patients the turnovers of 19S rheumatoid factor and IgM were studied simultaneously.

The turnover of IgM was similar to that reported for normal subjects and patients with other diseases: fractional catabolic rate 0·14–0·18, plasma and whole body T_½ 3·7–6·5 days, with 65–77% intravascular localization. The absolute catabolic rate for IgM was elevated (8–60 mg/kg/day).

The turnover of 19S rheumatoid factor isolated from serum was comparable in fractional catabolic rate (0·15–0·19) plasma and whole body T_½ (3·9–6·0 days) and intravascular localization (62–88%). No evidence of rapid catabolism of the `immune' elimination type was obtained.

     

The response of human leucocyte cultures to stimulation by tuberculin and phytohaemagglutinin measured by the uptake of radioactive thymidine

A method is described for assessing the lymphocyte transformation of human leucocyte cultures induced by phytohaemagglutinin (PHA) and tuberculin purified protein derivative (PPD) by the measurement of the uptake of [¹⁴C]thymidine. The thymidine uptake of the cultures has been expressed as percentage of the total activity added. The mean basal uptake of twenty-two unstimulated cultures was 0·96%. The mean uptake of twenty-four PHA stimulated cultures was 19·5%, whilst the mean uptake of eleven PPD stimulated cultures from seven subjects, who were strongly positive on skin testing to tuberculin, was 20·1%. A rise of thymidine uptake was detected in five out of six tuberculin negative subjects (mean uptake 3·8%). Hydrocortisone hemisuccinate in final concentrations of 5 and 50 μg/ml usually reduced the thymidine uptake of unstimulated and PHA and PPD stimulated cultures, though the total cell count was reduced in only four out of ten cultures. An increase of thymidine from 1 to 64 μg/culture resulted in twoto three-fold increase in the uptake of thymidine; further increase of thymidine added to the cultures to 256 μg/culture resulted in a decrease of thymidine uptake.     

The incidence of thyroglobulin antibodies and thyroid enlargement in a general practice in north-east England

Antibodies to thyroglobulin in a titre of 1:25 or more were found in 16·2% of women and 4·3% of men between the ages of 21 and 80 years in a random sample of the population from a general practice in the north-east of England. The incidence of antibodies was highest in the seventh decade in both sexes. High antibody titres (1:78125 or more) were found in 4·6% of women and 1·6% of men and it is suggested that this may represent the incidence of diffuse thyroiditis in the population.

Significant thyroid enlargement was found in 12% of women and 0·9% of men, the corrected incidence of goitre obtained by averaging the frequency of goitres in each decade between 21 and 80 years was 8·9% and 0·9% respectively.