0021-972X/78/4702-0249$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 47, No. 2 Printed in U.S.A.
Zoological Specificity of Human Thyroid-Stimulating Antibody* McGill University Clinic, Royal Victoria Hospital, Montreal, P.Q. H3A I Al Canada ABSTRACT. The zoological specificity of human thyroid-stimulating antibody (TSAb) that occurs in the blood in Graves' disease was examined by assessing its effect on the thyroid of the dog, guinea pig, calf, and mouse as well as that of man. With all but murine glands, thyroid stimulation was assayed by measuring the increase in the concentration of cAMP in the thyroid slices or fragments after 2 h of incubation in buffer containing TSAb. Effects on the thyroid of the mouse were monitored by the in vivo bioassay for LATS. Sera from 33 patients with Graves' disease were obtained and concentrates of TSAb were prepared by precipitation of IgG with 1.64 M (NH4)2SO4. These all stimulated the human thyroid, 13 were LATS-positive, and they variably affected the tissues of other species; of 27 tested, 14
iHE THYROID-stimulating antibody (TSAb) that occurs in the blood in Graves' disease is currently measured by various assay procedures. When assessed by its ability to stimulate the mouse thyroid in vivo, it is known as LATS (1). More recently techniques involving the binding to (2, 3), or stimulation of (4, 5), in vitro preparations of human thyroid have been described. In part, it is the high frequency of positive data when the latter procedures are used (2-5) and the comparatively low incidence of positive results in the LATS assay (6) that have led to the suggestion that the IgG measured in the mouse bioassay is different from that detected by the other procedures (4, 7). We have developed a sensitive procedure for the measurement of TSAb based on an
stimulated the thyroid of the dog, 8 out of 23 stimulated the thyroid of the guinea pig, and 12 out of 16 stimulated the gland of the calf. The more potent the TSAb as assayed with human tissue, the more likely was it to stimulate other species of thyroid; however, frequent exceptions occurred. In a separate analysis of 35 LATSpositive preparations of TSAb, correlation between the responses in the LATS and human thyroid slice assays was statistically significant (P < 0.001). The data are compatible with the view that stimulation by TSAb of nonhuman thyroids, including the murine as in the LATS bioassay, reflects cross-reaction of this immunoglobulin with an antigen that has sufficient similarity to the human molecule to be recognized by the human antibody. (J Clin Endocrinol Metab 47: 249, 1978)
increase in the concentration of cAMP in human thyroid slices incubated with the test material (8). From experience with this procedure we offered the hypothesis (8) that TSAb is an autoantibody to a human thyroid antigen and it cross-reacts, in some instances, with a similar but nonhuman molecule, e.g. in the mouse thyroid; such cross-reaction results in a positive bioassay in the mouse, i.e. the identification of LATS. In pursuing this hypothesis, data were accumulated to show that TSAb may stimulate the thyroid of several nonhuman species; the more potent a preparation of TSAb is in the homologous, i.e. human, system, the more likely is it to have heterologous effects.
Received October 31, 1977. Address requests for reprints to: M. Zakarija, M.D., Thyroid Research Laboratory, A.M.I. Research Building, 1033 Pine Avenue West, Montreal, P.Q. H3A 1A1 Canada. * A preliminary report of some of these data has been published {Recent Prog Horm Res 33: 29, 1977). This work was supported by grants from the Medical Research Council of Canada (MA-5190 and MT-884) and the USPHS (AM-04121). f Research Associate, Medical Research Council of Canada. 249
Materials and Methods Our procedure for measuring TSAb with the use of slices of normal human thyroid gland has been described in detail (8). In brief, grossly normal tissue that was obtained at operation for a thyroid nodule was brought in ice-cold 0.9% NaCl solution to the laboratory and sliced, and 5-15-mg portions (five or six per test group) were incubated for 2 h in Krebs-Ringer-bicarbonate buffer (Ca++ reduced to l k of original concentration) with 0.1% glucose, 0.2% human serum albumin, and 20 mM theophylline at
Downloaded from https://academic.oup.com/jcem/article-abstract/47/2/249/2678643 by University of Rhode Island user on 08 December 2018
MARGITA ZAKARIJA AND J. MAXWELL McKENZIEf
250
ZAKARIJA AND McKENZIE
Results Figure 1 illustrates the in vitro response of human thyroid slices to TSH, two preparations of TSAb, and, as control, normal human IgG. Vials containing the various batches of slices were removed at the times indicated. The maximum response to 50 mU TSH/ml was obtained within the first time period, viz. 7 min. Over 2 h there was only a slight decline in the concentration of cAMP in the control preparation. The TSAb known to be LATSpositive (serum 1) produced its peak effect in 30 min, whereas the slices exposed to the LATS-negative TSAb (serum 2) required 60 min before a statistically significant increase in the concentration of cAMP was seen and a plateau of effect was not clearly reached by even 2 h of incubation. Data of similar measurements of the effects of TSAb on the concentration of cAMP in slices of canine thyroid and fragments of cavian gland are listed in Table 1. Response to the IgG from patient 13 was statistically significant (P < 0.01) at 10 min for canine tissue and at 30, 60, and 120 min for both species. IgG from patient 12, tested only on cavian thyroid segments for establishing the optimal
1978 No 2
6TSH SERUM 1
4-
SERUM 2
2-
ICONT. TIME (min) 7 15
30
60
90
120
FIG. 1. cAMP in human thyroid slices: effects of TSH and of TSAb. Slices were incubated in vitro for the time periods and with the material shown. TSH was 50 mU/ml medium; TSAb (serums 1 and 2) was the (NH^SO* precipitate of globulins from sera obtained from two patients with hyperthyroidism of Graves' disease, and control (cont) was a similar preparation from the serum of a normal subject. Mean and SD of four observations per point are shown; the response to TSAb from serum 2 became statistically significant vs. control (P< 0.02) only after 60 min of incubation. Serum 1 was positive and serum 2 was negative in the LATS bioassay system.
time of incubation, significantly increased the concentration of cAMP by 30 min and the effect appeared to plateau by 60 min. In view of these findings, shown in Fig. 1 and Table 1, a routine of 2 h of incubation for thyroids of all four species (man, dog, guinea pig, and calf) was accepted for subsequent experiments. Table 2 lists results obtained with four preparations of TSAb known to be potent in the human slice assay and tested also on thyroids of dog, guinea pig, and calf, as well as in the mouse in vivo bioassay. TSAb from patient 8 significantly increased cAMP in the human, canine, cavian, and bovine tissues and was LATS-positive; from patient 27, the TSAb stimulated canine and cavian tissue but was ineffective on bovine and murine glands. While TSAb from patients 8, 10, and 11 enhanced the accumulation of cAMP in the thyroids of all five species, the degree of stimulation varied. For example, comparing responses to preparations 10 and 11 that were tested in single batches of tissues, the mean value obtained in each assay was greater with the latter for all species except for the guinea pig, where the effect was significantly less (P < 0.01) than with TSAb from patient 10. Also
Downloaded from https://academic.oup.com/jcem/article-abstract/47/2/249/2678643 by University of Rhode Island user on 08 December 2018
37 C in the presence of control or test material. The concentrations of cAMP in trichloroacetic acid extracts of the slices were subsequently measured by RIA using Schwartz-Mann kits. The test materials were crude preparations of IgG [obtained by precipitation with 1.64 M (NH4)2SO4 followed by dialysis against 0.9% NaCl solution and subsequent vacuum concentration] from serum of either patients with Graves' disease or normal control subjects, added as 0.3 ml (of a 3-fold concentrate over the initial volume of serum) to 0.3 ml buffer. Each batch of thyroid slices was tested for responsiveness with a standard concentration of ovine TSH, viz. 50 mU NIH-TSH-S7/ml incubation solution. Data were expressed as picomoles of cAMP per mg wet wt of tissue (weight determined before incubation) and differences between means were assessed by Student's t test. Similar studies were carried out with slices of thyroid from calf (glands obtained fresh at a local abbatoir) and dog and with fragments of guinea pig thyroid. The assay for LATS using mice was as previously described (8).
JCE & M Vol47
251
ZOOLOGICAL SPECIFICITY OF TSAb TABLE 1. Effects of TSAb in vitro on the thyroid glands of guinea pig and dog cAMP (pmol/mg wet wt, mean ± SD) Source of thyroid
Control Patient 13 Patient 12
10
30
60
120
0.92 ± 0.13 (3) 0.76 ± 0.08 (4) 0.53 ± 0.06 (4)c
0.62 ± 0.11 (4) 1.70 ± 0.08 (3)" 0.87 ± 0.06 (4)A
0.67 ± 0.03 (4) 3.18 ± 1.02 (4)" 1.33 ± 0.13 (3)"
0.72 ± 0.10 (4) 4.18 ± 1.71 (4)" 1.69 ± 0.07 (3)"
0.51 ± 0.04 (4) 0.34 ± 0.02 (4) 0.35 ± 0.02 (4) Control 0.43 ± 0.09 (4) Patient 13 0.72 ± 0.09 (4)" 0.58 ± 0.13 (4)" 1.47 ± 0.08 (3)" 1.58 ± 0.23 (4)" Fragments of thyroids from guinea pigs and slices of canine thyroids were incubated at 37 C in 0.6 ml buffer (see Materials and Methods) containing human IgG, equivalent to 1.5 times the concentration in the original sera. After the times indicated, tissue was removed and homogenized in ice-cold 6% trichloracetic acid for extraction of cAMP, which was subsequently measured by RIA. Statistical significance of difference of means from the corresponding control was calculated by Student's t test. Patients referred to by arabic numeral in this and Table 2 are the same as those listed by the same numbers in Table 3. Number of animals is in parentheses. " P < 0.001. A P
Vol. 47, No. 2 Printed in U.S.A.
Zoological Specificity of Human Thyroid-Stimulating Antibody* McGill University Clinic, Royal Victoria Hospital, Montreal, P.Q. H3A I Al Canada ABSTRACT. The zoological specificity of human thyroid-stimulating antibody (TSAb) that occurs in the blood in Graves' disease was examined by assessing its effect on the thyroid of the dog, guinea pig, calf, and mouse as well as that of man. With all but murine glands, thyroid stimulation was assayed by measuring the increase in the concentration of cAMP in the thyroid slices or fragments after 2 h of incubation in buffer containing TSAb. Effects on the thyroid of the mouse were monitored by the in vivo bioassay for LATS. Sera from 33 patients with Graves' disease were obtained and concentrates of TSAb were prepared by precipitation of IgG with 1.64 M (NH4)2SO4. These all stimulated the human thyroid, 13 were LATS-positive, and they variably affected the tissues of other species; of 27 tested, 14
iHE THYROID-stimulating antibody (TSAb) that occurs in the blood in Graves' disease is currently measured by various assay procedures. When assessed by its ability to stimulate the mouse thyroid in vivo, it is known as LATS (1). More recently techniques involving the binding to (2, 3), or stimulation of (4, 5), in vitro preparations of human thyroid have been described. In part, it is the high frequency of positive data when the latter procedures are used (2-5) and the comparatively low incidence of positive results in the LATS assay (6) that have led to the suggestion that the IgG measured in the mouse bioassay is different from that detected by the other procedures (4, 7). We have developed a sensitive procedure for the measurement of TSAb based on an
stimulated the thyroid of the dog, 8 out of 23 stimulated the thyroid of the guinea pig, and 12 out of 16 stimulated the gland of the calf. The more potent the TSAb as assayed with human tissue, the more likely was it to stimulate other species of thyroid; however, frequent exceptions occurred. In a separate analysis of 35 LATSpositive preparations of TSAb, correlation between the responses in the LATS and human thyroid slice assays was statistically significant (P < 0.001). The data are compatible with the view that stimulation by TSAb of nonhuman thyroids, including the murine as in the LATS bioassay, reflects cross-reaction of this immunoglobulin with an antigen that has sufficient similarity to the human molecule to be recognized by the human antibody. (J Clin Endocrinol Metab 47: 249, 1978)
increase in the concentration of cAMP in human thyroid slices incubated with the test material (8). From experience with this procedure we offered the hypothesis (8) that TSAb is an autoantibody to a human thyroid antigen and it cross-reacts, in some instances, with a similar but nonhuman molecule, e.g. in the mouse thyroid; such cross-reaction results in a positive bioassay in the mouse, i.e. the identification of LATS. In pursuing this hypothesis, data were accumulated to show that TSAb may stimulate the thyroid of several nonhuman species; the more potent a preparation of TSAb is in the homologous, i.e. human, system, the more likely is it to have heterologous effects.
Received October 31, 1977. Address requests for reprints to: M. Zakarija, M.D., Thyroid Research Laboratory, A.M.I. Research Building, 1033 Pine Avenue West, Montreal, P.Q. H3A 1A1 Canada. * A preliminary report of some of these data has been published {Recent Prog Horm Res 33: 29, 1977). This work was supported by grants from the Medical Research Council of Canada (MA-5190 and MT-884) and the USPHS (AM-04121). f Research Associate, Medical Research Council of Canada. 249
Materials and Methods Our procedure for measuring TSAb with the use of slices of normal human thyroid gland has been described in detail (8). In brief, grossly normal tissue that was obtained at operation for a thyroid nodule was brought in ice-cold 0.9% NaCl solution to the laboratory and sliced, and 5-15-mg portions (five or six per test group) were incubated for 2 h in Krebs-Ringer-bicarbonate buffer (Ca++ reduced to l k of original concentration) with 0.1% glucose, 0.2% human serum albumin, and 20 mM theophylline at
Downloaded from https://academic.oup.com/jcem/article-abstract/47/2/249/2678643 by University of Rhode Island user on 08 December 2018
MARGITA ZAKARIJA AND J. MAXWELL McKENZIEf
250
ZAKARIJA AND McKENZIE
Results Figure 1 illustrates the in vitro response of human thyroid slices to TSH, two preparations of TSAb, and, as control, normal human IgG. Vials containing the various batches of slices were removed at the times indicated. The maximum response to 50 mU TSH/ml was obtained within the first time period, viz. 7 min. Over 2 h there was only a slight decline in the concentration of cAMP in the control preparation. The TSAb known to be LATSpositive (serum 1) produced its peak effect in 30 min, whereas the slices exposed to the LATS-negative TSAb (serum 2) required 60 min before a statistically significant increase in the concentration of cAMP was seen and a plateau of effect was not clearly reached by even 2 h of incubation. Data of similar measurements of the effects of TSAb on the concentration of cAMP in slices of canine thyroid and fragments of cavian gland are listed in Table 1. Response to the IgG from patient 13 was statistically significant (P < 0.01) at 10 min for canine tissue and at 30, 60, and 120 min for both species. IgG from patient 12, tested only on cavian thyroid segments for establishing the optimal
1978 No 2
6TSH SERUM 1
4-
SERUM 2
2-
ICONT. TIME (min) 7 15
30
60
90
120
FIG. 1. cAMP in human thyroid slices: effects of TSH and of TSAb. Slices were incubated in vitro for the time periods and with the material shown. TSH was 50 mU/ml medium; TSAb (serums 1 and 2) was the (NH^SO* precipitate of globulins from sera obtained from two patients with hyperthyroidism of Graves' disease, and control (cont) was a similar preparation from the serum of a normal subject. Mean and SD of four observations per point are shown; the response to TSAb from serum 2 became statistically significant vs. control (P< 0.02) only after 60 min of incubation. Serum 1 was positive and serum 2 was negative in the LATS bioassay system.
time of incubation, significantly increased the concentration of cAMP by 30 min and the effect appeared to plateau by 60 min. In view of these findings, shown in Fig. 1 and Table 1, a routine of 2 h of incubation for thyroids of all four species (man, dog, guinea pig, and calf) was accepted for subsequent experiments. Table 2 lists results obtained with four preparations of TSAb known to be potent in the human slice assay and tested also on thyroids of dog, guinea pig, and calf, as well as in the mouse in vivo bioassay. TSAb from patient 8 significantly increased cAMP in the human, canine, cavian, and bovine tissues and was LATS-positive; from patient 27, the TSAb stimulated canine and cavian tissue but was ineffective on bovine and murine glands. While TSAb from patients 8, 10, and 11 enhanced the accumulation of cAMP in the thyroids of all five species, the degree of stimulation varied. For example, comparing responses to preparations 10 and 11 that were tested in single batches of tissues, the mean value obtained in each assay was greater with the latter for all species except for the guinea pig, where the effect was significantly less (P < 0.01) than with TSAb from patient 10. Also
Downloaded from https://academic.oup.com/jcem/article-abstract/47/2/249/2678643 by University of Rhode Island user on 08 December 2018
37 C in the presence of control or test material. The concentrations of cAMP in trichloroacetic acid extracts of the slices were subsequently measured by RIA using Schwartz-Mann kits. The test materials were crude preparations of IgG [obtained by precipitation with 1.64 M (NH4)2SO4 followed by dialysis against 0.9% NaCl solution and subsequent vacuum concentration] from serum of either patients with Graves' disease or normal control subjects, added as 0.3 ml (of a 3-fold concentrate over the initial volume of serum) to 0.3 ml buffer. Each batch of thyroid slices was tested for responsiveness with a standard concentration of ovine TSH, viz. 50 mU NIH-TSH-S7/ml incubation solution. Data were expressed as picomoles of cAMP per mg wet wt of tissue (weight determined before incubation) and differences between means were assessed by Student's t test. Similar studies were carried out with slices of thyroid from calf (glands obtained fresh at a local abbatoir) and dog and with fragments of guinea pig thyroid. The assay for LATS using mice was as previously described (8).
JCE & M Vol47
251
ZOOLOGICAL SPECIFICITY OF TSAb TABLE 1. Effects of TSAb in vitro on the thyroid glands of guinea pig and dog cAMP (pmol/mg wet wt, mean ± SD) Source of thyroid
Control Patient 13 Patient 12
10
30
60
120
0.92 ± 0.13 (3) 0.76 ± 0.08 (4) 0.53 ± 0.06 (4)c
0.62 ± 0.11 (4) 1.70 ± 0.08 (3)" 0.87 ± 0.06 (4)A
0.67 ± 0.03 (4) 3.18 ± 1.02 (4)" 1.33 ± 0.13 (3)"
0.72 ± 0.10 (4) 4.18 ± 1.71 (4)" 1.69 ± 0.07 (3)"
0.51 ± 0.04 (4) 0.34 ± 0.02 (4) 0.35 ± 0.02 (4) Control 0.43 ± 0.09 (4) Patient 13 0.72 ± 0.09 (4)" 0.58 ± 0.13 (4)" 1.47 ± 0.08 (3)" 1.58 ± 0.23 (4)" Fragments of thyroids from guinea pigs and slices of canine thyroids were incubated at 37 C in 0.6 ml buffer (see Materials and Methods) containing human IgG, equivalent to 1.5 times the concentration in the original sera. After the times indicated, tissue was removed and homogenized in ice-cold 6% trichloracetic acid for extraction of cAMP, which was subsequently measured by RIA. Statistical significance of difference of means from the corresponding control was calculated by Student's t test. Patients referred to by arabic numeral in this and Table 2 are the same as those listed by the same numbers in Table 3. Number of animals is in parentheses. " P < 0.001. A P
