|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1994 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1993 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1992 : ¥700,000 (Direct Cost : ¥700,000)
In the present study, we have developed a novel bioassay for the assessment of thyroid growth stimulating activity using cytochalasin B (CB) and tested immunoglobulins (IgGs) form patients with autoimmune thyroid diseases. The assay is based on the principle that growing cells during incubation with CB show an increased number of nuclei in a cell (N/C index), since CB,at appropriate concentrations, is known to inhibit cytoplasmic cleavage without affecting nuclear mitosis.
FRTL-5 thyroid cells were incubated with various thyroid stimulators in TSH-free medium containing 2 mg/I CB for 3 days. After the incubation, the cells were harvested in trypsin/EDTA to obtain single cell suspension, fixed, dropped onto a glass slide, stained and observed under a microscope to determine the N/C index.
Bovine TSH,forskolin, cholera toxin, or (Bu) 2cAMP increased the N/C index up to approximately 2.0 in a dose-dependent manner, suggesting that thyroid cell proliferation is attributable, at least, to the
increase in cAMP.IgGs not only from 27 patients with untreated goitrous Graves' disease but also from 14 patients with goitrous Hashimoto's thyroiditis elicited an increase in the N/C index, which exceeded the mean + 2SD of the values for 17 normal subjects (mean(]SY.+-。[))SD,1.063(]SY.+-。[))0.014). Four non-goitrous hypothyroid patients with primary myxedema displayd a normal N/C index. In Graves' disease, the N/C index did not correlate with TSAb activities but did correlate with estimated goiter size and TBII activities (both, P<0.05). IgGs containing blocking-type TSH-receptor antibody inhibited the TSH- or Graves' IgG-stimulated increase in N/C index, and the grade of inhibition correlated with TBII and TSBAb activities. The blocking-type antibody, however, did not influence the stimulatory effect of IgG from patients with Hashimoto's thyroiditis.
In conclusion, we have developed a sensitive and simple assay for thyroid growth simulating activity by using CB,and found that all tested patients with goitrous Graves' disease and goitrous Hashimoto's thyroiditis have thyroid growth stimulating immunoglobulin (TGI) whose activity does not correlate with TSAb. Less