Thyroid Microsomal Fraction Research Articles

Assessment of the Relation of Anti-TPO and TSH, T3 and T4 Levels between Some Subclinical Diabetes Patients in Iran

Background: As we know, around 28% of ills who had benign fibrocystic mastopathy mostly have shown antibodies of anti-TPO as well as around 80% of them that have shown the thyroid hypertrophy issue. The Assessment of thyroid peroxidase (TPO) as the main antigen agent of the thyroid microsomal fraction has enabled the progress of a sensitive and specific assay for the detection of the corresponding autoantibodies and other digestive fractions.
 Aim: Effect of gender, climate, and age on the ATPO, TSH, T3 and T4 rates were assessed as a goal.
 Materials and Methods: We evaluated the diagnostic validity of the anti-TPO, TSH, T3, and T4 assays and their relationships in 500 laboratory cases from Isfahan province with various types of thyroid and diabetes diseases and in controls. Age factor in the various levels of T3, T4, ATPO and TSH items is studied in the following research, which demonstrates a reduced rate in T4 factor of the 1st decade.
 Results: In the present study, patients with high TSH levels had high TPO tests. Patients with high levels of ATPO usually had high TSH, and in patients with low thyroid status, high TSH and high ATPO, TSH levels were normalized with levothyroxine tablets. Those with a high ATP content and normal TSH levels may have hypothyroidism in the future.
 Conclusion: We concluded that you should check the thyroid test every 6 months. This could be concluded from the following research that gender, age, race, and area all demonstrate some remarkable impacts on the levels of T4, T3, TSH, and ATPO.

Anti-thyroid peroxidase (anti-TPO) antibodies in thyroid diseases, non-thyroidal illness and controls. Clinical validity of a new commercial method for detection of anti-TPO (thyroid microsomal) autoantibodies

The identification of the thyroid peroxidase (TPO) as the main antigen of the thyroid microsomal fraction has enabled the development of a sensitive and specific assay for detection of the corresponding autoantibodies. We evaluated the diagnostic validity of the anti-TPO assay in 303 patients with different types of thyroid disease and in controls. Clearly elevated anti-TPO values (anti-TPO > 500 units/ml) were found in 59% of patients with thyroiditis but in none of the controls or the patients with non-thyroidal illness. The mean anti-TPO levels in these two control groups were 26 ± 31 units/ml (mean ± S.D.) and 39 ± 34 units/ml, respectively. The highest frequency of positive results (88%) was obtained in patients with autoimmune hypothyroidism (clinical diagnosis: Hashimoto's thyroiditis) followed by patients with Graves' disease (53%). With a cut-off point of 200 units/ml, a sensitivity of 96% was obtained for Hashimoto's thyroiditis and of 59% for Graves' disease with a specificity of 100% (50 cases). The new method (anti-TPO, Dynotest) was compared with three conventional methods (35 samples). The results for all measurements were in general agreement. In two cases the results were clearly discordant: one sample contained high anti-thyroglobulin antibody concentrations, the other was obtained from a patient with non-thyroidal illness. In both instances the ‘classical’ assays yielded false-positive results. Treatment of autoimmune hyperthyroidism resulted in a median decrease in anti-TPO levels of over 50% after reaching the euthyroid state ( P < 0.05), whereas in persistent hyperthyroidism no consistent changes were observed. In autoimmune hypothyroidism a marked variability in anti-TPO levels was noted. Some patients showed a clear decrease in anti-TPO levels during T4 substitution whereas in others no consistent changes were observed.

Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies.

MAb directed to the thyroid microsomal antigen have been developed. All bound to 101- and 107-kD bands in Western blot analysis using thyroid microsomal fraction as antigen. The MAb also bound to microsomal proteins immunoprecipitated by serum from patients having a high titer of anti-microsomal antibody but no antibodies to thyroglobulin or thyrotropin-stimulating hormone receptor. The pattern of binding was related to the amount of reducing agent. The 101- and 107-kD bands were increased by addition of dithiothreitol whereas, in its absence, numerous bands of higher molecular weight were present, suggesting a multimeric protein structure. Despite the inability to immunoprecipitate thyroid peroxidase (TPO) enzymatic activity, the MAb bound intensively in Western blot to denatured purified hog TPO and to denatured immunopurified human TPO. Trypsin digestion of the 101-107-kD antigen produced a doublet of 84-88 kD that was still immunoreactive with MAb. One of five polyclonal sera tested (with a microsomal antibody titer greater than 1/20,480 measured by the tanned red cell hemagglutination technique) also recognized the 84-88 kD trypsin fragments. Addition of V8 protease led to a disappearance of the 107-kD protein, but not the 101-kD protein, proving that this antigen is formed by two different polypeptides. The MAb bound strongly to thyroid epithelium, whereas binding to papillary carcinoma was absent or low and moderate for follicular and Hurthle cell carcinoma. This study indicates that the thyroid microsomal antigen and TPO are identical and are constituted of two different polypeptides. On SDS-PAGE the antigen appears as two contiguous bands which share common epitopes but are not identical, as proven by their size and difference in susceptibility to proteolytic digestion. The immunoreactivity of the molecule is highly dependent on a trypsin-sensitive site, which appears important in the recognition of the antigen by polyclonal sera and may have biological importance. The expression of microsomal antigenicity is variable among various thyroid carcinomas.

Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices.

Incubations of thyroid microsomes with radiolabeled dolichyl pyrophosphoryl oligosaccharide (Glc3Man9-GlcNAc2) under conditions optimal for the N-glycosylation of protein resulted in the release, by apparently independent enzymatic reactions, of two types of neutral glucosylated polymannose oligosaccharides which differed from each other by terminating either in an N-acetylglucosamine residue (Glc3Man9GlcNAc1) or a di-N-acetylchitobiose moiety (Glc3Man9GlcNAc2). The first mentioned oligosaccharide, which was released in a steady and slow process unaffected by the addition of EDTA, appeared to be primarily the product of endo-beta-N-acetylglucosaminidase action on newly synthesized glycoprotein and such an enzyme with a neutral pH optimum capable of hydrolyzing exogenous glycopeptides and oligosaccharides (Km = 18 microM) was found in the thyroid microsomal fraction. The Glc3Man9GlcNAc2 oligosaccharide, in contrast, appeared to originate from the oligosaccharide-lipid by a rapid hydrolysis reaction which closely paralleled the N-glycosylation step, progressing as long as oligosaccharide transfer to protein occurred and terminating when carbohydrate attachment ceased either due to limitation of lipid-saccharide donor or addition of EDTA. There was a striking similarity between oligosaccharide release and transfer to protein with lipid-linked Glc3Man9GlcNAc2 serving as a 10-fold better substrate for both reactions than lipid-linked Man9-8GlcNAc2. The coincidence of transferase and hydrolase activities suggest the possibility of the existence of one enzyme with both functions. The physiological relevance of oligosaccharide release was indicated by the formation of such molecules in thyroid slices radiolabeled with [2-3H]mannose. Large oligosaccharides predominated (12 nmol/g) and consisted of two families of components; one group terminating in N-acetylglucosamine, ranged from Glc1Man9GlcNAc1 to Man5GlcNAc1 while the other contained the di-N-acetylchitobiose sequence and included Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, and Man9GlcNAc2.

Leucocyte migration inhibition test in diabetes mellitus (author's transl)

The leucocyte migration inhibition test (LMT) using the agarose plate method introduced by Clausen is simple and highly reproducible. Using thyroglobulin, mitochondria and thyroid microsomal fractions as antigens, LMT was performed on ten patients with insulin dependent diabetics, twenty patients with insulin independent diabetics, eleven patients with Graves' diseases, ten patients with chronic thyroiditis and ten healthy controls. The migration index was expressed as a percentage of migration and calculated from the following formula; (formula: see text). Using a thyroid microsomal fraction, the mean migration index for the insulin dependent diabetics (93.2 +/- 11.6) was significantly lower than in the normal subjects (104.3 +/- 10.9) (0.025 less than p less than 0.05). Using thyroglobulin as an antigen for the insulin dependent diabetics and insulin independent diabetics, the mean migration indices (+/-SD) were 93.7+/-10.8 and 102.8+/-10.6 respectively. The corresponding values for the control group were 100.6+/-7.3. Using a mitochondria, MI values for insulin dependent and independent diabetics were 97.7+/-5.5 and 99.6+/-12.6 respectively, while MI value for the control group was 103.9+/-13.6. The mean migration indices obtained with the mitochondria and thyroglobulin were not significantly depressed when the diabetic groups were compared with the normal subjects.

Biosynthese in vitro d'iodothyronines a partir du diiodo-3,5- L-tyrosyl-diiodo-3,5- L-tyrosine

In vitro biosynthesis of iodothyronines from diiodo-3,5- L-tyrosyl-diiodo-3,5- L-tyrosine A comparative study of two types of in vitro synthesis of iodothyronines has been done from 3,5-diiodotyrosine and from diiodo-3,5- L-tyrosyl-diiodo-3,5- L-tyrosine (Tyr(I) 2-Tyr(I) 2) (equimolecular in tyrosyl rings). Incubations are made with rat thyroid gland minces in Eagle's medium or with thyroid microsomal fraction. Synthesis of thyroid hormones from Tyr(I) 2-Tyr(I) 2 is faster and more important than from diiodo-3,5- L-tyrosine (Tyr(I) 2). A mechanism of iodothyronine formation via Tyr(I) 2 - Tyr(I) 2 and different from the one occuring for Tyr(I) 2 is suggested.

Production of antibodies specifically binding triiodothyronine and thyroxine.

In an attempt to obtain antisera for radioimmunoassay of thyroid hormones, we immunized rabbits with various thyroid fractions, various conjugates of triiodothyronine (T3), or a charcoal-T3 complex. We examined the frequency of appearance of antibodies which bind T3 and/or thyroxine (T4) and the potency of these antibodies. Human thyroglobulin was the most effective antigen when injected in Freund's adjuvant but a poor one when injected in saline. Human thyroid microsomal fraction was more effective than thyroglobulin in saline, rabbit thyroid microsomal fraction, the various T3 conjugates, or the charcoal-T3 complex. Antisera produced by immunization against human thyroglobulin (HTg) possessed 2 distinct sets of binding sites, one which was more specific for T3 than T4 and one vice versa. In some antisera, the titer of the “T3-specific” site was higher, while in others the reverse was true, although rabbits had been immunized against the identical thyroglobulin. There was also no correlation between the T3-binding activity of a serum and its anti-HTg hemagglutinating titer. The potential application of these antisera in measurement of thyroid hormones by radioimmunoassay has been studied. The standard curve for T3 was linear from 0.1 to 10 ng/assay tube and cross reaction with T4 varied from 0.8 to 2.5%, depending upon the conditions used. Cross reaction with MIT, DIT, or tetraiodothyroacetic acid was negligible. Thyroglobulin did not displace labeled T3 when tested in amounts varying from 0.1 to 100 ng/assay tube. The standard curve for displacement of labeled T4 by unlabeled T4 was linear from 0.5 to 100 ng and cross reaction with T3 in 2 antisera thus tested was approximately 5 and 8%, indicating that concentrations of T3 likely to be present in serum would not interfere appreciably. These studies indicate that immunization against HTg may be a practical method for the production of thyronine-binding antibodies for radioimmunoassay of T4 and T3.

29. Comparative effects of immunization of rabbits with human thyroglobulin and human and rabbit thyroid microsomal fractions: G. N. Beall, M.D., I. J. Chopra, M.D., and D. H. Solomon, M.D., Torrance, Calif.

Dutch rabbits were immunized with human thyroglobulin and human and rabbit thyroid microsomal fractions. The animals were bled at intervals and their serum was assayed for thyroid-stimulating activity, thyroglobulin haemagglutinins, and total and free thyroxine (T4). Their thyroidal radio-iodine uptake and thyroid histology was also studied. Five out of ten animals immunized with human thyroglobulin developed histological evidence of thyroiditis but none had thyroid-stimulating activity in the serum. Only one out of twenty-six animals immunized with rabbit or human thyroid microsomal fractions had any histological abnormality in the thyroid, but eight had significant amounts of thyroid-stimulating activity in their serum. Although the frequency of the latter response was no greater after immunization with rabbit as compared to human thyroid microsomal fraction, there was a significant increase in serum free T4 in the group immunized with rabbit tissue. Thyroiditis and rabbit immunologic thyroid stimulator (RITS) formation appear to be completely separable phenomena, and RITS is not a by-product of thyroiditis. The absence of thyroid-stimulating activity in the serum of rabbits immunized with thyroglobulin is further evidence that thyroglobulin is not the important antigen leading to the production of this thyroid-stimulating globulin.

Comparative studies on the properties of LATS and TSH.

LATS activity was neutralized by pretreatment of mice with anti-IgG in vivo. Simultaneous injection of epsilon-amino caproic acid and hydrocortisone does not inhibit LATS action. Human thyroid-microsomal fractions neutralize LATS activity and TSH activity. The decrease in activity of LATS and TSH caused by thyroid microsomes correlates positively. Calf thyroid microsomes are much less active than human thyroid microsomes in absorbing LATS activity. Deoxycholate and sonification solubilize the LATS neutralizing activity. No visible precipitate formed between these solubilized fraction and LATS-IgG, using the Ouchterlony agar diffusion method. LATS did not differ from TSH in heat stability, and partially heat denatured LATS still had a long acting type response. Dissociation of a small rapidly acting molecule from LATS could not be obtained in high molar NaCI solution. LATS-IgG fragments digested by proteolytic enzymes (papain, pepsin, trypsin, and ficin) had a prolonged response like LATS. However, these fragments, in the presence of cysteine, either gave the same response after 2 hr and 8 hr, or gave a short acting response like TSH. The thyroid stimulating active site of the LATS active gamma globulin may be situated in Fab or Fab fragments having the antibody combining site. LATS did not stimulate the chicken thyroid, although TSH had this action. The proteolytic fractions of LATS-IgG also did not stimulate the chicken thyroid. No increase of gammaglobulin and/or IgG was found in some sera with a high LATS activity. Binding of LATS-IgG to mouse or human thyroid could not be visualized using the Coon's immunofluorescent method. These data support the concept that LATS is an IgG having a high specific activity of thyroid stimulating action.

Effects of immunization of baboons with human thyroid tissue.

ABSTRACT Rabbits immunized with human thyroid microsomal fraction develop LATS-like thyroid-stimulating activity in their serum, but no evidence of Graves' disease and no thyroiditis. We have immunized 5 baboons similarly. Three developed large amounts of thyroid-stimulating activity; 2 did not. All developed thyroglobulin antibodies. Thyroid function, serum T4-binding and ocular position remained essentially unchanged in all. Both the serum thyroid-stimulating activity and indices of thyroid function were suppressed by the administration of triiodothyronine to the baboons. The serum thyroid stimulator had all the characteristics of TSH: 2 hr/8 hr response ratio well over 1.0; a pattern of delayed elution from DEAE-cellulose; association with low molecular weight eluates from Sephadex Sephadex G-200; extractability from serum by ethanol salt solutions; and complete neutralization after incubation with antihuman TSH, contrasted with complete resistance to antihuman gamma globulin. All baboons developed his...

Observations on the role of circulating lymphocytes in thyroid auto-immunity.

Peripheral leukocytes from normal subjects and patients with Hashimoto's thyroiditis or Graves' disease were cultured in vitro. Induction of blast transformation and stimulation of tritium labeled thymidine incorporation into DNA were assessed in cultures in the absence of an antigenic stimulus, or after addition of phytohemagglutinin, human thyroid supernatant fraction, thyroid microsomal fraction, and monilia extract. Phytohemagglutinin and monilia extract produced a consistent stimulation of blast transformation and/or thymidine incorporation in cells from all 3 groups of subjects. The degree of response was not related to the metabolic status of the patient. Thyroid antigens did not depress reactivity to phytohemagglutinin. Neither thyroid supernatant fraction nor thyroid microsomal fraction stimulated blast transformation or thymidine incorporation in lymphocytes from normal subjects, or the patients with thyroid disease. The studies suggest that circulating lymphocytes do not play an important etiologic role in the pathogenesis of thyroid auto-immune reactions. Lymphocytes inside the thyroid gland may be more important in this process.

Stimulation of thiouracil binding and the iodination system in beef thyroid microsomes.

Abstract 1. 1. It has been shown previously by many investigators that beef thyroid microsomal fractions enzymically iodinate in vitro in the presence of glucose and glucose oxidase. [ 14 C]Thiouracil has now been found to be maximally bound to microsomal fractions under these same conditions. Both iodination and thiouracil binding are inhbited in a very similar pattern by thiols (mercaptoethanol, glutathione) as well as thioureylenes (1-methyl-2-mercaptoimidazole, thiourea). 2. 2. Both iodination and thiouracil binding still occur if glucose oxidase is replaced by 0.1 mM H 2 O 2 . 3. 3. Thiouracil has been shown previously 1 to react rapidly and at low concentrations with the sulfenyl iodide group in a model protein. This reaction of model sulfenyl iodide was inhibited by specific thiols and thioureylenes at concentrations comparable in all respect to that effective in the thyroidal system. 4. 4. After I 3 -pretreatment of the microsimal fraction the subsequent uptake of [ 14 C]thiouracil was increased, consistent with previously reported data on model sulfenyl iodides. The relative inhibition by thiols and thioureylenes of this [ 14 C]-thiouracil binding by iodine pretreated microsomal fractions was also characteristic of the sulfenyl iodide group. 5. 5. These data provide additional support for the hypothesis 1–3 that a sulfenyl iodide group takes part in the iodinating mechanism of the thyroid gland.

On the immunological nature of the long acting thyroid stimulator.

A series of experiments is reported designed to test the hypothesis that the long acting thyroid stimulator (LATS) of Graves' disease has antibody function. It was not possible to transfer LATS activity to new immunoglobulin G (IgG) molecules in a dialysis bath or bound to cellulose, thus supporting the idea that LATS is an integral part of the IgG molecule. LATS was consistently removed from serum by a thyroid microsomal fraction and eluted from the microsomes under conditions appropriate for dissociation of antigen-antibody complexes. The eluted material contained a much larger quantity of LATS in proportion to its IgG content than the original serum. These findings were consistent with the hypothesis that LATS is an antibody. However, mixtures of LATS-containing serum and thyroid microsomes did not fix complement.

Studies on Organ Specificity

Summary Serologic studies of human autoantibodies to thyroid specific antigens by tanned cell hemagglutination, immunofluorescent staining and complement fixation reveal the following relationships: Thyroglobulin antibodies occurring in the sera of patients with thyroiditis can best be demonstrated using human thyroid extract to coat the tanned cells. Monkey thyroid extract exhibits only little activity and there is almost no cross-reactions with thyroid extracts of rabbit and dog origin. Quantitative titration of the same sera using the immunofluorescent staining of the colloid reveals much lower titer than the tanned cell hemagglutination technique. On the other hand, species specific differences, although present, appear to be less marked.Human autoantibodies directed against the cytoplasm of thyroid cells cross the species lines more freely than the thyroglobulin antibodies as observed by immunofluorescent staining and complement fixation.Most thyroiditis sera react with the microsomes of the thyroid and not with those of any other organ when examined by means of the complement fixation test. However, an occasional serum was found which fixed complement not only in the presence of the thyroid microsomal fraction but also with the microsomal fraction of brain tissue. Absorption with thyroid microsomes completely removes the complement-fixing activity against both types of microsomes. Absorption with brain microsomes, on the other hand, removes only the brain reactive serum component but leaves the thyroid-specific one intact.In contrast to the results obtained by the complement fixation technique, immunofluorescent staining of the cytoplasm of thyroid cells with human antibodies seem to be specific for the thyroid and fail to cross-react with other organs as far as examined in this report.