Biomedical subjects

H Sugawa

Publications and source records attributed to H Sugawa.

At least 37 records · Page 2Linked to original sources

PubMed1996

High-dose intravenous steroid pulse therapy in thyroid-associated ophthalmopathy.

To evaluate the efficacy of high-dose intravenous steroid pulse followed by oral steroids in the treatment of thyroid-associated ophthalmopathy, we performed clinical assessment and measurement of retroorbital muscle enlargement in 27 patients before and after the therapy, and followed them up longitudinally. The mean duration of follow up is (mean +/- SD) 29.8 +/- 23.8 months (range 4-92). Diplopia disappeared in 10 patients and ameliorated in 11 patients. The degree of proptosis decreased in 15 patients and the fall in visual acuity improved in a third of the patients. The total ophthalmopathy index (OI) decreased from 7.0 +/- 1.9 to 3.0 +/- 1.5. The extraocular muscle enlargement (EME), expressed as the maximal ratio of extraocular muscle thickness to the diameter of the optic nerve, decreased from 2.33 +/- 0.56 to 1.27 +/- 0.26. No major side effects were found in any patient. The improvement in the eye disease was found immediately after the pulse therapy, prior to the start of the following therapy by oral steroids and/or orbital irradiation. Both of OI and EME decreased with time after the therapy and did not get worse after withdrawing oral steroids. The efficacy of the therapy evaluated by degrees of improvement in OI and in EME was significantly greater in females than in males. Although there was a significant positive correlation between initial OI and EME values and initial TBII and TSAb activities, a significant correlation was seen only between the degrees of improvement in EME and changes in TBII activity due to the therapy. The duration of eye disease, thyroid status, treatment with anti-thyroid drug, smoking and experience of previous treatment did not affect the efficacy of the present therapy. We conclude that high-dose intravenous steroid pulse therapy is effective and safe for thyroid-associated ophthalmopathy.

Adult↗

PubMed1995

Constitutive activation of the thyrotropin receptor by deletion of a portion of the extracellular domain.

Mutations involving the transmembrane domain of the thyrotropin receptor (TSHR) confer constitutive activation of the receptor and can cause human diseases. Naturally occurring activating mutations identified to date are located only in the transmembrane domain of the receptor. We now report a mutant involving the extracellular domain of the TSHR which also shows constitutive activation. This mutation is missing residues 339-367 located in the C-terminal portion of the extracellular domain. When expressed in COS-7 cells, the mutated TSHR (M3B) retained similar TSH binding ability to that of the wild-type receptor. However, the basal cAMP production without TSH stimulation in COS-7 cells transfected with M3B cDNA was significantly higher than that of COS-7 cells with wild-type receptor, indicating that the mutant receptor is constitutively activated. Our results provide new insight into the mechanism of receptor activation.

Animals↗

PubMed1995

Isolation of Epstein-Barr-virus-transformed lymphocytes producing IgG class monoclonal antibodies using a magnetic cell separator (MACS): preparation of thyroid-stimulating IgG antibodies from patients with Graves' disease.

In autoimmune diseases, IgG class autoantibodies are generally considered to be more pathognomonic than IgM class ones. Although Epstein-Barr virus (EBV)-transformation of lymphocytes is a useful method to obtain human monoclonal autoantibodies, it tends to result predominantly in IgM-producing cells. We depleted IgM+ cells before EBV-transformation with a Magnetic Cell Separator (MACS) in order to increase the chance of acquisition of cells producing IgG class anti-thyrotropin (TSH) receptor antibodies (TRAb). As a result, we obtained four independent B cell clones producing IgG class monoclonal thyroid-stimulating antibodies (TSAb) from three patients with Graves' disease. None of these clones showed any TSH binding inhibitor immunoglobulin (TBII) activity, suggesting independence of TSAb-producing lymphocytes from those producing TBII.

Antibodies, Monoclonal↗

PubMed1995

Statistical analysis of functional region(s) of the TSH receptor.

In this study, we apply recently developed methods to evaluate the thyrotropin receptor (TSH-R). These methods are called deviation (DEV) model, deviation decrease (DD) and DEV/DD analyses, and are based on deviation of amino acid sequences. A 3-dimensional structure model of TSH-R was graphically constructed, and found to possess a large central cavity (donut-like structure). The N-terminus was found to be in the center of the whole extracellular structure and to form a part of the bottom of the cavity. High DEV values indicate deviated amino acid compositions in the protein and were seen in 7 regions, 6 of which were found to be in regions with hydrophilic and acrophilic character. On the basis of the analysis of intra-molecular cis-acting relationships, 7 pairs of regions were presumed to be closely related. Further, when 3 exoplasmic loop lesions were analyzed similarly, 3 other regions were shown to have a close relationship with the cell surface. DEV/DD values were applied to predict the interface of TSH-R with trans-acting molecules such as TSH-R antibody or TSH. The regions in association with trans-acting molecules were seen in 14 regions, 11 of which included the high DEV regions. Both of the TSH-R specific regions in the N- and C-terminal side, especially the latter, were found to be the major components.

Amino Acid Sequence↗

PubMed1995

Inhibition of thyrocyte iodide uptake by H+K+ ATPase inhibitor, timoprazole.

The inhibitory effect of timoprazole on iodide uptake by cultured thyrocytes was observed using FRTL-5 cells. The inhibition was noticed to be dose dependent and was eliminated completely by rinsing. Timoprazole pretreated with acid had inhibitory effects, but [3H] leucine incorporation experiments indicated that acidified timoprazole inhibited protein synthesis to some degree. In addition, TSH-induced cAMP production was also inhibited by acidified timoprazole. Timoprazole pretreated in a neutral solution did not have any cytotoxic effects on TSH-induced cAMP production or protein synthesis at concentrations less than 1000 microM, suggesting that timoprazole by itself has specific effects on iodide uptake. The absence of any direct interaction of the drug with iodide was confirmed by separation of timoprazole and iodide on a C18 Sep-Pak column. It is concluded that timoprazole by itself could inhibit iodide transport through the thyroid cell membrane. This may provide a useful system in further investigating the mechanism of iodide transport.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1995

Thyroid-stimulating antibodies in sera from patients with Graves' disease are heterogeneous in epitope recognition.

Two synthetic peptides, P354-14 (amino acid nos. 354 to 367) and P338-16 (nos. 338 to 353), corresponding to the partial amino acid sequences of the hTSH receptor structure were studied for their ability to bind specifically serum IgGs from patients with Graves' disease and to inhibit thyroid stimulating TSH receptor antibody (TSH-R SAb) activity. IgG binding was measured by an ELISA using sera from 102 Graves', 20 Hashimoto patients, and 9 normal subjects. Both peptides showed significantly increased IgG binding of Graves' sera compared with those of Hashimoto and normal sera. There was a significant correlation (r = 0.529) between the amount of IgG bound by the two peptides, but neither of these values correlated well with their TSH-R SAb activity nor thyrotropin-binding inhibitor TSH receptor antibody (TSH-R IAb) activity. TSH-R SAb inhibiting effects of these peptides were then analysed by measuring TSH-R SAb activity after incubation with the peptides. Among eight Graves' IgGs tested the TSH-R SAb activity of three was inhibited by both peptides, two were inhibited only by P354-14 and three were not affected by either. These TSH-R SAb inhibiting effects were dose-dependent and reproducible. To confirm these findings, a peptide-sepharose gel affinity absorption study was performed. Eleven Graves' IgGs were applied to both peptide gels and the TSH-R SAb activity of the unabsorbed fraction was measured. The TSH-R SAb activity of five IgGs was strongly absorbed only by P354-14 and five others were absorbed by both peptides to an almost similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗

PubMed1995

Presence of heterogeneous thyroid-stimulating antibodies in sera from individual Graves' patients as shown by synthesized thyrotropin receptor peptide application: evidence showing two independent epitopes and a possible recognition of two epitopic regions by one antibody molecule.

To define the epitope(s) of stimulating thyrotropin receptor antibody (TSH-R-Sab), we synthesized 19 oligopeptides covering almost all amino acids of the extracellular domain of the human TSH-R and studied these effects on the inhibition of one TSH-R-Sab activity. Four of the 19 peptides encompassing residues 31-50 (P31-20), 91-119 (P91-29), 287-304 (P287-18) and 354-367 (P354-14) were found to show significant TSH-R-Sab inhibition and to have similar effects on the other three Graves' immunoglobulins. When these peptides were applied in combination with P354-14 only P287-18 revealed additional effects but the other two combinations did not. Furthermore, sequential addition of these peptide pairs confirmed the additional effects of P287-18 and P354-14. Sequential peptide-affinity gel studies were then performed. Most of the TSH-R-Sab activity in the unabsorbed fraction from P287-18 gel was absorbed to a subsequent P354-14 gel and the eluted fraction from P287-18 mostly remained unabsorbed by the P354-14 gel. On the other hand, most of the unabsorbed fraction from P91-29 gel remained unabsorbed even by the subsequent P354-14 gel. When a P354-14 affinity gel-purified TSH-R-Sab immunoglobulin was labeled and evaluated for its binding to FRTL-5 cells, additions of original immunoglobulin, P354-14 and P91-29 resulted in significant inhibition of the binding but P287-18 did not affect either. From these results, it was concluded that most of the individual Graves' immunoglobulins contain at least two heterogeneous moieties with TSH-R-Sab activity, one of which binds P354-14 and the other binds P287-18. Further, P354-14 and P91-29 were indicated to bind the same molecule of TSH-R-Sab immunoglobulin.

Absorption↗

PubMed1994

[A case report of primary triple cancers in the thyroid, stomach and rectum with evidence of variable oncoprotein expressions].

A case with primary triple cancers including thyroid cancer is reported. A 57-year old woman complaining of laryngeal discomfort was found to have an firm elastic lump on the right anterior neck. On 123I scan, the nodule in the right thyroid lobe accumulated considerable amounts of radioiodine as a warm nodule, while the remainder of the gland showed decreased uptake. Thyroid hormone levels remained within normal ranges. Cytological findings obtained by fine-needle aspiration biopsy showed papillary carcinoma. Right lobectomy was performed. The histological examinations revealed papillary carcinoma embedded within adenomatous thyroid tissue. It is probable that the surrounding adenomatous tissue accumulated radioiodine, since the warm nodule on 123I scan was larger than the size of the carcinoma. Examinations of the gastrointestinal tract revealed the presence of poorly differentiated adenocarcinoma in the stomach and well differentiated adenocarcinoma (carcinoma in adenoma) in the rectum. Expressions of ras p21 and p53 were examined immunohistochemically in these carcinoma tissues. The ras p21 product was clearly detected in not only the thyroid carcinoma but in a part of the surrounding adenomatous regions as well. Both ras p21 and p53 proteins were observed in the rectal cancer tissue. In contrast, these oncoproteins were not found in the gastric cancer tissue. In this case ras oncogene activation may be an early event in the tumorigenic process of the thyroid and rectum. However, different genetic alterations seem to occur during the development of these three carcinomas.

Adenocarcinoma↗

PubMed1994

Heterogeneity and diversity of IgM and IgG lupus anticoagulants in an individual with systemic lupus erythematosus.

From one patient with systemic lupus erythematosus retaining lupus anticoagulant (LAC), we established 6 Epstein-Barr virus-transformed human B cell clones secreting antibodies that affect the coagulation assay. Two and 4 of the clones secreted IgM and IgG antibodies, respectively. Although all 6 antibodies displayed anticardiolipin activity in ELISA, the increased binding activity in the presence of beta 2-glycoprotein I was limited only to the IgG antibodies. Five antibodies (two IgM and three IgG) had LAC activity which prolonged the activated partial thromboplastin time (APTT), whereas one IgG antibody shortened the APTT. Two of the IgG producing clones had an identical Ig heavy chain gene rearrangement despite their opposite effects on the coagulation assay. These results demonstrated the heterogeneity of LACs and diversity among their physiological functions.

Adult↗

PubMed1994

Variable regions of Ig heavy chain genes encoding antithyrotropin receptor antibodies of patients with Graves' disease.

We have established EBV-transformed human B cell clones producing monoclonal antithyrotropin receptor antibodies from two patients with Graves' disease. We then isolated and characterized Ig H chain genes of 5 B cell clones with the thyrotropin-binding inhibitor Ig (TBII) activity and 4 B cell clones with the thyroid-stimulating antibody (TSAb) activity. We found that VH gene families used in the 5 TBII clones were all VH-III, although those of the four TSAb clones were diverse, including VH-II, -III, -IV, all -V. Most of VH segments used in TBII and TSAb are commonly used in other autoantibodies and fetal liver repertoire. The frequency of somatic mutations in TBII was higher than that in TSAb. Inasmuch as the same germline VH segment (V3-23) was used for both TBII and TSAb, the frequency and position of somatic mutations may be important for generation of TBII and TSAb.

Amino Acid Sequence↗

PubMed1994

Preparation and characterization of monoclonal antithyrotropin receptor antibodies obtained from peripheral lymphocytes of hypothyroid patients with primary myxedema.

Anti-TSH receptor antibodies (TSH-R Ab), which have been detected in the serum of some patients with primary myxedema, are themselves considered to induce hypothyroidism. These are termed blocking-type TSH-R Ab (TSH-R BAb), because they inhibit adenylate cyclase stimulation by TSH on thyrocytes or nonthyroidal cells transfected with TSH-R complementary DNA. We prepared monoclonal TSH-R BAb and characterized them. Peripheral lymphocytes from three patients with primary hypothyroidism and potent TSH-R BAb were transformed by Epstein-Barr virus, and the culture supernatants were screened by TSH binding inhibitor immunoglobulin (TBII) assay. Twenty positive and 7 negative lymphocyte clones were obtained; their monoclonality was confirmed by Southern blot analysis, using an immunoglobulin (Ig) JH probe. These monoclonal antibodies were then tested for TSH-R BAb activity. TSH-R BAb activity ranged from 24.1-58.5% (normal range, < 24%) in all 20 TBII-positive clones and in 2 of 7 TBII-negative clones. An enzyme-linked immunosorbent assay showed that the Ig isotypes of these clones with TBII and/or TSH-R BAb activity were IgG in 8 and IgM in 14. Another enzyme-linked immunosorbent assay and Southern blot analysis of the light chains revealed that 13 clones had kappa-chains, whereas the light chains could not be determined in the other 9 clones. To summarize, 1) we obtained 22 clones that produced monoclonal TSH-R BAb, including 8 IgG-type clones. 2) The clones exhibited dominant usage of the kappa-chain. 3) Although all TBII clones had TSH-R BAb activity, their TBII and TSH-R BAb activities were not significantly correlated, and two TSH-R BAb clones did not show TBII activity.

Animals↗

PubMed1994

Recent progress in TSH receptor studies with a new concept of "autoimmune TSH receptor disease".

Recent progress in the studies of epitope analysis of the TSH-R against TSH-R Ab was reviewed extensively. Using both site-directed mutagenesis and synthetic TSH-R peptide, binding and/or action sites of TSH-R Ab have been known to be multiple and discontinuous, but the significance of two unique and TSH-R specific regions has been implicated. Further, the possible existence of heterogeneity among stimulatory TSH-R Ab has also been indicated. As for immunogenetic factors related to autoimmune thyroid diseases, studies on HLA analysis, TSH-R specific T lymphocyte analysis and VH analysis of TSH-R Ab were discussed. Of note was the negative association of HLA-DP w2 antigen in Japanese patients with Graves' disease and hypothyroidism due to blocking TSH-R Ab, and we proposed a new concept of autoimmune TSH receptor disease within autoimmune thyroid disease and emphasized possible critical roles of HLA-DP. Recent evidence of restricted usage of the Ig VH gene in cloned B lymphocytes from Graves' patients producing TSH-R Ab has also been presented.

Autoimmune Diseases↗

PubMed1993

Characterization of epidermal growth factor receptor in a rat thyroid cell line, FRTL-5.

A rat thyroid cell line FRTL-5 showed no significant binding of epidermal growth factor(EGF) either in the presence or absence of thyrotropin(TSH). Conditioned medium from FRTL-5 did not inhibit the specific binding of [125I]EGF to HeLa cell which has EGF receptors (EGF-R). Two bands of 170KD and 80KD obtained by immunoprecipitation with solubilized FRTL-5 and anti-EGF-R monoclonal antibody indicated autophosphorylation activity. The activity was enhanced by several thyroid cell growth factors including TSH. dibutyryl-cAMP(db-cAMP), insulin like growth factor I(IGF-I). Interleukin 1 beta (IL-1 beta) and phorbol ester. EGF alone enhanced the autophosphorylation activity. These data indicate that FRTL-5 possesses unique EGF-R with low affinity to EGF and that the tyrosine kinase of the receptor is activated during cell proliferation.

Animals↗

PubMed1993

Cloning of a human immunoglobulin gene fragment containing both VH-D and D-JH rearrangements: implication for VH-D as an intermediate to VH-D-JH formation.

In an Epstein-Barr virus-transformed human B cell line we found an unusual immunoglobulin heavy chain gene rearrangement. Restriction mapping and sequencing analysis led us to conclude that VH-D and D-JH recombination took place in a single allele. Both VH-D and D-JH complexes still had their recombination signal sequences adjacent and the DNA sandwiched by these two complexes retained a germ-line configuration, suggesting the potential for a secondary rearrangement resulting in a VH-D(-D)-JH formation. With this finding, we propose a novel pathway, in which the VH-D complex is an intermediate in the formation of a functional VH exon.

Amino Acid Sequence↗

PubMed1993

A thyroid cancer specific monoclonal antibody which recognizes cryptic epitope(s) of human thyroglobulin.

A monoclonal antibody (TCM-9) specific for human thyroid cancer but not for Graves' disease, adenoma or normal thyroid tissue was shown to recognize a 300 K protein but not to bind to native or mature human thyroglobulin (Tg). In this study, we investigated further the antigen recognized by TCM-9. When purified Tg was treated with periodate, dithiothreitol (DTT) and sodium dodecyl sulfate (SDS), immunoblotting of treated Tg with TCM-9 revealed an apparent enhancement of the staining only with DTT-treated Tg. Furthermore, the DTT-treated Tg was shown to bind dose-dependently to plates coated with TCM-9. We performed SDS-PAGE and immunoblot analyses on the immune complex obtained from a homogenate of thyroid cancer tissue which was preincubated with an excess of anti-Tg monoclonal antibody. When the protein was autoradiographed with [125I]TCM-9, a definite band was observed. The results indicate that TCM-9 is likely to be directed against a masked epitope of thyroglobulin which can be exposed after treatment with a reducing agent.

Adenocarcinoma↗

PubMed1993

Immunoglobulin G that interferes with thyroid-stimulating antibody measurements can be eliminated specifically by incubation with synthetic peptides corresponding to partial sequences of the human thyrotropin receptor.

Two IgG preparations out of more than 100 tested, distinct from the typical Graves' disease IgG, were shown specifically to enhance the cAMP production of FRTL-5 cells by the addition of a synthetic peptide, P-218, corresponding to the partial amino acid sequence from No. 354 to 367 of the h thyroid-stimulating hormone (TSH) receptor. IgG obtained from a patient with Graves' disease revealed a serial alteration of the enhancement; negative in July, 1989, potent in January, 1991, and weak in September 1991. During this time there was no remarkable change in the patient's serum protein components or TSH receptor antibody activities. A peptide with a completely reverse sequence of P-218 showed little effect, and P-218 in combination with bTSH or forskolin did not affect cAMP production by these ligands, and did not alter the inhibitory activity of thyroid-stimulation-blocking antibody. High concentrations of P-218 resulted in reduction of such enhancing effects of cAMP by thyroid-stimulating antibody. P-218 affinity chromatography showed almost complete absorption and recovery of thyroid-stimulating antibody and P-218 reactivity. In the 15 synthesized peptides with proximal sequences of P-218 (from 338 to 378), regions thought to be involved with the enhancement were defined as follows: 354-367 (P-218) is a critical unit; 354-357 and 364-367 are considered to be the essential sites; several amino acid extensions on both N- and C-terminal sides of P-218 show additional enhancement. In conclusion, evidence was shown to indicate the presence of IgG that interferes with thyroid-stimulating antibody measurements.

Amino Acid Sequence↗

PubMed1993

Apparent genetic difference between hypothyroid patients with blocking-type thyrotropin receptor antibody and those without, as shown by restriction fragment length polymorphism analyses of HLA-DP loci.

HLA types in Japanese patients with primary hypothyroidism were analyzed to see whether those with blocking-type TSH receptor antibody (TSH-R BAb M) differed genetically from those with idiopathic myxedema (IM). HLA typings of -A, -B, -C, -DR, and -DQ (73 antigens) were performed serologically, and those of -D and -DP (29 antigens) were analyzed by the restriction fragment length polymorphism method. Thirty patients were studied with TSH-R BAb M, and 28 with IM. The data were analyzed and compared with our previous results from 88 Graves' patients, 46 Hashimoto patients, and 186 control subjects. Overall, 192 patients with 4 autoimmune thyroid disorders showed a decrease in -Aw19 and an increase in -DQw4 (corrected P < 0.05) and significant associations of -Aw33, -Bw46, -Cw3, -DRw8, -DR9, and -DQw3. In TSH-R BAb M patients, increases in -B35, -Bw60, and -Dw8 and decreases in -DR4 and -DPw2 were seen, whereas IM patients showed increased -DPw2, -Bw61, and -Dw23. In comparisons between TSH-R BAb M and IM, the difference in -DPw2 was highly significant. HLA-B35 differed significantly in these 2 types of hypothyroidism. In conclusion, TSH-R BAb M patients have decreased frequency of -DPw2 and are genetically similar to Graves' disease, whereas IM patients are characterized by high frequency of -DPw2 and are genetically similar to Hashimoto's thyroiditis.

Adult↗

PubMed1992

Production and properties of novel human thyroid cancer specific monoclonal antibodies.

Monoclonal antibodies (TCM-7, -9 and -12) against human thyroid differentiated cancers were established by screening with human thyroid cancers, normal and benign thyroid tissue, and normal human serum protein. A monoclonal antibody (TCM-9) with strong specificity for human thyroid cancer but not for Graves' disease, adenoma or normal thyroid, was shown to recognize a 300 K protein but not to bind to native or mature human thyroglobulin. When TCM-9 was used in immunohistochemical staining tests on more than 30 types of non-thyroid lesions, no reactivity of TCM-9 was observed except with skin immature teratoma, lip squamous carcinoma and stomach adenocarcinoma, which revealed weak reactivities. TCM-9 also showed strong reactivity with two undifferentiated thyroid cancer cell lines and one tissue specimen. Thus TCM-9 is a novel monoclonal antibody against the thyroid cancer.

Adenocarcinoma↗