Biomedical subjects

T Akamizu

Publications and source records attributed to T Akamizu.

At least 55 records · Page 3Linked to original sources

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

Establishment and characterization of an antihuman thyrotropin (TSH) receptor-specific CD4+ T cell line from a patient with Graves' disease: evidence for multiple T cell epitopes on the TSH receptor including the transmembrane domain.

From the peripheral lymphocytes of a patient with Graves' disease, we established a T cell line using its reaction to a pool of 49 synthetic peptides corresponding to the entire human thyrotropin receptor (TSHR) sequence. This T cell line showed a specific response to the pool of peptides in a microproliferation assay (stimulation index: 4.8). Flow cytometry analysis revealed that the cell surface markers were CD4+ CD8-, T cell receptor (TcR) alpha beta+, and Tcr gamma delta-. To investigate T cell epitopes on TSHR, the T cell line reacted well against three groups: the N-terminal (amino acids 31-169) and C-terminal (338-420) regions of the extracellular domain and the N-terminal half (441-661) of the transmembrane domain of the receptor. This suggests a multiplicity of T cell epitopes on the TSHR, and was further supported by analysis of TcR gene expression in the cell line that showed the expression of 5 V alpha genes; V alpha-1, 2, 10, 20, and w25. In conclusion, the results of the present study indicated multiple T cell epitopes on the TSHR molecule including the transmembrane domain.

Adult↗

PubMed1995

Molecular studies on thyrotropin (TSH) receptor and anti-TSH receptor antibodies.

Cloning of TSHR gene and some subsequent studies using the gene were described. Enormous numbers of studies have been performed since the cloning of TSHR gene. Recent molecular studies on TSH receptor and TSHRAb gave various impacts on thyroidology and are resolving past problems. We mainly focused on regulation, processing and glycosylation, TSH- and TSHRAb binding sites, T cell epitopes, and signal transduction of TSHR. Furthermore, we isolated and characterized TSHRAb genes using lymphocytes producing monoclonal TSHRAb obtained from patients with Graves' disease and primary hypothyroidism. Thus, both antigen and antibody genes are cloned. Combined use of these genes will help to investigate the interactions between TSHR and TSHRAb, and may be expected to contribute to the understanding of molecular mechanisms underlying the pathogenesis of autoimmune thyroid disease as well as the physiology of the thyroid gland.

Amino Acid Sequence↗

PubMed1995

A hyperthyroid patient with Graves' disease who was strongly resistant to methimazole: investigation on possible mechanisms of the resistance.

We treated a patient who was hyperthyroid due to Graves' disease and strongly resistant to methimazole (MMI): in spite of good compliance, she needed 150 mg of MMI daily to control her hyperthyroidism. To elucidate the reasons of resistance to MMI, her serum and intrathyroidal MMI concentrations were determined by high pressure liquid chromatography (HPLC). After taking a 30 mg dose of MMI, she had a similar serum MMI concentration-time curve to that of a normal subject: drug malabsorption and rapid drug metabolism were not evident when studied after surgical treatment. After her serum containing MMI was incubated with Protein G, the MMI concentration of the fraction not bound to Protein G did not change significantly from that of untreated serum: the possibility of anti-MMI IgG antibody production was considered unlikely. Furthermore, the intrathyroidal concentration of MMI in a surgically obtained tissue specimen was 3 micrograms/g wet tissue and appeared to be comparable with those of other Graves' tissues reported. Considering that the patient had been taking 150 mg per day of MMI by the time of thyroidectomy, her intrathyroidal MMI concentration was relatively low, suggesting possible impairment of intrathyroidal MMI accumulation. The possibilities of impaired intrathyroidal actions and the severity of hyperthyroidism, especially high T3 levels, also remained as possible causes. In conclusion, here was a severely hyperthyroid patient who was poorly responsive to conventional doses of MMI, and impairment of thyroid uptake of MMI or of pathways after uptake were considered as possible mechanisms.

Adult↗

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↗

PubMed1995

[Recent progress in the diagnosis of thyroid diseases].

The diagnosis of thyroid diseases is mainly divided into three categories: morphological, functional and etiological diagnoses. Great progress has recently been seen in each field. For example, in the functional diagnosis, improvement of sensitivities and development of non-radioimmunoassays have been achieved in the measurement of hormone level. Especially, improvement of thyrotropin (TSH) measurement has greatly contributed to precise evaluation of thyroid function. In the etiological diagnosis, marked improvements have been made by genetic analyses. Mutations of genes of hormones, their receptors and other proteins related to thyroid function have been revealed as causes of various thyroid disorders, such as cretinism, hyperthyroidism and tumors. Immunological analyses also showed significant progress, i.e., improvement of anti-thyroglobulin and anti-thyroid peroxidase antibody measurements. It is also necessary for all three diagnostical approaches to be improved concomitantly in the future.

Humans↗

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

Possible difference in glycosylation of the thyrotropin receptor among species.

Residue 113 of the thyrotropin receptor (TSHR) is a possible asparagine-linked glycosylation site in the human TSHR, but not in rat or dog TSHR. Russo et al. (Mol Endocrinol 5:29-33) reported that mutation of this residue in the human TSHR diminished TSH binding activity after transfection. To investigate the difference in the role of residue 113 of the TSHR among species, we created a mutant at residue 113 in the rat TSHR, transfected Cos-7 cells with the mutant DNA and measured TSH binding and TSH- and Graves' IgG-stimulated cAMP and phosphoinositide signals. No difference was found in the activities of the mutant transfectant from the wild type receptor transfectant. These results might suggest a real difference in glycosylation of the TSHR among species.

Animals↗

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

Further studies of amino acids (268-304) in thyrotropin (TSH)--lutropin/chorionic gonadotropin (LH/CG) receptor chimeras: cysteine-301 is important in TSH binding and receptor tertiary structure.

Our previous study of chimeric TSH-LH/CG receptors showed that substituting amino acid residues 268-304 of the TSH receptor with homologous residues from the LH/CG receptor markedly decreased high affinity TSH binding as evidenced by ligand displacement assays [Akamizu et al. Endocr J 40:363-372, 1993]. Despite this change in TSH binding, there was a minimal change in TSH-stimulated activity in cAMP assays. To explain this dissociation of TSH binding and function, further characterization of individual residues within the 268-304 segment was performed. Five additional chimeric TSH-LH/CG receptors within this region were constructed by substituting corresponding residues of rat LH/CG receptor. After transfection in Cos-7 cells, TSH receptor activities of these chimeras were evaluated. A single point mutation of cysteine-301 to glutamate resulted in a mutant receptor that exhibited the same receptor bioactivity as the chimeric receptor with amino acids 268-304 substituted by LH/CG receptor residues: apparent low affinity TSH binding in ligand displacement assays but significant retention of the cAMP response to TSH. The four other chimeric substitutions yielded cDNAs coding for receptors that behaved like wild-type receptors. Western blot analyses using a TSH receptor-specific antibody showed that all five of the new mutants were synthesized and integrated in the membrane. These results suggest that cysteine-301 is the critical residue whose mutation accounts for the original chimeric results and the dissociation of binding and functional activity. The possibility must be considered that cysteine-301 is involved in disulfide bond formation and is important in tertiary structure and that conformational changes in the receptor result from its mutation.

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

The middle portion in the second cytoplasmic loop of the thyrotropin receptor plays a crucial role in adenylate cyclase activation.

We have examined the role of the 2nd cytoplasmic loop of the TSH receptor (TSHR) in TSH- and TSHR autoantibody-stimulated cAMP and inositol phosphate formation using mutants created by substituting sequences from the alpha 1- or beta 2-adrenergic receptors (AR). Unlike similar substitution mutants involving the 3rd cytoplasmic loop that lose agonist-induced inositol phosphate but not cAMP increase after transfection into Cos-7 cells, mutants involving the 2nd loop showed significant change in generating both signals. Mutant B525, which substitutes residues 525-527 with a comparable beta 2-AR sequence, exhibited a complete loss in TSH- or Graves' immunoglobulin G-increased cAMP signaling and a lesser loss in phosphoinositide signaling. This is a unique mutant in which cAMP response was completely lost in all those involving the 2nd or 3rd cytoplasmic loop. On the other hand, mutant B528, in which residues 528-532 are substituted with a comparable beta 2-AR sequence, exhibited the most profound loss in phosphoinositide signaling. Mutants involving portions surrounding residues 528-532 in the 2nd cytoplasmic loop had milder losses in agonist-increased phosphoinositide signaling and much lesser losses in agonist-increased cAMP generation. The transfection efficiency of all transfectants was the same. All transfectants with mutant or wild type TSHR had a similar amount and identical profile of TSHR mRNA in Northern blots and TSHR forms on Western blots. Thus, the 2nd cytoplasmic loop is important for agonist-induced cAMP as well as for phosphoinositide signal generation, whereas the 3rd loop appears to be important only for the latter. The most important determinant for agonist-increased cAMP signal generation is in the middle of the 2nd loop, around residues 525-527. In contrast, the determinants most critical for agonist-induced phosphoinositide signaling are also located in the middle of the 2nd loop, around residues 528-532, and those with less importance are broadly distributed.

Adenylyl Cyclases↗

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↗

PubMed1994

[Anti-thyrotropin (TSH) receptor antibody binding epitopes of TSH receptor: site-directed mutagenesis approach].

Anti-TSH receptor antibodies are thought to be involved in the expression of autoimmune thyroid diseases, especially Graves' disease and idiopathic myxedema. Cloning of TSH receptor gene allowed us to study aspects of its structure and function at the molecular level, including autoantibody-binding sites. The long extracellular domain of TSH receptor was presumed to contain antibody-binding site. Site-directed mutagenesis of this domain defined regions important for autoantibodies. The C-terminal region of the extracellular domain, residues 295-306, 387-395 and tyrosine 385 were determinants of the "blocking-type" antibody, which were present in patients with primary hypothyroidism. And the N-terminal region, residues 34-37, 40, 42-45 and 52-56 were the site of the "stimulatory-type" antibody interactions, important in patients with Graves' disease.

Amino Acid Sequence↗

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

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↗