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G C Huang

Publications and source records attributed to G C Huang.

34 records · Page 2Linked to original sources
PubMed1996

Human immunoglobulin G autoantibodies to the thyrotropin receptor from Epstein-Barr virus-transformed B lymphocytes: characterization by immunoprecipitation with recombinant antigen and biological activity.

The TSH receptor (TSH-R) is the target antigen for disease-related autoantibodies in Graves' disease and primary myxoedema, but the repertoire of the antibodies or the nature of the precise antigenic epitopes is not known. We have immortalized peripheral blood B cells from six different autoimmune thyroid disease patients with Epstein-Barr virus and selected IgG-producing B cells by magnetic selection on anti-IgG-coated beads. Purified recombinant insect cell-derived extracellular region of TSH-R was used to identify the positive wells for expansion in culture. Stable B cell lines (n = 11) were obtained, which after limiting dilution led to two stable B cell clones. B cell lines and clones secreted IgG antibody that were shown to react biochemically with metabolically labeled or in vitro translated, nascent extracellular region of TSH-R, giving strong, confirmatory evidence of the presence of anti-TSH-R antibody. Supernatants from lines contained thyroid-stimulating activity, thyroid-blocking activity (as assessed by inhibition of TSH-mediated cAMP stimulation), or both of these activities. Interestingly, antibodies with stimulating activity were generated from a primary myxoedema patient, and antibodies of blocking specificities were obtained from newly diagnosed thyrotoxic Graves' disease patients. Our results favor a fine balance between stimulating and blocking autoantibody activities in determining the clinical presentation observed in patients with autoimmune thyroid disease patients who have these antibodies present in their serum.

Antigens↗
PubMed1996

Monoclonal antibodies to the human TSH receptor: epitope mapping and binding to the native receptor on the basolateral plasma membrane of thyroid follicular cells.

We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.

Amino Acid Sequence↗
PubMed1995

Unaltered thyroid function in mice responding to a highly immunogenic thyrotropin receptor: implications for the establishment of a mouse model for Graves' disease.

Grave's disease (GD) is a common disorder characterized by the presence of autoantibodies to the thyrotropin receptor. In the past, the exceedingly low expression of the thyrotropin receptor on thyrocytes has not allowed its purification in quantities sufficient to investigate the establishment of an animal model for this disease. In this study, we have purified the 398-amino acid, extracellular region of the human thyrotropin receptor (TSH-R.E) from insect cells using recombinant baculovirus, and explored its immunopathogenic properties in H-2b,d,q,k,s strains of mice. The receptor preparation was highly immunogenic since it elicited strong specific proliferative T cell responses as well as IgG responses in all strains tested. In addition, hyperimmunization with TSH-R.E induced (i) serum antibodies that blocked the binding of 125I-TSH to its receptor, a common feature of GD autoantibodies; and (ii) IgG that reacted with a synthetic peptide (residues 32-54) from the N-terminus of the receptor, a region implicated in the binding of thyroid stimulating antibodies. In SJL animals only, a weak antibody response to two other thyroid antigens, thyroglobulin and thyroid peroxidase, was also observed. The presence of these antibodies, however, was not accompanied by a detectable alteration in thyroid function as assessed by the measurement of serum TSH, T4 and iodine levels. Also mononuclear infiltration of the thyroid gland or morphological changes compatible with an activation state of thyrocytes were not apparent in TSH-R-challenged mice. In contrast, mice treated with the anti-oxidant aminotriazole showed a dramatic increase in serum TSH levels and an activated follicular epithelium. These data demonstrate that a highly immunogenic TSH-R.E in mice does not necessarily provide a proper stimulus for the induction of a hyper- or hypothyroid status as defined by hormonal or histological criteria. Main reasons for the inability to induce receptor-specific antibodies that affect thyroid function such as those generated in GD are likely to be the inappropriate folding of the recombinant extracellular domain of the receptor, or the xenogeneic nature of the autoantigen.

Animals↗
PubMed1995

HLA-DR-restricted T cell lines from newly diagnosed type 1 diabetic patients specific for insulinoma and normal islet beta cell proteins: lack of reactivity to glutamic acid decarboxylase.

T cells reacting with pancreatic islet beta cell proteins play a pivotal role in the pathogenesis of type 1 diabetes in experimental animal models and man, although the islet cell autoantigens against which these T cells are directed remain to be characterized. We have previously shown the presence of disease-related antigens residing in the transplantable RIN insulinoma membranes which are recognized by T cells from diabetic NOD mice. We now report on the establishment of CD4+, T cell lines reacting with insulinoma membranes from six newly diagnosed type 1 diabetic patients. Detailed examination of T cell lines from two patients revealed that both the lines continued to react with normal islet cell proteins and, interestingly, were also stimulated by antigens present in brain microsomes. The two T cell lines showed reactivity with different molecular weight proteins of the insulinoma membranes and both the lines were histocompatibility-linked antigen (HLA)-DR restricted. Although the insulinoma membrane preparation is known to contain glutamic acid decarboxylase (GAD), none of the six T cell lines proliferates in response to purified GAD. These T cell lines will be valuable in characterizing novel islet beta cell antigens which are likely to be implicated in type 1 diabetes.

Adult↗
PubMed1994

Localization of a pernicious anaemia autoantibody epitope on the alpha-subunit of human H,K-adenosine triphosphatase.

Four cDNA fragments encoding different portions of the alpha-subunit of human H,K-adenosine triphosphatase (ATPase) were amplified by means of the polymerase chain reaction technique, ligated into the plasmid pGEX-2T, and expressed as glutathione S-transferase fusion proteins in Escherichia coli. The fragments A (residues 163-313), Ba (residues 360-797), Bb (residues 526-797), and C (residues 822-1031) together encompass 77% of the alpha-subunit and cover most of its cytosolic part. The reactivities of autoantibodies in the sera from patients with pernicious anaemia with the recombinant fusion proteins were analysed by immunoblotting. One autoantigenic epitope was found in the NH2-terminal part of the Ba fragment--that is, between residues 360 and 525. No epitope was detected in the other fragments. The Ba fragment was cleaved off from the glutathione S-transferase fusion protein by the action of thrombin and was then further purified. By means of enzyme-linked immunosorbent assay, 28 of 42 sera (67%) from patients with pernicious anaemia were positive against the purified Ba fragment. The present results provide a final proof that the human H,K-ATPase alpha-subunit is a major autoantigen in the parietal cell and that the major epitope is located between residues 360 to 525 on the cytosolic side of the secretory membrane.

Adult↗
PubMed1993

The thyrotrophin hormone receptor of Graves' disease: overexpression of the extracellular domain in insect cells using recombinant baculovirus, immunoaffinity purification and analysis of autoantibody binding.

Since the cloning of the TSH receptor (TSH-R), the target autoantigen of Graves' disease, the receptor has been expressed in a variety of eukaryotic cells to obtain a functional molecule. Despite this success, the levels of receptor expression have been marginally higher than the extremely low levels found in thyroid cells, preventing any progress on the purification of the molecule. In this study, the large extracellular region of the TSH-R, without the membrane spanning segments, has been expressed in insect cells using recombinant baculovirus to generate substantial quantities of the receptor protein. A monoclonal antibody previously generated to a bacterial TSH-R fusion protein was used to characterize and monitor the expression of the truncated receptor in insect cells. Two polypeptides of 63 and 49 kDa were recognized as the components of the truncated recombinant receptor. The 63 kDa protein was shown to be the glycosylated form of the smaller, 49 kDa, component. Expression in different insect cell lines showed that an increase in expression of approximately tenfold was apparent in High Five cells when compared with Sf21 cells. Very small quantities of the truncated receptor were secreted by the three insect cell lines examined, with the majority of the molecule being retained within the cells. Immunoaffinity purification of milligram quantities of the truncated receptor was achieved using the monoclonal antibody. The availability of the purified TSH-R has allowed the establishment of an enzyme-linked immunosorbent assay to measure autoantibodies in the sera of patients with Graves' disease. Although the truncated receptor interacts with autoantibodies, our results show that it does not bind TSH and differs in this respect from other glycoprotein hormone receptors.

Animals↗
PubMed1992

Expression of a human thyrotrophin receptor fragment in Escherichia coli and its interaction with the hormone and autoantibodies from patients with Graves' disease.

Graves' disease is an autoimmune thyroid disease characterized by the presence of pathogenic autoantibodies to the TSH receptor (TSH-R). By using polymerase chain reaction, the extracellular region of the human TSH-R cDNA has been amplified and used to prepare recombinant TSH-R (extracellular) protein fused with glutathione-S-transferase (GST). Purification of the recombinant TSH-R (extracellular)-GST fusion protein was achieved by preparative gel electrophoresis in SDS or by preparative isoelectric focusing in urea. Following removal of SDS by detergent exchange or urea by dialysis, the purified recombinant receptor preparations were assessed for binding to the hormone or to autoantibodies from Graves' disease patients. The purified recombinant receptor preparations fail to show any binding to the hormone or autoantibodies either by inhibition of binding assays or by immunoblotting. The results imply that the correct folding and/or post-translational modifications of the polypeptide chain which are not achieved in recombinant proteins produced in Escherichia coli may be important for the binding of the hormone or Graves' disease autoantibodies to the TSH-R. The recombinant receptor prepared in this manner will be useful for immunological and cellular investigations in patients with Graves' disease.

Autoantibodies↗
PubMed1991

Autoantibody stimulation of the human thyrotropin receptor: regulation of adenylate cyclase activity, thyroglobulin and thyroid peroxidase mRNA levels in primary cultures of Graves' thyroid tissue.

The effects of immunoglobulin preparations from hyperthyroid Graves' disease patients on primary cultures of thyroid cells have been studied at the mRNA level. Autoantibodies to the thyrotropin (TSH) receptor from these patients, which had been initially characterized by their ability to stimulate adenylate cyclase and inhibit the binding of radiolabelled TSH to thyroid membrane preparations, were studied for their effects on thyroglobulin and thyroid peroxidase mRNA levels. Incubation of thyroid cells with TSH receptor autoantibodies from different Graves' disease patients for 48 h led to time- and dose-dependent increases in the levels of thyroid peroxidase and thyroglobulin mRNA in primary cultures of thyrocytes. The incomplete correlation between G protein-linked adenylate cyclase activation and thyroid mRNA elevation indicates the possibility of the involvement of alternative second messenger pathways in the regulation of thyroid cell function and differentiation.

Adenylyl Cyclases↗
PubMed1991

[Eighty cases of alveolar hydatid disease treated with albendazole].

Alveolar hydatid disease occurs frequently in Xiji County of Ningxia. 80 cases of the disease were treated orally with albendazole from 1985 to 1988 and the short-term results proved satisfactory. The treatment consisted of two groups. Group I included 72 cases which were orally given 8-10 mg/kg.d of albendazole 2 to 3 times a day after each meal for 30 days and then stop taking the medicine for 10 days as one treatment course. The treatment continued 3-12 months comprising about 24 courses were given with the highest dosage of 412g. Group II included 8 cases who were given 15.6-20 mg/kg.d of the medicine for 14 days (one course) and every two weeks with a 10 days interval. The treatment of group II continued 3-6 months which totalled 19-24 courses with the highest dosage of 270g. All the treated patients tolerate well except that a few of them felt giddy and had diarrhoea which disappeared after the withdrawal of medicine. Through 3-5 years, follow-up observation, it was found that the gener alsymptoms of the treated patients improved and their body weight increased by 2-9.5 kg, some of them could take part in light physical labour. The jaundice index in 80% of them returned to normal. SGPT in 86.4% decreased to within normal range. The enlarged livers in half of the patients decreased by 1-6 cm. 23.7% of the patients were reexamined by B ultrasound scanning which revealed that their enlarged liver reduced in size while the liquifaction area and the shadows of calcification in the livers in 42.5% patients increased obviously.

Adolescent↗
PubMed1990

Molecular cloning of a human thyrotropin receptor cDNA fragment. Use of highly degenerate, inosine containing primers derived from aligned amino acid sequences of a homologous family of glycoprotein hormone receptors.

Autoantibodies to the thyrotropin (TSH) hormone receptor (TSH-R) are present in the sera of patients with thyroid autoimmune disease which are pathogenetic leading to hyperthyroidism of Graves' disease. Considerable interest has been focused on the cloning of the human TSH-R, which has until very recently, proven exceedingly difficult due to the very low receptor level expression on thyroid cells. We have used polymerase chain reaction and highly degenerate, inosine containing oligonucleotides derived from sequence alignments of the transmembrane regions 2 and 7 of a number of G-binding protein receptors including the lutropin/choriogonadotropin (LH/CG) receptors to amplify various cDNAs from human thyroid cDNA. Sequencing analysis of 27 different clones revealed that they fall into eight different groups. The very recent publication of the complete nucleotide sequence of the human TSH-R revealed that one of the groups (GT1) containing seven clones which had been sequenced belong to the human TSH-receptor. The sequence of all 7 GT1 clones was identical and in complete concordance with transmembrane regions 2 and 7 of the published TSH-R sequence. Our results show that by designing oligonucleotides to common transmembrane regions of G-binding proteins where the primers are biased in their sequence to the LH/CG receptors it is possible to amplify the TSH-R receptor sequence.

Amino Acid Sequence↗
PubMedDate not supplied

Controlled release effect of insecticide microcapsules and their results in common household insect pest control.

Since 1978 we have prepared microencapsulated insecticides by complex coacervation and interfacial polymerization for household insect pest control, fenthion being the main core element. A series of tests have been conducted against mosquitoes (Culex pipiens quinquefasciatus), cockroaches (Periplaneta americana) and bedbugs (Cimex lectularius), and the results indicate that microencapsulation can prolong persistent effects by 2-12 times, enhance safety and subdue offensive odour. Furthermore, the production cost is comparatively low.

Animals↗
PubMedDate not supplied

Eradiction of H. pylori results in regression of B-cell low grade gastric MALToma with evident B-symptoms.

Although it is well known that eradication of H. pylori may result in either complete or partial regression of low-grade B-cell mucosa-associated lymphoid tissue lymphoma (MALToma), it would be of clinical interest to determine whether the B-symptoms of patients with MALToma could be relieved by eradication of H. pylori. Here, we report on a 29 year-old female case with B-cell low-grade gastric MALToma with apparent B-symptoms. Her peripheral blood also disclosed large granular lymphocytes (LGL). The B-symptoms of this patient were quickly relieved within 2 weeks after starting an anti-H. pylori regimen; peripheral blood LGLs were clearly decreased as well. Complete regression of MALToma was determined 4 months after the anti-H. pylori regimen. Thereafter, the patient has been disease-free and in good general condition during a 2-year follow-up.

Adult↗