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J Furmaniak

Publications and source records attributed to J Furmaniak.

At least 19 recordsLinked to original sources
PubMed2006

Effects of a thyroid-stimulating human monoclonal autoantibody (M22) on functional activity of LH and FSH receptors.

OBJECTIVE: The glycoprotein hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyrotropin (TSH) show low-level cross-reactivity between their respective receptors (R). Patient serum autoantibodies to the thyrotropin receptor (TSHR) do not appear to cross-react with the luteinizing hormone receptor (LHR) or follicle-stimulating hormone receptor (FSHR), although the concentrations of autoantibody with which it is feasible to carry out experiments of this type are limited. Consequently, we have studied the effects of high doses of the thyroid-stimulating human monoclonal autoantibody (M22) on the LHR and FSHR. DESIGN: Chinese Hamster ovary (CHO) cells stably expressing the TSHR, LHR, and FSHR and purified M22 IgG preparations were used in the study. METHODS: CHO-TSHR, CHO-LHR, and CHO-FSHR cells were incubated with bovine TSH (0.1-25mU/mL), human recombinant chorionic gonadotropin (hCG; 0.5-10mU/mL) or human recombinant FSH (100-5000mU/mL) or with M22 IgG (0.001-5.0 microg/mL), and the extracellular cyclic AMP was measured by radioimmunoassay. RESULTS: Cyclic AMP levels increased in a dose-dependent manner after incubation of CHO-TSHR cells with TSH or M22 IgG, and on a molar basis the effects of TSH and M22 were similar. Cyclic AMP stimulation was not detectable in CHO-LHR and CHO-FSHR cells after incubation with M22 IgG, whereas incubation with hCG or FSH, respectively, caused dose-dependent cyclic AMP stimulation. On a molar basis, concentrations of M22 IgG approximately 100x those of FSH causing clear stimulation were ineffective with CHO-FSHR cells. Similarly, molar concentration of M22 IgG 20,000x those of hCG causing clear stimulation had no effect on CHO-LHR cells. CONCLUSIONS: This study shows that at relatively high concentrations, M22 IgG is unable to stimulate cyclic AMP levels in CHO-LHR or CHO-FSHR cells, suggesting that TSHR autoantibodies have greater specificity for the TSHR than TSH itself.

Animals↗
PubMed2005

Isolation and characterisation of a human monoclonal autoantibody to the islet cell autoantigen IA-2.

A hybridoma secreting a human monoclonal autoantibody to the islet cell autoantigen IA-2 was prepared from peripheral lymphocytes of a patient with type 1 diabetes and Graves' disease using EBV infection followed by fusion with a mouse/human hybrid cell line. The monoclonal antibody (M13) is an IgG1/kappa and in an immunofluorescence test M13 at 1 microg/mL showed islet cell antibody reactivity equivalent to 40 JDF units. M13 IgG bound (35)S-labelled IA-2 (26% at 100 microg/mL) and (125)I-labelled IA-2 (34% at 100 microg/mL) in an immunoprecipitation assay and reacted well with IA-2 in western blotting analysis. Amino acids 777-808 in the PTP domain of IA-2 were found to be important for M13 binding in an analysis using modified (35)S-labelled IA-2 proteins. M13 V region genes were from VH1-3, D3-22, JH4b, VKI DPK8/Vd+ and JK3 genes and showed a high replacement/silent mutation ratio for both the heavy (11.0) and the light (6.0) chain genes. Mouse monoclonal antibodies (mMAbs) reactive with at least three different epitopes within IA-2 aa 604-686 corresponding to the juxtamembrane domain were also obtained. F(ab')(2) or Fab from the mMAbs inhibited serum IA-2 autoantibody binding to IA-2 in 20/22 diabetic sera whereas M13 F(ab')(2) caused inhibition in only 6/22 sera. M13 is representative of some patient serum IA-2 autoantibodies and as such provides a useful tool to study autoimmune responses to IA-2.

Animals↗
PubMed2005

Characteristics of a monoclonal antibody to the thyrotropin receptor that acts as a powerful thyroid-stimulating autoantibody antagonist.

Analysis of nine mouse monoclonal antibodies (mAbs) to the thyrotropin receptor (TSHR) with TSH antagonist activity showed that only one of the mAbs (RSR B2) was an effective antagonist of the human thyroid stimulating autoantibody M22. Crystals of B2 Fab were analyzed by x-ray diffraction and a crystal structure at 3.3 A resolution was obtained. The surface charge and topography of the B2 antigen binding site were markedly different from those of the thyroid-stimulating mAb M22 and these differences might contribute to the different properties of the two mAbs. B2 (but not other mouse TSHR-specific mAbs) was also an effective antagonist of thyroid stimulating autoantibody activity in 14 of 14 different sera from patients with Graves' disease. 125I-labeled B2 bound to the TSHR with high affinity (2 x 10(10) L/mol) and patient serum TSHR autoantibodies inhibited labeled B2 binding to the receptor in a similar way to inhibition of labeled TSH binding (r = 0.75; n = 20). Furthermore, labeled B2 binding was inhibited by patient serum TSHR autoantibodies with TSH antagonist activity and also by mouse and human thyroid stimulating mAbs. Overall, mAb B2 is a powerful antagonist of thyroid stimulating autoantibodies (and TSH) thus resembling closely patient serum TSH antagonist TSHR autoantibodies. Furthermore, B2 might have potentially important in vivo applications when tissues containing the TSHR (including those in the orbit) need to be made unresponsive to stimulating autoantibodies.

Animals↗
PubMed2005

Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison's disease.

OBJECTIVE: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs) in vitro. DESIGN: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21. METHODS: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH. RESULTS: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21. CONCLUSIONS: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.

Addison Disease↗
PubMed2004

Characteristics of a human monoclonal autoantibody to the thyrotropin receptor: sequence structure and function.

The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described. Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation. Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation. Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22. These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity. Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation. The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain. The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction.

Animals↗
PubMed2003

Human monoclonal thyroid stimulating autoantibody.

A monoclonal autoantibody (MAb) with powerful thyroid stimulating activity has been produced from lymphocytes from a patient with Graves' disease. The autoantibody and its Fab fragment bind to the thyroid stimulating hormone (TSH) receptor (TSHR) with high affinity, inhibit labelled TSH binding to the receptor and stimulate cyclic AMP production in Chinese hamster ovary cells transfected with TSHR. TSHR autoantibodies with TSH agonist or antagonist activities from patients' serum samples are effective inhibitors of labelled monoclonal autoantibody binding to TSHR. Thus, the human monoclonal autoantibody has all the characteristics of serum TSHR autoantibodies. Its availability has important implications for new studies on the pathogenesis of Graves' disease.

Antibodies, Monoclonal↗
PubMed2003

Autoantibodies to steroidogenic enzymes in patients with premature ovarian failure with and without Addison's disease.

DESIGN: Adrenal cortex autoantibodies (ACA), steroid-producing cell autoantibodies (StCA) and autoantibodies (Abs) to steroidogenic enzymes in three groups of patients with premature ovarian failure (POF), 15 with autoimmune Addison's disease (AD), 26 with non-adrenal autoimmune diseases and 31 with isolated POF, have been assessed. METHODS: ACA and StCA were measured using an immunofluorescence technique. Abs to 21-hydroxylase (21-OH), to 17alpha-hydroxylase (17alpha-OH) and to cytochrome P450 side-chain cleavage (P450scc) were measured using an immunoprecipitation assay. RESULTS: Seventy-three percent of patients with POF and AD were positive for StCA, 93% for 17alpha-OH and/or P450scc Abs, 93% for ACA and 100% for 21-OH Abs. Among patients with POF and non-adrenal autoimmune diseases, 8% were positive for StCA, 12% for 17alpha-OH and/or P450scc Abs, and 8% and 12% for ACA and 21-OH Abs respectively. StCA, 17alpha-OH and/or P450scc Abs were all found in 10% of patients with isolated POF, and 13% had ACA and 21-OH Abs. All StCA-, 17alpha-OH- and/or P450scc Abs-positive patients were also positive for ACA and 21-OH Abs. Two patients with isolated POF who were ACA and 21-OH Ab positive developed AD 3 and 5 Years after the onset of POF. CONCLUSION: This study has shown that, when POF is associated with AD, StCA, 17alpha-OH and/or P450scc Abs are present in the majority of patients, while in the other two groups these Abs are detectable in a much lower proportion of patients. Measurement of ACA/21-OH Abs in some patients with POF may be important in identifying patients at risk of developing overt AD.

Addison Disease↗
PubMed2002

Isolation and characterization of human monoclonal autoantibodies to glutamic acid decarboxylase.

Production of human monoclonal autoantibodies to glutamic acid decarboxylase M(r) 65,000 (GAD65), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD65 autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD65 were produced using standard techniques. F(ab')2S from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to 125I-labelled GAD65 (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M(r) chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD65 of 2.2 x 10(9), 5.8 x 10(9), 1.3 x 10(10) mol/l(-1); affinities of the mouse monoclonals (n = 5) ranged from 1.1 x 10(8) to 5.4 x 10(10) mol/l(-1). The binding of each of the human monoclonals was inhibited by GAD6 F(ab')2 and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')2S suggesting that the epitopes for these antibodies were overlapping. Studies with GAD65/GAD67 chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to 125I-labelled GAD65. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD65 in the donor's serum and in the sera of patients with type-1 diabetes.

Addison Disease↗
PubMed2001

Antibodies to tissue transglutaminase: comparison of ELISA and immunoprecipitation assay in the presence and in the absence of calcium ions.

Endomysial antibodies are characteristic of coeliac disease and tissue transglutaminase (tTG) has been identified as a major component of the endomysial antigen. tTG autoantibodies were measured in sera from patients with coeliac disease using ELISA based on guinea pig tTG and immunoprecipitation assay (IPA) based on 35S-labelled human tTG produced in an in vitro transcription/translation system. In addition, the effect of calcium ions on the interaction between tTG autoantibodies in the two assays was studied. Under standard (i.e. Ca2+-free) conditions, 36/39 (92%) coeliac sera were positive for IgA tTG antibodies by ELISA and 34/39 (87%) sera were positive by IPA. Comparison of ELISA and IPA results showed three sera positive by ELISA but negative by IPA and one serum which was positive by IPA but negative by ELISA. Bland and Altman analysis of the correlation between the ELISA and IPA showed that the results for 37 out of 39 samples were in the agreement. The results by ELISA carried out without and with Ca2+ were in good agreement (r=0.99; n=39). IPA using Ca2+ containing buffer detected fewer samples compared to IPA using standard assay buffer however the results of the two assays also showed a good agreement (r=0.93; n=39). Our studies confirm that antibodies to tTG are good markers of coeliac disease and indicate that the autoantibody binding sites on tTG are formed in a way which is essentially independent of Ca2+.

Adolescent↗
PubMed2001

X-ray crystal structure of a monoclonal antibody that binds to a major autoantigenic epitope on thyroid peroxidase.

Thyroid peroxidase (TPO) catalyzes the production of thyroid hormones and is a major autoantigen in autoimmune thyroid disease (AITD). It is believed that the majority of TPO autoantibodies bind to an immunodominant region consisting of two overlapping domains. Precise location of these domains would help our understanding of the interaction between TPO and TPO autoantibodies. 4F5 is a mouse monoclonal antibody (IgG1, kappa) that reacts with high affinity (2.6 x 10(10) mol/L(-1)) with one of the major autoantigenic regions on TPO. Heavy chain genes of 4F5 were from the VH1 germline gene family, germline genes for the D region could not be assigned and the J region was from the JH2 germline. Light chain genes were from Vkappa4/5 and Jkappa2, germline gene families. The Fab fragment of 4F5 was prepared by papain digestion, purified, crystallized, and the structure solved to 1.9 A using molecular replacement. The refined structure had an R factor of 19.5% and a free R factor of 23.9%. Deduced amino acid sequence and amino acid sequence obtained from diffraction analysis were compared and used to finalize the 4F5 Fab model. Structural analysis indicated that the structure of 4F5 is that of a standard Fab and its combining site is flat and is rich in tyrosine residues. Comparison of the structure of 4F5 with that of a TPO autoantibody Fab, TR1.9 suggests that the two antibodies are unlikely to recognise the same structures on TPO.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Reactivity of thyrotropin receptor autoantibodies with the thyrotropin receptor on western blots.

Affinity purified recombinant human thyrotropin receptor (TSHR) was run on sodium dodecyl sulfate (SDS) gels and subjected to a renaturing and blotting procedure. Twenty sera from thyrotropin receptor autoantibodies (TRAb)-positive patients with a history of hyperthyroidism and 20 sera with high levels of TSH blocking activity were analyzed. Four of 20 sera with blocking-type of TRAb (i.e., TSH antagonist activity) were able to recognize the mature, fully glycosylated 120-kd form of the receptor on blots of gels run under reducing conditions. No sera recognized the 100-kd high mannose precursor form of the TSHR. Three of the four recognized a 74-kd band and 2 of the 4 recognized a 50-kd band. These bands are probably proteolytic cleavage fragments of the mature 120-kd TSHR. In the absence of reducing agent the same 4 of 20 sera described above together with a further serum sample (i.e., 5/20 in total) reacted with the 120-kd form of the receptor. No specific reaction with the TSHR was observed on Western blots with the remaining 15 sera with TSH blocking activity, nor with 20 sera from patients with a history of hyperthyroidism, nor with sera from 10 healthy blood donors, 10 Hashimoto sera (negative for TRAb) and 10 systemic lupus erythematosus sera. No clear differences were observed in the TRAb positive sera that were reactive and nonreactive on Western blots in terms of their ability to inhibit TSH binding or to immunoprecipitate 125I-labeled TSHR. Overall, our results indicate that the mature 120-kd form of the TSHR that is principally responsible for binding TSH is also responsible for binding TRAb (when this binding can be detected). These observations together with immunoprecipitation and TSH binding inhibition studies, emphasize the close relationship between the receptor's binding sites for TSH and TRAb.

Antibody Specificity↗
PubMed2000

Autoantibodies to IA-2 in insulin-dependent diabetes mellitus. Measurements with a new immunoprecipitation assay.

An immunoprecipitation assay for autoantibodies (Abs) to the human islet cell antigen IA-2 has been developed using 125I-labelled recombinant IA-2 expressed in E. coli. With this assay IA-2 Abs were detected in 103/217 (47%) of IDDM patients of different ages and with different disease duration. IA-2 Ab prevalence was higher in younger patients (at the age of 15 years or below) with the recent onset IDDM (64/113; 57%) compared to patients above the age of 15 years (11/25; 44%). One of 40 (2.5%) Graves' disease patients and five of 204 (2.5%) of NIDDM patients were also positive. IA-2 Abs were not detected in sera from patients with Hashimoto's thyroiditis (n=32), myasthenia gravis (n=20) or systemic lupus erythematosus (n=10). IA-2 Ab measurements based on 125I-labelled IA-2 showed a good correlation with the results of an immunoprecipitation assay based on 35S-labelled IA-2 produced in the in vitro transcription/translation system (r=0.78; n=113; p<0.001). Out of 217 IDDM sera which were tested for IA-2 Abs, 140 (65%) were positive for Abs to glutamic acid decarboxylase (GAD) and 166 (76%) were positive for Abs to IA-2 and/or Abs to GAD. In addition, Abs to IA-2, to GAD and to insulin were analysed in sera from recent onset IDDM patients who had not been treated with insulin (n=117). In all, 76/117 (65%) of these sera were positive for GAD Abs, 66/117 (56%) for IA-2 Abs, 45/117 (38%) for insulin Abs. However, 98/117 (84%) were positive for at least one of the three Abs confirming earlier observations on the complementarity of Ab testing in IDDM. Overall, the IA-2 Ab assay based on 125I-labelled recombinant IA-2 showed good sensitivity, precision and specificity which, combined with an easy and convenient protocol, makes it attractive for routine use.

Adolescent↗
PubMed2000

Epitope analysis of the human thyrotropin (TSH) receptor using monoclonal antibodies.

A panel of thyrotropin (TSH) receptor (TSHR) monoclonal antibodies (mAbs), produced using highly purified Chinese hamster ovary (CHO) cell-produced TSHR, has been used to study TSHR structure. All 41 mAbs recognized full-length TSHR containing complex carbohydrate (120 kDa), and 40 mAbs recognized full-length precursor-containing high mannose sugars (100 kDa). The mAbs also recognized TSHR cleavage products with three types of reactivity: type 1 mAbs reacting with bands at 70 kDa and 58 kDa, type 2 with bands at 70 kDa and 52 kDa, and type 3 with bands at 52 kDa and 40 kDa. Deglycosylation studies showed that the 70-kDa and 58-kDa bands contained complex carbohydrate, whereas the 52-kDa and 40-kDa bands were unglycosylated. These results are consistent with TSHR cleavage occurring at two sites. Cleavage at both sites gives rise to glycosylated A subunit (58 kDa) corresponding to the extracellular domain of the receptor and nonglycosylated B subunit (40 kDa) corresponding to the C-terminal transmembrane domain. Cleavage only at site 1 gives rise to the 58-kDa A subunit and a large B subunit (52 kDa). Cleavage only at site 2 gives rise to a large A subunit (70 kDa) and the B subunit (40 kDa). Four of the mAbs inhibited 125I-labeled TSH binding to solubilized full-length TSHR. TSH binding was inhibited by (a) two type 3 mAbs reactive with the N-terminal region of the B subunit (epitopes between amino acids 381 and 385 and between 380 and 418, respectively) and (b) two type 2 mAbs reactive with epitopes on the A subunit (between amino acids 246 and 260). These results together with previous studies on the direct binding of TSH to the TSHR A subunit suggest that at least two distinct regions of the TSHR sequence, including one region on the A subunit and one region on the B subunit, fold together to form part of a complex TSH binding site.

Amino Acid Sequence↗
PubMed2000

Recombinant human thyroid peroxidase produced in insect cells has similar properties to native human thyroid peroxidase.

Purified native human thyroid peroxidase (nTPO) isolated from thyroid tissue and recombinant (r)TPO produced in High Five insect cells have been compared. nTPO and rTPO were purified to about 95% homogeneity and showed similar UV and visual spectra and similar 412 nm per 280 nm absorbance ratios (0.4 for nTPO and 0.4 for rTPO). The nTPO and rTPO guaiacol oxidation enzyme activities were about 1,000 guaiacol units per milligram of protein. TPO autoantibody binding characteristics of nTPO and rTPO were analyzed in an assay based on 125I-labeled nTPO and precipitation with protein A. In the assay, the effect of unlabeled nTPO or rTPO on TPO autoantibody binding from 25 patients sera was studied. Unlabeled nTPO or rTPO (from 0 to 160 ng/mL) inhibited the binding of TPO autoantibodies in a dose-dependent manner in the case of each serum studied (from 100% in the absence of unlabeled TPO to 5%-10% in the presence of 160 ng/mL of TPO). The inhibition profile for each serum was essentially identical in the case of both TPO preparations. The effect of TPO autoantibodies on enzyme activity of rTPO was analyzed after incubation of rTPO with TPO autoantibody-positive serum immunoglobulin G (IgG) (n = 12), TPO monoclonal antibodies reactive with two different epitopes on the TPO, IgG (n = 3) from glutamic acid decarboxylase autoantibody positive patient sera, and IgG (n = 3) from healthy blood donors. Effective complexing of TPO by TPO autoantibodies was tested by precipitating the complexes with solid phase protein A and measuring the TPO enzyme activity in the resulting supernatants. These studies showed that the TPO enzyme activity was not affected by incubation with TPO autoantibody-positive IgG or monoclonal antibodies despite effective complexing of the autoantibodies with TPO. Overall, our studies demonstrate that nTPO and rTPO produced in insect cells are very similar in terms of enzyme activity, UV and visible spectra, and reactivity with autoantibodies. Furthermore, in our study, TPO autoantibodies did not appear to inhibit TPO enzyme activity.

Animals↗
PubMed1999

The interaction of TSH receptor autoantibodies with 125I-labelled TSH receptor.

Detergent-solubilized porcine TSH receptor (TSHR) has been labeled with 125I using a monoclonal antibody to the C-terminal domain of the receptor. The ability of sera containing TSHR autoantibody to immunoprecipitate the labeled receptor was then investigated. Sera negative for TSHR autoantibody (as judged by assays based on inhibition of labeled TSH binding to detergent-solubilized porcine TSHR) immunoprecipitated about 4% of the labeled receptor, whereas sera with high levels of receptor autoantibody immunoprecipitated more than 25% of the labeled receptor. The ability to immunoprecipitate labeled TSHR correlated well with ability of the sera to inhibit labeled TSH binding to the receptor (r = 0.92; n = 63), and this is consistent with TSHR autoantibodies in these samples being directed principally to a region of the receptor closely related to the TSH binding site. Preincubation of labeled TSHR with unlabeled TSH before reaction with test sera inhibited the immunoprecipitation reaction, providing further evidence for a close relationship between the TSHR autoantibody binding site(s) and the TSH binding site. This was the case whether the sera had TSH agonist (i.e., thyroid stimulating) or TSH antagonist (i.e., blocking) activities, thus, providing no clear evidence for different regions of the TSHR being involved in forming the binding site(s) for TSHR autoantibodies with stimulating and with blocking activities. The ability of TSHR autoantibodies to stimulate cyclic AMP production in isolated porcine thyroid cells was compared with their ability to immunoprecipitate labeled porcine TSHR. A significant correlation was observed (r = 0.58; n = 50; P < 0.001) and the correlation was improved when stimulation of cyclic AMP production was compared with inhibition of labeled TSH binding to porcine TSHR (r = 0.76). Overall, our results indicate that TSHR autoantibodies bind principally to a region on the TSHR closely related to the TSH binding site, and this seems to be the case whether the autoantibodies act as TSH agonists or antagonists.

Animals↗
PubMed1999

Adrenal-cortex autoantibodies and steroid-producing cells autoantibodies in patients with Addison's disease: comparison of immunofluorescence and immunoprecipitation assays.

Autoimmune Addison's disease and premature ovarian failure are characterized by the presence of organ-specific autoantibodies. The main adrenal and gonadal autoantigens have been identified and cloned, and the relationship between the autoantibodies detected by immunofluorescence techniques and those detected by the new assays using recombinant autoantigens needed to be investigated. We studied 165 patients with Addison's disease: 143 patients had different forms of autoimmune Addison's disease (13 with idiopathic premature ovarian failure) and 22 had nonautoimmune Addison's disease. Adrenal-cortex autoantibodies and steroid-producing cell autoantibodies were measured by the immunofluorescence techniques. Autoantibodies to steroid 21-hydroxylase, 17alpha-hydroxylase, and P450 side chain cleavage enzyme were measured by immunoprecipitation assay using 35S-labeled recombinant proteins. Adrenal-cortex autoantibodies and autoantibodies to 21-hydroxylase were found in 81% of the patients with autoimmune Addison's disease. None of the patients with nonautoimmune Addison's disease had adrenal-cortex autoantibodies or autoantibodies to 21-hydroxylase. A high association between these two markers in patients with different forms of autoimmune Addison's disease and in those with short- or long-standing disease was found. Steroid-producing cells autoantibodies were found in 26% of the patients with autoimmune Addison's disease, and autoantibodies to 17alpha-hydroxylase and/or P450 side chain cleavage enzyme in 36% of the patients. Steroid-producing cells autoantibodies were found in 11/13 (85%) of patients with idiopathic premature ovarian failure associated with autoimmune Addison's disease, and autoantibodies to 17alpha-hydroxylase and/or P450 side chain cleavage were found 12/13 (92%) of patients; the only case negative for all these three markers suffered from Turner's syndrome. Provided that a high standard of immunofluorescence technique is maintained, measurement of adrenal cortex autoantibodies or steroid-producing cells autoantibodies by either immunofluorescence or immunoprecipitation assay is essentially equivalent.

Addison Disease↗
PubMed1999

Analysis of carbohydrate residues on recombinant human thyrotropin receptor.

An investigation of the sugar groups on recombinant human TSH receptors (TSHR) expressed in CHO-K1 cells and solubilized with detergents is described. Western blotting studies with TSHR monoclonal antibodies showed that the receptor was present principally as two bands with approximate molecular masses of 120 and 100 kDa. Further blotting studies using lectins and/or involving treatment with different glycosidases indicated that the 100-kDa band contained about 16 kDa of high mannose-type sugars, and the 120-kDa band contained about 33 kDa of complex-type sugars. It was possible to separate the 120- and 100-kDa components of the TSHRs by lectin affinity chromatography. In particular, Galanthus nivalis lectin, which binds high mannose-type sugars, bound the 100-kDa band, but not the 120-kDa band, whereas Datura stramonium lectin, which binds complex-type sugars, bound the 120-kDa band, but not the 100-kDa band. 125I-Labeled TSH binding studies with the various lectin column fractions showed that TSH-binding activity was principally associated with the complex-type sugar containing the 120-kDa form of the receptor rather than the high mannose-containing 100-kDa form. During peptide chain glycosylation, high mannose-type sugar residues are attached first and then modified by the formation of complex type structures to form the mature glycoprotein. Our data suggest that in the case of the TSH receptor, this type of posttranslational processing has an important role in forming the TSH-binding site.

Animals↗