Biomedical subjects

M Platzer

Publications and source records attributed to M Platzer.

At least 55 records · Page 3Linked to original sources

PubMed1988

Activation of MHC-restricted rat T cells by cloned syngeneic thyrocytes.

We have previously demonstrated that rat thyrocytes express MHC class II Ag (RT1.B&D) in response to IFN-gamma. To determine whether MHC class II-positive thyrocytes can be recognized by MHC-restricted T cells, we used our clone of rat thyroid cells (1B-6) derived from the Fisher rat thyroid cell line (FRTL-5) and known to express MHC class II Ag in response to recombinant rat IFN-gamma. CD4+ and CD8+ normal syngeneic Fisher rat spleen T cells were selected by flow cytometry and averaged greater than 96% purity. We demonstrated that irradiated MHC class II-positive but not class II-negative 1B-6 thyrocytes stimulated CD4+ T cells in a primary sensitization reaction over 4 days. In contrast, CD8+ T cells had no response in similar experiments. This stimulation of CD4+ T cells was dose dependent for 1B-6 thyrocytes and was abrogated by anti-rat MHC class II mAb (MRC OX-6). Autoreactive (Fisher) and alloreactive (Buffalo) T cell lines and isolated CD4+ T cells derived from these lines, which were developed against Fisher rat spleen cells, similarly recognized MHC class II Ag expressed on 1B-6 cells but had no detectable response to 1B-6 MHC class II-negative thyrocytes or MHC class II-positive human thyroid cells. The CD4+ T cell recognition of 1B-6 cells via MHC class II Ag supports our previous data with autologous human thyroid T cell co-cultures and is indicative of an autospecific role for thyrocytes in the development of autoimmune thyroiditis.

Animals↗

PubMed1988

Condensation of vector DNA by the chromosomal protein HMG1 results in efficient transfection.

The aim of this study was the search for a method of vector packaging using natural chromatin constituents. The interaction of the chromosomal non-histone protein HMG1 with a vector plasmid (pLTEneo) was studied by sedimentation analysis and electron microscopy at physiological salt concentration. At high protein input the complexes exist in a condensed, monodisperse form sedimenting with 80 S irrespective of the supercoiled or relaxed conformation of DNA. Saturation binding is already observed at much lower input ratios. Dilution of 80 S complexes results in decondensation of the complexes. In the decondensed complex form, HMG1 binds in a bead-like manner to specific DNA regions. Condensation by HMG1 is sufficient to introduce the vector into mammalian cells without the need for unphysiological additives. The transfection rates were similar to or even higher than those obtained by the calcium phosphate coprecipitation technique.

Animals↗

PubMed1988

Retention of cyclic AMP response to TSH in a cloned human thyrocyte/T cell hybridoma (HY2-15).

Our observation of a human T cell leukemia cell (Molt 4) demonstrating low affinity thyroid-stimulating hormone (TSH) responses, as evidenced by generation of cyclic AMP, led us to test Molt 4 cells as a suitable partner for immortalizing high affinity TSH receptors present on human thyroid cells. Therefore, we generated a hybridoma (HY2-15) by a fusion between thyroid monolayer cells from a patient with Graves' disease, and a hypoxanthine-aminopterin-thymidine (HAT)-sensitive variant of this human T cell leukemia line, Molt 4-8AGR. The hybrid nature of HY2-15 was confirmed by DNA histograms using propidium iodide and flow cytometry. Karyotyping showed the HY2-15 cells to have five sets of chromosomes and human leukocyte antigen (HLA) class I determination revealed the presence of an additional HLA class I antigen (A2) not present on the Molt 4 partner cells. The established, cloned, hybridoma cells showed a greater than 30-fold increase in cyclic AMP release after stimulation with bovine TSH (bTSH, 1 mU/ml) with a minimum detectable stimulating dose of less than 10 microU/ml bTSH. However, no other thyroid-specific functions could be detected. Furthermore, HY2-15 cells failed to express HLA class II antigens either constitutively or in response to recombinant human gamma interferon (IF) and a variety of other stimuli, data similar to the Molt 4 partner cells but in contrast to human thyroid cells which show high sensitivity to gamma IF. The preservation of highly sensitive TSH responsiveness in a proliferating cell offers a unique approach to the study of human TSH receptor function.

Cell Division↗

PubMed1988

HLA-DR gene expression in a proliferating human thyroid cell clone (12S).

We have used a retroviral vector carrying the adenovirus E1A oncogene and the neomycin phosphotransferase gene to establish a human thyroid-derived cell line that exhibits TSH-mediated cAMP generation as well as the differential expression of HLA class II antigens in response to recombinant gamma-interferon. Twenty-two-week gestation, histologically confirmed, human fetal thyroid was collagenase digested, cultured as a monolayer, and infected directly with 12S or 13S E1A-containing retrovirus constructs. Infected clones (n = 30) were selected in a hormone-supplemented medium containing bovine TSH (bTSH; 1 mU/ml), 10% fetal bovine serum, and 0.5 mg/ml G418 antibiotic. A rapidly growing clone (designated 12S) was chosen for detailed analysis over 18 months of continuous culture. The 12S clone was sensitive to less than 10 microU/ml bTSH when assessed by extracellular accumulation of cAMP, but TSH had no influence on 72-h incorporation of [3H]thymidine. Clone 12S responded to recombinant human gamma-interferon (1-10(4) U/ml) by induction of HLA DR alpha-chain-specific mRNA and the surface expression of HLA-DR antigen detected by fluorescein isothiocyanate-labeled monoclonal antibody to nonpolymorphic HLA-DR regions using flow cytometry. These studies indicate the potential for immortalizing human thyroid cells for use as targets of anti-TSH receptor immune responses and for long term studies of human throcyte HLA gene regulation.

Adult↗

PubMed1987

Cloning the Fisher rat thyroid cell line (FRTL-5): variability in clonal growth and 3,'5'-cyclic adenosine monophosphate response to thyrotropin.

To investigate the stability of FRTL-5 cells we cloned stock cells by limiting dilution in a 6-hormone medium with bovine TSH (bTSH) (10 mU/ml) (6H medium). One third of the wells with irradiated fibroblast feeder cells developed FRTL-5 colonies. One hundred and twenty clones were obtained, of which 29 (8%) developed sufficiently for functional analysis. Clones were tested for growth responses to bTSH stimulation as evidenced by 72-h [3H]thymidine uptake and TSH receptor activation by cAMP generation. Clonal growth responses to bTSH were of three types: dose-related growth increase, absence of growth stimulation, and stimulation by bTSH in concentrations up to 100 microU/ml and inhibition of growth above 100 microU/ml. All clones tested showed evidence of extracellular cAMP accumulation in response to bTSH. However, sensitivity to bTSH varied from 1 to 100 microU/ml and maximum cAMP secretion with 1 mU/ml bTSH varied from 2 to 13 pmol/ml. Whereas certain clones showed high sensitivity to bTSH with respect to both growth and cAMP responses (e.g. 1B-6), there were clones which showed disparity in this relationship, as evidenced by poor growth dependency but high cAMP responses or by growth stimulation yet insensitivity with respect to cAMP secretion. These data demonstrate that the FRTL-5 line contains cells with variable responsiveness to bTSH. Whereas the FRTL-5 line is heterogeneous and subject to developmental variation, cloning by limiting dilution allows the derivation of highly bTSH-sensitive cells, such as 1B-6, from the stock cultures.

Animals↗

PubMed1987

Induction of rat thyroid cell MHC class II antigen by thyrotropin and gamma-interferon.

Major histocompatibility (MHC) class II antigen expression by thyroid epithelial cells has been widely implicated in the pathogenesis of autoimmune thyroid disease. We have examined rat MHC (RT1) class II antigen gene regulation in 1B-6 cells, cloned in our laboratory from the Fisher rat thyroid line FRT-L5. 1B-6 cells are TSH dependent for growth and proliferation, and are responsive to more than 5 microU/ml bovine TSH (bTSH) in terms of extracellular cAMP accumulation. Recombinant rat gamma interferon (gamma IF; 1-1000 U/ml) treatment for 5 days together with bTSH (10(3) microU/ml) was able to induce class II antigen expression in up to 90% of 1B-6 cells, as detected by a murine monoclonal antibody to rat MHC class II antigen (FITC-OX-6). Total cellular RNA was examined in Northern blot analyses using an HLA-DR alpha-chain-specific RNA probe, which shares 78% sequence homology with the rat RT1.D alpha-chain gene. A 1.4-kilobase mRNA transcript was detected in gamma IF-stimulated cells, but not in untreated cells. Dose-response studies of MHC class II gene expression, using a cytoplasmic RNA slot blot technique for alpha-chain mRNA levels and FITC-OX 6 monoclonal antibody for detection of MHC class II antigen expression, indicated 1B-6 sensitivity to gamma IF in the range of 10-100 U/ml (in the presence of 10(3) microU/ml bTSH) and to bTSH in the range of 10-100 microU/ml (in the presence of 10(2) U/ml gamma IF). These data demonstrate dual control of MHC class II antigen gene expression by gamma IF and bTSH in a differentiated rat thyroid cell. Clone 1B-6 represents a powerful means of analyzing reproducibly and in long term studies the regulation of thyroid cell MHC class II gene expression.

Animals↗

PubMed1987

Lymphokine regulation of HLA-DR gene expression in human thyroid cell monolayers.

Studies were conducted to examine the regulation of HLA class II gene expression in human thyroid cells in vitro. Normal human thyroid cells cultured in the absence of lectin or gamma-interferon stimulation lacked detectable HLA-DR cell surface antigen, although low levels of DR alpha-chain-specific mRNA were present. Cyclosporine A, known to inhibit lymphokine production, inhibited basal as well as lectin-mediated increases in levels of DR alpha-chain-specific mRNA and DR surface antigen expression on normal human thyrocytes. Cyclosporine had no effect on the induction of DR antigen gene expression by recombinant gamma-interferon. These data suggested that lectin enhancement of DR antigen expression in human thyroid cells may be mediated by a lymphokine(s) produced in primary human thyroid cell monolayers. This suggestion was confirmed by studies that demonstrated the abrogation of lectin responsiveness by antibody directed against gamma-interferon. Indirect immunofluorescence studies using flow cytometric analyses identified 1.6 +/- 0.2% (mean +/- SD) of cells in primary thyroid cultures as T lymphocytes, a potential source of lymphokine production. Cells derived from thyroid follicular adenomas and carcinomas demonstrated reduced lectin-mediated increases in DR antigen expression compared to normal thyroid cells. DR expression could be enhanced in these lectin-treated cells, however, by T cell coculture. Dose-response studies demonstrated that human thyroid cells were as sensitive to gamma-interferon induction of DR antigen expression as human monocyte/macrophages. These results indicate that human thyroid cell HLA-DR antigen gene expression is sensitive to low levels of lymphokines, such as gamma-interferon; an intrathyroidal T cell population, which may serve as a source of lymphokine(s), remains associated with thyroid epithelial cells in primary thyroid cultures; and lymphokine-thyroid cell interactions may be implicated in the immunopathology of human autoimmune thyroid disease.

Antigens, Neoplasm↗

PubMed1986

hCG-induced TSH receptor activation and growth acceleration in FRTL-5 thyroid cells.

Our previous studies have indicated specificity cross-over between LH/hCG and the TSH receptor. We have now analyzed the ability of the TSH-dependent Fisher Rat thyroid cell line (FRTL-5) to proliferate in a differentiated state under the influence of highly purified hCG (hCG-CR121). TSH receptor activation and growth induction were observed after 7 days suspension from a TSH-induced growth phase. The effect of 10 ug hCG-CR121 was equivalent to 500 +/- 43 (mean +/- SEM) uIU of human TSH (2nd IRP, 80/558) with reference to growth stimulation, as judged by 72-h [3H]-thymidine uptake and to approximately 15 +/- 35 uIU human TSH with reference to receptor activation as judged by cyclic AMP accumulation. These data demonstrate specificity cross-over by hCG for the TSH-dependent FRTL-5 cell line and suggest that hCG has greater growth-stimulating than thyroid-stimulating potential when compared with human TSH.

Animals↗

PubMed1986

Restricted heterogeneity and T cell dependence of human thyroid autoantibody immunoglobulin G subclasses.

Twenty-five sera from patients with autoimmune thyroiditis, positive for thyroglobulin (hTg) and/or thyroid microsomal autoantibodies (M-Ab), were assessed by specific micro-ELISA to determine thyroid autoantibody immunoglobulin G (IgG) subclass distribution. Of the 25 sera, 22 were positive for M-Ab. All but 1 sample had restricted heterogeneity confined to IgG1 and/or IgG4 subclasses. The contribution of each subclass to an individual autoantibody titer varied from 100% IgG1 to 100% IgG4. Sixteen of the 25 sera had detectable hTg-Ab, and the majority also were restricted to IgG1 and IgG4, with similar distributions occurring among subclasses. In all, only 5 sera had hTg/M-Ab in IgG subclasses 2 and/or 3. T cell control of pokeweed mitogen-stimulated IgG subclass secretion was analyzed using increasing numbers of T cells in ratios of T to non-T from 0:1 to 10:1. In normal subjects, IgG1, but not IgG 2, 3, or 4, had T cell dependence, as evidenced by enhancement and inhibition of IgG1 secretion as the number of T cells increased. T cell suppressor dysfunction was apparent in patients with autoimmune thyroiditis, as demonstrated by the reduced ability of patient T cells (n = 6), compared with normal T cells (n = 6), to suppress total IgG1 subclass secretion. These data indicate 1) restricted heterogeneity of human thyroid autoantibodies, principally to IgG1 and IgG4; 2) T cell dependence of only IgG1 secretion in vitro; and 3) a T cell defect in patients with autoimmune thyroid disease. The possibility of IgG4 thyroid autoantibodies being under less stringent T cell regulatory control questions the likely importance of thyroid-specific suppressor T cell dysfunction in the etiology of autoimmune thyroid disease.

Autoantibodies↗

PubMed1986

The T cell suppressor defect in autoimmune thyroiditis: evidence for a high set 'autoimmunostat'.

We examined T cell function in patients with autoimmune thyroiditis in relation to mitogen-stimulated thyroglobulin autoantibody secretion and used total IgG and IgM secretion as a measure of non-specific polyclonal activation. Control subjects (n = 8) showed enhanced immunoglobulin secretion (both IgG and IgM) in the presence of increasing autologous T cells up to a ratio of 4:1 T:non-T followed by suppression of such secretion when the number of T cells was increased still further (the T cell suppressor effect). Patients (n = 8) demonstrated similar enhancement of immunoglobulin secretion when compared to normals but the T cell suppressor effect was markedly reduced. Autoantibodies to human thyroglobulin antigen, measured by specific ELISA techniques, were detected in cultures from five patients and demonstrated a similar pattern of enhancement but reduced suppression as observed with the immunoglobulin assays. However, normal suppression of immunoglobulin and thyroglobulin autoantibody secretion in such patients was obtained with the use of allogeneic normal T cells. These data confirm the presence of a T cell suppressor defect in autoimmune thyroiditis but indicate that the abnormality is not restricted to the control of thyroid autoantibodies. Such T cell dysfunction may allow the abnormal amplification of a distinct and separate primary event and may be viewed as an abnormal set point for a hypothetical 'autoimmunostat'.

Autoantibodies↗

PubMed1985

T-cell sensitization to autologous thyroid cells and normal non-specific suppressor T-cell function in Graves' disease.

We have employed a syngeneic system utilizing thyroid cell monolayers initiated following thyroid surgery co-cultured with autologous T cells to demonstrate T cell autosensitization in patients with Graves' disease. Antigen-induced blastogenesis was monitored using 24 h [3H]thymidine uptake. Control experiments with 5 d cultured normal human thyroid cells from tissue around benign adenomata showed no evidence of syngeneic T cell autosensitization. Human thyroid cells alone were unable to incorporate [3H]thymidine in the presence of bTSH. In three of four experiments with Graves' thyroid cells there was significant induction of autologous T cell blastogenesis with a mean stimulation index of 220%. In parallel experiments we explored the non-specific helper and suppressor T cell function of these and similar patients with Graves' disease. In normal controls (n = 6) increasing numbers of T cells added to a constant number of B cells (consisting of a T cell depleted peripheral mononuclear cell preparation) showed a marked helper effect measured as increasing IgG secretion. As the ratio of T:B cells increased above 4:1 there was a suppression of IgG secretion. One of two hyperthyroid Graves' patients was observed to have deficient T cell function as demonstrated by lack of IgG suppression. The remaining five patients (all but one of whom were euthyroid at the time of testing) had results similar to the controls indicating normal suppressor T cell function in this disease. Such data showed that patients with Graves' disease possessed circulating T cells which exhibited autosensitization to syngeneic thyroid cell surface antigens, a phenomenon not demonstrable in control individuals. Furthermore, this specific T cell autosensitization did not interfere with non-specific T cell function as judged by its influence on IgG secretion.

Adult↗

PubMed1985

Short- and long-term evaluation of normal and abnormal human thyroid cells in monolayer culture.

We have investigated the TSH responsiveness of normal and abnormal human thyroid cells cultured in the short term with high serum concentrations and for up to 6 months in a low serum, chemically-defined, medium. Cells from normal human thyroid tissue (n = 9), multinodular goitre (n = 6), benign follicular adenomata (n = 6), and differentiated thyroid carcinoma (n = 3) formed confluent monolayers which were sensitive to bovine TSH (bTSH) in concentrations greater than 25 microU/ml when assessed by the intracellular response of cyclic AMP at 7 d of culture. Such sensitivity was less than that observed with a continuously proliferating thyroid cell line (FRTL-5) derived from Fisher rat thyroid and which responded to concentrations of bTSH as low as 5-10 microU/ml. Human cells derived from iodine/antithyroid drug treated Graves' thyroid tissue (n = 6) were less sensitive than normal cells requiring up to 500 microU/ml bTSH to increase intracellular cyclic AMP and poorly differentiated thyroid cancer cells (n = 3) failed to respond to bTSH. Long-term human thyroid cultures of normal and follicular adenoma cells in the chemically-defined medium used for the FRTL-5 cells had absent fibroblast growth and continued in monolayer form without significant follicle formation. These cells remained highly sensitive to bTSH stimulation when tested after 4, 13, and 26 weeks of continuous culture. All such cell preparations failed to proliferate under conditions which favoured the rapid growth of the rat thyroid cells. These data demonstrated that while thyroid cell culture conditions described in the literature do not permit proliferation of human thyroid cells, they do allow an assessment of their functional state in vitro which may lead to a further understanding of thyroid cell pathophysiology.

Adenoma↗

PubMed1984

Plasmid rescue - a tool for reproducible recovery of genes from transfected mammalian cells?

The efficient rescue of plasmids containing the thymidine kinase gene (tk) of Herpes simplex virus type I from genetically transformed mouse cells by transformation of bacteria is described. Rescued plasmids contain insertions of calf DNA used as a carrier in the transfection but usually lack portions of plasmid DNA. Deletions generally concern the region spanning from around the PvuII site of pBR322 to within the tetracycline resistance coding sequence, whereas the extent of tk sequence deletion varies, depending on the site of its integration (BamHI or PvuII) into the plasmid. Modelling the rescue process by transformation of bacteria with a mixture of original plasmids and sheared mouse cell DNA clearly demonstrates that deletions are caused by the presence of the mammalian DNA and they probably occur during re-transformation of bacteria before the onset of tetracycline gene expression. Plasmids lacking the Tcr region are reproducibly rescuable without deletion. Methods for reproducible re-isolation of transferred genes from mammalian cells are discussed.

Animals↗

PubMed1984

Thyroglobulin secretion by human thyroid cells after monolayer culture--comparison of normal and adenomatous cells.

We have analysed immunoassayable thyroglobulin (hTg) secretion from both normal and abnormal isolated human thyroid cells after short-term monolayer culture. All cells were sensitive to more than 10 microU/ml bTSH when assessed by intracellular cyclic AMP accumulation in the presence of a phosphodiesterase inhibitor. hTg release was stimulated in all cells by bTSH in a dose related manner and with a detectable response within 24 h. Basal hTg secretion rates were greater in cells derived from benign follicular adenomata (range 1.1-2.2 ng/10(5) cells/h, n = 4) than in normal human thyroid cells (range 0.1-0.65 ng). Therefore it appears likely that hTg secretion by adenomatous thyroid cells was a likely contributor to increased serum hTg in patients with single follicular adenomata. We conclude that simple human thyroid cell monolayers have potential for the further study of hTg secretion and its control by TSH.

Adenoma↗

PubMed1983

Studies on the SV40-like papovavirus SV40-GBM. II. Molecular cloning in Escherichia coli of variant DNA molecules from glioblastoma-derived virus.

The complete DNA genomes of the SV40-like GBM virus (GBM1), isolated from a human glioblastoma multiforme, and of two discrete classes of GBM DNA molecules that appear following three passages in CV-1 monkey cells at low multiplicities (GBM3-H and GBM3-L), were cloned in Escherichia coli using plasmid vector pBR322. The cloned viral DNAs were characterized (i) by digestion of the chimeric plasmid DNAs with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose with that of similarly digested uncloned DNA, (ii) by hybridization of digested chimeric plasmid DNAs to 32P-labeled uncloned GBM DNA, and (iii) by electron microscopy. In restriction enzyme analysis the cloned GBM DNAs showed the same cleavage pattern as the uncloned DNAs, indicating that no major insertions or deletions were present. The electrophoretic data were confirmed by electron microscopic heteroduplex analysis.

Cloning, Molecular↗

PubMed1983

Studies on the SV40-like papovavirus SV40-GBM. I. Genomic analysis by restriction endonucleases and electron microscopy after propagation in CV-1 monkey cells.

Infection of CV-1 monkey cells with SV40-GBM, a papovavirus isolated from a human glioblastoma multiforme, resulted in the appearance of defective viral DNA molecules. In contrast to SV40 wild-type, two main types of variant DNA molecules could be found after three viral passages at multiplicities of infection of about 10. The molecules of one variant DNA (GBM3-L) were about 19% shorter than the GBM3-H DNA molecules and the DNA of the original GBM isolate, as demonstrated by electron microscopy. Restriction enzyme analysis revealed that GBM3-L DNA had lost both the EcoRI and the HpaII cleavage sites which are located in the late viral genome region. Furthermore, SV40 GBM3-L did not possess the two PvuII sites which are located in the late genome region, and a portion of the GBM3-H and GBM3-L DNA molecules had lost the unique KpnI site. Heteroduplex analysis verified that the rearrangements in the GBM3-L DNA are located only in the late region of this DNA. The possible differences between SV40 wild-type and SV40-GBM are discussed on the basis of these results.

Animals↗

PubMed1983

Graves' immunoglobulins protect the human TSH receptor: further evidence for TSH receptor antibodies in Graves' disease.

We have investigated whether membrane-bound TSH and Graves' immunoglobulins (Ig) were able to inhibit the action of the disulphide-reducing agent dithiothreitol (DTT) which reduced the binding of 125I-bTSH to thyroid membranes. Human thyroid (20,000 g fraction) was incubated with increasing concentrations of cold bTSH, washed and resuspended in 10 mmol/lDTT for 60 min at 37 degrees C. After the DTT was removed, 70-80% of the receptor-bound bTSH dissociated with 2 mol/l NaCl. In the absence of cold bTSH, 80% of the binding of receptor-purified 125I-bTSH was inhibited after membrane exposure to DTT but in the presence of bTSH-occupied receptor sites there was a dose related protection of the receptors, with 100% protection following incubation with 30 mU/ml bTSH. Like bTSH, immunoglobulin fractions from Graves' sera caused a time-dependent reduction in 125I-bTSH binding-inhibition after incubation and washing of thyroid membranes prior to ligand binding. Approximately 25-40% of this reduction was reversible by exposure to 2 mol/l NaCl. After pre-binding normal Ig to thyroid membranes, and subsequent incubation with DTT, there was no protection of TSH receptors. However, each of the Graves' Ig examined (n = 4) was able to provide protection of the TSH receptor binding sites in proportion to their derived TSH receptor occupancy. Receptor-bound bTSH was, therefore, able to protect the human TSH binding site from disulphide reduction. In addition, Graves' Ig, but not normal Ig, contained antibodies which were able to protect the TSH receptor binding sites in the same way as bTSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies↗

PubMed1983

Functionality of thyroid-stimulating antibodies assessed by cryopreserved human thyroid cell bioassay.

Immunoglobulin preparations from patients with autoimmune thyroid disease may inhibit the binding of 125I-labeled bovine TSH to solubilized TSH receptors without exhibiting concurrent thyroid stimulation. Such data have emphasized the continuing need for assay of biological function in addition to binding inhibition. We have employed a convenient technique using normal human thyroid cells cultured in monolayers for 7 days and then cryopreserved in liquid N2. Thyroid stimulation was assessed by measuring intracellular cAMP accumulation in a modified hypotonic Hank's Balanced Salt Solution using approximately 30,000 cells/well. Forty-five consecutive unselected immunoglobulin samples from hyperthyroid Graves' patients were assayed at 10 mg/ml protein for 2 h. Thirty-two (71%) of these untreated samples and 3 of 13 (23%) samples from treated euthyroid Graves' patients caused significantly greater stimulation than normal Ig prepared and assayed simultaneously. Forty-four percent of the positive samples were of low titer, causing less than a 200% increase in basal cAMP levels. One high titer sample (12,000% increase) had even greater bioactivity when serially diluted, thus suggesting the presence of an inhibitor, a phenomenon not observed in 20 lower titer and negative preparations. These data indicate the convenience and usefulness of a cryopreserved thyroid cell bioassay and emphasize that only low titers of thyroid-stimulating antibody occur in the peripheral circulation of many Graves' patients.

Adult↗