RESUMO
A cell line derived from the Fisher rat thyroid (FRT), that does not have functional TSH receptor, was stably transfected with the cDNA of the human TSH receptor (h TSH-R). In wild FRT cells TSH (1-1000 mU/l) was unable to increase cAMP production, while 10-10000 nmol/l forskolin elicited a 10-30 fold cAMP stimulation. Two of the transfected clones were responsive to TSH in terms of cAMP production. In particular, the FRT-R3 transfected clone showed the highest sensitivity to the hormone with a 10 fold cAMP increase over the basal at 100 mU/l TSH. The Northern blot analysis using a 2.4 kbp cDNA probe for the hTSH-R showed a band corresponding to the mRNA of TSH receptor in FRT-R3 cells, but not in wild FRT cells. In both cell types TSH was ineffective in stimulating growth assayed by 3H-thymidine incorporation into DNA. Hybridization with a probe for thyroperoxidase on polymerase chain reaction products after reverse transcription of mRNA showed that FRT-R3, as well as FRT cells, do not have a transcript for thyroperoxidase. In conclusion, the data reported in this paper show that the insertion of the hTSH-R cDNA in the genome of poorly differentiated rat thyroid cells results in the recovery of TSH-dependent adenylate cyclase, but not other differentiated thyroid cell functions.
Assuntos
DNA Complementar/genética , Genoma , Receptores da Tireotropina/genética , Glândula Tireoide/citologia , Transfecção , Adenilil Ciclases/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Humanos , Ratos , Ratos Endogâmicos F344RESUMO
A role of thyroid autoimmunity in the pathogenesis of myxedematous endemic cretinism was suggested by reports indicating the presence of thyroid growth-blocking antibodies in the sera of these patients. To check this hypothesis, we searched for TSH receptor antibodies with thyroid growth-blocking or adenylate cyclase (AC)-inhibiting (TSH-blocking) activity in immunoglobulin G (IgG) from 18 euthyroid and 21 hypothyroid endemic cretins living in Italy and Peru. Among hypothyroid cretins, 12 had no palpable goiter. Stages I-III goiters were present in 12 of 18 euthyroid cretins. Controls included 25 euthyroid nongoitrous subjects living in the same endemic regions as cretins, and 10 normal subjects from an iodine-sufficient area. IgG from 4 selected patients with autoimmune atrophic thyroiditis and from 2 neonates with sporadic transient congenital hypothyroidism due to maternal TSH-blocking antibodies were included in the study. The blocking effect of the IgG was assessed in FRTL-5 cells by measuring TSH-stimulated [3H]thymidine incorporation, DNA accumulation, and AC activation. A radioreceptor assay was used to detect TSH-binding inhibiting antibodies (TBIAb). No IgG from hypothyroid endemic cretins without goiter contained TBIAb or inhibited TSH-stimulated cell growth or AC activation. The effect of IgG from hypothyroid nongoitrous cretins did not differ from that produced by IgG from hypothyroid cretins with goiter, euthyroid cretins with or without goiter, or normal controls. In contrast to these results, IgG from patients with autoimmune atrophic thyroiditis and from neonates with sporadic transient congenital hypothyroidism contained TBIAb that inhibited both TSH-stimulated cell growth and AC activation. In conclusion, our results indicate that, similar to other types of endemic cretinism, hypothyroid endemic cretins without goiter do not have TSH receptor antibodies able to inhibit TSH-stimulated thyroid cell growth or function. These observations argue against a role of humoral thyroid autoimmunity in the development of myxedematous endemic cretinism.
Assuntos
Autoimunidade , Hipotireoidismo Congênito/imunologia , Glândula Tireoide/imunologia , Adolescente , Adulto , Idoso , Formação de Anticorpos , Autoanticorpos/análise , Autoanticorpos/imunologia , Divisão Celular/fisiologia , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Glândula Tireoide/patologia , Tireotropina/imunologia , Tireotropina/fisiologiaRESUMO
Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) were used to develop an assay to detect thyroid autoantibodies blocking the TSH-dependent cAMP production (TSHBAb). The study group included 38 patients with goitrous Hashimoto's thyroiditis (HT) and 47 subjects with atrophic thyroiditis (AT). In the HT group, 8 patients had subclinical hypothyroidism (HT-SH) and 30 had overt hypothyroidism (HT-H). Thirty normal subjects served as controls. Immunoglobulin G (IgG) was prepared from serum by double chromatography on DEAE-Sephadex. CHO-R cells were seeded in 96-well plates and were cultured for 48 h before the assay in RPMI-1640 medium plus 1 mmol/L glutamine, 10% fetal calf serum, and 0.4 g/L geneticin. In the assay for TSHBAb, CHO-R cells were incubated with IgG alone (0.5-2 mg/ml), TSH alone (0.2-625 mU/L), or IgG plus TSH; all samples were diluted in hypotonic medium containing 0.5 mmol/L isobutylmethylxanthine (IBMX). After 2 h of incubation at 37 degrees C in 5% CO2-95% air atmosphere, TSH-stimulation was quantified by measuring extracellular cAMP by a RIA. IgGs from normal subjects did not significantly modify the stimulation of adenylate cyclase produced by TSH, the results obtained ranging between -30% and +18% (mean +/- SD = -3 +/- 14%). All IgGs producing an inhibition greater than 2SD from the mean of controls (> 25%) were considered positive for blocking antibodies. TSHABAb were detected in 1/8 (12.5%) patients with HT-SH, in 7/30 (23.3%) with HT-H and 16/47 (34.0%) patients with AT.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anticorpos/análise , Células CHO/imunologia , Receptores da Tireotropina/genética , Tireotropina/efeitos dos fármacos , Tireotropina/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ligação Competitiva , Células CHO/fisiologia , Linhagem Celular , Criança , Células Clonais/fisiologia , Cricetinae , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Doenças da Glândula Tireoide/diagnóstico , Glândula Tireoide/citologia , Tireotropina/antagonistas & inibidores , TransfecçãoRESUMO
The [3H]thymidine incorporation assay in FRTL-5 cells was used to measure thyroid growth-stimulating antibody in the purified immunoglobulin G (IgG) fraction of patients with endemic nontoxic goiter (grade I-III) living in Italy (n = 34) or Peru (n = 37). IgG of euthyroid nongoitrous subjects living in the same endemic area (n = 25) and from an area of sufficient iodine intake were used as controls. Bovine TSH (10 mU/L) and thyroid-stimulating antibody of Graves' disease produced a significant increase in [3H]thymidine incorporation and DNA content in FRTL-5 cells. IgG from Italian or Peruvian patients with endemic goiter produced a small increase in [3H]thymidine incorporation in FRTL-5 cells (131 +/- 54% and 165 +/- 57%, respectively), which was indistinguishable from that obtained with IgG from normal nongoitrous subjects residing in endemic or nonendemic areas (167 +/- 80% and 161 +/- 36%, respectively). For comparison 18 of 25 (72%) IgG of hyperthyroid patients with Graves' disease produced clear-cut increases in [3H]thymidine incorporation (1142 +/- 1065%) and DNA content (219%) in FRTL-5 cells. IgG from patients with endemic goiter, at variance with Graves' IgG, did not cause an increase in DNA in FRTL-5 cells. All Graves' IgG that stimulated [3H]thymidine incorporation in FRTL-5 cells also stimulated cAMP production in this culture system, whereas no adenylate cyclase stimulation was produced by IgG from patients with endemic goiter. The prevalence of thyroglobulin antibody and thyroperoxidase antibody in endemic goiter patients did not differ from that in control subjects residing in the same iodine-deficient area. Our data show that sera of endemic goiter patients are devoid of thyroid growth-stimulating antibody and thyroid-stimulating antibody activities. These observations argue against a direct role of thyroid autoimmunity in the development of goiter in iodine-deficient areas.
Assuntos
Autoanticorpos/análise , Bócio Endêmico/imunologia , Imunoglobulina G/análise , Glândula Tireoide/imunologia , Adulto , Idoso , DNA/biossíntese , Feminino , Humanos , Imunoglobulinas Estimuladoras da Glândula Tireoide , Masculino , Pessoa de Meia-IdadeRESUMO
Some authors have suggested a role of autoimmunity in the pathogenesis of iodine deficiency disorders (IDD). For this purpose we have searched for thyroid adenylate cyclase stimulating antibody (TSAb) and thyroid growth stimulating antibody (TGSAb) in patients with endemic goiter (EG) and endemic cretinism (EC). Immunoglobulins G preparations (IgGs) were tested in FRTL-5 cells. TSAb were calculated as percent of cAMP increase over basal production and TGSAb were expressed as percent of increase of 3H-thymidine incorporation and DNA content in FRTL-5 cells. Our results show that IgGs from goitrous patients were devoid of TSAb and TGSAb activities, while in the same conditions IgGs from patients with Graves' disease had the ability to stimulate cAMP production and 3H-thymidine incorporation in FRTL-5 cells. These data argue against a direct role of TSAb and TGSAb in the pathogenesis of IDD.
Assuntos
Doenças Autoimunes/imunologia , Bócio Endêmico/imunologia , Glândula Tireoide/imunologia , Adenilil Ciclases/metabolismo , Adulto , Autoanticorpos/farmacologia , Linhagem Celular , Feminino , Doença de Graves/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulinas Estimuladoras da Glândula Tireoide , Insulina/farmacologia , Masculino , Glândula Tireoide/enzimologia , Tireotropina/farmacologiaRESUMO
Spores of the strain NCIB 8122 of Bacillus cereus have been depleted of coats by treatment with 0.1% sodium dodecyl sulfate--200 mM 2-mercaptoethanol--0.5 M NaCl (pH 9.6). The coat-depleted spores did not show any decrease in viability, heat resistance, refractility, dipicolinic acid content, or specific activities of several protoplastic enzymes. The germinative response of the coat-depleted spores to adenosine and several analogues thereof was found qualitatively similar to that obtained with intact spores. However, germination kinetics appeared to be affected by coat removal, since germination rate measured as loss of refractility was eight times slower even at inducer concentrations 10-fold higher than those required to promote optimal germination response of intact spores. Loss of heat resistance, on the other hand, was hardly affected by coat removal. These results suggest that, even though spore coats are not essential for the triggering reaction, they are required for a rapid evolution of the later events in the germination process.