RESUMEN

The etiology of autoimmune thyroid diseases is unclear; however, the extreme female predominance suggests that sex hormones may have a pathogenic role. 2-Methoxyestradiol (2-ME) is present in the serum of women during the ovulatory and luteal phases of the menstrual cycle, and during pregnancy. We investigated the actions of 2-ME and estrogen on thyroid follicular cells. 2-ME induced dramatic changes in cell morphology and decreased the viability of the cells, as well as disrupted the structural integrity of cultured thyroid follicles. Flow cytometric analysis showed that 2-ME halted cell proliferation by arresting the cells in the G2/M cell-cycle compartment. Prolonged exposure to 2-ME led to apoptosis and to increased release of the autoantigen thyroid peroxidase (TPO). 17beta-estradiol failed to produce a similar effect even in 40-fold molar excess to 2-ME. Co-treatment with estrogen receptor antagonists did not alter the 2-ME effect, indicating that 2-ME was not operating through a classic nuclear estrogen receptor. In conclusion, this study indicates that 2-ME induces G2/M cycle arrest, apoptosis and the disruption of thyroid follicles. This process results in the release of thyroid antigens that may play a role in high incidence of thyroid autoantibodies and autoimmune thyroid disease in women.

Asunto(s)

Apoptosis/efectos de los fármacos , Estradiol/análogos & derivados , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , 2-Metoxiestradiol , Animales , Autoantígenos/metabolismo , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Estradiol/metabolismo , Estradiol/farmacología , Estrógenos/metabolismo , Femenino , Humanos , Yoduro Peroxidasa/inmunología , Yoduro Peroxidasa/metabolismo , Masculino , Embarazo , Ratas , Tiroiditis Autoinmune/etiología

RESUMEN

DNA in viruses and cells exists in highly condensed, tightly packaged states. We have undertaken an in vitro study of the kinetics of DNA condensation by the trivalent cation hexaammine cobalt (III) with the aim of formulating a quantitative, mechanistic model of the condensation process. Experimental approaches included total intensity and dynamic light scattering, electron microscopy, and differential sedimentation. We determined the average degree of condensation, the distribution of condensate sizes, and the fraction of uncondensed DNA as a function of reaction time for a range of [DNA] and [Co(NH(3))(3+)(6)]. We find the following: (1) DNA condensation occurs only above a critical [Co(NH(3))(3+)(6)] for a given DNA and salt concentration. At the onset of condensation, [Co(NH(3))(3+)(6)]/[DNA-phosphate] is close to the average value of 0.54, which reflects the 89-90% charge neutralization criterion for condensation. (2) The equilibrium weight average hydrodynamic radius of the condensates first decreases, then increases with increasing [Co(NH(3))(3+)(6)] as they undergo a transition from intramolecular (monomolecular) to intermolecular (multimolecular) condensation. However, is insensitive to [DNA]. (3) The uncondensed DNA fraction decays approximately exponentially with time. The equilibrium uncondensed DNA fraction and relaxation time decrease with increasing [Co(NH(3))(3+)(6)] but are insensitive to [DNA]. (4) The condensation rate in its early stages is insensitive to [DNA] but proportional to [Co(NH(3))(3+)(6)](xs) = [Co(NH(3))(3+)(6)] - [Co(NH(3))(3+)(6)](crit). (5) Data for low [DNA] and low [Co(NH(3))(3+)(6)] at early stages of condensation are most reliable for kinetic modeling since under these conditions there is minimal clumping and network formation among separate condensates. A mechanism with initial monomolecular nucleation and subsequent bimolecular association and unimolecular dissociation steps with rate constants that depend on the number of DNA molecules in the condensate, accounts reasonably well for these observations.

Asunto(s)

Cobalto/metabolismo , ADN/química , ADN/metabolismo , Cinética , Microscopía Electrónica , Plásmidos/química , Plásmidos/metabolismo , Dispersión de Radiación , Ultracentrifugación

RESUMEN

Thyrocyte apoptosis signaled through the Fas receptor has been proposed as a mechanism for the cytotoxicity observed in thyroiditis, but the role the Fas pathway plays in thyroid cancer is not known. We examined Fas expression in thyroid tissue derived from patients with papillary carcinoma and follicular cancer. More intense immunohistological staining for the Fas protein was observed on papillary cancer cells as compared with adjacent normal follicles. To further characterize the expression of Fas in papillary cancer, paired normal and cancerous thyroid tissues were obtained at thyroidectomy from several donors, digested, and placed into cell culture. Messenger RNA was analyzed by ribonuclease protection assays, and protein was identified by flow cytometry. Fas expression was detected at levels up to 3-fold higher in cancerous thyrocytes compared with paired normal cells. To determine whether the expressed Fas antigen was functional, thyrocytes were treated with a monoclonal IgM anti-Fas antibody (clone CH11; Upstate Biotechnology, Inc., Lake Placid, NY) in the presence of interferon-gamma and cycloheximide. Whereas both normal and cancerous thyrocytes were induced to die after this treatment, the cancerous thyrocytes were more sensitive to anti-Fas antibody. This work demonstrates that the Fas antigen is expressed and functional on papillary thyroid cancer cells and this may have potential therapeutic significance.

Asunto(s)

Carcinoma Papilar/inmunología , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Tiroides/inmunología , Receptor fas/genética , Adenocarcinoma Folicular/inmunología , Anticuerpos Monoclonales/farmacología , Apoptosis , Citometría de Flujo , Humanos , Inmunoglobulina M/farmacología , Inmunohistoquímica , ARN Mensajero/análisis , Receptor fas/análisis , Receptor fas/fisiología

RESUMEN

Human thyrocytes are resistant to Fas-mediated programmed cell death (PCD). It has been reported that a labile protein inhibitor is involved in the protection of thyrocytes from PCD, and its action can be reversed by incubation of thyrocytes with cycloheximide (CHX) during treatment with agonist anti-Fas Ab. Fas-associated phosphatase-1 (FAP-1) is a protein that has been shown to interact with the negative regulatory domain of Fas and block Fas-mediated apoptosis in FAP-1 transfected Jurkat cells. We investigated the possibility that FAP-1 might be involved in protection against Fas-mediated PCD in human thyrocytes. FAP-1 mRNA was detected in primary thyrocytes using a ribonuclease protection assay. The presence of FAP-1 protein was confirmed by immunohistochemical staining and flow cytometry using a polyclonal anti-FAP-1 Ab. FAP-1 protein also disappeared from thyroid cells in response to CHX. To determine whether FAP-1 is a functional inhibitor of PCD in thyrocytes, we incubated thyrocytes with synthetic SLV (Ac-SLV) tripeptide to compete with Fas for interaction with FAP-1. Thyrocytes treated with Ac-SLV tripeptide showed significantly increased cell death as compared to cells treated with control tripeptide. In addition, in the presence of a suboptimal concentration of CHX, the Ac-SLV tripeptide yielded a strong, synergistic increase in Fas-mediated PCD as compared to thyrocytes treated with control tripeptide. These results implicate FAP-1 as a regulator of Fas-induced PCD in thyrocytes.

Asunto(s)

Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Expresión Génica , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , Glándula Tiroides/metabolismo , Apoptosis , Cicloheximida/farmacología , Citometría de Flujo , Humanos , Inmunohistoquímica , Proteína Fosfatasa 1 , Inhibidores de la Síntesis de la Proteína/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 13 , ARN Mensajero/análisis , Glándula Tiroides/química , Receptor fas/fisiología

RESUMEN

To determine whether programmed cell death in thyroid follicular cells can be related to activation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway, we examined the expression and function of this pathway in primary thyroid follicular cells and a papillary thyroid carcinoma cell line in vitro. Despite the expression of TRAIL receptors death receptor 4 and death receptor 5, purified TRAIL could not induce programmed cell death (PCD) in any of the thyroid follicular cells examined. However, pre-incubation with cycloheximide before TRAIL facilitated the induction of rapid and massive PCD. This suggested that despite the presence of a labile inhibitor of the TRAIL pathway, TRAIL could mediate PCD under appropriate conditions. To determine whether there were sources of TRAIL in the thyroid that could interact with thyroid follicular cell TRAIL receptors, RNase protection assays were used to determine TRAIL mRNA expression. TRAIL message was expressed in intrathyroidal lymphocytes isolated from a patient with thyroiditis, and unexpectedly, thyroid follicular cells themselves could be induced to express abundant TRAIL message in the presence of the inflammatory cytokines interferon gamma, tumor necrosis factor alpha, and interleukin 1beta. Furthermore, the papillary thyroid carcinoma cell line could be induced to kill the TRAIL-sensitive lymphoma cell line BJAB through a TRAIL-dependent mechanism.

Asunto(s)

Apoptosis/fisiología , Receptores del Factor de Necrosis Tumoral/genética , Glándula Tiroides/metabolismo , Citocinas/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Mediadores de Inflamación/metabolismo , Papiloma/metabolismo , Papiloma/patología , ARN Mensajero/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/fisiología , Glándula Tiroides/citología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Células Tumorales Cultivadas

RESUMEN

The occurrence of apoptosis in thyroid follicular cells induced by Fas activation has been a subject of much debate. This is due, in part, to the fact that no physiologically relevant treatment conditions have been reported to cause rapid and extensive Fas-mediated apoptosis in thyroid cells, whereas treatment with the protein synthesis inhibitor cycloheximide prior to Fas activation allows for massive cell death. This indicates that the Fas signaling pathway is present but that its function is blocked in the overwhelming majority of cultured thyroid cells. To reconcile the conflicting reports, we set out to identify physiologically relevant conditions in which rapid, massive thyroid cell apoptosis in response to Fas activation could be demonstrated. We determined that susceptibility to Fas-activated apoptosis could be influenced by certain combinations of inflammatory cytokines. Although no single cytokine was effective, pretreatment of thyroid cells with the combination of gamma-interferon and either tumor necrosis factor-alpha or interleukin 1beta allowed for massive Fas-mediated apoptosis. Susceptibility to Fas-induced death correlated with an increase in expression of a tunicamycin-inhibitable high molecular weight form of Fas but not with aggregate expression of Fas.

Asunto(s)

Apoptosis/efectos de los fármacos , Interferón gamma/farmacología , Interleucina-1/farmacología , Glándula Tiroides/patología , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/metabolismo , Células Cultivadas , Interacciones Farmacológicas , Humanos , Glándula Tiroides/metabolismo

RESUMEN

The origin of the various forms of autoimmune thyroiditis remains unclear. Most investigations into the pathogenesis of these disorders have focused on immune abnormalities that might lead to an autoimmune response. However, no unique immune response to thyroid autoantigens has been identified that either is limited to patients with thyroiditis or is absolutely correlated with clinical disease expression. CD8 T-cell-mediated cytotoxicity is thought to be a major cause of thyroid follicular cell damage in thyroiditis. This damage is produced in part through the induction of apoptosis in thyroid cells. Recent studies have demonstrated that programmed cell death is regulated in thyroid cells and that a major pathway for immune-mediated apoptosis, the Fas pathway, is blocked by labile inhibitors in a manner that could prevent cytotoxicity. This review also examines several other types of regulation of apoptotic pathways in thyrocytes. We hypothesize that the regulation of programmed cell death pathways in the thyroid may alter the expression of autoimmune thyroid diseases by modifying the susceptibility of thyroid cells to immune-mediated apoptosis.

Asunto(s)

Apoptosis/inmunología , Tiroiditis Autoinmune/patología , Humanos , Tiroiditis Autoinmune/etiología , Tiroiditis Autoinmune/inmunología

RESUMEN

To determine whether thyroid cell apoptosis observed in autoimmune thyroid disease could be related to activation of the Fas pathway, we examined the expression and function of Fas on thyroid follicular cells in vitro. Fas messenger RNA was found to be present using two different techniques and was expressed at equal levels in thyrocytes cultured either in the presence or absence of TSH. Fas antigen protein expression was demonstrated by Western blot of thyroid cell lysates and by immunohistochemical staining of thyrocytes, and the amount of Fas protein present did not appear to vary regardless of culture conditions. Despite expressing substantial amounts of Fas protein, thyrocytes treated with anti-Fas monoclonal antibody failed to undergo apoptosis. The addition of either interferon-gamma or interleukin-1beta to the anti-Fas-treated cell cultures also did not promote apoptotic signaling through this pathway. In contrast, the concomitant administration of cycloheximide allowed the induction of apoptosis through the activation of Fas in thyrocytes. These results suggest that Fas is constitutively expressed in thyrocytes, but that the induction of apoptosis through the Fas pathway is blocked by a labile protein inhibitor.

Asunto(s)

Apoptosis/fisiología , Glándula Tiroides/citología , Glándula Tiroides/fisiología , Receptor fas/fisiología , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Cicloheximida/farmacología , Estabilidad de Medicamentos , Femenino , Humanos , Masculino , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas/antagonistas & inhibidores , ARN Mensajero/metabolismo , Glándula Tiroides/metabolismo , Receptor fas/genética , Receptor fas/metabolismo

RESUMEN

To define the autoantibody epitopes in amino acids 513-633 of thyroid peroxidase (TPO), a region frequently recognized in thyroiditis, cDNA sequences coding for peptide fragments of this region were amplified and ligated into pMalcRI and pGEX vectors for expression as recombinant fusion proteins. Western blots and enzyme-linked immunosorbent assay were then used to examine the reactivity in sera from 45 Hashimoto's and 47 Graves' disease patients. Two autoantibody epitopes within TPO amino acids 589-633 were identified; 16 of 35 patients reactive to TPO513-633 recognized the epitope of TPO592-613, while 6 patients recognized the epitope of TPO607-633. Eleven other patients with thyroiditis and two with Graves' disease recognized only the whole 589-633 fragment, and this response accounted for the Hashimoto's disease specificity. An amino acid sequence comparison of TPO592-613 with analogous regions of other peroxidase enzymes revealed significant differences in this area, and the substitution of even a single amino acid in one of the epitopes markedly decreased the binding affinity of autoantibodies. Additionally, the exclusive recognition by patients of only one of the epitopes within this region suggests a genetic restriction of the autoantibody response.

Asunto(s)

Autoanticuerpos/sangre , Epítopos/química , Enfermedad de Graves/inmunología , Yoduro Peroxidasa/química , Yoduro Peroxidasa/inmunología , Tiroiditis Autoinmune/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Arginina , Autoanticuerpos/biosíntesis , Secuencia de Bases , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Femenino , Enfermedad de Graves/sangre , Humanos , Yoduro Peroxidasa/biosíntesis , Lactoperoxidasa/química , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Peroxidasa/química , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Homología de Secuencia de Aminoácido , Tiroiditis Autoinmune/sangre

RESUMEN

DNA molecules condense into compact structures in the presence of a critical concentration of multivalent cations. To probe the contribution of electrostatic forces to condensation, we used mixtures of water with methanol (MeOH), ethanol (EtOH), and isopropanol (iPrOH) to vary the dielectric constant epsilon from 80 to 50. The condensation of pUC18 plasmids by hexaammine cobalt (III), Co(NH3)(3+)6, was monitored by total intensity and dynamic light scattering, electron microscopy, and CD. The total scattering intensity increased as epsilon went from 80 to 70, and the decreased as epsilon decreased further. Ultraviolet spectrophotometry confirmed that the loss of intensity at low epsilon was not due to the particles' settling out of solution. The rate as well as the extent of condensation increased as epsilon was lowered from 80 to 70, and also depended on the species of alcohol (MeOH < EtOH < iPrOH). The hydrodynamic radii RH of the particles, however, remained roughly the same at 300-350 A and was independent of the species of alcohol. RH increased below epsilon = 70. The critical concentration of Co(NH3)6(3+) required to induce DNA condensation decreased from 21 microM to about 16 microM as the dielectric constant decreased from 80 to 70, and decreased moderately with the nonpolarity of the alcohol. The fraction of DNA charge neutralized at the onset of DNA condensation was calculated by a modification of Manning's two-variable counterion condensation theory to be 0.90 +/- 0.01, independent of epsilon. By electron microscopy we observed that the condensed particles changed from about 93% toroids at epsilon = 80 to 89% rods at epsilon = 70 and 98% rods at epsilon = 65. At epsilon lower than 65, DNA collapsed into a network of multistranded fibers. The morphology of condensed DNA particles, whether toroids, rods, or fibers, was independent of the alcohol species. CD spectra in ethanol-water mixtures indicated that both closed circular and linearized plasmids were in the B conformation when condensed with Co(NH3)6(3+) at epsilon > or = 70, although the closed circular molecules exhibited a weak psi-DNA spectrum. A transition from the B to A form took place between epsilon = 70 and 60, well above the normal dielectric constant of epsilon = 40 for this transition, indicating that ethanol and Co(NH3)6(3+) synergistically promote the B-A transition. We interpret these results to mean that alcohols have both electrostatic and structural effects on DNA, leading to three regimes of condensation. At the lowest alcohol concentrations the B conformation is stable and condensation is relatively slow, allowing time for the packing adjustments necessary to form toroids.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Cobalto , ADN/química , Conformación de Ácido Nucleico , Plásmidos/química , 1-Propanol , Alcoholes , Secuencia de Bases , Dicroismo Circular , Electroquímica , Etanol , Metanol , Microscopía Electrónica , Oligodesoxirribonucleótidos , Plásmidos/ultraestructura , Fármacos Sensibilizantes a Radiaciones , Dispersión de Radiación , Solventes , Agua

RESUMEN

Thyroid peroxidase (TPO) is an important enzyme in the production of thyroid hormone and one of the major autoantigens in autoimmune thyroid disease. The gene for human thyroid peroxidase encodes a single 933 amino acid polypeptide chain. However, several reports have suggested that it exists in both high- and low-molecular-weight forms and the exact structure of the native enzyme is not known. We examined the structure of TPO using two monoclonal antibodies against different portions of TPO, a polyclonal mouse antiserum raised against a 300 amino acid fragment of TPO and autoantibodies directed against TPO obtained from patients with autoimmune thyroid disease. Western blots performed under nonreducing conditions identified three bands of approximately 220-230 kDa and two bands of 105 and 110 kDa that appeared to be immunologic TPO. After reduction, the TPO activity migrated as a smear of bands from 105 to 110 kDa, suggesting that the higher molecular weight form of the enzyme is a disulfide-linked dimer. Patients with autoimmune thyroid disease showed higher rates of recognition of the dimer than the reduced monomer when serologic reactivity was analyzed by Western blots. Eighty-three percent (40 of 48) of patients with Graves' disease and 76% (34 of 45) of Hashimoto's disease patients recognized the dimer form of TPO, while 48% (23 of 48) of Graves' and 60% (27 of 45) of Hashimoto's patients recognized reduced monomer TPO, even though both forms were denatured with SDS. Antibodies against different portions of the TPO chain all bound to the 105 kDa bands, indicating that the TPO chain is not bisected during posttranslational processing.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Yoduro Peroxidasa/química , Yoduro Peroxidasa/inmunología , Adolescente , Adulto , Anciano , Anticuerpos Monoclonales , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Western Blotting , Membrana Celular/enzimología , Niño , Enfermedad de Graves/inmunología , Humanos , Microsomas/enzimología , Persona de Mediana Edad , Peso Molecular , Pruebas de Precipitina , Desnaturalización Proteica , Glándula Tiroides/enzimología , Tiroiditis Autoinmune/inmunología

RESUMEN

In order to examine the specificity of the autoantibody response to thyroid peroxidase (TPO, previously identified as thyroid microsomal antigen) in autoimmune thyroid disease, we examined reactivity of sera from 45 Hashimoto's and 48 Graves' patients to native thyroid microsomes, denatured and reduced human TPO and several recombinant fragments of human TPO corresponding to amino acids 457-933 of the native protein. Both Graves' and Hashimoto's sera bound native, denatured and reduced TPO at significantly greater rate than normal controls, and no differences were noted between the two disorders in binding to these forms of the autoantigen. Binding was also noted to two recombinant fragments of TPO, corresponding to amino acids 513-633 and 633-933 in TPO. The frequency of autoantibodies to the TPO AA(633-933) region was not significantly different in Hashimoto's vs. Graves' disease patients (58% vs. 65% respectively), and appeared to relate to evidence of glandular inflammation in the Graves' patients (presence of anti-thyroglobulin antibodies and elevated anti-microsomal antibody levels). In contrast, antibodies to the TPO AA(513-633) fragment were significantly more common and of higher titer in Hashimoto's vs. Graves' disease patients, and did not correlate with any measure of glandular inflammation. These results identify two specific regions of TPO autoantibody binding and indicate that there are differences in the autoantibody response to TPO in Hashimoto's and Graves' diseases.

Asunto(s)

Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/inmunología , Enfermedad de Graves/inmunología , Yoduro Peroxidasa/inmunología , Tiroiditis Autoinmune/inmunología , Adolescente , Adulto , Anciano , Análisis de Varianza , Secuencia de Bases , Western Blotting , Niño , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Microsomas/enzimología , Microsomas/inmunología , Persona de Mediana Edad , Datos de Secuencia Molecular , Glándula Tiroides/ultraestructura , Hormonas Tiroideas/sangre

RESUMEN

A recent report has identified a new autoantigen called D1 that appears to be associated with Graves' ophthalmopathy and is expressed in the thyroid and eye muscle. To better characterize the tissue specificity and disease relevance of this antigen, we evaluated the expression of D1 RNA in various human tissues using a reverse transcriptase polymerase chain reaction assay. These studies indicate a wide tissue distribution of the messenger RNA for this antigen, including the thyroid, eye muscle, parathyroid, spleen, skeletal muscle, and uterus. There were variations in the relative amounts of specific message for D1 in the different tissues, with the uterus, thyroid, and eye muscle having the greatest amount of product per microgram of total RNA. A maltose binding protein-D1 fusion protein was expressed in Escherichia coli, purified, and used to assess serologic reactivity to D1 by Western blot. Autoantibodies to this antigen were noted in 19 of 24 (78%) of Hashimoto's disease patients, 26 of 41 (63%) of Graves' disease patients, and in 9 of 17 (53%) of normal controls. Sixty percent of Graves' disease patients with clinical ophthalmopathy had antibodies to D1, as did 63% of Graves' patients without signs or symptoms of clinical ophthalmopathy. There was no correlation between reactivity to D1 and either clinical measures of hyperthyroidism or antibody titers to thyroid peroxidase or thyroglobulin. The presence of autoantibodies to this antigen in patients with Hashimoto's disease, in Graves' disease patients without ophthalmopathy and in normal controls indicate that serologic recognition of this antigen is not restricted to patients with ophthalmopathy. In addition, the expression of messenger RNA for this antigen in multiple types of cells questions the tissue specificity of this autoantigen.

Asunto(s)

Autoantígenos/biosíntesis , Enfermedades Autoinmunes/inmunología , Enfermedad de Graves/inmunología , Tiroiditis Autoinmune/inmunología , Adulto , Autoanticuerpos/biosíntesis , Autoanticuerpos/inmunología , Autoantígenos/genética , Autoantígenos/inmunología , Secuencia de Bases , Western Blotting , Oftalmopatías/etiología , Oftalmopatías/inmunología , Femenino , Enfermedad de Graves/complicaciones , Humanos , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión , Glándula Tiroides/inmunología

RESUMEN

Recent reports have suggested that Yersinia enterocolitica proteins encoded by a 72-kilobase virulence plasmid (known as release proteins and now identified as YOP2-5) are antigens recognized specifically by patients with Graves' disease and of potential etiological importance in this disorder. To examine this hypothesis, we evaluated immune responses to YOP in patients with autoimmune thyroid disease and in normal controls. Humoral responses to Yersinia were assessed using Western blots of crude Y. enterocolitica membrane proteins, Yersinia release proteins (YOP2-5), and human thyrocyte membranes. Twenty-four of 25 Graves' and 10 of 18 Hashimoto's patients showed reactivity with the release proteins, primarily the 67-, 46-, 36-, and 25-kilodalton bands. However, 17 of 24 normal subjects also demonstrated serological reactivity to the release proteins, and the pattern of reactivity of these sera was similar to that in the thyroid patients. No correlation was noted between serological reactivity to the release proteins and thyroid hormone levels. Patients and controls with serological reactivity to YOP also showed reactivity with Yersinia membranes. In addition to the serological studies, cellular immune responses were determined by peripheral blood mononuclear cell proliferation assays. Cellular reactivity to the release proteins was present in four of five Graves' and both Hashimoto's patients tested, but also in two of six nonthyroid illness patients with serological immunity to the release proteins. Intrathyroidal lymphocytes obtained from two Graves' patients demonstrated marked proliferation in response to the release proteins. These results indicate that there is no unique pattern of serological reactivity against Yersinia membranes or the release proteins in patients with autoimmune thyroid diseases and suggest that any causal relationship between Yersinia infection and Graves' disease may be related to T-cell immunity.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Enfermedad de Graves/inmunología , Yersinia enterocolitica/inmunología , Proteínas Bacterianas/metabolismo , Western Blotting , Humanos , Inmunidad Celular , Activación de Linfocitos , Unión Proteica , Serotipificación , Tirotropina/metabolismo , Tirotropina/farmacología , Yersinia enterocolitica/clasificación

RESUMEN

Recent reports have disagreed on the nature of the autoantibody epitopes in thyroid peroxidase (TPO). We used immunoprecipitation of recombinant human TPO constructs to determine if localized autoantibody binding sites exist in this autoantigen. In vitro transcription and translation of TPO cDNA fragments yielded 35S-labeled products consisting of either full-length protein (933 amino acids) or N-terminal peptides of 631, 455, and 120 amino acids. Immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the Hashimoto's sera consistently precipitated the full-length and the 631 amino acid products, but not the shorter N-terminal peptides. An additional construct resulting in a full-length TPO peptide with an internal deletion of amino acids 4-455 was also made, and this product was also precipitated by the Hashimoto's sera. A fusion protein consisting of maltose binding protein followed by amino acids 456-933 of human TPO was produced in Escherichia coli and subjected to Western blot analysis using the Hashimoto's sera. The Hashimoto's sera reacted with the MalTose binding protein TPO (MBP/TPO) fusion protein, but not a control fusion protein (MBP/LacZ alpha). Together, these results indicate the presence of localized autoantibody epitopes in the portion of the human TPO molecule from amino acids 456 to 933, with at least one binding site located between amino acids 456 and 631.

Asunto(s)

Autoanticuerpos/inmunología , Epítopos/análisis , Yoduro Peroxidasa/inmunología , Western Blotting , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Humanos , Pruebas de Precipitina , Tiroiditis Autoinmune/enzimología , Transfección

Asunto(s)

Microscopía de Túnel de Rastreo , Ácidos Nucleicos/química , Microscopía de Túnel de Rastreo/instrumentación , Microscopía de Túnel de Rastreo/métodos , Manejo de Especímenes

Asunto(s)

ADN/química , Conformación de Ácido Nucleico , Cinética , Rayos Láser , Luz , Dispersión de Radiación , Soluciones

Asunto(s)

ADN/ultraestructura , Conformación de Ácido Nucleico , Microscopía/métodos

Asunto(s)

ADN/ultraestructura , Polinucleótidos/química , ARN/ultraestructura , Animales , Bovinos , Microscopía de Túnel de Rastreo , Conformación de Ácido Nucleico , Poli A-U/química , Polidesoxirribonucleótidos/química

RESUMEN

The scanning tunnelling microscope (STM) is capable of atomic resolution of highly conductive materials. Whether biological molecules can be visualized to the same extent remains an open question, but remarkable progress in the past year confirms the possibility of seeing the fine structure of nucleic acids, proteins, membranes and viruses, and provides evidence that their dynamic interactions can be monitored under conditions approximating to those of the native environment.

Asunto(s)

Biotecnología/instrumentación , Microscopía de Túnel de Rastreo/métodos , Microscopía de Túnel de Rastreo/instrumentación