RESUMEN
Experimental autoimmune ovarian disease (AOD) is a T cell-mediated chronic inflammatory disease that may lead to premature ovarian failure. Autoimmune disease can be suppressed by oral administration of autoantigens leading to tolerance. One of the major mechanisms of oral tolerance is induction of regulatory CD4+ T cells that can mediate active suppression by producing immunomodulatory cytokines. However, the role of oral tolerance as a treatment for experimental AOD has received little attention. Therefore, the purpose of this study was to examine the conditions necessary to produce oral tolerance in experimental AOD in B6AF1 female mice. In this study, mice received different doses of peptides of the mouse zona pellucida 3 (pZP3) via gastric intubation for 7 times. After 4 times of oral administration, AOD was induced by immunization with pZP3. The optimal tolerating regimen for oral administration of pZP3 in mice was 10 microg, which decreased morbidity of oophoritis compared to the control group. In this moderate-dose therapeutic group (MD), alterations in the estrous cycle were normalized and CD4+ T cells that were CD25+ increased while those that were CD25- decreased. The severity of autoimmune oophoritis and the titer of ZP autoantibodies were also significantly reduced. These findings suggest that oral administration of pZP3 may be successfully used as an oral tolerance strategy for suppression of AOD.
Asunto(s)
Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteínas del Huevo/inmunología , Glicoproteínas de Membrana/inmunología , Ooforitis/inmunología , Péptidos/administración & dosificación , Péptidos/inmunología , Receptores de Superficie Celular/inmunología , Administración Oral , Animales , Autoanticuerpos/sangre , Enfermedades Autoinmunes/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Modelos Animales de Enfermedad , Proteínas del Huevo/administración & dosificación , Estro , Femenino , Tolerancia Inmunológica , Glicoproteínas de Membrana/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Receptores de Superficie Celular/administración & dosificación , Glicoproteínas de la Zona PelúcidaRESUMEN
OBJECTIVE: To explore the molecular mechanism of neural tube defects (NTDs) caused by hyperglycemia and thiadiazole and the antagonistic effect of taurine. METHODS: The pregnant mice were divided into hyperglycemia groups, thiadiazole group, taurine groups and control groups. The mRNA and the protein of Pax3 or Cx43 gene were detected respectively by reverse transcription-polymerase chain reaction assay and immunohistochemical method. RESULTS: As compared with mice treated by thiadiazole-stomach-perfusing, NTDs were significantly increased from mice treated with glucose-injection when blood glucose levels were >or= 13.4 mmol/L. Elevated glucose and thiadiazole could cause changes in Pax3 and Cx43 expression. Hyperglycemia had stronger developmental toxicity on mice embryos. Expression of Pax3 (mRNA 0.97 +/- 0.20, protein 0.11 +/- 0.02) in hyperglycemia group was significantly decreased, while expression of Cx43 (mRNA 7.05 +/- 1.63, protein 0.94 +/- 0.05) was significantly increased, and the relationship of dose-effect was demonstrated. In the thiadiazole group, the expression of Cx43 (mRNA 6.96 +/- 0.73, protein 0.92 +/- 0.12) was significantly stronger than control groups, but there were no significant differences in expression of Pax3 between thiadiazole and its control groups. Both of their teratogenicity could be antagonized by taurine. CONCLUSIONS: This study suggests that congenital malformation associated with diabetic pregnancy is caused by disruption of regulatory genes, Pax3 and Cx43 expression in embryo in response to elevated glucose. Thiadiazole can only disturb the regulation of Cx43 gene causing NTDs. Taurine can correct the disruption caused by the two teratogens.
Asunto(s)
Hiperglucemia/tratamiento farmacológico , Defectos del Tubo Neural/genética , Taurina/farmacología , Animales , Conexina 43/genética , Conexina 43/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hiperglucemia/inducido químicamente , Hiperglucemia/complicaciones , Inmunohistoquímica , Masculino , Ratones , Defectos del Tubo Neural/embriología , Defectos del Tubo Neural/etiología , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiadiazoles/toxicidad , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: To find out the molecular mechanisms of togenesis, especially neural tube defects (NTDs) caused by hyperglycemia and thiadiazole. METHODS: Mice were raised with a ratio of 2:1 between females and males. Forty-five pregnant female mice were randomly divided into 5 groups: blank control group (without any treatment), high-glucose group (treated with subcutaneous injection of 25% glucose), glucose-control group (n = 12, treated with subcutaneous injection of 5% glucose), thiadiazole group (treated with gastric perfusion of thiadiazole), and thiadiazole-control group (n = 11, treated with the solvent of thiadiazole). The blood sugar was examined among the mice of the first three groups on the 8th gestational day. All of the mice were killed on the 15th gestational day and the embryos were taken. The existence of teras was observed. RT-PCR was used to detect the expression of Pax3 and of Cx43 genes and immunohistochemistry was used to examine the Pax3 and Cx43 proteins in the embryos. RESULTS: The blood sugar of the pregnant mice in the high-glucose group was 13.4 mmol/L +/- 0.8 mmol/L, significantly higher than those in the 2 control groups (4.9 mmol/L +/- 0.4 mmol/L and 4.8 mmol/L +/- 0.4 mmol/L respectively, both P < 0.01). The teras rate, absorbed embryo rate and still embryo rate in high-glucose group were significantly higher than those in the 2 control groups (all P < 0.01). The embryo weight of the high-glucose group was significantly lower. The expression of Pax3 in the hyperglycemia group was significantly lower than those in the 2 control groups (both P < 0.01), while the expression of Cx43 in high-glucose group was significantly higher than those in the 2 control groups (both P < 0.01). In the thiadiazole group, the expression of Pax3 was not significantly different from, and the expression of Cx43 was significantly higher than those in the 2 control groups. CONCLUSION: Hyperglycemia is induced in nondiabetic pregnant mice by subcutaneous glucose injection, and elevated glucose appears to be critical in the diabetic embryopathy. Hyperglycemic episodes disturb the essential embryonic control genes: decrease the expression of Pax3 gene and increase the expression of Cx43 gene, thus causing congenital defects. Thiadiazole induces NTDs by increasing the expression of Cx43.
Asunto(s)
Conexina 43/genética , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/metabolismo , Hiperglucemia/complicaciones , Defectos del Tubo Neural/metabolismo , Factores de Transcripción , Animales , Femenino , Masculino , Ratones , Defectos del Tubo Neural/etiología , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Embarazo , ARN Mensajero/análisisRESUMEN
OBJECTIVE: To investigate the clinical feasibility of fetal sex determination by using nested polymerase chain reaction (PCR) to detect cell-free fetal DNA in the urine of pregnant women. METHODS: The urine specimens of 40 healthy predelivery women were centrifuged. The DNA in the specimens of supernatant was extracted by using of QIAamp Viral RNA Mini Kits. Nested PCR was used to specifically amplify the SRY gene, 300 bp in length, on Y chromosome in order to detect the fetal DNA from the women bearing male fetuses. One specimen of normal man's urine and one specimen of non-pregnant woman's urine were used as controls for each specimen from pregnant women. The results were confirmed by the sexes of neonates examined after birth. RESULTS: Amplified band of SRY was found in 6 specimens. Sex examination after delivery showed 21 male neonates and 19 female neonates. The positive rate of detection of SRY gene was 28.6% (6/21) and the false negative rate was 71.4%, and false positive rate was 0. Five out of the 6 cases with positive results showed positive 2 times and one out of the 6 cases only showed positive at the second time. CONCLUSION: There is cell-free fetal DNA in the urine of pregnant women. Nested PCR is useful in detection of fetal sex, however, still with a rather low positive rate.