RESUMEN
BACKGROUND AND OBJECTIVE: Female sex hormones are elevated and are potential host response modifiers during pregnancy. Modulation of immune responses by estrogen and progesterone may be responsible for periodontal inflammation. Therefore, we aimed to investigate the role of ß-estradiol and progesterone in human monocyte immune responses, at cellular and molecular levels, to identify their role as a possible immunological link between pregnancy and periodontal disease. MATERIAL AND METHODS: Primary human monocytes were purified from human peripheral blood mononuclear cells by adherent method. Expression of Toll-like receptor (TLR) 2, 4 and CD14 was analyzed by flow cytometry. TLR2, TLR4, cyclooxygenase-2 (COX2), nuclear factor-kappa B (NF-κB) and NF-κB inhibitor-alpha mRNA expressions were measured using real-time reverse transcriptase-polymerase chain reaction and prostaglandin E2 secretion was assayed by enzyme-linked immunosorbent assay. NF-κB expression was also examined by immunofluorescence. Western blotting was performed to determine the activation of mitogen-activated protein kinase pathway. RESULTS: We report herein that both ß-estradiol and progesterone significantly reduced TLR2 expression at both protein and mRNA levels but had less of an effect on TLR4 expression in primary human monocytes. We also found that the hormones decreased monocyte cell surface protein expression of CD14. Significantly, ß-estradiol and progesterone dose-dependently downregulated monocyte expression of COX2 mRNA. Pretreatment monocytes with ß-estradiol or progesterone reduced effects of Porphyromonas gingivalis lipopolysaccharide (LPS) on COX2 mRNA expression and decreased prostaglandin E2 secretion by the monocytes. Furthermore, we demonstrated that both ß-estradiol and progesterone inhibited P. gingivalis LPS-induced NF-κB signaling pathway through the upregulation of NF-κB inhibitor-alpha expression. However, neither ß-estradiol nor progesterone altered the phosphorylation of the p38, the extracellular signal-regulated kinase 1/2 and the c-Jun N-terminal activated kinase in P. gingivalis LPS-stimulated monocytes. Thus, the inhibitory effects of these hormones on the response of human monocytes to P. gingivalis LPS appear to be independent on mitogen-activated protein kinase signaling pathway. CONCLUSION: The results of the present study suggest that ß-estradiol and progesterone could influence the immune response of human monocytes to periodontal pathogens and this process may have a role in the clinical manifestations of periodontal disease associated with pregnancy.
Asunto(s)
Hormonas Esteroides Gonadales/farmacología , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Porphyromonas gingivalis/metabolismo , Receptores Toll-Like/efectos de los fármacos , Receptores Toll-Like/inmunología , Proliferación Celular/efectos de los fármacos , Ciclooxigenasa 2/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Regulación hacia Abajo , Estradiol/farmacología , Estrógenos/farmacología , Femenino , Humanos , Leucocitos Mononucleares , Receptores de Lipopolisacáridos/efectos de los fármacos , Receptores de Lipopolisacáridos/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , Enfermedades Periodontales , Fosforilación , Porphyromonas gingivalis/inmunología , Embarazo , Progesterona/farmacología , ARN Mensajero/análisis , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/efectos de los fármacos , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Regulación hacia ArribaRESUMEN
The non-catalytic region of tyrosine kinase (Nck) is proposed to play an essential role in T cell activation. However, evidence based on functional and biochemical studies has brought into question the critical function of Nck. Therefore, the aim of the present work was to investigate the role of Nck in T cell activation. To study this, the human Jurkat T cell line was used as a model for human T lymphocytes. The short interfering (si) RNA targeting Nck1 gene was used with electroporation to knock-down Nck1 protein expression in Jurkat T cells. Primary human CD4 T cells were also transfected with the siRNA of Nck1. The results showed that decreased Nck1 protein expression did not affect the apoptosis of the transfected Jurkat T cells compared with control siRNA-transfected cells and non-transfected cells. Upon CD3ε/CD28 stimulation, knock-down of Nck1 in Jurkat T cells caused a decrease in CD69 expression and in interleukin (IL)-2 secretion. Similarly, knock-down of Nck1 in primary CD4 T cells also caused decreased CD69 expression. However, no significant alterations of CD69 and IL-2 expression were found upon phytohaemagglutinin (PHA)/phorbol myristate acetate (PMA) stimulation. Knock-down of Nck1 had no effect on the proliferation of Jurkat T cells stimulated with either PHA or anti-T cell receptor (TCR) monoclonal antibody (C305). The reduced Nck1 expression in Jurkat cells was also associated with a reduced phosphorylation of extracellular regulated kinase (Erk)1 and Erk2 proteins upon CD3ε/CD28 stimulation. In conclusion, the decreased Nck1 protein in Jurkat T cells resulted in an impairment of TCR-CD3-mediated activation involving a defective Erk phosphorylation pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Linfocitos T CD4-Positivos/inmunología , Células Jurkat/inmunología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Oncogénicas/fisiología , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Inmunidad Adaptativa/inmunología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/genética , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/metabolismo , Electroporación , Humanos , Interleucina-1/biosíntesis , Interleucina-1/genética , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Lectinas Tipo C/biosíntesis , Lectinas Tipo C/genética , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/genética , Fosforilación , Fitohemaglutininas/farmacología , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Interleukin (IL)-17 is a proinflammatory cytokine with pleiotropic activities including inducing neovascularization and production of proangiogenic molecules. As pregnancy outcome depends on the balance of Th1-like/Th2-like cytokines and an increased blood supply to the fetoplacental unit, the expression of IL-17 mRNA and protein in human placental tissues was investigated. IL-17 mRNA was expressed by purified cytokeratin-positive term placental trophoblast cells, HLA-G+ extravillous trophoblast cells and placental macrophages (Hofbauer cells). IL-17 localized in both cyto- and syncytiotrophoblasts of normal term pregnancy, spontaneous miscarriage and in molar pregnancy. In spontaneous miscarriage and molar pregnancy extravillous trophoblast cells were consistently immunoreactive for IL-17. IL-17 expression in human placenta may play a key role in angiogenesis and/or immunoregulation in the establishment of pregnancy.
Asunto(s)
Interleucina-17/metabolismo , Placenta/metabolismo , Adolescente , Adulto , Células Cultivadas , Femenino , Expresión Génica , Humanos , Placenta/química , Embarazo , Proteínas Gestacionales/análisis , ARN Mensajero/análisisRESUMEN
Complete hydatidiform moles are totally paternally derived and represent complete allografts that might be expected to provoke maternal immune rejection. Our previous and other studies have shown expression of Fas by increased numbers of activated decidual CD4(+) T cells in both complete and partial molar pregnancy as well as increased FasL(+) expression by molar trophoblasts compared with trophoblasts in normal pregnancies. As the Fas/FasL system represents a major apoptotic pathway that can play a role in immune privilege, the aim of this study was to investigate whether apoptosis of decidual immune cells, particularly T cells, could be responsible for maternal immune tolerance in molar pregnancy. Using terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labelling (TUNEL), a significant increase in TUNEL(+) cells was demonstrated in decidua associated with partial (P = 0.0052) and complete (P = 0.0096) hydatidiform mole compared with normal early pregnancy. Co-labelling immunoperoxidase studies showed that the TUNEL(+) cells in both normal and molar pregnancies were not activated CD45RO(+) immune cells, CD3(+) T cells, CD56(+) uterine natural killer (NK) cells or CD14(+) CD68(+) macrophages. Double immunohistochemical labelling with antiactive caspase-3 and leucocyte markers confirmed the lack of leucocyte apoptosis. Double immunostaining with anticytokeratin to detect trophoblast and M30 CytoDeath, which detects a neoepitope of cytokeratin 18 revealed after caspase-mediated cleavage, revealed apoptotic extravillous trophoblast cells within decidual tissue. We conclude that there is no evidence that apoptosis of decidual leucocytes plays a role in maintaining maternal tolerance in either normal or molar pregnancy.
Asunto(s)
Apoptosis/inmunología , Decidua/inmunología , Mola Hidatiforme/inmunología , Tolerancia Inmunológica/inmunología , Leucocitos/inmunología , Neoplasias Uterinas/inmunología , Antígenos CD/análisis , Caspasa 3 , Caspasas/análisis , Decidua/patología , Precursores Enzimáticos/análisis , Femenino , Humanos , Técnicas para Inmunoenzimas/métodos , Etiquetado Corte-Fin in Situ/métodos , Queratinas/análisis , Células Asesinas Naturales/inmunología , Embarazo , Linfocitos T/inmunologíaRESUMEN
To clarify the Fas and Fas-ligand status of normal and molar trophoblast, the expression of Fas and FasL by placental trophoblast populations in partial and complete hydatidiform moles was compared with that in normal first trimester and term pregnancies using an avidin-biotin peroxidase technique on frozen and formalin-fixed paraffin-embedded placental tissues with both monoclonal and polyclonal antibodies. The TUNEL technique was used to detect apoptotic cells in the same tissues. The immunoreactivity for Fas and Fas-ligand was comparable with both monoclonal and polyclonal antibodies on frozen as well as paraffin-embedded sections. In normal early and molar pregnancy there was strong FasL expression by villous cytotrophoblast and syncytiotrophoblast. However, there were significant differences in FasL expression by trophoblast subpopulations in both early and term normal pregnancy and between the same trophoblast subpopulation at different gestations, with FasL staining generally being weaker at term. Strong FasL staining by cytotrophoblast cells in the distal parts of cell columns contrasted with unstained cytotrophoblast in the proximal part of columns. Distinct trophoblast subpopulations in partial hydatidiform mole also differentially expressed FasL with reduced FasL expression in proliferating syncytiotrophoblast. In contrast there was no differential FasL expression in complete hydatidiform mole, all trophoblast subpopulations strongly expressing FasL. Unlike the differential expression of FasL there were no differences in Fas expression by trophoblast populations in normal early or term placental tissues. Fas expression was reduced in villous cytotrophoblast at term. Differential expression of Fas by different trophoblast subpopulations was noted in partial and complete hydatidiform mole. In complete mole villous cytotrophoblast and syncytiotrophoblast stained strongly compared with proliferating trophoblast. Using TUNEL labelling apoptosis was rarely detected in placental trophoblast. Differential Fas and FasL expression by trophoblast subpopulations in normal and pathological pregnancy does not appear to be related to apoptosis of trophoblast.
Asunto(s)
Apoptosis , Mola Hidatiforme/metabolismo , Glicoproteínas de Membrana/metabolismo , Trofoblastos/metabolismo , Receptor fas/metabolismo , Adulto , Recuento de Células , Vellosidades Coriónicas/metabolismo , Vellosidades Coriónicas/patología , Proteína Ligando Fas , Femenino , Humanos , Mola Hidatiforme/patología , Técnicas para Inmunoenzimas , Etiquetado Corte-Fin in Situ , Embarazo , Primer Trimestre del Embarazo , Trofoblastos/citología , Trofoblastos/patologíaRESUMEN
This paper evaluated the effect of VKP in the neonates by oral route with different dosages compared to the standard parenteral route giving a single dose at birth. Two hundred and thirty-six healthy, breast-fed infants were divided into 4 groups receiving vitamin K1 1 mg intramuscularly and 2, 3, 5 mg orally during 2-4 hours after birth. The vitamin K dependent clotting factors were measured by the thrombotest at the age of 2 weeks and 4-6 weeks. The result showed no statistical differences among these 4 groups regarding the mean prothrombin complex level and the number of PC deficient subjects. Vitamin K prophylaxis in the newborn babies by 2 mg oral route would be benefit and can be applied routinely as well as 1 mg parenteral route to prevent both HDN and APCD syndrome particularly in breast fed infants. The routine practice of giving vitamin K1 prophylaxis 2 mg orally or 0.5-1 mg intramuscularly should be recommended to all newborn infants. Giving VKP by oral route is practical for developing countries because of simple way of administration, low cost, low toxicity, as well as high efficacy.