RESUMO
Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRß, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo- and in vitro-produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development.
Assuntos
Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Animais , Bovinos , Células Cultivadas , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Oócitos/fisiologia , Oogênese/genética , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismoRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disorder of the connective tissue with a wide and heterogeneous spectrum of manifestations, with renal and neurological involvement usually related to worse prognosis. SLE more frequently affects females of reproductive age, and a high prevalence and renal manifestation seem to be associated with non-European ethnicity. The present study aims to investigate candidate loci to SLE predisposition and evaluate the influence of ethnic ancestry in the disease risk and clinical phenotypic heterogeneity of lupus at onset. Samples represented by 111 patients and 345 controls, originated from the city of Belém, located in the Northern Region of Brazil, were investigated for polymorphisms in HLA-G, HLA-C, SLC11A1, MTHFR, CASP8 and 15 KIR genes, in addition to 89 Amerindian samples genotyped for SLC11A1. We also investigated 48 insertion/deletion ancestry markers to characterize individual African, European and Amerindian ancestry proportions in the samples. Predisposition to SLE was associated with GTGT deletion at the SLC11A1 3'UTR, presence of KIR2DS2 +/KIR2DS5 +/KIR3DS1 + profile, increased number of stimulatory KIR genes, and European and Amerindian ancestries. The ancestry analysis ruled out ethnic differences between controls and patients as the source of the observed associations. Moreover, the African ancestry was associated with renal manifestations.
Assuntos
Proteínas de Transporte de Cátions/genética , Etnicidade/genética , Marcadores Genéticos , Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/genética , Polimorfismo Genético , Receptores KIR/genética , Adulto , Idade de Início , Brasil , Cidades , Feminino , Frequência do Gene , Humanos , Lúpus Eritematoso Sistêmico/etnologia , Masculino , Receptores KIR3DS1/genéticaRESUMO
In the present study we investigated the role of nitric oxide (NO) in the effector mechanisms of host defense against Cryptococcus neoformans in vivo. Our results showed an increase of NO produced by the peritoneal macrophages from 14-days infected rats compared with normal rats. These cells were capable of killing C. neoformans to a greater extent than macrophages from noninfected rats (80% vs 20%, respectively). The killing of C. neoformans by infected cells was efficiently inhibited (80% to 35%, P < 0.001) by adding aminoguanidine (AG) to the cultures. We observed that in vivo administration of AG to the infected animals efficiently inhibited the metabolism producing NO and failed to affect that of normal animals. When the NO synthase (NOS) was inhibited in vivo in the infected animals, a marked increase of the fungi charge in the organs was observed with respect to the normal animals treated with AG. We also observed that the course of the infection is drastically modified after the inhibition of NO production because all the animals infected and treated with AG died from cryptococcosis before 20 days postinfection (p.i.). These results indicate that NO is a crucial molecule in the effector mechanisms in this infection model.
Assuntos
Criptococose/imunologia , Cryptococcus neoformans/imunologia , Óxido Nítrico/imunologia , Animais , Criptococose/metabolismo , Criptococose/patologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Guanidinas/farmacologia , Técnicas In Vitro , Pulmão/imunologia , Pulmão/patologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
We investigated the proliferative response to mitogens of spleen mononuclear (Spm) cells from Cryptococcus neoformans-infected rats. We determined reactive oxygen intermediates (ROI) and nitric oxide (NO) production by peritoneal and Spm cells, and evaluated the correlation of the proliferative response with NO and ROI production. The proliferative response of Spm cells from infected rats dramatically decreased at 14 and 21 days postinfection (PI). The unresponsiveness of Spm cells from 14-day infected rats was not abrogated by the addition of L-NAME and AG, indicating that NO is not involved in the antiproliferative response of experimental cells. When SOD, catalase, and indomethacin were added to the cultures, the suppression was still observed, indicating that ROI and prostaglandins are not involved in the unresponsiveness of lymphocytes. The proliferative response of lymphocytes from 14-day infected rats was significantly improved when cultures were made in the presence of Con A and exogenous IL-2. Additionally, a purified T-rich fraction from infected rats cultured with control macrophages recovered the normal proliferative response. This result indicates that macrophages from infected rats mediate the unresponsiveness of lymphocytes, probably by reducing the ability of lymphocytes to secrete IL-2.
Assuntos
Criptococose/metabolismo , Ativação Linfocitária/fisiologia , Subpopulações de Linfócitos/imunologia , Macrófagos Peritoneais/fisiologia , Óxido Nítrico/fisiologia , Baço/citologia , Animais , Antioxidantes/farmacologia , Catalase/farmacologia , Concanavalina A/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Guanidinas/farmacologia , Indometacina/farmacologia , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Lipopolissacarídeos/farmacologia , Subpopulações de Linfócitos/citologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Antagonistas de Prostaglandina/farmacologia , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Taxa Secretória/efeitos dos fármacos , Superóxido Dismutase/farmacologiaRESUMO
The biochemical basis of peritoneal cell cytotoxicity for Cryptococcus neoformans was studied by measuring the killing of the yeast by peritoneal resident cells and peritoneal exudate cells obtained from normal and proteose-peptone-injected animals, respectively. Both cell populations killed C. neoformans to an equivalent extent after 3 h incubation. Exudate cells showed anti-cryptococcal activity from the first hour of incubation, while no killing was observed with resident cells before 3 h. Both cell populations triggered a respiratory burst in response to opsonized C. neoformans as indicated by the fact that killing of the yeast was inhibited by scavengers of reactive oxygen intermediates (ROI). C. neoformans susceptibility to H2O2 and hydroxyl radicals in cell-free systems is demonstrated by incubating a yeast suspension with different concentrations of H2O2 and Fenton's reagents, respectively. These results suggest that oxygen metabolites play an active role in C. neoformans killing.