RESUMO
The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.
Assuntos
Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , Imunoglobulina G/biossíntese , Receptores Fc/biossíntese , Receptores Fc/genética , Regulação para Cima/imunologia , Animais , Afinidade de Anticorpos/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Bovinos , Galinhas , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Antígenos de Histocompatibilidade Classe I/fisiologia , Humanos , Imunidade Materno-Adquirida , Imunoglobulina G/metabolismo , Imunoglobulinas/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Plasmócitos/imunologia , Plasmócitos/metabolismo , Ligação Proteica/imunologia , Receptores Fc/fisiologia , Regulação para Cima/genéticaRESUMO
The aim of this study was to develop a method to generate identical twins and triplets with predicted gender. As a first step toward that aim, single blastomeres obtained from EGFP expressing eight-cell stage embryos and either diploid or tetraploid host embryos were used to compose chimera. We could follow the fate of EGFP expressing diploid blastomere derived cells in 3.5- and 4.5-day-old chimera embryos in vitro. We found that the diploid blastomere-derived cells had significantly higher chance to contribute to the inner cell mass if tetraploid host embryos were applied. After that, we developed a quick and reliable multiplex PCR strategy for sex diagnosis from single blastomeres by simultaneous amplification of the homologous ZFX and ZFY genes. By composed chimeras using single blastomeres, derived from sexed eight-cell stage embryos and a tetraploid host embryo, we could get preplanned sex newborns, wholly derived from these blastomeres. Among these mice, identical twins and a triplet were identified by microsatellite analysis. Unlike clones produced by nuclear transfer, these mice are identical at both the nuclear as well as mitochondrial DNA level. Therefore, the tetraploid embryo complementation method to produce monozygotic twins and triplets could be a valuable tool both in biomedical and agricultural applications.
Assuntos
Blastômeros/fisiologia , Quimera/embriologia , Tamanho da Ninhada de Vivíparos , Análise para Determinação do Sexo , Animais , DNA Mitocondrial/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Poliploidia , GêmeosRESUMO
Leukemia inhibitory factor (LIF) is a multifunctional cytokine with an important role during early embryonic development, implantation, and as an inhibitor of murine embryonic stem cell differentiation. It exerts its effects by binding to the leukemia inhibitory factor receptor, a heterodimer of two transmembrane proteins, the specific leukemia inhibitory factor receptor subunit, and the common gp130. A partial cDNA clone coding for the membrane-bound form of the specific rabbit leukemia inhibitory factor receptor was isolated from the genital ridge of 13.5 days postcoitum fetus. Fluorescent in situ hybridization analysis revealed that the rabbit leukemia inhibitory factor receptor gene is located on chromosome OCU11p11.1. It has been shown that the membrane-bound rabbit leukemia inhibitory factor receptor mRNA is expressed during embryo implantation but not at earlier developmental stages. Rabbit embryonic stem cell-like line establishment is improved in the presence of LIF, and those cells express both leukemia inhibitory factor and its receptor. The withdrawal of leukemia inhibitory factor results the differentiation of embryonic stem cell-like cells to beating myocardial-like cells. Our findings suggest that the self-renewal mechanism is similar in mouse and rabbit embryonic stem cells, and expands our knowledge on the role of the LIF-LIFR signal pathway in early rabbit embryogenesis and rabbit embryonic stem cell establishment.
Assuntos
Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/fisiologia , Receptores de OSM-LIF/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Técnicas de Cultura de Células , Cromossomos/genética , Clonagem Molecular , Células-Tronco Embrionárias/metabolismo , Humanos , Fator Inibidor de Leucemia/fisiologia , Camundongos , Dados de Sequência Molecular , Coelhos , Receptores de OSM-LIF/genética , Alinhamento de Sequência , Transdução de SinaisRESUMO
Here we report that improved reproductive technologies combined with an efficient microinjection method and in vitro cultivation medium enabled us to create germ line chimeric rabbits. To follow the fate of the chimeric embryo a blastomere marked with the human blood coagulation factor VIII (hFVIII) transgene was microinjected into a morula stage wild type embryo. The degree of chimerism in different tissues was estimated by real-time PCR and was found to be in the range of 0.1-42%. Among the four chimeric animals, one was identified as a chromosomal intersex and two were germline chimeras.