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2.
Artigo em Inglês | MEDLINE | ID: mdl-30351228

RESUMO

Recombinant adeno associated viruses (rAAV) have become an important tool for the delivery of gene therapeutics due to long-standing safety and success in clinical trials. Since humans often become exposed to AAVs and develop anti-AAV antibodies (Abs), a potential impediment to the success of gene therapeutics is neutralization of the viral particle before it has had a chance to bind and enter target cells to release the transgene. Identification of subjects with preexisting Abs having neutralizing potential, and exclusion of such subjects from clinical studies is expected to enhance drug efficacy. In vitro cell-based reporter assays are most often employed to determine the level of neutralizing antibodies in a given population. Such assays measure the ability of the Abs to prevent viral binding and entry into cells by engaging epitopes on the viral capsid involved in host cell receptor binding. In general, cell-based assays are low throughput and labor intensive and may suffer from high variability and low sensitivity issues. In contrast, enzyme-linked immunosorbent assays (ELISAs) are simpler, less variable, and have higher throughput. Demonstrating a correlation between neutralizing Abs assessed by a cell-based assay and total binding Abs measured in an ELISA will enable the use and substitution of the latter for screening and exclusion of subjects. In this work, we describe the development of a highly sensitive, specific, robust, and reproducible chemiluminescent ELISA method for the detection of total anti-AAV9 Abs. Using this method, we analyzed the prevalence of preexisting anti-AAV9 Abs in 100 serum samples from heart disease patients. Analysis of neutralizing Abs in the same samples using an in vitro cell-based assay showed a strong correlation between total anti-AAV9 Abs and neutralizing Abs, indicating the feasibility of using the total Ab ELISA in the future for patient screening and exclusion.

3.
Cell Metab ; 24(2): 223-33, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27508871

RESUMO

The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRß-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area.


Assuntos
Movimento Celular , Imidazóis/efeitos adversos , Imidazóis/farmacologia , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Receptores X do Fígado/agonistas , Neutrófilos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Tecido Adiposo/metabolismo , Adolescente , Adulto , Animais , Movimento Celular/efeitos dos fármacos , Colesterol/sangue , Colesterol/metabolismo , Voluntários Saudáveis , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Imidazóis/uso terapêutico , Contagem de Leucócitos , Lipoproteínas/sangue , Macaca fascicularis , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Sistema Fagocitário Mononuclear/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Triglicerídeos/sangue , Adulto Jovem
4.
Mol Reprod Dev ; 76(1): 11-21, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18425777

RESUMO

Cells that morphologically and functionally resemble male germ cells can be spontaneously derived from ES cells. However, this process is inefficient and unpredictable suggesting that the expression pattern of male germ cell associated genes during spontaneous ES cell differentiation does not mimic the in vivo profiles of the genes. Thus, in the present study, the temporal profile of genes expressed at different stages of male germ cell development was examined in differentiating ES cells. The effect of all-trans retinoic acid (RA) which is a known inducer of primordial germ cell (PGC) proliferation/survival in vitro and testosterone which is required for spermatogenesis in vivo on the expression of these genes was also determined. Each of the 12 genes analyzed exhibited one of four temporal expression patterns in untreated differentiating ES cells: progressively decreased (Dppa3, Sycp3, Msy2), initially low and then increased (Stra8, Sycp1, Dazl, Act, Prm1), initially decreased and then increased (Piwil2, Tex14), or relatively unchanged (Akap3, Odf2). RA-treated cells exhibited increased expression of Stra8, Dazl, Act, and Prm1 and suppressed expression of Dppa3 compared to untreated controls. Furthermore, testosterone increased expression of Stra8 while the combination of RA and testosterone synergistically increased expression of Act. Our findings establish a comprehensive profile of male germ cell gene expression during spontaneous differentiation of murine ES cells and describe the capacity of RA and testosterone to modulate the expression of these genes. Furthermore, these data represent an important first step in designing a plausible directed differentiation protocol for male germ cells.


Assuntos
Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testosterona/farmacologia , Tretinoína/farmacologia , Animais , Biomarcadores , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Masculino , Camundongos
5.
Methods ; 45(2): 172-81, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18593614

RESUMO

The study of germ cell-specific gene regulation in vitro is challenging. Here we report that the promoter of the oocyte-specific gene, Gdf9, is active in a population of cultured murine embryonic stem cells (ES) which have a phenotype resembling oocytes. The promoter region of the murine Gdf9 coupled to enhanced green fluorescent protein (eGFP) was stably transfected into XX mouse ES cells. eGFP was expressed only in oocytes of chimeric mice generated from the transfected XX ES cells. The transfected ES cells were examined when cultured on feeder layers or as embryoid bodies. Large eGFP-positive cells, surrounded by a structure resembling a zona pellucida appeared transiently in cultures of the ES cells on feeder layers. Surprisingly, they were detectable on days 1 and 2 of culture but virtually absent on day 3. Addition of leukemia inhibitory factor (LIF) to the media significantly increased the number of eGFP-positive cels resembling oocytes. Quantitative-time PCR demonstrated a parallel increase in Gdf9 and Zp3 mRNA with changes in the abundance of eGFP-positive cells. In embryoid body cultures, eGFP-positive cells appeared transiently and then re-appeared in regional clusters after 30-45 days of culture. These findings demonstrate that a population of cultured murine ES cells contain the transcriptional machinery to drive expression of an oocyte-specific gene, and that those cells phenotypically resemble oocytes.


Assuntos
Células-Tronco Embrionárias/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Oócitos/fisiologia , Animais , Proteína Morfogenética Óssea 15 , Técnicas de Cultura de Células , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Fator 9 de Diferenciação de Crescimento , Fator Inibidor de Leucemia/farmacologia , Camundongos , Oócitos/metabolismo , Fenótipo , Regiões Promotoras Genéticas/genética , Transfecção/métodos
6.
J Biol Chem ; 279(26): 27621-32, 2004 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-15056665

RESUMO

A-kinase anchoring proteins (AKAPs) function to target protein kinase A (PKA) to specific locations within the cell. AKAPs are functionally identified by their ability to bind the type II regulatory subunits (RII) of PKA in an in vitro overlay assay. We previously showed that follicle-stimulating hormone (FSH) induces the expression of an 80-kDa AKAP (AKAP 80) in ovarian granulosa cells as they mature from a preantral to a preovulatory phenotype. In this report, we identify AKAP 80 as microtubule-associated protein 2D (MAP2D), a low molecular weight splice variant of the neuronal MAP2 protein. MAP2D is induced in granulosa cells by dexamethasone and by FSH in a time-dependent manner that mimics that of AKAP 80, and immunoprecipitation of MAP2D depletes extracts of AKAP 80. MAP2D is the only MAP2 protein present in ovaries and is localized to granulosa cells of preovulatory follicles and to luteal cells. MAP2D is concentrated at the Golgi apparatus along with RI and RII and, based on coimmunoprecipitation results, appears to bind both RI and RII in granulosa cells. Reduced expression of MAP2D resulting from treatment of granulosa cells with antisense oligonucleotides to MAP2 inhibited the phosphorylation of cAMP-response element-binding protein. These results suggest that this classic neuronal RII AKAP is a dual RI/RII AKAP that performs unique functions in ovarian granulosa cells that contribute to the preovulatory phenotype.


Assuntos
Proteínas de Transporte/metabolismo , Células da Granulosa/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Encéfalo/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Diferenciação Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dexametasona/farmacologia , Feminino , Hormônio Foliculoestimulante/farmacologia , Expressão Gênica/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células da Granulosa/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/ultraestrutura , Fosforilação , Isoformas de Proteínas , Ratos , Ratos Sprague-Dawley , Receptores do LH/metabolismo
7.
J Biol Chem ; 278(9): 7167-79, 2003 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-12493768

RESUMO

In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca(2+) channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca(2+) channel blocker, and chelation of extracellular Ca(2+). These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.


Assuntos
Hormônio Foliculoestimulante/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sulfonamidas , Animais , Northern Blotting , Western Blotting , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Núcleo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Células da Granulosa/metabolismo , Isoquinolinas/farmacologia , Sistema de Sinalização das MAP Quinases , Microscopia de Fluorescência , Modelos Biológicos , Ovário/enzimologia , Fosforilação , Testes de Precipitina , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ratos , Ratos Sprague-Dawley , Ovinos , Transdução de Sinais , Fatores de Tempo , Tirosina/química
8.
Endocrinology ; 143(8): 2986-94, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12130564

RESUMO

LH receptor activation leads to the phosphorylation/activation of p42/44 MAPK in preovulatory granulosa cells. As the LH receptor can activate both adenylyl cyclase and phospholipase C, we hypothesized that the LH receptor could elicit phosphorylation of p42/44 MAPK through activation of protein kinase A (PKA) and/or protein kinase C (PKC). Preovulatory granulosa cells in serum-free primary cultures were treated with ovulatory concentrations of human chorionic gonadotropin (hCG), an LH receptor agonist, with or without various inhibitors. The PKA inhibitor H89 as well as the myristoylated PKA inhibitor peptide PKI strongly inhibited hCG-stimulated p42/44 MAPK phosphorylation, whereas the PKC inhibitor GF109203X had no effect on p42/44 MAPK phosphorylation. LH receptor-stimulated phosphorylation of cAMP response element-binding protein (CREB), histone H3, and MAPK kinase (MEK) was also strongly inhibited by H89 and not by GF109203X. The extent of PKC activation was assessed in preovulatory granulosa cells using three criteria: translocation of PKC isoforms to the membrane fraction, phosphorylation of a known PKC substrate, and autophosphorylation of PKC delta on an activation-related site. By all three criteria PKCs were partially activated before hCG stimulation, and hCG treatment failed to elicit further PKC activation, in vitro or in vivo. Taken together, these results indicate that, under primary culture conditions where physiological levels of signaling proteins are present, hCG signals to activate MEK, p42/44 MAPK, CREB, and histone H3 in a predominantly PKA-dependent and PKC-independent manner. Unexpectedly, PKCs were partially activated in the absence of LH receptor activation, and LH receptor activation did not elicit further detectable PKC activation.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinase 1 , Proteínas de Membrana , Proteína Quinase C/fisiologia , Receptores do LH/fisiologia , Animais , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Ativação Enzimática , Glucosidases , Histonas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley
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