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1.
Arthritis Rheumatol ; 71(12): 1976-1984, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31313532

RESUMO

A meeting was organized to bring together multiple stakeholders involved in the testing and authorization of new medications for juvenile idiopathic arthritis (JIA) to discuss current issues surrounding clinical trials and access to new medications for children and adolescents with JIA. The Childhood Arthritis and Rheumatology Research Alliance invited representatives of regulatory agencies (Food and Drug Administration and European Medicines Agency), and major pharmaceutical companies with JIA-approved products or products in development, patient and parent representatives, representatives of an advocacy organization (Arthritis Foundation), and pediatric rheumatology clinicians/investigators to a 1-day meeting in April 2018. The participants engaged in discussion regarding issues in clinical trials. As the pharmacologic options to treat inflammatory arthritis rapidly expand, registration trial designs to test medications in JIA patients must adapt. Many methodologies successfully used in the recent past are no longer feasible. The pool of patients meeting entry criteria who are willing to participate is shrinking while the number of medications to be tested is growing. Suggested solutions included proposing innovative clinical trial methods to regulatory agencies, as well as open discussions among stakeholders. Ensuring that new medications are authorized in a timely manner to meet the needs of JIA patients worldwide is critical. Approaches should include open dialog between regulatory agencies, pharmaceutical companies, and other stakeholders to develop and implement novel study designs, including patient and clinician perspectives to define meaningful trial outcomes, and changing existing study plans.

2.
PLoS Med ; 16(5): e1002800, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31067237

RESUMO

BACKGROUND: Treatment decision-making regarding immunosuppressive therapy is challenging for individuals with lupus. We assessed the effectiveness of a decision aid for immunosuppressive therapy in lupus nephritis. METHODS AND FINDINGS: In a United States multicenter, open-label, randomized controlled trial (RCT), adult women with lupus nephritis, mostly from racial/ethnic minority backgrounds with low socioeconomic status (SES), seen in in- or outpatient settings, were randomized to an individualized, culturally tailored, computerized decision aid versus American College of Rheumatology (ACR) lupus pamphlet (1:1 ratio), using computer-generated randomization. We hypothesized that the co-primary outcomes of decisional conflict and informed choice regarding immunosuppressive medications would improve more in the decision aid group. Of 301 randomized women, 298 were analyzed; 47% were African-American, 26% Hispanic, and 15% white. Mean age (standard deviation [SD]) was 37 (12) years, 57% had annual income of <$40,000, and 36% had a high school education or less. Compared with the provision of the ACR lupus pamphlet (n = 147), participants randomized to the decision aid (n = 151) had (1) a clinically meaningful and statistically significant reduction in decisional conflict, 21.8 (standard error [SE], 2.5) versus 12.7 (SE, 2.0; p = 0.005) and (2) no difference in informed choice in the main analysis, 41% versus 31% (p = 0.08), but clinically meaningful and statistically significant difference in sensitivity analysis (net values for immunosuppressives positive [in favor] versus negative [against]), 50% versus 35% (p = 0.006). Unresolved decisional conflict was lower in the decision aid versus pamphlet groups, 22% versus 44% (p < 0.001). Significantly more patients in the decision aid versus pamphlet group rated information to be excellent for understanding lupus nephritis (49% versus 33%), risk factors (43% versus 27%), medication options (50% versus 33%; p ≤ 0.003 for all); and the ease of use of materials was higher in the decision aid versus pamphlet groups (51% versus 38%; p = 0.006). Key study limitations were the exclusion of men, short follow-up, and the lack of clinical outcomes, including medication adherence. CONCLUSIONS: An individualized decision aid was more effective than usual care in reducing decisional conflict for choice of immunosuppressive medications in women with lupus nephritis. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02319525.


Assuntos
Técnicas de Apoio para a Decisão , Imunossupressores/uso terapêutico , Nefrite Lúpica/tratamento farmacológico , Educação de Pacientes como Assunto , Participação do Paciente , Adulto , Comportamento de Escolha , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Alfabetização em Saúde , Humanos , Imunossupressores/efeitos adversos , Nefrite Lúpica/etnologia , Nefrite Lúpica/imunologia , Pessoa de Meia-Idade , Folhetos , Resultado do Tratamento , Estados Unidos/epidemiologia
3.
Trends Biotechnol ; 27(5): 266-76, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19339068

RESUMO

Microscopy has always been an obligate tool in the field of developmental biology, a goal of which is to elucidate the essential cellular and molecular interactions that coordinate the specification of different cell types and the establishment of body plans. The 2008 Nobel Prize in chemistry was awarded 'for the discovery and development of the green fluorescent protein, GFP' in recognition that the discovery of genetically encoded fluorescent proteins (FPs) has spearheaded a revolution in applications for imaging of live cells. With the development of more-sophisticated imaging technology and availability of FPs with different spectral characteristics, dynamic processes can now be live-imaged at high resolution in situ in embryos. Here, we review some recent advances in this rapidly evolving field as applied to live-imaging capabilities in the mouse, the most genetically tractable mammalian model organism for embryologists.


Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Imunofluorescência/métodos , Proteínas Luminescentes/metabolismo , Camundongos/metabolismo , Microscopia de Fluorescência/métodos , Animais , Imunofluorescência/tendências , Microscopia de Fluorescência/tendências
4.
Genesis ; 47(5): 330-6, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19358158

RESUMO

To simultaneously follow multiple subcellular characteristics, for example, cell position and cell morphology, in living specimens requires multiple subcellular labels. Toward this goal, we generated dual-tagged mouse embryonic stem (ES) cells constitutively expressing differentially localized, spectrally distinct, genetically encoded fluorescent protein fusions. We have used human histone H2B fusions to fluorescent proteins to mark chromatin. This provides a descriptor of cell position, division, and death. An additional descriptor of cell morphology is achieved by combining this transgene with select lipid-modified fluorescent protein fusions that mark the plasma membrane. Using this strategy, wewere able to live image cellular dynamics in three dimensions over time both in cultured ES cells and in mouse embryos generated using dual-tagged ES cells. This study, therefore, presents the feasibility of applying multiple spectrally and subcellularly distinct fluorescent protein reporters for live imaging studies in ES cells and mouse embryos. Furthermore, the increasing availability of spectral variant fluorescent proteins along with the development of methods that permit improved spectral separation now facilitate multiplexing of fluorescent reporters to provide readouts of a variety of anatomical and physiological behaviors simultaneously in living specimens.


Assuntos
Membrana Celular/metabolismo , Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Transgenes/genética , Animais , Agregação Celular , Linhagem Celular , Quimera , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Estudos de Viabilidade , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Plasmídeos/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transfecção/métodos
5.
Dev Dyn ; 235(9): 2549-58, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16708394

RESUMO

Alpha-fetoprotein (Afp) is the most abundant serum protein in the developing embryo. It is secreted by the visceral endoderm, its derivative yolk sac endoderm, fetal liver hepatocytes, and the developing gut epithelium. The abundance of this protein suggested that Afp gene regulatory elements might serve to effectively drive reporter gene expression in developing endodermal tissues. To this end, we generated transgenic mouse lines Tg(Afp-GFP) using an Afp promoter/enhancer to drive expression of green fluorescent protein (GFP). Bright GFP fluorescence allowed the visualization, in real time, of visceral endoderm, yolk sac endoderm, fetal liver hepatocytes, and the epithelium of the gut and pancreas. Comparison of the localization of green fluorescence with that of endogenous Afp transcripts and protein indicated that the regulatory elements used to generate these mouse lines directed transgene expression in what appeared to be all Afp-expressing cells of the embryo, but only in a subset of fetal liver cells. The bright GFP signal permitted flow cytometric analysis of fetal liver hepatocytes. These mice represent a valuable resource for live imaging as well as identification, quantitation, and isolation of cells from the primitive and definitive endoderm lineages of the developing mouse embryo.


Assuntos
Endoderma/metabolismo , Proteínas de Fluorescência Verde/genética , alfa-Fetoproteínas/genética , Animais , Blastocisto/metabolismo , Técnicas de Cultura Embrionária , Endoderma/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Hepatócitos/metabolismo , Camundongos , Camundongos Transgênicos , Gravidez , Proteínas Recombinantes/genética , Saco Vitelino/embriologia , Saco Vitelino/metabolismo
6.
Genome Biol ; 7(2): 205, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16522220

RESUMO

The 3.5-day-old blastocyst-stage mouse embryo consists of two tissues and contains approximately 60 cells. This tiny structure has now been observed to express nearly 600 genes in a sex-specific fashion, including at least one gene (Rhox/Pem) expressed only in females from their paternal X chromosome.


Assuntos
Blastocisto/fisiologia , Regulação da Expressão Gênica , Animais , Feminino , Masculino , Camundongos , Família Multigênica , Caracteres Sexuais
7.
Reprod Fertil Dev ; 18(1-2): 99-107, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16478607

RESUMO

The class Mammalia is composed of approximately 4800 extant species. These mammalian species are divided into three subclasses that include the monotremes, marsupials and eutherians. Monotremes are remarkable because these mammals are born from eggs laid outside of the mother's body. Marsupial mammals have relatively short gestation periods and give birth to highly altricial young that continue a significant amount of 'fetal' development after birth, supported by a highly sophisticated lactation. Less than 10% of mammalian species are monotremes or marsupials, so the great majority of mammals are grouped into the subclass Eutheria, including mouse and human. Mammals exhibit great variety in morphology, physiology and reproduction. In the present article, we highlight some of this remarkable diversity relative to the mouse, one of the most widely used mammalian model organisms, and human. This diversity creates challenges and opportunities for gamete and embryo collection, culture and transfer technologies.


Assuntos
Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Desenvolvimento Fetal , Genitália Feminina/fisiologia , Células Germinativas/fisiologia , Mamíferos/fisiologia , Reprodução/fisiologia , Animais , Blastocisto , Feminino , Genitália Feminina/anatomia & histologia , Humanos , Masculino , Monotremados/fisiologia , Oviparidade/fisiologia , Placenta/anatomia & histologia , Placenta/fisiologia , Gravidez , Especificidade da Espécie
8.
Nat Protoc ; 1(3): 1145-53, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17406396

RESUMO

The production of mouse chimeras is a common step in the establishment of genetically modified animal strains. Chimeras also provide a powerful experimental tool for following cell behavior during both prenatal and postnatal development. This protocol outlines a simple and economical technique for the production of large numbers of mouse chimeras using traditional diploid morula<-->diploid embryonic stem (ES) cell aggregations. Additional steps are included to describe the procedures necessary to produce specialized tetraploid chimeras using tetraploid morula<-->diploid ES cell aggregations. This increasingly popular form of chimera produces embryos of nearly complete ES cell derivation that can be used to speed transgenic production or ask developmental questions. Using this protocol, mouse chimeras can be generated and transferred to pseudopregnant surrogate mothers in a 5-d period.


Assuntos
Quimera/genética , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/citologia , Modelos Animais , Mórula/citologia , Animais , Quimera/embriologia , Camundongos , Mórula/fisiologia
9.
Dev Biol ; 288(1): 150-9, 2005 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16246322

RESUMO

Tetraploid (4n) mouse embryos die at variable developmental stages. By examining 4n embryos from F2 hybrid and outbred mice, we show that 4n developmental potential is influenced by genetic background. The imprinted inactivation of an X chromosome-linked eGFP transgene in extraembryonic tissues occurred correctly in 4n embryos. A decrease of the cleavage rate in 4n preimplantation embryos compared to diploid (2n) embryos was revealed by real-time imaging, using a histone H2b:eGFP reporter. It has previously been known that mouse chimeras produced by the combination of diploid (2n) embryos with embryonic stem (ES) cells result in mixtures of the two components in epiblast-derived tissues. In contrast, the use of 4n host embryos with ES cells restricts 4n cells from the embryonic regions of chimeras, resulting in mice that are believed to be completely ES-derived. Using H2b:eGFP transgenic mice and ES cells, the behavior of 4n cells was determined at single cell resolution in 4n:2n injection and aggregation chimeras. We found a significant contribution of 4n cells to the embryonic ectoderm at gastrulation in every chimera analyzed. We show that the transition of the embryonic regions from a chimeric tissue to a predominantly 2n tissue occurs after gastrulation and that tetraploid cells may persist to midgestation. These findings suggest that the results of previously published tetraploid complementation assays may be influenced by the presence of tetraploid cells in the otherwise diploid embryonic regions.


Assuntos
Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/genética , Poliploidia , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quimera/genética , Quimera/metabolismo , Compensação de Dosagem (Genética) , Embrião de Mamíferos/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Cromossomo X , Inativação do Cromossomo X
10.
Semin Cell Dev Biol ; 15(5): 619-29, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15271307

RESUMO

The class mammalia is composed of approximately 4800 extant species. This class is divided into three subclasses, the prototheria (monotremes), metatheria (marsupials), and eutheria. Surprisingly, there is relatively little knowledge about germ layer and axis formation in mammalian species. Most knowledge about these embryonic processes has been obtained from one species, the mouse, Mus musculus. Here we discuss major variations in germ layer and axis formation among mammals. We suggest that more studies of embryonic development in diverse mammalian species are required for an understanding of germ layer and axis formation to provide insights into human biology and disease.


Assuntos
Padronização Corporal/fisiologia , Gástrula/fisiologia , Mamíferos/embriologia , Animais , Blastocisto/fisiologia , Humanos , Camundongos , Oócitos/fisiologia , Organizadores Embrionários/fisiologia
11.
Dev Dyn ; 228(4): 751-66, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14648853

RESUMO

Spontaneous duplication of the mammalian genome occurs in approximately 1% of fertilizations. Although one or more whole genome duplications are believed to have influenced vertebrate evolution, polyploidy of contemporary mammals is generally incompatible with normal development and function of all but a few tissues. The production of tetraploid (4n) embryos has become a common experimental manipulation in the mouse. Although development of tetraploid mice has generally not been observed beyond midgestation, tetraploid:diploid (4n:2n) chimeras are widely used as a method for rescuing extraembryonic defects. The tolerance of tissues to polyploidy appears to be dependent on genetic background. Indeed, the recent discovery of a naturally tetraploid rodent species suggests that, in rare genetic backgrounds, mammalian genome duplications may be compatible with the development of viable and fertile adults. Thus, the range of developmental potentials of tetraploid embryos remains in large part unexplored. Here, we review the biological consequences and experimental utility of tetraploid mammals, in particular the mouse.


Assuntos
Biologia do Desenvolvimento/métodos , Técnicas Genéticas , Genoma , Poliploidia , Animais , Núcleo Celular/metabolismo , Embrião de Mamíferos/metabolismo , Camundongos , Modelos Biológicos , Zigoto/metabolismo
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