RESUMO
OBJECTIVE: To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. DESIGN: Prospective embryo cohort study. SETTING: Academic center and private in vitro fertilization (IVF) clinic. PATIENT(S): Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. INTERVENTION(S): Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. MAIN OUTCOME MEASURE(S): Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. RESULT(S): Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (â¼4 copies) and mtDNA (â¼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. CONCLUSION(S): Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin.
Assuntos
Blastocisto/metabolismo , Meios de Cultura/metabolismo , DNA Mitocondrial/metabolismo , DNA/metabolismo , Testes Genéticos , Infertilidade Masculina/terapia , Reação em Cadeia da Polimerase/métodos , Valor Preditivo dos Testes , Diagnóstico Pré-Implantação/métodos , Injeções de Esperma Intracitoplásmicas , Adulto , Cromossomos Humanos Y , DNA/genética , Variações do Número de Cópias de DNA , Impressões Digitais de DNA , DNA Mitocondrial/genética , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Feminino , Fertilidade , Dosagem de Genes , Marcadores Genéticos , Humanos , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/fisiopatologia , Masculino , Gravidez , Reprodutibilidade dos TestesRESUMO
Inflammation is an important and appropriate host response to infection or injury. However, dysregulation of this response, with resulting persistent or inappropriate inflammation, underlies a broad range of pathological processes, from inflammatory dermatoses to type 2 diabetes and cancer. As such, identifying new drugs to suppress inflammation is an area of intense interest. Despite notable successes, there still exists an unmet need for new effective therapeutic approaches to treat inflammation. Traditional drug discovery, including structure-based drug design, have largely fallen short of satisfying this unmet need. With faster development times and reduced safety and pharmacokinetic uncertainty, drug repositioning - the process of finding new uses for existing drugs - is emerging as an alternative strategy to traditional drug design that promises an improved risk-reward trade-off. Using a zebrafish in vivo neutrophil migration assay, we undertook a drug repositioning screen to identify unknown anti-inflammatory activities for known drugs. By interrogating a library of 1280 approved drugs for their ability to suppress the recruitment of neutrophils to tail fin injury, we identified a number of drugs with significant anti-inflammatory activity that have not previously been characterized as general anti-inflammatories. Importantly, we reveal that the ten most potent repositioned drugs from our zebrafish screen displayed conserved anti-inflammatory activity in a mouse model of skin inflammation (atopic dermatitis). This study provides compelling evidence that exploiting the zebrafish as an in vivo drug repositioning platform holds promise as a strategy to reveal new anti-inflammatory activities for existing drugs.
Assuntos
Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Animais , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/farmacocinética , Avaliação Pré-Clínica de Medicamentos , Humanos , Peixe-ZebraRESUMO
In addition to satisfying the metabolic demands of cells, mitochondrial metabolism helps regulate immune cell function. To date, such cell-intrinsic metabolic-immunologic cross-talk has only been described operating in cells of the immune system. Here we show that epidermal cells utilize fatty acid ß-oxidation to fuel their contribution to the immune response during cutaneous inflammation. By live imaging metabolic and immunological processes within intact zebrafish embryos during cutaneous inflammation, we uncover a mechanism where elevated ß-oxidation-fuelled mitochondria-derived reactive oxygen species within epidermal cells helps guide matrix metalloproteinase-driven leukocyte recruitment. This mechanism requires the activity of a zebrafish homologue of the mammalian mitochondrial enzyme, Immunoresponsive gene 1. This study describes the first example of metabolic reprogramming operating within a non-immune cell type to help control its contribution to the immune response. Targeting of this metabolic-immunologic interface within keratinocytes may prove useful in treating inflammatory dermatoses.
Assuntos
Movimento Celular , Epiderme/patologia , Ácidos Graxos/metabolismo , Inflamação/patologia , Leucócitos/patologia , Metaloproteinase 9 da Matriz/metabolismo , Animais , Dermatite Atópica/patologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Glucocorticoides/metabolismo , Larva/microbiologia , Macrófagos/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Morfolinos/farmacologia , Infiltração de Neutrófilos/efeitos dos fármacos , Oxirredução , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Receptores de Glucocorticoides/metabolismo , Salmonelose Animal/metabolismo , Transdução de Sinais , Análise de Sobrevida , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-ZebraRESUMO
We have identified a novel family of human major histocompatibility complex (MHC) class I genes. This MHC class I related gene family is defined by 10 members, among which 6 encode potentially functional glycoproteins. The 180-kb cluster containing them has been generated by serial duplication and minimal diversification of an ancestral prototype. They are not located within the MHC on 6p21.3, but near the tip of its long arm at q24.2-q25.3, close to the human equivalent of the mouse H2-linked t-complex, a subchromosomal region syntenic to a segment of mouse chromosome 10 harboring the orthologous MHC class I related retinoic acid early transcript loci, Raet1a-d. Hence we have named the identified loci RAET1E-N. Human RAET1 products are all devoid of the membrane-proximal immunoglobulin-like alpha3 domain and most, but not all, are predicted to remain membrane-anchored via glycosylphosphatidylinositol linkage and are shown to display an atypical pattern of polymorphism. RAET1 transcripts are absent from hematopoietic tissues, but largely expressed in tumors. The involvement of orthologous mouse RAET1A-D/H60 in natural killer and T-cell activation through NKG2D engagement augurs a similar function for the human RAET1 proteins.