RESUMEN
The reproducibility of research using laboratory animals requires reliable management of their quality, in particular of their genetics, health and environment, all of which contribute to their phenotypes. The point at which these biological materials are transferred between researchers is particularly sensitive, as it may result in a loss of integrity of the animals and/or their documentation. Here, we describe the various aspects of laboratory animal quality that should be confirmed when sharing rodent research models. We also discuss how repositories of biological materials support the scientific community to ensure the continuity of the quality of laboratory animals. Both the concept of quality and the role of repositories themselves extend to all exchanges of biological materials and all networks that support the sharing of these reagents.
Asunto(s)
Investigadores , Animales , Humanos , Reproducibilidad de los ResultadosRESUMEN
Matrix metalloproteinases (MMPs) are potential biomarkers for disease activity in inflammatory bowel disease (IBD). However, clinical trials targeting MMPs have not succeeded, likely due to poor understanding of the biological functions of individual MMPs. Here, we explore the role of MMP-19 in IBD pathology. Using a DSS-induced model of colitis, we show evidence for increased susceptibility of Mmp-19-deficient (Mmp-19(-/-)) mice to colitis. Absence of MMP-19 leads to significant disease progression, with reduced survival rates, severe tissue destruction, and elevated levels of pro-inflammatory modulators in the colon and plasma, and failure to resolve inflammation. There was a striking delay in neutrophil infiltration into the colon of Mmp-19(-/-) mice during the acute colitis, leading to persistent inflammation and poor recovery; this was rescued by reconstitution of irradiated Mmp-19(-/-) mice with wild-type bone marrow. Additionally, Mmp-19-deficient macrophages exhibited decreased migration in vivo and in vitro and the mucosal barrier appeared compromised. Finally, chemokine fractalkine (CX3CL1) was identified as a novel substrate of MMP-19, suggesting a link between insufficient processing of CX3CL1 and cell recruitment in the Mmp-19(-/-) mice. MMP-19 proves to be a critical factor in balanced host response to colonic pathogens, and for orchestrating appropriate innate immune response in colitis.
Asunto(s)
Quimiocina CX3CL1/metabolismo , Colitis/inmunología , Colon/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Macrófagos/inmunología , Metaloproteinasas de la Matriz Secretadas/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Colitis/inducido químicamente , Citocinas/metabolismo , Sulfato de Dextran , Progresión de la Enfermedad , Humanos , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Mucosa Intestinal/patología , Metaloproteinasas de la Matriz Secretadas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/genéticaRESUMEN
Lecithin:retinol acyltransferase (LRAT) is the major enzyme responsible for retinol esterification in the mammalian body. LRAT exhibits specific activity in the cells with active retinol metabolism where it converts retinols into retinyl esters, which represents the major storage form of retinol. Besides hepatic stellate cells in the liver, LRAT appears to have a key physiologic role in several other tissues. In this study, we generated a transgenic reporter mouse expressing green fluorescence protein (EGFP) under the control of region containing -1166 bps from promoter upstream from the putative transcriptional start site and 262 bps downstream of this start. Transgenic reporter mice exhibited specific expression in eyes and testes. In eyes, expression of EGFP-reporter is found in lens and lens epithelium and fibers from embryo to adulthood. In testes, LRAT-EGFP reporter is expressed both in Sertoli and in spermatocytes marking initiation of spermatogenesis in prepubertal mice. Our data show that the examined LRAT regulatory region is sufficient to achieve strong and selective expression in the eye and testes but not in liver and other organs.
Asunto(s)
Aciltransferasas/genética , Cristalino/metabolismo , Meiosis/genética , Espermatocitos/ultraestructura , Testículo/metabolismo , Animales , Epitelio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Genotipo , Proteínas Fluorescentes Verdes , Masculino , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Células de Sertoli/metabolismo , Espermatogénesis , Transcripción Genética/genética , Vitamina A/metabolismoRESUMEN
Matrix metalloproteinases (MMPs), responsible for extracellular matrix remodelling and processing of numerous soluble and cell-surface proteins, appear to play important roles in pathogenesis of gastrointestinal diseases. MMPs influence migration of inflammatory cells, mucosal destruction, matrix deposition and degradation. In this study, we analysed the expression of MMP-19 in the main forms of gastrointestinal diseases including inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease, and colorectal carcinoma. We identified prominent MMP-19 expression in unaffected areas of intestinal epithelia and macrophages but not in other cells or tissues. Abundant expression of MMP-19 was also found in the endothelium of blood and lymphatic vessels of inflamed intestinal tissue. High MMP-19 immunoreactivity was also associated with macrophages in inflamed areas and myenteric plexuses. In comparison to the intestinal epithelium, all these cell types and compartments appeared to express MMP-19 irrespective of the disease pathogenesis and progression. Intestinal epithelia exhibited striking differential immunoreactivity for MMP-19. While immunoreactivity of monoclonal antibody recognizing the propeptide domain declined in virtually all IBD and colorectal carcinoma samples, other polyclonal antibodies against the hinge region and propetide domain did not show such an obvious decrease. Additional Western blotting analysis revealed that the antibodies against MMP-19 recognize differently processed forms of this MMP. The disappearance of immunoreactivity of the monoclonal anti-propeptide domain antibody does not mean down-regulation of MMP-19, but processing of the immature form. As this processing likely leads to the activation of this MMP, the differential staining pattern may be an important sign of disease progression.
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Progresión de la Enfermedad , Enfermedades Gastrointestinales/enzimología , Enfermedades Gastrointestinales/patología , Metaloproteinasas de la Matriz Secretadas/metabolismo , Procesamiento Proteico-Postraduccional , Adulto , Anciano , Anticuerpos/metabolismo , Colon/enzimología , Colon/patología , Femenino , Células HCT116 , Humanos , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Intestino Delgado/enzimología , Intestino Delgado/patología , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Mutations extended the host range of the polyvalent bacteriophage 812 of the family Myoviridae in up to 95 % of Staphylococcus aureus strains and 43 % of strains of different coagulase-positive and -negative Staphylococcus species. Mutational changes in the genome of several host-range mutants of phage 812 were identified. Host-range mutant 812F1 harbors a deletion in endolysin gene that arose together with intron excision. Four mutants (812i, 812b, 812p, 812F3) harbor deletion in the structural gene orf8 that results from a genome rearrangement associated with intron insertion. This rearrangement was also detected in the genome of the closely related phages U16 and phi131. Another intron was discovered in the recA812 gene in these four mutants. An insertion was found in a non-coding region of the restriction fragment PstI-O of three mutants (812b, 812F3, 812g) and phages U16 and phi131. The above results contribute to the explanation of genetic factors affecting the host range of polyvalent staphylococcal bacteriophages.
Asunto(s)
Bacteriófagos/genética , Genoma Viral , Mutación , Staphylococcus aureus/virología , Secuencia de Aminoácidos , Secuencia de Bases , Endopeptidasas/química , Endopeptidasas/genética , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , ARN Viral/química , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Proteínas de la Cola de los Virus/química , Proteínas de la Cola de los Virus/genéticaRESUMEN
Conserved genomic sequences distinctive of Staphylococcus aureus phage types 3A, 11, 77, 187 and Twort, representative of phage serogroups A, B, F, L and D, were identified and characterized. PCR primers designed for the above sequences were used for development of a multiplex PCR assay which enabled us not only to classify all phages of the International Typing Set plus 16 additional phages, but also to detect prophages in S. aureus genomes. One to four different prophages were unambiguously detected in experimentally lysogenized S. aureus strains, and substantial variation in prophage content was found in 176 S. aureus clinical strains of different provenance. In addition, by using a comparative genomics approach, all the prophages in the S. aureus genomes sequenced to date could be revealed and classified.