Biomedical subjects

Martin J Evans

Publications and source records attributed to Martin J Evans.

4 recordsLinked to original sources

PubMed2006

TRACP Influences Th1 pathways by affecting dendritic cell function.

UNLABELLED: TRACP, a marker of osteoclasts, is also expressed by cells of the immune system. We identified a novel function for TRACP in the dendritic cell. DCs from TRACP knockout mice have impaired maturation and trigger reduced Th1 responses in vivo. We postulate that TRACP has an important role in the presentation of antigens to T cells. INTRODUCTION: TRACP is highly expressed by osteoclasts, activated macrophages, and dendritic cells (DCs). Knockout mice lacking TRACP have an intrinsic defect in osteoclastic resorption and macrophages that display abnormal immunomodulatory responses and cytokine secretion profiles. Our aim in this study was to investigate the significance of TRACP in the inductive phase of the immune response by examining dendritic cells from TRACP(-/-) mice. MATERIALS AND METHODS: Maturational state and function of leukocyte subsets in mice was assessed by flow cytometry. The ability of the immune system to respond to nonspecific activation and to specific antigen was assessed by delayed type hypersensitivity and the presence of isotype-specific serum antibody in vivo and T-cell proliferation and cytokine production in vitro. RESULTS: The ability of lipopolysaccharide (LPS) to upregulate MHC II and CD80 in DCs from TRACP(-/-) mice was reduced compared with wildtype mice, although production of IL-10 by DCs from TRACP-deficient animals was increased. T- and B-cell responses not involving antigen presentation (anti-CD3, TNP-ficoll) were normal in TRACP(-/-) mice, but responses to T-dependent antigens were impaired. Specifically, TRACP(-/-) mice had defective delayed hypersensitivity responses to picryl chloride and reduced proliferative responses to ovalbumin compared with wildtype mice. In response to ovalbumin, but not anti-CD3, T cells from TRACP(-/-) mice produced less interferon-gamma (IFN-gamma), but there was no difference in IL-4 production: TRACP(-/-) mice also produced less ovalbumin (OVA)-specific IgG2a after immunization. CONCLUSIONS: The finding that DCs from TRACP(-/-) mice have impaired maturation and defective Th1 responses shows that TRACP is important for polarizing responses in naïve T cells to antigen-presented dendritic cells.

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PubMed2004

Gene expression changes during cataract progression in Sparc null mice: differential regulation of mouse globins in the lens.

PURPOSE: Sparc/osteonectin is a hydroxyapatite, calcium and, collagen binding protein, implicated in tissue morphogenesis, cell proliferation, and repair. Sparc null mice develop sub-cortical posterior cataract with eventual rupture of the lens. We wished to correlate genotype with phenotype in these mice via analysis of gene expression pattern changes leading to disease. METHODS: We carried out microarray analysis of adult lenses from Sparctm1cam knockout mice on two strain backgrounds of varying phenotypic severity at two time points, 4 and 9 months. Labelled cDNA from Sparctm1cam knockout and age, strain, and sex matched control lenses was hybridized with HGMP NIA 15,000 clone set arrays. Differential expression was confirmed using semi-quantitative RT-PCR. RESULTS: We have confirmed differential expression of 54 genes. Most notably, 5 of the mouse globin genes, Hbb-b1, Hbb-b2, Hba, Hba-x, and Hbb-y and an EST, C79876, were significantly downregulated in 9-month old Sparc null mice from two genetic backgrounds at different stages of disease. Another downregulated gene, EraF, is involved in folding of globin proteins. Immune response components, including various members of the complement cascade, were upregulated in lenses with advanced cataract. CONCLUSIONS: Five mouse globins show persistent downregulation as a result of Sparc loss. We speculate as to possible roles of this phenomenon on pathogenesis of cataract in these mice. Other confirmed genes allow extension of previous models of cataract development in Sparc null mice.

Animals↗

PubMed2003

Expression profiling and gene discovery in the mouse lens.

PURPOSE: Defects in the development and physiology of the lens can result in cataracts (opacification of the lens), which are currently treatable only by surgical removal. The lens is also an excellent system for understanding fundamental biological processes such as cellular differentiation and ageing. Here, microarrays have been used to gain insights into global patterns of gene expression in the mouse lens. Lens gene expression compared to non-lens tissues has been investigated in order to identify genes preferentially expressed in the lens and lenses of different ages have been compared to identify differentially regulated genes. METHODS: Genes expressed in the lens were identified using mouse GeneFilters microarrays (GF400; ResGen). Each array comprises 5,184 mouse cDNAs representing sequence-verified known genes and uncharacterized ESTs spotted onto a nylon membrane. Target RNA (33P labeled) from lens and non-lens samples was hybridized to the arrays. The proportion of genes involved in various biological processes was investigated using Onto-Express to search for GeneOntology terms associated with them. Differential gene expression was investigated using K-means clustering analysis. Expression of known and uncharacterized genes selected from the arrays was investigated further using semi-quantitative RT-PCR. RESULTS: 1,668 genes were expressed in one or more of newborn, 7 day old, and adult mouse lenses at levels significantly above background. Raw data and bioinformatics data relating to these genes have been published herein. There were 543 (33%) known genes, 124 (7%) had some similarity to known genes, 400 (24%) were functionally uncharacterized, and the remaining 601 (36%) genes were novel (matching only existing ESTs). Onto-Express identified genes involved in various biological processes including several categories containing greater numbers of genes than would be expected by chance, such as transcription regulation and G-protein coupled receptor signaling genes. Semi-quantitative RT-PCR confirmed preferential expression of several genes in the lens compared to non-lens tissues and genes exhibiting significantly higher expression in the 7 day lens compared to either adult or newborn lenses. Expression in the lens of 10 genes involved in apoptosis was also confirmed and, intriguingly, expression of hemoglobin isoforms (Hba-a1, Hba-X, Hbb-b1, Hbb-b2, and Hbb-Y) was confirmed using isotype specific primers. Finally, we confirmed the expression in the lens of all additional novel, uncharacterized and known genes tested. CONCLUSIONS: The present work has provided insights into global patterns of gene expression in the lens and the expression of a significant number of genes has been confirmed using semi-quantitative RT-PCR. Genes preferentially expressed in the lens compared to non-lens tissues have been identified as well as genes differentially expressed between lenses at different ages. Gene expression profiling and gene discovery in the lens are essential prerequisites for future functional studies aimed at gaining insights into the potential roles of these genes in lens development, maturation, physiology, and pathogenesis (using targeted mutagenesis in mice, for instance).

Animals↗

PubMed2002

Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since approximately 5% of all mutant CF alleles are stop mutations, it can be calculated that approximately 10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr-/-) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr-/- hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.

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