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  • 1
    Electronic Resource
    Electronic Resource
    Purification and characterization of defensins from cystic fibrosis sputum (1997)
    Springer
    Inflammation research 46 (1997), S. 98-102 
    Details
    ISSN: 1420-908X
    Keywords: Key words: Defensin — Cystic fibrosis — Neutrophil — Airway epithelial cell — Sputum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. Objective and Design: Cystic fibrosis (CF) sputum contains large numbers of neutrophils whose most abundant granule proteins are defensins. Within phagolysosomes, defensins kill microbes; however, extracellular defensins can be toxic to human cells. To begin to explore the possibility that defensins damage CF airways, this study examines the concentration and properties of defensins in CF sputum. ¶Materials: As a source of biological material in which to assay levels of defensins, purulent sputum was collected from persons with CF hospitalized with exacerbations of bronchitis. Purulent CF sputum was also a source of material for purification of defensins. ¶Methods: Defensin concentration in the cell-free component of CF sputum was measured by immunoassay. Defensins acid-extracted from sputum were purified by Sephacryl S-200 gel filtration chromatography and reverse phase-high pressure liquid chromatography (HPLC). Toxicity of CF defensins was tested by incubating the purified defensins with a line of CF tracheal cells cultured in serum-free medium. ¶Results: In 5 patients with CF, sputum defensin levels ranged from 300 to 〉 1600 〈mu〉g/ml, higher than levels previously reported in any human secretion or fluid and greatly exceeding concentrations toxic to mammalian cells in vitro. HPCL-purified CF sputum defensins were pure as judged by acid urea-PAGE and N-terminal sequencing, which revealed a mixture of defensins-1, -2 and -3 at ratios of ∼4 : 2 : 1. At 〉 10 〈mu〉g/ml the purified mixture was toxic for a line of CF tracheal cells cultured in serum-free medium, as judged by reductions in cell numbers and increased permeability to trypan blue. ¶Conclusions: This study suggests that defensins in CF sputum are intact and sufficiently abundant that they may damage airway epithelium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s000110050114
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  • 2
    Electronic Resource
    Electronic Resource
    Genetic deficiency of human mast cell a-tryptase (2002)
    Oxford, UK : Blackwell Science Ltd
    Clinical & experimental allergy 32 (2002), S. 0 
    Details
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background Human α- and β-tryptases are proteases secreted by mast cells. Beta (but not α) tryptases are implicated in asthma. Genes encoding both types of tryptases cluster on chromosome 16p13.3.Objective This study examines the hypothesis, generated from mapping data, that α-alleles compete with some β-alleles at one locus and that an adjacent locus contains β-alleles exclusively. This hypothesis predicts that β-alleles outnumber α and that some genomes lack α genes altogether.Methods To test this hypothesis, we developed PCR-based techniques to distinguish α from β genes. We then genotyped genomic DNA from individuals and tryptase-expressing cell lines.Results In support of our hypothesis, we find that α-tryptase deficiency affects 80/274 (29%) of individuals surveyed. The genotype of the α-deficient individuals is ββββ, due to inheritance of four β genes. The percentage of the population with the mixed genotypes ααββ and αβββ is 21% and 50%, respectively. Accounting for all α- and β-alleles at the tandem loci on 16p13.3, overall α-allele frequency is only 0.23, with β-alleles considerably outnumbering α as hypothesized. In samples of defined ethnicity, α deficiency affects 45% of Caucasians, but a much lower percentage of other backgrounds, including African-Americans and Asians. Examination of cell lines reveals that HMC-1 and U-937 lack α-genes; thus, lack of α transcripts in these cells is due to absence of α-genes rather than β-selective transcription. By contrast, α-transcribing Mono Mac 6 and KU812 cells contain α- and β-genes.Conclusions Genetic α-tryptase deficiency is common and varies strikingly between ethnic groups. Because β-tryptases are implicated in allergic disorders, inherited differences in α/β-genotype may affect disease susceptibility, severity and response to tryptase inhibitor therapy.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2222.2002.01416.x
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  • 3
    Electronic Resource
    Electronic Resource
    Tissue-selective mast cell reconstitution and differential lung gene expression in mast cell-deficient KitW-sh/KitW-sh sash mice (2005)
    Oxford, UK : Blackwell Science Ltd
    Clinical & experimental allergy 35 (2005), S. 0 
    Details
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background Mast cell-deficient KitW/KitW-v mice are an important resource for studying mast cell functions in vivo. However, because they are compound heterozygotes in a mixed genetic background and are infertile, they cannot be crossed easily with other mice.Objective To overcome this limitation, we explored the use of KitW-sh/KitW-sh mice for studying mast cell biology in vivo.Results These mice are in a C57BL/6 background, are fertile and can be bred directly with other genetically modified mice. Ten-week-old KitW-sh/KitW-sh are profoundly mast cell-deficient. No mast cells are detected in any major organ, including the lung. Gene microarrays detect differential expression of just seven of 16 463 genes in lungs of KitW-sh/KitW-sh mice compared with wild-type mice, indicating that resting mast cells regulate expression of a small set of genes in the normal lung. Injecting 107 bone marrow-derived mast cells (BMMC) into tail veins of KitW-sh/KitW-sh mice reconstitutes mast cell populations in lung, stomach, liver, inguinal lymph nodes, and spleen, but not in the tongue, trachea or skin. Injection of BMMC into ear dermis or peritoneum reconstitutes mast cells locally in these tissues. When splenectomized KitW-sh/KitW-sh mice are intravenously injected with BMMC, mast cells circulate longer and are found more often in the liver and inguinal lymph nodes, indicating that the spleen acts as a reservoir for mast cells following injection and limits migration to some tissues.Conclusion In summary, these findings show that mast cell-deficient KitW-sh/KitW-sh mice possess unique attributes that favour their use for studying mast cell functions in vivo.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2222.2005.02136.x
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  • 4
    Electronic Resource
    Electronic Resource
    Proteinase-activated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase (1999)
    Oxford, UK : Blackwell Publishing Ltd
    Experimental dermatology 8 (1999), S. 0 
    Details
    ISSN: 1600-0625
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Proteinase-activated receptor-2 (PAR-2) is a G-protein coupled receptor. Tryptic proteases cleave PAR-2 exposing a tethered ligand (SLIGKV), which binds and activates the receptor. Although PAR-2 is highy expressed by cultured keratinocytes and is an inflammatory mediator, its precise localization in the normal and inflamed human skin in unknown, and the proteases that activate PAR-2 in the skin have not been identified. We localized PAR-2 in human skin by immunohistochemistry, examined PAR-2 expression by RT-PCR and RNA blotting, and investigated PAR-2 activation by mast cell tryptase. PAR-2 was localized to keratinocytes, especially in the granular layer, to endothelial cells, hair follicles, myoepithelial cells of sweat glands, and dermal dendritic-like cells. PAR-2 was also highly expressed in keratinocytes and endothelial cells of inflamed skin. PAR-2 mRNA was detected in normal human skin by RT-PCR, and in cultured human keratinocytes and dermal microvascular endothelial cells by Northern hybridization. Trypsin, tryptase and a peptide corresponding to the tethered ligand (SLIGKVNG2) increased [Ca2+]i in keratinocytes, measured using Fura-2/AM. Although tryptase-containing mast cells were sparsely scattered in the normal dermis, they were numerous in the dermis in atopic dermatitis, and in the dermis, dermal-epidermal border, and occasionally within the lower epidermis in psoriasis. Tryptase may activate PAR-2 on keratinocytes and endothelial cells during inflammation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1600-0625.1999.tb00383.x
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