Allergy of the respiratory tract

Corrigan, Chris J.

doi:10.1016/0952-7915(92)90065-m

Cited by 8 publications

(4 citation statements)

References 76 publications

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“…Purification and stimulation of T cells.. PBMC from a maximum of 4 ml heparinized blood of children, were purified by density centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). 2 The children ranged from 0-4 years of age and were healthy or with allergic asthma. These PBMC were incubated (30 min, 4C) with monoclonal antibodies specific for monocytes (My4, 100 l.tl (40 l.tg/ml) per 1 x 107 pelleted cells, Coulter Cytometry, FL), B cells (CD19, 100 lal (100 btg/ml) per 1 x 10 r pelleted cells, Coulter) and natural Miler cells (CD16 and CD56PE, both 50 1 (100 l.tg/ml) per 1 x 107 pelleted cells, Becton Dickinson, Mountain View, CA).…”

Section: Methodsmentioning

confidence: 99%

“…5'-ACTCTGTGCACCGAGTT-GACCGTAA-3' (exon 2), antisense: 5'-TCTCATGATCTGCTITAG-CCTTTCC-3' (exon 4), (product size 300 bp) 32 IFN-7 sense: 5'-TTTAATGCAGGTCATTCA-GATG-3' (exon 1-2) antisense: 5'-CAGGGATGCTTCTTCGA-CCTCGAAAC-3' (exon 4) (product size 388 bp) 2 We developed the HPRT (human hypoxanthine phosphoribosyl transferase) primerset from the genomic structure, by selecting primers on exon 2 and 6. In this way, the product including the intron sequences will be too large (3.5 kB) to be amplified by PCR.…”

Section: Methodsmentioning

confidence: 99%

“…To make such studies feasible, T cells were purified from peripheral blood. 2 The amount of peripheral blood obtained from young children is usually small, and the number of T cells that can be purified is therefore limited. Moreover, cytokine gene expression is usually transient and cytokine mRNA levels generally occur at low abundance.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, such analysis requires sensitive procedures. 2 The polymerase chain reaction (PER) 22'23 is a sensitive method for the detection of gene expression. Here we describe optimizations of the PCR method to analyse cytokine gene expression occurring at low abundance and from a limited number of cells, in a semi-quantitative way.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of cyt0kine gene expression in stimulated T cells of small children by semi‐quantitative PCR

Koning

Baert

Groot

et al. 1995

Mediators of Inflammation

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Only limited amounts of peripheral blood samples can be obtained from small children. Therefore, a polymerase chain reaction (PCR) aided analysis of cytokine gene expression by PBMC or T cells is a valuable tool. We present a combination of procedures to obtain an accurate estimation of the expression of the cytokines IL-4 and IFN-γ. This can be performed on T cells purified from blood samples of up to 5 ml in volume from children aged 0–4 years with allergic asthma and atopic dermatitis. This procedure includes multiple sampling of PCR products to determine the linear phase of the PCR; inter-experiment correction using a helper T-cell clone, expressing both IL-4 and IFN-γ; interpatient correction by comparing the expression of a housekeeping gene (HPRT); and finally the development of specific software to analyse densitometric data obtained by scanning photographs of agarose gels, separating PCR products. In this way it is possible to study cytokine gene expression from a very small amount of material.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Analysis of cyt0kine gene expression in stimulated T cells of small children by semi‐quantitative PCR

Koning

Baert

Groot

et al. 1995

Mediators of Inflammation

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The cytokines NAP-1 (IL-8), MCP-1, IL-1 beta, and GRO in rabbit inflammatory skin lesions produced by the chemical irritant sulfur mustard

et al. 1996

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Developing and healing dermal inflammatory lesions were produced in rabbits by the topical application of dilute sulfur mustard (SM), the military vesicant. In tissue sections of such lesions, cells containing the mRNA of important cytokines were identified with in situ hybridization techniques. These cytokines were neutrophil attractant/activation protein-1 (NAP-1 (also called IL-8), monocyte chemoattractant (activating) protein 1 (MCP-1), interleukin 1 (beta) (IL-1 (beta)), and GRO (a growth factor and chemokine). Mononuclear cells (mainly macrophages and activated fibroblasts) contained the mRNA of all four of these cytokines. A higher percentage of cytokine-producing mononuclear cells (macrophages and activated fibroblasts) was present in lesions at 2 days (their peak size) than at 6 days, when they were almost healed. Granulocytes emigrated from the bloodstream, passed through the lesions, and were the major constituent of the protective crust. This sequence correlated with the distribution of cells able to produce NAP-1: At 2 days and 6 days, the mononuclears that contained messenger RNA for this granulocyte chemoattractant were found mainly in the upper part of the dermis. At 2 days and 6 days, cells containing the mRNA of IL-1, a primary cytokine, were also found predominantly in the upper dermis, i.e., nearest the site of injury. In contrast, mononuclears containing the mRNA of MCP-1 (a monocyte chemoattractant), and the mRNA of GRO (a granulocyte chemoattractant) were more equally distributed throughout the dermis. SM stimulated hair follicle epithelial cells to up-regulate GRO mRNA and, to a lesser degree, NAP-1 mRNA. Apparently, the irritation produced by SM directly or indirectly induces such epithelial cells to manufacture these growth factors. In the rabbit, hair follicles are known to be the main source of new epithelial cells after the covering epithelium has been destroyed. Therefore, GRO is probably a major autocrine-paracrine stimulus for such repair. A brief review of the role of cytokines in dermal inflammation is presented.

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Chamber for testing metered-dose propellant-driven aerosols of immunologically relevant proteins

Brown

Pickrell

1994

Journal of Immunological Methods

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Allergy of the respiratory tract

Cited by 8 publications

References 76 publications

Analysis of cyt0kine gene expression in stimulated T cells of small children by semi‐quantitative PCR

Analysis of cyt0kine gene expression in stimulated T cells of small children by semi‐quantitative PCR

The cytokines NAP-1 (IL-8), MCP-1, IL-1 beta, and GRO in rabbit inflammatory skin lesions produced by the chemical irritant sulfur mustard

Chamber for testing metered-dose propellant-driven aerosols of immunologically relevant proteins

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