A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats

Siegling, Angela; Lehmann, Manfred; Platzer, Cornelia; Emmrich, Frank; Volk, Hans‐Dieter

doi:10.1016/0022-1759(94)90139-2

Cited by 173 publications

(75 citation statements)

References 8 publications

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“…Competitive PCR was performed as described [40]. Briefly, a multispecific competitor fragment for various rat cytokines and house-keeping genes was added to the PCR reaction (1 pg/PCR reaction).…”

Section: Rt-pcr and Competitive Pcrmentioning

confidence: 99%

Oxidative stress stimulates IL‐4 and IL‐6 production in mast cells by an APE/Ref‐1‐dependent pathway

Frossi

Carli

Daniël³

et al. 2003

Eur J Immunol

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Section: Rt-pcr and Competitive Pcrmentioning

confidence: 99%

Oxidative stress stimulates IL‐4 and IL‐6 production in mast cells by an APE/Ref‐1‐dependent pathway

Frossi

Carli

Daniël³

et al. 2003

Eur J Immunol

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“…Oligonucleotide primer pairs for the 5Ј and 3Ј rat cytokine regions were selected based on published sequences (20). To compare the relative level of each cytokine in different samples, competitors for IL-2, IFN-␥, IL-4, IL-10, and ␤-actin were constructed, and the competitive template RT-PCR amplification was performed, as described (4,20).…”

Section: Pcr Primers/competitive Template Rt-pcrmentioning

confidence: 99%

Regulatory Cells Potentiate the Efficacy of IL-4 Gene Transfer by Up-Regulating Th2-Dependent Expression of Protective Molecules in the Infectious Tolerance Pathway in Transplant Recipients

Ritter

Kato

et al. 2000

The Journal of Immunology

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We have previously shown that the tolerant state in allograft recipients can be maintained and perpetuated by an “infectious” T cell-dependent regulatory mechanism. Hence, 1) treatment of LEW rats with RIB-5/2, a CD4 nondepleting mAb, produces indefinite survival of LBNF1 cardiac allografts; 2) donor-specific tolerance can be then transferred by spleen cells into new cohorts of test allograft recipients; and 3) putative regulatory CD4+ Th2-like cells are instrumental in this tolerance model. We now report on studies aimed at exposing mechanisms underlying the infectious tolerance pathway, with emphasis on the interactions between intragraft adenovirus-IL-4 gene transfer and systemic infusion of regulatory cells from tolerant hosts. Unlike individual treatment regimens, adjunctive therapy with adenovirus-IL-4 and suboptimal doses of regulatory spleen cells was strongly synergistic and extended donor-type test cardiac allograft survival to about 2 mo. RT-PCR-based expression of intragraft mRNA coding for IL-2 and IFN-γ remained depressed, whereas that of IL-4 and IL-10 reciprocally increased selectively in the combined treatment group, data supported by ELISA studies. In parallel, only adjunctive treatment triggered intragraft induction of molecules with anti-oxidant (HO-1) and anti-apoptotic (Bcl-xL/Bag-1) but not with pro-apoptotic (CPP-32) functions, both in the early and late posttransplant phases. Hence, systemic infusion of regulatory cells potentiates the effects of local adenovirus-IL-4 gene transfer in transplant recipients. Th2-driven up-regulation of protective molecule programs at the graft site, such as of anti-oxidant HO-1 and/or anti-apoptotic Bcl-xL and Bag-1, may contribute, at least in part, to the maintenance of the infectious tolerance pathway in transplant recipients.

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“…We used a semiquantitative reverse transcriptasepolymerase chain reaction (RT-PCR) method which determines the level of IFN-gene expression after reverse transcription of mRNA by comparing the ratio of PCR amplicons generated by IFN-cDNA and an endogenous standard gene ( -actin). This PCR-based method has earlier been shown to discriminate between two-fold differences in gene expression [22]. In the NK cell fraction, IFN-mRNA accumulated in response to IL-2, whereas IFN-mRNA was not detected in NK cells admixed with monocytes.…”

Section: Pre-translational Inhibition Of Nk Cell Production Of Ifn-mentioning

confidence: 99%

Histaminergic regulation of interferon-gamma (IFN-γ) production by human natural killer (NK) cells

Asea¹,

Hansson²,

Czerkinsky³

et al. 1996

Clinical and Experimental Immunology

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SUMMARYMonocytes, recovered from human peripheral blood by counter-current centrifugal elutriation, effectively inhibit the production of IFN-°by CD3 ÿ /56 NK cells in response to IL-2. This study aimed at defining the nature of the inhibitory signal, particularly the importance of monocyte-derived reactive metabolites of oxygen. It was found that monocytes recovered from patients with chronic granulomatous disease (CGD), a condition characterized by deficient NADPH-oxidase activity of phagocytes, did not inhibit IFN-°production by NK cells. Further, catalase, a scavenger of hydrogen peroxide, completely reversed the inhibitory signal, whereas scavengers of the superoxide anion, hypohalous acids, the hydroxyl radical, or nitric oxide synthesis inhibitors such as L-NMMA were ineffective. Inhibition of IFN-°production was operating on a pre-translational level, as indicated by the inability of enriched NK cells to accumulate IFN-°mRNA in the presence of elutriated monocytes. Hydrogen peroxide, at micromolar concentrations, reconstituted the inhibition of IFN-°production when added to enriched NK cells. Histamine, a biogenic amine which inhibits the generation of reactive oxygen metabolites in monocytes, abrogated the inhibition of IFN-°production in NK cells; by this mechanism, histamine strongly synergized with IL-2 to induce IFN-°in mixtures of NK cells and monocytes. The synergizing effect of histamine was specifically mediated by H 2 -type histamine receptors. We conclude that: (i) the induction of IFN-°mRNA in NK cells is effectively down-regulated by products of the oxidative metabolism of monocytes; and (ii) histamine effectively enhances IFN-°production by preventing monocyte-induced oxidative damage to NK cells.

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A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats

Cited by 173 publications

References 8 publications

Oxidative stress stimulates IL‐4 and IL‐6 production in mast cells by an APE/Ref‐1‐dependent pathway

Oxidative stress stimulates IL‐4 and IL‐6 production in mast cells by an APE/Ref‐1‐dependent pathway

Regulatory Cells Potentiate the Efficacy of IL-4 Gene Transfer by Up-Regulating Th2-Dependent Expression of Protective Molecules in the Infectious Tolerance Pathway in Transplant Recipients

Histaminergic regulation of interferon-gamma (IFN-γ) production by human natural killer (NK) cells

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