Quantitation of bovine TNF-α mRNA by reverse transcription and competitive polymerase chain reaction amplification

Bienhoff, S.E.; Allen, Gary K.

doi:10.1016/0165-2427(94)05292-z

Cited by 11 publications

(4 citation statements)

References 13 publications

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“…Similar results were obtained with cells from a different donor. Rapid enhanced levels of cytokine gene transcription followed by a decrease in the levels of cytokinespecific mRNA transcripts has also been reported for human and other animal species (4,17,19,21,28,32). The enhanced cytokine gene transcription levels observed in our study were specific in the sense that they correlated with the detection of cytokine biological activity in corresponding macrophage cul- Cytokine mRNA expression was also detected in unstimulated control cells.…”

supporting

confidence: 83%

“…PCR is a technique by which the nucleic acid in any sample can be specifically amplified by up to 10 6 -fold prior to attempting to detect it (33). Over the past few years, a reverse transcription (RT) step has been used in conjunction with PCR amplification for the detection of a variety of RNA molecules from different sources (4,6,9,15,18,19,21,31,32,38). In the study described in this report, porcine-specific cytokine nucleic acid probes were generated, and an RT-PCR assay, standardized with the endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, was developed and was used to measure the relative levels of expression of porcine TNF-␣, IL-6, and IL-1␤ mRNAs in porcine macrophages.…”

mentioning

confidence: 99%

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Cloning of porcine cytokine-specific cDNAs and detection of porcine tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-1 beta gene expression by reverse transcription PCR and chemiluminescence hybridization

Vézina

Roberge

Fournier

et al. 1995

Clin Diagn Lab Immunol

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A reverse transcription PCR assay with porcine cytokine-specific primers was developed to clone cDNA fragments and generate cDNA probes that were specific for porcine tumor necrosis factor alpha (TNF-␣), interleukin 6 (IL-6), and IL-1␤. The specificities of the cDNA PCR products were confirmed by sequence analysis on the basis of known porcine cytokine gene sequences. The reverse transcription PCR assay was also used to study cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated and control unstimulated porcine alveolar macrophages. The cDNA products were analyzed in ethidium bromide-stained agarose gels, and the transcription level of each cytokine was determined relative to the endogenous glyceraldehyde-3phosphate dehydrogenase (GAPDH) RNA level of each cytokine by measuring the intensity of the chemiluminescence hybridization signals by densitometric scanning. Various levels of cytokine mRNAs were detected in both LPS-stimulated and control unstimulated cells. Thus, TNF-␣ mRNA levels were enhanced in the cell cultures stimulated for 6 h with LPS compared with those in control cell cultures. No differences in TNF-␣ transcription levels between LPS-stimulated and control cells were observed after incubation for 24 or 55 h. Enhancements of IL-6 and IL-1␤ mRNA levels were also observed in the cultures stimulated with LPS for 6 and 24 h compared with the cytokine mRNA levels in control cell cultures. The presence of cytokine mRNA transcripts in the LPS-stimulated macrophage cultures correlated with the detection of these soluble cytokines by the bioassays. In contrast, no soluble cytokine was detected in control macrophage culture supernatants in the presence of cytokine mRNA transcripts.

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supporting

confidence: 83%

mentioning

confidence: 99%

Cloning of porcine cytokine-specific cDNAs and detection of porcine tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-1 beta gene expression by reverse transcription PCR and chemiluminescence hybridization

Vézina

Roberge

Fournier

et al. 1995

Clin Diagn Lab Immunol

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“…Enhancements of porcine IL-1␤ and TNF-␣ mRNA transcription levels in alveolar macrophage were observed after 6 and/or 16 h of stimulation with MCWE. Such enhanced levels of cytokine gene transcription have also been reported for endotoxin-and MDP-stimulated immune cells from humans and other animal species (7,16,18,20,26,31). IL-1␤ transcription level enhancement in blood-derived adherent cell cultures, and presumably the secretion of the protein, may very well account, as mentioned above, for the potentiating effect of MCWE on the blastogenic activities in control and PHA-and ConA-stimulated porcine blood lymphocyte cultures observed in this study.…”

Section: Discussionsupporting

confidence: 55%

Modulatory effect of mycobacterium cell wall extract (Regressin) on lymphocyte blastogenic activity and macrophage cytokine gene transcription in swine

Vézina

Archambault

1997

Clin Diagn Lab Immunol

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Mycobacterium cell wall extract (MCWE) (Regressin) contains trehalose dimycolate and muramyl dipeptide, both of which have immunomodulatory properties. The goal of this study was to evaluate the effect of MCWE on the in vitro peripheral blood lymphocyte blastogenic activities to mitogens phytohemagglutinin (PHA) and concanavalin A (ConA) in 6-to 8-week-old piglets. The effect of MCWE on alveolar macrophage tumor necrosis factor alpha (TNF-␣) and interleukin-1␤ (IL-1␤) gene transcription, as determined by a reverse transcription-PCR assay standardized with the endogenous glyceraldehyde-3-phosphate dehydrogenase gene, was also investigated. The results show enhanced blastogenic lymphocyte activities to mitogens PHA and ConA in MCWE-exposed cell cultures compared to those of control cell cultures. The enhanced blastogenic activity effect of MCWE was dose dependent. The cell background activity (spontaneous [ 3 H]thymidine incorporation) of lymphocyte cultures was also significantly increased in the presence of MCWE, thereby demonstrating a lymphocyte mitogenic effect of MCWE. Cytokine gene transcription analysis showed that the TNF-␣ transcript levels in alveolar macrophage cell cultures stimulated with MCWE for 6 or 16 h were enhanced compared with those in control cell cultures. An enhancement of IL-1␤ mRNA levels in cell cultures stimulated for 16 h with MCWE, compared with those in control cell cultures, was also observed. The overall results demonstrate that MCWE can stimulate lymphocyte functional activity and cytokine mRNA expression in swine, thereby indicating its potential use as a clinical immunotherapeutic agent.

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“…Equal amounts of RNA were used from donor cells from each animal to analyze cytokine levels. For TNF-␣, PCR was performed using the GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR Kit (Perkin-Elmer, Foster City, CA) as previously described [24]. Reverse transcription was performed using the bovine specific TNF primer AGGGCGATGA TCCCAAAGTA GACC and 50 ng RNA per reaction at 70°C for 30 min.…”

Section: Rt-pcr Southern Analysis Of Ifn-␥ and Tnf-␣ Mrna Expressionmentioning

confidence: 99%

Role of CD8+ and WC-1+ γ/δ T cells in resistance to Mycobacterium bovis infection in the SCID-bo mouse

Smith

Kreeger

Alvarez

et al. 1999

Journal of Leukocyte Biology

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The role of various effector T cell populations in the bovine immune response toMycobacterium bovis infection is poorly understood. This is largely due to the difficulties associated with performing in vivo challenge studies in the natural host species. In this report, we utilized a fetal bovine-severe combined immunodeficient (SCID-bo) xenochimeric mouse model to study the protective role of two putative effector cell types, CD8 ؉ T cells and a subpopulation of ␥/␦ T cells that express WC-1, a member of the cysteine-rich scavenger receptor superfamily (CRSR). We demonstrate that CD8 ؉ T cells play a key role in protection and contribute substantially to bovine IFN-␥ mRNA levels at 30 days post-infection. The role of WC-1 bearing cells to protection was less definitive but our results suggest that this population may play a pivotal role early in infection. Granuloma architecture was altered in anti-WC-1 (ILA29) but not anti-CD8 (ILA51) -treated animals, suggesting that this population may be involved in recruitment of various cell types to sites of infection. J. Leukoc. Biol. 65: 28-34; 1999.

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Quantitation of bovine TNF-α mRNA by reverse transcription and competitive polymerase chain reaction amplification

Cited by 11 publications

References 13 publications

Cloning of porcine cytokine-specific cDNAs and detection of porcine tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-1 beta gene expression by reverse transcription PCR and chemiluminescence hybridization

Cloning of porcine cytokine-specific cDNAs and detection of porcine tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-1 beta gene expression by reverse transcription PCR and chemiluminescence hybridization

Modulatory effect of mycobacterium cell wall extract (Regressin) on lymphocyte blastogenic activity and macrophage cytokine gene transcription in swine

Role of CD8+ and WC-1+ γ/δ T cells in resistance to Mycobacterium bovis infection in the SCID-bo mouse

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