Macrophage-derived chemokine gene expression in human and macaque cells: mRNA quantification using NASBA technology.
Macrophage-derived chemokine (MDC) is a CC-chemokine that inhibits infection by both macrophage- and T cell-tropic strains of HIV-1. This suppressor activity has led to great interest in fully characterizing the role of MDC in the pathogenesis of HIV-1 infection. Methods for the quantitation of constitutive levels of MDC protein in vivo are lacking. In this report, we describe the development and performance of a NASBA-based assay for the quantification of MDC mRNA expression in human and macaque cells. Although the constitutive in vivo levels of MDC mRNA in macaque and human T lymphocytes were low, in vitro activation of these cells greatly increased MDC transcription. Levels in the human and macaque cells were comparable under all conditions tested. Positive correlations between MDC transcription and protein expression were observed. The results indicate that this assay is extremely sensitive and reproducible over a five log dynamic range, and effectively quantifies MDC mRNA in resting and activated T cells. This assay may therefore permit characterization of the role of MDC in HIV-1/SIV pathogenesis, and in vaccine-induced immune responses.
Romano
J W
JW
Advanced BioScience Laboratories, Inc., 5510 Nicholson Ln., Kensington, MD 20895, USA. jromano@ablinc.com
Shurtliff
R N
RN
Grace
M
M
Lee
E M
EM
Ginocchio
C
C
Kaplan
M
M
Pal
R
R
D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism / Q000821:virology; D002460:Cell Line; D054422:Chemokine CCL22; D019742:Chemokines, CC / Q000096:biosynthesis* / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005821:Genetic Techniques; D006679:HIV Seropositivity / Q000097:blood; D006801:Humans; D008213:Lymphocyte Activation; D008251:Macaca; D010957:Plasmids / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D015302:Simian Immunodeficiency Virus / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic
C515482:CCL22 protein, human; D054422:Chemokine CCL22; D019742:Chemokines, CC; D018076:DNA, Complementary; D012333:RNA, Messenger