Quantitative assessment of cell adhesion molecule gene expression in endomyocardial biopsy specimens from cardiac transplant recipients using: Competitive polymerase chain reaction

Background. Adhesion of leukocytes to vascular endothelium is an early step in cardiac allograft rejection leading to migration of lymphocytes into pa renchymal tissues. Cell adhesion molecule (CAM) protein expression appears to increase as a result of rejection. The relationship of CAM gene expressi on to rejection is less well defined. The goal of this study was to define cell adhesion molecule gene expression in relation to the presence of acute cellular rejection in endomyocardial biopsies from cardiac transplant reci pients. Methods. To quantitatively assess intercellular adhesion molecule-1 (ICAM-1 ), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin gene expressio n, we developed a competitive PCR system using nonhomologous DNA fragments (MIMICs) with complementary sequences to CAM gene-specific primers as inter nal standards. MIMIC fragments with known concentrations were mixed in seri al dilutions with constant amounts of cDNA from the biopsy specimens and am plified with common primers under the same polymerase chain reaction condit ions. The relative CAM cDNA concentrations were determined by comparing the density of MIMIC to target cDNA bands on agarose gel. ICAM-1, VCAM-1, and E-selectin mRNA concentrations were analyzed from 38 cardiac transplant bio psies divided into 3 groups according to ISHLT rejection grade: group 1-gra de 0 (n=13); group 2-grade 1A or 1B (n=13); group S-grade 3A (n=12). Glycer aldehyde-3-phosphate dehydrogenase (a constitutive gene) was quantified in the same way as CAMs to normalize the relative levels of CAMs. Results. The results expressed as mean (1x10(-3) pM) (+/-SEM) in groups 1, 2, and 3, respectively, were: ICAM-1; 5+/-1; 57+/-4*; 64+/-13*, VCAM-1; 0.8 +/-0.1; 6+/-1**; 9+/-1*, E-selectin; 0.4+/-0.2; 0.8+/-0.2; 0.4+/-0.1 (*P<0. 001 versus group 1; **P<0.01 versus group 1). Conclusions. ICAM-1 and VCAM-1 gene expression was increased during rejecti on in endomyocardial biopsy specimens. Competitive polymerase chain reactio n can be used to quantitatively assess gene expression in biopsy specimens from patients.