X P Zhang1, S E Kelemen, H J Eisen. 1. Department of Medicine, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Abstract
BACKGROUND: Adhesion of leukocytes to vascular endothelium is an early step in cardiac allograft rejection leading to migration of lymphocytes into parenchymal tissues. Cell adhesion molecule (CAM) protein expression appears to increase as a result of rejection. The relationship of CAM gene expression 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 recipients. METHODS: To quantitatively assess intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin gene expression, we developed a competitive PCR system using nonhomologous DNA fragments (MIMICs) with complementary sequences to CAM gene-specific primers as internal standards. MIMIC fragments with known concentrations were mixed in serial dilutions with constant amounts of cDNA from the biopsy specimens and amplified with common primers under the same polymerase chain reaction conditions. 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 biopsies divided into 3 groups according to ISHLT rejection grade: group 1-grade 0 (n=13); group 2-grade 1A or 1B (n=13); group 3-grade 3A (n=12). Glyceraldehyde-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 rejection in endomyocardial biopsy specimens. Competitive polymerase chain reaction can be used to quantitatively assess gene expression in biopsy specimens from patients.
BACKGROUND: Adhesion of leukocytes to vascular endothelium is an early step in cardiac allograft rejection leading to migration of lymphocytes into parenchymal tissues. Cell adhesion molecule (CAM) protein expression appears to increase as a result of rejection. The relationship of CAM gene expression 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 recipients. METHODS: To quantitatively assess intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin gene expression, we developed a competitive PCR system using nonhomologous DNA fragments (MIMICs) with complementary sequences to CAM gene-specific primers as internal standards. MIMIC fragments with known concentrations were mixed in serial dilutions with constant amounts of cDNA from the biopsy specimens and amplified with common primers under the same polymerase chain reaction conditions. 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 biopsies divided into 3 groups according to ISHLT rejection grade: group 1-grade 0 (n=13); group 2-grade 1A or 1B (n=13); group 3-grade 3A (n=12). Glyceraldehyde-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 rejection in endomyocardial biopsy specimens. Competitive polymerase chain reaction can be used to quantitatively assess gene expression in biopsy specimens from patients.