Screening of a HUVEC cDNA library with transplant-associated coronary artery disease sera identifies RPL7 as a candidate autoantigen associated with this disease.

A HUVEC cDNA library was screened with sera from two patients who had developed transplant-associated coronary artery disease (TxCAD) following cardiac transplantation. A total of six positive clones were isolated from a primary screen of 40 000 genes. Subsequent DNA sequence analysis identified these to be lysyl tRNA synthetase, ribosomal protein L7, ribosomal protein L9, beta transducin and TANK. Another gene whose product could not be identified showed homology to a human cDNA clone (DKFZp566M063) derived from fetal kidney. Full-length constructs of selected genes were expressed as his-tag recombinant fusion proteins and used to screen a wider patient base by ELISA to determine prevalence and association with TxCAD. Of these ribosomal protein L7 showed the highest prevalence (55.6%) with TxCAD sera compared to 10% non-CAD.

Strain specific variation in cytokine regulated ICAM-1 expression by rat brain-endothelial cells.

Cytokine induced levels of ICAM-1 expressed by rat brain-endothelial cells were quantitated by enzyme immunoassay in response to stimulation by TNF-alpha in the presence or absence of IFN-gamma. The rat strains investigated differ in their susceptibility to experimental allergic encephalomyelitis; significantly less ICAM-1 was induced by BEC derived from the resistant PVG strain as compared to the susceptible LEW strain with both cytokine combinations. In contrast, despite the difference in disease susceptibility, equivalent levels of ICAM-1 were induced between the LEW and BN strain. Furthermore, evidence for a synergistic interaction of both TNF-alpha and IFN-gamma was observed in the BN strain. The results are discussed with relevance to the disease profile of each strain.

Strain specific variation in IFN-gamma inducible lymphocyte adhesion to rat brain endothelial cells.

We have examined the interferon-gamma (IFN-gamma) induced increase in lymphocyte adhesion to rat brain endothelial cells (BEC) in the experimental allergic encephalomyelitis (EAE) susceptible LEW and resistant PVG strain. A significant increase in adhesion of mitogen activated lymphocytes could be demonstrated by stimulating LEW BEC with 50 U/ml IFN-gamma for 24 h. In contrast the same treatment failed to induce a significant increase in lymphocyte adhesion in the PVG strain. Flow cytometric analysis of lymphocyte integrin expression indicated a marked increase in the number of cells expressing both LFA-1 and VLA-4 following mitogen activation and was similar between the LEW and PVG strain. Depletion of LFA-1 and VLA-4 positive lymphocytes resulted in an equivalent inhibition of adhesion to BBB-EnC indicating that the adherent population was comprised predominantly of the VLA-4 + /LFA-1 + phenotype. We conclude that this strain variation in the IFN-gamma activation of BEC may be related to the disease phenotypes of these strains.

Strain-specific variation in constitutive and inducible expression of MHC class II, class I and ICAM-1 on rat cerebral endothelium.

Strain variation in levels of inducible major histocompatibility complex (MHC) class II expression by rat cerebral endothelium has previously been reported. Using primary cell cultures of rat cerebral endothelium from PVG (RT1c), LEW (RT1l), PVG.LEW (RT1l) and PVG.AGUS (RT1lv?) strains it was determined that variation in levels of inducible MHC class II expression between strains can be accounted for by both cis-acting elements within the MHC region and by trans-acting elements outside the MHC. In addition it was determined that levels of constitutive MHC class I expression varied between PVG (RT1c) and LEW (RT1l) strains which can be attributed to cis-acting elements within the MHC region. Furthermore, while levels of constitutive class I expression vary between PVG (RT1c) and LEW (RT1l) endothelium we could find no difference in the interferon-gamma (IFN-gamma) inducible expression of class I between these two strains. In contrast the inducibility of intercellular adhesion molecule-1 (ICAM-1) in response to IFN-gamma was found to differ between PVG and LEW endothelium. Significant levels of ICAM-1 are induced on LEW cerebral endothelium after 24 hr exposure to 50 U/ml IFN-gamma. However, no significant induction of ICAM-1 could be demonstrated on PVG, or BN cerebral endothelium after the same exposure to IFN-gamma. Induction of ICAM-1 by IFN-gamma precedes MHC class II by at least 24 hr and its persistence is proportional to the concentration of IFN-gamma used. We suggest that the rat MHC region (RT1) contains elements which control the levels of constitutive class I expression and inducible class II expression in response to IFN-gamma, but that other non-RT1 genes influence the inducibility of MHC class II on rat cerebral endothelial cells. This observation, together with the finding that ICAM-1 expression is not significantly increased in response to IFN-gamma on PVG or BN endothelium, suggests that IFN-gamma responsiveness by these strains differs from LEW.