Objective: We have attempted to demonstrate the induction of inducible nitric oxide synthase in human vascular tissue and define the capacity of different cytokines to induce this enzyme. Methods: Segments of human arteries were stimulated with lipopolysaccharide (10 mu g/ml), interleukin-1 beta (5 U/ml), tumor necrosis factor-alpha (10 U/ml), and interferon-gamma (200 U/ml). Cytokines were either used alone or in certain combinations, as well as in the presence of L-N-G-monomethyl-arginine (100 mu mol/l) or cycloheximide (1 mu mol/l). Induction was assessed by measurement of mRNA expression, immunocytochemical localisation of the expressed protein, nitric oxide synthase activity and levels of nitrite, a product of nitric oxide formation. Results: PCR analysis showed the presence of mRNA for iNOS in stimulated samples which could be inhibited by cycloheximide. There was positive staining with an antibody against human iNOS in the media of stimulated vessel segments. Stimulated segments were also shown to contain Ca2+-independent nitric oxide synthase activity. The cytokines and Lipopolysaccharide together gave a significant rise in levels of nitrite in the medium after 36 and 48 h, which was inhibited by L-N-G-monomethyl-arginine and cycloheximide. Only interferon-gamma incubated alone was capable of increasing nitrite levels. This effect was enhanced by co-incubation with either interleukin-1 beta, tumor necrosis factor-alpha or lipopolysaccharide. Conclusion: We have shown that increased production of nitrite by human vascular tissue in response to cytokines is associated with induction of iNOS as shown at the molecular and protein levels, and further supported by the presence of increased Ca2+-independent nitric oxide synthase activity following cytokine stimulation. (C) 1998 Elsevier Science B.V. All rights reserved.

AIMS To investigate the role of angiotensin converting enzyme (ACE) (I/D) gene polymorphism in the development of coronary sclerosis after cardiac transplantation. METHODS AND RESULTS Eighty cardiac transplant recipients (44 transplant associated coronary artery disease; 36 non-transplant associated coronary artery disease) and their donors were genotyped by polymerase chain reaction. The allele frequencies of the recipients in the transplant associated coronary artery disease and non-transplant associated coronary artery disease groups (I = 0.47 and 0.48, D = 0.53 and 0.52, respectively) did not differ significantly between the groups. However, there was a negative association between the frequency of the I allele in the donor and the development of transplant associated coronary artery disease. The D allele in the donor population of the non-transplant associated coronary artery disease group had a significantly (P < 0.01) lower frequency (0.35) than either the transplant associated coronary artery disease group (0.53) or that of the general population (0.57). Other factors analysed were recipient family history, cholesterol levels, age, sex and body mass index, donor age and acute rejection, of which the significant (P < 0.05) factors were acute rejection and sex of the recipient. CONCLUSION These results suggest that the ACE genotype of the donor organ may be an additional risk factor for the development of coronary artery disease following cardiac transplantation and that tissue rather than circulating ACE could be implicated in the pathogenesis of this disease.

The observations of 2 types of CD4+ T cells (Th1 and Th2), which can be distinguished by their different cytokine profiles, has led to the possibility that analysis of cytokine profiles produced locally within transplanted allografts could be predictive of rejection or acceptance of that graft. We have investigated the expression of IL-2 and TNF beta (Th1 type cytokines), IL-4 and IL-10 (Th2 type cytokines), and the proinflammatory cytokines TNF alpha and IL-1 beta in sequential endomyocardial biopsies collected from 12 cardiac transplant recipients during the first 4 months after transplantation, by the analysis of RNA extracted from each biopsy by reverse transcriptase-polymerase chain reaction. The results obtained were compared with histopathological and clinical indicators of rejection. IL-2 was found in all severe (grade 3), in 57% of moderate (grade 2), in 21% of mild (grade 1) rejection, and in only 1 nonrejection (subsequently progressing to grade 3), where rejection was classified by routine histology. IL-4 and IL-10 were absent from grade 3 rejection, but present in 24% (IL-4) and in 17% (IL-10) of mild rejection and in a single nonrejecting biopsy, respectively. IL-4 was found in 2 cases of moderate rejection, and IL-10 in 1 case of moderate rejection. Statistical analysis showed that the presence of IL-2 positively correlated with both mild and moderate rejection, while IL-4 correlated with mild rejection (P < or = 0.05). IL-1 beta, TNF alpha, and TNF beta were found in both rejecting and nonrejecting biopsies, with no significant differences between the histological grades. Our results suggest that in the human situation, IL-2 and IL-4 may indeed be important in the modulation of rejection.