Cell adhesion molecules in clinical renal transplantation.

Leukocyte adhesion molecules are critically involved at a number of stages in immune and inflammatory responses, and their importance in the response to a renal allograft has been recognized for some years. They are involved in antigen presentation, in the cascade of events leading to extravasation of leukocytes into the allograft, in the subsequent migration of leukocytes through the extracellular matrix, and in the interactions between effector and target cells. Thus the adhesion molecules are highly attractive targets for therapeutic intervention in organ transplantation. Strategies have been explored to exploit the involvement of adhesion molecules in ischemia/reperfusion injury, allograft rejection, and the induction of immunological tolerance. Furthermore, the expression of a number of adhesion molecules is regulated by cytokines, and elevated levels may be detected both in transplant biopsies and as soluble forms measured in serum and urine. It has been proposed that these changes in levels might provide useful information in the diagnosis of allograft rejection and differentiation from other causes of graft dysfunction.

Late reflush in clinical renal transplantation. Protection against delayed graft function not observed.

Mechanical flushing of cadaveric kidneys with organ preservation fluid immediately before transplantation has been reported to be associated with improved early graft function. We report here the results of a prospective randomized controlled study of cadaveric renal transplantation after late reflush with organ preservation fluid in which no benefit with respect to delayed graft function was observed and, indeed, the protocol may have been harmful. The study was terminated after recruitment of only 18 patients (9 to each arm) because postreperfusion biopsies of reflushed kidneys contained unusual features, including abnormal cellular debris within the tubules or eosinophilic proteinaceous material within Bowman's capsule. These features were not present in the control kidneys. Acute tubular necrosis and biopsy-proven acute rejection episodes were more frequently seen in the reflushed kidneys, but at 1 year there was no significant difference in the function of the surviving grafts.

Cross-species reactivity of a panel of antibodies with monkey and porcine tissue.

The continuing shortage of organs available for transplantation limits the number of patients able to benefit from this highly successful form of therapy. Interest in alternative sources of organs has now turned towards the pig because of its physiological similarity to human. There is a requirement therefore for reagents not only for research purposes but possibly for studying xenotransplants in the clinical situation in the future. In this study, we have concentrated on determining the cross-species reactivity of a large panel of antibodies directed against human leukocyte markers, testing peripheral blood leukocytes and also including renal tissue to determine non-leukocyte cross-reactivity. A total of 63 out of 127 antibodies cross-reacted with cynomolgus monkey cells. Twenty of these antibodies stained similar populations of leukocytes to human, whereas the remaining 43 reacted with clearly different populations. The majority of antibodies (108/127) were unreactive with porcine leukocytes, reflecting the evolutionary differences between pig and man. Of the 19 antibodies cross-reactive with porcine cells, seven reacted with similar proportions of leukocytes to human, whereas the remaining 12 antibodies stained entirely different populations. The most interesting, and potentially most useful, antibodies were four that reacted with human, cynomolgus monkey and porcine tissue in a similar manner, suggesting that the epitopes recognized are present on similar molecules. These antibodies were directed against CD29 (MEM1O1A, K20) and CD18 (BU87, 7E4), the common beta1- and beta2-integrin subunits respectively. This study demonstrates that there are antigens common to cynomolgus monkey, pig and man that react with currently available antibodies. Nevertheless, when determining cross-species reactivity of human antibodies, it is important to consider the possibility that there may be additional non-leukocyte reactivity in other tissues.

Ischemia/reperfusion injury in human kidney transplantation: an immunohistochemical analysis of changes after reperfusion.

Organs used for transplantation undergo varying degrees of cold ischemia and reperfusion injury after transplantation. In renal transplantation, prolonged cold ischemia is strongly associated with delayed graft function, an event that contributes to inferior graft survival. At present, the pathophysiological changes associated with ischemia/reperfusion injury in clinical renal transplantation are poorly understood. We have performed an immunohistochemical analysis of pre- and postreperfusion biopsies obtained from cadaver (n = 55) and living/related donor (LRD) (n = 11) renal allografts using antibodies to adhesion molecules and leukocyte markers to investigate the intragraft changes after cold preservation and reperfusion. Neutrophil infiltration and P-selectin expression were detected after reperfusion in 29 of 55 (53%) and 24 of 55 (44%) cadaver renal allografts, respectively. In marked contrast, neutrophil infiltration was not observed in LRD allografts, and only 1 of 11 (9%) had an increased level of P-selectin after reperfusion. Immunofluorescent double-staining demonstrated that P-selectin expression resulted from platelet deposition and not from endothelial activation. No statistically significant association was observed between neutrophil infiltration and P-selectin expression in the glomeruli or intertubular capillaries despite the large number of cadaver renal allografts with postreperfusion changes. Neutrophil infiltration into the glomeruli was significantly associated with long cold ischemia times and delayed graft function. Elevated serum creatinine levels at 3 and 6 months after transplantation were also associated with the presence of neutrophils and platelets after reperfusion. Our results suggest that graft function may be influenced by early inflammatory events after reperfusion, which can be targeted for future therapeutic intervention.

Cadaver versus living donor kidneys: impact of donor factors on antigen induction before transplantation.

BACKGROUND: It is widely recognized that living-related donor (LRD) renal allografts have a higher overall graft survival than cadaver donor transplants. We tested the hypothesis that part of this is attributable to LRD kidneys being obtained under optimal conditions from healthy donors, whereas cadaveric kidneys may have experienced injury as a result of inflammatory events around the time of brain death. METHODS: We have performed a comparative immunohistochemical analysis of pretransplant donor biopsies from cadaveric (N = 65) and LRD (N = 29) kidneys to determine any differences that may predispose them to subsequent damage. Cryostat sections were stained with antibodies to leukocytes, adhesion molecules, and human leukocyte antigen (HLA)-DR antigens, and the expression was assessed semiquantitatively. RESULTS: High levels of endothelial E-selectin and proximal tubular expression of HLA-DR antigens, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 were detected in biopsies from cadaveric kidneys, whereas expression of these markers was markedly reduced in LRD kidneys. High levels of tubular antigen expression were significantly associated with traumatic death, prolonged ventilation, and episodes of infection in cadaver donors. Furthermore, the expression of pretransplant tubular antigens in cadaver donor kidneys was significantly associated with early acute rejection following transplantation, suggesting that such kidneys are predisposed to subsequent immune-mediated attack following transplantation. CONCLUSIONS: These results may explain, in part, the superior outcome of LRD allografts compared with cadaver renal allografts.

Nested polymerase chain reaction with sequence-specific primers typing for HLA-A, -B, and -C alleles: detection of microchimerism in DR-matched individuals.

It is widely accepted that donor leukocytes survive within the recipient periphery after blood transfusion or solid organ transplantation. The significance of this microchimerism remains unclear, partially because of the insecurity of assays used to detect the donor-derived material. The techniques used to detect donor-derived DNA within recipient peripheral blood rely largely on major histocompatibility complex class II polymorphism. We and others have shown that the sensitivity of polymerase chain reaction with sequence-specific primers (PCR-SSP) typing for HLA class II alleles can be increased 100-fold by the addition of a primary amplification step (nested PCR-SSP). We have now extended this technique to encompass typing for HLA class I alleles, thereby adding flexibility to microchimerism testing by enabling testing of recipients HLA-DR matched with their donors. However, the high level of sensitivity achieved with the technique (1:100,000) leads to a concomitant decrease in the specificity that results in the amplification of unexpected products, a phenomenon we encountered in the development of our nested PCR-SSP typing system for HLA class II alleles. We describe here how it is possible to compensate for these anomalies by including multiple testing of a pretransfusion sample that acts as a specificity control, establishing a rigorous baseline for subsequent analysis.

Endothelial cell protection against ischemia/reperfusion injury by lecithinized superoxide dismutase.

BACKGROUND: Organs used for transplantation may experience long periods of cold ischemic preservation and consequently oxygen free radical-mediated damage following reperfusion. Lecithinized superoxide dismutase (lec-SOD) is a novel free radical scavenger that has been shown to bind with high affinity to cell membranes. The aim of this study was to determine whether lec-SOD bound to endothelial cells under organ preservation conditions to mediate direct antioxidant activity at the endothelial cell surface and thus offer protection against the harmful effects of ischemia/reperfusion injury. METHODS: An in vitro study was performed on large vessel endothelial cells (HUVEC) and a human microvascular endothelial cell line HMEC-1, to investigate the potential therapeutic benefits of incorporating lec-SOD into organ preservation solution. A cold hypoxia/reoxygenation system was developed to examine lec-SOD binding affinity to endothelial cells, protection against hypoxia/reoxygenation-induced cell death, and neutrophil adhesion. RESULTS: Lec-SOD bound to endothelial cells with higher affinity than unmodified recombinant human superoxide dismutase (rhSOD) and significantly protected both HUVEC and HMEC-1 from cell death following 27 hours of cold hypoxia (P < 0.01). Furthermore, neutrophil adhesion to the endothelium stimulated by hypoxia and reoxygenation was significantly inhibited by treatment with lec-SOD but not by lecithin or rhSOD (P < 0.01). Analysis by flow cytometry demonstrated that E-selectin and ICAM-1 were up-regulated by hypoxia/reoxygenation that was inhibited in part by lec-SOD. CONCLUSIONS: The results from this study suggest that incorporation of lec-SOD into organ preservation solutions provides effective protection to endothelial cells against cold ischemia and reperfusion injury following transplantation.