Comparison of HTK and hypertonic citrate to intraarterial cooling in human non-heart-beating kidney donors.

Intraarterial cooling (IAC) of non-heart-beating donors (NHBD) for renal donation requires a cheap, low-viscosity solution. HTK contains a high hydrogen ion buffer level that theoretically should reduce the observable acidosis associated with ongoing anaerobic metabolism. A retrospective comparison of all retrieved NHBD kidneys as well as of viability on the Organ Recovery Systems Lifeporter machine perfusion circuit was performed with respect to the preservation solution HTK or Marshall's HOC. Forty-two NHBD kidneys (19 HTK and 23 HOC) were machine perfused between February 2004 and May 2005. Most of the HTK kidneys were obtained from uncontrolled donors (12 vs 5; Fisher exact test, P = .01). As a consequence, the glutathione-s-transferase viability assay (411 vs 292 IU/L, P = .12) and the lactate concentrations (2.33 vs 1.94 mmol/L, P = .13) were higher among the HTK cohort. There was evidence of greater buffering capacity in HTK, since the lactate:hydrogen ion ratios were consistently lower during the first 2 perfusion hours (1 hour P = .03, 2 hour P = .02). A linear regression analysis confirmed that this was related to the IAC solution (ANCOVA, P < .001). All controlled donor kidneys passed viability testing and were transplanted. In contrast, 83% (10/12) of the uncontrolled donor kidneys preserved with HTK passed the viability test and were transplanted, compared with only 20% (1/5) of the HOC-treated comparators (Fisher exact test, P = .03). It may be concluded that the postulated advantages of improved pH buffering with HTK appear to have clinical relevance.

Routine intraoperative ureteric stenting for kidney transplant recipients.

BACKGROUND: Major urological complications (MUCs) after kidney transplantation contribute to patient morbidity and compromise graft function. The majority arise from the vesico-ureteric anastomosis and present early after transplantation. Ureteric stents have been successfully used to treat such complications. A number of centres have adopted a policy of universal prophylactic stenting, at the time of graft implantation, to reduce the incidence of urine leaks and ureteric stenosis. Stents are associated with specific complications and some centres advocate a policy of only stenting selected anastomoses. OBJECTIVES: To examine the benefits and harms of routine ureteric stenting to prevent urological complications in kidney transplant recipients. SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials (CENTRAL in The Cochrane Library), MEDLINE, EMBASE, reference lists of articles, books and abstracts and contacted companies, authors and experts to identify relevant randomised controlled trials (RCTs). SELECTION CRITERIA: All RCTs and quasi-RCTs were included in our meta-analysis. DATA COLLECTION AND ANALYSIS: Four reviewers assessed the trials for quality against four criteria (allocation concealment, blinding, intention-to-treat and completeness of follow-up). The primary outcome was the incidence of MUCs. Further outcomes of interest were graft and patient survival and the incidence of adverse events (urinary tract infection (UTI), haematuria, irritative symptoms, pain and stent migration). Statistical analyses were performed using the random effects model and the results expressed as relative risk (RR) with 95% confidence intervals (CI). MAIN RESULTS: Seven RCTs (1154 patients) of low or moderate quality were identified. The incidence of MUCs was significantly reduced (RR 0.24, 95% CI 0.07 to 0.77, P = 0.02, NNT 13) by universal prophylactic stenting. This was dependent on whether the same surgeon performed, or was in attendance, during the operations. Two patients lost their grafts to infective urinary tract complications in the stented group. UTIs, in general, were more common in stented patients (RR 1.49, 95% CI 1.04 to 2.15) unless the patients were prescribed cotrimoxazole 480 mg/d: in which case the incidence was equivalent (RR 0.97, 95% CI 0.71 to 1.33). Stents appeared generally well tolerated, although trials using longer stents (>/= 20 cm) for longer periods (> 6 weeks) had more problems with encrustation and migration. AUTHORS' CONCLUSIONS: Routine prophylactic stenting reduces the incidence of MUCs. Trials comparing selective stenting and universal prophylactic stenting, whilst difficult to design and analyse, would address the unresolved quality of life and economic issues.

Development of a flow cytometric assay to quantify lymphocyte adhesion to cytokine-stimulated human endothelial and biliary epithelial cells.

The adhesive interaction between T lymphocytes and parenchymal cells is of importance for many processes of the cellular immune response. This adhesion is regulated by the activation status of the T cell and by cytokines in the microenvironment which can alter adhesion molecule expression by endothelial and epithelial cells. In this study results from an isotopic adhesion assay were compared with those from a flow cytometric assay in order to determine which was most appropriate for the investigation of lymphocyte adhesion to human umbilical vein endothelial cells (HUVEC) and intrahepatic biliary epithelial cells (HIBEC). Treatment of both these cell types with the proinflammatory cytokines interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) significantly upregulated expression of intercellular adhesion molecule-1 (ICAM-1). Treatment with TNF-alpha also induced endothelial cells to express vascular cell adhesion molecule-1 (VCAM-1). The isotopic assay demonstrated increased adhesion of lymphoblasts to HUVEC which had been stimulated with cytokines for 15 h but failed to detect major changes in adhesion following 72 h of cytokine treatment of HUVEC or HIBEC. However, the flow cytometric assay reproducibly demonstrated increased adhesion following cytokine treatment for both these time periods; these increases corresponded with the changes in adhesion molecule expression by cytokine-stimulated HUVEC and HIBEC targets. The differences in apparent adhesion measured by the two assays after cytokine stimulation for 72 h may be explained by cytokine-induced changes in the morphology and confluency of cultured cells. Results of the isotopic assay are proportional to the total number of lymphoid cells bound by the cultured target cells and will be distorted by changes in effective target cell area. The flow cytometric assay measures the mean number of lymphoid cells bound by each target cell and is independent of the total binding area. It is concluded that the flow cytometric assay is more suitable than the isotopic technique for following time-dependent changes in the adhesion of leukocytes to cytokine-stimulated target cells.

Examination of the mechanism by which heparin antagonizes activation of a model endothelium by interferon-gamma (IFN-gamma).

IFN-gamma increases the potential immunogenicity of vascular endothelial cells by up-regulation of intercellular adhesion molecule-1 (ICAM-1) and class I MHC antigen expression and by induction of class II MHC antigens and certain chemokines. In this study the mechanism by which the glycosaminoglycan (GAG) heparin antagonizes the activation of a model endothelium by IFN-gamma was investigated. Radioligand binding assays demonstrated that total binding of 125I-IFN-gamma to the EAhy.926 endothelial hybridoma cell line was reduced in the presence of heparin or heparan sulphate (HS); the structurally dissimilar GAG chondroitin sulphate had no effect. Treatment of the cells with chlorate, a metabolic inhibitor of GAG sulphation, was found to reduce both the subsequent binding of IFN-gamma and its ability to induce expression of class II MHC antigens. Treatment with heparinase II dramatically reduced the binding of IFN-gamma, while chondroitin ABC lyase had no effect. A cationic peptide from the C-terminal region of IFN-gamma was also found to reduce binding of intact IFN-gamma to the cells. These results appear to demonstrate that IFN-gamma is sequestered at the surface of endothelial cells by electrostatic interaction between specific basic amino acid residues and sulphated domains on HS, the most abundant endothelial GAG. This interaction is competitively inhibited by heparin, which is structurally related to HS. These observations are consistent with the model that IFN-gamma is bound by membrane-associated HS before engagement with the high-affinity receptor and signal transduction. Inhibition of the interaction between proinflammatory cytokines and membrane-associated GAG molecules may provide a mechanism for inducing clinically useful immunosuppression.

Endothelial production of MCP-1: modulation by heparin and consequences for mononuclear cell activation.

Heparin is a polyanionic glycosaminoglycan (GAG) that can bind with high affinity to a range of cytokines including interferon-gamma (IFN-gamma) and members of the chemokine superfamily. This GAG also possesses immunomodulatory activity in vivo and can antagonize the capacity of IFN-gamma to induce class II MHC antigen expression, and to up-regulate intercellular adhesion molecule-1, by cultured endothelial cells. Previous studies have shown that binding to cell-surface heparan sulphate is essential for optimal activity of IFN-gamma and that free heparin competitively inhibits this sequestration process. The present study was performed to increase our understanding of the immunosuppressive activity of heparin by investigation of potential antagonism of the production and function of monocyte chemotactic peptide-1 (MCP-1), a chemokine important for mononuclear leucocyte recruitment across vascular endothelium. It was found that mixture of heparin with IFN-gamma inhibited up-regulation of the signal transducer and activator of transcription protein, STAT-1 produced normally by treatment of endothelial cells with IFN-gamma. An inhibition of MCP-1 production was observed that was specifically caused by mixture of IFN-gamma with heparin-like, and therefore cytokine-binding, GAGs. It was also shown that mixture of heparin-like GAGs with MCP-1 inhibited the rapid tyrosine phosphorylation of phosphatidylinositol 3-kinase which is normally produced by treatment of mononuclear leucocytes with this chemokine. Blockade of this intracellular signalling event was associated with a reduction in the normal transendothelial migration response towards MCP-1. Results from this study indicate that soluble, heparin-like GAGs can block IFN-gamma-dependent up-regulation of MCP-1 production by cultured endothelial cells, and can also antagonize the leucocyte-activating and migration-promoting properties of pre-existing MCP-1. These activities may contribute to the immunomodulatory properties of heparin.

Role of glycosaminoglycans (GAGs) in regulation of the immunogenicity of human vascular endothelial cells.

Heparan sulphate is a common glycosaminoglycan component of proteoglycans present on the luminal surface of vascular endothelium. It has been proposed that an important function of these molecules is the sequestration of a range of proinflammatory and proadhesive cytokines. Such cytokines play a vital role during lymphocyte recruitment from the blood at sites of inflammation. In this study it is shown that the effects of interferon-gamma (IFN-gamma), but not of tumour necrosis factor-alpha (TNF-alpha), are inhibited by treatment with soluble heparin. Specifically, heparin was shown to inhibit the induction of class II MHC antigens and the up-regulation of intercellular adhesion molecule-1 (ICAM-1) produced by treatment of cultured human endothelial cells with IFN-gamma. Furthermore, it was shown that heparin blocked the enhanced adhesion of T lymphocytes to IFN-gamma-treated endothelial cells. Investigation of the inhibitory effects of other GAG molecules demonstrated a requirement for heparin-like structural domains as chondroitin sulphate was unable to inhibit the function of IFN-gamma. These results may explain reported immunosuppressive properties of heparin, and are consistent with the model that heparin may compete with cell surface GAGs to bind IFN-gamma, thereby reducing effective biological activity.