John L. R. Forsythe

Management of biliary tract complications after orthotopic liver transplantation.

Abstract:  Introduction:  Despite improved survival, biliary complications remain a significant cause of morbidity following orthotopic liver transplantation. The aim of this study was to review the incidence, treatment and optimum management pathway of biliary complications at the Scottish Liver Transplant Unit.

Glucocorticoids Acutely Increase Brown Adipose Tissue Activity in Humans, Revealing Species-Specific Differences in UCP-1 Regulation.

Summary The discovery of brown adipose tissue (BAT) in adult humans presents a new therapeutic target for metabolic disease; however, little is known about the regulation of human BAT. Chronic glucocorticoid excess causes obesity in humans, and glucocorticoids suppress BAT activation in rodents. We tested whether glucocorticoids regulate BAT activity in humans. In vivo, the glucocorticoid prednisolone acutely increased 18fluorodeoxyglucose uptake by BAT (measured using PET/CT) in lean healthy men during mild cold exposure (16°C–17°C). In addition, prednisolone increased supraclavicular skin temperature (measured using infrared thermography) and energy expenditure during cold, but not warm, exposure in lean subjects. In vitro, glucocorticoids increased isoprenaline-stimulated respiration and UCP-1 in human primary brown adipocytes, but substantially decreased isoprenaline-stimulated respiration and UCP-1 in primary murine brown and beige adipocytes. The highly species-specific regulation of BAT function by glucocorticoids may have important implications for the translation of novel treatments to activate BAT to improve metabolic health.

Characterization of donor-directed antibody class in the post-transplant period using flow cytometry in renal transplantation

Over the past few years there has been increasing awareness of the importance of humoral mechanisms in the rejection of renal transplants. In this study we have monitored the development of antibodies directed against donor T and B lymphocytes using the sensitive flow cytometric technique. Forty-two cadaveric renal transplants were studied both before and for a maximum of 14 days after transplantation. Donor cells were separated from spleen on the day of transplantation and stored in liquid nitrogen until required. The dual colour flow cytometric assay was used to detect IgG or IgM directed againts donor T or B lymphocytes. Using AB sera as controls, results were expressed as relative median fluorescence (RMF) and then correlated with the clinical performance of the grafts. Significant associations were found between the incidence of donor-directed antibodies and the development of clinical rejection. The magnitude of the rise in antibody levels was also related to graft performance. In patients showing severe graft rejection, high levels of antibodies of the IgG class developed before the clinical diagnosis of rejection was made. The routine use of this test allows the prediction of impending severe rejection to be made and may have important implications for immunosuppressive therapy.

RENAL ALLOGRAFT REJECTION : POSSIBLE INVOLVEMENT OF ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY

We have demonstrated that serum from appropriately sensitized patients can contain IgG antibodies that bind to cultured renal epithelial cells. The presence of such antibodies on the surface of renal cells enables otherwise nonlytic PBMC to lyse these renal cells by an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. Experiments involving cell-sorting and specific complement-mediated lysis showed that the ADCC effector cells were of the CD3 -ve, C16 +ve phenotype characteristic of NK cells. In this report it is argued that an ADCC mechanism may be of importance in mediating chronic renal cell damage in the absence of acute allograft rejection.

Renal allograft rejection: induction and function of adhesion molecules on cultured epithelial cells.

The interaction of graft‐infiltrating immune cells with donor parcnchymal cells is an important early event in allograft rejection. This binding is stabilized by interaction of antigen‐independent ‘adhesion’ molecules expressed on the two cell types. As lhe level ofexpressionofthese molecules can be altered during inflammation, a series of experiments was prcrformed to examine the effects of the inflammiiiory cytokines intcrfcron‐gamma (IFN‐;); ind tumour necrosis factor‐alpha (TNF‐a) on adhesion molecules expressed by cultured human renal tubular epithelial cells. These cells constitutively expressed ICAM‐l and LFA‐. Incubation with IFN‐y increased expression of ICAM‐i but had no significant effect on expression of LFA‐3 (<005). Incuhation with TNF‐a increased expression of both ICAM‐1 and LFA‐3; IFN‐ysynergizcd withTNF‐ot to further augment expression of these molecules. Peripheral blood lymphocyles (PBL) showed an enhanced binding to allogeneic renal epithelial cell monolayers which had been pretreated with IFN‐yorTNF‐x. MoAbs specific for ICAM‐l or its ligand LFA‐1 inhibited adhesion of PBL lo either IFN‐;‐ or TNF‐apretreated renal cells. By contrast, antibodies specific for LFA‐3 or its ligand CD2 only significantly blocked PBL adhesion to renal cells which had been pretrealed with TNF‐x. Combination of antibodies spccitic lor multiple components of the adhesion systems produced greater inhibition of adhesion than was produced by any single MoAb. These results suggest thai the inliammatory cytokines IFN‐7 and TNF‐a up‐regulatc expression of functional lCAM‐1 and LFA‐3 molecules which can augment ihe binding of potentially graft‐damaging lymphoid cells to renal tubular epithelial eells.

The Role of Vascular Endothelial Growth Factor in the Kidney in Health and Disease

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen, angiogenic factor and enhancer of vascular permeability. Expressed in the epithelial cells of the developing glomerulus and tubular epithelium, VEGF plays an important role in the development and maintenance of the early vasculature of the kidney. Here, we review the available literature regarding the expression and function of VEGF both in the developing and healthy adult kidney. Furthermore, we highlight how VEGF expression is altered in the diseased kidney and how this modulated expression may impact on and reflect underlying functional changes occurring during the disease process. As discussed, many controversial issues remain, particularly concerning the role of VEGF in the diseased kidney. That VEGF has been proposed as a potential future therapeutic target for the management of some renal diseases requires first that the precise role of VEGF in the normal kidney and various renal pathologies be further and more clearly defined.

Renal allograft rejection: the temporal relationship and predictive value of plasma TNF (alpha and beta), IFN-gamma and soluble ICAM-1

Abstract  Recently, close interactions have been described between the tumour necrosis factors alpha and beta (TNF‐alpha and beta), interferon‐gamma (INF‐gamma) and intercellular adhesion molecule‐1 (ICAM‐1) in T‐cell mediated immune activation. During the process of renal graft rejection, the properties of these cytokines to act as powerful stimulators of macrophages, to upregulate class II MHC expression and to stabilise cell‐to‐cell binding make them of great potential interest. The aim of the present study was to determine the plasma levels of each cytokine and soluble ICAM‐1 in 16 renal allograft recipients. We examined plasmas of patients for the first 2 weeks after transplantation and correlated results with the clinical pattern of rejection. Our data suggest an immunopathologic involvement of TNF‐alpha, TNF‐beta and slCAM‐1 in renal allograft rejection and showed that there was a significant elevation in plasma concentrations of these parameters 2 or 3 days prior to the diagnosis of clinical rejection. Rises in INF‐gamma did not appear to be significant with regard to rejection as very high levels were found in patients showing no evidence of clinical rejection.

Commercially available recombinant sonic hedgehog up-regulates Ptc and modulates the cytokine and chemokine expression of human macrophages: an effect mediated by endotoxin contamination?

The Sonic hedgehog (Shh) signalling pathway plays an important role in developmental patterning and proliferation. Recent evidence suggests that Shh also plays a role in the development of the immune system. Here, we demonstrate that components of the Shh signalling pathway are expressed in human macrophages and that the receptor for Shh, Ptc, is up-regulated by a commercially available recombinant preparation of Shh (CArShh). Further, we report that the addition of CArShh up-regulates the production of IL-6, IL-8, MCP-1, IP-10, MIG and RANTES by macrophages, an effect enhanced by the presence of fetal calf serum in the culture medium. In contrast, TGF-beta, TNF-alpha, IL-1b, IL-12 and IL-10 production were not modulated by CArShh and VEGF was minimally up-regulated even in the presence of serum. The up-regulation of these cytokines and chemokines was abrogated by CD14 inhibition and polymixin B, but not reliably inhibited by the specific Shh pathway inhibitor cyclopamine. These results suggest that, although components of the Shh signalling pathway are expressed in macrophages, the modulation of macrophage cytokine and chemokine effector function seen in response to commercially available rShh results from low levels of endotoxin contained within the CArShh preparations employed to explore the effects of Shh in vitro.

Xenotransplantation: an examination of the adhesive interactions between human lymphocytes and porcine renal epithelial cells

Specific adhesion molecules stabilize the binding between lymphocytes and antigen bearing cells; this intercellular adhesion is vital to both the affector and effector phases of an immune response. It is not known whether adhesion molecules and their counter-receptors can form the cross-species interactions that will facilitate human T cell recognition of xenogeneic porcine target cells. In this report it is demonstrated that a higher proportion of mitogen-activated than of resting human lymphocytes adhere to cultured porcine renal epithelial cells. Furthermore, antibody blocking experiments demonstrated that at least part of this cell-cell binding is stabilized by the human adhesion molecules LFA-1 (lymphocyte function-associated antigen-1) and the alpha 4-containing integrins. It is possible that this capacity for cross-species adhesion will play a role during the cell-mediated rejection of clinical porcine xenografts.

Reducing renal injury during transplantation

Damage sustained by an ischaemic kidney is reduced by cooling the organ. For this reason kidneys are rapidly cooled during the retrieval operation and preserved at low temperature before implantation. When the kidney is removed from cold storage for implantation into the recipient it gradually rewarms (second warm ischaemic time) and a prolonged second warm ischaemic time has been shown to be a cause of acute tubular necrosis following transplantation. The temperature rise in a kidney during implantation has been poorly investigated and little work to minimize that rise has been carried out. This study investigates, in an animal model, the changes that occur in the core temperature of kidneys during the second warm ischaemic time. A jacket has been designed which greatly reduces the rate of kidney rewarming during simulated operative conditions. Kidneys unprotected by the test system showed a rapid rise in temperature from a mean of 1 degrees C to a mean of 20 degrees C after 45 min, compared with those kidneys placed in the protective jacket in which the temperature rose to a mean of only 8 degrees C in the same time. The jacket is not bulky and is simple to use. Maintaining a low kidney core temperature during the second warm ischaemic time will reduce injury to the kidney and should be part of routine clinical practice.