S. Thiru

CYCLOSPORIN A IN PATIENTS RECEIVING RENAL ALLOGRAFTS FROM CADAVER DONORS

Seven patients on dialysis with renal failure received transplants from mismatched cadaver donors and were treated with cyclosporin A (CyA), initially as the sole immunosuppressive agent. CyA was effective in inhibiting rejection but there was clear evidence of both nephrotoxicity and hepatotoxicity. A cyclophosphamide analogue was added to the CyA treatment in six of the patients. Five patients are out of hospital with functioning allografts, and two of these have received no steroids. One patient required an allograft nephrectomy because of pyelonephritis in the graft. Another died of systemic aspergillus and candida infection. Further careful study of this potentially valuable drug will by required before it can be recommended in clinical practice.

Life-supporting Pig-to-primate Renal Xenotransplantation Using Genetically Modified Donors

BACKGROUND In order to circumvent the complement-mediated hyperacute rejection of discordant xenografts, a colony of pigs transgenic for the human regulator of complement activity, human decay-accelerating factor (hDAF), has been produced. METHODS Seven kidneys from hDAF transgenic pigs and six kidneys from nontransgenic control pigs were transplanted into cynomolgus monkeys; both native kidneys were removed during the same operation. The recipient animals were immunosuppressed with cyclosporine, steroids, and cyclophosphamide. RESULTS In the transgenic group, the median survival time was 13 days (range, 6-35 days); the median survival time in the control group was 6.5 days (range, 0.3-30 days). There were no cases of hyperacute rejection in the transgenic group, and the two longest-surviving kidneys in this group showed no evidence of rejection on histological examination. In contrast, all control kidneys underwent antibody-mediated rejection, one demonstrating hyperacute rejection and the others acute vascular rejection. CONCLUSION This study demonstrates that (i) a kidney from an hDAF transgenic pig can support the life of a primate for up to 35 days (and also shows the basic physiological compatibility between the pig and nonhuman primate); (ii) nontransgenic kidneys are not routinely hyperacutely rejected; and (iii) the presence of hDAF on the kidney confers some protection against acute vascular rejection. Improved immunosuppression and immunological monitoring may enable extended survival.

Cyclosporine: Five Years' Experience in Cadaveric Renal Transplantation

Using a retrospective analysis we compared 79 recipients of cadaveric renal allografts who were treated with cyclosporine as the sole initial immunosuppressant and 29 concurrent transplant recipients treated with conventional immunosuppressants. The cyclosporine-treated group had a slightly higher actuarial patient survival at four years (86 per cent v. 76 per cent). Actuarial graft survival at four years was 70 per cent in the cyclosporine group, as compared with 62 per cent in the conventionally immunosuppressed group. The incidence of acute rejection episodes was 62.1 per cent in the former and 65.5 per cent in the latter. Nephrotoxicity was managed either by reduction of the dose of cyclosporine or by conversion to conventional immunosuppression. Monitoring of trough serum levels of cyclosporine facilitated its administration. Serum creatinine levels have been higher in cyclosporine-treated patients with functioning grafts, but graft deterioration has not occurred after more than three years. Cyclosporine provides adequate immunosuppression for patients with renal allografts. At four years, the rates of patient and graft survival remain superior to those with conventional immunosuppression. In 41 per cent of patients the use of steroids was completely avoided. The longer-term results of this powerful immunosuppressive agent are not yet known.

Alemtuzumab (CAMPATH 1H) Induction Therapy in Cadaveric Kidney Transplantation—Efficacy and Safety at Five Years

Alemtuzumab is a powerful lymphocyte depleting antibody currently being evaluated in solid organ transplantation. This paper describes 5‐year results of a single center study of alemtuzumab as induction in renal transplantation.

Phase I study of an engineered aglycosylated humanized CD3 antibody in renal transplant rejection

BACKGROUND The potential therapeutic benefits of CD3 monoclonal antibodies, such as OKT3, have been limited by their immunogenicity and their propensity to activate a severe cytokine release syndrome. This has constrained the clinical use of OKT3 to the treatment of acute rejection episodes of organ allografts. METHODS We have humanized a rat CD3 antibody and created a single amino acid substitution in position 297 of the IgG1 heavy chain to prevent glycosylation and, consequently, binding of the therapeutic antibody to Fc receptors and to complement. This antibody has been given as first line antirejection therapy in nine kidney transplant recipients with biopsy-proven acute rejection episodes. RESULTS None of the patients demonstrated any antiglobulin response nor any significant cytokine release syndrome. Seven of the nine showed evidence of resolution of their rejection, although some patients experienced re-rejection. CONCLUSIONS These findings suggest that CD3 antibodies can be engineered to lose their toxicity while retaining their potency as immunosuppressants. Nonactivating humanized CD3 monoclonal antibodies now merit further investigation in the management of transplant patients and in therapy of autoimmune diseases.

TNFR1- and TNFR2-mediated signaling pathways in human kidney are cell type-specific and differentially contribute to renal injury

In normal kidney, TNFR1 is expressed in glomerular and peritubular capillary EC, and some tubular cells, and colocalizes with inactive apoptosis signal‐regulating kinase‐1 (ASK1) phosphorylated at serine 967. Biopsies of rejecting or ischemic renal allografts, which show both tubular cell injury and proliferation, display down‐regulation of TNFR1 and activation of ASK1 as well as up‐regulation of TNFR2 on tubular cells, where it colocalizes with phosphorylated endothelial/epithelial tyrosine kinase (Etk). We have exploited receptor‐selective muteins and evaluated phosphorylation of receptor‐specific kinases to study TNF responses in situ. In organ culture, a TNFR1‐specific mutein changes phosphorylation of ASK1 to threonine 845, indicative of kinase activation. A TNFR2‐specific mutein down‐regulates TNFR1 in glomerular EC, up‐regulates TNFR2 and Etk in tubular cells, and induces phosphorylation of Etk. Wild‐type TNF induces TNFR2 and Etk and activates both ASK1 and Etk but does not down‐regulate TNFR1. Wild‐type TNF and TNFR1‐specific mutein trigger tubular cell apoptosis whereas wild‐type TNF and TNFR2‐specific mutein induce tubular cells to express proliferating cell nuclear antigen. Differential activation of ASK1 and Etk by regulated TNFRs in patient‐derived materials provides an explanation for diverse and opposing responses to TNF at distinct sites, and an in situ bioassay of TNFR signaling Al‐Lamki, R. S., Wang, J., Vandenabeele, P., Bradley, J. A., Thiru, S., Luo, D., Min, W., Pober, J. S., Bradley, J. R. TNFR1‐ and TNFR2‐mediated signaling pathways in human kidney are cell type‐specific and differentially contribute to renal injury. FASEB J. 19, 1637–1645 (2005)

Expression of tumor necrosis factor receptors in normal kidney and rejecting renal transplants.

Activation of the TNF signal transduction cascade is initiated by the interaction of TNF with either of two cell surface receptors, TNFR-1 and TNFR-2. The levels and regulation of expression of these two receptors has been extensively analyzed in cultured cells, but little is known of TNFR expression in situ. We analyzed the expression of TNFR-1 and -2 in normal human renal kidney and in renal transplants undergoing acute cellular rejection. Immunohistochemistry and immunogold electron microscopy indicated a strong expression of TNFR-1 on the endothelium of glomeruli of normal kidney. Immunogold colocalization for TNFR-1 and a marker of the trans-Golgi network (TGN-46) demonstrated TNFR-1 within the Golgi complex in endothelial cells in normal kidney, confirming our previous studies with cultured cells. TNFR-1 expression was lost in glomeruli from acutely rejecting kidney, but TNFR-1 was detected in abundance on infiltrating leukocytes in the interstitium of allografts with acute rejection. In contrast, TNFR-2 was demonstrated predominantly in epithelial cells of distal convoluted tubule (DCT) in acute rejection kidney near TNF-expressing leukocytes. TNF was absent in normal kidney, but present in rejecting allograft. TNF was found in infiltrating leukocytes and in adjacent tubular epithelial cells. In situ hybridization showed TNFR-1 mRNA within the endothelium of the glomeruli and of a few arterioles in normal kidney, whereas TNFR-2 mRNA was seen in tubular epithelial cells of the DCT in acute transplant rejection. These data reveal that there is both differential expression and regulation of the two TNF receptors in human kidney.

Maintenance triple immunosuppression with cyclosporin A, mycophenolate sodium and steroids allows prolonged survival of primate recipients of hDAF porcine renal xenografts

Abstract:  To date, the best results in life‐supporting pig‐to‐primate renal xenotransplantation have been obtained in recipients exposed to long‐term immunosuppression with cyclophosphamide. As this agent is frequently associated with side‐effects, we have explored the potential of a mycophenolate sodium‐based maintenance immunosuppression in this model. Human decay‐accelerating factor (hDAF) transgenic kidneys were transplanted into splenectomized and bilaterally nephrectomized cynomolgus monkeys immunosuppressed with mycophenolate sodium, cyclosporin A and steroids, and exposed to a brief induction course with cyclophosphamide (up to four doses). After transplantation, the primates were monitored daily for biochemical and haematological evaluations and for the measurements of haemolytic anti‐pig antibodies (APA). A detailed histological analysis of each explanted graft was also performed. All the animals showed very poor initial graft function but survived for up to 51 days. In contrast to our previous studies in xenograft recipients on long‐term immunosuppression with cyclophosphamide, minimal or no circulating xeno‐directed antibodies, as measured by the evaluation of APA titres, were detected in this series although some degree of acute humoral rejection was observed in all the explanted grafts and was the primary cause of graft failure. Furthermore, in addition to areas of humorally mediated graft damage, we have observed for the first time areas with exclusive and prominent infiltration by CD2+ and CD8+ mononuclear cells presenting patterns compatible with tubulitis, glomerulitis and arteritis, which we have called acute cellular xenograft rejection (ACXR). In addition, CD68+ infiltrating macrophages and CD20+ B‐cells were also present. This study demonstrates that a triple maintenance immunosuppression with mycophenolate sodium, cyclosporin A and steroids is a viable alternative to a cyclophosphamide‐based immunosuppression to obtain prolonged survival of porcine organs transplanted into primates. However, a more stringent control of antibody forming cells remains essential to further extend the survival of xenografts in this model. In addition, the use of the immunosuppressive regimen reported here in the primate is associated with the occurrence of a new category of cell‐mediated xenograft injury (ACXR) whose significance has yet to be clarified.

TL1A Both Promotes and Protects from Renal Inflammation and Injury

Death receptor 3 (DR3), a member of the TNF receptor (TNFR) superfamily, is induced in human renal tubular epithelial cells (TEC) in response to injury. This study examined the expression and actions of TL1A, the principal ligand for DR3. In histologically normal tissue from biopsy or nephrectomy specimens of renal allografts, TL1A mRNA and protein were expressed in vascular endothelial cells but not in TEC. In specimens of acute or antibody-mediated allograft rejection, vascular endothelial cells and infiltrating leukocytes expressed increased TL1A mRNA and protein, but TEC expressed TL1A protein without mRNA, consistent with uptake of exogenous ligand. Addition of TL1A to organ cultures of human or mouse kidney caused activation of NF-kappaB, expression of TNFR2, activation of caspase-3, and apoptosis in TEC. Inhibition of NF-kappaB activation increased TL1A-mediated caspase-3 activation and apoptosis of TEC, but it did not reduce the induction of TNFR2. In organ culture of DR3-deficient mouse kidneys, addition of TL1A induced TNFR2 but did not activate NF-kappaB and did not increase apoptosis of TEC. These data suggest that TL1A may contribute to renal inflammation and injury through DR3-mediated activation of NF-kappaB and caspase-3, respectively, but that an unidentified receptor may mediate the NF-kappaB-independent induction of TNFR2 in TEC.

Interleukin-4 Gene Expression in Mercury-Induced Autoimmunity

Mercuric chloride (HgCl2) induces autoitnmunity in Brown Norway (BN) rats, with necrotizing vasculitis in the gut. Circumstantial evidence implicates the Th2 subset of CD4+ T lymphocytes, which produces IL‐4. We developed a quantitative polymerase chain reaction (PCR) technique to quantify IL‐4 gene expression. A phagemid containing rat IL‐4 cDNA was modified to act as the template for a synthetic RNA construct; a known amount of synthetic RNA was added to total RNA from spleen and caecum of BN rats at various times after HgCl2, followed by reverse transcriptase PCR. IL‐4 gene expression increased markedly in spleen and caecum after HgCl2. Splenic levels peaked by 10 days at approximately five‐times baseline, then returned towards normal as the autoimmune response was spontaneously regulated. Caecal IL‐4 expression peaked at 48 h, at which time we observed a previously unreported early phase of tissue injury, with necrotizing vasculitis qualitatively similar to that reported previously in the later phases of the model. These data support a key role for IL‐4 in this experimental model of autoimmunity. The quantitative PCR technique can be modified for analysis of other cytokines, allowing further investigation of the role of T ceil subsets in this model.