Proteomics for rejection diagnosis in renal transplant patients: where are we now?

Rejection is one of the key factors that determine the long-term allograft function and survival in renal transplant patients. Reliable and timely diagnosis is important to treat rejection as early as possible. Allograft biopsies are not suitable for continuous monitoring of rejection. Thus, there is an unmet need for non-invasive methods to diagnose acute and chronic rejection. Proteomics in urine and blood samples has been explored for this purpose in 29 studies conducted since 2003. This review describes the different proteomic approaches and summarizes the results from the studies that examined proteomics for the rejection diagnoses. The potential limitations and open questions in establishing proteomic markers for rejection are discussed, including ongoing trials and future challenges to this topic

Assay for free secretory component and methods for monitoring organ rejection

Methods of monitoring and detecting the early onset of organ injury incident rejection of a organ rejection in an animal are disclosed. The described methods are capable of distinguishing organ rejection injury from other organ tissue damage in the animal. Free secretory component levels in an animal biological fluid (e.g., bile, urine, blood, amniotic fluid) may be used to identify organ rejection in an animal. Multiple and single organ transplant patients may be monitored and diagnosed according to the claimed methods. Biological fluids, such as blood, (serum) or urine, are analyzed immunologically using a particularly adapted ELISA which are then compared to an FSC control concentration to identify elevated FSC values. Animals with test FSC above FSC control concentrations are diagnosed as having an ongoing organ rejection episode. The detection of congenital renal dysfunction in utero is also provided according to the present invention through the measurement of FSC in the amniotic fluid. The described methods are specific for indicating organ rejection tissue injury, and distinguish kidney rejection tissue injury, in particular, from other causes of kidney injury, such as cyclosporin toxicity, urinary tract infection, and urinary obstruction and toxicity (incident to immunosuppressive therapy with cyclosporin). A kit for use in the identification of an organ rejection episode in a patient through measurement of FSC in a biological sample is also provided.Board of Regents, University of Texas Syste

Experimental rat models of chronic allograft nephropathy: a review

Chronic allograft nephropathy (CAN) is the leading cause of late allograft loss after renal transplantation (RT), which continues to remain an unresolved problem. A rat model of CAN was first described in 1969 by White et al. Although the rat model of RT can be technically challenging, it is attractive because the pathogenesis of CAN is similar to that following human RT and the pathological features of CAN develop within months as compared with years in human RT. The rat model of RT is considered as a useful investigational tool in the field of experimental transplantation research. We have reviewed the literature on studies of rat RT reporting the donor and recipient strain combinations that have investigated resultant survival and histological outcomes. Several different combinations of inbred and outbred rat combinations have been reported to investigate the multiple aspects of transplantation, including acute rejection, cellular and humoral rejection mechanisms and their treatments, CAN, and potential targets for its prevention

Biomarkers in solid organ transplantation: establishing personalized transplantation medicine.

Technological advances in molecular and in silico research have enabled significant progress towards personalized transplantation medicine. It is now possible to conduct comprehensive biomarker development studies of transplant organ pathologies, correlating genomic, transcriptomic and proteomic information from donor and recipient with clinical and histological phenotypes. Translation of these advances to the clinical setting will allow assessment of an individual patient's risk of allograft damage or accommodation. Transplantation biomarkers are needed for active monitoring of immunosuppression, to reduce patient morbidity, and to improve long-term allograft function and life expectancy. Here, we highlight recent pre- and post-transplantation biomarkers of acute and chronic allograft damage or adaptation, focusing on peripheral blood-based methodologies for non-invasive application. We then critically discuss current findings with respect to their future application in routine clinical transplantation medicine. Complement-system-associated SNPs present potential biomarkers that may be used to indicate the baseline risk for allograft damage prior to transplantation. The detection of antibodies against novel, non-HLA, MICA antigens, and the expression of cytokine genes and proteins and cytotoxicity-related genes have been correlated with allograft damage and are potential post-transplantation biomarkers indicating allograft damage at the molecular level, although these do not have clinical relevance yet. Several multi-gene expression-based biomarker panels have been identified that accurately predicted graft accommodation in liver transplant recipients and may be developed into a predictive biomarker assay

Non-invasive monitoring of renal transplant recipients: Urinary excretion of soluble adhesion molecules and of the complement-split product C4d

Background: The number of inducible adhesion molecules known to be involved in cell-mediated allograft rejection is still increasing. In addition, recent data describe complement activation during acute humoral allograft rejection. The aim of this study was to assess whether specific molecules from either pathway are excreted into urine and whether they can provide useful diagnostic tools for the monitoring of renal transplant recipients. Methods: Urinary concentrations of soluble adhesion molecules (sICAM-1, sVCAM-1) and of the complement degradation product C4d were determined by standardized ELISA technique in 75 recipients of renal allografts and 29 healthy controls. Patient samples were assigned to four categories according to clinical criteria: group 1: acute steroid-sensitive rejection (ASSR, n=14), group 2: acute steroid-resistant rejection (ASRR, n=12), group 3: chronic allograft dysfunction (CAD, n=20) and group 4: stable graft function (SGF, n=29). Results: All patients with rejection episodes (groups 1-3) had significantly higher values of urinary sC4d compared with healthy controls and patients with stable graft function (p<0.05). The urinary levels of sVCAM-1 were significantly higher in group 2 (ASRR) compared with all other groups (p<0.001). Uniformly low amounts of s-VCAM-1 and complement-split product C4d were excreted by healthy controls (group 0). In contrast, urinary sICAM-1 concentration in healthy controls was almost as high as in group 2 (ASRR) whereas patients with a stable functioning graft (group 4) excreted significantly less sICAM-1 (p<0.05). Conclusion: The evaluation of sVCAM-1 and sC4d excretion in urine can provide a valuable tool with regard to the severity and type of allograft rejection. With respect to long-term allograft survival, serial measurements of these markers should have the potential to detect rejection episodes and prompt immediate treatment. Copyright (C) 2003 S. Karger AG, Basel

The histopathological changes associated with allograft rejection and drug toxicity in renal transplant recipients maintained on FK506: Clinical significance and comparison with cyclosporine

The histopathological changes in 51 renal allograft biopsies from patients immunosuppressed with FK506 were compared with those seen in 30 needle biopsies obtained from patients on cyclosporine. The frequency and severity of rejection episodes were similar in both groups. Tubular vacuolation and myocyte vacuolation were found to be useful morphological markers to monitor short-term drug toxicity associated with both drugs. Long-term administration of FK506 led to striped interstitial fibrosis and arteriolar hyalinosis, similar to that previously documented for cyclosporine. One case each of hemolytic uremic syndrome and necrotizing arteriopathy was noted in patients receiving FK506. FK506 and cyclosporine are structurally unrelated compounds; hence the parallelism observed in their nephrotoxicity profile suggests that the interactions of these drugs with renal tissue involves the operation of two different initial signal-transducing mechanisms, ultimately activating the same final metabolic pathways

Cellular Requirements for Renal Allograft Rejection

Graft rejection remains a major problem in clinical renal transplantation despite recent improvements in immunosuppressive therapy. While it is accepted that T lymphocytes play an essential role in acute rejection, the relative contributions of the different effector pathways have not been established. The aim of this thesis was to examine the cellular effector mechanisms of renal allograft rejection in the rat. In initial experiments, the characteristic features of unmodified rejection were observed in a serial immunohistological study of rejecting DA allografts transplanted into PVG recipients. The progressive mononuclear cell infiltration of the grafts initially comprised predominantly CD4 lymphocytes and subsequently CD8 cells. In addition to T cells, many of the infiltrating cells were of a phenotype consistent with NK cells and macrophages. This was associated with a striking increase in the expression, within the graft, of donor MHC class I and II antigens, together with the early disappearance from the graft of class II positive donor interstitial cells. Mononuclear cells harvested from the grafts and spleens of these animals displayed moderate levels of in vitro allospecific cytotoxicity against donor strain ConA blasts as well as high levels of nonspecific cytotoxicity against the NK susceptible Y3 target. Following on from these basic observations, subsequent experiments investigated the ability of CD4 or CD8 T lymphocyte subpopulations (prepared by negative selection) to mediate allogeneic kidney damage in different experimental models. One approach was to examine the ability of adoptively transferred lymphocyte subpopulations to cause renal allograft rejection in rats rendered lymphocyte deficient by a sublethal dose of whole body gamma irradiation. Acutely irradiated (8.5 Gy) Lewis recipients were unable to reject DA renal allografts unless reconstituted with syngeneic lymph node cells (LNC). Whereas transfer of 5x10e6 naive Lewis T lymphocytes rapidly restored graft rejection, similar numbers of either CD4 or CDS lymphocytes were relatively ineffective. Immunohistological examination of day 7 allografts in reconstituted recipients revealed, in all groups, a moderate leukocytic infiltrate of similar phenotypic composition, irrespective of the phenotype of the reconstituting cells, and with broad similarity to the infiltrate observed in unmodified rejection. When harvested infiltrating cells and splenocytes were tested in functional assays, only effector cells from CD4+CD8 T cell reconstituted animals, and not those from animals receiving either separated subpopulation, demonstrated allospecific cytotoxicity. Interestingly, splenocytes from all animals (including unreconstituted rats) showed nonspecific cytotoxic activity against Y3, but graft infiltrating cells from the same groups showed none. Collectively, these experiments suggested that both T cell subpopulations were necessary for optimal graft rejection, and that in this particular strain and model, graft rejection correlated with specific cytotoxic I cell lysis rather than nonspecific cytotoxic activity. The second approach examined the ability of T cell subpopulations to mediate allogeneic tissue damage in the renal graft versus host reaction (GVHR). Renal subcapsular injection of parental CD4 lymphocytes in F1 recipients was sufficient to produce, by day 7, a grossly observable renal GVHR, while CD8 lymphocytes (even if specifically sensitised) were ineffective. CD4 lymphocytes required the essential participation of radiosensitive F1 host, bone marrow derived cells to cause tissue damage. This occurred in the absence of demonstrable specific T cell lysis and appeared to be a DTH reaction. Experiments with PVG recombinant rats showed that an isolated MHC class II, but not a class I incompatibility was sufficient to provoke a response. The final group of experiments examined the ability of adoptively transferred lymphocyte subpopulations to restore renal allograft rejection in the congenitally athymic PVG-rnu/rnu rat. CD4 lymphocytes alone were able to restore the first-set rejection response, while CD8 cells alone (naive or specifically sensitised) were ineffective, although the addition of CD8 cells to the inoculum had a synergistic effect on the ability of CD4 cells to restore rejection. Immunohistological studies revealed moderate cellular infiltration of non-rejecting grafts and increased cellular infiltration of the rejecting grafts, with a significant increase in the number of MRC 0X8 positive cells. An interesting finding was the presence, in unmodified PVG-rnu/rnu rats, of extrathymically derived cells with wide alloreactivity as detected by in vitro cytotoxicity assays against a range of allogeneic ConA blasts and NK susceptible targets. Treatment of the effector cells with anti-asialo GM1, and cold target inhibition assays together suggested the presence of populations of atypical, widely reactive NK cells, with the additional ability to preferentially recognise a specific target. These cells were also present in the rejecting grafts of CD4 reconstituted recipients. Overall therefore, the transfer of CD8 lymphocytes alone was insufficient to cause tissue damage in allogeneic kidneys in any of the experimental models studied. In contrast, CD4 lymphocytes alone were able to cause extensive parenchymal damage in the renal GVHR, and were sufficient to initiate allograft rejection in athymic recipients, but required the additional presence of CD8 cells to restore rejection in acutely irradiated animals

Tissue typing by unidirectional mixed lymphocyte culture. 3. The relationship of in vitro lymphocyte compatibility to renal allograft rejection

We applied unidirectional MLC test to renal allograft in dogs, and investigated the correlation between the growth rates of MLC reaction and the intensity of rejection of the kidney transplants or the postoperative renal function. It was concluded that the grade of rejection became three plus (+ + +) when the rate of blastformation was more than 18 %, while it became one plus when the rate was less than 15 %. The rate of blast. formation was closely correlated with the strength of rejection of kidney transplants. However, the postoperative renal function was not always correlated with the mixed lymphocyte reaction.</p