A proteomic analysis of allograft rejection in rats after liver transplantation

In order to understand the allograft rejection in orthotopic liver transplantation (OLT), an allograft re- jection rat model was established and studied by proteomic approach. The protein expression profiles of liver tissues were acquired by fluorescence two-dimensional difference gel electrophoresis (2D DIGE) that incorporated a pooled internal standard and reverse fluorescent labeling method. The expression levels of 27 protein spots showed significant changes in acute rejection rats. Among these spots, 19 were identified with peptide mass fingerprinting using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) after tryptic in-gel digestion. The results of the present paper could be helpful for our better understanding of allograft rejection in organ transplantation.     

A proteomic analysis of allograft rejection in rats after liver transplantation

In order to understand the allograft rejection in orthotopic liver transplantation (OLT), an allograft rejection rat model was established and studied by proteomic approach. The protein expression profiles of liver tissues were acquired by fluorescence two-dimensional difference gel electrophoresis (2D DIGE) that incorporated a pooled internal standard and reverse fluorescent labeling method. The expression levels of 27 protein spots showed significant changes in acute rejection rats. Among these spots, 19 were identified with peptide mass fingerprinting using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) after tryptic in-gel digestion. The results of the present paper could be helpful for our better understanding of allograft rejection in organ transplantation.     

Prediction of acute cellular renal allograft rejection by urinary metabolomics using MALDI-FTMS

The present study investigated small molecule analysis of urinary samples as a noninvasive method to detect acute cellular renal allograft rejection. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used to analyze 15 urinary samples from transplant patients with different grades of biopsy showing improved clinical acute cellular rejection (ACR) and 24 urinary samples from 8 transplant patients without evidence of rejection. Seven small molecules demonstrated highly successful diagnostic performance (m/z): 278.1 (t = 3.398, p = 0.004), 293.0 (t = 2.169, p = 0.048), 294.1 (t = 2.154, p = 0.05), 382.2 (t = 2.961, p = 0.010), 383.3 (t = 2.270, p = 0.040), 402.2 (t = 2.994, p = 0.010), 424.0 (t = 2.644, p = 0.019). Kidney transplant patients with ACR could be distinguished from those without ACR using four individual small molecules with a specificity of 100%. In conclusion, the combination of MALDI-FTMS technology with a clear definition of patient groups can detect urine small molecule associated with ACR.     

De novo expression of MHC class II molecules by microglial cells during acute rat renal allograft rejection

The expression of MHC class I and class II molecules in the cerebral cortex of rats was investigated at daily intervals from day 3 to day 6 after fully allogeneic (DA→LEW) and isogeneic (LEW→LEW) kidney transplantation. MHC class II molecules were temporarily induced on the previously negative microglial cells and on the endothelia of arterioles and venules during acute rejection. On the endothelia of all brain vessels MHC class I expression was enhanced. MHC class I⁺ cells with microglial cell morphology were discernible within the diffusely MHC class I⁺ brain parenchyma. In contrast, the brain parenchyma of isograft recipients and untreated control animals did not express detectable levels of MHC molecules. In conclusion, we demonstrate that a strong immune reaction in the periphery is able to activate microglial cells in the central nervous system.     

Detection of renal allograft rejection by computer.

A computer program incorporating an adaptation of a statistical method, the multiprocess Kalman filter, was used to detect changes in trends of plasma creatinine and urea concentrations. In 28 recipients of renal allografts a definite deterioration in renal function was identified retrospectively on 32 occasions by an experienced renal physician independently of the statistical analysis. The computer identified 31 of these 32 episodes using creatinine and urea results, and 29 using creatinine alone. Dysfunction was identified by the computer significantly earlier (p less than 0.05) than by the clinician and a median of one day earlier (p less than 0.02) than treatment was actually initiated. The computer identified dysfunction on 11 out of 1259 days when the clinician did not suspect rejection. These 11 episodes may have had a pathological importance, though no clinical diagnosis was made. This computer method is useful for immediate analysis of incoming results and for timing events either prospectively or retrospectively.     

Differential proteomic analysis of renal cell carcinoma tissue interstitial fluid

Renal cell carcinoma (RCC), the most common type of kidney cancer, currently has no biomarker of clinical utility. The present study utilized a mass spectrometry-based proteomics workflow for identifying differentially abundant proteins in RCC by harvesting shed and secreted proteins from the tumor microenvironment through sampling tissue interstitial fluid (TIF) from radical nephrectomies. Matched tumor and adjacent normal kidney (ANK) tissues were collected from 10 patients diagnosed with clear cell RCC. One-hundred thirty-eight proteins were identified with statistically significant differential abundances derived by spectral counting in tumor TIF when compared to ANK TIF. Among those proteins with elevated abundance in tumor TIF, nicotinamide n-methyltransferase (NNMT) and enolase 2 (ENO2) were verified by Western blot and selected reaction monitoring (SRM). The presence of ENO2 and thrombospondin-1 (TSP1) were verified as present and at elevated abundance in RCC patient serum samples as compared to a pooled standard control by enzyme-linked immunosorbent assay (ELISA), recapitulating the relative abundance increase in RCC as compared with ANK TIF.     

HLA-DRw6 and renal allograft rejection.

HLA-DRw6-positive patients are "high responders" to certain renal allograft antigens. A study was therefore conducted of the outcome of 247 first renal allografts in 74 DRw6-positive and 173 DRw6-negative recipients. The effectiveness of matching for HLA-DR determinants in both groups was also analysed. The one-year graft survival in DRw6-positive patients was 59% as compared with 75% in DRw6-negative recipients (p = 0.012). A striking difference between the two groups was that HLA-DR matching significantly improved renal allograft survival only in the DRw6-positive patients. In those patients the one-year survival of HLA-DR-identical grafts was 95% as compared with only 38% for 2-DR mismatched grafts (p = 0.009). In DRw6-negative patients only a slight beneficial effect of HLA-DR matching was observed (83% versus 72% at one year for the 0-DR and 2-DR mismatched grafts, respectively) (p greater than 0.05). These findings are clear evidence that DRw6-positive patients (about a quarter of the patients on the waiting list of Eurotransplant) should be given HLA-DR-identical kidney transplants only.     

Characterization of in vivo-activated allospecific T lymphocytes propagated from human renal allograft biopsies undergoing rejection

To evaluate in situ lymphocyte responses in cell-mediated immune tissue injury, we have developed an approach for propagation of human allospecific T lymphocytes directly from tissue biopsies. We have utilized renal allograft tissue obtained from eight patients undergoing cellular rejection. Needle biopsy tissue was cultured in medium containing interleukin 2 (IL 2), including recombinant-DNA-produced IL 2. In each case, lymphoblasts migrated out of the tissue and increased in numbers, especially adjacent to the tissue. In two cases in which there was no cellular infiltrate present in the biopsy, no lymphocytes proliferated in vitro. Instead, fibroblasts eventually filled the wells from these allograft biopsies. The continued presence of the allograft tissue enhanced the viability and growth of the lymphoblasts in cultures from rejecting allografts. The isolated lymphoblasts had surface markers of mature OKT3+ lymphocytes of either OKT4+ or OKT8+ subsets. OKT8+ cells predominated. There was variability (41 to 97%) in the percentage of T lymphoblasts that bore surface HLA-DR antigens. In assays of lymphoblasts obtained from eight separate renal allografts, there was donor-specific cytotoxicity, and in all but two of the cases there was donor-induced proliferation. The specificity of the cytotoxic reaction was tested by using 51Cr-labeled, PHA-stimulated target cells prepared from a panel of HLA-typed donors. Proliferation was tested after 48 hr in the presence of mitomycin C-treated peripheral blood mononuclear cells as stimulator cells by using only 10(4) responder T lymphoblasts. Of particular note was that the cytotoxicity of the isolated lymphoblasts showed specificity against both "private" HLA class I alloantigens (of the allograft donor) as well as "public" cross-reacting epitopes. This method permits the propagation and functional characterization of in vivo-activated T lymphoblasts that are obtained from the actual sites of immune-mediated injury. Preliminary studies of other tissues with diverse inflammatory processes indicate the possible widespread applicability of obtaining in vivo-activated lymphocytes.     

Metabolic markers for differentiation between renal allograft rejection and immunosuppressant toxicity in rat urine

Introduction

Although current immunosuppressive protocols have dramatically improved 1-year survival of kidney transplants, there has been less progress in terms of long-term graft survival over the last two decades. The key to avoiding late graft loss is early diagnosis and differentiation between anti-allograft immune processes and immunosuppressant toxicity (IS-Tox). Modern bioanalytical technologies have opened new opportunities for the development of sensitive and specific diagnostic tools. There is an immediate need for biomarkers that are able to differentiate between renal allograft rejection and immunosuppressant toxicity.

Objective

To test our hypothesis that changes of metabolite patterns in urine have the potential to serve as a non-invasive combinatorial biomarker that can differentiate between allograft immune reactions and IS-Tox.

Methods

We used ¹H-NMR spectroscopy and Luminex multiplexing for metabolic profiling of rat urine and the analysis of protein biomarkers in urine and plasma, respectively, to compare the effects of chronic allograft rejection in a Fisher-to-Lewis rat transplant model with IS-Tox induced by cyclosporine, tacrolimus and/or sirolimus in Lewis rats.

Results

Our results showed that, while IS-Tox caused changes in metabolite patterns that are typically associated with proximal tubule damage, rejection caused more profuse changes not specifically focused on a particular kidney region. Moreover, metabolite pattern changes were more sensitive than changes in protein markers that were evident only during the later stages of rejection.

Conclusion

The present study provides first proof-of-concept that longitudinal monitoring of urine metabolite markers has the potential to differentiate between early renal allograft rejection and immunosuppressant nephrotoxicity.

     

Evaluation of biomarker discovery approaches to detect protein biomarkers of acute renal allograft rejection

Management of host responses to allografts by immunosuppressive therapy is the cornerstone of transplantation medicine, but it is still deficient in one important element: biomarkers that are readily accessible and predict the fate of the transplant early, specifically, and reliably. Using a Brown Norway (BN)-to-Lewis rat renal allograft model of kidney transplantation, this study aims at evaluating two proteomic approaches to discover biomarkers for acute rejection: SELDI-MS technology and 2D gel electrophoresis combined with mass spectrometry. Several novel potential serum biomarkers have been identified for follow up. Overall, the conclusion is that apparently at the serum protein level, dramatic changes only occur at a stage where kidney function is already severely affected. Multivariate analysis of serum profiles suggests that there is an ensemble of subtle changes, comprising a proteomic signature of acute rejection at an early stage, a more detailed evaluation of which might provide novel opportunities for the diagnosis of acute rejection. Profiling of the excreted proteins indicates that urine might even present the earliest signs of the rejection process.     

Cellular immunity in allograft rejection: role of lymphocyte subpopulations and T-cell subsets in rat cardiac allograft rejection

In this study, cellular requirements for rejection are examined by the use of adoptive transfer assays in the ACI to Lewis cardiac allograft model. The findings show that adoptive transfer of 1 x 10(8) spleen cells (SpL), 5 x 10(7) T-cells, and 2 x 10(7) helper T-cells (W3/25+) obtained from normal, nonsensitized donors restores acute ACI graft rejection in sublethally irradiated (750 rad) Lewis recipients. In contrast, reconstitution with 2 x 10(7) cytotoxic T-cells (0X8+) does not restore first-set graft rejection. Reconstitution of the irradiated recipients with either W3/25+ or 0X8+ T-cells obtained from specifically sensitized syngeneic donors resulted in acute rejection. The W3/25+ T-cell subset was significantly more potent (P less than 0.01) in effecting rejection on a per-cell basis. Adoptive transfer of SpL, T-cells, and 0X8+ T-cells obtained from sensitized rats led to accelerated cardiac allograft rejection in the naive secondary recipients while W3/25+ T-cells did not. This study suggests that although the W3/25+ T-cells alone have the capacity to initiate first-set graft rejection, both W3/25+ and 0X8+ subsets appear to be critical to the completion of rejection of heart allografts. We also examined the capacity of adoptively transferred B-cells from sensitized donors to influence graft rejection. Our findings suggest that while B-cells fail to restore the capacity for graft rejection in irradiated recipients, they can, however, present MHC antigens to the secondary naive host thus causing allosensitization which results in accelerated rejection of a subsequent graft.     

Possible effect of time on renal allograft rejection.

The change in plasma creatinine concentrations from decreasing values after successful renal transplantation to increasing values after the onset of rejection occurs as a sudden event. Twenty-two such episodes in 16 renal allograft recipients were studied by extrapolating sequential measurements of plasma creatinine concentrations to see when the change occurred. Seventeen of the episodes occurred between 2300 and 1100 and the rest at other times. This difference was significant. The results suggest that rejection is more common at night and apparently has a circadian rhythm, being likely to first influence creatinine clearance at around 0600.     

Comparison of high-dose intravenous methylprednisolone with low-dose oral prednisolone in acute renal allograft rejection in children.

Two corticosteroid regimens were compared in a randomised, prospective study of 48 consecutive acute rejection episodes occurring at least one month after transplantation in 22 children who had received renal allografts. The higher dose schedule (intravenous methylprednisolone 600 mg/m2 daily for three days) was no more effective than the lower (oral prednisolone 3 mg/kg daily for three days) in reversing rejection, being successful in 70% as opposed to 72% of episodes. Few major side effects were seen with either treatment, but unpleasant sensations were reported much more frequently in the group given intravenous methylprednisolone; this regimen was much more disruptive of the patient''s life. Oral prednisolone in the dosage described is as effective as about 10 times that dose of intravenous methylprednisolone; it is much cheaper and is viewed as less unpleasant by patients.     

Immunological monitoring of renal transplant recipients to predict acute allograft rejection following the discontinuation of tacrolimus

Background

Transplant patients would benefit from reduction of immunosuppression providing that graft rejection is prevented. We have evaluated a number of immunological markers in blood of patients in whom tacrolimus was withdrawn after renal transplantation. The alloreactive precursor frequency of CD4+ and CD8+ T cells, the frequency of T cell subsets and the functional capacity of CD4+CD25+FoxP3+ regulatory T cells (Treg) were analyzed before transplantation and before tacrolimus reduction. In a case-control design, the results were compared between patients with (n = 15) and without (n = 28) acute rejection after tacrolimus withdrawal.

Principal Findings

Prior to tacrolimus reduction, the ratio between memory CD8+ T cells and Treg was higher in rejectors compared to non-rejectors. Rejectors also had a higher ratio between memory CD4+ T cells and Treg, and ratios <20 were only observed in non-rejectors. Between the time of transplantation and the start of tacrolimus withdrawal, an increase in naive T cell frequencies and a reciprocal decrease of effector T cell percentages was observed in rejectors. The proportion of Treg within the CD4+ T cells decreased after transplantation, but anti-donor regulatory capacity of Treg remained unaltered in rejectors and non-rejectors.

Conclusions

Immunological monitoring revealed an association between acute rejection following the withdrawal of tacrolimus and 1) the ratio of memory T cells and Treg prior to the start of tacrolimus reduction, and 2) changes in the distribution of naive, effector and memory T cells over time. Combination of these two biomarkers allowed highly specific identification of patients in whom immunosuppression could be safely reduced.     

Deficiency of 5-lipoxygenase accelerates renal allograft rejection in mice.

Acute renal allograft rejection is associated with alterations in renal arachidonic acid metabolism, including enhanced synthesis of leukotrienes (LTs). LTs, the products of the 5-lipoxygenase (5-LO) pathway, are potent lipid mediators with a broad range of biologic activities. Previous studies, using pharmacological agents to inhibit LT synthesis or activity, have implicated these eicosanoids in transplant rejection. To further investigate the role of LTs in acute graft rejection, we transplanted kidneys from CByD2F1 mice into fully allogeneic 129 mice that carry a targeted mutation in the 5lo gene. Unexpectedly, allograft rejection was significantly accelerated in 5-LO-deficient mice compared with wild-type animals. Despite the marked reduction in graft survival, the 5lo mutation had no effect on the hemodynamics or morphology of the allografts. Although LTB4 levels were reduced, renal thromboxane B2 production and cytokine expression were not altered in 5-LO-deficient allograft recipients. These findings suggest that, along with their proinflammatory actions, metabolites of 5-LO can act to enhance allograft survival.     

Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates.

CD154 is the ligand for the receptor CD40. This ligand-receptor pair mediates endothelial and antigen-presenting cell activation, and facilitates the interaction of these cells with T cells and platelets. We demonstrate here that administration of a CD154-specific monoclonal antibody (hu5C8) allows for renal allotransplantation in outbred, MHC-mismatched rhesus monkeys without acute rejection. The effect persisted for more than 10 months after therapy termination, and no additional drug was required to achieve extended graft survival. Indeed, the use of tacrolimus or chronic steroids seemed to antagonize the anti-rejection effect. Monkeys treated with antibody against CD154 remained healthy during and after therapy. The mechanism of action does not require global depletion of T or B cells. Long-term survivors lost their mixed lymphocyte reactivity in a donor-specific manner, but still formed donor-specific antibody and generated T cells that infiltrated the grafted organ without any obvious effect on graft function. Thus, therapy with antibody against CD154 is a promising agent for clinical use in human allotransplantation.