Human Renal Allograft Recipients Research Articles

Three‐year follow‐up of human transplanted kidneys by diffusion‐weighted MRI and blood oxygenation level‐dependent imaging

To prospectively determine the 3-year stability and potential changes of functional parameters in renal allograft recipients obtained from diffusion-weighted imaging (DWI) and blood oxygenation level-dependent (BOLD) MRI. Nine renal allograft recipients underwent DWI and BOLD-MRI twice, once 7 ± 3 months after transplantation, and again 32 ± 2 months after the first MRI. DWI yielded an apparent diffusion coefficient (ADC) and the perfusion contribution (F(P) ). BOLD imaging yielded R2, providing an estimation of renal oxygenation. Coefficients of variation between (CV(b) ) and within subjects (CV(w) ) were calculated. The parameters were stable after 32 months in eight of the nine patients, who had well-functioning allografts. Mean diffusion values were very similar in the first and second scan. CV(w) and CV(b) for ADC values were less than 3.5% and 5.9%, respectively, in cortex and medulla, but were higher for F(P) (15%-18%). CV(w) and CV(b) of R2 were also low (medulla: CV(w) = 10.8%, CV(b) = 11.4%; cortex: CV(w) and CV(b) = 7.2%). R2 increased significantly (P = 0.035) in cortex but not in medulla, suggesting reduced cortical oxygen content. One subject with decreased glomerular filtration rate demonstrated strongly altered parameters. In the absence of graft dysfunction, DWI and BOLD imaging yield consistent results over 3 years in stable human renal allograft recipients.

An Experimental Model of Acute Humoral Rejection of Renal Allografts Associated with Concomitant Cellular Rejection

Acute humoral rejection (AHR), which occurs in up to 8% of kidney transplant recipients, is a significant cause of renal allograft dysfunction and loss. More efficacious treatment modalities are needed to eliminate or curtail alloantibody production and its deleterious effects on the kidney. The availability of animal models mimicking human AHR is essential to understand its pathophysiology and develop new treatment strategies. Using a mouse kidney transplant model, we demonstrate that presensitization of recipients with donor skin grafts results in rejection of subsequent renal allografts. All presensitized mice developed renal failure 8.6 +/- 4.3 days after engraftment, with serum creatinine values near 100 micromol/dl. Graft histology revealed mild, diffuse, interstitial, mononuclear cell infiltrates; prominent peritubular capillary inflammatory cell margination; patchy interstitial hemorrhage; interstitial edema; and focal glomerular fibrin deposition. Complement (C3d) deposition was diffuse and prominent in peritubular capillaries. Serum analysis demonstrated high levels of circulating alloantibodies with broad cross-reactivity to many MHC haplotypes. The clinical setting and histological findings of our model strongly resemble AHR, which is frequently associated with cellular rejection, a situation commonly encountered in human renal allograft recipients. This animal model provides a valuable tool to study the pathogenesis of AHR, its relationship to cellular alloimmunity, its contribution to graft injury, and the effects of various potential therapeutic interventions.

Pharmacodynamic effects of everolimus on anti-CD3 antibody-stimulated T-lymphocyte proliferation and interleukin-10 synthesis in stable kidney-transplant patients

Everolimus (rapamycin derivative, RAD) is a new immunosuppressive drug that prevents allograft rejection. Herein, the pharmacodynamics of everolimus in human renal-allograft recipients is evaluated. Single doses of everolimus (0.75–10 mg), combined with a maintenance immunosuppressive therapy based on CyA, decreased lymphocyte proliferation. In addition, the effect of multiple doses of everolimus (0.75–10 mg) given daily for 21 days, to stable renal-allograft patients ( n = 11), was investigated. Everolimus treatment resulted in immediate inhibition (25–55%) of lymphocyte proliferation in renal-allograft recipients; values returning to baseline by 14 days after cessation of everolimus treatment. Placebo-treated patients showed no decrease in lymphocyte proliferation. Interestingly, everolimus reduced IL-10 synthesis by 20–60% in renal-allograft recipients. Phagocytosis rates were not changed by everolimus. In vitro, everolimus inhibited lymphocyte proliferation and IL-10 synthesis dose dependently in anti-CD3 mAb and LPS stimulated peripheral blood mononuclear cell cultures derived from human volunteers.

Following Anti-CD25 Treatment, A Functional CD4+CD25+ Regulatory T-Cell Pool Is Present in Renal Transplant Recipients

Daclizumab, a humanized antibody directed against the alpha-chain of the interleukin-2 receptor (CD25), has shown efficacy in the prevention of acute rejection following organ transplantation. However, anti-CD25 therapy can be expected to affect not only alloreactive effector T cells, but also CD4(+)CD25(+) regulatory T (Treg) cells that are shown to play an important role in the induction of transplantation tolerance. Therefore, the size and function of the Treg pool in human renal allograft recipients after single-dose daclizumab administration was investigated in this study. Approximately 8 weeks after administration, daclizumab was cleared from the circulation and the Treg population then present appeared not different from that observed before transplantation. Functional analysis revealed that the Treg possessed a normal capacity to suppress mixed lymphocyte reactions in vitro. These data indicate that after daclizumab therapy a Treg population, normal in number and function, is present in the peripheral blood of renal transplant recipients.

Results from a Human Renal Allograft Tolerance Trial Evaluating T-Cell Depletion with Alemtuzumab Combined with Deoxyspergualin

Perioperative lymphocyte depletion induces allograft tolerance in some animal models, but in humans has only been shown to reduce immunosuppressive requirements. Without maintenance immunosuppression, depleted human renal allograft recipients experience rejection characterized by infiltration of the allograft with monocytes and macrophages. T-cell depletion combined with a brief course of deoxyspergualin (DSG), a drug with inhibitory effects on monocytes and macrophages, induces tolerance in nonhuman primates. We therefore performed a trial to determine if lymphocyte depletion with alemtuzumab combined with DSG would induce tolerance in humans. Five recipients of live donor kidneys were treated perioperatively with alemtuzumab and DSG and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically, by flow cytometry, and by protocol biopsies analyzed immunohistochemically and with real-time polymerase chain reaction. Results were compared to previously studied patients receiving alemtuzumab alone or standard immunosuppression. Despite profound T-cell depletion and therapeutic DSG dosing, all alemtuzumab/DSG patients developed reversible rejection that was similar in timing, histology, and transcriptional profile to that seen in patients treated with alemtuzumab alone. Chemokine expression was marked prior to and during rejections. We conclude that treatment with alemtuzumab and DSG does not induce tolerance in humans. Chemokine production may not be adequately suppressed using this approach.

CC-Chemokine RANTES Is Increased in Serum and Urine in the Early Post-Transplantation Period of Human Renal Allograft Recipients

Background: The chemokine RANTES is a potent chemoattractant for T cells and monocytes that has been shown to enhance inflammation. The aim of our study was to investigate whether RANTES is upregulated within the early post-transplantation period that may influence short-time allograft function rate. Methods: Serum and urine samples from transplanted renal allograft recipients (n = 17) were obtained from specimens taken for diagnostic reasons. Four patients developed biopsy-proven rejection episodes within the first month. Time course of RANTES was studied within the first 12 days after renal transplantation using ELISA technique. Data were tested for significances between patients with rejection and without rejection, compared to healthy volunteers as controls, and correlated with clinical data. Results: In the control group RANTES concentration was 37.2 ± 2.7 ng/ml (serum) and 8.1 ± 1.3 pg/ml (urine), respectively. In transplanted recipients serum RANTES was significantly upregulated up to 132 ± 28 ng/ml on day 1 after transplantation and remained elevated within the first 12 days (n = 17). Time course of urine RANTES demonstrated elevated concentrations with 754 ± 115 pg/ml on day 1 followed by an continuous decrease to 22.3 ± 7 pg/ml on day 12 (n = 17). No significant differences could be detected between patients with rejection and without rejection episodes. Conclusions: In contrast to data of other urinary marker molecules (like IL-6), there are no significant differences between the rejection and non-rejection group. RANTES is therefore not suitable for early detection of rejection. Nevertheless, serum and urine RANTES concentrations were highly elevated in freshly transplanted renal allograft recipients reflecting an activated immune system.

Alloreactivity in renal transplant recipients with and without chronic allograft nephropathy.

The pathogenesis of chronic allograft nephropathy (CAN) involves both immunologic (antigen-dependent) and nonimmunologic (antigen-independent) mechanisms. In order to provide further insight into the immunologic basis of this disease, a cross-sectional analysis of cellular and humoral immunity in human renal allograft recipients with or without deteriorating renal function and biopsy proven CAN was performed. Interferon-gamma enzyme-linked immunosorbent spot assays were used to assess cellular immunity to donor, or fully mismatched third-party stimulator cells (direct pathway), and to synthetic peptides derived from donor HLA molecules (indirect pathway). Anti-HLA antibodies were evaluated by flow cytometry using HLA-coated beads. Both the mean frequencies of donor-reactive peripheral blood lymphocytes and the number of individuals who responded to donor antigens per group were statistically higher in CAN patients versus control subjects (P < 0.02). Calculated ratios of donor/third-party enzyme-linked immunosorbent spot responses showed mean values of 2.61 +/- 3.0 in the CAN group, with ratios of 0.50 to 0.72 +/- 0.42 in control subjects (P < 0.001), confirming that direct, donor-specific cellular immunity predominated in patients with CAN. Fifty percent of CAN patients studied exhibited donor peptide reactivity compared with only 28.6% in control subjects. Finally, 33% of patients in the CAN group developed new posttransplantation anti-HLA antibodies compared with only 4% in the control group (P < 0.05). Overall, the results suggest that persistent cell-mediated and humoral alloimmunity contribute to the development of CAN and further demonstrate that anti-donor immunity in patients with CAN is heterogeneous. Immune monitoring to predict long-term outcome should include multiple measures of cellular and humoral immunity.

Long-term dietary l-arginine supplementation attenuates proteinuria and focal glomerulosclerosis in experimental chronic renal transplant failure

Glomerular endothelial nitric oxide synthase expression is decreased in humans during acute rejection and chronic renal transplant failure (CRTF). This may contribute to vascular damage through changes in the renal hemodynamics and enhanced endothelial adhesion of leukocytes and platelets. Dietary supplementation of l-arginine may increase endothelial NO production, thereby protecting the vascular wall and improving renal hemodynamics. We tested the hypothesis that long-term l-arginine supplementation attenuates the development of CRTF in an experimental model for renal transplantation. In the Fisher 344 to Lewis rat model for renal transplantation, renal function and histology of untreated rats was compared with rats receiving l-arginine in the drinking water (10 g/L), starting 2 days before transplantation. Every 4 weeks systolic blood pressure was measured and serum and urine were collected for measurement of nitrite and nitrate (NO x ), creatinine, and proteinuria. At 34 weeks the histological renal damage was assessed by scoring focal glomerulosclerosis and measurement of alpha-smooth muscle actin (α-SMA) expression. Urinary NO x was significantly increased in treated animals. Proteinuria was significantly lower in l-arginine-treated animals from week 24 onward ( p<0.05). Plasma creatinine and creatinine clearance did not differ between the groups. The focal and segmental glomerulosclerosis (FGS) score (max 400) at week 34 was also significantly lower in treated rats arbitrary U (20±21 vs 61±67 arbitrary U; p<0.05). The expression of α-SMA was lower in l-arginine-treated rats than in untreated rats (1.93±0.8% area surface vs 3.64±2.5% area surface). In conclusion, in this experimental model for CRTF, l-arginine administration significantly reduced FGS and proteinuria, without affecting renal function. Our data suggest that dietary l-arginine supplementation attenuates progression of CRTF and may therefore be an additional therapeutic option in human renal allograft recipients.

Regulatory functions of alloreactive Th2 clones in human renal transplant recipients

Chronic allograft rejection is the major clinical problem in organ transplantation. There is evidence that indirect T cell recognition of donor-specific HLA peptides may play an important role in the immunopathogenesis of chronic allograft rejection. We have recently shown that HLA allopeptide-specific T cell clones generated from renal transplant recipients with chronic allograft nephropathy are of the Th1 phenotype, while those from stable patients are Th2. There is evidence in experimental animal models of autoimmunity and transplantation that Th2 cells may function to regulate immune responses, but the biological relevance of these observations in humans has not been reported. The purpose of this study was to investigate the putative regulatory functions of alloreactive human Th2 clones. HLA-DR allopeptide-specific Th1 and Th2 cell clones were generated from peripheral blood lymphocytes of human renal allograft recipients with chronic allograft nephropathy (CAN) or with stable renal function (SRF), respectively. An in vitro co-culture system showed that the proliferative responses of Th1 clones from patients with CAN were significantly inhibited by the Th2 clones in response to the donor-derived HLA allopeptides. In addition, co-culture of the Th2 clones inhibited cytokine production (IFN-gamma) by the Th1 clones in response to the donor-specific peptides. The regulatory functions of Th2 clones were antigen-specific since they only occurred when both the Th1 and Th2 clones were reactive to the same HLA-DR allopeptide, and were mediated by IL-4 and IL-10. This is the first demonstration, to our knowledge, indicating that Th2 cells may function to regulate indirect Th1 alloimmune responses that are critical for the progression of CAN in humans.

Early Human Renal Allograft Fibrosis: Cellular Mediators

Background: The development of early renal interstitial fibrosis (IF) in renal allografts is likely to depend on multiple factors. We studied retrospectively renal biopsies from cadaveric human renal allografts, transplanted from 1996 to 1998, with the intention of detecting early fibrotic changes and determining the underlying cellular mediators. We studied 23 transplant patients whose 46 renal biopsies were analysed, including a donor biopsy taken routinely at implantation from each patient and 23 follow-up biopsies, taken as clinically indicated over a period of 3 months following transplantation. Methods: Histological evaluation of induction and progression of fibrosis relied on point count analysis of conventional (Masson’s trichrome/MT) and immunohistochemical staining for collagen III and IV, and alpha-smooth muscle actin (α-SMA) as a marker of myofibroblast differentiation. Mast cells (MC) were counted in sections stained with an anti-human mast cells tryptase monoclonal antibody. Activated macrophages as well as total, helper and cytotoxic T-lymphocytes were identified on frozen sections by direct immunofluorescence using mouse anti-CD71, CD3, CD4 and CD8 antibodies respectively. Eosinophils (E) were counted in hematoxylin and eosin (HE)-stained sections. Changes in interstitial fibrosis (IF) scores were evaluated and correlated with myofibroblasts, MC, E and lympho-monocytic cells. Results: We noted a significant increase in IF over a 3 months period following transplantation. There was also a significant increase in α-SMA⁺ cells, MC and E counts from implantation to follow-up biopsies. Similarly, there was a significant increase in interstitial infiltration by T-lymphocytes (modal category = 2 versus 0, p = 0.012) but not by macrophages. MC at implantation and follow-up were found to be predictive of IF (immunostainable collagen III) at follow-up (R² = 0.510, p = 0.023 and p = 0.030). Further, the predictive value for total T-lymphocyte infiltration at follow-up was also significant (R² = 0.617, p = 0.036). A strong correlation was found between α-SMA⁺ cells and MC counts at implantation (r = 0.7259, p < 0.001) and in follow-up biopsies (r = 0.5183, p < 0.01). However, there was no correlation between E counts and either α-SMA⁺ cells or MC either at implantation or in follow-up biopsies. Based on changes in interstitial immunostainable α-SMA, our patients were divided arbitrarily into 2 groups; group 1 (n = 12) with >100% increase in α-SMA and group 2 (n = 11) with <100% increase. Group 1 patients differed significantly from group 2 regarding the degree of MC infiltration at follow-up (t = 0.4519, p < 0.05) with the mean increase in MC count from implantation to follow-up biopsies being +5.1 cells/high power field (HPF) in group 1 and +0.8 cell/HPF in group 2 (p = 0.0237). MC counts in group 1 were associated with a higher modal category (greater than one) of cytotoxic: helper T-lymphocytes ratio compared to group 2 (2:1 versus 1:1 respectively). Conclusion: Multiple cells may contribute to early interstitial fibrosis in a subgroup of human renal allograft recipients with MC playing a major role.

Pharmacodynamic monitoring of lymphocyte proliferation and TGF-β1 expression at cyclosporine a (CyA) trough levels (C 0) and 2 hours after intake (C 2) of CyA in human renal allograft recipients

Pharmacodynamic monitoring of lymphocyte proliferation and TGF-β1 expression at cyclosporine a (CyA) trough levels (C 0) and 2 hours after intake (C 2) of CyA in human renal allograft recipients

FTV 720A mediates reduction of lymphocyte counts in human renal allograft recipients by an apoptosis-independent mechanism

FTV 720A mediates reduction of lymphocyte counts in human renal allograft recipients by an apoptosis-independent mechanism

CD3 ANTIBODY-INDUCED IL-10 IN RENAL ALLOGRAFT RECIPIENTS

The first administration of CD3 monoclonal antibodies, such as anti-human CD3 (OKT3), induces a massive release of several cytokines, including tumor necrosis factor alpha (TNF-alpha), interferon (IFN)-gamma, interleukin (IL)-2, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor. Cytokine levels in patient's sera were measured by specific ELISA. In vitro cultures were performed using OKT3-stimulated peripheral blood mononuclear cells and/or whole blood from patients and normal controls. Here we describe that OKT3 administration to human renal allograft recipients also leads to a significant release of IL-10. Contrasting with most OKT3-induced cytokines, such as TNF-alpha whose release is transient, IL-10 levels show a more progressive increase, they peak only by 4-8 hr after the first OKT3 injection and persist longer. Thus, significant IL-10 levels are still detectable at the time of the second and the third OKT3 injection. Administration of corticosteroids, 1 hr before the first OKT3 injection, significantly reduced both TNF-alpha and IL-10 release. Experiments were performed to evaluate the source(s) of IL-10 and its (their) influence on the initial T-cell activation. When stimulated in culture with soluble OKT3, the production of IL-10 was dependent on the cooperation between T lymphocytes and monocytes. It is important that, as assessed through the use of a specific neutralizing antibody, the endogenous IL-10 produced in the co-culture system exerted a negative feed-back on the release of the other pro-inflammatory CD3-induced cytokines, which was reproducible. These results are supportive of a major role of IL-10 in the down-modulation of the OKT3-triggered T-cell activation cascade.

THE TEMPORAL RELATIONSHIP BETWEEN THE CELLULAR AND HUMORAL IMMUNE RESPONSES IN THE PATHOGENESIS OF TRANSPLANT VASCULAR SCLEROSIS

249 Humoral responses against the allografts have long been contented to play a cardinal role in the initiation and perpetuation of morphological changes pathognomonic of transplant vascular sclerosis (TVS). Studies in human recipients of cadaveric renal allografts have suggested that the presence of anti-donor antibodies in the sera of transplant patients must be considered as a premonition for the eventual development of TVS. However, despite these observations, the temporal relationship between cell and antibody-mediated immune responses in the precise evolvement of this lesion is as yet ambiguous. Methods: To address this issue further, we transplanted aortic allografts across MHC disparate C57B1/10 (H2b) → C3H/HeJ (H2k) allogeneic barrier (n=24). Aorta transplanted across syngeneic animals (C3H → C3H) served as a control (n=24). Animals were not immunosuppressed. At d1, 3, 6, 10, 15, 20 and 30 post-transplantation (PTx), the grafts were harvested and examined for microscopic and immunohistochemical aberrations. To delineate the role of Th1 and Th2-specific cytokines in the genesis of this lesion, RT-PCR was performed using mRNA isolated from grafts harvested at variable time PTx. Additionally, using recipient's splenocytes and sera, α-donor cellular and humoral immunity was determined by MLR and microcytotoxicity assays respectively. Results: No morphological alterations were witnessed in syngeneic grafts harvested at various times PTx. On the contrary, mononuclear cell infiltration in the intima was first encountered in aortic allografts harvested at d6 PTx. It resolved completely by d10 post-implantation with evidence of residual patchy endothelial cell denudation and necrosis. By d20 PTx, patchy intimal thickening (which was largely due to proliferation of α-smA+ cells) became evident which progressively worsened such that, by d30 PTx, it acquired a diffuse concentric appearance characteristic of TVS. Contrary to humoral responses, α-donor proliferative responses were also heightened at d6 PTx, only to abate thereafter. Detectable titers of α-donor cytotoxic antibodies were first encountered at d20 PTx and were markedly increased (1/800) in sera obtained at d30 post-implantation. Interestingly, with the exception of IL-10, the mRNA levels of all other cytokines examined (IL-2, IL-4 and γ-IFN) were elevated in the allografts harvested at d10-15 (and beyond) PTx. Conclusions: Taken together these data suggest that antibody-mediated cytotoxicity is involved in the perpetuation but not the initiation of changes characteristic of TVS. The latter is possibly accomplished by cell-mediated immune reactivity. Additionally, the presence of markedly elevated levels of IL-10 mRNA alone at d3 and d6 PTx in allogeneic but not syngeneic grafts, suggest that this cytokine may play an important role in the precipitous self-resolution of acute cellular rejection.

NON-INVASIVE MOLECULAR DIAGNOSIS OF ACUTE REJECTION OF RENAL ALLOGRAFTS: BETA-2-MICROGLOBULIN (β2m) mRNA IS HYPEREXPRESSED IN PERIPHERAL BLOOD MONONUCLEAR CELLS OBTAINED FROM PATIENTS WITH ACUTE REJECTION.

15 β2m, coexpressed with Class I MHC antigens on all types of immune cells (T, B, NK and APCs), is upregulated following activation of immune cells. Thus, quantification of the level of expression of β2m mRNA has the potential to identify cellular activation and clonal expansion that are prerequisites for allograft rejection. We therefore formulated and tested the hypothesis that β2m mRNA levels would distinguish biopsy-proven acute renal allograft rejection from other histological diagnoses. We also tested the hypothesis that β2m mRNA levels in peripheral blood mononuclear cells (PBM), obtained from patients with biopsy proven acute rejection, would be higher than those in PBM obtained from patients without biopsy evidence of rejection. Our hypotheses were tested by designing and developing β2m mRNA specific competitor DNA templates for quantification by competitive RT-PCR. Our investigation of the significance of β2m mRNA levels in allograft recipients revealed the following: ▸ A significant association between intrarenal β2m mRNA expression and biopsy confirmed acute rejection (p=0.0003, Fisher's exact test). ▸ Significantly higher levels of β2m mRNA levels in rejecting allografts (n=31) compared to non-rejecting allografts (n=16)(p=0.01, t test). ▸ A significant association between β2m mRNA expression in PBM and acute rejection (p=0.004, Fisher's exact test). ▸ Significantly higher levels of β2m mRNA in PBM obtained from patients with acute rejection (n=23), compared to PBM obtained from patients without acute rejection (n=13) (p=0.02, t test). The sensitivity, specificity, positive predictive value (PPV) and the negative predictive value (NPV) of β2m mRNA levels in biopsies or in PBM are summarized below. (Table)TableOur first time investigation of β2m mRNA levels in human renal allograft recipients has identified a non-invasive and clinically useful molecular test for the diagnosis of acute allograft rejection in humans.

MHC ALLOPEPTIDE REACTIVE T CELL CLONES GENERATED FROM HUMAN RENAL ALLOGRAFT RECIPIENTS: INDIRECT ALLORECOGNITION IN LONG TERM ENGRAFTMENT

9 T cells obtained from patients with chronic allograft dysfunction are primed to recognize and respond to donor-specific MHC allopeptides in vitro. Class II MHC allopeptide-specific Th1 clones generated from rejecting animals exhibit a restricted TCR Vβ repertoire and transfer delayed type hypersensitivity responses in vivo indicating, the important role they play in the genesis of rejection. In order to evaluate the role of T cells primed by donor specific MHC peptides in chronic allograft dysfunction, T cell lines and clones were generated from peripheral blood lymphocytes (PBLs) of human renal allograft recipients, all of whom had been transplanted for more than one year. All patients were maintained on the same immunosuppressive protocol. Nine patients mismatched for MHC class II specificities for which peptides were available were studied. The patients were divided into 2 groups; 1. chronic allograft dysfunction (n=4) and 2. stable graft function (n=5). T cell lines were generated from PBLs by repeated stimulation with the relevant mismatched class II MHC allopeptide presented by self antigen-presenting cells. The mean proliferative response of T cells lines from "rejecting" patients was significantly greater than that of the lines from "stable" patients; (10743.0±1834.2 versus 1175.4±127.3 cpm, p<0.0001). The production of cytokines generated in response to the allopeptides was evaluated by ELISA and RNAse protection assays. Cell lines from the "rejecting" group produced the Th1 cytokine IFN-γ but not the Th2 cytokine IL-4 in culture supernatants. One of the cell lines produced TGF-β. In contrast, the "stable" group produced IL-4 but not IFN-γ in response to re-challenge with the allopeptides. These data were confirmed by RNAse protection assay. T cell clones were then generated by limiting dilution and were phenotyped using flow cytometric analysis. T cell clones from both groups were all found to be CD4+. RT-PCR transcript analysis with specific human TCR Vβ primers showed that the clones expressed a restricted TCR Vβ repertoire with some overlaps. The mean proliferative response of the "rejecting" T cell clones was also significantly greater than that of the "stable" clones; (20352.0±2678.7 versus 2873.2±348.1 cpm, p<0.0001). Interestingly, without exception, analysis of 49 T cell clones showed that all the clones from rejecting patients produced IFN-γ but not IL-4, while clones from stable patients produced IL-4 but not IFN-γ. This is the first demonstration, at the clonal level, that chronic allograft rejection in humans is associated with the presence of CD4+ MHC allopeptide-specific T cell clones that express a Th1 phenotype. In contrast, MHC allopeptide-specific T cell clones generated in the absence of rejection are associated with a Th2 phenotype that may be protective. These data provide an interesting insight into the potential mechanisms underlying chronic allograft dysfunction and more importantly suggest an innovative approach to immune surveillance.

Polyclonal Antilymphocyte Sera Induce Immune Activation in Human Renal Allograft Recipients

Polyclonal Antilymphocyte Sera Induce Immune Activation in Human Renal Allograft Recipients

"Heterophile" antigen in sera of human recipients of renal allografts.

It was noted that many sera of patients with renal allograft produce distinct precipitation lines in gel diffusion tests with about 20% of infectious mononucleosis sera. The antibodies in infectious mononucleosis sera were of IgM isotope, but, interestingly, they could be removed by guinea pig kidney homogenate, which indicated that the reactions studied were of the Hanganutziu-Deicher rather than of the Paul-Bunnell type. This contention was strengthened by the fact that positive transplantation sera reacted also with standard serum with Hanganutziu-Deicher antibodies. Thus far, the presence of the antigen in the transplantation sera could not be related to the clinical status of the patients, however, the antigen was noted primarily in those sera that did not contain heterophile transplantation antibodies. It was proposed that the antigen detected in the transplantation sera was an altered tissue antigen released from the grafted organ. Besides, interactions between two serum samples from the same patient were noted in immunodiffusion tests. These reactions occurred very seldom and were unrelated to heterophile transplantation antigens or antibodies.

The role of mycophenolate mofetil in clinical renal transplantation.

Mycophenolate mofetil (MM) is the ester derivative of mycophenolic acid (MPA), which exerts immunosuppressive activity by inhibiting de novo purine biosynthesis. Following animal models and pilot studies in human renal allograft recipients that confirmed MM's ability to prevent and half rejection episodes, 3 large multicenter studies of 500 patients each were conducted in North America, Europe, and Australia to compare MM with azathioprine or placebo, all drugs being used in conjunction with prednisone and cyclosporine. The results demonstrated a statistically significant reduction in episodes of allograft rejection in the MM groups with a concomitant reduction in the use of antirejection therapy, especially antilymphocyte antibody therapy. Adverse-event profiles were similar for all groups, although there were slightly more gastrointestinal and tissue-invasive cytomegalovirus (CMV) episodes in the MM groups. It is concluded that MM, which recently received Food and Drug Administration (FDA) approval in the United States, should be an important addition to the transplant physician's immunosuppressive armamentarium.

Potential Applications of Therapeutic Drug Monitoring of Sirolimus Immunosuppression in Clinical Renal Transplantation

Sirolimus is a potent immunosuppressive agent with a novel mechanism of action. It inhibits the transduction of cytokine signals necessary for the proliferation and maturation of T cells. Because sirolimus blocks a broad spectrum of cytokine signals, it seems logical to use it as an adjunct to CsA-based immunosuppression. The high degree of synergy between these two agents, as suggested by the rigorous median-effect analysis, has been confirmed by a reduced rate of rejection episodes among human renal allograft recipients. However, Phase I studies document wide interindividual variation in the pharmacokinetic parameters of 26 stable renal transplant patients, thereby suggesting that optimal therapy may require monitoring of drug concentrations, which is a task that has been somewhat simplified by the good correlation of trough level to AUC. Development of a monoclonal antibody assay system may simplify the monitoring of drug concentrations further. Additional studies of sirolimus will be required to determine the therapeutic concentrations and ratios of sirolimus to CsA that provide optimal immunosuppression, and to assess the possibility of a steroid-free regimen.