Lack of benefit of early protocol biopsies in renal transplant patients receiving TAC and MMF: a randomized study.

We conducted a randomized, multicenter study to determine whether treatment of subclinical rejection with increased corticosteroids resulted in beneficial outcomes in renal transplant patients receiving tacrolimus (TAC), mycophenolate mofetil (MMF) and prednisone. One hundred and twenty-one patients were randomized to biopsies at 0,1,2,3 and 6 months (Biopsy arm), and 119 to biopsies at 0 and 6 months only (Control arm). The primary endpoint of the study was the prevalence of the sum of the interstitial and tubular scores (ci + ct)> 2 (Banff) at 6 months. Secondary endpoints included clinical and subclinical rejection and renal function. At 6 months, 34.8% of the Biopsy and 20.5% of the Control arm patients had a ci + ct score >or= 2 (p = 0.07). Between months 0 and 6, clinical rejection episodes were 12 in 10 Biopsy arm patients and 8 in 8 Control arm patients (p = 0.44). Overall prevalence of subclinical rejection in the Biopsy arm was 4.6%. Creatinine clearance at 6 months was 72.9 +/- 21.7 in the Biopsy and 68.90 mL/min +/- 18.35 mL/min in the Control arm patients (p = 0.18). In conclusion, we found no benefit to the procurement of early protocol biopsies in renal transplant patients receiving TAC, MMF and prednisone, at least in the short term. This is likely due to their low prevalence of subclinical rejection.

Transplant glomerulopathy, late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause.

To define the relative frequency of phenotypes of transplant glomerulopathy, we retrospectively reviewed the findings in 1036 biopsies for clinical indications from 1320 renal transplant patients followed in our clinics between 1997 and 2005. Transplant glomerulopathy, defined by double contours of glomerular basement membranes (D), was diagnosed in 53 biopsies (5.1%) from 41 patients (3.1%) at a median of 5.5 years post-transplant (range 3.8-381 months). In cases with D, we studied the frequency of circulating anti-HLA alloantibody (A), peritubular capillary basement membrane multilayering (B) and peritubular capillary C4d deposition (C). B was present in 48 (91%) of D biopsies. C4d staining by indirect immunofluorescence was detected in 18 of 50 D biopsies studied (36%). By Flow PRA Screening or ELISA, A was detected in 33 (70%) in 47 D cases with available sera, of which 28/33 or 85% were donor-specific. Class II (13/33) or class I and II (17/33) were more common than class I (3/33) antibodies. Thus 73% of transplant glomerulopathy has evidence of alloantibody-mediated injury (A and/or C), with ABCD and ABD being the common phenotypes in biopsies for cause. The remaining 27%, mostly BD, may be a different disease or a stage in which A and C are undetectable.

Identifying the patient at risk for post-transplant lymphoproliferative disorder.

Post-transplant lymphoproliferative disorders (PTLD) are a recognized complication of the immunosuppression required to prevent allograft rejection, occurring in 1-20% of recipients of solid organ transplants. Several factors greatly increase the risk of developing PTLD early post-transplant in any individual recipient. Epstein-Barr virus (EBV) infection is critical in the pathogenesis of the majority of these cases. Pre-transplant EBV seronegativity increases the incidence of PTLD 10- to 75-fold over that of EBV-seropositive recipients. Other risk factors include very young recipient age, cytomegalovirus infection or mismatching (donor positive-recipient negative), aggressive immunosuppression with conventional biologic agents, and the type of organ transplanted. In contrast, the risk of developing PTLD late in the post-transplant course does not appear to be influenced by the type of immunosuppressive agents employed, but rather by the duration of any immunosuppression. The role of EBV in late PTLD is also less certain, as a greater proportion of lesions are not associated with evidence of EBV infection. As the understanding of these risk factors has expanded, opportunities exist to target those populations at highest risk for the development of PTLD for aggressive monitoring and pre-emptive or prophylactic therapy. It is hoped that implementation of such strategies will render early PTLD a preventable complication of transplantation.

Interferon-gamma gene expression during acute graft-versus-host disease: relationship to MHC induction and tissue injury.

The pathogenetic role of interferon-gamma (IFN-gamma) in acute graft-versus-host disease (GVHD) was examined in a murine model. IFN-gamma gene expression was evaluated by northern blotting and mRNA in situ hybridization. The temporal and tissue specific patterns of IFN-gamma gene expression were related to the patterns of major histocompatibility complex (MHC) antigen induction and of tissue injury. Markedly increased levels of IFN-gamma transcripts were seen in the spleen during the early lymphoproliferative phase and coincided with widespread MHC induction in non-lymphoid tissues. Increased IFN-gamma transcripts were also found in the non-lymphoid target tissues during the phase of subsequent tissue injury. These findings support a role for IFN-gamma in leading to widespread MHC induction during acute GVHD and suggest that IFN-gamma may also contribute to target tissue injury during acute GVHD.

On the influence of sample size on the prognostic accuracy and reproducibility of renal transplant biopsy.

INTRODUCTION: The minimal specimen size necessary for accurate interpretation of a renal biopsy has not been identified. We attempted such a determination by three different analyses of a collection of biopsies performed in renal transplants. METHODS: First, we studied the influence of three lesions (glomerulosclerosis, arteriolar hyalinosis, interstitial fibrosis/tubular atrophy) in 199 baseline biopsies, obtained at time of transplantation, on transplant outcome. Secondly, we compared the results from the three lesions in baseline biopsy with those from 114 subsequent core biopsies in the same patients. Thirdly, we compared the two baseline biopsies obtained in 118 paired kidneys in cadaver transplantation where both kidneys were used. RESULTS: For statistically significant prediction of outcome from glomerulosclerosis, we found that a specimen containing at least 25 glomeruli was needed in the baseline biopsy. Arteriolar hyalinosis predicted outcome independent of sample size, but became less important than percentage glomerulosclerosis in predicting outcome if only samples containing more than 25 glomeruli were considered. Interstitial fibrosis/tubular atrophy did not predict the outcome of a kidney, independent of sample size. When comparing baseline with subsequent core biopsies, or with paired baseline biopsies, at least 14 glomeruli were necessary to allow even moderate reproducibility of glomerulosclerosis (Cohen's kappa > 0.25) and to allow statistical significance (P < 0.05). The reproducibility of arteriolar hyalinosis was not dependent on sample size but was reproducible in 80% of paired baseline biopsies, and in 67% of the comparison of the baseline with core biopsy. Both precision and significance was lost if sample numbers were reduced by including only larger samples. There was no reproducibility in any study of interstitial fibrosis/tubular atrophy when comparing either baseline with subsequent biopsy, or paired baseline biopsies. SUMMARY: Much larger biopsy samples are necessary than has generally been assumed in order for glomerulosclerosis rates to be reproducible or predictive of outcome. Arteriolar hyalinosis is prognostically important and shows good reproducibility independent of sample size. Interstitial fibrosis/tubular atrophy appear useless as predictors, being of no prognostic importance and lacking reproducibility. Our finding clarifies some of the discrepancies found by different investigators regarding the importance of renal biopsy in predicting prognosis. Preliminary, our data indicate that samples containing fewer than 25 glomeruli are unreliable in determining outcome based on glomerulosclerosis. The importance of our findings which are based only on chronic lesions, with respect to acute changes, is unknown.

Serologic responses to hepatitis C virus in solid organ transplant recipients.

BACKGROUND: Hepatitis C (HCV) infection is known to have been transmitted by both blood transfusion and donor organs. We sought to determine the historical incidence of donor- and transfusion-acquired HCV infection in kidney transplant (RTx) and heart transplant (HTx) recipients at our center and to study the kinetics of seroconversion to HCV. METHODS: A bank of sera collected from organ donors (388 RTx and 88 HTx) who received allografts between January 1984 and April 1992 was screened for anti-HCV using a third generation enzyme immunoassay. Recipient sera collected before transplant (preTx), at 1 year after transplant, and at last follow-up were tested. Fresh follow-up sera on all surviving anti-HCV-positive (+) RTx and HTx, all anti-HCV-negative (-) HTx, and a subset of 85 anti-HCV- RTx were assayed for HCV RNA using an reverse transcriptase-polymerase chain reaction assay. RESULTS: Twenty-four of 388 RTx (6.2%) and 2 of 88 HTx (2.3%) were anti-HCV+ preTx. Eight of 218 (3.7%) organ donors were anti-HCV+. Six of the seven (85.7%) anti-HCV+ donors with adequate recipient follow-up transmitted HCV infection to one or more recipients. Nineteen of 313 RTx (6.1%) and 8 of 72 HTx (11.1%) with follow-up > or =1 year seroconverted to anti-HCV. One of 85 (1.2%) anti-HCV- RTx and 3 of 44 (6.8%) anti-HCV-HTx were HCV RNA+ when tested at last follow-up. Five cases of de novo HCV infection occurred after the introduction of first generation anti-HCV screening of donors. Persistent viremia (HCV RNA+) at last follow-up was observed in 70.6% (12/17) RTx anti-HCV+ preTx. Fourteen of 15 (93.3%) RTx and 9 of 9 (100%) HTx with de novo HCV infection had persistent viremia. Seroconversion was more delayed in HTx than RTx (P=0.0572, log-rank Mantel-Cox statistic) although both groups demonstrated an impaired humoral response to HCV when compared with the immunocompetent host. CONCLUSIONS: Organ donor- and transfusion-acquired HCV infection was common in RTx and HTx transplanted before the introduction of second generation anti-HCV screening in 1992. Serologic responses to HCV are often delayed and sometimes absent in these patients. Assays for HCV RNA should be considered as a screening test for the detection of HCV infection in this population. Serologic responses to HCV were more impaired in HTx compared with RTx, which may reflect the more intensive immunosuppressive regimens given to HTx at our center.

Post-transplant lymphoproliferative disorder in renal allograft recipients. Clinical experience and risk factor analysis in a single center.

Post-transplant lymphoproliferative disorder (PTLD) is a well-recognized complication of solid organ transplantation. The University of Alberta Renal Transplant Program had not experienced a case of PTLD occurring in the early post-transplant period until March 1989. Since then, 4 patients have developed this complication. To identify the major risk factors for the recent appearance of PTLD, a retrospective analysis was carried out on 162 cadaveric renal transplants performed between July 1987 and December 1990. Four cases of polymorphic PTLD were seen. Two patients presented with fatal disseminated disease. Two others developed PTLD confined to the renal allograft; both are disease free at > 24 months of follow-up. Seventy-two (44.4%) of the cadaveric transplant recipients had received Minnesota antilymphocyte globulin (MALG) induction therapy during the study period. Twenty-four of these also received OKT3 for steroid-resistant rejection. Of the 4 patients with PTLD, 3 had received both MALG induction and OKT3; the remaining patient had received MALG induction only. The incidence of PTLD in the MALG/OKT3 group was 12.5%, which is significantly higher than that of patients receiving other immunosuppressive regimes (0.7%, P = 0.015). The incidence of PTLD was also significantly greater in the 13 patients at risk for primary EBV infection compared to the EBV seropositive patients (23.1 vs. 0.7%, P = 0.002). Only 2 seronegative patients received sequential MALG/OKT3; both developed PTLD. Thus, the population most at risk is that receiving potent antilymphocyte preparations in the setting of primary EBV infection. Allograft involvement with PTLD must be considered in the differential diagnosis of allograft dysfunction, as early diagnosis may permit the successful management of this complication.

Regulation of IFN-gamma expression in vivo. IFN-gamma up-regulates expression of its mRNA in normal and lipopolysaccharide-stimulated mice.

Immune responses to such stimuli as tissue injury, infection, and allografting result in localized IFN-gamma expression. Autoregulation of cytokine expression has been described for some cytokines in vitro, but whether this occurs in vivo is unknown. We therefore examined the role of murine IFN-gamma in the regulation of its own expression in vivo after stimulation with bacterial LPS. This agent is known to induce IFN-gamma expression in both spleen and kidney in a T cell-independent, cyclosporine-sensitive manner. We found that concomitant administration of a neutralizing mAb to IFN-gamma inhibited not only the MHC expression induced by LPS but also the increased IFN-gamma mRNA expression, suggesting an autoregulatory role for IFN-gamma. Inhibition was dose dependent and observed in both spleen and kidney. The effect was not seen with a neutralizing anti-IL-3 mAb, demonstrating specificity. The inhibition of IFN-gamma mRNA expression by the anti-IFN-gamma mAb occurred in both T cell-deficient athymic nude mice and their normal controls, suggesting that the autoamplification of IFN-gamma mRNA in vivo is T cell independent. Administration of rIFN-gamma to unstimulated mice induced IFN-gamma mRNA expression in spleen and kidney, supporting the conclusion that IFN-gamma up-regulates expression of its mRNA. Exposure of resting murine splenocytes to concentrations of rIFN-gamma as low as 10 U/ml in vitro induced expression of IFN-gamma mRNA. Thus, in vivo IFN-gamma may participate in an autoregulatory loop to amplify the amount of IFN-gamma expressed both at the site of local inflammation and at remote sites. This would have relevance in the mechanism by which the host defends itself against and prevents dissemination of an infectious agent.

Regulation of IFN-gamma and tumor necrosis factor-alpha expression in vivo. Effects of cycloheximide and cyclosporine in normal and lipopolysaccharide-treated mice.

Despite accumulating information about cytokine expression in vitro, relatively little is known about the regulation and biologic relevance of these mediators in vivo. In order to study the effects of inhibition of protein synthesis and cyclosporine in vivo, we made use of systemically administered LPS, which induces the expression of a variety of cytokines. The expression of IFN-gamma and TNF-alpha mRNA in normal and LPS-treated mice was examined by Northern blot analysis and amplification using the polymerase chain reaction. IFN-gamma activity was monitored using the biologic end point of MHC induction. TNF-alpha activity in serum was assessed using a L929 cytotoxicity assay. Messenger RNA for IFN-gamma and TNF-alpha could not be reliably detected by Northern analysis in spleens or kidneys of normal mice. After treatment with cycloheximide, a protein synthesis inhibitor, IFN-gamma and TNF-alpha mRNA could be detected in both sites in otherwise normal mice. The level of both IFN-gamma and TNF-alpha mRNA increased after LPS, although the temporal patterns of expression were different. The concurrent administration of cycloheximide led to marked superinduction of both cytokine mRNA levels. Similar effects were seen in T cell-deficient nude mice, suggesting that these responses are T cell independent. Cyclosporin A blocked induction of IFN-gamma in a dose dependent manner, but failed to significantly inhibit TNF-alpha mRNA or protein expression. Thus at least part of the immunosuppressive effect of cyclosporin A in vivo may be caused by its ability to inhibit the expression of certain cytokine genes, as has been found in vitro systems. However, the cellular target for this effect may extend to cell populations other than T cells.

The regulation of expression of MHC products in mice. Factors determining the level of expression in kidneys of normal mice.

Cytokines, especially IFN, have been shown to increase MHC expression above basal levels in many immunologic processes, but their contribution to normal MHC expression is unknown. Environmental stimuli, such as bacterial LPS, may provoke the secretion of cytokines which could influence MHC expression in the normal host. We therefore studied the factors influencing the normal expression of MHC products in mice, by using kidney as a representative nonlymphoid tissue. Considerable variation in MHC expression was observed between otherwise normal individuals by using indirect immunoperoxidase staining and a radiolabeled antibody binding assay. An intact T cell compartment was not necessary for normal levels of expression; in athymic nude mice and mice with severe combined immunodeficiency disease, MHC expression was similar to or greater than that of controls. Neither a course of broad spectrum antibiotics nor genetically determined resistance to LPS influenced the level of normal expression. Mice raised under germ-free conditions, on a chemically defined diet also had similar levels of MHC expression when compared with mice maintained under conventional conditions. However, when germfree mice were placed in a conventional colony, induction of both class I and II MHC products occurred. Thus, although exposure to bacterial flora or other sources of endotoxin was not required for normal MHC expression, a change in flora can up-regulate expression, probably by inducing the secretion of cytokines from non-T cells. Treatment with cyclosporine or anti-IFN antibodies produced, at most, a small reduction in the level of renal MHC expression. Therefore in some mice, a small component of MHC expression may reflect the secretion of cytokines in response to stimuli such as bacterial LPS. However, most MHC expression in nonlymphoid tissues is constitutive and independent of a physiologic IFN response.

A view from the groove: peptide binding by MHC molecules and the implications for regional immune responses.

A significant event in immunology occurred in 1987 with the publication of the three dimensional structure of a class I major histocompatibility complex (MHC) product as determined by X ray crystallography. The crystal structure revealed a groove created by the first and second domains of the molecule which could be the antigen (Ag) binding site. Subsequent analyses have suggested that a similar groove exists in the class II molecule. The present view will focus on the implications of this new knowledge for understanding regional immune responses. In particular, we shall discuss how the occupation of the groove by different peptides in different tissues implies that a component of T cell recognition of MHC may be tissue specific.

Multiple low dose streptozotocin induces systemic MHC expression in mice by triggering T cells to release IFN-gamma.

Multiple low dose streptozotocin (STZ) is believed to induce immunologically mediated islet cell necrosis. We sought to establish whether this agent also increased MHC expression, as has been reported for other diabetes models. STZ (40 mg/kg/day for 5 days) produced increased levels of both class I and II MHC products in kidney, liver, heart, and pancreas. Class I expression was induced in renal tubular cells, Kupffer cells, hepatocytes, occasional cardiac myocytes, and cells within the pancreatic islet. In contrast, class II products were increased in dendritic cells, renal tubules, and cells within the pancreatic islet. Steady state mRNA levels for class I, class II, and beta 2-microglobulin correlate closely with the level of MHC products measured by radiolabeled antibody binding, suggesting that changes in MHC expression reflect changes in gene transcription. The effect is T cell dependent and inhibitable by cyclosporine. IFN-gamma is an essential mediator of the MHC induction; administration of a neutralizing antibody blocks the increase in expression. Furthermore, this antibody attenuates the hyperglycemic response to STZ, demonstrating a pathogenic role for IFN-gamma in mediating beta cell damage. We conclude that the MHC induction observed after low dose STZ is due to immunologic mechanisms, in particular the release of lymphokines such as IFN-gamma from T cells. The release of IFN-gamma and changes in MHC expression may be relevant to the injury seen with this agent.