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The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v-lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics-technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

In the last decade, there has been a growing recognition of alloantibody responses in organ transplantation, but phenotypes related to antibody-mediated rejection (ABMR) remain incompletely defined. This article reviews recent molecular studies in kidney allograft tissues that decipher molecular burden and mechanisms of ABMR, leading to discovery of a new phenotype ‘C4d-negative ABMR’.

We studied whether de novodonor-specific antibodies (DSA) in sera from patients undergoing kidney transplant biopsies associate with specific histologic lesions in the biopsy and prognosis. DSA were assessed in 145 patients at the time of biopsy between 7 days to 31 years posttransplant. DSA was detected in 54 patients (37), of which 32 represented de novoDSA. De novoDSA was more frequent in patients having late biopsies (34) versus early biopsies (4), and was usually either against class II alone or class I and II but rarely against class I alone. Microcirculation inflammation (glomerulitis, capillaritis) and damage (glomuerulopathy, capillary basement membrane multilayering), and C4d staining were associated with de novoDSA. However, the degree of scarring, arterial fibrosis and tubulo-interstitial inflammation did not correlate with the presence of de novoDSA. De novoDSA correlated with reduced graft survival after the biopsy. Thus, de novoDSA at the time of a late biopsy for clinical indication is primarily against class II, and associates with microcirculation changes in the biopsy and subsequent graft failure. We propose careful assessment of de novoDSA, particularly against class II, be performed in all late kidney transplant biopsies.

Anti-HLA alloantibody is a risk factor for graft loss, but does not indicate which kidneys are experiencing antibody-mediated rejection (ABMR). C4d staining in biopsies is specific for ABMR but insensitive. We hypothesized that altered expression of endothelial genes due to alloantibody acting on the microcirculation would be sensitive indicator of ABMR. We identified 119 endothelial-associated transcripts (ENDATs) from literature, and studied their expression by microarrays in 173 renal allograft biopsies for cause. Mean ENDAT expression was increased in all rejection but was higher in ABMR than in T-cell-mediated rejection and correlated with histopathologic lesions of ABMR, and alloantibody. Many individual ENDATs were increased in ABMR and predicted graft loss. Kidneys with high ENDATs and antibody showed increased lesions of ABMR and worse prognosis in comparison to controls. Only 40% of kidneys with high ENDAT expression and chronic ABMR or graft loss were diagnosed by C4d positivity. High ENDAT expression with antibody predicts graft loss with higher sensitivity (77% vs. 31%) and slightly lower specificity (71% vs. 94%) than C4d. The results were validated in independent set of 82 kidneys. High renal endothelial transcript expression in patients with alloantibody is indicator of active antibody-mediated allograft damage and poor graft outcome.

Transcripts expressed in cytotoxic T lymphocytes (CTL) have mechanistic and diagnostic importance in transplantation. We used microarrays to select CTL-associated transcripts (CATs) expressed in human CD4CTL, CD8CTL and NK cells, excluding transcripts expressed in B cells, monocytes and kidney. This generated three transcript sets: CD4-associated, CD8-associated and NK-associated. Surprisingly, many CATs were expressed in effector memory cells e.g. granzyme BGZMB, interferon-?IFNG. Transcript expression was very similar between CD4and CD8CTL. There were no transcripts highly selective for CD4CTL or CD8CTL: for example, cytotoxic molecule transcripts (perforin, granzymes, granulysin) were shared between CD8CTL and CD4CTL although expression remained higher in CD8CTL. Transcripts that differentiated between CD8CTL and CD4CTL were primarily those shared between CD8CTL and NK cells (e.g. NK receptors KLRC1, KLRC3, KLRD1, KLRK1). No transcripts could differentiate CD4CTL from CD8CTL but NK cell-associated transcripts could differentiate NK cells from CTL. This study serves as a foundation for the interpretation of CATs in rejecting allografts and highlights the extensive sharing of CATs among CD4CTL, CD8CTL and effector memory T cells.

Organ allografts deficient in interferon-? (Ifng) or major histocompatibility complex (MHC) class I products develop accelerated necrosis when rejection develops, depending on perforin and granzymes. Thus Ifng-induced donor class I products deliver inhibitory signals to host inflammatory cells. We used microarrays to investigate whether Ifng-induced donor class I products also control inflammation patterns in mouse kidney allografts. Compared to wild-type (WT) allografts, many transcripts were increased in both Ifng-deficient allografts (Ifng-suppressed transcripts GSTs) and class I-deficient allografts (class I-suppressed transcripts CISTs), with 73 overlap between GSTs and CISTs. Some GSTs and CISTs reflected increased necrosis, including known injury-induced transcripts. However, many GSTs and CISTs were independent of perforin, granzymes and necrosis, and were associated with alternative macrophage activation (AMA) (e.g. arginase I Arg1, macrophage elastase Mmp12 and macrophage mannose receptor 1 Mrc1). AMA transcripts were induced despite absence of host interleukin (IL)4 and IL13 receptors. The AMA inducer may be activins, whose genes (inhibin A InhbA and inhibin B InhbB) were increased in all allografts with AMA. We conclude that in allograft rejection, Ifng acts via donor Ifng receptors (Ifngr) to induce donor class Ia and Ib products, which engage host inflammatory cells to limit perforin-granzyme-mediated damage and prevent AMA associated with inhibition of activin expression. Thus, Ifng may control T helper type 2 (Th2) cell inflammation by induction of class I products.

Interferon-? (Ifng) protects organ allografts: mouse kidney allografts lacking Ifng receptors rapidly fail with massive ischemic necrosis around days 5 to 7, reflecting microcirculation failure. We hypothesized that Ifng protects the graft by preventing perforin-granzyme-mediated cytotoxic damage to the microcirculation by inducing class Ia andor Ib products. We transplanted kidney allografts lacking Ifng receptors into various knockout hosts. The necrosiscongestion phenotype did not require host B cells or IL-4 and IL-13 receptors, but required the T-cell alloresponse: it did not occur if the hosts were syngeneic or T-cell deficient. However, host perforin-granzyme mechanisms were required: no necrosis developed if hosts lacked either perforin or granzymes A and B. The ability of Ifng to protect the allograft required donor class I products: allografts lacking class I products due to Tap1 or ?2 microglobulin deficiency developed a similar necrosis-congestion phenotype at day 7 despite Ifng receptors being present. Thus when host cytotoxic T cells infiltrate organ allografts, Ifng prevents their perforin-granzyme mechanism from compromising the microcirculation by a mechanism requiring donor class Ia or Ib products. We propose that donor class Ia or Ib products are needed to trigger inhibitory receptors on effector T cells.

The usual phenotype of clinical kidney allograft rejection is infiltration by lymphocytes and macrophages and evolution of histologic Banff lesions, particularly tubulitis, which indicate parenchymal injury. Using Affymetrix microarrays, we evaluated the relationship between the evolution of pathologic lesions and the transcriptome. We studied CBA/J into C57Bl/6 mouse kidney allografts in which one host kidney is left in place to permit observation of lesion development. Histology was dominated by early infiltration by mononuclear cells from day 3 and slower evolution of tubulitis after day 7. We defined a set of cytotoxic T lymphocyte-associated transcripts (CATs) on the basis of expression in purified cytotoxic T lymphocytes (CTL) and in a mixed lymphocyte culture, and absence in normal kidney. CATs were detectable by day 3 and highly expressed by day 5 in rejecting kidneys, with a median signal 14% of that in CTL, compared to 4% in isografts and normal kidneys, and persisted through day 42. Lack of mature B cells had little effect on CAT expression, confirming that CATs reflect T-cell-mediated rejection. Expression of CATs was established before diagnostic lesions and remained remarkably consistent through day 42 despite massive alterations in the pathology, and probably reflects T cells recruited to the graft.

The usual phenotype of clinical kidney allograft rejection is infiltration by lymphocytes and macrophages and evolution of histologic Banff lesions, particularly tubulitis, which indicate parenchymal injury. Using Affymetrix microarrays, we evaluated the relationship between the evolution of pathologic lesions and the transcriptome. We studied CBA/J into C57Bl/6 mouse kidney allografts in which one host kidney is left in place to permit observation of lesion development. Histology was dominated by early infiltration by mononuclear cells from day 3 and slower evolution of tubulitis after day 7. We defined a set of cytotoxic T lymphocyte‐associated transcripts (CATs) on the basis of expression in purified cytotoxic T lymphocytes (CTL) and in a mixed lymphocyte culture, and absence in normal kidney. CATs were detectable by day 3 and highly expressed by day 5 in rejecting kidneys, with a median signal 14% of that in CTL, compared to 4% in isografts and normal kidneys, and persisted through day 42. Lack of mature B cells had little effect on CAT expression, confirming that CATs reflect T‐cell‐mediated rejection. Expression of CATs was established before diagnostic lesions and remained remarkably consistent through day 42 despite massive alterations in the pathology, and probably reflects T cells recruited to the graft.

The hallmark of failing renal transplants is tubular atrophy and interstitial fibrosis (TA/IF). Injury to tubular epithelial cells (TEC) could contribute to fibrogenesis via epithelial–mesenchymal transition (EMT). We examined the features of EMT in renal transplants that developed TA/IF. Biopsies from 10 allograft kidneys with impaired function and TA/IF and 10 biopsies from transplants with stable function were compared to their implantation biopsies. Relative to implantation biopsies, TEC in TA/IF kidneys showed loss of epithelial markers (E-cadherin, cytokeratin) with altered distribution. Some TEC also showed new cytoplasmic expression of mesenchymal markers vimentin, S100A4, and alpha smooth muscle actin (α-SMA) and collagen synthesis marker heat shock protein (HSP-47), both in deteriorating and atrophic tubules. Double immunostaining showed coexpression of cytokeratin and vimentin, S100A4 and HSP-47, indicating intermediate stages of EMT in TA/IF. These changes were absent or much less in transplants with stable function. EMT features in the TA/IF group correlated with serum creatinine (vimentin, S100A4, HSP-47), history of T-cell-mediated rejection (cytokeratin, S100A4) and proteinuria (cytokeratin). These findings support a model in which the TEC damage induces loss of epithelial features and expression of fibroblast features, as a common pathway of deterioration by either immunologic or nonimmunologic processes.

Although long-term survival after kidney transplantation is critically dependent on maintaining stable allograft function, few studies have examined renal allograft function over time. Using pooled data from 10 278 consecutive transplants at five centers, we calculated slopes of estimated glomerular filtration rates (eGFR) measured after 1, 6 and 12 months in 9515, 8861 and 7359 patients surviving ≥1, ≥6 and ≥12 months, respectively. Slopes of eGFR progressively diminished for patients transplanted during 1984–1989, 1990–1993, 1994–1998 and 1999–2002 (analysis of variance p < 0.0001 and p = 0.1245 for slopes measured after 1 and 6 months, respectively). Slopes measured after 12 months were less in the most recent era: −2.2 ± 7.2 mL/min/1.73 m2/year, −2.3 ± 6.6 mL/min/1.73 m2/year, −2.4 ± 7.4 mL/min/1.73 m2/year and −1.4 ± 10.9 mL/min/1.73 m2/year, respectively, p = 0.0058. Slopes measured after 1, 6 and 12 months each were less for transplantations during 1999–2002, after adjusting for multiple transplantation characteristics (p < 0.0001). Similarly, in Cox proportional hazards analysis, the risk (95% CI) for a 25% reduction in eGFR was 0.92 (0.85–1.01), p = 0.0736 during 1990–1994; 0.94 (0.82–1.08), p = 0.4111 during 1995–1998 and 0.78 (0.64–0.95), p = 0.0110 during 1999–2002 (compared to 1984–1989). We conclude that the rate of decline in allograft function after kidney transplantation has improved, suggesting that stable, long-term function may be achievable.

Some features of kidney transplants with dysfunction overlap the lesions of aging, such as tubular atrophy and interstitial fibrosis (TA/IF) without major glomerular abnormalities. Somatic cell limitations could contribute to deterioration in aging and disease states. Since expression of p16INK4a, a cell cycle inhibitor associated with somatic cell senescence in vitro, is induced in aged kidney, we studied whether kidneys with dysfunction and TA/IF manifested increased p16INK4a expression. We performed p16INK4a immunostaining on transplanted kidneys and native kidneys with chronic renal diseases. At implantation, transplants manifested little TA/IF, and nuclear p16INK4a immunostaining was consistent with age. However, transplants biopsied for abnormal function displaying TA/IF showed strong nuclear and cytoplasmic p16INK4a staining, beyond the amount predicted for age. Both atrophic and non-atrophic nephrons displayed increased p16INK4a, suggesting that it was not simply a feature of atrophy. Epithelial p16INK4a staining was not increased in transplants with good function, but was increased in diseased native kidneys. The finding of increased p16INK4a expression in renal transplants and diseased kidneys with TA/IF and impaired function supports the concept that some cell senescence changes that accompany aging are also induced by injury and disease stresses. Thus, aging, injury and disease may share common pathways involving somatic cell senescence.

Some features of kidney transplants with dysfunction overlap the lesions of aging, such as tubular atrophy and interstitial fibrosis (TA/IF) without major glomerular abnormalities. Somatic cell limitations could contribute to deterioration in aging and disease states. Since expression of p16INK4a, a cell cycle inhibitor associated with somatic cell senescence in vitro, is induced in aged kidney, we studied whether kidneys with dysfunction and TA/IF manifested increased p16INK4aexpression. We performed p16INK4aimmunostaining on transplanted kidneys and native kidneys with chronic renal diseases. At implantation, transplants manifested little TA/IF, and nuclear p16INK4aimmunostaining was consistent with age. However, transplants biopsied for abnormal function displaying TA/IF showed strong nuclear and cytoplasmic p16INK4astaining, beyond the amount predicted for age. Both atrophic and non-atrophic nephrons displayed increased p16INK4a, suggesting that it was not simply a feature of atrophy. Epithelial p16INK4astaining was not increased in transplants with good function, but was increased in diseased native kidneys. The finding of increased p16INK4aexpression in renal transplants and diseased kidneys with TA/IF and impaired function supports the concept that some cell senescence changes that accompany aging are also induced by injury and disease stresses. Thus, aging, injury and disease may share common pathways involving somatic cell senescence.

Ageing of the kidney is a problem of clinical and basic interest. The problem of renal dysfunction and end-stage renal disease is a major burden on the health system, and old donor age is a major limitation on the use of donor organs and on survival of transplanted kidneys. Moreover, stresses linked to nephropathies, postoperative stress, inflammation and allograft rejection can lead to premature senescence of renal cells thus accelerating organ atrophy. Age-related and disease or stress-related nephron loss could reflect both the limited ability of epithelial cells to repair and replicate in the face of environmental stresses, and limitations on the number of cell replications caused by telomere shortening. Therefore, elucidating cellular senescence mechanisms is relevant to kidney diseases and kidney transplantation.

IFN-γ is produced by cytotoxic T lymphocytes (CTL) but can also decrease CTL generation. We used γ-R1-deficient (GRKO) and γ-deficient (GKO) mice to study the effects of γ in MLC on the generation of CTL activity and CTL number, IL-2 production and cell proliferation. CTL activity was increased in MLC when GRKO responders or GKO stimulators and responders were used, compared to wild-type (WT) MLC. The number of cells displaying the CTL phenotype (CD3+, CD8+, CD25+) was also increased, accompanied by increased IL-2 production and proliferation. Combinations of WT or GRKO CD4+ T cells with WT or GRKO CD8+ T cells as responders showed that γ mostly affects CD4+ T cells to limit CTL generation. Intracellular staining indicated that IL-2 production was largely by CD4+ T cells. Moreover, addition of IL-2 to WT responders mimicked GKO CTL generation and activity, whereas neutralizing IL-2 decreased CTL activity in GRKO and WT responders. Thus γ reduces CTL generation in alloimmune responses largely by limiting proliferation of IL-2 producing CD4+ T cells. This creates a feedback loop in which effectors produce γ that limits IL-2 production which in turn limits CTL generation.

The Seventh Banff Conference on Allograft Pathology was held June 14–18, 2003 in Aberdeen, Scotland representing the latest iteration of the international consensus meeting, which develops worldwide standards for interpretation of allograft biopsies. The meeting is an important force behind standardized slide interpretation to strengthen endpoints in international clinical trials. Of participants polled 87% reported that they would alter clinical practice as a direct consequence of the meeting and its content. Advances were made in many areas including tubulitis mechanisms, real-time polymerase chain reaction (PCR) gene analysis and microarrays in rejection diagnosis, tolerance/accommodation/immunomodulation, the role of monocytes and macrophages in rejection and C4d as a marker for antibody-mediated rejection. A provisional scoring system for peritubular capillary inflammatory cell accmulation in antibody-mediated rejection was presented for testing, as well as plans for a nephrectomy study to determine specificity of vascular lesions of rejection. Future meetings are planned for 2005 (Edmonton), 2007 and 2009, with active ongoing Internet discussion between meetings.

The Seventh Banff Conference on Allograft Pathology was held June 14–18, 2003 in Aberdeen, Scotland representing the latest iteration of the international consensus meeting, which develops worldwide standards for interpretation of allograft biopsies. The meeting is an important force behind standardized slide interpretation to strengthen endpoints in international clinical trials. Of participants polled 87% reported that they would alter clinical practice as a direct consequence of the meeting and its content. Advances were made in many areas including tubulitis mechanisms, real-time polymerase chain reaction (PCR) gene analysis and microarrays in rejection diagnosis, tolerance/accommodation/immunomodulation, the role of monocytes and macrophages in rejection and C4d as a marker for antibody-mediated rejection. A provisional scoring system for peritubular capillary inflammatory cell accmulation in antibody-mediated rejection was presented for testing, as well as plans for a nephrectomy study to determine specificity of vascular lesions of rejection. Future meetings are planned for 2005 (Edmonton), 2007 and 2009, with active ongoing Internet discussion between meetings.

Organ allograft rejection is strongly associated with the presence of alloreactive cytotoxic T cells but the role of cytotoxicity in the pathologic lesions is unclear. Previous studies showed that the principal lesions of kidney rejection – interstitial infiltration, tubulitis, and endothelial arteritis – are T-cell-dependent and antibody-independent. We studied the role of cytotoxic granule components perforin and granzymes A and B in the evolution of the T-cell-mediated lesions of mouse kidney transplant rejection. By real-time RT-PCR, allografts rejecting in wild-type hosts at days 5, 7, 21, and 42 showed massively elevated and persistent expression of perforin and granzymes A and B, but evolution of tubulitis and arteritis did not correlate with increasing granzyme or perforin expression. Allografts transplanted into hosts with disrupted genes for perforin or granzymes A and B showed no change in tubulitis, arteritis, or MHC induction. Thus the development of the histologic lesions diagnostic of T-cell-mediated kidney transplant rejection are associated with but not mediated by perforin or granzyme A or B. Together with previous graft survival studies, these results indicate that the granule-associated cytotoxic mechanisms of T cells are not the effectors of T-cell-mediated allograft rejection.

We examined our renal transplant population for glomerular diseases demonstrated on biopsy between January 1993 and April 2002, focusing on transplant glomerulopathy (TGP). Of 1156 patients followed in our clinics during this period, glomerular disease was diagnosed in 132 cases (11.4%). Glomerulonephritis was diagnosed in 86 transplants (7.4%), with IgA nephropathy (IgAN) being the commonest diagnosis [32 cases (2.8%)]. Thirty-one cases (2.7%) of biopsy-proven TGP were analyzed for associated factors compared with 27 cases (2.3%) of recurrent IgAN. Transplant glomerulopathy was less frequent with mycophenolate mofetil (MMF) and/or tacrolimus, whereas recurrent IgAN showed no such tendency (P= 0.02). Peritubular capillary (PTC) C4d deposition was observed in six of 24 cases (25%) with TGP but none with recurrent IgAN (P= 0.02). Peritubular capillary basement membrane (BM) multilayering was significantly greater in TGP (4.92 ± 2.94) than in recurrent IgAN (1.86 ± 1.04) (P< 0.001). The graft survival of TGP was worse than recurrent IgAN (P= 0.05). The association of TGP with BM multilayering and C4d deposits in PTC suggests a generalized disorder of the graft microcirculation and its BM, owing to antibody-mediated rejection in at least some cases. Transplant glomerulopathy has a serious prognosis but is less frequent in patients on newer immunosuppression, unlike recurrent IgAN.

The natural history and pathogenesis of the pathologic lesions that define rejection of kidney transplants have not been well characterized. We studied the evolution of the pathology of rejection in mouse kidney allografts, using four strain combinations across full major histocompatibility complex (MHC) plus nonMHC disparities, to permit more general conclusions. Interstitial infiltrate, MHC induction, and venulitis appeared by day 5, peaked at day 7–10, then stabilized or regressed by day 21. In contrast, tubulitis, arteritis, and glomerulitis were absent or mild at days 5 and 7, but progressed through day 21, indicating separate regulation and homeostatic control of these lesions. Edema, hemorrhage, and necrosis also increased through day 21. All lesions were T-dependent, failing to develop in T-cell-deficient hosts. Allografts into immunoglobulin-deficient hosts manifested typical infiltration, MHC induction, and tubulitis at days 7 and 21, indicating that these lesions are alloantibody-independent. However at day 21 kidneys rejecting in immunoglobulin-deficient hosts showed decreased edema, arteritis, venulitis, and necrosis.

Experimental liver allografts undergo spontaneous acceptance despite undergoing rejection during the first few weeks post transplant. We explored the role of interferon-γ (IFN-γ) in the spontaneous acceptance of mouse liver allografts. Strain of mouse (CBA) liver allografts transplanted into normal BALB/c mice developed histologic changes typical of rejection that spontaneously regressed, permitting long-term survival of these allografts similar to that of syngeneic grafts. In contrast, CBA liver allografts in IFN-γ-deficient hosts manifested not only infiltration but also hemorrhage and necrosis, with no survival beyond 14 days. Despite differences in survival, local expression of cytotoxic T-cell genes in the transplant was not increased in IFN-γ-deficient hosts, but livers in interferon-γ-deficient mice (GKO) hosts displayed much less induction of major histocompatibility complex (MHC) class I and II expression. To determine whether the difference in survival was secondary to the direct effects of IFN-γ on the liver, we transplanted livers from IFN-γ-receptor-deficient mice into normal hosts. Liver allografts lacking IFN-γ receptors also developed hemorrhage and necrosis with minimal induction of MHC expression. Thus IFN-γ mediates a direct effect on rejecting liver allografts that reduces hemorrhage and necrosis, induces MHC expression, and is absolutely required for spontaneous acceptance.

Antibody-mediated rejection (AbAR) is increasingly recognized in the renal allograft population, and successful therapeutic regimens have been developed to prevent and treat AbAR, enabling excellent outcomes even in patients highly sensitized to the donor prior to transplant. It has become critical to develop standardized criteria for the pathological diagnosis of AbAR. This article presents international consensus criteria for and classification of AbAR developed based on discussions held at the Sixth Banff Conference on Allograft Pathology in 2001. This classification represents a working formulation, to be revisited as additional data accumulate in this important area of renal transplantation.

Left ventricular assist devices (LVAD) may improve cardiac function. The pathogenesis of this phenomenon, called ‘reverse remodelling’, is not completely elucidated. To examine the hypothesis that LVAD support eliminates tissue stress by reducing local hypoxia, the distribution of heme oxygenase‐1 (HO‐1), a stress protein inducible by hypoxia, was examined in vivoand in vitro. The immunoreactivity for HO‐1 was semi‐quantitatively analysed in left ventricular tissue of 23patients (14 dilated cardiomyopathy (DCM), six ischaemic heart disease (IHD), three myocarditis/congenital heart disease) with end‐stage heart failure before and after LVAD support, while two unused donor hearts served as controls. Control hearts stained almost negative for HO‐1, while failing hearts showed immunoreactivity mainly in cardiomyocytes, but also in endothelial cells, some smooth muscle cells and fibroblasts. Hearts with IHD showed significantly higher HO‐1 immunoreactivity than hearts with DCM or myocarditis/congenital heart disease. After LVAD support, the HO‐1 content decreased significantly in the DCM and IHD group and was significantly higher in the subendocardium than in the subepicardium. In vitro, under hypoxic conditions, neonatal rat cardiomyocytes showed an increase of HO‐1 protein content up to sixfold above the normal level, which returned to normal values after normoxic cultivation. Mechanical support reduces the HO‐1 content of the failing heart and HO‐1 is inducible in vitrounder hypoxia and is reversible under normoxia. This supports the concept that restoration of cardiac normoxia by mechanical unloading, particularly in the subendocardium, may be in part responsible for the phenomenon of ‘reverse remodelling’. Copyright © 2002 John Wiley & Sons, Ltd.

The successful kidney transplant must withstand the stresses of the transplant process and the posttransplant course and maintain itself over many years. Allograft nephropathy is the pathology most frequently associated with late renal graft loss. Allograft nephropathy is defined by graft dysfunction and a characteristic but unspecific pathology, with tubular atrophy, interstitial fibrosis, and fibrous intimal thickening of blood vessels. A number of early factors lead to the development of allograft nephropathy—older donor age, brain death, prolonged preservation time, and rejection—but drug toxicity, hypertension, and other recipient factors can contribute as time passes. Allograft nephropathy is the result of these cumulative injuries plus the inherent ability of the kidney to resist and repair injury. This overview highlights some nonimmunologic injuries important in the course of transplantation and in the evolution of allograft nephropathy.

I propose a set of definable entities in the renal transplant course, eliminating the need for the term ‘chronic rejection’. The status of a renal transplant can be defined by the presence and extent of rejection (T-cell-mediated or antibody-mediated); allograft nephropathy (parenchymal atrophy, fibrosis, and fibrous intimal thickening in arteries); transplant glomerulopathy; specific diseases; and factors which could accelerate progression. The level of function and the slope of the loss of function should be separately determined. This approach can be applied both in research and in clinical practice, and can be adapted to other organ transplants.

The immunosuppressive activity of cyclosporine is mediated by inhibiting calcineurin phosphatase. However, calcineurin is widely distributed in other tissues. We examined the degree of calcineurin inhibition by cyclosporine in various tissues. In vitro,the cyclosporine concentration inhibiting 50% (IC50) of calcineurin was ∼ 10 ng/mL in human and mouse leukocytes suspensions. In vitroand in vivoIC50s of cyclosporine in homogenates of mouse kidney, heart, liver, testis, and spleen were also comparable (9–48 ng/mL). The maximum calcineurin inhibition by cyclosporine varied, from 83 to 95% of calcineurin activity in spleen, kidney, liver, and testis to 60% in heart and only 10% in brain. Maximum calcineurin inhibition was increased by the addition of cyclophilin A, indicating that cyclophilin concentrations were limiting in some tissues, at least in this assay. Western analysis of mouse tissues showed significantly less cyclophilin in heart than other tissues. cyclosporine concentrations per weight of tissue protein were highest in kidney and liver and lowest in brain and testis after oral dosing, with intermediate levels in spleen, heart, and whole blood. Thus each cyclosporine dose produces rapid and widespread inhibition of calcineurin in tissues, with differences in total susceptibility of each tissue.

In transplant rejection interferon (IFN)-γ regulates the recipient immune response but also acts directly on IFN-γ receptors in the graft. We investigated these direct actions by comparing rejecting kidneys from donors lacking IFN-γ receptors (GRKO mice) or control donors (129Sv/J) in CBA recipients. Beginning day 5, 129Sv/J kidneys displayed high major histocompatibility complex (MHC) expression, progressive infiltration by inflammatory cells, but no thrombosis and little necrosis, even at day 21. GRKO kidneys showed increasing fibrin thrombi in small veins, peritubular capillary congestion, hyaline casts, and patchy parenchymal necrosis, progressing to near total necrosis at day 10. Terminal dUTP nick-end labeling assays were positive only in the interstitial infiltrate, confirming that massive cell death in GRKO transplants was not apoptotic. Paradoxically, GRKO kidneys showed little donor MHC induction and less inflammatory infiltration. Both GRKO and 129Sv/J allografts evoked vigorous host immune responses including alloantibody and mRNA for cytotoxic T cell genes (perforin, granzyme B, Fas ligand), and displayed similar expression of complement inhibitors (CD46, CD55, CD59). GRKO kidneys displayed less mRNA for inducible nitric oxide synthase and monokine inducible by IFN-γ but increased heme oxygenase-1 mRNA. Thus IFN-γ acting on IFN-γ receptors in allografts promotes infiltration and MHC induction but prevents early thrombosis, congestion, and necrosis.

As the molecular basis of the immune response is elucidated, the opportunities for intervention with agents with established sites of action increase. With conventional small molecular drugs, major sites of action have been identified, such as inhibition of calcineurin, target of rapapmycin, and inosine monophosphate dehydrogenase. However, relating these effects to the actions and toxicities of the drugs remains incomplete. The focus now is on further characterizing these important signaling pathways. The proteins targeting surface molecules such as CD3, CD40L, B7, and others are better understood. The accumulated experience with these interventions furthers our understanding of these agents and the human immune response. The failure of a promising agent in some trials may reveal an incomplete understanding of the targeted biologic process in vivo. Studies with gene knockout mice also help us better understand the roles of these molecules and the molecular basis of how these agents might work. Curr Opin Organ Transplant 2000, 5268–275 © 2000 Lippincott Williams & Wilkins, Inc.

The immunosuppressive drugs cyclosporine (CsA) and tacrolimus (FK506 or FK) are qualitatively similar but differ in molar potency. Both drugs sterically inhibit the phosphatase activity of calcineurin (CN) but differ in molar potency. In our study we explored whether differential inhibition of CN explained the differences in molar potency of FK versus CsA.

With the development of new immunosuppressive agents, the focus of anti-rejection therapy has shifted from prevention of acute allograft rejection to an emphasis on sufficient immunosuppression with minimal toxicity. Mycophenolate mofetil (MMF) is a recently developed immunosuppressive drug, which acts to inhibit T and B cell proliferation by blocking the production of guanosine nucleotides required for DNA synthesis. It also prevents the glycosylation of adhesion molecules that are involved in attachment of lymphocytes to endothelium and potentially in leukocyte infiltration of an allograft during an immune response. High-quality randomized clinical trials have demonstrated that MMF, when used with cyclosporine (CsA) and steroids, reduces the frequency and severity of acute rejection episodes in kidney and heart transplants, improves patient and graft survival in heart allograft recipients and increases renal allograft survival at 3 years. It has also been effective in reversing acute and resistant rejection episodes in heart, kidney and liver recipients. The ability of MMF to facilitate sparing of other immunosuppressive agents, particularly in CsA-related nephrotoxicity, is also promising. By permitting reduction in CsA doses, MMF may stabilize or improve renal graft function in patients with CsA-related nephrotoxicity or chronic allograft nephropathy. Early results of phase I and II trials evaluating MMF therapy in liver and combined pancreas/kidney transplant recipients are encouraging. The main adverse effects associated with oral or intravenous MMF are gastrointestinal and hematologic in nature. Although the direct costs of using MMF vs. azathioprine (AZA) are higher, the decreased incidence and treatment of acute rejection in patients treated with MMF supports its use as a cost-effective option during the first year following transplantation.

The pathogenetic role of interferon-γ (IFN-γ) in acute graft-versus-host disease (GVHD) was examined in a murine model. IFN-γ gene expression was evaluated by northern blotting and mRNA in situ hybridization. The temporal and tissue specific patterns of IFN-γ gene expression were related to the patterns of major histocompatibility complex (MHC) antigen induction and of tissue injury. Markedly increased levels of IFN-γ transcripts were seen in the spleen during the early lymphoproliferative phase and coincided with widespread MHC induction in non-lymphoid tissues. Increased IFN-γ transcripts were also found in the non-lymphoid target tissues during the phase of subsequent tissue injury. These findings support a role for IFN-γ in leading to widespread MHC induction during acute GVHD and suggest that IFN-γ may also contribute to target tissue injury during acute GVHD. Copyright © 1999 John Wiley & Sons, Ltd.

Cyclosporine (CsA) acts by inhibiting the phosphatase calcineurin (CN), but the time course and extent of inhibition in vivo are unknown. We examined the effect of single oral CsA doses on CN activity in humans and mice in vivo.

Poor initial function is common in cadaveric renal transplantation, and is usually attributed to acute tubular necrosis (ATN) brought about by ischemia during harvesting/implantation. However, this is often an assumption rather than a specific diagnosis. Recently, in 4 kidneys from 2 cadaver donors, we found evidence of severe endothelial injury, prior to exposure to cyclosporine or other known endothelial toxins. The biopsies at the time of completion of the transplant revealed apparent loss of glomerular endothelial cells on light microscopy, corresponding on electron microscopy to shrinkage of the endothelial cells away from the basement membranes of the capillary loops. Extensive microvascular thrombi were present. All 4 grafts displayed impaired initial function, which partially recovered with time. The finding of these unusual lesions in both kidneys from each of 2 donors suggested donor factors, although the only factor common to both donors was massive brain disruption. Thus, in the differential diagnosis of poor initial function in kidneys transplanted from cadaver donors, one should consider renal endothelial injury, which could lead to microthrombus formation, abnormal vasomotion, and functional impairment in the transplant.

In many studies of renal transplant recipients, acute tubular necrosis has been shown to predispose to a higher rate of graft loss, apparently due to rejection, but the mechanism of this effect is unknown. One possibility is an increased immunogenicity of the graft. To study this possibility, we examined the expression of major histocompatibility complex antigens in kidneys damaged by ischemia, using a mouse model of ischemic ATN. ATN was produced in the left kidney of male CBA mice by temporary clamping of the vascular pedicle for up to 60 min. Class I and II MHC expression was quantified by the extent of binding of monoclonals in radioimmunoassay, after 1 to 35 days in both kidneys. MHC induction was localized by indirect immunoperoxidase staining. Specific steady state mRNA for β2microglobulin and class II were quantified by northern blotting using 32P-labeled probes. Changes in MHC expression were assessed by comparing the ischemically injured left kidney to the control right kidney. By day 1, ATN was evident by histology but there was no change in MHC expression. By day 3, class I was increased in the left kidney by 3–6-fold over the right. In tissue sections, the class I increase was localized to tubular epithelial cells. Starting on day 7 and persisting to day 35, class II was increased by 1.5 to 3 times for the ischemic kidney over the control, primarily in interstitial cells but also in tubular cells. This increase in class II was associated with the appearance of Thy 1.2-positive cells in the interstitial areas. Increased antigen expression was preceded by increased steady state mRNA. Thus unilateral ischemic ATN causes increased MHC expression in tubular cells and the accumulation of an inflammatory infiltrate, both of which may contribute to the increased rate of rejection and graft loss in ischemically injured kidneys.

In renal transplantation, preformed cytotoxic antibody against donor HLA class I antigens causes hyper-acute rejection of renal allografts, but its pathogenic significance when it develops in the posttransplant period is unknown. In the present studies we describe the clinical and pathologic features of patients with rejection associated with anti-class I. In the course of 400 consecutive cadaveric renal transplants, 7 patients were identified who had antibody against donor class I HLA antigens in association with atypical but distinctive patterns of rejection. All 7 were presensitized. In 3 patients, the transplant had been inadvertently performed with a positive donor-specific T cell crossmatch. In the remaining 4, the T cell crossmatch on current sera was negative but became positive posttransplant. The clinical picture was deterioration of graft function with rapid onset of oliguria, apparently due to acute tubular necrosis, but with persistence of blood flow demonstrable by radioisotope scan studies. Renal histology showed that the typical lesions observed in cell-mediated rejection, such as tubulitis and interstitial infiltration, were absent. Granular complement deposition (6), polymorphonuclear infiltration (6), and endothelial injury in the microvasculature (6) were common, and mononuclear infiltrates were absent (2) or not prominent (4). In 3 patients the glomerular changes resembled a picture of hemolytic uremic syndrome, with capillary fibrin thrombi and widening of the subendothelial space. IgG staining was negative. The pathologic features suggest that anti—class I antibody appearing or persisting in the early posttransplant period injures the endothelium of the microvasculature, with the clinical presentation different from that of hyperacute rejection. Particularly in sensitized patients, rapid deterioration in function, leading to a picture of acute tubular necrosis, with pathologic features of endothelial injury in the microcirculation, should suggest the diagnosis of anti-class I-mediated rejection.

The origins of Ia antigens in perfused mouse kidney were investigated. Three possible sources were considered: leukocytes in residual blood which was trapped in kidney, bone marrow-derived cells resident in kidney, and nonbone marrow-derived renal parenchymal or vascular cells. Leukocytes in trapped blood seemed to make no significant contribution to renal Ia expression because (1) perfused kidney had approximately as much Ia as nonperfused kidney, even though the perfusion reduced the blood content by 90; (2) the estimated number of leukocytes in trapped blood was at least three orders of magnitude less than that needed to account for Ia expression by kidney; and (3) perfused kidney, volume for volume, absorbed more anti-Ia than did whole blood, so that no amount of blood contamination could account for all renal Ia expression. Thus, most Ia in kidney must be on resident cells, either bone marrow-derived or parenchymal. To demonstrate bone marrow-derived Ia-positive cells, we created radiation chimeras of (B10 × B10.D2)F1bone marrow into B10 hosts. Ia of (B10 × B10.D2)F1bone marrow donor origin was easily detectable in kidneys of these chimeras at 4 months. However, we also demonstrated Ia of nonbone marrow donor origin in chimera kidney: long-term B10.A into (BALB/c × A)F1chimeras and C57BL/6 into (C57BL/6 × DBA/2) F1chimeras continued to express renal Ia of bone marrow recipient origin. Thus, some renal Ia is produced by bone marrow-derived cells, and some is produced by cells which are nonmarrow derived (or are marrow derived but are, resistant to replacement in bone marrow chimeras). The cells expressing Ia in kidney were unlikely to be thymus derived because anti-Thy-1.2 was not absorbable by the same kidney preparations which absorbed anti-Ia.

A new radioisotope assay for the quantitative assessment of macrophage-alloantibody-target cell interaction was studied. The assay, abrogation of complement lysis (ACL), is based on the observation that antibody-coated cells incubated with macrophages become resistant to complement lysis. The degree of reduction in complement lysis, as measured by 51Cr release, can therefore be used to assess the extent of macrophage engulfment of the target cells. Previous incubation of macrophages with antigen-antibody complexes inhibited ACL, confirming that Fc receptors were necessary for ACL. However, the temperature dependence of ACL, and the inability of many types of Fc receptor-bearing nonphagocytic cell populations to mediate ACL indicated that macrophage effector function after Fc receptor binding was essential for ACL. Microscopic assessment confirmed that the macrophage activity producing most of the ACL was engulfment of the cells, although certain observations suggested that a nonphagocytic effect of macrophages on the target cells may be detectable in ACL under certain circumstances. Macrophage activation strongly influenced the degree of ACL and of phagocytosis, the chief effect being not on the absolute number, but on the percentage of target cells engulfed by macrophages. The simplicity, objectivity, and rapidity of ACL suggest that this will be a useful technique for exploring the role of macrophages in humoral alloimmunity.

Two nonphagocytic effects of macrophages on antibodycoated lymphocyte target cells were studied: antibody-dependent cell-mediated cytotoxicity (ADCC) and abrogation of complement lysis (ACL) by nonphagocytic mechanisms. Macrophages did not mediate ADCC, and actually inhibited K cell-mediated ADCC, presumably by direct interactions with the antibody-coated target cells. These interactions were studied in the ACL assay previously described, in which macrophages, antibody, and 51Cr-labelled target cells were incubated for 1 hr. Then complement lysis (as measured by 51Cr release) was performed to assess the status of the target cells. The nonphagocytic component of ACL could be distinguished from the phagocytic component by the addition of a second antibody during the complement lysis phase. This procedure revealed that some of the target cells which were resistant to the original antibody were susceptible to lysis by a second antibody and were therefore not phagocytized. Such cells had apparently been stripped of their antibody and the associated antigen by the macrophages. In support of this interpretation, specific antigen alterations were demonstrable on these stripped cells under certain conditions. These alterations were produced more consistently when the macrophages were less than maximally stimulated, and were detected better by guinea pig complement than by rabbit complement. The mechanism of stripping may involve inactivation, redistribution, or removal of target cell-bound antibody by the macrophages. Possible in vivo roles for the stripping mechanism, for example, in the enhancement of tumours or allografts, are discussed.

Mycophenolate mofetil is a new immunosuppressive agent recently released for clinical use, in conjunction with steroids and cyclosporine, for the prevention of acute rejection in renal transplant patients. It inhibits the enzyme inosine monophosphate dehydrogenase, which is essential for de-novo synthesis of guanine nucleotides. The result is lymphocyte-selective arrest of cell division, with little effect on other tissues. In clinical trials in renal transplant, its efficacy and safety have been established mycophenolate mofetil reduces acute rejection episodes by approximately 50, with the principal toxicity being gastrointestinal, and perhaps some increase in cytomegalovirus infection. Overall, mycophenolate mofetil (plus cyclosporine and steroids) has a toxicity profile similar to that of azathioprine but with considerably increased efficacy. Despite its cost, mycophenolate mofetil is likely to be used in many renal transplant centres to reduce acute rejection, especially in the first year. Future trials are necessary to establish its long-term role in renal transplantation and its usefulness in the transplantation of other organs.

The 38th attempt at allotransplantation of a human long is described in a patient with injury due to smoke inhalation. The innovative features in the procedure included prolonged support with an extracorporeal membrane oxygenator during and for four days following transplantation, pharmacologic control of platelet function with sulfinpyrazone, continuous monitoring with a fiberoptic ear oximeter, and pretreatment of the transplanted lung with cytotoxic drugs. The patient survived until the 18th postoperative day, with no evidence of tissue rejection, but he died following ischemic disruption of the bronchial anastomosis. We conclude that the major determinant in the future of human lung transplantation is related to the establishment of a bronchial arterial supply to the transplanted bronchus.

Cyclosporine (CsA) inhibits cytokine transcription by preventing the activation of key promoter sites, in particular the binding of nuclear factor of activated T cells (NFAT) to the IL-2 NFAT site and the “P” site in IL-4. To identify potential NFAT-like sites in the IFN-γ promoter, we sought areas of homology with the known sites in other promoters. In the promoter region of the mouse and human IFN-γ gene, we identified two repeats of a consensus sequence ATTTCCnnT, designated P1 and P2 because of their homology to the calcium-inducible and CsA-sensitive “P” sequences in the IL-4 promoter. In electrophoretic mobility shift assay (EMSA), a probe containing the second P sequence “P2” in the human IFN-γ gene bound nuclear proteins from stimulated, but not unstimulated, human T cells. The cytosol of unstimulated cells contained similar binding activity that decreased after stimulation, indicating that this binding activity translocated to the nucleus after stimulation. CsA inhibited nuclear translocation. Competition studies demonstrated that oligomers containing the sequences P1 and P2 in IFN-γ gene, the NFAT site in the IL-2 gene, and the IL-4 P site competed with the P2 probe for protein binding, whereas an oligomer containing mutations in the P2 site did not. Addition of anti-NFAT antiserum altered protein binding to P2, indicating that the proteins binding were either identical or related to NFAT. Stimulation of T cells transfected with constructs containing three copies of the P2 sequence enhanced CAT activity in response to ionomycin, and this effect was blocked by CsA. These results suggest that the P2 sequence, and probably the P1 sequence, in the IFN-γ promoter are NFAT binding sites and contribute to the calcium inducibility and CsA sensitivity of IFN-γ production.

During studies to investigate the localization of Ia antigens in normal mouse kidney, a monoclonal antibody Mab specific for IAbby microcytotoxicity criteria was found to crossreact with a tissue antigen present in the glomeruli and vasculature of murine tissues, irrespective of their Ia haplotype, as well as in human kidneys. Immunofluorescence and immunoperoxidase studies of frozen tissue sections revealed that the antigen responsible for the crossreaction was present in the cytoplasm of glomerular epithelial cells and of smooth muscle of blood vessels. The immunofluorescence pattern of primary cultures of human glomerular epithelial cells and of a glioblastoma cell line indicated that the crossreactive antigen forms part of the cytoskeleton, and resembled the pattern observed with a monoclonal against vimentin, one of the intermediate filaments. Thus this Mab, which is directed against mouse alloantigen Ia.20 in microcytotoxicity was found to crossreact with a cytoskeletal component present in the mouse, as well as in human glomerular epithelial and smooth muscle cells.

We noted previously that ischemic acute tubular necrosis ATN induces local expression of MHC products in renal epithelium. The present investigations were conducted to establish the role of IFN in the regulation of MHC antigen expression in ATN and to explore the changes in cytokine and growth factor expression induced by ischemic renal injury. We produced unilateral ischemic ATN in mice by clamping the left renal pedicle. MHC class I and II steady state mRNA induction was assessed by northern blot analysis, and MHC product was quantified by the extent of binding of radiolabeled monoclonals to tissue homogenates. The steady state mRNA levels for IFN, IL2, IL10, and granulocytemacrophage CSF were assessed by reverse transcriptase polymerase chain reaction and the levels for transforming growth factor1 and preproepidermal growth factor ppEGF were assessed by Northern blot analysis. In the injured kidneys, steady state mRNA levels for IFN, IL2, IL10, granulocytemacrophage CSF, and transforming growth factor1 were increased, whereasppEGF mRNA was markedly decreased. The MHC expression was inhibited by treatment of mice with an antiIFN mAb R46A2. Murine EGF, administered in an attempt to accelerate recovery, did not reduce the cytokine and MHC changes. These data indicate that ischemic injury, and possibly other forms of injury, triggers a complex circuit of proinflammatory cytokines. This “injury response” could be relevant to clinical renal transplants, where ATN is associated with poor graft outcome.

Cryopreservation is an effective method of islet storage that can facilitate clinical trials of islet transplantation. In the present study we examined the effect of cryopreservation on the survival of islet allografts and the quantity of islet MHC antigen expression. Islets isolated from CBA/J (H-2k) mice were transplanted into streptozotocin-induced diabetic BALB/c (H-2d) mice treated with or without antilymphocyte serum (ALS). Frozen/thawed (F/T) grafts were cooled slowly to −40oC, stored at −196oC, and thawed rapidly. Fresh and F/T isograft controls reversed diabetes promptly and maintained normoglycemia > 100 days. Allografts of fresh and F/T islets induced normoglycemia initially, but graft failure ensued at 18.2 ± 1.5 and 16.4 ± 1.9 days, respectively. ALS treatment prolonged allograft survival significantly to 35.3 ± 3.9 and 37.5 ± 6.3 days for fresh and F/T islets, respectively. Following cryopreservation, the quantity of class I antigen expression was reduced by 40, while the quantity of class II expression was variable. These data indicate that murine islet MHC class I expression is reduced after cryopreservation. This decrease was not associated with altered survival of allogeneic grafts.