Supplemental hydrogen sulfide in models of renal transplantation after cardiac death.

BACKGROUND: The increasing use of kidneys from donations after cardiac death (DCD) for renal transplantation is hindered by negative outcomes owing to organ injury after prolonged warm and cold ischemia-reperfusion. Recently, hydrogen sulfide (H2S) has shown cytoprotective effects against ischemia-reperfusion injury; however, its effectiveness in the context of DCD renal transplantation is unknown. METHODS: We tested a novel 30-day in vivo syngeneic murine model of DCD renal transplantation, in which the donor kidney was clamped for 30 minutes and stored for 18 hours in cold University of Wisconsin (UW) solution or UW with 150 µM sodium hydrogen sulfide (UW + NaHS) before transplantation. We also tested a 7-day in vivo porcine model of DCD renal autotransplantation, in which the left kidney was clamped for 60 minutes and preserved for 24 hours using hypothermic perfusion with UW or UW + 150 µM NaHS before autotransplantation. We collected blood and urine samples periodically, and collected kidney samples at the end point for histopathology and quantitative reverse transcription polymerase chain reaction. RESULTS: Rats that received H2S-treated kidneys showed significantly higher survival, faster recovery of graft function and significantly lower acute tubular necrosis than controls. Pig kidneys perfused with UW + NaHS showed significantly higher renal blood flow and lower renal resistance than control kidneys after 24 hours of perfusion. After autotransplantation, pigs that received H2S-treated kidneys showed significantly lower serum creatinine on days 1 and 7 after transplantation. Rat and pig kidneys treated with H2S also showed more protective gene expression profiles than controls. CONCLUSION: Our findings support the potential use of H2S-supplemented UW solution during cold storage as a novel and practical means to improve DCD graft survival and function.

Cyclophilin D Regulates the Nuclear Translocation of AIF, Cardiac Endothelial Cell Necroptosis and Murine Cardiac Transplant Injury.

Ischemia-reperfusion injury (IRI) is an inevitable consequence of organ transplant procedure and associated with acute and chronic organ rejection in transplantation. IRI leads to various forms of programmed cell death, which worsens tissue damage and accelerates transplant rejection. We recently demonstrated that necroptosis participates in murine cardiac microvascular endothelial cell (MVEC) death and murine cardiac transplant rejection. However, MVEC death under a more complex IRI model has not been studied. In this study, we found that simulating IRI conditions in vitro by hypoxia, reoxygenation and treatment with inflammatory cytokines induced necroptosis in MVECs. Interestingly, the apoptosis-inducing factor (AIF) translocated to the nucleus during MVEC necroptosis, which is regulated by the mitochondrial permeability molecule cyclophilin D (CypD). Furthermore, CypD deficiency in donor cardiac grafts inhibited AIF translocation and mitigated graft IRI and rejection (n = 7; p = 0.002). Our studies indicate that CypD and AIF play significant roles in MVEC necroptosis and cardiac transplant rejection following IRI. Targeting CypD and its downstream AIF may be a plausible approach to inhibit IRI-caused cardiac damage and improve transplant survival.

An Addition of U0126 Protecting Heart Grafts From Prolonged Cold Ischemia-Reperfusion Injury in Heart Transplantation: A New Preservation Strategy.

BACKGROUND: Ischemia-reperfusion injury (IRI) is the major cause of primary graft dysfunction in organ transplantation. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in cell physiological and pathological processes including IRI. This study aims to investigate whether inhibition of ERK signaling with U0126 can prevent prolonged cold IRI in heart transplantation. METHODS: Rat cardiac cell line H9c2 cells were treated with U0126 before exposure to hypothermic hypoxia/reoxygenation (H/R) conditions. The effect of U0126 on H9c2 cells in response to H/R stress was determined by measuring cell death, reactive oxygen species production, mitochondrial membrane potential, and ERK signaling activation. Mouse syngeneic heterotopic heart transplantation was conducted, where a donor heart was preserved in the University of Wisconsin (UW) solution supplemented with U0126 for 24 hours at 4°C before transplantation. Heart graft function, histopathologic changes, apoptosis, and fibrosis were measured to assess IRI. RESULTS: Phosphorylated ERK was increased in both in vitro H/R-injured H9c2 cells and in vivo heart grafts with IRI. Pretreatment with U0126 inhibited ERK phosphorylation and prevented H9c2 cells from cell death, reactive oxygen species generation, and mitochondrial membrane potential loss in response to H/R. Preservation of donor hearts with U0126-supplemented solution improved graft function and reduced IRI by reductions in cell apoptosis/death, neutrophil infiltration, and fibrosis of the graft. CONCLUSIONS: Addition of U0126 to UW solution reduces ERK signal activation and attenuates prolonged cold IRI in a heart transplantation model. ERK inhibition with U0126 may be a useful strategy to minimize IRI in organ transplantation.

Loss of receptor interacting protein kinases 3 and caspase-8 augments intrinsic apoptosis in tubular epithelial cell and promote kidney ischaemia-reperfusion injury.

BACKGROUND: Ischaemia-reperfusion injury (IRI) is associated with programmed cell death that promotes inflammation and organ dysfunction. Necroptosis is mediated by members of receptor interacting protein kinases (RIPK1/3). Inhibition of RIPK1/3 provides a pro-survival benefit in kidney IRI. Caspase-8 initiates apoptosis and contributes to IRI. We studied whether inhibiting both RIPK3 and caspase-8 would provide an additional benefit in kidney IRI. METHODS: A clamp was applied to the left kidney pedicle for 45 min followed by right kidney nephrectomy. Kidney and serum from wild type, RIPK3-/- , and RIPK3-/- caspase-8-/- double knockout (DKO) mice were collected post-IRI for assessment of injury. Tubular epithelial cells (TEC) isolated from wild type, RIPK3-/- , and DKO mice were treated with interferons-γ and interleukin-1ß to induce apoptotic death. RESULTS: Kidney IRI of DKO mice did not show improvement over RIPK3-/- mice. We have found that DKO triggered 'intrinsic' apoptosis in TEC in response to interleukin-1ß and interferons-γ. Up-regulation of the B-cell lymphoma 2 (Bcl-2)-associated death promoter, the Bcl-2-homologous antagonist killer and Bcl-2-associated X protein and enhanced activation of caspase-3 and 9 were found in DKO TEC. TEC infected with Murine cytomegalovirus that encodes multiple cell death inhibitors resist to death. CONCLUSION: We show that the deletion of both RIPK3 and caspase-8 does not provide additive benefit in IRI or TEC death and may enhance injury by up-regulation of intrinsic apoptosis. This suggests blocking multiple death pathways may be required for the prevention of kidney IRI clinically.

Mitochondrial permeability regulates cardiac endothelial cell necroptosis and cardiac allograft rejection.

Transplantation is invariably associated with programmed cell death including apoptosis and necrosis, resulting in delayed graft function and organ rejection. We have demonstrated the contribution of necroptosis to mouse microvascular endothelial cell (MVEC) death and transplant rejection. Organ injury results in the opening of mitochondrial permeability transition pores (mPTPs), which can trigger apoptotic molecules release that ultimately results in cell death. The effect of mPTPs in the necroptotic pathway remains controversial; importantly, their role in transplant rejection is not clear. In this study, tumor necrosis factor-α triggered MVECs to undergo receptor-interacting protein kinase family (RIPK1/3)-dependent necroptosis. Interestingly, inhibition of mPTP opening could also inhibit necroptotic cell death. Cyclophilin-D (Cyp-D) is a key regulator of the mPTPs. Both inhibition and deficiency of Cyp-D protected MVECs from necroptosis (n = 3, P < .00001). Additionally, inhibition of Cyp-D attenuated RIPK3-downstream mixed-lineage kinase domain-like protein phosphorylation. In vivo, Cyp-D-deficient cardiac grafts showed prolonged survival in allogeneic BALB/c mice posttransplant compared with wild-type grafts (n = 7, P < .0001). Our study results suggest that the mPTPs may be important mechanistic mediators of necroptosis in cardiac grafts. There is therapeutic potential in targeting cell death via inhibition of the mPTP-regulating molecule Cyp-D to prevent cardiac graft rejection.

Over-expression of growth differentiation factor 15 (GDF15) preventing cold ischemia reperfusion (I/R) injury in heart transplantation through Foxo3a signaling.

Ischemia reperfusion (I/R) injury which inevitably occurs during heart transplantation is the major factor leading to organ failure and graft rejection. In order to develop new therapies to prevent I/R injury, we used both a murine heart transplantation model with 24 hour cold I/R and an in vitro cell culture system to determine whether growth differentiation factor 15 (GDF15) is a protective factor in preventing I/R injury in heart transplantation and to further investigate underlying mechanisms of I/R injury. We found that cold I/R caused severe damage to the endocardium, epicardium and myocardium of heart grafts from wild type C57BL/6 mice, whereas grafts from GDF15 transgenic (TG) mice showed less damage as demonstrated by decreased cell apoptosis/death, decreased neutrophils infiltration and the preservation of the normal structure of the heart. Over-expression of GDF15 reduced expression of phosphorylated RelA p65, pre-inflammatory and pro-apoptotic genes while it enhanced Foxo3a phosphorylation in vitro and in vivo. Over-expression of GDF15 inhibited cell apoptosis/death and reduced neutrophil infiltration. In conclusion, this study, for the first time, demonstrates that GDF15 is a promising target for preventing cold I/R injury in heart transplantation. This study also shows that the resultant protective effects are mediated by the Foxo3 and NFκB signaling pathways.

Necroptosis Is Involved in CD4+ T Cell-Mediated Microvascular Endothelial Cell Death and Chronic Cardiac Allograft Rejection.

BACKGROUND: Despite advances in immunosuppressive therapies, the rate of chronic transplant loss remains substantial. Organ injury involves various forms of cell death including apoptosis and necrosis. We now recognize that early injury of cardiac transplants involves a newly described form of programmed necrotic cell death, termed necroptosis. Because this involves receptor-interacting protein (RIP) kinase 1/3, this study aimed to establish the role of RIP3 in chronic cardiac allograft rejection. METHODS: We used major histocompatibility complex class II mismatched C57BL/6N (H-2; B6) or B6.RIP3 (H-2; RIP3) mice to B6.C-H-2 (H2-Ab1; bm12) mouse cardiac transplantation. Microvascular endothelial cells (MVEC) were developed from B6 and RIP3 cardiac grafts. RESULT: CD4 T cell-mediated cardiac graft rejection is inhibited using RIP3 deficient donor grafts, with reduced cellular infiltration and vasculopathy compared with wild type cardiac grafts. Alloreactive CD4 T cell-mediated MVEC death involves TNFα, Fas ligand (FasL) and granzyme B. Although necroptosis and release of danger molecule high-mobility group box 1 are eliminated by the absence of RIP3, CD4 T cells had attenuated MVEC death through granzyme B and FasL. CONCLUSIONS: CD4 T cell-mediated MVEC death involves in TNFα, FasL and granzyme B. Necroptotic cell death and release of the danger molecule may promote inflammatory responses and transplant rejection. Although loss of RIP3 does not eliminate alloimmune responses, chronic graft injury is reduced. RIP3 is an important therapeutic target but additional granzyme and caspases inhibition is required for sufficiently improving long-term graft survival.

Attenuating Ischemia-Reperfusion Injury in Kidney Transplantation by Perfusing Donor Organs With siRNA Cocktail Solution.

BACKGROUND: Ischemia-reperfusion (I/R) injury is the major cause of delayed renal graft function in kidney transplantation. To date, there are no effective therapeutic approaches for preventing I/R injury. We previously reported that treatment of animals with small interference RNA (siRNA) would prevent warm I/R injury in nontransplant models and cold I/R injury in heart transplantation. In the present study, we further explore the feasibility of protecting grafts from extended cold I/R injury as applied to kidney transplantation by downregulating I/R-associated genes using siRNA. METHODS: Donor kidneys were intra-arterially perfused with siRNA containing solution during donor excision and preserved in siRNA containing solution. The siRNA-treated donor organs were then implanted into syngeneic recipient mice, and the 2 original kidneys were removed from the recipient. The effect of siRNA solution on extended cold I/R injury was determined by assessing renal function, histopathological change, cell apoptosis, and inflammation. RESULTS: The perfused siRNA solution knocked down the expression of complement 3, RelB, and Fas in the kidney at the mRNA and protein levels. Administration of siRNA solution reduced the levels of blood urea nitrogen and serum creatinine as compared with control groups. The siRNA cocktail decreased cell apoptosis and histopathological changes in the kidney and prolonged graft survival. The siRNA cocktail also reduced the expression of proinflammatory cytokines, IL-6, and TNFα. CONCLUSIONS: In conclusion, this is the first demonstration that perfusing donor organs with an siRNA cocktail solution can induce gene silencing in the kidney and prevent kidneys from extended cold I/R injury in kidney transplantation, highlighting the promise of the clinical application of siRNA-based therapies in the preservation of donor organs.

Natural killer cells play a critical role in cardiac allograft vasculopathy in an interleukin-6--dependent manner.

BACKGROUND: Approximately 50% of cardiac transplants fail in the long term, and currently, there are no specific treatments to prevent chronic rejection. In the clinic, donor cardiac graft ischemia time is limited to within a few hours and correlates with delayed graft function and organ failure. It is still unknown how ischemic injury negatively influences allograft function over the long term despite advances in immunosuppression therapy. METHODS: Allogeneic cardiac grafts were stored at 4 °C for 4 hr before being transplanted into T/B cell-deficient Rag(-/-) mice or T/B/natural killer (NK) cell-deficient γc(-/-)Rag(-/-) mice. Grafts were harvested 60 days after transplantation and indicators of chronic allograft vasculopathy (CAV) were quantified. RESULTS: We have found that cold ischemia of cardiac grafts induces CAV after transplantation into Rag1(-/-) mice. Interestingly, cold ischemia-induced CAV posttransplantation was not seen in T/B/NK cell-deficient γc(-/-)Rag(-/-) mice. However, cardiac grafts in γc(-/-)Rag(-/-) mice that received an adoptive transfer of NK cells developed CAV, supporting the role of NK cells in CAV development. Analysis of various cytokines that contribute to NK cell function revealed high interleukin (IL)-6 expression in cardiac grafts with CAV. In addition, IL-6-deficient cardiac grafts did not develop CAV after transplantation into allogeneic Rag(-/-) mice. CONCLUSION: These data demonstrate that cold ischemia and NK cells play critical roles in the development CAV. Natural killer cells and injured grafts may play a reciprocal role for CAV development in an IL-6-independent manner. Specific therapeutic strategies may be required to attenuate NK cell contribution to chronic cardiac rejection.

Lack of renal dopamine D5 receptors promotes hypertension.

Disruption of the dopamine D(5) receptor gene in mice increases BP and causes salt sensitivity. To determine the role of renal versus extrarenal D(5) receptors in BP regulation, we performed cross-renal transplantation experiments. BP was similar between wild-type mice and wild-type mice transplanted with wild-type kidneys, indicating that the transplantation procedure did not affect BP. BP was lower among D(5)(-/-) mice transplanted with wild-type kidneys than D(5)(-/-) kidneys, demonstrating that the renal D(5) receptors are important in BP control. BP was higher in wild-type mice transplanted with D(5)(-/-) kidneys than wild-type kidneys but not significantly different from syngenic transplanted D(5)(-/-) mice, indicating the importance of the kidney in the development of hypertension. On a high-salt diet, all mice with D(5)(-/-) kidneys excreted less sodium than mice with wild-type kidneys. Transplantation of a wild-type kidney into a D(5)(-/-) mouse decreased the renal expression of AT(1) receptors and Nox-2. Conversely, transplantation of a D(5)(-/-) kidney into a wild-type mouse increased the expression of both, suggesting that both renal and extrarenal factors are important in the regulation of AT(1) receptor and Nox-2 expression. These results highlight the role of renal D(5) receptors in BP homeostasis and the pathogenesis of hypertension.

Regulatory T-cell generation and kidney allograft tolerance induced by mesenchymal stem cells associated with indoleamine 2,3-dioxygenase expression.

BACKGROUND: The immunoregulatory properties of mesenchymal stem cells (MSCs) have been observed in vitro and in vivo. However, the underlying mechanisms of this immunomodulation remain undefined. Recent research demonstrated that MSCs express the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO), known to suppress T-cell responses. This study was designed to address whether MSCs induce kidney allograft tolerance and whether IDO contributes to the immunoregulatory functions of MSCs in vivo. METHODS: MSCs (1×10(6), intravenously) from wild-type (WT-MSCs) or IDO knockout (IDO(-/-)-MSCs) C57BL/6 mice were injected into BALB/c recipients 24 hr after receiving a life-supporting orthotopic C57BL/6 renal graft. RESULTS: WT-MSC-treated recipients achieved allograft tolerance with normal histology and undetectable antidonor antibody levels. Tolerant recipients demonstrated increased circulating kynurenine levels and significantly high frequencies of tolerogenic dendritic cells. They also exhibited significantly impaired CD4+ T-cell responses consisting of decreased donor-specific proliferative ability and a Th2-dominant cytokine shift. In addition, high frequencies of CD4+CD25+Foxp3+ regulatory T cells (Tregs) were found in recipient spleens and donor grafts, with antibody-induced CD25+ cell depletion confirming the critical role of Tregs in the MSC-induced tolerance. Interestingly, renal allograft recipients treated with WT MSCs concomitant with the IDO inhibitor 1-methyl-tryptophan, or those treated with IDO(-/-)-MSCs alone, were unable to achieve allograft tolerance--revealing that functional IDO was necessary for the immunosuppression observed with WT-MSC treatment. CONCLUSIONS: IDO secreted by MSCs was responsible, at least in part, for induction of kidney allograft tolerance through generation of Tregs. This study supports the clinical application of MSCs in transplantation.

Free bone graft attenuates acute rejection and in combination with cyclosporin a leads to indefinite cardiac allograft survival.

We report on a novel approach aimed at preventing acute vascular rejection (AVR), one of the major unresolved hurdles of clinical transplantation. In a C3H-to-BALB/c heterotopic heart transplant model, we demonstrate that free bone transplantation combined with cyclosporin A suppresses antidonor Ab responses, induces indefinite cardiac allograft survival (>100 days), and preserves graft architecture. In contrast, untreated- or cyclosporin A alone-treated recipients rejected their cardiac grafts on days 7.7 +/- 0.6 and 15.5 +/- 1.1, respectively, with graft histology indicative of AVR. Splenic dendritic cells from nonrejecting recipients expressed low levels of MHC II, CD40, and CD86, reduced ability to stimulate donor cell proliferation, and augmented IL-10 production of responding T cells in vitro. Adoptive transfer of dendritic cells from long-term surviving recipients 1 day before cardiac grafting was able to confer hyporesponsiveness to naive BALB/c recipients of cardiac allografts. To determine whether graft survival was associated with hematopoietic or stromal elements of the transplanted free bone, we administered isolated bone marrow mononuclear cells or free bone that was irradiated to deplete hematopoietic elements. Although bone marrow mononuclear cells had no effect on cardiac graft survival, irradiated free bone transplantation was capable of prolonging graft survival. Most interestingly, the prolongation effect was Ag nonspecific, because third party irradiated bone graft was also effective. Due to the fact that current immunosuppressive approaches are clinically ineffective at preventing AVR, this study provides promise for further investigations of BM components as a means of addressing a currently unmet medical need.

Reduction of Foxp3-expressing regulatory T cell infiltrates during the progression of renal allograft rejection in a mouse model.

BACKGROUND: Regulatory T (Treg) cells are the immune suppressors in the maintenance of immune homeostasis and tolerance to self and non-self antigens, and may have therapeutic potential in the treatment of transplant rejection in patients. However, Treg cell development and action are poorly understood in transplantation. In this study, the association of CD4(+)Foxp3(+) infiltrates within renal allograft tissue with graft survival was investigated in a mouse model. METHODS: Kidney donors from C57BL/6J mice (H-2(b)) were transplanted to bilaterally nephrectomized Balb/c recipient mice (H-2(d)). Treg cells were examined with FACS and immunohistochemical staining. RESULTS: Here we showed that without any immunosuppressive regimen, kidney allografts were mostly rejected from 20 to 60 days after transplantation. During the progression of allograft rejection Foxp3(+) Treg phenotype infiltrates were significantly diminished, while intragraft expression of TGF-beta1, IL-6, IL-17 and IL-23 was up-regulated. The regulatory function of CD4(+)CD25(+) infiltrates was confirmed by their suppressive activity in mixed lymphocyte reaction. Further in vitro studies indicated that primary renal tubular epithelial cell (TEC) cultures produced high levels of IL-6 in response to allogeneic lymphocyte or IL-17 stimulation, and neutralization of IL-6 increased CD4(+)CD25(+)Foxp3(+) cells in co-cultures with TEC. CONCLUSION: Diminution of Foxp3(+) Treg infiltrates associates with renal allograft rejection, and neutralization of IL-6 activity enhances Foxp3(+) Treg cell differentiation. Our findings suggest that increase in intragraft IL-6 may down-regulate infiltrating Foxp3(+) Treg cells.

Establishment of duodenojejunal bypass surgery in mice: a model designed for diabetic research.

We have developed a mouse duodenojejunal bypass (DJB) surgical model that is for studying the effects of bariatric surgery on glucose homeostasis and has potential to impact clinical therapy of diabetes. The operation consists of using the majority of the duodenum and proximal part of the jejunum for biliopancreatic diversion. The distal end of the jejunum is anastomosed in an end-to-end fashion to the remaining proximal end of the duodenum just distal to the pylorus. The biliopancreatic secretions are diverted into the distal jejunum through an end-to-side anastomosis. We performed 10 DJB operations in C57BL/6 mice, with a 100% survival rate. The surgery had no effect on the growth or feeding patterns of the animals. The intestinal mucosa showed normal histology and function. This study confirms that it is technically possible to perform DJB surgery in mice. This mouse model can be used in the study of surgical treatment for type II diabetes.

Induction of indefinite cardiac allograft survival correlates with toll-like receptor 2 and 4 downregulation after serine protease inhibitor-1 (Serp-1) treatment.

BACKGROUND: Innate immunity provides obstacles to successful organ transplantation, which cannot be prevented by cyclosporine (CsA). Here we have determined the potential of a myxoma viral serpin, Serp-1, with proven anti-inflammatory and antiatherogenic actions, to modulate innate immunity and contribute synergistically with CsA in the prevention of acute cardiac allograft rejection. METHODS: Brown-Norway rat hearts were heterotopically transplanted into Lewis rats and given either a monotherapy treatment of Serp-1, a subtherapeutic dose of CsA, or the two drugs in combination. RESULTS: A brief treatment of Serp-1 alone, or a subtherapeutic dose of CsA, resulted in a marked decrease in intragraft macrophage infiltration and downregulation of toll-like receptor (TLR)-2, TLR4 and MyD88 at 48 hours posttransplantation, which was associated with significantly reduced numbers of mature dendritic cells. A significant reduction in intragraft T-lymphocyte infiltration was observed with both Serp-1 monotherapy and Serp-1 and CsA combination therapy, with the combination treatment achieving indefinite graft survival (>100 days) with normal histology. The CsA monotherapy group displayed partially reduced lymphocyte infiltration compared to the untreated controls, but failed to inhibit early innate immune graft recognition events such as macrophage infiltration and TLR 2, TLR4, and MyD88, and was ultimately unsuccessful in preventing rejection (36.3+/-7.8 days). CONCLUSION: Observed suppressive effects of Serp-1 on early innate immune response components such as TLR-2 and 4, and on adaptive responses such as T-cell intragraft infiltration suggests that Serp-1 may modulate the transition from innate to adaptive immunity, exhibiting a synergistic effect on allograft survival when used in combination with a subtherapeutic dose of CsA.

Inhibition of terminal complement components in presensitized transplant recipients prevents antibody-mediated rejection leading to long-term graft survival and accommodation.

Ab-mediated rejection (AMR) remains the primary obstacle in presensitized patients following organ transplantation, as it is refractory to anti-T cell therapy and can lead to early graft loss. Complement plays an important role in the process of AMR. In the present study, a murine model was designed to mimic AMR in presensitized patients. This model was used to evaluate the effect of blocking the fifth complement component (C5) with an anti-C5 mAb on prevention of graft rejection. BALB/c recipients were presensitized with C3H donor skin grafts 7 days before heart transplantation from the same donor strain. Heart grafts, transplanted when circulating anti-donor IgG Abs were at peak levels, were rejected in 3 days. Graft rejection was characterized by microvascular thrombosis and extensive deposition of Ab and complement in the grafts, consistent with AMR. Anti-C5 administration completely blocked terminal complement activity and local C5 deposition, and in combination with cyclosporine and short-term cyclophosphamide treatment, it effectively prevented heart graft rejection. These recipients achieved permanent graft survival for >100 days with normal histology despite the presence of systemic and intragraft anti-donor Abs and complement, suggesting ongoing accommodation. Furthermore, double-transplant experiments demonstrated that immunological alterations in both the graft and the recipient were required for successful graft accommodation to occur. These data suggest that terminal complement blockade with a functionally blocking Ab represents a promising therapeutic approach to prevent AMR in presensitized recipients.

Double-negative T cells, activated by xenoantigen, lyse autologous B and T cells using a perforin/granzyme-dependent, Fas-Fas ligand-independent pathway.

The ability to control the response of B cells is of particular interest in xenotransplantation as Ab-mediated hyperacute and acute xenograft rejection are major obstacles in achieving long-term graft survival. Regulatory T cells have been proven to play a very important role in the regulation of immune responses to self or non-self Ags. Previous studies have shown that TCRalphabeta+CD3+CD4-CD8- (double-negative (DN)) T cells possess an immune regulatory function, capable of controlling antidonor T cell responses in allo- and xenotransplantation through Fas-Fas ligand interaction. In this study, we investigated the possibility that xenoreactive DNT cells suppress B cells. We found that DNT cells generated from wild-type C57BL/6 mice expressed B220 and CD25 after rat Ag stimulation. These xenoreactive B220+CD25+ DNT cells lysed activated, but not naive, B and T cells. This killing, which took place through cell-cell contact, required participation of adhesion molecules. Our results indicate that Fas ligand, TGF-beta, TNF-alpha, and TCR-MHC recognition was not involved in DNT cell-mediated syngenic cell killing, but instead this killing was mediated by perforin and granzymes. The xenoreactive DNT cells expressed high levels of granzymes in comparison to allo- or xenoreactive CD8+ T cells. Adoptive transfer of DNT cells in combination with early immune suppression by immunosuppressive analog of 15-deoxyspergualin, LF15-0195, significantly prolonged rat heart graft survival to 62.1 +/- 13.9 days in mice recipients. In conclusion, this study suggests that xenoreactive DNT cells can control B and T cell responses in perforin/granzyme-dependent mechanisms. DNT cells may be valuable in controlling B and T cell responses in xenotransplantation.

Endothelin-mediated oncofetal fibronectin expression in chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy is a sclerotic process characterized by an increased extracellular matrix (ECM) protein deposition. Fibronectin (FN) is a major component of ECM. FN has been reported to undergo alternative splicing and produce several isoforms including the extra domain-B (ED-B) containing embryonic isoform. In the present study, we investigated ED-B FN expression in chronic allograft nephropathy and its relationship with endothelins (ET). METHODS: To establish chronic allograft nephropathy, allografts were performed between Fisher 344 --> Lewis rats. Allograft recipients were then randomly divided into two groups, allografts and allografts treated with ET receptor antagonist bosentan. Lewis --> Lewis recipients were used as isograft controls. Grafts were harvested at 30, 90 and 140 days for histological and gene expression analyses with respect to ED-B FN, ET-1 and transforming growth factor-beta1 (TGF-beta1) mRNA. ED-B FN protein levels were assessed by immunohistochemical analysis. Additionally, we analyzed human renal allograft biopsies. RESULTS: Our data demonstrates that rat chronic allograft nephropathy is associated with progressive upregulation of ED-B FN mRNA and protein. ET-1 and TGF-beta1 mRNA were also upregulated. Treatment of allograft recipient rats with bosentan prevented upregulation of ED-B FN and TGF-beta1. We further show that total FN, ED-B FN, ET-1 and TGF-beta1 mRNA expression were upregulated in human chronic allograft nephropathy specimens. CONCLUSION: Results obtained from both human and rat renal allograft tissues suggest that expression of ED-B FN is upregulated in chronic allograft nephropathy and such upregulation is mediated via ET-1 and its interaction with TGF-beta1.

Prevention of chronic renal allograft rejection by SERP-1 protein.

BACKGROUND: In previous studies we have demonstrated that Serp-1, a myxoma virus encoded serine protease inhibitor, dramatically inhibits neointimal hyperplasia in vascular injury and aortic transplant models. Here we examined the effect of peritransplant Serp-1 administration on chronic renal allograft rejection. METHODS: Rat renal transplants were performed with sequential recipient sacrifice on postoperative days 2, 10 and 140 to examine both the acute and chronic effects of Serp-1 in recipient rats. RESULTS: Serp-1 administration reduced early posttransplant injury (POD 2) with less acute tubular and vascular necrosis. This translated into a reduction of the characteristic late stage changes of chronic rejection (POD 140), with significantly decreased glomerulosclerosis and neointimal hyperplasia. Effects of Serp-1 treatment were already evident as early as POD 2 with markedly decreased levels of TGF-beta mRNA witnessed at both the early and late time points (POD 2, 10 and 140). CONCLUSION: We have demonstrated that peritransplant Serp-1 viral protein decreased early injury and allowed reduced chronic rejection in a rat renal model. Recipients treated with Serp-1 are associated with a decrease in TGF-beta mRNA levels in the allografts suggesting that the serine protease inhibitor may inhibit TGF-beta transcription and its profibrotic effects.

Renal tubular epithelial cell self-injury through Fas/Fas ligand interaction promotes renal allograft injury.

Tubular epithelial cells (TECs) coexpress Fas and Fas ligand (FasL), which could influence renal allograft injury. While TECs can resist apoptosis by Fas antibody, TEC apoptosis by contact with adjacent TECs has not been studied. Fas expression increased in TECs with cytokine treatment (IFN-gamma, TNF-alpha) while abundant FasL levels were not altered. Apoptosis (Annexin-V, DNA fragmentation) occurred in cytokine-treated TECs monolayers from C3H-HeJ mice by 24 h, but was absent in similarly treated TECs from Fas-deficient (lpr) or FasL-mutant (gld) mice, suggesting that 'self injury' occurred through Fas/FasL. Membrane labeling of TECs in cocultures confirmed that FasL-bearing TECs induced apoptosis when in contact with Fas-bearing TECs. Culturing TECs with allogeneic C57BL/6 (H-2b) splenocytes resulted in apoptosis and elimination of C3H-HeJ TECs by 48 h, with enhanced survival and reduced apoptosis using lpr or gld TECs. In a renal allograft model, survival of C57BL/6 recipients was greater (p < 0.05) and renal function improved (p < 0.001) using C3H-lpr or C3H-gld (H-2 k) donor kidneys compared with C3H-HeJ kidneys. These data demonstrate for the first time that cytokine-activated TECs can injure TECs through expression of functional FasL and Fas. We suggest that inhibition of TEC-TEC 'self injury' may be a novel strategy to augment renal allograft survival.