Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys.

We transplanted kidneys from alpha1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GalT-KO pigs to humans.

Tolerogenic dendritic cells transferring hyporesponsiveness and synergizing T regulatory cells in transplant tolerance.

Dendritic cells are among the most potent antigen-presenting cells and are important in the development of both immunity and tolerance. Tolerogenic dendritic cell (Tol-DC) is a key factor in the induction and maintenance of tolerance during transplantation. However, the precise mechanism and direct evidence of in vivo immune modulation remain unclear. In the present study, we identified critical roles of immune modulation on transplant tolerance through interactions between Tol-DCs and regulatory T (Treg) cells. Tol-DCs remained in an immature state and were insensitive to maturation stimuli. Tol-DCs in tolerant recipients heightened the expression of indoleamine 2,3-dioxygenase (IDO) that induced allogeneic T-cell apoptosis. Adoptive transfer of Tol-DCs isolated from primary tolerant recipients resulted in augmentation of CD4(+)CD25(+)CTLA4(+) Treg cells and prolonged graft survival in secondary allogeneic heart transplantation and synergized with Treg cells to induce tolerance in secondary recipients. This study indicates that Tol-DC offers two functions during the process of tolerogenesis: suppression of anti-donor T-cell responses through production of IDO and interaction with Treg cells, which provides a framework for future research into tolerance induction.

The serpin saga; development of a new class of virus derived anti-inflammatory protein immunotherapeutics.

Serine proteinase inhibitors, also called serpins, are an ancient grouping of proteins found in primitive organisms from bacteria, protozoa and horseshoe crabs and thus likely present at the time of the dinosaurs, up to all mammals living today. The innate or inflammatory immune system is also an ancient metazoan regulatory system, providing the first line of defense against infection or injury. The innate inflammatory defense response evolved long before acquired, antibody dependent immunity. Viruses have developed highly effective stratagems that undermine and block a wide variety of host inflammatory and immune responses. Some of the most potent of these immune modifying strategies utilize serpins that have also been developed over millions of years, including the hijacking by some viruses for defense against host immune attacks. Serpins represent up to 2-10 percent of circulating plasma proteins, regulating actions as wide ranging as thrombosis, inflammation, blood pressure control and even hormone transport. Targeting serpin-regulated immune or inflammatory pathways makes evolutionary sense for viral defense and many of these virus-derived inhibitory proteins have proven to be highly effective, working at very low concentrations--even down to the femptomolar to picomolar range. We are studying these viral anti-inflammatory proteins as a new class of immunomodulatory therapeutic agents derived from their native viral source. One such viral serpin, Serp-1 is now in clinical trial (conducted by VIRON Therapeutics, Inc.) for acute unstable coronary syndromes (unstable angina and small heart attacks), representing a 'first in class' therapeutic study. Several other viral serpins are also currently under investigation as anti-inflammatory or anti-immune therapeutics. This chapter describes these original studies and the ongoing analysis of viral serpins as a new class of virus-derived immunotherapeutic.

Prolongation of cardiac allograft survival by inhibition of ERK1/2 signaling in a mouse model.

BACKGROUND: It has been demonstrated that in vitro the presence of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling inhibitor suppresses T cell activation and Th1 development. However, pharmacological interference of ERK1/2 signaling by administration of its small molecule inhibitor has not been tested as a therapeutic target in the prevention of allograft rejection. METHODS: The immunosuppressive effect of targeting ERK1/2 signaling was tested on cardiac allograft survival in C57BL/6 (H-2b) to Balb/c (H-2d) murine model using PD98059 inhibitor. Phosphorylation/activation of ERK 1/2 and STAT6 proteins were assessed by Western blot. RESULTS: Blockade of ERK1/2 using PD98059 had significant immunosuppressive effect and prolonged survival of mouse cardiac allografts from 8.3+/-0.5 days (vehicle) to 12.6+/-1.3 days (100 mg/kg PD98059; P<0.0001). Combination therapy of PD98059 (100 mg/kg) with cyclosporine (CsA, 15 mg/kg for 20 days) additionally enhanced graft survival (34.4+/-1.2 days) compared to CsA (14.9+/-1.1 days; P<0.0001) or PD98059 monotherapy (P<0.0001). Attenuation of graft rejection by PD98059 correlated to reduction of intragraft ERK phosphorylation and leukocyte infiltration, and to increase in interleukin (IL)-4 or decrease in interferon-gamma production within the grafts. In vitro inhibition of ERK1/2 by PD98059 promoted Th2 differentiation by upregulation IL-4 production but not altering IL-4 stimulating STAT6 pathway. CONCLUSION: Targeting ERK1/2 signaling results in suppression of alloimmune responses by an unique mechanism that involves Th1/Th2 skewing, suggesting a therapeutic potential of inhibition of ERK1/2 signaling for transplant rejection, particularly in combination with CsA.

Long-term limb allograft survival using a short course of anti-CD45RB monoclonal antibody, LF 15-0195, and rapamycin in a mouse model.

BACKGROUND: The purpose of this study was to determine if a short course of monoclonal antibody (mAb) against CD45RB, LF 15-0195, and rapamycin would achieve long-term survival by inducing tolerance in a mouse limb transplant model. METHODS: Group 1 (n=9) consisted of nine isogenic (C57BL/6) transplants. Group 2 (n=3) included C57BL/6-to-BALB/c transplants receiving no drug therapy. Group 3 mice (n=4) were treated with mAb (3 mg/kg) and LF (2 mg/kg), and Group 4 (n=13) was treated with mAb, LF, and rapamycin (2 mg/kg). Both treatment groups received drug treatment for only 14 days posttransplantation. Animals were sacrificed if they displayed evidence of rejection or when deemed to be tolerant (defined as >day 100). RESULTS: All isografts had normal histology and graft function on day 100. Untreated C57BL/6-to-BALB/c allografts developed acute rejection within 10 days. The combination of mAb and LF prolonged allograft survival to a mean of 39+/-7 days. In Group 4, two animals had to be sacrificed at days 28 and 76 due to acute urinary retention. Transplant tolerance was achieved in 8 of the remaining 11 animals with a mean survival time of 100+/-12 days. Donor specific tolerance was demonstrated through permanent acceptance of skin grafts from the donor strain and rejection of skin grafts from C3H mice. Three Group 4 animals showed clinical and histological signs of mild, chronic rejection. Dendritic cells isolated from tolerant recipients exerted a suppressive effect in mixed lymphocyte reaction. CONCLUSION: A short course of anti-CD45RB mAb and LF 15-0195 prolonged limb allograft survival. The addition of rapamycin induced limb allograft tolerance which is associated with the generation of tolerogenic dendritic cells that suppressed T-cell proliferation.

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.

Regulation of B- and T-cell mediated xenogeneic transplant rejection by interleukin 12.

BACKGROUND: Xenotransplantation may provide a solution to the increasing shortage of donor organs. Acute vascular rejection and cell-mediated rejection remain the primary barriers to successful xenotransplantation. In animal models where acute vascular rejection can be attenuated, xenografts succumb to cell-mediated rejection. The mechanisms of acute vascular rejection and cell-mediated rejection are poorly understood. METHODS: Using a heterotopic rat-to-mouse cardiac transplantation model, we demonstrate that IL-12p40 attenuates both allogeneic and xenogeneic acute vascular rejection pathology by suppressing B-cell activation and anti-rat isotype switching. To study the mechanism of xenogeneic cell-mediated rejection, we use B-cell deficient mice that only develop cell-mediated rejection pathology. To elucidate the role of IL-12 in cell-mediated rejection, we generated B cell/ IL-12p40 double knockout mice. RESULTS: We demonstrate that xenogeneic cell-mediated rejection is mediated by CD4+ T cells, and is accompanied by elevated FasL and granzyme mRNA expression. Strikingly, by generating B cell/IL-12p40 double knockout mice, we demonstrate that xenogeneic cell-mediated rejection is IL-12p40 dependent. In contrast, we demonstrate that allogeneic cellular rejection is IL-12p40 independent. CONCLUSIONS: We conclude that IL-12 plays a dual role in xenotransplantation by driving xenogeneic CD4+ T cell responses but suppressing both allogeneic and xenogeneic B cell responses. Therefore, the mechanism of allogeneic and xenogeneic transplantation rejection is differentially regulated by IL-12.

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.

Prevention of renal ischemic injury by silencing the expression of renal caspase 3 and caspase 8.

BACKGROUND: Apoptotic pathways mediated by caspases play a critical role in renal ischemia-reperfusion injury (IRI). Downregulation of the caspase cascade, using small interfering RNA (siRNA) to silence the expression of caspase 3 and caspase 8, may have substantial therapeutic potential for limiting renal injury. METHODS: IRI was induced in mice by clamping of the renal vein and artery for 25 or 35 min at 37 degrees C. Caspase 3 and caspase 8 (caspase 3/8) siRNA was administrated by hydrodynamic injection. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to analyze the gene silencing efficacy, and the therapeutic effects of siRNA were evaluated by renal function analysis, histological examination, and overall survival of mice suffering from IRI. RESULTS: In this study, we have shown, using quantitative PCR, that IRI is associated with increased levels of renal caspase 3/8 mRNA. Mice treated with caspase 3/8 siRNA showed a significant down-regulation in kidney expression of caspase 3/8 at both, transcriptional and protein levels. Kidney function in IRI was protected by siRNA therapy, as levels of blood urea nitrogen and creatinine were significantly reduced in mice treated with siRNA. Histological examination demonstrated that tissue injury caused by IRI was significantly reduced as a result of caspase 3/8 siRNA treatment. Furthermore, survival data showed that more than 70% of mice in siRNA-treated groups survived until the end of the eight-day observation period. CONCLUSION: Herein, we have demonstrated the therapeutic potential of using siRNA to knock down the expression of caspases and prevent acute renal injury.

Protection of renal ischemia injury using combination gene silencing of complement 3 and caspase 3 genes.

BACKGROUND: Ischemia/reperfusion (I/R) injury occurs in clinical kidney transplantation, which results in graft dysfunction and rejection. It has been documented that I/R injury is associated with complement activation and renal cell apoptosis. The purpose of this study was to develop a strategy to prevent I/R injury using small interfering RNA (siRNA) that target complement 3 (C3) and caspase 3 genes. METHODS: siRNA-expression vectors were constructed to target C3 and caspase 3 genes. Gene silencing efficacy was assessed using real-time polymerase chain reaction. In vivo gene silencing was performed by hydrodynamic injection with C3 and caspase 3 siRNA. Renal I/R injury was induced through clamping the renal vein and artery for 25 min. I/R injury was evaluated using kidney histopathology, blood urea nitrogen (BUN), serum levels of creatinine, and survival. RESULTS: Effective gene silencing was first confirmed in vitro. Notably upregulated expression of C3 and caspase 3 genes was observed from 2 to 48 hr after I/R injury, which were effectively and specifically inhibited by C3 and caspase 3 siRNA. In comparison with control mice, serum levels of creatinine and BUN were also significantly decreased in C3 and caspase 3 siRNA-treated mice. Furthermore, the therapeutic effect of siRNA was assessed in a severe, lethal I/R injury experiment, in which siRNA treatment significantly reduced mortality. Tissue histopathology showed an overall reduction in injury area in siRNA-treated mice. CONCLUSIONS: This is the first demonstration that renal I/R injury can be prevented through silencing the complement gene and apoptosis gene, highlighting the potential for siRNA-based clinical therapy.

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy.

BACKGROUND: The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. METHODS: Seven baboons received life-supporting kidney transplants from hDAF transgenic pigs. Anti-Gal antibodies were neutralized by GAS914 or TPC (a Gal PEG glycoconjugate polymer). Group 1 (n=5) underwent a conventional immunosuppressive therapy with FK506, rabbit anti-thymocyte serum/immunoglobulin, mycophenolate mofetil, and steroids. Group 2 (n=2) received an anti-humoral immunity regimen with LF15-0195, Rituxan and cobra venom factor in addition to ATG, FK506 and steroids. Levels of anti-non-Gal antibodies and their mediated complement-dependent cytotoxic activities (CDC) were detected by flow cytometry using Gal knockout (k/o) pig lymphocytes (LC) or endothelial cells (EC) as targets. RESULTS: Continuous infusion of GAS914/TPC significantly reduced anti-Gal antibodies. In Group 1, four of five baboons developed severe acute humoral xenograft rejection (AHXR) and the rejection was associated with either a high level of preformed anti-non-Gal IgG or a marked elevation in induced anti-non-Gal IgG and IgM. Sera collected at the time of AHXR had a high level of CDC to porcine LC/EC from Gal k/o animals. The intensive anti-humoral therapy in Group 2 completely inhibited both anti-Gal and non-Gal antibody production and prevented AHXR. However, this therapy was not well tolerated by the baboons. CONCLUSION: In a pig-to-baboon kidney transplant model, both preformed and induced anti-non-Gal antibodies are strongly associated with the pathogenesis of AHXR when anti-Gal antibodies are neutralized.

The influence of baseline expression of human decay accelerating factor transgene on graft survival and acute humoral xenograft rejection.

BACKGROUND: Transgenic pigs expressing human decay accelerating factor (hDAF) have been widely used as donors in various non-human primate transplant models. Despite the use of similar immunosuppressive protocols, there is marked variation in graft survival among centres. The present study was undertaken to determine whether the level of hDAF expression in the pig kidney correlates with the degree of rejection and duration of graft survival. METHODS: hDAF transgenic pigs were provided from two suppliers: Guelph Imutran Centre (G) and Harlan Sprague Dawley (H). Following a bilateral nephrectomy, a single hDAF pig kidney was implanted in the baboon, which was subsequently treated with conventional immunosuppressive protocols. The pig's contralateral kidney was collected to provide baseline data. The severity of acute humoral xenograft rejection (AHXR) was graded as stage I-III. hDAF expression was measured using morphologic analysis comparing the contralateral and grafted kidneys at the endpoint. RESULTS: Baseline hDAF expression in kidneys from pigs provided by supplier G was significantly higher than that from supplier H (P<0.01). Furthermore, the survival of baboons receiving grafts from G pigs was significantly longer than those receiving grafts from H pigs (P<0.05). In addition, reduction of hDAF expression at the endpoint was associated with a higher degree of AHXR. Severe apoptosis or necrosis was found in grafts with AHXR II-III. CONCLUSIONS: Pig kidneys from different suppliers have variable baseline hDAF expression, which may have an influence on graft survival. Reduced expression of hDAF in the terminal graft was associated with the severity of rejection.

Prevention of acute vascular rejection by a functionally blocking anti-C5 monoclonal antibody combined with cyclosporine.

BACKGROUND: Inhibition of the complement cascade at C5 prevents formation of pro-inflammatory molecules C5a and C5b-9, which play a key role in allograft rejection. The present study was undertaken to determine whether blocking terminal complement with anti-C5 monoclonal antibody (mAb) alone or combined with cyclosporine (CsA) would prevent acute vascular rejection (AVR) in a mouse cardiac allograft model. METHODS: C3H mouse hearts were transplanted into BALB/c mice and randomized into five groups with the following treatments: (1) no treatment; (2) CsA alone; (3) control mAb; (4) anti-C5 mAb alone; and (5) anti-C5 mAb and CsA. RESULTS: Allografts in untreated or control mAb-treated recipients were rapidly rejected at 8.0+/-0.6 and 8.2+/-0.8 days, respectively. These grafts exhibited typical AVR, characterized by vasculitis, hemorrhage, and thrombosis. A high level of complement activity was also demonstrated in these animals. High-dose CsA was not able to inhibit complement activation or AVR, and grafts were rejected in 15.5+/-1.1 days. Anti-C5 monotherapy completely inhibited complement activation and attenuated AVR, but grafts were rejected in 8.3+/-0.5 days by acute cellular rejection. In contrast, a combination of anti-C5 mAb and CsA successfully achieved indefinite graft survival (>100 days). This combined therapy completely inhibited terminal complement activation and prevented both humoral- and cellular-mediated rejection. CONCLUSIONS: Combination therapy of anti-C5 mAb and CsA achieves indefinite graft survival in a mouse cardiac allograft model. These data suggest that inhibition of terminal complement using anti-C5 mAb may be an effective therapeutic adjunct to prevent AVR in clinical transplantation.

Immunosuppression with a combination of pg490-88 and a subtherapeutic dose of FK506 in a canine renal allograft model.

BACKGROUND: PG490-88 is a water soluble, semisynthetic derivative of a novel compound PG490 (triptolide) purified from the Chinese herb Tripterygium Wilfordii Hook F. In this study, we evaluated the immunosuppressive effect of PG490-88 alone or combined with FK506 in a dog renal transplantation model. METHODS: Recipient and donor male beagle dogs were obtained from different breeders to ensure MHC mismatching. PG490-88 and/or FK506 were administered orally based on protocol design. RESULTS: All dogs in the untreated group developed acute vascular rejection with a median survival time of 6 days. The grafts from this group presented with massive hemorrhage, IgM, IgG, and C4c deposition. Administration of PG490-88 0.06 mg/kg/day significantly prolonged graft survival to a median survival time of 11 days (P=0.038, vs. control). Treatment with FK506 0.3 mg/kg/day did not prolong graft survival with a median survival time of 9 days. Although FK506 0.6 mg/kg/day significantly prolonged survival, this dose was not tolerated by the dogs. The combination of PG 0.06 mg/kg/day and FK506 0.3 mg/kg/day significantly prolonged survival to a median survival time of 15 days (P=0.017, vs. control). Compared to the untreated control group, the pattern of acute humoral rejection was attenuated in renal allografts treated with PG490-88 and/or FK506. C4c deposition was significantly decreased in renal allografts treated with PG490-88 monotherapy and combination therapy. CONCLUSIONS: PG490-88 alone and combined with low dose FK506 significantly prolonged renal allograft survival in a dog model. This agent attenuated acute humoral rejection by inhibiting complement activation and T-cell infiltration.

LF15-0195 generates tolerogenic dendritic cells by suppression of NF-kappaB signaling through inhibition of IKK activity.

LF15-0195 (LF) is a potent, less toxic analog of the immunosuppressant 15-deoxyspergualine, which we previously reported to prevent graft rejection and to induce permanent tolerance in a murine cardiac transplantation model. However, the underlying mechanism of action of LF required elucidation. In this study, dendritic cells (DC) treated with LF before activation with tumor necrosis factor alpha (TNF-alpha)/lipopolysaccharide (LPS) failed to express maturation markers (major histocompatibility complex II, CD40, CD86) and interleukin-12. LF prevented, in a concentration-dependent manner, the activation and nuclear translocation of nuclear factor-kappaB (NF-kappaB) in DC following addition of TNF-alpha/LPS. Yet-activated and active IkappaB kinases (IKKs) were inhibited in cells pretreated with LF, thereby preventing the phosphorylation of IkappaB and release of NF-kappaB, a key regulator of genes associated with the maturation of DC. LF-induced inhibition of IKK activity was reversed in a dose-dependent manner by the overexpression of IKK. The T helper cell type 2 (Th2) differentiation of naïve T cells promoted by LF-treated DC in vitro correlates with Th2 polarization observed in transplant recipients made tolerant by LF. These data demonstrated that LF-induced blockade of NF-kappaB signaling at the level of IKK promoted the generation of tolerogenic DC that inhibited Th1 polarization and increased Th2 polarization in vitro and in vivo.

Current advances in xenotransplantation.

BACKGROUND: Transplantation of organs is a well-known and accepted life-saving procedure for end-stage kidney, liver, heart and lung diseases. The insufficient number of donor organs limits the application of this technique and leads to unnecessary loss of life. Experimental techniques such as xenotransplantation are extremely important to determine new methods of creating organ availability. DATA SOURCES: A literature search of Pubmed database was conducted and research articles reviewed. RESULTS: Xenotransplantation is a progressive field of research. Human complement regulatory protein (hDAF) transgenic pigs and new immunosuppressive strategies that reduce xenoreactive alphagal antibodies, have decreased rates of acute vascular rejection. Transplantation of alpha-1, 3-galactosyltransferase knock-out pig organs into baboons has resulted in the longest graft survival to date. Coagulation pathways have been identified as having a role in graft rejection. In vitro studies of porcine endogenous retroviruses (PERVs) show encouraging results that zoonosis will be less hindering to xenotransplantation than once thought. CONCLUSIONS: Several recent advances in xenotransplantation research have brought this technique closer to clinical application. The Ethics Committee of the International Xenotransplantation Association has made recommendations to ensure maintenance of ethical standards.Advancement will depend on the development of pig models, novel immunosuppressive strategies to target the innate immune system, and new ways to create donor specific tolerance. Prevention of rejection and transmission of infectious agents remain unresolved issues. In the future, it is feasible that xenotransplantation will be used to resolve this medical dilemma.

Induction of RNA interference in dendritic cells.

Dendritic cells (DC) reside at the center of the immunological universe, possessing the ability both to stimulate and inhibit various types of responses. Tolerogenic/regulatory DC with therapeutic properties can be generated through various means of manipulations in vitro and in vivo. Here we describe several attractive strategies for manipulation of DC using the novel technique of RNA interference (RNAi). Additionally, we overview some of our data regarding yet undescribed characteristics of RNAi in DC such as specific transfection strategies, persistence of gene silencing, and multi-gene silencing. The advantages of using RNAi for DC genetic manipulation gives rise to the promise of generating tailor-made DC that can be used effectively to treat a variety of immunologically mediated diseases.

LF 15-0195, a novel immunosuppressive agent prevents rejection and induces operational tolerance in a mouse cardiac allograft model.

BACKGROUND: LF 15-0195 (LF) is a new analogue of 15-deoxyspergualin (DSG) that is less toxic and more potent than DSG. The present study was undertaken to determine (1). the dose response of LF monotherapy, (2). its ability to induce tolerance, and (3) its interaction with cyclosporine (CsA), FK 506 (FK), and rapamycin (RAPA). METHODS: Varying doses of LF were administered to determine dose-dependent effects on graft survival in a C57BL/6 to BALB/c heterotopic heart allograft mouse model. Transplanting-donor and third-party skin grafts into long-term survivors were used to assess the tolerance status. CsA, FK, and RAPA were combined with LF to determine their interactive effects on graft survival. RESULTS: The efficacy and toxicity of LF was dose dependent. High-dose LF monotherapy (>2 mg/kg) induced donor-specific operational tolerance, but it was associated with high mortality. Simultaneous administration of high-dose calcineurin inhibitors (CsA FK) prevented tolerance induced by LF. In contrast, a short course of LF combined with a subtherapeutic dose of CsA FK achieved indefinite survival of C57/BL6 cardiac allografts. RAPA and LF had a synergistic effect in induction of tolerance. CONCLUSIONS: The efficacy and toxicity of LF were dose dependent. A short course of LF significantly reduced the requirement of CsA or FK to prevent rejection. RAPA and LF had synergy in induction of tolerance. These data indicate that LF may be a promising agent that warrants further studies in nonhuman primate models of transplantation.

Chemokine-binding viral protein M-T7 prevents chronic rejection in rat renal allografts.

M-T7 is a myxoma virus-encoded protein that has been found to bind and disrupt human chemokine gradients. This study examined whether purified M-T7 could prevent chronic rejection in a rat renal allograft model. Fisher F344 renal allografts were transplanted into Lewis rats. Recipients were randomly grouped into two groups: control animals treated with cyclosporine alone and animals treated with cyclosporine combined with low-, medium- and high-dose M-T7 viral protein. The survival rate was not significantly different between allograft groups. Renal allografts treated with high-dose M-T7 demonstrated a significant reduction in tubular atrophy, glomerular atrophy, vascular hyalinization, cortical scarring, and lymphocyte infiltration. Morphometric analyses demonstrated that the high-dose M-T7 group also showed a significantly decreased amount of glomerulosclerosis and transplant arteriosclerosis. These data demonstrate for the first time that the immunoregulatory viral protein M-T7 can effectively attenuate chronic rejection in rat renal allografts.

Attenuation of acute xenograft rejection by short-term treatment with LF15-0195 and monoclonal antibody against CD45RB in a rat-to-mouse cardiac transplantation model.

BACKGROUND: We previously reported that Lewis rat hearts transplanted into BALB/c mice developed typical acute vascular rejection (AVR). The present study was undertaken to determine the efficacy of LF15-0195, a new analogue of 15-deoxyspergualin, in the prevention of AVR and to determine whether a combination of LF15-0195 and CD45RB monoclonal antibody (mAb) would have a synergistic effect in prolonging xenograft survival. METHODS: We transplanted 2-week-old Lewis rat hearts into BALB/c mice, followed by experimental immunosuppressive regimens. Control groups were either untreated or treated with mAb monotherapy (100 microg/mouse, days -1 to 7, intravenously). Experimental groups were either treated with LF15-0195 (2 mg/kg, days -1 to 14, subcutaneously) or with LF15-0195 combined with mAb at monotherapeutic doses. RESULTS: Heart xenografts in both untreated and mAb-treated BALB/c recipients were rejected at 6.0+/-0.7 days and 8.5+/-1.3 days, respectively, with typical features of AVR, characterized by hemorrhage, fibrin deposition, thrombosis, and massive accumulations of anti-rat IgG and IgM. Serum xenoreactive antibodies (xAbs) were also markedly elevated in these animals. In contrast, LF15-0195 monotherapy significantly prolonged graft survival to 19.3+/-0.7 days. Notably, xAbs were significantly decreased and graft rejection was of a cell-mediated nature instead of AVR. When mAb was combined with LF15-0195, graft survival was further increased to 65.2+/-9.1 days. Antibody production and T-cell infiltration were significantly inhibited at terminal stages of graft survival. Sequential studies on days 6 and 14 demonstrated that LF15-0195, either alone or combined with mAb, completely inhibited antibody production. However, intragraft infiltration by Mac-1+ cells in LF15-0195-treated recipients was similar to that of untreated recipients. CONCLUSIONS: LF15-0195 effectively attenuated AVR by markedly inhibiting antidonor xAb production. Treatment with a combination of LF15-0195 and CD45RB mAb also significantly reduced T-cell infiltration and should be studied further to evaluate its efficacy in nonhuman primate subjects.