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Hybrid-excited reluctance machine (HERM) with both dc field and permanent-magnet (PM) excitations has a robust structure and flexible flux regulation capability, which is an emerging integrated starter-generator (ISG) candidate for electric vehicle application. In order to improve the torque density, a novel high-order-harmonic winding design (HHWD) is proposed. The key is to abandon the traditional winding design (WD) which makes use of fundamental field excitation harmonic to adopt a novel WD which utilizes high-order field excitation harmonic. In this way, the no-load back electromotive force (EMF) can be enhanced by 42.7% and average torque can be improved by 38.3%. In this article, machine configuration and operation principle are illustrated in detail. Using the finite element analysis (FEA), the electromagnetic performance of HERMs with traditional WD and HHWD are compared, which testifies the improvement of HHWD.

Inflammation posttransplant is directly linked to cell death programs including apoptosis and necrosis. Cell death leads to the release of cellular contents which can promote inflammation. Targeting of these pathways should be an effective strategy to prevent transplant rejection. Toll-like receptor 3 (TLR3) is emerging as a major endogenous sensor of inflammation. In this study, we assessed the role of TLR3 on cell death and transplant rejection. We showed that TLR3 is highly expressed on mouse microvascular endothelial cell (ECs) and the endothelium of cardiac grafts. We demonstrated that TLR3 interacting with dsRNA or self-RNA triggered apoptosis and necroptosis in ECs. Interestingly, TLR3-induced necroptosis led mitochondrial damage. Inhibition of the mitochondrial membrane permeability molecule Cyclophilin D prevented necroptosis in ECs. In vivo, endothelium damage and activities of caspase-3 and mixed lineage kinase domain-like protein were inhibited in TLR3–/–cardiac grafts compared with C57BL/6 grafts posttransplant (n= 5, p< .001). Importantly, TLR3–/–cardiac grafts had prolonged survival in allogeneic BALB/c mice (mean survival = 121 ± 67 vs. 31 ± 6 days of C57BL/6 grafts, n= 7, p= .002). In summary, our study suggests that TLR3 is an important cell death inducer in ECs and cardiac grafts and thus a potential therapeutic target in preventing cardiac transplant rejection.

Inflammation posttransplant is directly linked to cell death programs including apoptosis and necrosis. Cell death leads to the release of cellular contents which can promote inflammation. Targeting of these pathways should be an effective strategy to prevent transplant rejection. Toll‐like receptor 3 (TLR3) is emerging as a major endogenous sensor of inflammation. In this study, we assessed the role of TLR3 on cell death and transplant rejection. We showed that TLR3 is highly expressed on mouse microvascular endothelial cell (ECs) and the endothelium of cardiac grafts. We demonstrated that TLR3 interacting with dsRNA or self‐RNA triggered apoptosis and necroptosis in ECs. Interestingly, TLR3‐induced necroptosis led mitochondrial damage. Inhibition of the mitochondrial membrane permeability molecule Cyclophilin D prevented necroptosis in ECs. In vivo, endothelium damage and activities of caspase‐3 and mixed lineage kinase domain‐like protein were inhibited in TLR3−/−cardiac grafts compared with C57BL/6 grafts posttransplant (n= 5, p< .001). Importantly, TLR3−/−cardiac grafts had prolonged survival in allogeneic BALB/c mice (mean survival = 121 ± 67 vs. 31 ± 6 days of C57BL/6 grafts, n= 7, p= .002). In summary, our study suggests that TLR3 is an important cell death inducer in ECs and cardiac grafts and thus a potential therapeutic target in preventing cardiac transplant rejection. This study demonstrates that toll‐like receptor 3 is an important mechanistic mediator of necroptosis and apoptosis in endothelial cells and an effective target for prevention of cardiac transplant rejection.

Heart transplant has been accepted as the standard treatment for end-stage heart failure. Because of its susceptibility to ischemia–reperfusion injury, the heart can be preserved for only 4 to 6 hours in cold static preservation solutions. Prolonged ischemia time is adversely associated with primary graft function and long-term survival. New strategies to preserve donor hearts are urgently needed. We demonstrate that AP39, a mitochondria-targeting hydrogen sulfide donor, significantly increases cardiomyocyte viability and reduces cell apoptosis/death after cold hypoxia/reoxygenation in vitro. It also decreases gene expression of proinflammatory cytokines and preserves mitochondria function. Using an in vivo murine heart transplant model, we show that preserving donor hearts with AP39-supplemented University of Wisconsin solution (n = 7) significantly protects heart graft function, measured by quantitative ultrasound scan, against prolonged cold ischemia–reperfusion injury (24 hours at 4°C), along with reducing tissue injury and fibrosis. Our study demonstrates that supplementing preservation solution with AP39 protects cardiac grafts from prolonged ischemia, highlighting its therapeutic potential in preventing ischemia–reperfusion injury in heart transplant.

Heart transplant has been accepted as the standard treatment for end‐stage heart failure. Because of its susceptibility to ischemia–reperfusion injury, the heart can be preserved for only 4 to 6 hours in cold static preservation solutions. Prolonged ischemia time is adversely associated with primary graft function and long‐term survival. New strategies to preserve donor hearts are urgently needed. We demonstrate that AP39, a mitochondria‐targeting hydrogen sulfide donor, significantly increases cardiomyocyte viability and reduces cell apoptosis/death after cold hypoxia/reoxygenation in vitro. It also decreases gene expression of proinflammatory cytokines and preserves mitochondria function. Using an in vivo murine heart transplant model, we show that preserving donor hearts with AP39‐supplemented University of Wisconsin solution (n = 7) significantly protects heart graft function, measured by quantitative ultrasound scan, against prolonged cold ischemia–reperfusion injury (24 hours at 4°C), along with reducing tissue injury and fibrosis. Our study demonstrates that supplementing preservation solution with AP39 protects cardiac grafts from prolonged ischemia, highlighting its therapeutic potential in preventing ischemia–reperfusion injury in heart transplant. Preserving donor hearts with mitochondria‐targeted H S donor AP39–supplemented preservation solution prevents heart grafts from prolonged cold ischemia–reperfusion injury in heart transplantation by protecting mitochondrial integrity and reducing oxidative stress, cell apoptosis/death, and fibrosis.

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. Mitochondria membrane permeability is an important mechanistic mediator of necroptosis in endothelial cells and an effective target for prevention of cardiac transplant 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.

NK cells participate in ischemia reperfusion injury (IRI) and transplant rejection. Endogenous regulatory systems may exist to attenuate NK cell activation and cytotoxicity in IRI associated with kidney transplantation. A greater understanding of NK regulation will provide insights in transplant outcomes and could direct new therapeutic strategies. Kidney tubular epithelial cells (TECs) may negatively regulate NK cell activation by their surface expression of a complex family of C-type lectin-related proteins (Clrs). We have found that Clr-b and Clr-f were expressed by TECs. Clr-b was upregulated by inflammatory cytokines TNFα and IFNγ in vitro. Silencing of both Clr-b and Clr-f expression using siRNA resulted in increased NK cell killing of TECs compared to silencing of either Clr-b or Clr-f alone (p<0.01) and when compared to control TECs (p<0.001). NK cells treated in vitrowith soluble Clr-b and Clr-f proteins reduced their capacity to kill TECs (p<0.05). Hence, NK cell cytotoxicity can be inhibited by Clr proteins on the surface of TECs. Our study suggests a synergistic effect of Clr molecules in regulating NK cell function in renal cells and this may represent an important endogenous regulatory system to limit NK cell-mediated organ injury during inflammation.

Kwok et al report that necroptic endothelial cell death results in the release of HMGB1 that promotes inflammatory responses, and when necroptic death is prevented by RIP3 deficiency in combination with granzyme and caspase inhibition, allograft survival is improved.

The authors report that the intra-arterial perfusion of siRNA to knock down the expression of C3, RelB and Fas in donor kidneys protected them from extended cold ischemia reperfusion injury and prolonged their survival in mice posttransplant. Supplemental digital content is available in the text.

Chronic allograft injury remains the leading cause of late kidney graft loss despite improvements in immunosuppressive drugs and a reduction in acute T cell–mediated rejection. We have recently demonstrated that natural killer (NK) cells are cytotoxic to tubular epithelial cells and contribute to acute kidney ischemia-reperfusion injury. The role of NK cells in kidney allograft rejection has not been studied.

Dendritic cells (DCs) are crucial regulators of immunity and important in inducing and maintaining tolerance. Here, we investigated the potential of a novel DC-immunomodulating agent, soluble CD83 (sCD83), in inducing transplant tolerance.

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.

Tolerogenic dendritic cells (Tol-DCs) play a critical role in inducing and maintaining tolerance. Recognizing that both T-cell inactivation and activation are contingent on signals provided by DCs and that graft-specific activated T cells are major mediators of transplant rejection, we aimed to create an environment favoring Tol-DCs with a novel reagent, human soluble CD83 (hsCD83).

Chronic allograft nephropathy (CAN), the most common cause of late kidney allograft failure, is not effectively prevented by immunosuppressive regimens. Activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) via MEK mediates actions of various growth factors, including transforming growth factor (TGF)-β1, which plays a key role in CAN. Hence, we tested the therapeutic potential of MEK-ERK1/2 signaling disruption to prevent CAN. Kidneys from C57BL/6J (H-2b) mice were transplanted to bilaterally nephrectomized BALB/c (H-2d) mice. At 14 days after transplantation, the recipients were subjected to 28 days of treatment with the MEK inhibitor CI-1040. All six CI-1040-treated allografts survived, while two of seven grafts in the vehicle-treated group were lost. At the end of the experiment, the function and structure of grafts in the CI-1040-treated group were significantly preserved, as indicated by lower levels of serum creatinine or blood urea nitrogen than in the vehicle-treated group [30 ± 6 vs. 94 ± 39 μM creatinine (P= 0.0015) and 22 ± 8 vs. 56 ± 25 mM BUN (P= 0.0054)] and reduced CAN in the CI-1040-treated group compared with vehicle controls (CAN score = 4.2 vs. 10.3, P= 0.0119). The beneficial effects induced by CI-1040 were associated with reduction of ERK1/2 phosphorylation and TGFβ1 levels in grafts. Also, CI-1040 potently suppressed not only TGFβ biosynthesis in kidney cell cultures but also antiallograft immune responses in vitro and in vivo. Our data suggest that interference of MEK-ERK1/2 signaling with a pharmacological agent (e.g., CI-1040) has therapeutic potential to prevent CAN in kidney transplantation.

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.

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.

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-BFN expression in chronic allograft nephropathy and its relationship with endothelins (ET).

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.

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.

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.

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-γ, TNF-α) 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.

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-γ, TNF-α) 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 lpror gldTECs. In a renal allograft model, survival of C57BL/6 recipients was greater (p < 0.05) and renal function improved (p < 0.001) using C3H-lpror 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.

MT7 is a myxoma virusencoded protein that has been found to bind and disrupt human chemokine gradients. This study examined whether purified MT7 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 highdose MT7 viral protein. The survival rate was not significantly different between allograft groups. Renal allografts treated with highdose MT7 demonstrated a significant reduction in tubular atrophy, glomerular atrophy, vascular hyalinization, cortical scarring, and lymphocyte infiltration. Morphometric analyses demonstrated that the highdose MT7 group also showed a significantly decreased amount of glomerulosclerosis and transplant arteriosclerosis. These data demonstrate for the first time that the immunoregulatory viral protein MT7 can effectively attenuate chronic rejection in rat renal allografts.

Intimal expansion by vascular smooth muscle cells (SMCs) is a characteristic feature of graft vascular disease. Whether graft intimal SMCs arise from donor or recipient tissue is not well established but has important pathogenetic implications. We examined for the presence of male cells in the expanded intima of sex-mismatched mouse aortic allografts (C57BL/6-to-BALB/c) at 30 or 60 days after transplant by in situhybridization using a Y-chromosome probe. Study groups included male-to-female allografts, female-to-male allografts, and female-to-female allografts in recipients previously engrafted with male bone marrow. Although intimal expansion developed in all allografts, male-to-female allografts lacked Y-chromosome-positive intimal cells. In contrast, such cells were abundant in female-to-male allografts and most of these cells co-labeled for smooth muscle α-actin by immunostain. Female-to-female allografts in recipients with male bone marrow showed a limited number of intimal Y-chromosome-positive cells. However, none of these clearly co-labeled for smooth muscle α-actin and their numbers declined throughout time, consistent with graft-infiltrating inflammatory cells. We conclude that intimal expansion of mouse aortic allografts is mediated by SMCs that originated from the recipient. There was little evidence of their derivation from the bone marrow, suggesting instead the adjacent host aorta as the primary source of intimal SMCs.

Abstract: Endothelins (ET) are a family of vasoactive peptides that play an important role in several disorders affecting kidneys. In this study we investigated the expressions of ET-1, ET-3, and their receptors, ETA and ETB, in a rat chronic renal transplant rejection model. Renal allografts were performed (F344 → Lewis) with bilateral nephrectomy in recipients. For isograft control, lewis → lewis transplantations were performed. All recipients were sacrificed 140 days after transplantation and the grafts were analyzed histologically. ET-1 and ET-3 protein expression in grafts was measured by immunohistochemistry and ELISA. Semiquantitative RT-PCR methods were used for mRNA levels of ET-1, ET-3, ETA and ETB. No evidence of chronical rejection was manifested in isografts. The allografted rats showed proteinuria and increased serum creatinine levels. Histologically, renal allografts showed atrophy and sclerosis of the glomeruli, cortical scarring and vascular intimal thickening. Immunohistochemically, ET-1 and ET-3 were localized in the convoluted tubules, collecting ducts, endothelium and smooth muscle cells of the large blood vessels. Significantly increased staining for ET-1 and ET-3 were found in allografts compared to isografts. Simultaneously, ELISA for ET-1 and ET-3 showed elevated protein concentrations in allografts compared to isografts. Allografts showed significantly increased ET-1- and ET-3 mRNA compared to isografts. On the other hand, a significant down regulation of the ETA mRNA was noted, and the ETB mRNA remained unchanged. The data from the present study suggest that alteration of ET system may be of importance in the pathogenesis of chronic renal transplant rejection.

AbstractEndothelins (ET) are a family of vasoactive peptides that play an important role in several disorders affecting kidneys. In this study we investigated the expressions of ET‐1, ET‐3, and their receptors, ETAand ETB, in a rat chronic renal transplant rejection model. Renal allografts were performed (F344 → Lewis) with bilateral nephrectomy in recipients. For isograft control, lewis → lewis transplantations were performed. All recipients were sacrificed 140 days after transplantation and the grafts were analyzed histologically. ET‐1 and ET‐3 protein expression in grafts was measured by immunohistochemistry and ELISA. Semiquantitative RT‐PCR methods were used for mRNA levels of ET‐1, ET‐3, ETAand ETBNo evidence of chronical rejection was manifested in isografts. The allografted rats showed proteinuria and increased serum creatinine levels. Histologically, renal allografts showed atrophy and sclerosis of the glomeruli, cortical scarring and vascular intimal thickening. Immunohistochemically, ET‐1 and ET‐3 were localized in the convoluted tubules, collecting ducts, endothelium and smooth muscle cells of the large blood vessels. Significantly increased staining for ET‐1 and ET‐3 were found in allografts compared to isografts. Simultaneously, ELISA for ET‐1 and ET‐3 showed elevated protein concentrations in allografts compared to isografts. Allografts showed significantly increased ET‐1‐ and ET‐3 mRNA compared to isografts. On the other hand, a significant down regulation of the ETAmRNA was noted, and the ETBmRNA remained unchanged. The data from the present study suggest that alteration of ET system may be of importance in the pathogenesis of chronic renal transplant rejection.

Intrigued by the finding that a monoclonal antibody (mAb) directed against the B exon of restricted CD45 (CD45RB mAb) induced renal allograft tolerance in the mouse model, we hypothesized that CD45RB mAb may prevent xenograft rejection.We explored the role of CD45RB mAb in preventing xenograft rejection in rat-to-mouse kidney and heart transplant models.

Clinical intestinal transplantation has been plagued by frequent and severe graft rejection. It has been proposed that the major histocompatibility complex (MHC) antigens might play a critical role in this process owing to their extensive expression on enterocytes and mucosa-associated immune cells.