Dynamics of necroptosis in kidney ischemia-reperfusion injury.

Necroptosis, a pathway of regulated necrosis, involves recruitment and activation of RIPK1, RIPK3 and MLKL, leading to cell membrane rupture, cell death and release of intracellular contents causing further injury and inflammation. Necroptosis is believed to play an important role in the pathogenesis of kidney ischemia-reperfusion injury (IRI). However, the dynamics of necroptosis in kidney IRI is poorly understood, in part due to difficulties in detecting phosphorylated MLKL (pMLKL), the executioner of the necroptosis pathway. Here, we investigated the temporal and spatial activation of necroptosis in a mouse model of unilateral warm kidney IRI, using a robust method to stain pMLKL. We identified the period 3-12 hrs after reperfusion as a critical phase for the activation of necroptosis in proximal tubular cells. After 12 hrs, the predominant pattern of pMLKL staining shifted from cytoplasmic to membrane, indicating progression to the terminal phase of necroptotic cell death. Mlkl-ko mice exhibited reduced kidney inflammation at 12 hrs and lower serum creatinine and tubular injury at 24 hrs compared to wild-type littermates. Interestingly, we observed increased apoptosis in the injured kidneys of Mlkl-ko mice, suggesting a relationship between necroptosis and apoptosis in kidney IRI. Together, our findings confirm the role of necroptosis and necroinflammation in kidney IRI, and identify the first 3 hrs following reperfusion as a potential window for targeted treatments.

Characterisation of transgenic pigs expressing a human T cell-depleting anti-CD2 monoclonal antibody.

BACKGROUND: Pig islet xenotransplantation is a potential treatment for type 1 diabetes. We have shown that maintenance immunosuppression is required to protect genetically modified (GM) porcine islet xenografts from T cell-mediated rejection in baboons. Local expression of a depleting anti-CD2 monoclonal antibody (mAb) by the xenograft may provide an alternative solution. We have previously reported the generation of GGTA1 knock-in transgenic pigs expressing the chimeric anti-CD2 mAb diliximab under an MHC class I promoter (MHCIP). In this study, we generated GGTA1 knock-in pigs in which MHCIP was replaced by the ß-cell-specific porcine insulin promoter (PIP), and compared the pattern of diliximab expression in the two lines. METHODS: A PIP-diliximab knock-in construct was prepared and validated by transfection of NIT-1 mouse insulinoma cells. The construct was knocked into GGTA1 in wild type (WT) porcine fetal fibroblasts using CRISPR, and knock-in cells were used to generate pigs by somatic cell nuclear transfer (SCNT). Expression of the transgene in MHCIP-diliximab and PIP-diliximab knock-in pigs was characterised at the mRNA and protein levels using RT-qPCR, flow cytometry, ELISA and immunohistochemistry. Islets from MHCIP-diliximab and control GGTA1 KO neonatal pigs were transplanted under the kidney capsule of streptozotocin-diabetic SCID mice. RESULTS: NIT-1 cells stably transfected with the PIP-diliximab knock-in construct secreted diliximab into the culture supernatant, confirming correct expression and processing of the mAb in ß cells. PIP-diliximab knock-in pigs showed a precise integration of the transgene within GGTA1. Diliximab mRNA was detected in all tissues tested (spleen, kidney, heart, liver, lung, pancreas) in MHCIP-diliximab pigs, but was not detectable in PIP-diliximab pigs. Likewise, diliximab was present in the serum of MHCIP-diliximab pigs, at a mean concentration of 1.8 µg/mL, but was not detected in PIP-diliximab pig serum. An immunohistochemical survey revealed staining for diliximab in all organs of MHCIP-diliximab pigs but not of PIP-diliximab pigs. Whole genome sequencing (WGS) of a PIP-diliximab pig identified a missense mutation in the coding region for the dixilimab light chain. This mutation was also found to be present in the fibroblast knock-in clone used to generate the PIP-diliximab pigs. Islet xenografts from neonatal MHCIP-diliximab pigs restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated expression of diliximab in situ. CONCLUSION: Diliximab was widely expressed in MHCIP-diliximab pigs, including in islets, consistent with the endogenous expression pattern of MHC class I. Further investigation is required to determine whether the level of expression in islets from the MHCIP-diliximab pigs is sufficient to prevent T cell-mediated islet xenograft rejection. The unexpected absence of diliximab expression in the islets of PIP-diliximab pigs was probably due to a mutation in the transgene arising during the generation of the knock-in cells used for SCNT.

Sex, T Cells, and the Microbiome in Natural ABO Antibody Production in Mice.

BACKGROUND: "Natural" ABO antibodies (Abs) are produced without known exposure to A/B carbohydrate antigens, posing significant risks for hyperacute rejection during ABO-incompatible transplantation. We investigated anti-A "natural" ABO antibodies versus intentionally induced Abs with regard to the need for T-cell help, the impact of sex, and stimulation by the microbiome. METHODS: Anti-A was measured by hemagglutination assay of sera from untreated C57BL/6 wild-type (WT) or T cell-deficient mice of both sexes. Human ABO-A reagent blood cell membranes were injected intraperitoneally to induce anti-A Abs. The gut microbiome was eliminated by maintenance of mice in germ-free housing. RESULTS: Compared with WT mice, CD4 + T-cell knockout (KO), major histocompability complex-II KO, and αß/γδ T-cell receptor KO mice produced much higher levels of anti-A nAbs; females produced dramatically more anti-A nAbs than males, rising substantially with puberty. Sensitization with human ABO-A reagent blood cell membranes did not induce additional anti-A in KO mice, unlike WT. Sex-matched CD4 + T-cell transfer significantly suppressed anti-A nAbs in KO mice and rendered mice responsive to A-sensitization. Even under germ-free conditions, WT mice of several strains produced anti-A nAbs, with significantly higher anti-A nAbs levels in females than males. CONCLUSIONS: Anti-A nAbs were produced without T-cell help, without microbiome stimulation, in a sex- and age-dependent manner, suggestive of a role for sex hormones in regulating anti-A nAbs. Although CD4 + T cells were not required for anti-A nAbs, our findings indicate that T cells regulate anti-A nAb production. In contrast to anti-A nAbs, induced anti-A production was T-cell dependent without a sex bias.

The birth of Dolly and xenotransplantation 25 years on.

A number of reviews have been written recently celebrating the 25th anniversary of the birth of Dolly the cloned sheep and the effect this breakthrough has had on various fields of research. However, arguably the biggest impact Dolly has had is on the field of xenotransplantation, described here based on our own experience and that of others.

Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) impacts TGF-ß1 responses: insights into cardiac fibrosis and function following myocardial infarction.

Extracellular purine nucleotides and nucleosides released from activated or injured cells influence multiple aspects of cardiac physiology and pathophysiology. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; CD39) hydrolyzes released nucleotides and thereby regulates the magnitude and duration of purinergic signaling. However, the impact of CD39 activity on post-myocardial infarction (MI) remodeling is incompletely understood. We measured the levels and activity of ectonucleotidases in human left ventricular samples from control and ischemic cardiomyopathy (ICM) hearts and examined the impact of ablation of Cd39 expression on post-myocardial infarction remodeling in mice. We found that human CD39 levels and activity are significantly decreased in ICM hearts (n = 5) compared with control hearts (n = 5). In mice null for Cd39, cardiac function and remodeling are significantly compromised in Cd39-/- mice following myocardial infarction. Fibrotic markers including plasminogen activator inhibitor-1 (PAI-1) expression, fibrin deposition, α-smooth muscle actin (αSMA), and collagen expression are increased in Cd39-/- hearts. Importantly, we found that transforming growth factor ß1 (TGF-ß1) stimulates ATP release and induces Cd39 expression and activity on cardiac fibroblasts, constituting an autocrine regulatory pathway not previously appreciated. Absence of CD39 activity on cardiac fibroblasts exacerbates TGF-ß1 profibrotic responses. Treatment with exogenous ectonucleotidase rescues this profibrotic response in Cd39-/- fibroblasts. Together, these data demonstrate that CD39 has important interactions with TGF-ß1-stimulated autocrine purinergic signaling in cardiac fibroblasts and dictates outcomes of cardiac remodeling following myocardial infarction. Our results reveal that ENTPD1 (CD39) regulates TGF-ß1-mediated fibroblast activation and limits adverse cardiac remodeling following myocardial infarction.NEW & NOTEWORTHY We show that CD39 is a critical modulator of TGF-ß1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-ß1-induced CD39 expression generates a negative feedback loop that attenuates cardiac fibroblast activation. In the absence of CD39 activity, collagen deposition is increased, elastin expression is decreased, and diastolic dysfunction is worsened. Treatment with ecto-apyrase attenuates the TGF-ß1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis.

Xenotransplantation of Genetically Modified Neonatal Pig Islets Cures Diabetes in Baboons.

Xenotransplantation using porcine donors is rapidly approaching clinical applicability as an alternative therapy for treatment of many end-stage diseases including type 1 diabetes. Porcine neonatal islet cell clusters (NICC) have normalised blood sugar levels for relatively short periods in the preclinical diabetic rhesus model but have met with limited success in the stringent baboon model. Here we report that NICC from genetically modified (GM) pigs deleted for αGal and expressing the human complement regulators CD55 and CD59 can cure diabetes long-term in immunosuppressed baboons, with maximum graft survival exceeding 22 months. Five diabetic baboons were transplanted intraportally with 9,673 - 56,913 islet equivalents (IEQ) per kg recipient weight. Immunosuppression consisted of T cell depletion with an anti-CD2 mAb, tacrolimus for the first 4 months, and maintenance with belatacept and anti-CD154; no anti-inflammatory treatment or cytomegalovirus (CMV) prophylaxis/treatment was given. This protocol was well tolerated, with all recipients maintaining or gaining weight. Recipients became insulin-independent at a mean of 87 ± 43 days post-transplant and remained insulin-independent for 397 ± 174 days. Maximum graft survival was 675 days. Liver biopsies showed functional islets staining for all islet endocrine components, with no evidence of the inflammatory blood-mediated inflammatory reaction (IBMIR) and minimal leukocytic infiltration. The costimulation blockade-based immunosuppressive protocol prevented an anti-pig antibody response in all recipients. In conclusion, we demonstrate that genetic modification of the donor pig enables attenuation of early islet xenograft injury, and in conjunction with judicious immunosuppression provides excellent long-term function and graft survival in the diabetic baboon model.

Overcoming perioperative inflammation as a hurdle for successful preclinical orthotopic cardiac xenogeneic transplantations - particular in regard of the mandatory use of heart-lung machines.

INTRODUCTION: After orthotopic cardiac xenotransplantation, the combination of both the inflammatory responses to the exposure of a recipient to the xenogeneic organ and the use of cardiopulmonary bypass has been assumed to cause detrimental side effects. These have been described not only to affect the transplanted organ (heart) itself, but also the recipient's lungs. In this article, we summarize how these possible detrimental processes can be minimized or even avoided. METHODS: Data from eight pig-to-baboon orthotopic cardiac xenotransplantation experiments were analyzed with a special focus on early (within the first week) postoperative organ dysfunction and systemic inflammatory responses. Non-ischemic heart preservation and the careful management of the heart-lung machine were deemed essential to guarantee not only the immediate function of the transplanted xenogeneic organ but also the prompt recovery of the recipient. RESULTS: After weaning from cardiopulmonary bypass, very low catecholamine amounts were needed to ensure an adequate pump function and cardiac output. Central venous oxygen saturation and serum lactate levels remained within normal ranges. All animals were successfully weaned from ventilation within the first postoperative hours. Serum parameters of the transplants and native kidneys and livers were initially slightly elevated or always normal, as were hemoglobin, LDH, and platelet measurements. Markers of systemic inflammation, C-reactive protein, and IL-6 were slightly elevated, but the reactions caused no lasting damage. CONCLUSION: Consistent short-term and long-term results were achieved after orthotopic cardiac pig-to-baboon transplantation without detrimental inflammatory responses or signs of multiorgan failure. In comparison to allogeneic procedures, non-ischemic heart preservation was important for successful immediate organ function, as was the management of the heart-lung machine. Thus, we believe that genetically modified porcine hearts are ready for use in the clinical setting.

A potent truncated form of human soluble CR1 is protective in a mouse model of renal ischemia-reperfusion injury.

The complement system is a potent mediator of ischemia-reperfusion injury (IRI), which detrimentally affects the function and survival of transplanted kidneys. Human complement receptor 1 (HuCR1) is an integral membrane protein that inhibits complement activation by blocking the convertases that activate C3 and C5. We have previously reported that CSL040, a truncated form of recombinant soluble HuCR1 (sHuCR1), has enhanced complement inhibitory activity and improved pharmacokinetic properties compared to the parent molecule. Here, we compared the capacity of CSL040 and full-length sHuCR1 to suppress complement-mediated organ damage in a mouse model of warm renal IRI. Mice were treated with two doses of CSL040 or sHuCR1, given 1 h prior to 22 min unilateral renal ischemia and again 3 h later. 24 h after reperfusion, mice treated with CSL040 were protected against warm renal IRI in a dose-dependent manner, with the highest dose of 60 mg/kg significantly reducing renal dysfunction, tubular injury, complement activation, endothelial damage, and leukocyte infiltration. In contrast, treatment with sHuCR1 at a molar equivalent dose to 60 mg/kg CSL040 did not confer significant protection. Our results identify CSL040 as a promising therapeutic candidate to attenuate renal IRI and demonstrate its superior efficacy over full-length sHuCR1 in vivo.

Combination of Antioxidant Enzyme Overexpression and N-Acetylcysteine Treatment Enhances the Survival of Bone Marrow Mesenchymal Stromal Cells in Ischemic Limb in Mice With Type 2 Diabetes.

Background Therapy with mesenchymal stem cells remains a promising but challenging approach to critical limb ischemia in diabetes because of the dismal cell survival. Methods and Results Critical limb ischemia in type 2 diabetes mouse model was used to explore the impact of diabetic limb ischemia on the survival of bone marrow mesenchymal stromal cells (bMSCs). Inhibition of intracellular reactive oxygen species was achieved with concomitant overexpression of superoxide dismutase (SOD)-1 and glutathione peroxidase-1 in the transplanted bMSCs, and extracellular reactive oxygen species was attenuated using SOD-3 overexpression and N-acetylcysteine treatment. In vivo optical fluorescence imaging and laser Doppler perfusion imaging were used to track cell retention and determine blood flow in diabetic ischemic limb, respectively. Survival of the transplanted bMSCs was significantly decreased in diabetic ischemic limb compared with the control. In vitro study indicated that advanced glycation end products, not high glucose, significantly decreased the proliferation of bMSCs and increased their apoptosis associated with increased reactive oxygen species production and selective reduction of SOD-1 and SOD-3. In vivo study demonstrated that concomitant overexpression of SOD-1, SOD-3, and glutathione peroxidase-1, or host treatment with N-acetylcysteine, significantly enhanced in vivo survival of transplanted bMSCs, and improved critical limb ischemia in diabetic mice. Combination of triple antioxidant enzyme overexpression in bMSCs with host N-acetylcysteine treatment further improved bMSC survival with enhanced circulatory and functional recovery from diabetic critical limb ischemia. Conclusions Simultaneous suppression of reactive oxygen species from transplanted bMSCs and host tissue could additively enhance bMSC survival in diabetic ischemic limb with increased therapeutic efficacy in diabetes.

Circulating Endothelial Progenitor Cells Are Preserved in Female Mice Exposed to Ambient Fine Particulate Matter Independent of Estrogen.

Males have a higher risk for cardiovascular diseases (CVDs) than females. Ambient fine particulate matter (PM) exposure increases CVD risk with increased reactive oxygen species (ROS) production and oxidative stress. Endothelial progenitor cells (EPCs) are important to vascular structure and function and can contribute to the development of CVDs. The aims of the present study were to determine if sex differences exist in the effect of PM exposure on circulating EPCs in mice and, if so, whether oxidative stress plays a role. Male and female C57BL/6 mice (8-10 weeks old) were exposed to PM or a vehicle control for six weeks. ELISA analysis showed that PM exposure substantially increased the serum levels of IL-6 and IL-1ß in both males and females, but the concentrations were significantly higher in males. PM exposure only increased the serum levels of TNF-α in males. Flow cytometry analysis demonstrated that ROS production was significantly increased by PM treatment in males but not in females. Similarly, the level of circulating EPCs (CD34+/CD133+ and Sca-1+/Flk-1+) was significantly decreased by PM treatment in males but not in females. Antioxidants N-acetylcysteine (NAC) effectively prevented PM exposure-induced ROS and inflammatory cytokine production and restored circulating EPC levels in male mice. In sharp contrast, circulating EPC levels remained unchanged in female mice with PM exposure, an effect that was not altered by ovariectomy. In conclusion, PM exposure selectively decreased the circulating EPC population in male mice via increased oxidative stress without a significant impact on circulating EPCs in females independent of estrogen.

Pathways to Clinical Cardiac Xenotransplantation.

Heart transplantation is the only long-lasting lifesaving option for patients with terminal cardiac failure. The number of available human organs is however far below the actual need, resulting in substantial mortality of patients while waiting for a human heart. Mechanical assist devices are used to support cardiac function but are associated with a high risk of severe complications and poor quality of life for the patients. Consistent success in orthotopic transplantation of genetically modified pig hearts into baboons indicates that cardiac xenotransplantation may become a clinically applicable option for heart failure patients who cannot get a human heart transplant. In this overview, we project potential paths to clinical cardiac xenotransplantation, including the choice of genetically modified source pigs; associated requirements of microbiological, including virological, safety; optimized matching of source pig and recipient; and specific treatments of the donor heart after explantation and of the recipients. Moreover, selection of patients and the regulatory framework will be discussed.

Blockade of the G-CSF Receptor Is Protective in a Mouse Model of Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is a complex inflammatory process that detrimentally affects the function of transplanted organs. Neutrophils are important contributors to the pathogenesis of renal IRI. Signaling by G-CSF, a regulator of neutrophil development, trafficking, and function, plays a key role in several neutrophil-associated inflammatory disease models. In this study, we investigated whether targeting neutrophils with a neutralizing mAb to G-CSFR would reduce inflammation and protect against injury in a mouse model of warm renal IRI. Mice were treated with anti-G-CSFR 24 h prior to 22-min unilateral renal ischemia. Renal function and histology, complement activation, and expression of kidney injury markers, and inflammatory mediators were assessed 24 h after reperfusion. Treatment with anti-G-CSFR protected against renal IRI in a dose-dependent manner, significantly reducing serum creatinine and urea, tubular injury, neutrophil and macrophage infiltration, and complement activation (plasma C5a) and deposition (tissue C9). Renal expression of several proinflammatory genes (CXCL1/KC, CXCL2/MIP-2, MCP-1/CCL2, CXCR2, IL-6, ICAM-1, P-selectin, and C5aR) was suppressed by anti-G-CSFR, as was the level of circulating P-selectin and ICAM-1. Neutrophils in anti-G-CSFR-treated mice displayed lower levels of the chemokine receptor CXCR2, consistent with a reduced ability to traffic to inflammatory sites. Furthermore, whole transcriptome analysis using RNA sequencing showed that gene expression changes in IRI kidneys after anti-G-CSFR treatment were indistinguishable from sham-operated kidneys without IRI. Hence, anti-G-CSFR treatment prevented the development of IRI in the kidneys. Our results suggest G-CSFR blockade as a promising therapeutic approach to attenuate renal IRI.

Xeno-organ donor pigs with multiple genetic modifications - the more the better?

The number of donated human organs and tissues for patients with terminal organ failure falls far short of the need. Alternative sources, such as organs and tissues from animals, are therefore urgently required. During the past few years, major progress has been made in the development of genetically multi-modified donor pigs, and their organs have been shown to be safe and efficacious in life-supporting transplantation models into non-human primates, paving the way to clinical xenotransplantation studies. Here, we summarize recent developments in pig genome engineering and discuss efforts to develop the optimum donor pig for xenotransplantation. In addition, we speculate on how many genetic modifications may be required for initial xenotransplantation clinical trials.

Xenotransplantation in Australia: Development of the regulatory process.

In this commentary, we present a brief history of the development of a national regulatory framework for xenotransplantation clinical research in Australia, including the reasons behind the imposition of a 5-year moratorium in 2005 and its subsequent lifting. We conclude with a summary of current relevant guidelines and standards.

High-fat diet selectively decreases bone marrow lin- /CD117+ cell population in aging mice through increased ROS production.

Bone marrow (BM) stem cells (BMSCs) are an important source for cell therapy. The outcome of cell therapy could be ultimately associated with the number and function of donor BMSCs. The present study was to evaluate the effect of long-term high-fat diet (HFD) on the population of BMSCs and the role of reactive oxygen species (ROS) in aging mice. Forty-week-old male C57BL/6 mice were fed with HFD for 3 months with regular diet as control. Experiments were repeated when ROS production was reduced in mice treated with N-acetylcysteine (NAC) or using mice overexpressing antioxidant enzyme network (AON) of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase. BM and blood cells were analyzed with flowcytometry for lineage negative (lin- ) and Sca-1+ , or lin- /CD117+ , or lin- /CD133+ cells. Lin- /CD117+ cell population was significantly decreased with increased intracellular ROS and apoptosis and decreased proliferation in BM, not in blood, in HFD-treated mice without change for Sca-1+ or CD133+ cell populations in BM or blood. NAC treatment or AON overexpression effectively prevented HFD-induced intracellular ROS production and reduction of BM lin- /CD117+ population. These data suggested that long-term HFD selectively decreased BM lin- /CD117+ cell population in aging mice through increased ROS production.

Pig endothelial protein C receptor is functionally compatible with the human protein C pathway.

BACKGROUND: Endothelial protein C receptor (EPCR) plays an anticoagulant and anti-inflammatory role by promoting the activation of protein C by thrombin bound to thrombomodulin (TBM). Incompatibility between pig TBM and human/primate thrombin is thought to contribute to dysregulated coagulation in pig-to-primate organ xenografts, and expression of human TBM (hTBM) in pigs has shown benefit in preclinical models. However, it is not known whether there are incompatibilities-or molecular barriers-between endogenous pig EPCR (pEPCR) and transgenically expressed human TBM. AIM: To clone and express pEPCR, and determine its function in the human protein C pathway in vitro. METHODS: Pig endothelial protein C receptor cDNA was generated from pig lung RNA by RT-PCR. Primate COS-7 transfectants expressing various combinations of human and pig TBM and EPCR were incubated with human thrombin and human protein C, and tested for TBM cofactor activity. RESULTS: The predicted protein sequence of pEPCR shared 72.3% amino acid sequence identity with hEPCR, and residues critical for protein C binding were conserved. COS-7 cells transfected with hEPCR, pEPCR or vector showed minimal TBM cofactor activity (0.13 ± 0.04, 0.13 ± 0.02 and 0.14 ± 0.06 U, respectively). The cofactor activity of hTBM-transfected cells (1.18 ± 0.29 U) was 8-fold higher than vector-transfected cells (P = .004) and further increased 4-fold and 3-fold by co-transfection with hEPCR (5.01 ± 1.12 U, P = .004) or pEPCR (3.73 ± 0.65 U, P = .003), respectively. CONCLUSIONS: Our data show that pEPCR is largely compatible with the human TBM/thrombin complex, when expressed on COS-7 cells in vitro, promoting the activation of human protein C. These findings suggest that endogenous pEPCR will enhance the activity of transgenic hTBM in the xenograft setting.