RESUMEN
Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:ß-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galß1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.
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Escherichia coli/fisiología , Inmunoglobulina M/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/transmisión , Plasmodium/fisiología , Polisacáridos/inmunología , Adulto , Animales , Anopheles/parasitología , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Autoantígenos/inmunología , Línea Celular Tumoral , Niño , Escherichia coli/clasificación , Escherichia coli/inmunología , Femenino , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Tracto Gastrointestinal/microbiología , Vida Libre de Gérmenes , Humanos , Inmunoglobulina M/sangre , Malaria Falciparum/microbiología , Malaria Falciparum/parasitología , Ratones , Plasmodium/clasificación , Plasmodium/crecimiento & desarrollo , Plasmodium/inmunología , Plasmodium falciparum/inmunología , Plasmodium falciparum/fisiología , Esporozoítos/inmunología , Receptor Toll-Like 9/agonistasRESUMEN
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.
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Clonación de Organismos , Técnicas de Transferencia Nuclear , Animales , Ovinos , Trasplante HeterólogoRESUMEN
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.
RESUMEN
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.
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Infarto del Miocardio , Factor de Crecimiento Transformador beta1 , Humanos , Ratones , Animales , Factor de Crecimiento Transformador beta1/metabolismo , Remodelación Ventricular , Miocardio/metabolismo , Fibrosis , Fibroblastos/metabolismo , Colágeno/metabolismoRESUMEN
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.
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Trasplante de Corazón , Trasplantes , Animales , Trasplante de Corazón/métodos , Máquina Corazón-Pulmón , Inflamación , Papio , Porcinos , Trasplante Heterólogo/métodosRESUMEN
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.
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Enfermedades Renales/tratamiento farmacológico , Sustancias Protectoras/farmacología , Receptores de Factor Estimulante de Colonias de Granulocito/antagonistas & inhibidores , Daño por Reperfusión/tratamiento farmacológico , Animales , Quimiocinas/metabolismo , Activación de Complemento/efectos de los fármacos , Creatinina/sangre , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Inflamación/sangre , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Enfermedades Renales/sangre , Enfermedades Renales/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Daño por Reperfusión/sangre , Daño por Reperfusión/metabolismo , Urea/sangreRESUMEN
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.
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Células Progenitoras Endoteliales/efectos de los fármacos , Células Progenitoras Endoteliales/metabolismo , Material Particulado/toxicidad , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/metabolismo , Citocinas/sangre , Células Progenitoras Endoteliales/patología , Estrógenos/metabolismo , Femenino , Mediadores de Inflamación/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ovariectomía , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factores SexualesRESUMEN
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.
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Regulación Gubernamental , Trasplante Heterólogo/normas , AustraliaRESUMEN
Gene editing using clustered regularly interspaced short palindromic repeats/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off-target mutations in gene-edited pigs remains largely undefined. We have previously generated GGTA1 knock-in pigs for xenotransplantation using FokI-dCas9, a variant of Cas9 that is reported to reduce the frequency of off-target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of FokI-dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using GRIDSS variant-calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock-in and wild-type pigs. Platypus variant-calling software was used to detect single-nucleotide variations (SNVs) and small insertions/deletions (indels). GRIDSS analysis confirmed the precise integration of one copy of the knock-in construct in the gene-edited cells. Three additional SVs were detected by GRIDSS: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the CALD1 gene on chromosome 18. These mutations were not associated with plausible off-target sites, and were not detected in a second line of knock-in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off-target activity. Platypus identified 1375 SNVs/indels after quality filtering, but none of these were located in proximity to potential off-target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from FokI-dCas9-edited cells. Our results demonstrate that FokI-dCas9 is capable of high-fidelity gene editing with negligible off-target or undesired on-target mutagenesis.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Biología Computacional/métodos , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Edición Génica/métodos , Mutación/genética , Animales , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Análisis Mutacional de ADN , Estudios de Factibilidad , Sus scrofa , Trasplante Heterólogo , Secuenciación Completa del GenomaRESUMEN
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.
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Animales Modificados Genéticamente/inmunología , Células Endoteliales/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Proteína C/metabolismo , Animales , Coagulación Sanguínea/fisiología , Receptor de Proteína C Endotelial/genética , Porcinos , Trasplante Heterólogo/métodosRESUMEN
Endothelial progenitor cells (EPCs) are a group of heterogeneous cells in bone marrow (BM) and blood. Ischaemia increases reactive oxygen species (ROS) production that regulates EPC number and function. The present study was conducted to determine if ischaemia-induced ROS differentially regulated individual EPC subpopulations using a mouse model concomitantly overexpressing superoxide dismutase (SOD)1, SOD3 and glutathione peroxidase. Limb ischaemia was induced by femoral artery ligation in male transgenic mice with their wild-type littermate as control. BM and blood cells were collected for EPCs analysis and mononuclear cell intracellular ROS production, apoptosis and proliferation at baseline, day 3 and day 21 after ischaemia. Cells positive for c-Kit+ /CD31+ or Sca-1+ /Flk-1+ or CD34+ /CD133+ or CD34+ /Flk-1+ were identified as EPCs. ischaemia significantly increased ROS production and cell apoptosis and decreased proliferation of circulating and BM mononuclear cells and increased BM and circulating EPCs levels. Overexpression of triple antioxidant enzymes effectively prevented ischaemia-induced ROS production with significantly decreased cell apoptosis and preserved proliferation and significantly increased circulating EPCs level without significant changes in BM EPC populations, associated with enhanced recovery of blood flow and function of the ischemic limb. These data suggested that ischaemia-induced ROS was differentially involved in the regulation of circulating EPC population.
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Antioxidantes/metabolismo , Células Progenitoras Endoteliales/metabolismo , Células Progenitoras Endoteliales/patología , Miembro Posterior/metabolismo , Miembro Posterior/patología , Isquemia/metabolismo , Isquemia/patología , Animales , Apoptosis/fisiología , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Proliferación Celular/fisiología , Células Cultivadas , Glutatión Peroxidasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especies Reactivas de Oxígeno/metabolismo , Células Madre/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Ischemia-reperfusion injury (IRI) is the outcome of an inflammatory process that is triggered when an organ undergoes a transient reduction or cessation of blood flow, followed by re-establishment of perfusion. In the clinical setting, IRI contributes to significant acute kidney injury, patient morbidity and mortality, and adverse outcomes in transplantation. Tubular cell death by necrosis and apoptosis is a central feature of renal IRI. Recent research has challenged traditional views of cell death by identifying new pathways in which cells die in a regulated manner but with the morphologic features of necrosis. This regulated necrosis (RN) takes several forms, with necroptosis and ferroptosis being the best described. The precise mechanisms and relationships between the RN pathways in renal IRI are currently the subject of active research. The common endpoint of RN is cell membrane rupture, resulting in the release of cytosolic components with subsequent inflammation and activation of the immune system. We review the evidence and mechanisms of RN in the kidney following renal IRI, and discuss the use of small molecule inhibitors and genetically modified mice to better understand this process and guide potentially novel therapeutic interventions.
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Lesión Renal Aguda/patología , Trasplante de Riñón/efectos adversos , Túbulos Renales/patología , Microvasos/patología , Daño por Reperfusión/patología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/prevención & control , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Ensayos Clínicos Fase II como Asunto , Modelos Animales de Enfermedad , Células Epiteliales/patología , Ferroptosis/efectos de los fármacos , Ferroptosis/genética , Humanos , Fallo Renal Crónico/cirugía , Túbulos Renales/citología , Ratones , Ratones Transgénicos , Microvasos/efectos de los fármacos , Necroptosis/efectos de los fármacos , Necroptosis/genética , Necrosis/etiología , Necrosis/patología , Oxazepinas/farmacología , Oxazepinas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Ensayos Clínicos Controlados Aleatorios como Asunto , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Flujo Sanguíneo Regional/efectos de los fármacos , Flujo Sanguíneo Regional/genética , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/etiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Resultado del Tratamiento , Triazoles/farmacología , Triazoles/uso terapéuticoRESUMEN
Deficiency in the membrane-bound complement regulators CD55 and CD59 exacerbates renal ischemia-reperfusion injury (IRI) in mouse models, but the effect of increasing CD55 and CD59 activity has not been examined. In this study, we investigated the impact of overexpression of human (h) CD55 ± hCD59 or treatment with soluble rhCD55 in a mouse model of renal IRI. Unilaterally nephrectomised mice were subjected to 18 (mild IRI) or 22 min (moderate IRI) warm renal ischemia, and analyzed 24 h after reperfusion for renal function (serum creatinine and urea), complement deposition (C3b/c and C9), and infiltration of neutrophils and macrophages. Transgenic mice expressing hCD55 alone were protected against mild renal IRI, with reduced creatinine and urea levels compared with wild type littermates. However, the renal function of the hCD55 mice was not preserved in the moderate IRI model, despite a reduction in C3b/c and C9 deposition and innate cell infiltration. Mice expressing both hCD55 and hCD59, on the other hand, were protected in the moderate IRI model, with significant reductions in all parameters measured. Wild type mice treated with rhCD55 immediately after reperfusion were also protected in the moderate IRI model. Thus, manipulation of CD55 activity to increase inhibition of the C3 and C5 convertases is protective against renal IRI, and the additional expression of hCD59, which regulates the terminal complement pathway, provides further protection. Therefore, anti-complement therapy using complement regulatory proteins may provide a potential clinical option for preventing tissue and organ damage in renal IRI.
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Antígenos CD55/genética , Antígenos CD55/uso terapéutico , Antígenos CD59/genética , Enfermedades Renales/terapia , Daño por Reperfusión/terapia , Animales , Antígenos CD55/inmunología , Activación de Complemento , Creatinina/sangre , Humanos , Enfermedades Renales/inmunología , Enfermedades Renales/fisiopatología , Macrófagos/inmunología , Ratones , Ratones Transgénicos , Neutrófilos/inmunología , Daño por Reperfusión/inmunología , Daño por Reperfusión/fisiopatología , Urea/sangreRESUMEN
PURPOSE OF REVIEW: The use of genetically modified donor pigs has been integral to recent major advances in xenograft survival in preclinical nonhuman primate models. CRISPR-Cas9 gene editing technology has dramatically accelerated the development of multimodified pigs. This review examines the current and projected impact of CRISPR-Cas9-mediated donor modification on preventing rejection and potentially promoting tolerance of porcine xenografts. RECENT FINDINGS: CRISPR-Cas9 has been used to engineer several genetic modifications relevant to xenotransplantation into pigs, including glycosyltransferase knockouts (GGTA1, CMAH, ß4GALNT2, A3GALT2 and combinations thereof), other knockouts (SLA-I, ULBP1, PERV and GHR), and one knock-in (anti-CD2 monoclonal antibody transgene knocked into GGTA1). Although the use of these pigs as donors in preclinical nonhuman primate models has been limited to a single study to date, in-vitro analysis of their cells has provided invaluable information. For example, deletion of three of the glycosyltransferases progressively decreased the binding and cytotoxicity of preexisting immunoglobulin G and immunoglobulin M in human sera, suggesting that this 'triple-KO' pig could be a platform for clinical xenotransplantation. SUMMARY: CRISPR-Cas9 enables the rapid generation of gene-edited pigs containing multiple tailored genetic modifications that are anticipated to have a positive impact on the efficacy and safety of pig-to-human xenotransplantation.
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Anticuerpos Heterófilos/genética , Sistemas CRISPR-Cas/inmunología , Trasplante Heterólogo/métodos , Animales , Animales Modificados Genéticamente , Humanos , PorcinosRESUMEN
Since purinergic signaling was discovered in the early 1970s, it has been shown that extracellular nucleotides, and their derivative nucleosides, are released in a regulated or unregulated manner by cells in various challenging settings and then bind defined purinergic receptors to activate intricate signaling networks. Extracellular ATP plays a role based on different P2 receptor subtypes expressed on specific cell types. Sequential hydrolysis of extracellular ATP catalyzed by ectonucleotidases (e.g. CD39, CD73) is the main pathway for the generation of adenosine, which in turn activates P1 receptors. Many studies have demonstrated that extracellular ATP signaling functions as an important dynamic regulatory pathway to coordinate appropriate immune responses in various pathological processes, including intracellular infection, host-tumor interaction, pro-inflammation vascular injury, and transplant immunity. ATP receptors and CD39 also participate in related clinical settings. Here, we review the latest research in to the development of promising clinical treatment strategies.
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Adenosina Trifosfato/metabolismo , Espacio Extracelular/metabolismo , Receptores Purinérgicos/metabolismo , Transducción de Señal , Adenosina/metabolismo , Animales , Humanos , Hidrólisis , Modelos Biológicos , Nucleotidasas/metabolismoRESUMEN
OBJECTIVE: Antiphospholipid syndrome (APS) is a systemic autoimmune disorder of young adults associated with devastating pregnancy complications (recurrent miscarriages, preeclampsia and low birth weight) and vascular complications including thrombosis. The key components implicated in pathogenesis of APS are the complement cascade and tissue factor (TF) activity causing inflammation and coagulation. Purinergic signalling involving catabolism of ATP to adenosine by cell-surface enzymes CD39 and CD73 has anti-inflammatory and anti-thrombotic effects. We studied whether activities of CD39 and CD73 are important in preventing the development of miscarriages in APS. METHODS: We studied frequency of miscarriages and decidual pathology following passive transfer of human aPL-ab to pregnant wildtype mice, and mice deficient in CD39 and CD73, and also transgenic mice exhibiting 2-3X higher CD39 activity. RESULTS: aPL-ab infusion in pregnant CD39-or CD73-knockout mice triggers an increase in miscarriages, associated with increased TF expression and complement deposition as well as elevated oxidative stress and pro-inflammatory TNF-α and IL-10 expression within the placental decidua. In contrast, aPL-ab induced miscarriages are prevented in mice over-expressing CD39, with reduced decidual TF expression and C3d deposition, diminished lipid peroxidation (4-hydroxynonenal or 4-HNE positive lipid adducts), and reduced TNF-α expression. CONCLUSION: We demonstrate a protective role for CD39 in APS and provide rationale for both the development of endothelial cell-targeted soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease.
Asunto(s)
Aborto Espontáneo/inmunología , Anticuerpos Antifosfolípidos/metabolismo , Antígenos CD/metabolismo , Síndrome Antifosfolípido/inmunología , Apirasa/metabolismo , Complicaciones del Embarazo/inmunología , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Adulto , Animales , Antígenos CD/genética , Apirasa/genética , Complemento C3d/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunización Pasiva , Inflamación , Mediadores de Inflamación/metabolismo , Peroxidación de Lípido , Ratones , Ratones Noqueados , Ratones Transgénicos , Embarazo , Tromboplastina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: Ischemia-reperfusion (I/R) injury is a major clinical problem linked to vascular surgery. Currently, no drugs to prevent or to treat I/R injury are approved for clinical use. C1 inhibitor (C1 INH) is known to reduce activation of the plasma cascade systems that are involved in the pathophysiologic process of I/R injury. The aim of this study was therefore to investigate the effect of C1 INH on complement deposition and endothelial cell activation in a rat model of hind limb I/R injury. METHODS: Male Wistar rats (wild type, bred at the central animal facility, University of Bern), weighing 250 to 320 g, were used. The rats underwent 2-hour ischemia and 24-hour reperfusion by unilateral clamping of the femoral artery and additional use of a tourniquet. Five groups were divided according to intravenous treatment 5 minutes before ischemia: 50 IU/kg C1 INH (n = 5); 100 IU/kg C1 INH (n = 7); vehicle control (n = 5); nontreated control (n = 7); and normal, healthy control without intervention (n = 4). At the end, muscle edema, tissue viability, and histologic features were assessed. Deposition of immunoglobulin M, C1r, C4d, and fibrin and expression of plasminogen activator inhibitor 1, heparan sulfate (HS), E-selectin, and vascular cell adhesion molecule 1 were evaluated by fluorescence staining. In addition, high-mobility group box 1 protein was measured in plasma. RESULTS: Edema formation was reduced by C1 INH at two dosages, mirrored by improved histologic injury scores and preserved muscle viability. Deposition of immunoglobulin M, C4d, and fibrin was significantly decreased by 100 IU/kg C1 INH compared with nontreated controls. Pretreatment with 100 IU/kg C1 INH also significantly reduced HS shedding and expression of plasminogen activator inhibitor 1 as well as plasma levels of high-mobility group box 1 protein. CONCLUSIONS: Pretreatment with both 50 and 100 IU/kg C1 INH attenuated reperfusion injury of rat hind limbs. Pretreatment with 100 IU/kg also preserved the endothelial HS layer as well as the natural, profibrinolytic phenotype of the endothelium. Prevention of endothelial cell activation by C1 INH may therefore be a promising strategy to prevent I/R injury in the clinical setting of peripheral vascular diseases and elective surgery on extremities.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Proteína Inhibidora del Complemento C1/farmacología , Inactivadores del Complemento/farmacología , Células Endoteliales/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Daño por Reperfusión/prevención & control , Animales , Complemento C1r/metabolismo , Complemento C4b/metabolismo , Modelos Animales de Enfermedad , Selectina E/metabolismo , Edema/inmunología , Edema/metabolismo , Edema/patología , Edema/prevención & control , Células Endoteliales/metabolismo , Células Endoteliales/patología , Fibrina/metabolismo , Proteína HMGB1/metabolismo , Heparitina Sulfato/metabolismo , Miembro Posterior , Inmunoglobulina M/metabolismo , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Fragmentos de Péptidos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Ratas Wistar , Daño por Reperfusión/inmunología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Supervivencia Tisular/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The last few years have seen significant progress in xenotransplantation. Porcine xenograft survival in preclinical models continues to improve, accompanied by the adjustment of immunosuppression to more clinically realistic levels. The rapid uptake of CRISPR/Cas9 technology has accelerated the generation of new knock-in and knockout pigs, including animals null for the endogenous retrovirus PERV. This brief review presents a personal view of recent developments in the field.
Asunto(s)
Retrovirus Endógenos/patogenicidad , Xenoinjertos , Investigación , Trasplante Heterólogo , Animales , Supervivencia de Injerto/fisiología , Humanos , Sus scrofa/virología , PorcinosRESUMEN
Adenosine is considered the canonical ligand for the adenosine 2B receptor (A2BR). A2BR is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A2BR signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A2BR. The computational modeling suggested that AMP interacts with more favorable energy to A2BR compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A2BR, indicating preferential signaling via the Gq pathway. Therefore, a putative AMP-A2BR interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential Gq activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI.