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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. [ABSTRACT FROM AUTHOR]

Genetic modification of the source pig offers the opportunity to improve the engraftment and survival of islet xenografts. The type of modification can be tailored to the transplant setting; for example, intraportal islet xenografts have been shown to benefit from the expression of anticoagulant and anti-inflammatory transgenes, whereas cytoprotective transgenes are probably more relevant for encapsulated islets. The rapid development of pig genetic engineering, particularly with the introduction of genome editing techniques such as CRISPR-Cas, has accelerated the generation of new pig lines with multiple modifications. With pre-clinical testing in progress, it is an opportune time to consider any implications of genetic modification for the conditions for undertaking clinical trials. Obviously, the stringent requirements to fulfill designated pathogen-free status that are applied to wild-type pigs will apply equally to genetically modified ( GM) source pigs. In addition, it is important from a safety perspective that the genetic modifications are characterized at the molecular level (e.g., integration site, absence of off-target mutations), the phenotypic level (e.g., durability and stability of transgene expression), and the functional level (e.g., protection of islets in vitro or in vivo, absence of detrimental effects on insulin secretion). The assessment of clinical trial protocols using GM pig islets will need to be performed on a case-by-case basis, taking into account a range of factors including the particular genetic modification(s) and the site and method of delivery. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Renal fibrosis, the key histopathological lesion in the development and progression of chronic kidney disease (CKD), has been the focus of much research in recent decades. The growing burden of CKD in both developed and developing nations highlights a need for novel therapies to halt the progression of renal disease. Insights into the pathogenesis of renal fibrosis and the key cellular and molecular mediators have been critical in the process of identifying potential targets of therapy. Adenosine signaling is an innate biological autocrine and paracrine cellular signaling pathway involving several key mediators: ectonucleotidases, adenosine, and adenosine receptors. Short-term activation of the adenosine A2A and A2B receptors decreases inflammation, which precedes renal fibrosis. However, in conditions of persistent, excessive adenosine exposure, such as in patients born with adenosine deaminase (ADA) deficiency, adenosine signaling via A2B receptor promotes renal fibrosis, as seen in chronic inflammation. This review will describe the increasingly recognized complex role of adenosine signaling in the development of renal fibrosis. We will speculate how the knowledge gained may be employed in the search for more effective therapies based on these complex signaling pathways. [ABSTRACT FROM AUTHOR]

Renal fibrosis, the key histopathological lesion in the development and progression of chronic kidney disease (CKD), has been the focus of much research in recent decades. The growing burden of CKD in both developed and developing nations highlights a need for novel therapies to halt the progression of renal disease. Insights into the pathogenesis of renal fibrosis and the key cellular and molecular mediators have been critical in the process of identifying potential targets of therapy. Adenosine signaling is an innate biological autocrine and paracrine cellular signaling pathway involving several key mediators: ectonucleotidases, adenosine, and adenosine receptors. Short-term activation of the adenosine A2A and A2B receptors decreases inflammation, which precedes renal fibrosis. However, in conditions of persistent, excessive adenosine exposure, such as in patients born with adenosine deaminase (ADA) deficiency, adenosine signaling via A2B receptor promotes renal fibrosis, as seen in chronic inflammation. This review will describe the increasingly recognized complex role of adenosine signaling in the development of renal fibrosis. We will speculate how the knowledge gained may be employed in the search for more effective therapies based on these complex signaling pathways. [ABSTRACT FROM AUTHOR]

Xenotransplantation using pigs as donors offers the possibility of eliminating the chronic shortage of donor kidneys, but there are several obstacles to be overcome before this goal can be achieved. Preclinical studies have shown that, while porcine renal xenografts are broadly compatible physiologically, they provoke a complex rejection process involving preformed and elicited antibodies, heightened innate immune cell reactivity, dysregulated coagulation, and a strong T cell-mediated adaptive response. Furthermore, the susceptibility of the xenograft to proinflammatory and procoagulant stimuli is probably increased by cross-species molecular defects in regulatory pathways. To balance these disadvantages, xenotransplantation has at its disposal a unique tool to address particular rejection mechanisms and incompatibilities: genetic modification of the donor. This review focuses on the pathophysiology of porcine renal xenograft rejection, and on the significant genetic, pharmacological, and technical progress that has been made to prolong xenograft survival. [ABSTRACT FROM AUTHOR]

Schmelzle M, Cowan PJ, Robson SC. Which anti-platelet therapies might be beneficial in xenotransplantation? Xenotransplantation 2011; 18: 79-87. © 2011 John Wiley & Sons A/S. Xenotransplantation could provide an unlimited and elective supply of grafts, once mechanisms of graft loss and vascular injury are better understood. The development of α-1,3-galactosyltransferase gene-knockout (GalT-KO) swine with the removal of a dominant xeno-antigen has been an important advance; however, delayed xenograft and acute vascular reaction in GalT-KO animals persist. These occur, at least in part, because of humoral reactions that result in vascular injury. Intrinsic molecular incompatibilities in the regulation of blood clotting and extracellular nucleotide homeostasis between discordant species may also predispose to thrombophilia within the vasculature of xenografts. Although limited benefits have been achieved with currently available pharmacological anti-thrombotics and anti-coagulants, the highly complex mechanisms of platelet activation and thrombosis in xenograft rejection also require potent immunosuppressive interventions. We will focus on recent thromboregulatory approaches while elucidating appropriate anti-platelet mechanisms. We will discuss potential benefits of additional anti-thrombotic interventions that are possible in transgenic swine and review recent developments in pharmacological anti-platelet therapy. [ABSTRACT FROM AUTHOR]

The article focuses on the manipulation of the xenotransplantation method to pigs wherein unmatched, untested and immunosuppressing possibilities in the operation are overcome. It notes the potential genetic modification of the pig using the employed technology which include removing particular pig characteristics and adding human characteristics to the pig tissue. It mentions the manipulation of the power of genetic engineering making the pig donor tissue to look like human allograft.

Xenotransplantation using porcine donors holds tremendous promise for alleviating the chronic shortage of human organ donors. However, transplantation from pigs into higher primates triggers a vigorous immune response involving complement activation and thrombosis. Hyperacute rejection can be prevented by using donors in which GalT, the gene responsible for the predominant target of anti-pig natural antibodies, has been deleted. Unfortunately, the adaptive response to GalT knockout (KO) organs has been difficult to immunosuppress. The focus has shifted to identifying additional genetic modifications that will extend xenograft survival beyond the several months currently achievable. Which gene(s) should be added to the GalT KO background is open to debate. Overexpression of a complement regulatory factor(s) seems a logical first choice, supported by recent in vitro data but not yet validated in preclinical models. The next obvious candidate would be an anticoagulant protein(s) to deal with the dysregulated coagulation that is almost invariably associated with xenograft rejection. Several potentially useful molecules have been identified, although this study is yet to be translated to the pig. Our understanding of the mechanisms of GalT KO xenograft rejection continues to grow, providing a rational basis for tailoring further genetic modification of the donor and immunosuppression of the recipient.Immunology and Cell Biology (2009) 87, 203–208; doi:10.1038/icb.2008.107; published online 20 January 2009 [ABSTRACT FROM AUTHOR]

Xenotransplantation using porcine donors holds tremendous promise for alleviating the chronic shortage of human organ donors. However, transplantation from pigs into higher primates triggers a vigorous immune response involving complement activation and thrombosis. Hyperacute rejection can be prevented by using donors in which GalT, the gene responsible for the predominant target of anti‐pig natural antibodies, has been deleted. Unfortunately, the adaptive response to GalT knockout (KO) organs has been difficult to immunosuppress. The focus has shifted to identifying additional genetic modifications that will extend xenograft survival beyond the several months currently achievable. Which gene(s) should be added to the GalT KO background is open to debate. Overexpression of a complement regulatory factor(s) seems a logical first choice, supported by recent in vitro data but not yet validated in preclinical models. The next obvious candidate would be an anticoagulant protein(s) to deal with the dysregulated coagulation that is almost invariably associated with xenograft rejection. Several potentially useful molecules have been identified, although this study is yet to be translated to the pig. Our understanding of the mechanisms of GalT KO xenograft rejection continues to grow, providing a rational basis for tailoring further genetic modification of the donor and immunosuppression of the recipient. [ABSTRACT FROM AUTHOR]

The article describes how pig genes may be modified and outlines various considerations on gene modifications and reactions like the instant blood-mediated inflammatory reaction (IBMIR). Accordingly, the considerations of gene modifications suggests that gene modification is a must for islet cell xenotransplantation to be effective therapy. The article also discusses the advancement of technology and its effect to gene modification.

Acute humoral rejection remains the major barrier to long-term pig-to-primate xenograft survival, and microvascular thrombosis is a critical element of the rejection process. It appears that persistent endothelial cell activation and injury, by even low levels of anti-graft antibodies, eventually overwhelm the cellular anticoagulant defences and promote the development of thrombotic microangiopathy. Porcine endothelium may be particularly vulnerable because of cross-species molecular incompatibilities affecting the function of thrombomodulin and possibly TFPI. Recent data from small animal models suggest that transgenic overexpression of anti-thrombotic molecules on xenograft endothelium is capable of inhibiting intravascular thrombosis and preventing acute humoral rejection. In conjunction with existing genetic modifications (e.g. Gal KO, hDAF), this is a promising strategy to move xenotransplantation to the clinic. [ABSTRACT FROM AUTHOR]

Abstract: It is highly likely that successful pig-to-human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell-specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM-1 promoter was weak in liver and non-uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM-2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty-seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM-2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species-specific gene expression in pigs. [ABSTRACT FROM AUTHOR]

Xenotransplantation offers a potential solution to the shortfall in donor organs for human transplantation. This review describes the barriers to xenotransplantation and the progress that has been made towards making it a clinical reality. Data from preclinical pig-to-primate cardiac and pulmonary xenografts are highlighted. (Heart, Lung and Circulation 2002; 11: 32-41). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article discusses the third World Health Organization (WHO) Global conference on regulatory requirements for Xenotransplantation clinical trials that was held in Changsha, China on 12–14 December, 2018. It mentions recent advances in the development of technologies for solid organ xenotransplantation; and also mentions Dr. Megan Sykes reviewed the current technologies and studies in the development of tolerance and use of regulatory T cells.

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 β‐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 β 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Transplantation of human embryonic stem cell (ESC) derived beta‐like cells and xenotransplantation of porcine islets are two potential cures for type 1 diabetes (T1D). We have previously reported the isolation of porcine ESCs and have also separately shown that islets from genetically modified pigs can effectively cure diabetes in a preclinical baboon model. Here we discuss the possibility of combining these technologies to produce gene‐edited porcine ESC‐derived islets as an alternative for treating T1D, which may offer several advantages compared with these approaches. [ABSTRACT FROM AUTHOR]

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 β1 (TGF-β1) 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-β1 profibrotic responses. Treatment with exogenous ectonucleotidase rescues this profibrotic response in Cd39−/− fibroblasts. Together, these data demonstrate that CD39 has important interactions with TGF-β1-stimulated autocrine purinergic signaling in cardiac fibroblasts and dictates outcomes of cardiac remodeling following myocardial infarction. Our results reveal that ENTPD1 (CD39) regulates TGF-β1-mediated fibroblast activation and limits adverse cardiac remodeling following myocardial infarction. NEW & NOTEWORTHY We show that CD39 is a critical modulator of TGF-β1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-β1-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-β1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Diabetes mellitus encompasses two distinct disease processes: autoimmune Type 1 (T1D) and nonimmune Type 2 (T2D) diabetes. Despite the disparate aetiologies, the disease phenotype of hyperglycemia and the associated complications are similar. In this paper, we discuss the role of the CD39-adenosinergic axis in the pathogenesis of both T1D and T2D, with particular emphasis on the role of CD39 and CD73. [ABSTRACT FROM AUTHOR]

Diabetes mellitus encompasses two distinct disease processes: autoimmune Type 1 (T1D) and nonimmune Type 2 (T2D) diabetes. Despite the disparate aetiologies, the disease phenotype of hyperglycemia and the associated complications are similar. In this paper, we discuss the role of the CD39-adenosinergic axis in the pathogenesis of both T1D and T2D, with particular emphasis on the role of CD39 and CD73. [ABSTRACT FROM AUTHOR]

Background: Despite overcoming xenograft hyperacute rejection (HAR), Gal (galactose-α1,3-galactose) expression may not be completely eliminated from the α1,3-galactosyltransferase gene knockout (Gal KO) pig because of alternative galactosyltransferases. Whether low levels of “residual” Gal are still susceptible to either complement fixing or non-complement fixing antibody beyond the HAR barrier remains unknown. Furthermore, it would be impossible to analyze the immune response specific to low-level Gal in a xenograft setting given the multitude of xenoantigens that could induce a recipient response. To investigate this question, we therefore used a skin graft model in BALB/c mice where the sole difference between donor and recipient was the expression of Gal, where rejection is caused by passively administered anti-Gal monoclonal antibody and where HAR does not occur. Methods: Gal expression over time was examined by immunohistochemistry in wildtype-to-Gal KO skin grafts. Graft rejection in response to passively administered anti-Gal monoclonal antibody at early and late time points was studied to determine changes in susceptibility to antibody. To independently test the effect of reduced Gal expression on antibody-mediated rejection, we used two separate lines of α1,2-fucosyltransferase transgenic mice as skin donors in the model. These mice have known reduced but different levels of Gal as determined by flow cytometry on peripheral blood leukocytes. Results: Gal expression on skin grafts diminished with time with a corresponding reduction in susceptibility to antibody-mediated rejection. Skin grafts at day 30 (n = 7) and 150 (n = 11) had a rejection rate of 100% and 45% respectively in response to non-complement fixing anti-Gal antibody administered to the recipient. Similar results were demonstrated with a complement fixing anti-Gal antibody. When α1,2-fucosyltransferase transgenic mice skin was used in the model, the line with lowest level of Gal expression was resistant to antibody-induced rejection with a rate 0% (n = 9) vs. 60% (n = 5) in the alternative line with relatively more Gal expressed but still much less than normal mice. Conclusions: Resistance to anti-Gal antibody-mediated damage in the model was observed in skin grafts 100 to 150 days post-grafting but not earlier and was associated with a reduction in Gal expression. It is possible that below a threshold level of Gal expression, the grafts were not susceptible to anti-Gal antibody. [ABSTRACT FROM AUTHOR]

Focuses on transgenic strategies for overcoming complement-mediated hyperacute rejection of a xenotransplant. Shortage of human donor organs, tissues and cells; Use of pronuclear microinjection to produce pigs containing up to three transgenes.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-GCSFR, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Adenosine is considered the canonical ligand for the adenosine 2B receptor (A 2B R). A 2B R is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A 2B R 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 A 2B R. The computational modeling suggested that AMP interacts with more favorable energy to A 2B R compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A 2B R, indicating preferential signaling via the G q pathway. Therefore, a putative AMP-A 2B R interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The anaphylaxis response is frequently associated with food allergies, representing a significant public health hazard. Recently, exposure to tick bites and production of specific IgE against α-galactosyl (α-Gal)-containing epitopes has been correlated to red meat allergy. However, this association and the source of terminal, non-reducing α-Gal-containing epitopes have not previously been established in Brazil. Here, we employed the α-1,3-galactosyltransferase knockout mouse (α1,3-GalT-KO) model and bacteriophage Qβ-virus like particles (Qβ-VLPs) displaying Galα1,3Galβ1,4GlcNAc (Galα3LN) epitopes to investigate the presence of α-Gal-containing epitopes in the saliva of Amblyomma sculptum , a species of the Amblyomma cajennense complex, which represents the main tick that infests humans in Brazil. We confirmed that the α-1,3-galactosyltransferase knockout animals produce significant levels of anti-α-Gal antibodies against the Galα1,3Galβ1,4GlcNAc epitopes displayed on Qβ-virus like particles. The injection of A. sculptum saliva or exposure to feeding ticks was also found to induce both IgG and IgE anti-α-Gal antibodies in α-1,3-galactosyltransferase knockout mice, thus indicating the presence of α-Gal-containing epitopes in the tick saliva. The presence of α-Gal-containing epitopes was confirmed by ELISA and immunoblotting following removal of terminal α-Gal epitopes by α-galactosidase treatment. These results suggest for the first known time that bites from the A. sculptum tick may be associated with the unknown etiology of allergic reactions to red meat in Brazil. [ABSTRACT FROM AUTHOR]

The International Xenotransplantation Association has updated its original 'Consensus Statement on Conditions for Undertaking Clinical Trials of Porcine Islet Products in Type 1 Diabetes,' which was published in Xenotransplantation in 2009. This update is timely and important in light of scientific progress and changes in the regulatory framework pertinent to islet xenotransplantation. Except for the chapter on 'informed consent,' which has remained relevant in its 2009 version, all other chapters included in the initial consensus statement have been revised for inclusion in this update. These chapters will not provide complete revisions of the original chapters; rather, they restate the key points made in 2009, emphasize new and under-appreciated topics not fully addressed in 2009, suggest relevant revisions, and communicate opinions that complement the consensus opinion. Chapter 1 provides an update on national regulatory frameworks addressing xenotransplantation. Chapter 2 a, previously Chapter 2, suggests several important revisions regarding the generation of suitable source pigs from the perspective of the prevention of xenozoonoses. The newly added Chapter 2b discusses conditions for the use of genetically modified source pigs in clinical islet xenotransplantation. Chapter 3 reviews porcine islet product manufacturing and release testing. Chapter 4 revisits the critically important topic of preclinical efficacy and safety data required to justify a clinical trial. The main achievements in the field of transmission of all porcine microorganisms, the rationale for more proportionate recipient monitoring, and response plans are reviewed in Chapter 5. Patient selection criteria and circumstances where trials of islet xenotransplantation would be both medically and ethically justified are examined in Chapter 6 in the context of recent advances in available and emerging alternative therapies for serious and potentially life-threatening complications of diabetes. It is hoped that this first update of the International Xenotransplantation Association porcine islet transplant consensus statement will assist the islet xenotransplant scientific community, sponsors, regulators, and other stakeholders actively involved in the clinical translation of islet xenotransplantation. [ABSTRACT FROM AUTHOR]

Background: Survival of vascularized xenografts is dependent on pre-emptive inhibition of the xenoantibody response against galactosyltransferase knockout (GTKO) porcine organs. Our analysis in multiple GTKO pig-to-primate models of xenotransplantation has demonstrated that the anti-non-gal-α-1,3-gal (anti-non-Gal) xenoantibody response displays limited structural diversity. This allowed our group to identify an experimental compound which selectively inhibited induced anti-non-Gal IgM xenoantibodies. However, because this compound had an unknown safety profile, we extended this line of research to include screening small molecules with known safety profiles allowing rapid advancement to large animal models. Methods: The NIH clinical collections of small molecules were screened by ELISA for their ability to inhibit xenoantibody binding to GTKO pig endothelial cells. Serum collected from non-immuno-suppressed rhesus monkeys at day 14 post-injection with GTKO pig endothelial cells was utilized as a source of elicited xenoantibody for initial screening. Virtual small molecule screening based on xenoantibody structure was used to assess the likelihood that the identified small molecules bound xenoantibody directly. As a proxy for selectivity, ELISAs against tetanus toxoid and the natural antigens laminin, thyroglobulin, and single-stranded DNA (ssDNA) were utilized to assess the ability of the identified reagents to inhibit additional antibody responses. The identified inhibitory small molecules were further tested for their ability to inhibit xenoantibody elicited in multiple settings, including rhesus monkeys pre-treated with an anti-non-Gal selective anti-idiotypic antibody, non-immunosuppressed rhesus monkeys immunized with wild-type fetal pig isletlike cell clusters, and non-immunosuppressed baboons transplanted with GTKO multiple transgenic pig kidneys. Results: Four clinically relevant small molecules inhibited anti-non-Gal IgM binding to GTKO pig endothelial cells in vitro. Three of these drugs displayed a limited region of structural similarity suggesting they may inhibit xenoantibody by a similar mechanism. One of these, the anti-hypertensive agent clonidine, displayed only minimal inhibition of antibodies elicited by vaccination against tetanus toxoid or pre-existing natural antibodies against laminin, thyroglobulin, or ssDNA. Furthermore, clonidine inhibited elicited anti-non-Gal IgM from all animals that demonstrated a xenoantibody response in each experimental setting. Conclusions: Clinically relevant small molecule drugs with known safety profiles can inhibit xenoantibody elicited against non-Gal antigens in diverse experimental xenotransplantation settings. These molecules are ready to be tested in large animal models. However, it will first be necessary to optimize the timing and dosing required to inhibit xenoantibodies in vivo. [ABSTRACT FROM AUTHOR]

Aims: Some environmental insults, such as fine particulate matter (PM) exposure, significantly impair the function of stem cells. However, it is unknown if PM exposure could affect the population of bone marrow stem cells (BMSCs). The present study was to investigate the effects of PM on BMSCs population and related mechanism(s). Main Metheods: PM was intranasally distilled into male C57BL/6 mice for one month. Flow cytometry with antibodies for BMSCs, Annexin V and BrdU ware used to determine the number of BMSCs and the levels of their apoptosis and proliferation in vivo. Phosphorylated Akt (P-Akt) level was determined in the BM cells with western blotting. Intracellular reactive oxygen species (ROS) formation was quantified using flow cytometry analysis. To determine the role of PM-induced ROS in BMSCs population, proliferation, and apotosis, experiments were repeated using N-acetylcysteine (NAC)-treated wild type mice or a triple transgenic mouse line with overexpression of antioxidant network (AON) composed of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase-1 with decreased in vivo ROS production. Key Findings: PM treatment significantly reduced BMSCs population in association with increased ROS formation, decreased P-Akt level, and inhibition of proliferation of BMSCs without induction of apoptosis. NAC treatment or AON overexpression with reduced ROS formation effectively prevented PM-induced reduction of BMSCs population and proliferation with partial recovery of P-Akt level. Significance: PM exposure significantly decreased the population of BMSCs due to diminished proliferation via ROS-mediated mechanism (could be partially via inhibition of Akt signaling). [ABSTRACT FROM AUTHOR]

Background/Aims: Bone marrow (BM)-derived endothelial progenitor cells (EPCs) play a critical role in angiogenesis and vascular repair. Some environmental insults, like fine particulate matter (PM) exposure, significantly impair cardiovascular functions. However, the mechanisms for PM-induced adverse effects on cardiovascular system remain largely unknown. The present research was to study the detrimental effects of PM on EPCs and explore the potential mechanisms. Methods: PM was intranasal-distilled into male C57BL/6 mice for one month. Flow cytometry was used to measure the number of EPCs, apoptosis level of circulating EPCs and intracellular reactive oxygen species (ROS) formation. Serum TNF- α and IL-1β were measured using ELISA. To determine the role of PM-induced ROS in EPC apoptosis, PM was co-administrated with the antioxidant N-acetylcysteine (NAC) in wild type mice or used in a triple transgenic mouse line (TG) with overexpression of antioxidant enzyme network (AON) composed of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase (Gpx-1) with decreased in vivo ROS production. Results: PM treatment significantly decreased circulating EPC population, promoted apoptosis of EPCs in association with increased ROS production and serum TNF-α and IL-1β levels, which could be effectively reversed by either NAC treatment or overexpression of AON. Conclusion: PM exposure significantly decreased circulating EPCs population due to increased apoptosis via ROS formation in mice. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Summary 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:β-galactoside-α1-3-galactosyltransferase ( α1,3GT ) gene, which ablated the expression of the Galα1-3Galβ1-4GlcNAc-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. PaperFlick [ABSTRACT FROM AUTHOR]

Background Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate ( NHP) models, and particularly using genetically engineered donors. Methods In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/ hCD55/ hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model. Results Corneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals. Conclusions Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression. [ABSTRACT FROM AUTHOR]

Background Xenotransplantation of porcine organs holds promise of solving the human organ donor shortage. The use of α-1,3-galactosyltransferase knockout (GTKO) pig donors mitigates hyperacute rejection, while delayed rejection is currently precipitated by potent immune and hemostatic complications. Previous analysis by our laboratory suggests that clotting factor VIII (FVIII) inhibitors might be elicited by the structurally restricted xenoantibody response which occurs after transplantation of either pig GTKO/ hCD55/ hCD59/ hHT transgenic neonatal islet cell clusters or GTKO endothelial cells. Methods A recombinant xenoantibody was generated using sequences from baboons demonstrating an active xenoantibody response at day 28 after GTKO/ hCD55/ hCD59/ hHT transgenic pig neonatal islet cell cluster transplantation. Rhesus monkeys were immunized with GTKO pig endothelial cells to stimulate an anti-non-Gal xenoantibody response. Serum was collected at days 0 and 7 after immunization. A two-stage chromogenic assay was used to measure FVIII cofactor activity and identify antibodies which inhibit FVIII function. Molecular modeling and molecular dynamics simulations were used to predict antibody structure and the residues which contribute to antibody-FVIII interactions. Competition ELISA was used to verify predictions at the domain structural level. Results Antibodies that inhibit recombinant human FVIII function are elicited after non-human primates are transplanted with either GTKO pig neonatal islet cell clusters or endothelial cells. There is an apparent increase in inhibitor titer by 15 Bethesda units (Bu) after transplant, where an increase greater than 5 Bu can indicate pathology in humans. Furthermore, competition ELISA verifies the computer modeled prediction that the recombinant xenoantibody, H66K12, binds the C1 domain of FVIII. Conclusions The development of FVIII inhibitors is a novel illustration of the potential impact the humoral immune response can have on coagulative dysfunction in xenotransplantation. However, the contribution of these antibodies to rejection pathology requires further evaluation because 'normal' coagulation parameters after successful xenotransplantation are not fully understood. [ABSTRACT FROM AUTHOR]

Background Promising developments in porcine islet xenotransplantation could resolve the donor pancreas shortage for patients with type 1 diabetes. Using α1,3-galactosyltransferase gene knockout (GTKO) donor pigs with multiple transgenes should extend xenoislet survival via reducing complement activation, thrombus formation, and the requirement for exogenous immune suppression. Studying the xenoantibody response to GTKO/ hCD55/ hCD59/ hHT islets in the pig-to-baboon model, and comparing it with previously analyzed responses, would allow the development of inhibitory reagents capable of targeting conserved idiotypic regions. Methods We generated IgM heavy and light chain gene libraries from 10 untreated baboons and three baboons at 28 days following transplantation of GTKO/ hCD55/ hCD59/ hHT pig neonatal islet cell clusters with immunosuppression. Flow cytometry was used to confirm the induction of a xenoantibody response. IgM germline gene usage was compared pre- and post-transplant. Homology modeling was used to compare the structure of xenoantibodies elicited after transplantation of GTKO/ hCD55/ hCD59/ hHT pig islets with those induced by GTKO and wild-type pig endothelial cells without further genetic modification. Results IgM xenoantibodies that bind to GTKO pig cells and wild-type pig cells were induced after transplantation. These anti-non-Gal antibodies were encoded by the IGHV3-66*02 (Δ28%) and IGKV1-12*02 (Δ25%) alleles, for the immunoglobulin heavy and light chains, respectively. IGHV3-66 is 86.7% similar to IGHV3-21 which was elicited by rhesus monkeys in response to GTKO endothelial cells. Heavy chain genes most similar to IGHV3-66 were found to utilize the IGHJ4 gene in 85% of V-D regions analyzed. However, unlike the wild-type response, a consensus complementary determining region 3 was not identified. Conclusions Additional genetic modifications in transgenic GTKO pigs do not substantially modify the structure of the restricted group of anti-non-Gal xenoantibodies that mediate induced xenoantibody responses with or without immunosuppression. The use of this information to develop new therapeutic agents to target this restricted response will likely be beneficial for long-term islet cell survival and for developing targeted immunosuppressive regimens with less toxicity. [ABSTRACT FROM AUTHOR]

Background B-cell depletion significantly extends survival of α-1,3-galactosyltranferase knockout ( GTKO) porcine organs in pig-to-primate models. Our previous work demonstrated that the anti-non-Gal xenoantibody response is structurally restricted. Selective inhibition of xenoantigen/xenoantibody interactions could prolong xenograft survival while preserving B-cell-mediated immune surveillance. Methods The anti-idiotypic antibody, B4N190, was selected from a synthetic human phage display library after enrichment against a recombinant anti-non-Gal xenoantibody followed by functional testing in vitro. The inhibitory small molecule, JMS022, was selected from the NCI diversity set III using virtual screening based on predicted xenoantibody structure. Three rhesus monkeys were pre-treated with anti-non-Gal-specific single-chain anti-idiotypic antibody, B4N190. A total of five monkeys, including two untreated controls, were then immunized with GTKO porcine endothelial cells to initiate an anti-non-α-1,3-Gal (non-Gal) xenoantibody response. The efficacy of the inhibitory small molecule specific for anti-non-Gal xenoantibody, JMS022, was tested in vitro. Results After the combination of in vivo anti-id and in vitro small molecule treatments, IgM xenoantibody binding to GTKO cells was reduced to pre-immunization levels in two-thirds of animals; however, some xenoantibodies remained in the third animal. Furthermore, when treated with anti-id alone, all three experimental animals displayed a lower anti-non-Gal IgG xenoantibody response compared with controls. Treatment with anti-idiotypic antibody alone reduced IgM xenoantibody response intensity in only one of three monkeys injected with GTKO pig endothelial cells. In the one experimental animal, which displayed reduced IgM and IgG responses, select B-cell subsets were also reduced by anti-id therapy alone. Furthermore, natural antibody responses, including anti-laminin, anti-ss DNA, and anti-thyroglobulin antibodies were intact despite targeted depletion of anti-non-Gal xenoantibodies in vivo indicating that selective reduction of xenoantibodies can be accomplished without total B-cell depletion. Conclusions This preliminary study demonstrates the strength of approaches designed to selectively inhibit anti-non-Gal xenoantibody. Both anti-non-Gal-specific anti-idiotypic antibody and small molecules can be used to selectively limit xenoantibody responses. [ABSTRACT FROM AUTHOR]

Abstract: It is often desirable to co-express a reporter protein with a potential therapeutic protein, to verify correct targeting of an expression strategy. Vectors containing a viral self-processing 2A sequence have been reported to drive equimolar expression of two or more transgenes from a single promoter. Here, we report on the co-expression of a secreted antibody fragment and an intracellular reporter protein, enhanced yellow fluorescent protein from lentiviral shuttle plasmids by inserting a furin-2A (F2A) sequence between the two cDNAs, in two different orientations, in the expression cassette. We show that the order of these two transgenes relative to the F2A sequence affects expression levels. Reduced expression of each transgene positioned downstream of F2A, compared with upstream of F2A, was observed (p<0.05). Moreover, protein expression from double-cDNA plasmids was significantly lower than from their corresponding single transgene counterparts (p<0.05). [Copyright &y& Elsevier]

Background: Xenotransplantation could provide a solution to the donor shortage that is currently the major barrier to solid-organ transplantation. The ability to breed pigs with multiple genetic modifications provides a unique opportunity to explore the immunologic challenges of pulmonary xenotransplantation. Methods: Explanted lungs from wild-type and 3 groups of genetically modified pigs were studied: (i) α1,3-galactosyltransferase gene knockout (GTKO); (ii) GTKO pigs expressing the human complementary regulatory proteins CD55 and CD59 (GTKO/CD55-59); and (iii) GTKO pigs expressing both CD55-59 and CD39 (GTKO/CD55-59/CD39). The physiologic, immunologic and histologic properties of porcine lungs were evaluated on an ex vivo rig after perfusion with human blood. Results: Lungs from genetically modified pigs demonstrated stable pulmonary vascular resistance and better oxygenation of the perfusate, and survived longer than wild-type lungs. Physiologic function was inversely correlated with the degree of platelet sequestration into the xenograft. Despite superior physiologic profiles, lungs from genetically modified pigs still showed evidence of intravascular thrombosis and coagulopathy after perfusion with human blood. CONCLUSIONS: The ability to breed pigs with multiple genetic modifications, and to evaluate lung physiology and histology in real-time on an ex vivo rig, represent significant advances toward better understanding the challenges inherent to pulmonary xenotransplantation. [ABSTRACT FROM AUTHOR]

Background Immunological and histopathological features in pig-to-primate renal xenotransplantation are widely studied. Only limited data have been reported about clinicopathological findings in primate recipients of life-supporting renal xenografts. In human medicine, proteinuria represents a common complication in kidney transplantation and is associated with impaired graft survival. The detection of low molecular weight proteins of tubular origin is considered an early method for predicting potential graft rejection. In this study, the presence and the significance of quantitative and qualitative proteinuria were evaluated in xenotransplanted non-human primates in which kidney function was supported only by the transplanted organ. Methods Eight bilaterally nephrectomized cynomolgus monkeys ( Macaca fascicularis) were transplanted with a single kidney from α1,3-galactosyltransferase gene-knockout ( GTKO) pigs transgenic for human CD39, CD55, CD59, and α1,2-fucosyltransferase. In addition to hematological and biochemical analyses, quantitative and qualitative analysis of proteinuria was evaluated by urinary protein-to-creatinine ratio ( UPC ratio) and sodium dodecyl sulfate-agarose gel electrophoresis ( SDS- AGE), respectively. Results The main hematological and biochemical changes recorded after transplantation were a progressive anemia and a severe and progressive decrease in total proteins. In urine samples, the UPC ratio was low before transplantation and increased after transplantation. Similarly, SDS- AGE was negative before transplantation, but bands consistent with mixed (i.e., tubular and glomerular) proteinuria were observed in all samples collected post-transplantation. Conclusions The study of clinicopathological changes in cynomolgus monkey renal xenograft recipients provides a valid help in monitoring the health conditions in the post-transplant period. Moreover, the evaluation of UPC ratio and the use of SDS- AGE technique in urine samples of cynomolgus monkey renal xenograft recipients may be considered a valid, inexpensive, and less time-consuming method than more sophisticated techniques in monitoring proteinuria. Proteinuria and presence of low molecular weight ( LMW) proteins were consistently found in urine after transplantation, independent of fluctuations in renal function. [ABSTRACT FROM AUTHOR]

Background Glutaraldehyde fixation does not guarantee complete tissue biocompatibility in current clinical bioprosthetic heart valves ( BHVs). Particularly, circulating anti-α Gal human antibodies increase significantly from just 10 days after a BHV implantation. The inactivation of such epitope should be mandatory to meet the requirements for a perspectively safe clinical application; nevertheless, its quantitative assessment in commercially available BHVs has never been carried out. Methods In this investigation, seven different models of BHVs were tested. The number of epitopes was determined with reference to a standard α Gal source by an ELISA test. The presence of xenoantigen was subsequently confirmed by immunofluorescence analysis. Porcine tissue, knockout for the α Gal epitopes, was used as negative control. Results Epic™ valve was the only model among those tested, in which the α Gal antigen appeared to be completely shielded. Composite Trifecta™ valve exhibited conflicting results: cusps of bovine pericardial tissue were devoid of reactive α Gal epitopes, while the stent cover strip of porcine pericardium still maintained 30% of active antigens originally present in native tissue. All other tested BHVs express an α Gal amount not significantly different from that exhibited by porcine Mosaic® valve (5.2 ± 0.6 × 1010 each 10 mg of tissue). Conclusions For the first time, the quantitative evaluation of the α Gal epitope in heart valve bioprostheses, already in clinical practice for about 40 yrs, was finally determined. Such quantification might provide indications of biocompatibility relevant for the selection of bioprosthetic devices and an increase in the confidence of the patient. It might become a major quality control tool in the production and redirection of future investigation in the quest for α Gal-free long-lasting substitutes. [ABSTRACT FROM AUTHOR]

Islet allograft survival limits the long-term success of islet transplantation as a potential curative therapy for type 1 diabetes. A number of factors compromise islet survival, including recurrent diabetes. We investigated whether CD39, an ectonucleotidase that promotes the generation of extracellular adenosine, would mitigate diabetes in the T cell-mediated multiple low-dose streptozotocin (MLDS) model. Mice null for CD39 (CD39KO), wild-type mice (WT), and mice overexpressing CD39 (CD39TG) were subjected to MLDS. Adoptive transfer experiments were performed to delineate the efficacy of tissue-restricted overexpression of CD39. The role of adenosine signaling was examined using mutant mice and pharmacological inhibition. The susceptibility to MLDS-induced diabetes was influenced by the level of expression of CD39. CD39KO mice developed diabetes more rapidly and with higher frequency than WT mice. In contrast, CD39TG mice were protected. CD39 overexpression conferred protection through the activation of adenosine 2A receptor and adenosine 2B receptor. Adoptive transfer experiments indicated that tissue-restricted overexpression of CD39 conferred robust protection, suggesting that this may be a useful strategy to protect islet grafts from T cell-mediated injury. [ABSTRACT FROM AUTHOR]