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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]

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.

Abstract: Transplantation exposes vascularized grafts to several potential injuries, including ischemia-reperfusion injury and rejection. These processes are associated with, at least in part, extracellular nucleotide-triggered (type 2 purinergic receptor) inflammatory responses that if left unchecked might compromise the long-term function and survival of the graft. Modulation of type 2 purinergic receptor–mediated signaling occurs by ectoenzymes that hydrolyze nucleotides to adenosine, which in turn activate type-1 purinergic receptors, and play a critical role in limiting inflammation and thrombosis. CD39 is the prototype nucleoside triphosphate diphosphohydrolase and is highly expressed on the endothelium. Different levels of CD39 expression by the vasculature influence the fate of the transplanted organ in ischemia-reperfusion injury and transplant rejection models. Additional roles for extracellular nucleotides/adenosine have also been defined within the immune system. Moreover, CD39 is a phenotypic marker of regulatory T cells, and through the generation of adenosine, contributes to the suppressive capabilities of these cells in vitro and in vivo. Our experimental models suggest that purinergic mechanisms may provide an integration point for the vascular and immunological responses in transplantation. CD39, the dominant ectonucleotidase, receptors for nucleotide mediators and adenosine are expressed in a regulated manner on both vascular and immune cells. We predict that administration of soluble CD39 and adenosine agonists or the targeted expression of CD39 through genetically modified organs or cells may have future therapeutic application in transplant-associated and other vascular diseases. [Copyright &y& Elsevier]

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]

Abstract 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. Clinical Relevance Our animal model reveals a range of pathologic changes seen in patients with elective surgery on extremities requiring prolonged arterial occlusion. Pretreatment with C1 inhibitor shows the potential to reduce the extent of reperfusion injury by preventing endothelial cell damage. Our results may lead to novel, targeted therapies that could be used alone or in combination with other reperfusion strategies to improve clinical care. [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]

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]

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]

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]

Summary: Many pathogenic bacteria have sophisticated mechanisms to interfere with the mammalian immune response. These include the disruption of host extracellular ATP levels that, in humans, is tightly regulated by the nucleoside triphosphate diphosphohydrolase family (NTPDases). NTPDases are found almost exclusively in eukaryotes, the notable exception being their presence in some pathogenic prokaryotes. To address the function of bacterial NTPDases, we describe the structures of an NTPDase from the pathogen Legionella pneumophila (Lpg1905/Lp1NTPDase) in its apo state and in complex with the ATP analog AMPPNP and the subtype-specific NTPDase inhibitor ARL 67156. Lp1NTPDase is structurally and catalytically related to eukaryotic NTPDases and the structure provides a basis for NTPDase-specific inhibition. Furthermore, we demonstrate that the activity of Lp1NTPDase correlates directly with intracellular replication of Legionella within macrophages. Collectively, these findings provide insight into the mechanism of this enzyme and highlight its role in host-pathogen interactions. [Copyright &y& Elsevier]

Abstract: Signalling actions of extracellular nucleotides via P2 receptors influence cellular function in most tissues. In the inner ear, P2 receptor signaling is involved in many processes including the regulation of hearing sensitivity and the cochlea’s response to noise stress. CD39 (NTPDase1/ENTPD1) is an ectonucleotidase (ecto-nucleoside triphosphate diphosphohydrolase) that can hydrolyse purine and pyrimidine nucleoside tri- and di-phosphates to generate monophosphate nucleosides. Mice null for Cd39 exhibit major alterations in haemostasis and profound alterations in inflammatory and thrombotic reactions. Studies in the cochlea have suggested the involvement of purinergic-type signals that could be modulated by CD39 in regulation of cochlear blood flow and also auditory neurotransmission. This study aimed to determine the auditory phenotype of adult Cd39 null mice on the C57BL6 background. Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were unaffected in Cd39-deficient mice across the range of test frequencies, suggesting normal neural and outer hair cell function. Mutant mice also showed little difference to wild type mice in vulnerability to acoustic trauma. Gene expression analysis of other membrane-bound NTPDases with comparable hydrolytic activity demonstrated an up-regulation of Entpd2 and Entpd8 in the cochleae of Cd39 deficient mice. These findings suggest that Cd39 deletion alone does not adversely affect cochlear function, possibly as compensatory up-regulation of other surface located NTPDases may offset predicted alterations in cochlear homeostasis. [Copyright &y& Elsevier]

Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different [ABSTRACT FROM AUTHOR]

Abstract: Glycosylation of cell surface proteins is important in thymocyte maturation. In particular, the level of sialylation of key glycoproteins such as CD45 is believed to play a major role in regulating TCR signaling, adhesion and apoptosis of developing thymocytes. We show here that transgenic expression of human α1–2 fucosyltransferase (hFUT1) in mice resulted in a marked shift from sialylation to fucosylation of thymocyte glycoproteins. This was associated with a significant reduction in thymocyte number, an increased rate of apoptosis in double positive and single positive thymocytes, and a maturation arrest at TCR-dependent developmental transitions reminiscent of CD45 deficiency. Indeed, CD45RB dimerization was elevated in hFUT1 thymocytes, consistent with its hyposialylation, and there was a corresponding increase in phosphorylation of the TCR-associated protein Lck. However, contrary to the reduced TCR signaling in CD45 null mice, basal and stimulated TCR signaling was higher in hFUT1 thymocytes than in wild type thymocytes. Our results therefore demonstrate that aberrant expression of a single glycosyltransferase can profoundly affect thymopoiesis, although the relative involvement of CD45-dependent and -independent mechanisms is yet to be determined. [Copyright &y& Elsevier]