Serp-2 preserves tissue viability after ischemia-reperfusion injury. (a) Representative images of sham operated or LIRI-induced mice treated with saline, Serp-2 or M-T7 with 2× or 10× objectives (20× and 100× magnification, respectively). 10× objective image regions are indicated by black boxes in the 2× objective images. Scale bars represent 1000 μm (2× obj.) and 200 μm (10× obj.). Infarcted tissue is indicated with black arrows. (b) Relative measure of infarct thickness in livers of LIRI-induced mice treated with saline, Serp-2 or M-T7. Statistics calculated by one-way ANOVA with Fisher's PLSD post-hoc test (N = 2-5 mice per group)

Serp-2 preserves tissue viability after ischemia-reperfusion injury. (a) Representative images of sham operated or LIRI-induced mice treated with saline, Serp-2 or M-T7 with 2× or 10× objectives (20× and 100× magnification, respectively). 10× objective image regions are indicated by black boxes in the 2× objective images. Scale bars represent 1000 μm (2× obj.) and 200 μm (10× obj.). Infarcted tissue is indicated with black arrows. (b) Relative measure of infarct thickness in livers of LIRI-induced mice treated with saline, Serp-2 or M-T7. Statistics calculated by one-way ANOVA with Fisher's PLSD post-hoc test (N = 2-5 mice per group)

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Background: Ischemia-reperfusion injury (IRI) is an antigen-independent, innate immune response to arterial occlusion and ischemia with subsequent paradoxical exacerbation after reperfusion. IRI remains a critical problem after vessel occlusion and infarction or during harvest and surgery in transplants. After transplant, liver IRI (LIRI) contribu...

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... next investigated the effect of Serp-2 or M-T7 treatment on maintenance of liver viability after ischemiareperfusion. When compared to sham-operated mice, histopathology clearly demonstrates a significant increase in hepatocyte necrosis in saline-treated mice. This tissue necrosis was ameliorated by treatment with Serp-2, but not by M-T7 ( Fig. 2A). Regions of the liver affected by IRI that developed evidence for infarction and scarring had significantly reduced infarct areas after treatment with Serp-2, but not with M-T7 when compared to saline at 24 h follow up ( Fig. 2B; p = 0.0016). At this point in our study, we determined that M-T7 was not effective in this model and thus ...
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... necrosis in saline-treated mice. This tissue necrosis was ameliorated by treatment with Serp-2, but not by M-T7 ( Fig. 2A). Regions of the liver affected by IRI that developed evidence for infarction and scarring had significantly reduced infarct areas after treatment with Serp-2, but not with M-T7 when compared to saline at 24 h follow up ( Fig. 2B; p = 0.0016). At this point in our study, we determined that M-T7 was not effective in this model and thus focused on the mechanism and therapeutic benefit of Serp-2. Independent histopathological analysis by pathologists blinded to treatments, indicated that numerous indicators of liver viability were improved with Serp-2 treatment ...
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... immune modulating protein, did not provide any survival advantage, reduction in acute injury markers nor preservation of tissue viability ( Figs. 1 and 2). We also note that not only do mice treated with Serp-2 survive, but their livers display significant reductions in necrosis and overall pathology when compared to saline-treated mice ( Fig. 2 and Table 2). In investigating a potential physiologic mechanism for Serp-2-dependent survival advantage in this model, we note that in addition to reduced infarct scarring of the liver, areas of infarct had smaller inflammatory cell infiltrates, identified as macrophages by immunohistochemistry (Fig. 3). While we cannot specifically ...

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... In addition, the liver sections were analyzed according to the scoring system of Suzuki that assesses three main injury outcomes, congestion, vacuolization and necrosis (Suzuki et al., 1993). High levels of necrosis were evident in the livers of mice subjected to IR, consistent with previous findings (Yaron et al., 2019), and this was significantly reduced in the IR animals receiving AC pretreatment ( Figure 7B). In all analyses the readers were blinded to the treatment groups. ...
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Ceramide is a bioactive signaling lipid involved in the pathogenesis of numerous diseases. It also plays an important role in ischemia reperfusion (IR) injury via activation of inflammatory/oxidative stress-stimulated signaling pathways, resulting in tissue damage. Acid ceramidase is a lipid hydrolase that modulates the levels of ceramide, and as such has a potential therapeutic role in many human diseases where ceramide has been implicated. Here we investigated the therapeutic potential of recombinant acid ceramidase in a murine model of hepatic IR injury. Serum ALT, AST, and LDH activities, as well as oxidative stress (MDA) and inflammatory (MCP-1) markers, were increased in mice subjected to IR compared to a sham group. In contrast, these elevations were significantly lower in an IR group pretreated with a single injection of acid ceramidase. Histological examination by two different assessment criteria also revealed that acid ceramidase pretreatment alleviated IR-induced hepatocyte damage, including reduced evidence of cell death and necrosis. In addition, elevated ceramide and sphingosine levels were observed in the IR group compared to sham, and were markedly reduced when pretreated with acid ceramidase. In contrast, the levels of the protective signaling lipid, sphingosine-1-phosphate (S1P), were reduced following IR and elevated in response to acid ceramidase pretreatment. These changes in sphingolipid levels could be correlated with changes in the activities of several sphingolipid-metabolizing enzymes. Overall, these results indicated that sphingolipid changes were an important pathologic component of hepatic IR injury, and that acid ceramidase administration ameliorated these lipid changes and other downstream pathologic changes.
... In a related study, Zhang et al. described that A1AT-dependent protection of islet viability after cytokine-and streptozotocininduced diabetes in mice was in part due to dramatic reduction of beta cell apoptosis (175) Ischemia-reperfusion injury is characterized by a transient loss of blood and oxygen to a tissue, followed by a period of reoxygenation which paradoxically accelerates damage caused during the hypoxic period (184). Ischemia-reperfusion injury can occur in any tissue, whether by pathogenic etiology or by complications of surgical procedure, and there is an unmet need for novel therapeutics to address the condition (185)(186)(187)(188). Moldthan et al. first demonstrated the therapeutic efficacy of A1AT therapy in a rat model of ischemic stroke which resulted in a drastic reduction of infarct volume and preservation of sensory motor system function (189). ...
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The making and breaking of clots orchestrated by the thrombotic and thrombolytic serine protease cascades are critical determinants of morbidity and mortality during infection and with vascular or tissue injury. Both the clot forming (thrombotic) and the clot dissolving (thrombolytic or fibrinolytic) cascades are composed of a highly sensitive and complex relationship of sequentially activated serine proteases and their regulatory inhibitors in the circulating blood. The proteases and inhibitors interact continuously throughout all branches of the cardiovascular system in the human body, representing one of the most abundant groups of proteins in the blood. There is an intricate interaction of the coagulation cascades with endothelial cell surface receptors lining the vascular tree, circulating immune cells, platelets and connective tissue encasing the arterial layers. Beyond their role in control of bleeding and clotting, the thrombotic and thrombolytic cascades initiate immune cell responses, representing a front line, “off-the-shelf” system for inducing inflammatory responses. These hemostatic pathways are one of the first response systems after injury with the fibrinolytic cascade being one of the earliest to evolve in primordial immune responses. An equally important contributor and parallel ancient component of these thrombotic and thrombolytic serine protease cascades are the ser ine p rotease in hibitors, termed serpins . Serpins are metastable suicide inhibitors with ubiquitous roles in coagulation and fibrinolysis as well as multiple central regulatory pathways throughout the body. Serpins are now known to also modulate the immune response, either via control of thrombotic and thrombolytic cascades or via direct effects on cellular phenotypes, among many other functions. Here we review the co-evolution of the thrombolytic cascade and the immune response in disease and in treatment. We will focus on the relevance of these recent advances in the context of the ongoing COVID-19 pandemic. SARS-CoV-2 is a “respiratory” coronavirus that causes extensive cardiovascular pathogenesis, with microthrombi throughout the vascular tree, resulting in severe and potentially fatal coagulopathies.
... Inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD domain) deficiency, IL-1β blocking antibody injection [43], injection of Serp 2 (a virus-derived serine protease and pan-caspase inhibitor) [47], and caspase 1/caspase 4 substrate Gasdermin D deficiency [48] lead to protection against liver IRI [49]. Caspase 1 deficient mice are less susceptible to acetaminophen-induced liver injury [50]. ...
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Ischemia reperfusion injury (IRI) during liver transplantation increases morbidity and contributes to allograft dysfunction. There are no therapeutic strategies to mitigate IRI. We examined a novel hypothesis: caspase 1 and caspase 11 serve as danger-associated molecular pattern (DAMPs) sensors in IRI. By performing microarray analysis and using caspase 1/caspase 11 double-knockout (Casp DKO) mice, we show that the canonical and non-canonical inflammasome regulators are upregulated in mouse liver IRI. Ischemic pre (IPC)- and post-conditioning (IPO) induce upregulation of the canonical and non-canonical inflammasome regulators. Trained immunity (TI) regulators are upregulated in IPC and IPO. Furthermore, caspase 1 is activated during liver IRI, and Casp DKO attenuates liver IRI. Casp DKO maintained normal liver histology via decreased DNA damage. Finally, the decreased TUNEL assay-detected DNA damage is the underlying histopathological and molecular mechanisms of attenuated liver pyroptosis and IRI. In summary, liver IRI induces the upregulation of canonical and non-canonical inflammasomes and TI enzyme pathways. Casp DKO attenuate liver IRI. Development of novel therapeutics targeting caspase 1/caspase 11 and TI may help mitigate injury secondary to IRI. Our findings have provided novel insights on the roles of caspase 1, caspase 11, and inflammasome in sensing IRI derived DAMPs and TI-promoted IRI-induced liver injury.
... Myxoma virus (MYXV) is a leporipoxvirus with well-known strict species-specificity and hosttropism to the European rabbit (Oryctolagus cuniculus) [16]. We have demonstrated the safety and immunotherapeutic efficacy of several MYXV immune modulators in a wide array of preclinical models [17][18][19][20][21][22][23][24][25][26][27]. M-T7 is an MYXV-derived immune modulator with broad chemokine-binding activity and proven therapeutic potential in inflammation-related diseases. ...
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Complex dermal wounds represent major medical and financial burdens, especially in the context of comorbidities such as diabetes, infection and advanced age. New approaches to accelerate and improve, or "fine tune" the healing process, so as to improve the quality of cutaneous wound healing and management, are the focus of intense investigation. Here, we investigate the topical application of a recombinant immune modulating protein which inhibits the interactions of chemokines with glycosaminoglycans, reducing damaging or excess inflammation responses in a splinted full-thickness excisional wound model in mice. M-T7 is a 37 kDa-secreted, virus-derived glycoprotein that has demonstrated therapeutic efficacy in numerous animal models of inflammatory immunopathology. Topical treatment with recombinant M-T7 significantly accelerated wound healing when compared to saline treatment alone. Healed wounds exhibited properties of improved tissue remodeling, as determined by collagen maturation. M-T7 treatment accelerated the rate of peri-wound angiogenesis in the healing wounds with increased levels of TNF, VEGF and CD31. The immune cell response after M-T7 treatment was associated with a retention of CCL2 levels, and increased abundances of arginase-1-expressing M2 macrophages and CD4 T cells. Thus, topical treatment with recombinant M-T7 promotes a pro-resolution environment in healing wounds, and has potential as a novel treatment approach for cutaneous tissue repair.
... In other models, Serp-2 was found to suppress inflammation and improve outcomes in models of aortic transplant and carotid cuff compression injury [16][17][18]. Based on the overlap of the targeted protease pathways (inflammasome and apoptosis), Serp-2 was tested as a putative therapeutic to prevent liver injury and improve outcomes after 90 min of partial (70%) warm ischemia-reperfusion injury [19]. Using this model, we found that treatment with recombinant purified Serp-2 improves 10-day survival, ameliorates acute injury as measured by circulating liver markers, and preserves liver viability. ...
... We previously tested this model with purified recombinant Serp-2 protein [19]. To produce the recombinant protein, the coding sequence for Serp-2 was tagged with an N-terminal 10Â-histidine affinity tag and transformed into HeLa cells with a vaccinia/T7 vector [17,23]. ...
Chapter
Ischemia-reperfusion injury (IRI) drives early and long-term damage to organs as well as compounding damage from acute transplant rejection and surgical trauma. IRI initiates an aggressive and prolonged inflammation leading to tissue injury, organ failure, and death. However, there are few effective therapeutic interventions for IRI. The destructive inflammatory cell activity in IRI is part of an aberrant innate immune response that triggers multiple pathways. Hence, immune-modulating treatments to control pathways triggered by IRI hold great therapeutic potential. Viruses, especially large DNA viruses, have evolved highly effective immune-modulating proteins for the purpose of immune evasion and to protect the virus from the host immune defenses. A number of these immune-modulating proteins have proven therapeutically effective in preclinical models, many with function targeting pathways known to be involved in IRI. The use of virus-derived immune-modulating proteins thus represents a promising source for new treatments to target ischemia-reperfusion injury. Laboratory small animal models of IRI are well established and are able to reproduce many aspects of ischemia-reperfusion injury seen in humans. This chapter will discuss the methods used to perform the IRI procedure in mice, as well as clinically relevant diagnostic tests to evaluate liver injury and approaches for assessing histological damage while testing novel immune modulating protein treatments.
... In mouse models of aortic allograft transplants, Serp-2 significantly reduced inflammation and intimal hyperplasia, again with no detected side effects [50,51]. In a model of partial 70% warm ischemiareperfusion injury in the liver (LIRI), Serp-2 treatment given systemically also improved survival over 10 days, reduced necrotic damage of the liver and lowered acute markers of liver damage [61]. Surprisingly, caspase-1, caspase-3 and caspase-8 activation were not suppressed, suggesting an alternative mechanism of protection potentially by inhibition of circulating inflammatory proteases. ...
... Continuous infusion of vMIP-II decreased infiltrating hematogenous cells at the site of injury in a spinal cord contusion injury in rats, with associated reductions in neuronal loss and gliosis [150]. However, this study did not include an assessment of locomotor function, thus it remains to be seen whether chemokine antagonism with vMIP-II improves functional recovery, as seen with the poxvirus chemokine modulator M-T7 [61]. Gene transfer of vMIP-II by direct injection of plasmid DNA improved cardiac allograft survival when hearts were placed in the subcutaneous position of the ear pinnae of CBA/J recipients [151]. ...
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Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly effective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have "performed the R&D", developing highly effective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with efficacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics.
... Moreover, specific inhibitors of inflammasome need to be examined under conditions of different warm ischemia times and percentages. A recent study shows that treatment with Serp-2, a virus-derived inhibitor of apoptosis and inflammasome, regulates the levels of caspase-1, 8 and 10, improving the survival of mice submitted to 90 min of partial (70%) warm ischemia [128]. Interestingly, a study by Yang et al., using an experimental model of hepatic I/R based on 45 min of 70% warm ischemia, showed that Z-VD-fmk, a pan-caspase inhibitor (including caspase-1), had no effect on I/R injury or on the number of TUNEL-positive cells and staining pattern (nucleus and cytosol) [129]. ...
... Interestingly, a study by Yang et al., using an experimental model of hepatic I/R based on 45 min of 70% warm ischemia, showed that Z-VD-fmk, a pan-caspase inhibitor (including caspase-1), had no effect on I/R injury or on the number of TUNEL-positive cells and staining pattern (nucleus and cytosol) [129]. The authors noted the minor role of apoptosis, thus contesting a relevant role for inflammasome or pyroptosis in hepatic I/R, at least in conditions of 90 min of partial (70%) warm ischemia [128] or 30 min of total warm I/R injury [126]. Data suggest that future research should be focused on detailing the type of cell death (necrosis, apoptosis and/or pyroptosis) and the signaling mechanisms of cell death, to identify specific targets for attenuating hepatic I/R injury. ...
Article
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Ischemia-reperfusion injury is an important cause of liver damage occurring during surgical procedures including hepatic resection and liver transplantation, and represents the main underlying cause of graft dysfunction and liver failure post-transplantation. To date, ischemia-reperfusion injury is an unsolved problem in clinical practice. In this context, inflammasome activation, recently described during ischemia-reperfusion injury, might be a potential therapeutic target to mitigate the clinical problems associated with liver transplantation and hepatic resections. The present review aims to summarize the current knowledge in inflammasome-mediated inflammation, describing the experimental models used to understand the molecular mechanisms of inflammasome in liver ischemia-reperfusion injury. In addition, a clear distinction between steatotic and non-steatotic livers and between warm and cold ischemia-reperfusion injury will be discussed. Finally, the most updated therapeutic strategies, as well as some of the scientific controversies in the field will be described. Such information may be useful to guide the design of better experimental models, as well as the effective therapeutic strategies in liver surgery and transplantation that can succeed in achieving its clinical application.
Article
Liver ischaemia–reperfusion injury (LIRI), a local sterile inflammatory response driven by innate immunity, is one of the primary causes of early organ dysfunction and failure after liver transplantation. Cellular damage resulting from LIRI is an important risk factor not only for graft dysfunction but also for acute and even chronic rejection and exacerbates the shortage of donor organs for life-saving liver transplantation. Hepatocytes, liver sinusoidal endothelial cells and Kupffer cells, along with extrahepatic monocyte-derived macrophages, neutrophils and platelets, are all involved in LIRI. However, the mechanisms underlying the responses of these cells in the acute phase of LIRI and how these responses are orchestrated to control and resolve inflammation and achieve homeostatic tissue repair are not well understood. Technological advances allow the tracking of cells to better appreciate the role of hepatic macrophages and platelets (such as their origin and immunomodulatory and tissue-remodelling functions) and hepatic neutrophils (such as their selective recruitment, anti-inflammatory and tissue-repairing functions, and formation of extracellular traps and reverse migration) in LIRI. In this Review, we summarize the role of macrophages, platelets and neutrophils in LIRI, highlight unanswered questions, and discuss prospects for innovative therapeutic regimens against LIRI in transplant recipients.
Chapter
Myxoma virus (MYXV) has proven to be an effective candidate for oncolytic virotherapy in many preclinical cancer models. As a nonhuman pathogen, MYXV does not need to overcome any preexisting antiviral immunity, and its DNA cannot integrate into the host genome, making it an extremely safe vector. Moreover, the large dsDNA genome of MYXV allows the insertion of multiple transgenes and the design of engineered recombinant oncolytic viruses (OVs) with enhanced immunostimulatory or other desired properties. In this chapter, we describe detailed protocols for the generation and characterization of transgene-armed recombinant MYXV vectors.
Chapter
Viral diseases, whether of animals or humans, are normally considered as problems to be managed. However, in Australia, two viruses have been used as landscape-scale therapeutics to control European rabbits (Oryctolagus cuniculus), the preeminent invasive vertebrate pest species. Rabbits have caused major environmental and agricultural losses and contributed to extinction of native species. It was not until the introduction of Myxoma virus that effective control of this pest was obtained at a continental scale. Subsequent coevolution of rabbit and virus saw a gradual reduction in the effectiveness of biological control that was partially ameliorated by the introduction of the European rabbit flea to act as an additional vector for the virus. In 1995, a completely different virus, Rabbit hemorrhagic disease virus (RHDV), escaped from testing and spread through the Australian rabbit population and again significantly reduced rabbit numbers and environmental impacts. The evolutionary pressures on this virus appear to be producing quite different outcomes to those that occurred with myxoma virus and the emergence and invasion of a novel genotype of RHDV in 2014 have further augmented control. Molecular studies on myxoma virus have demonstrated multiple proteins that manipulate the host innate and adaptive immune response; however the molecular basis of virus attenuation and reversion to virulence are not yet understood.