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| DAH Serp-1 and Serp-1m5 treatments reduced M1 macrophage and neutrophil numbers in lung sections after pristane-induced DAH, but did not significantly affect M2 macrophage counts, CD3+ cell, or CD4+ cell infiltration. (A) Representative micrographs (40×) of iNOS + IHC staining in lungs at 14 days post-pristine injection. (B) Serp-1 and Serp-1m5 group had significantly lower numbers of M1 macrophage counts marked by positive staining for iNOS IHC staining (Serp-1, p = 0.0350; Serp-1m5, p = 0.0053) when compared to the saline treatment group. (C) The Serp-1m5 treated group had significantly lower numbers of Ly6G+ neutrophils (Serp-1, p = 0.0371; Serp-1m5, p = 0.004). Serp-1 treatment had a strong trend toward reduced neutrophil counts, but did not reach significance. The number of Arg-1 + M2 macrophages (D), CD3+ T cells (E), or CD4+ T cells (F) were not statistically different among the three groups, although a non-significant trend toward increased Arg+ cells was seen. *p < 0.05, **p < 0.01.

| DAH Serp-1 and Serp-1m5 treatments reduced M1 macrophage and neutrophil numbers in lung sections after pristane-induced DAH, but did not significantly affect M2 macrophage counts, CD3+ cell, or CD4+ cell infiltration. (A) Representative micrographs (40×) of iNOS + IHC staining in lungs at 14 days post-pristine injection. (B) Serp-1 and Serp-1m5 group had significantly lower numbers of M1 macrophage counts marked by positive staining for iNOS IHC staining (Serp-1, p = 0.0350; Serp-1m5, p = 0.0053) when compared to the saline treatment group. (C) The Serp-1m5 treated group had significantly lower numbers of Ly6G+ neutrophils (Serp-1, p = 0.0371; Serp-1m5, p = 0.004). Serp-1 treatment had a strong trend toward reduced neutrophil counts, but did not reach significance. The number of Arg-1 + M2 macrophages (D), CD3+ T cells (E), or CD4+ T cells (F) were not statistically different among the three groups, although a non-significant trend toward increased Arg+ cells was seen. *p < 0.05, **p < 0.01.

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Diffuse alveolar hemorrhage (DAH) is one of the most serious clinical complications of systemic lupus erythematosus (SLE). The prevalence of DAH is reported to range from 1 to 5%, but while DAH is considered a rare complication there is a reported 50-80% mortality. There is at present no proven effective treatment for DAH and the therapeutics that...

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... characterized the proinflammatory M1 macrophage polarization in the mouse lung tissue sections by IHC staining for iNOS (iNOS+). As shown in Figure 5, the lungs of mice treated by Serp-1 and Serp-1m5 treatments had significantly lower numbers of iNOS+ ...
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... p = 0.0350; Serp-1m5, p = 0.0053). Additionally, lung tissues treated with Serp-1 and Serp-1m5 also have significantly less detected numbers of Ly6G+ neutrophils than the saline treatment group (Figure 5C; Serp-1, p = 0.0371; Serp-1m5, p = 0.004). We also performed IHC staining for arginase-1 (Arg-1) to characterize the anti-inflammatory M2 macrophages (Figure 5D), but no statistical differences among these three groups were observed for Arg-1+ M2 macrophage. ...
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... lung tissues treated with Serp-1 and Serp-1m5 also have significantly less detected numbers of Ly6G+ neutrophils than the saline treatment group (Figure 5C; Serp-1, p = 0.0371; Serp-1m5, p = 0.004). We also performed IHC staining for arginase-1 (Arg-1) to characterize the anti-inflammatory M2 macrophages (Figure 5D), but no statistical differences among these three groups were observed for Arg-1+ M2 macrophage. Arg-1+ M2 macrophage staining detected a nonsignificant trend toward increased numbers. ...
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... M2 macrophage staining detected a nonsignificant trend toward increased numbers. Serp-1 and Serp-1m5 treatment groups have a trend to increased CD3+ T cells (Figure 5E) when compared to saline treated mice but this does not achieve significance (p = 0.3595). There were no identified changes in different in CD4+ T helper cell staining either (Figure 5F; p = 0.1015). ...
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... and Serp-1m5 treatment groups have a trend to increased CD3+ T cells (Figure 5E) when compared to saline treated mice but this does not achieve significance (p = 0.3595). There were no identified changes in different in CD4+ T helper cell staining either (Figure 5F; p = 0.1015). ...

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... Treatment with purified native Serp-1 has demonstrated both acute and long-term efficacy in modulating inflammation in a wide range of inflammatory disorders and injuries, including atherosclerosis, transplant, wound healing, and spinal cord injury [39][40][41][42]. More recently, a modified Serp-1 protein, PEGSerp-1, with a longer halflife (~8 h), was shown to reduce inflammation and fibrosis in healing corneal wounds, and reduced macrophage invasion of alveoli in a mouse model of diffuse alveolar hemorrhage [43][44][45]. Based on these previous studies, we examined whether the pegylated version of the viral Serp-1 protein, PEGSerp-1, would ameliorate the chronic inflammatory pathology of DMD. ...
... Serp-1 was endotoxin-free, as detected by limulus amebocyte lysate (LAL) assay. Serp-1 was incubated with mPEG-NHS (5 K) (Nanocs Inc., #PG1-SC-5k-1, New York, NY, USA) in PBS (pH 7.8) at 4 • C overnight to modify the protein according to standard PEGylation protocols [43]. PEGylated-Serp-1 was purified by FPLC using an ÄKTA pure protein purification system with Superdex-200 [43]. ...
... Serp-1 was incubated with mPEG-NHS (5 K) (Nanocs Inc., #PG1-SC-5k-1, New York, NY, USA) in PBS (pH 7.8) at 4 • C overnight to modify the protein according to standard PEGylation protocols [43]. PEGylated-Serp-1 was purified by FPLC using an ÄKTA pure protein purification system with Superdex-200 [43]. ...
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Duchenne muscular dystrophy is an X-linked disease afflicting 1 in 3500 males that is characterized by muscle weakness and wasting during early childhood, and loss of ambulation and death by early adulthood. Chronic inflammation due to myofiber instability leads to fibrosis, which is a primary cause of loss of ambulation and cardiorespiratory insufficiency. Current standard of care focuses on reducing inflammation with corticosteroids, which have serious adverse effects. It is imperative to identify alternate immunosuppressants as treatments to reduce fibrosis and mortality. Serp-1, a Myxoma virus-derived 55 kDa secreted glycoprotein, has proven efficacy in a range of animal models of acute inflammation, and its safety and efficacy has been shown in a clinical trial. In this initial study, we examined whether pegylated Serp-1 (PEGSerp-1) treatment would ameliorate chronic inflammation in a mouse model for Duchenne muscular dystrophy. Our data revealed a significant reduction in diaphragm fibrosis and increased myofiber diameter, and significantly decreased pro-inflammatory M1 macrophage infiltration. The M2a macrophage and overall T cell populations showed no change. These data demonstrate that treatment with this new class of poxvirus-derived immune-modulating serpin has potential as a therapeutic approach designed to ameliorate DMD pathology and facilitate muscle regeneration.
... A PEGylated version of the Myxomavirus derived SERPIN Serp-1 has also been developed demonstrating improved efficacy in a mouse model of diffuse alveolar hemorrhage (259). Serp-1 is a broad acting SERPIN with anti-inflammatory and antifibrinolytic activities (260). ...
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Appropriate activation of coagulation requires a balance between procoagulant and anticoagulant proteins in blood. Loss in this balance leads to hemorrhage and thrombosis. A number of endogenous anticoagulant proteins, such as antithrombin and heparin cofactor II, are members of the serine protease inhibitor (SERPIN) family. These SERPIN anticoagulants function by forming irreversible inhibitory complexes with target coagulation proteases. Mutations in SERPIN family members, such as antithrombin, can cause hereditary thrombophilias. In addition, low plasma levels of SERPINs have been associated with an increased risk of thrombosis. Here, we review the biological activities of the different anticoagulant SERPINs. We further consider the clinical consequences of SERPIN deficiencies and insights gained from preclinical disease models. Finally, we discuss the potential utility of engineered SERPINs as novel therapies for the treatment of thrombotic pathologies.