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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) contributes to increased acute and chronic rejection and graft loss. Tissue loss during LIRI has been attributed to local macrophage activation and invasion with excessive inflammation together with hepatocyte apoptosis and necrosis. Inflammatory and apoptotic signaling are key targets for reducing post-ischemic liver injury. Myxomavirus is a rabbit-specific leporipoxvirus that encodes a suite of immune suppressing proteins, often with extensive function in other mammalian species. Serp-2 is a cross-class serine protease inhibitor (serpin) which inhibits the inflammasome effector protease caspase-1 as well as the apoptotic proteases granzyme B and caspases 8 and 10. In prior work, Serp-2 reduced inflammatory cell invasion after angioplasty injury and after aortic transplantation in rodents. In this report, we explore the potential for therapeutic treatment with Serp-2 in a mouse model of LIRI. Methods Wildtype (C57BL/6 J) mice were subjected to warm, partial (70%) hepatic ischemia for 90 min followed by treatment with saline or Serp-2 or M-T7, 100 ng/g/day given by intraperitoneal injection on alternate days for 5 days. M-T7 is a Myxomavirus-derived inhibitor of chemokine-GAG interactions and was used in this study for comparative analysis of an unrelated viral protein with an alternative immunomodulating mechanism of action. Survival, serum ALT levels and histopathology were assessed 24 h and 10 days post-LIRI. Results Serp-2 treatment significantly improved survival to 85.7% percent versus saline-treated wildtype mice (p = 0.0135), while M-T7 treatment did not significantly improve survival (p = 0.2584). Liver viability was preserved by Serp-2 treatment with a significant reduction in serum ALT levels (p = 0.0343) and infarct scar thickness (p = 0.0016), but with no significant improvement with M-T7 treatment. Suzuki scoring by pathologists blinded with respect to treatment group indicated that Serp-2 significantly reduced hepatocyte necrosis (p = 0.0057) and improved overall pathology score (p = 0.0046) compared to saline. Immunohistochemistry revealed that Serp-2 treatment reduced macrophage infiltration into the infarcted liver tissue (p = 0.0197). Conclusions Treatment with Serp-2, a virus-derived inflammasome and apoptotic pathway inhibitor, improves survival after liver ischemia-reperfusion injury in mouse models. Treatment with a cross-class immune modulator provides a promising new approach designed to reduce ischemia-reperfusion injury, improving survival and reducing chronic transplant damage. Electronic supplementary material The online version of this article (10.1186/s12950-019-0215-1) contains supplementary material, which is available to authorized users.

Aims Atrial fibrillation (AF) ablation is associated with increased circulating markers of inflammation. Innate immune or inflammation pathways up-regulate mononuclear cell responses and may increase the risk for recurrent arrhythmia. Chemokines and serine protease coagulation pathways both activate innate immune responses. Here, we measured inflammatory markers in peripheral blood samples from patients after cryoballoon and/or radiofrequency pulmonary vein isolation and assessed the capacity for the inhibition of chemokine and serine protease pathways to block cell activation. Methods Markers of inflammation were measured in 55 patients immediately before and one day after AF ablation. Peripheral blood mononuclear cells (PBMCs) isolated from 19 patients were further tested for responsiveness to two anti-inflammatory proteins ex vivo using fluorescence assays and RT-qPCR analysis of gene expression. Results White blood cells (WBC), C-reactive protein, fibrinogen and troponin T levels were significantly elevated after ablation. PBMCs isolated from the circulating blood had increased activation with Phorbol 12-myristate 13-acetate. Cell activation, as measured by membrane fluidity, was blunted after treatment with a broad-spectrum chemokine modulating protein, M-T7, which interferes with chemokine/glycosaminoglycan (GAG) interactions, but not by Serp-1, a serine protease inhibitor (serpin) that targets both thrombotic and thrombolytic pathway proteases. Differential gene expression changes in the apoptotic pathway were identified with M-T7 and Serp-1. Conclusions Patients undergoing AF ablation have significantly increased inflammatory markers. Inhibition of chemokine signaling, but not serine proteases, reduced the activation of monocytes isolated from patients, in vitro. Targeting chemokines have the potential to reduce post-ablation activation of circulating leukocytes.

Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1-P1' bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR(-/-)). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b(+) monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.

The American Heart Association supports an association between periodontal diseases and atherosclerosis but not a causal association. This study explores the use of the integrin β6 −/− mouse model to study the causality. We investigated the ability of a polymicrobial consortium of Porphyromonas gingivalis , Treponema denticola , Tannerella forsythia , and Fusobacterium nucleatum to colonize the periodontium and induce local and systemic inflammatory responses. Polymicrobially infected Itgβ6 −/− mice demonstrate greater susceptibility to gingival colonization/infection, with severe gingival inflammation, apical migration of the junctional epithelium, periodontal pocket formation, alveolar bone resorption, osteoclast activation, bacterial invasion of the gingiva, a greater propensity for the bacteria to disseminate hematogenously, and a strong splenic T cell cytokine response. Levels of atherosclerosis risk factors, including serum nitric oxide, oxidized low-density lipoprotein, serum amyloid A, and lipid peroxidation, were significantly altered by polybacterial infection, demonstrating an enhanced potential for atherosclerotic plaque progression. Aortic gene expression revealed significant alterations in specific Toll-like receptor (TLR) and nucleotide-binding domain- and leucine-rich-repeat-containing receptor (NLR) pathway genes in response to periodontal bacterial infection. Histomorphometry of the aorta demonstrated larger atherosclerotic plaques in Itgβ6 −/− mice than in wild-type (WT) mice but no significant difference in atherosclerotic plaque size between mice with polybacterial infection and mice with sham infection. Fluorescence in situ hybridization demonstrated active invasion of the aortic adventitial layer by P. gingivalis . Our observations suggest that polybacterial infection elicits distinct aortic TLR and inflammasome signaling and significantly increases local aortic oxidative stress. These results are the first to demonstrate the mechanism of the host aortic inflammatory response induced by polymicrobial infection with well-characterized periodontal pathogens.

Early damage to transplanted organs initiates excess inflammation that can cause ongoing injury, a leading cause for late graft loss. The endothelial glycocalyx modulates immune reactions and chemokine-mediated haptotaxis, potentially driving graft loss. In prior work, conditional deficiency of the glycocalyx-modifying enzyme N-deacetylase-N-sulfotransferase-1 (Ndst1f/f TekCre+) reduced aortic allograft inflammation. Here we investigated modification of heparan sulfate (HS) and chemokine interactions in whole-organ renal allografts. Conditional donor allograft Ndst1 deficiency (Ndst1−/−; C57Bl/6 background) was compared to systemic treatment with M-T7, a broad-spectrum chemokine-glycosaminoglycan (GAG) inhibitor. Early rejection was significantly reduced in Ndst1−/− kidneys engrafted into wildtype BALB/c mice (Ndst1+/+) and comparable to M-T7 treatment in C57Bl/6 allografts (P Ndst1−/− allografts, while M-T7 point mutants with modified GAG-chemokine binding displayed a range of anti-rejection activity. CD3+ T cells (P Ndst1 deficiency exhibit significantly reduced acute rejection, comparable to systemic chemokine-GAG inhibition. Modified disaccharides in engrafted organs correlate with reduced rejection. Altered disaccharides in engrafted organs provide markers for rejection with potential to guide new therapeutic approaches in allograft rejection.

Inflammatory bowel disease (IBD) and Clostridium difficile infection cause gastrointestinal (GI) distension and, in severe cases, toxic megacolon with risk of perforation and death. Herpesviruses have been linked to severe GI dilatation. MHV-68 is a model for human gamma herpesvirus infection inducing GI dilatation in interleukin-10 (IL-10)-deficient mice but is benign in wildtype mice. MHV-68 also causes lethal vasculitis and pulmonary hemorrhage in interferon gamma receptor-deficient (IFN&gamma, R&minus, /&minus, ) mice, but GI dilatation has not been reported. In prior work the Myxomavirus-derived anti-inflammatory serpin, Serp-1, improved survival, reducing vasculitis and pulmonary hemorrhage in MHV-68-infected IFN&gamma, mice with significantly increased IL-10. IL-10 has been investigated as treatment for GI dilatation with variable efficacy. We report here that MHV-68 infection produces severe GI dilatation with inflammation and gut wall degradation in 28% of INF&gamma, R-/- mice. Macrophage invasion and smooth muscle degradation were accompanied by decreased concentrations of T helper (Th2), B, monocyte, and dendritic cells. Plasma and spleen IL-10 were significantly reduced in mice with GI dilatation, while interleukin-1 beta (IL-1&beta, ), IL-6, tumor necrosis factor alpha (TNF&alpha, ) and INF&gamma, increased. Treatment of gamma herpesvirus-infected mice with exogenous IL-10 prevents severe GI inflammation and dilatation, suggesting benefit for herpesvirus-induced dilatation.

Serpins in the mammalian body are highly potent serine protease inhibitors which modulate both thrombotic and thrombolytic pathway activation, with direct and indirect crosstalk with immune and inflammatory pathways. In this review, we discuss mammalian and viral serpins as regulators of coagulation and inflammation. We focus first on the thrombotic and thrombolytic serine proteases and known interactions between these protease cascades and elements of the innate immune response. Serpin-mediated regulation of the thrombotic pathway is then discussed, with emphasis on those serpins that have been evaluated as potential new drugs. Finally the potential of viral serpins that target the coagulation and thrombolytic cascades as potential therapeutics for anti-inflammatory properties is discussed from basic molecular activity to studies in animal models. The studies discussed range from thrombosis and hemorrhage to vascular disease and transplant rejection and finally to sepsis and clinical studies in humans. In conclusion, these unique proteins, the serpin family, now have demonstrated therapeutic potential for a wide variety of inflammatory diseases in both animal and human studies and represent a new approach for drug development.

Inflammatory responses now have a defined central role in cancer cell growth, invasion, and metastases. Anti-inflammatory proteins from viruses target key stages in immune response pathways and have potential as novel therapeutics for cancer, including highly potent virus-derived inhibitors of protease, chemokine, cytokine, and apoptotic cascades that have been identified. Serine proteases, in addition to their conventional roles in thrombosis, thrombolysis, and apoptotic pathways, are essential regulators of inflammation and are associated with developing cancers. Chemokines drive other inflammatory response pathways with central roles in cell invasion and activation as well as establishing the microenvironment of tumors, modulating immune cell infiltration, cancer cell proliferation, metastasis, and angiogenesis. This review focuses on the mechanisms of action and potential for application of viral immunomodulatory proteins as anticancer therapeutics.

Aim: Ser ine p rotease in hibitors ( Serpins ) regulate key clotting and inflammatory pathways. Two anti-inflammatory Serpins , neuroserpin (NSP) and Serp-1 markedly reduce arterial inflammation and plaque growth in a rodent aortic transplant vasculitis model after single dose infusion. NSP is a mammalian serpin that inhibits thrombolytic proteases and Serp-1 is a virus-derived serpin that inhibits both thrombotic and thrombolytic proteases. We postulated that proteolytic cleavage of the reactive site loop (RSL) produces active peptide derivatives with expanded functions. 8 peptide sequences encompassing predicted cleavage sites for Serp-1 and NSP were synthesized and tested in mouse aortic allograft transplant and herpesvirus (MHV68) vasculitis models. The main aim was to assess therapeutic potential for RSL peptides derived from NSP and Serp-1 in vascular inflammation. Methods: We assessed the capacity of 8 predicted serpin RSL peptides, 4 from Serp-1 and 4 from NSP to reduce vascular inflammation. 40 C57BL/6 mice had aortic allograft transplant with a single IV injection of each single peptide. 29 IFN-γR KO mice had MHV-68 infection and were treated for 10 days with S-2 (ISRMAV), S-7 (GTTASSDTAITLIPR), Inverse S-7 (RPILTIATDSSATTG), S-8 (MAVLYPQVIVDHPFFFLIRNR) peptides, or Saline control. Results: RSL peptides with R or RN sequences, but not RM, and with 0-+1 charge significantly reduced vascular inflammatory cell invasion and plaque growth after aortic transplant from C57Bl/6 donor to BALB/c recipient transplant mice (5-6 transplants/peptide) (P < 0.01). S-1, S-3, S-7 & S-8 peptides reduced inflammation and plaque in aortic transplant. Hydrophobicity and negative charge in S-6 peptide increased thrombosis. S-7 peptide also significantly improved survival in a lethal MHV68 infection vasculitic model (150 days survival; P < 0.043), while inactive S-2 peptide, Inverse S-7 and S-8 had no effect (P = NS; 40 days) (P< 0.043). Conclusions: In conclusion, a series of serpin RSL peptides were synthesized based upon natural protease cleavage sites. RSL peptides have demonstrated significant efficacy in reducing inflammatory vasculitis after transplant or after lethal mouse herpesviral (MHV) infections, extending serpin regulatory activity.

Introduction: Periodontal disease (PD) is a polymicrobial dysbiotic chronic inflammatory disease produced by a complex subgingival microbiome, and is linked to systemic inflammatory atherosclerotic vascular disease (ASVD). American Heart Association supported the association between PD and ASVD but not causal association. Prior reports investigated the associations between these two diseases PD and ASVD. But the complex signaling molecules and mechanisms establishing a causal association are not well defined. Methods: To investigate how polybacterial infections initiate atherosclerotic plaque growth, ApoE null mice were orally infected with a polybacterial consortium of 4 well-characterized periodontal pathogens Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum . We assessed PD characteristics, hematogenous dissemination of bacteria, peripheral T cell response, serum inflammatory cytokines, atherosclerosis risk factors, atherosclerotic plaque development, and alteration of aortic gene expression. Results: Polybacterial infections have enhanced gingival colonization and significantly induced alveolar bone loss ApoE null mice. Polybacterial infections were found to be invasive with hematogenous dissemination into heart and aorta. Polybacterial infection-induced significantly higher levels of serum risk factors (oxidized LDL, nitric oxide), altered lipid profiles (cholesterol, triglycerides, chylomicrons, VLDL) as well as accelerated aortic plaque formation in ApoE null mice. Polybacterial infection is associated with significant decreases in Apoa1, Apob, Birc3, Fga, FgB genes that are associated with atherosclerosis. Gingival infections after 12 weeks had modified levels of inflammatory molecules, with decreased Fas ligand, IL-13, SDF-1 and increased chemokine RANTES. At 24 weeks of chronic infection additional changes in inflammatory molecules were induced with reduced KC, MCSF, and enhanced GM-CSF, IFNγ, IL-1β, IL-13, IL-4, IL-13, lymphotactin, and RANTES. Conclusions: This is the first study demonstrating differences in the host immune response to a polybacterial infection with atherosclerotic lesion progression in a mouse model.

Background: Transplant allograft vasculopathy (TAV) is the leading cause for late transplant organ loss. TAV is induced by activation and invasion of inflammatory monocytes and T cells in response to chemokine binding to glycosaminolycans (GAGs) in the endothelial glycocalyx. We investigated the effect of inhibiting heparin sulfate (HS-GAG) / chemokine interactions on rejection in a renal transplant model with (a) heparin sulfate HS-GAG deficient (Ndst1 -/- ) donor mice and (b) a virus-derived chemokine modulating protein M-T7, that is blocks C, CC and CXC interaction with GAGs. Methods: BALB/c renal allograft recipients receiving either Ndst1 -/- (C57BL/6 background) or C57Bl/6 wild type (WT) donor kidneys (n = 48 each) were treated for 10 days with either Saline, M-T7 or one of three M-T7 point mutations (100 ng/gm) with differing inhibitory actions for HS-GAG / chemokine interaction. No other immunosuppressants were added. Results: Ndst1 -/- kidneys had 10X reduced HS content and markedly reduced sulfation compared to WT kidneys. Overall acute rejection and vasculitis scores were significantly reduced in Ndst1 -/- donor kidneys (P < 0.0001). In WT donors, M-T7 significantly reduced rejection and vasculitis (P < 0.0001), but interestingly, lost activity in Ndst1 -/- allografts. CD3+ cells were significantly reduced (P < 0.0001) in Ndst1 -/- donor grafts and with M-T7 treatment in WT transplants and TH17+ cells were significantly reduced in spleen cell isolates. M-T7 point mutation E 209 I retained activity in both Ndst1 -/- and WT donors while but R 171 E was active only in WT. The third mutant F 137 D was inactive. PCR array analysis detected significant decreases in IL-4, NOS2 and CC chemokine 20, in Ndst1 -/- donor transplants or in WT transplants with M-T7 or M-T7 mutant treatments with significant reductions in TAV. Conclusions: A critical role played by HS-GAG / chemokine interactions in the DONOR allograft transplant is demonstrated by these studies. Significant decreases in acute rejection and inflammatory vasculitis are seen in Ndst1 -/- donors with saline treatment and after treatment of C57Bl/6 WT donor grafts with M-T7. HS-GAG composition of donor organs has a potential decisive effect on transplant viability.

Periodontal disease (PD) develops from a synergy of complex subgingival oral microbiome, and is linked to systemic inflammatory atherosclerotic vascular disease (ASVD). To investigate how a polybacterial microbiome infection influences atherosclerotic plaque progression, we infected the oral cavity of ApoE null mice with a polybacterial consortium of 4 well-characterized periodontal pathogens, Porphyromonas gingivalis, Treponema denticola, Tannerealla forsythia and Fusobacterium nucleatum, that have been identified in human atherosclerotic plaque by DNA screening. We assessed periodontal disease characteristics, hematogenous dissemination of bacteria, peripheral T cell response, serum inflammatory cytokines, atherosclerosis risk factors, atherosclerotic plaque development, and alteration of aortic gene expression. Polybacterial infections have established gingival colonization in ApoE null hyperlipidemic mice and displayed invasive characteristics with hematogenous dissemination into cardiovascular tissues such as the heart and aorta. Polybacterial infection induced significantly higher levels of serum risk factors oxidized LDL (p < 0.05), nitric oxide (p < 0.01), altered lipid profiles (cholesterol, triglycerides, Chylomicrons, VLDL) (p < 0.05) as well as accelerated aortic plaque formation in ApoE null mice (p < 0.05). Periodontal microbiome infection is associated with significant decreases in Apoa1, Apob, Birc3, Fga, FgB genes that are associated with atherosclerosis. Periodontal infection for 12 weeks had modified levels of inflammatory molecules, with decreased Fas ligand, IL-13, SDF-1 and increased chemokine RANTES. In contrast, 24 weeks of infection induced new changes in other inflammatory molecules with reduced KC, MCSF, enhancing GM-CSF, IFNγ, IL-1β, IL-13, IL-4, IL-13, lymphotactin, RANTES, and also an increase in select inflammatory molecules. This study demonstrates unique differences in the host immune response to a polybacterial periodontal infection with atherosclerotic lesion progression in a mouse model.

Tannerella forsythia is a Gram-negative anaerobic organism that inhabits the subgingival cavity and initiates connective tissue destruction and alveolar bone resorption in periodontal disease (PD). PD is a chronic immunoinflammatory disease and has been linked to several systemic diseases including atherosclerosis. This study evaluated the effects of a chronic oral infection with T. forsythia ATCC 43037 on the induction of PD, inflammatory markers and atherosclerosis risk factors in hyperlipidemic ApoE(null) mice. Mice were orally infected for 12 and 24 weeks prior to euthanasia. Bacterial colonization of the oral cavity and bacteremia was confirmed via isolation of genomic DNA from oral plaque and tissues. Oral infection elicited significantly elevated levels of serum IgG and IgM antibodies and alveolar bone resorption compared to control mice. Tannerella forsythia-infected mice had increased serum amyloid A, and significantly reduced serum nitric oxide when compared to controls. Tannerella forsythia chronic infection also significantly increased serum lipoproteins suggesting altered cholesterol metabolism and potential for aortic inflammation. Despite enhanced acute phase reactants and altered lipid profiles, T. forsythia infection was associated with decreased aortic plaque. This study investigates the potential of a known periodontal bacterial pathogen found in atherosclerotic plaque in humans to accelerate atherosclerosis in hyperlipdemic mice.

The American Heart Association supports an association between periodontal disease (PD) and atherosclerotic vascular disease (ASVD) but does not as of yet support a causal relationship. Recently, we have shown that major periodontal pathogens Porphyromonas gingivalis and Treponema denticola are causally associated with acceleration of aortic atherosclerosis in ApoEnull hyperlipidemic mice. The aim of this study was to determine if oral infection with another significant periodontal pathogen Fusobacterium nucleatum can accelerate aortic inflammation and atherosclerosis in the aortic artery of ApoEnull mice. ApoEnull mice (n = 23) were orally infected with F. nucleatum ATCC 49256 and euthanized at 12 and 24 weeks. Periodontal disease assessments including F. nucleatum oral colonization, gingival inflammation, immune response, intrabony defects, and alveolar bone resorption were evaluated. Systemic organs were evaluated for infection, aortic sections were examined for atherosclerosis, and inflammatory markers were measured. Chronic oral infection established F. nucleatum colonization in the oral cavity, induced significant humoral IgG (P=0.0001) and IgM (P=0.001) antibody response (12 and 24 weeks), and resulted in significant (P=0.0001) alveolar bone resorption and intrabony defects. F. nucleatum genomic DNA was detected in systemic organs (heart, aorta, liver, kidney, lung) indicating bacteremia. Aortic atherosclerotic plaque area was measured and showed a local inflammatory infiltrate revealed the presence of F4/80+ macrophages and CD3+ T cells. Vascular inflammation was detected by enhanced systemic cytokines (CD30L, IL-4, IL-12), oxidized LDL and serum amyloid A, as well as altered serum lipid profile (cholesterol, triglycerides, chylomicrons, VLDL, LDL, HDL), in infected mice and altered aortic gene expression in infected mice. Despite evidence for systemic infection in several organs and modulation of known atherosclerosis risk factors, aortic atherosclerotic lesions were significantly reduced after F. nucleatum infection suggesting a potential protective function for this member of the oral microbiota.

Serine protease inhibitors, termed serpins, regulate myriad physiological processes in the mammalian body from thrombotic and thrombolytic pathways to inflammation, angiogenesis, hormone transport, and hypertension. The large percentage of serpins among the plasma proteins in the circulating blood as in the case of plasminogen activators, the functional redundancy of serpins, and also the debilitating serpinopathies of antithrombin III (SERPINC1), neuroserpin (SERPINI1), and alpha-1 antitrypsin (SERPINA1) provide evidence of the importance of serpins and their widespread impact in normal physiological homeostasis. Inflammation, also termed innate immunity, interacts closely with and both regulates and is regulated by thrombotic and thrombolytic serine proteases. Activation of the coagulation proteases is, in turn, controlled by serpins. Apoptosis is also modulated by serpins with cross-class inhibitory activity for cysteine and serine proteases. Excessive inflammation and cell death processes are now recognized as interacting with the thrombotic and thrombolytic proteases. Viruses have evolved to communicate and control these processes by encoding their own serpins, which confer on them the ability to evade host immune defenses. This chapter provides an introduction to the viral serpins derived from poxvirus origins that have been shown not only to be essential for successful viral infection but, in some cases, as for Serp-1 and Serp-2, to have the potential to mitigate inflammatory disease in animal models. Serp-1 has in fact been successfully tested in a small phase 2A clinical trial in unstable angina patients with coronary stent implants. A tandem discussion of mammalian serpins with actions similar to those of the viral serpins is also presented to emphasize potential evolutionary relationships between viral and mammalian serpins. The anti-inflammatory serpins hold the potential to be effective in disease states such as atheroma, sepsis, cancer, and wound healing given that these conditions are all associated with aberrant inflammatory responses and with dysregulation of thrombotic, thrombolytic, and apoptotic protease cascades. The capacity of viral serpins to provide antiviral protection by modulating the virome as well as possible therapeutic effects of serpin metabolites in inflammation will also be discussed. In summary, viral serpins have evolved over many millions of years and provide a unique and highly potent reservoir for both the study of serpin modulation of inflammatory responses and for new therapeutic approaches to inflammatory and even infectious diseases.

Giant cell arteritis (GCA) and Takayasu's disease are inflammatory vasculitic syndromes (IVS) causing sudden blindness and widespread arterial obstruction and aneurysm formation. Glucocorticoids and aspirin are mainstays of treatment, predominantly targeting T cells. Serp-1, a Myxomavirus-derived serpin, blocks macrophage and T cells in a wide range of animal models. Serp-1 also reduced markers of myocardial injury in a Phase IIa clinical trial for unstable coronary disease. In recent work, we detected improved survival and decreased arterial inflammation in a mouse Herpesvirus model of IVS. Here we examine Serp-1 treatment of human temporal artery (TA) biopsies from patients with suspected TA GCA arteritis after implant (TAI) into the aorta of immunodeficient SCID (severe combined immunodeficiency) mice. TAI positive for arteritis (GCApos) had significantly increased inflammation and plaque when compared to negative TAI (GCAneg). Serp-1 significantly reduced intimal inflammation and CD11b+ cell infiltrates in TAI, with reduced splenocyte Th1, Th17, and Treg. Splenocytes from mice with GCApos grafts had increased gene expression for interleukin-1 beta (IL-1β), IL-17, and CD25 and decreased Factor II. Serp-1 decreased IL-1β expression. In conclusion, GCApos TAI xenografts in mice provide a viable disease model and have increased intimal inflammation as expected and Serp-1 significantly reduces vascular inflammatory lesions with reduced IL-1β.

Stromal derived factor-1 alpha (SDF-1alpha) and its receptor CXCR4 have been shown to play a role in the systematic movement of hematopoietic stem cells (HSC) in the fetal and adult stages of hematopoiesis. Under certain physiological conditions liver oval cells can participate in the regeneration of the liver. We have shown that a percentage of oval cells are of hematopoietic origin. Others have shown that bone marrow derived stem cells can participate in liver regeneration as well. In this study we examined the role of SDF-1alpha and its receptor CXCR4 as a possible mechanism for oval cell activation in oval cell aided liver regeneration. In massive liver injury models where oval cell repair is involved hepatocytes up-regulate the expression of SDF-1alpha, a potent chemoattractant for hematopoietic cells. However, when moderate liver injury occurs, proliferation of resident hepatocytes repairs the injury. Under these conditions SDF-1alpha expression is not up-regulated and oval cells are not activated in the liver. In addition, we show that oval cells express CXCR4, the only known receptor for SDF-1alpha. Lastly, in vitro chemotaxis assays demonstrated that oval cells migrate along a SDF-1alpha gradient which suggests that the SDF-1alpha/CXCR4 interaction is a mechanism by which the oval cell compartment could be activated and possibly recruit a second wave of bone marrow stem cells to the injured liver. In conclusion, these experiments begin to shed light on a possible mechanism, which may someday lead to a better understanding of the hepatic and hematopoietic interaction in oval cell aided liver regeneration.

Treponema denticola is a predominantly subgingival oral spirochete closely associated with periodontal disease and has been detected in atherosclerosis. This study was designed to evaluate causative links between periodontal disease induced by chronic oral T. denticola infection and atherosclerosis in hyperlipidemic ApoE −/− mice. ApoE −/− mice ( n = 24) were orally infected with T. denticola ATCC 35404 and were euthanized after 12 and 24 weeks. T. denticola genomic DNA was detected in oral plaque samples, indicating colonization of the oral cavity. Infection elicited significantly ( P = 0.0172) higher IgG antibody levels and enhanced intrabony defects than sham infection. T. denticola -infected mice had higher levels of horizontal alveolar bone resorption than sham-infected mice and an associated significant increase in aortic plaque area ( P ≤ 0.05). Increased atherosclerotic plaque correlated with reduced serum nitric oxide (NO) levels and increased serum-oxidized low-density lipoprotein (LDL) levels compared to those of sham-infected mice. T. denticola infection altered the expression of genes known to be involved in atherosclerotic development, including the leukocyte/endothelial cell adhesion gene ( Thbs4 ), the connective tissue growth factor gene ( Ctgf ), and the selectin-E gene ( Sele ). Fluorescent in situ hybridization (FISH) revealed T. denticola clusters in both gingival and aortic tissue of infected mice. This is the first study examining the potential causative role of chronic T. denticola periodontal infection and vascular atherosclerosis in vivo in hyperlipidemic ApoE −/− mice. T. denticola is closely associated with periodontal disease and the rapid progression of atheroma in ApoE −/− mice. These studies confirm a causal link for active oral T. denticola infection with both atheroma and periodontal disease.

1Division of Cardiovascular Medicine, Department of Medicine, University of Florida, Gainesville, FL, USA 2Department of Molecular Genetics and Microbiology, University of Florida, USA 3Departments of Oral Biology and Dental Research, College of Dentistry, USA 4Department of English, College of Liberal Arts and Sciences, University of Florida, USA 5Professor of English and Distinguished Teaching Scholar, USA 6University of Paris Descartes and Centre Nationale de la Recherche Scientifique, Paris, France 7Division of Gastroenterology, Department of Medicine, University of Florida, USA 8Pathology Department, University of Florida, USA

Atherosclerotic vascular disease is a leading cause of myocardial infarction and cerebrovascular accident, and independent associations with periodontal disease (PD) are reported. PD is caused by polymicrobial infections and aggressive immune responses. Genomic DNA of Porphyromonas gingivalis, the best-studied bacterial pathogen associated with severe PD, is detected within atherosclerotic plaque. We examined causal relationships between chronic P. gingivalis oral infection, PD, and atherosclerosis in hyperlipidemic ApoEnull mice. ApoEnull mice (n = 24) were orally infected with P. gingivalis for 12 and 24 weeks. PD was assessed by standard clinical measurements while the aorta was examined for atherosclerotic lesions and inflammatory markers by array. Systemic inflammatory markers serum amyloid A, nitric oxide, and oxidized low-density lipoprotein were analyzed. P. gingivalis infection elicited specific antibodies and alveolar bone loss. Fluorescent in situ hybridization detected viable P. gingivalis within oral epithelium and aorta, and genomic DNA was detected within systemic organs. Aortic plaque area was significantly increased in P. gingivalis-infected mice at 24 weeks (P

Lethal viral infections produce widespread inflammation with vascular leak, clotting, and bleeding (disseminated intravascular coagulation [DIC]), organ failure, and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by an inflammatory cytokine storm and also can induce systemic inflammation with loss of normal ser ine p rotease in hibitor (serpin) regulation. Myxomavirus secretes a potent anti-inflammatory serpin, Serp-1, that inhibits clotting factor X (fX) and thrombolytic tissue- and urokinase-type plasminogen activators (tPA and uPA) with anti-inflammatory activity in multiple animal models. Purified serpin significantly improved survival in a murine gammaherpesvirus 68 (MHV68) infection in gamma interferon receptor (IFN-γR) knockout mice, a model for lethal inflammatory vasculitis. Treatment of MHV68-infected mice with neuroserpin, a mammalian serpin that inhibits only tPA and uPA, was ineffective. Serp-1 reduced virus load, lung hemorrhage, and aortic, lung, and colon inflammation in MHV68-infected mice and also reduced virus load. Neuroserpin suppressed a wide range of immune spleen cell responses after MHV68 infection, while Serp-1 selectively increased CD11c + splenocytes (macrophage and dendritic cells) and reduced CD11b + tissue macrophages. Serp-1 altered gene expression for coagulation and inflammatory responses, whereas neuroserpin did not. Serp-1 treatment was assessed in a second viral infection, mouse-adapted Zaire ebolavirus in wild-type BALB/c mice, with improved survival and reduced tissue necrosis. In summary, treatment with this unique myxomavirus-derived serpin suppresses systemic serine protease and innate immune responses caused by unrelated lethal viral infections (both RNA and DNA viruses), providing a potential new therapeutic approach for treatment of lethal viral sepsis.

Background Glucocorticoids are a principal treatment for giant cell arteritis (GCA), with some studies reporting predominate effect on T helper17 (Th17) cell activity in GCA, with lesser effects on other inflammatory cells. Serp-1 is a 55kDa myxomaviral serpin that modifies macrophage, Th1, and Th17 responses in aortic transplants in animal models. Objectives We have analyzed the effects of a virus-derived anti-inflammatory ser ine p rotease in hibitor ( serpin ) on human temporal artery (TA) biopsies isolated from patients with suspected GCA. Methods Using a newly developed “Aortic Window Xenopatch” model, human TA biopsy sections were divided implanted as full thickness grafts into the abdominal aorta of SCID mice in parallel and tested for response to Serp-1, with and without human PBMC infusion (N = 32). Results TA sections positive for arteritis (GCA pos ) displayed significantly increased inflammatory plaque when compared to negative sections (GCA neg ). Serp-1 reduced plaque in GCA pos sections (+ cells after PBMC infusion with reduced TNF-α expression in spleen, but not CD3 + , CCR6 + , nor CD86 + . Splenocytes from mice with GCA pos grafts had increased interleukin-1beta (Il-1β), IL-17, and CD25 expression, while IL-1β was significantly reduced by Serp-1.Serp-1 also markedly reducedt gene expression for the FII, FXa-PAR2, tPA, uPA in splenocytes (P Conclusions Treatment with a virus-derived serpin, significantly reduces inflammation and plaque thickness in human GCA pos TA xenograft implants with associated reductions in macrophage and Th1 and Th17 responses in mice.

Background Lethal viral infections produce widespread inflammation with vascular leaking, clotting and bleeding (DIC), and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by a cytokine storm and also induce systemic inflammation. Normal host ser ine p roteases in hibitor ( serpin ) regulation is lost. Serp-1 is a potent myxomavirus anti-inflammatory protein, which inhibits factor X (fX), tPA and uPA in vitro . Serp-1 reduces arterial inflammation and atherogenesis in many animal models and has been successfully tested in patients with unstable plaque and stent implants. Methods and results Purified Serp-1 protein treatment significantly improved survival in lethal murine Gammaherpesvirus-68 (MHV68) infection in interferon gamma receptor (IFNγR) knock-out mice (N = 72 mice; P < 0.02) when given for 10 up to 30 days after infection. Treatment with a mammalian serpin, neuroserpin, that inhibits only tPA and uPA, was ineffective (N = 30 mice; P = NS). Serp-1 reduced viral antigen, lung hemorrhage, and aortic, lung and colon inflammation in MHV68 infected mice. Neuroserpin suppressed a wide swath of immune cell responses, while Serp-1 selectively increased CD11c+ splenocytes (macrophage and dendritic cells) and reduced CD11b+ tissue macrophage. Serp-1 significantly increased the IL-10 gene expression in aorta and plasma protein, whereas neuroserpin decreased IL-10. Serp-1 mediated survival in MHV68 infected mice was lost after treatment with IL-10 receptor antibody immediately post infection (N = 12 mice; P =0.014). Conclusions Serp-1, a unique myxomavirus-deried serpin, improves survival after lethal mouse herpesviral infection, reduces viral load, hemorrhage and arterial inflammation. Serp-1 provides a potential new therapeutic approach for lethal viral sepsis and DIC through altered FX and IL-10.

Modification of the tumor microenvironment by inflammatory cells represents a newly recognized driving force in cancer with a critical role in tumor invasion, growth, angiogenesis, and metastasis. Increased serine proteases in the thrombolytic cascade, specifically urokinase-type plasminogen activator and its receptor, correlate with inflammatory cell invasion, angiogenesis, cancer growth and invasion. Inflammation in pancreatic cancer is linked with myeloid-derived suppressor cell activity and cancer progression. Myxomavirus is a complex DNA virus encoding highly potent immune modulators such as Serp-1, which inhibits uPA, tPA, plasmin and coagulation factor X. Serp-1 has also been demonstrated to inhibit angiogenesis in a chicken chorioallantoic membrane (CAM) model. To explore the potential use of anti-inflammatory, anti-angiogenesis proteins that target thrombotic and thrombolytic as well as inflammatory pathways, for cancer treatment, severe combined immunodeficient (SCID) mice engrafted with human cancer cells were treated with Serp-1 and neuroserpin, a selective mammalian inhibitor of thrombolytic proteases or control saline. Proteins were tested for efficacy in preventing human pancreatic and breast cancer cell growth in NOD/SCID mice (n=67 total) at 4 weeks after implantation. Serp-1 and neuroserpin inhibited pancreatic cancer Hs766t (n=27; P=0.03 and P=0.01, respectively), but not breast cancer MDA231 growth (n=11) when compared to saline controls (N =5). Serpin inhibition of pancreatic tumor growth was associated with decreased MDSC as shown by flow cytometry analysis of splenocyte (P=0.009). Serpin-mediated anti-inflammatory activity in pancreatic tumor growth was also demonstrated by reduced macrophage infiltration in tumors (P=0.001). Furthermore, RT-PCR array analysis revealed a five-fold decrease in the expression of inducible nitric oxide synthase (iNOS), which has been shown to play important role in tumor angiogenesis. Conclusions Anti-inflammatory protease inhibitors, virus-derived Serp-1 and mammalian neuroserpin, may provide new treatment approaches by targeting the angiogenesis and inflammatory responses that support cancer cell growth.

Modification of the tumor microenvironment by inflammatory cells represents a newly recognized driving force in cancer with critical roles in tumor invasion, growth, angiogenesis, and metastasis. Increased thrombolytic cascade serine proteases, specifically urokinase-type plasminogen activator and its receptor, correlate with inflammatory cell migration, pancreatic cancer growth, invasion and unfavorable outcomes. Inflammation in pancreatic cancer is linked with myeloid-derived suppressor cell (MDSC) activity and cancer progression. Myxomavirus is a complex DNA virus encoding highly potent immune modulators. Serp-1 and M-T7 are two such secreted anti-inflammatory myxomaviral proteins. Serp-1 inhibits uPA, plasmin and coagulation factor X while M-T7 inhibits C, CC, and CXC chemokines. We have explored the potential use of these viral proteins for treatment of a range of human cancer isolates engrafted in severe combined immunodeficient (SCID) mice. Engrafted tumors were treated with either Serp-1, neuroserpin, a related mammalian serpin that inhibits thrombolytic proteases, or M-T7. Serp-1 and neuroserpin inhibited growth of the pancreatic cancer cell line Hs766t (P=0.03 and P=0.01, respectively) at 4 weeks after implantation. Serp-1 also inhibited growth of a second pancreatic cancer cell line MIA PaCa-2 in mice (P=0.02). Growth of the human breast cancer line MDA231 was not inhibited by Serp-1. M-T7, in contrast, did not alter growth of any of the cancer cell lines tested after implant into SCID mice. Serpin inhibition of pancreatic tumor growth was associated with a significant decrease in splenocyte MDSC counts by flow cytometry (P=0.009), without detected change in other splenocyte subpopulations. Serp-1 and NSP treatment also significantly reduced macrophage infiltration in tumors (P=0.001). In summary two anti-inflammatory serpins reduced inflammatory macrophage invasion and pancreatic tumor cell growth, suggesting potential therapeutic efficacy.

Vasculitic syndromes cause devastating arterial occlusion with associated end organ ischemia and significant morbidity. Treatment for vasculitic syndromes remains very limited. Gamma 68 herpesvirus (g68HV) infection in interferon gamma receptor deficient (IFNγR -/- ) mice provides a good working model of artery inflammation, inducing early death with associated inflammatory cell invasion. Myxomavirus serpin, Serp-1, inhibits tPA, uPA, plasmin (thrombolytic) and fXa (thrombotic) proteases, reducing inflammation in a wide range of animal models whereas mammalian neuroserpin (NSP) inhibits tPA and uPA and reduced myocardial damage in clinical trial of unstable angina and stent implant. To assess the capacity of serpins to block inflammatory vasculopathy development, γ68HV infected IFNγR -/- mice were treated with either Serp-1 (100ng/g/100μl), NSP (100ng/g/100μl) or saline (100ul) .Serp-1 treatment given by daily intraperitoneal injections (i.p.) for the first 10 days post infection (6 - 12.5 X 106 PFU), significantly prolonged survival in mice (IFNγR -/- ) (P ≤ 0.048). Saline treated γ68HV infected mice survived up to 42 days, whereas Serp-1 treated mice survived up to 55 days (N=60 mice, 6-12 mice per group). NSP treatment (100ng/gm i.p. X 10 days) produced an opposite trend toward increased mortality (P ≤ 0.079; N=16). In a subsequent study, prolonged daily treatment with Serp-1 (100ng/gm i.p.) given for the first 30 days post infection, markedly prolonged survival in γ68HV infected INFγR -/- mice for up to 150 days (P ≤ 0.032, N =10). Treatment with Serp-1, that was started 3 weeks post γ68HV infection, produced a non-significant trend toward improved survival (P=0.685; N = 12 mice), whereas treatment begun at 1 week post infection again significantly improved survival (P ≤ 0.020; N=12 mice). Serp-1 treatment significantly reduced inflammatory cell invasion in the aortic adventitia ( p < 0.010) and lung ( p < 0.047), NSP significantly increased inflammatory cell invasion when compared to Serp-1 treatment in infected mice in the lung interstitial layer (p ≤ 0.005). At 10 days follow up the plasma cytokine interleukin-1beta (IL-1β) was significantly reduced by Serp-1 treatment (p ≤ 0.049), Treatment with the NSP reduced plasma cytokine TNFα ( p ≤ 0.022), IFNγ (p ≤ 0.022), and IL-4 (p ≤ 0.022) in γ68HV infected IFNγR -/- mice (N=14).Gene expression was significantly increased in Serp-1 treated mice for regulatory serpins in the thrombolytic cascade, specifically PAI-1 (p ≤ 0.020) and unexpectedly NSP (p ≤ 0.032), gene expression was significantly reduced for E selectin (p ≤ 0.048) and increased for IL-10 (p ≤ 0.031), and addressin (p ≤ 0.002) with Serp-1 treatment at 10 days . Compared to the saline treated mice, NSP treatment lead to decreased arterial IL-17 and IL-10 gene expression (p ≤ 0.006 and 0.041, respectively) and increased PAI-2 (p ≤ 0.021) . When comparing differences in gene expression for Serp-1 and NSP; Serp-1 treatment increased gene expression for IL-10 (p ≤ 0.001), bFGF (p < 0.022), Addressin (p < 0.001), PAI-1 (p < 0.021), and NSP (p < 0.009) and NSP reduced IL-6 (p < 0.016) and PAI-2 (p < 0.016). Conclusion: Serp-1 but not NSP produced an unexpected improved survival after γ68HV infection with associated rebalancing of the coagulation and thrombolytic cascades as well as modulating inflammatory responses. Anti-inflammatory serpin provides a potential new therapeutic approach to virus induced vasculitis.

Serine protease inhibitors (Serpins) play an important role in regulating a wide array of diverse biological activities, representing up to 2-10% of circulating plasma proteins. The serpin suicide inhibitors regulate coagulation (thrombosis and thrombolysis), neurotrophic factors, hormone transport, complement and inflammation, angiogenesis, hormone transport, and blood pressure among many other biological reactions. Select serpins have been associated with progression or remission of selected cancers, making them valuable for therapeutic or diagnostic use. Plasminogen activator inhibitor-1 (PAI-1), the main regulator of thrombolysis, has the potential to either reduce or accelerate tumor growth but blockade of PAI-1 has recently been reported to reduce cancer cell migration, proliferation and survival through modulating the function of urokinase-type plasminogen activator receptor. Maspin is a non-inhibitory serpin, that increases cancer cell sensitivity to apoptosis and inhibits cancer cell migration thus providing a serpin that blocks tumor gorwth. Pigment epithelium derived factor (PEDF) has potent anti-angiogenesis activity and also promotes cancer cell apoptosis. Among other serpins, the mammalian serpin, neuroserpin, and the myxomavirus derived serpin, Serp-1 are under investigation in our lab for their potential tumor-suppressive functions. Further study on the efficacy and mechanisms of serpin mediated anti-cancer activity is warranted in order to develop new serpin-based approaches in cancer therapy.

Over the past 19 years, we have developed a novel myxoma virus-derived anti-inflammatory serine protease inhibitor, termed a serpin, as a new class of immunomodulatory therapeutic. This review will describe the initial identification of viral serpins with anti-inflammatory potential, beginning with preclinical analysis of viral pathogenesis and proceeding to cell and molecular target analyses, and successful clinical trial. The central aim of this review is to describe the development of two serpins, Serp-1 and Serp-2, as a new class of immune modulating drug, from inception to implementation. We begin with an overview of the approaches used for successful mining of the virus for potential serpin immunomodulators in viruses. We then provide a methodological overview of one inflammatory animal model used to test for serpin anti-inflammatory activity followed by methods used to identify cells in the inflammatory response system targeted by these serpins and molecular responses to serpin treatment. Finally, we provide an overview of our findings from a recent, successful clinical trial of the secreted myxomaviral serpin, Serp-1, in patients with unstable inflammatory coronary arterial disease.

Deregulation of insulin-like growth factor-1 receptor (IGF-1R) and focal adhesion kinase (FAK) signaling pathways plays an important role in cancer cell proliferation and metastasis. In pancreatic cancer cells, the crosstalk and compensatory mechanisms between these two pathways reduce the efficacy of the treatments that target only one of the pathways. Ablation of IGF-1R signaling by siRNA showed minimal effects on the survival and growth of pancreatic cancer cells. An increased activity of FAK pathway was seen in these cells after IGF-1R knockdown. Further inhibition of FAK pathway using Y15 significantly decreased cell survival, adhesion, and promoted apoptosis. The combination of Y15 treatment and IGF-1R knockdown also showed significant antitumor effect in vivo. The current study demonstrates the importance of dual inhibition of both these signaling pathways as a novel strategy to decrease both in vitro and in vivo growth of human pancreatic cancer.

The interaction of focal adhesion kinase (FAK) and insulin-like growth factor-1 receptor (IGF-1R) plays an important role in cancer cell survival. Targeting this interaction with small molecule drugs could be a novel strategy in cancer therapy. By a series of pull-down assays using GST-tagged FAK fragments and His-tagged IGF-1R intracellular fragments, we showed that the FAK-NT2 (a.a. 127-243) domain directly interacts with the N-terminal part of the IGF-1R intracellular domain. Overexpressed FAK-NT2 domain was also shown to co-localize with IGF-1R in pancreatic cells. Computational modeling was used to predict the binding configuration of these two domains and to screen for small molecules binding to the interaction site. This strategy successfully identified a lead compound that disrupts FAK/IGF-1R interaction.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is overexpressed in many types of tumors, including pancreatic cancer, and plays an important role in cell adhesion and survival signaling. Pancreatic cancer is a lethal disease and is very resistant to chemotherapy, and FAK has been shown recently to assist in tumor cell survival. Therefore, FAK is an excellent potential target for anti-cancer therapy. We identified a novel small molecule inhibitor (1,2,4,5-Benzenetetraamine tetrahydrochloride, that we called Y15) targeting the main autophosphorylation site of FAK and hypothesized that it would be an effective treatment strategy against human pancreatic cancer. Y15 specifically blocked phosphorylation of Y397-FAK and total phosphorylation of FAK. It directly inhibited FAK autophosphorylation in a dose- and time-dependent manner. Furthermore, Y15 increased pancreatic cancer cell detachment and inhibited cell adhesion in a dose-dependent manner. Y15 effectively caused human pancreatic tumor regression in vivo, when administered alone and its effects were synergistic with gemcitabine chemotherapy. This was accompanied by a decrease in Y397-phosphorylation of FAK in the tumors treated with Y15. Thus, targeting the Y397 site of FAK in pancreatic cancer with the small molecule inhibitor, 1,2,4,5-Benzenetetraamine tetrahydrochloride, is a potentially effective treatment strategy in this deadly disease.

To examine the role of the brain stem melanocortin system in long-term energy regulation, we assessed the effects of overproduction of proopiomelanocortin (POMC) in the caudal brain stem of F344xBN rats with adult-onset obesity. Recombinant adeno-associated viral vector encoding POMC gene was delivered to the nucleus of solitary tract (NTS) in the hindbrain, and food intake, body weight, glucose and fat metabolism, brown adipose tissue thermogenesis, and mRNA levels of neuropeptides and melanocortin receptors were assessed. POMC delivery resulted in sustained reduction in food intake and body weight over 42 days and improved insulin sensitivity. At death, in recombinant adeno-associated viral vector-POMC-treated rats vs. control rats, α-melanocyte-stimulating hormone in NTS increased nearly 21-fold, whereas hypothalamic α-melanocyte-stimulating hormone remained unchanged. Visceral adiposity decreased by 37%; tissue triglyeride content diminished by 26% and 47% in liver and muscle, respectively; serum triglyeride and nonesterified fatty acids were reduced by 35% and 34%, respectively; phosphorylation of acetyl-CoA carboxylase was elevated by 63% in soleus muscle; brown adipose tissue uncoupling protein 1 increased by 30%; and melanocortin 3 receptor expression declined by 60%, whereas neuropeptide Y, agouti-related protein, and MC4 receptor mRNA levels were unchanged in the NTS. In conclusion, POMC overexpression in the NTS produces a characteristic unabated hypophagia that is uniquely different from the anorexic tachyphylaxis following POMC overexpression in the hypothalamus. The sustained anorectic response may result from absence of compensatory elements in the NTS, such as increased agouti-related protein expression, suggesting melanocortin activation of the brain stem may be a viable strategy to alleviate obesity.

Stromal cell-derived factor-1 (SDF-1) is known to play an essential role in the regulation of stem/progenitor cell trafficking. During hepatic stem, or oval, cell activation, SDF-1 has been reported to be up-regulated within the liver, implying a possible role in oval cell-aided liver regeneration. In the present study, SDF-1 expression was knocked down in the liver of 2-acetylaminofluorene/partial hepatectomy-treated rats using short interfering RNA delivered by recombinant adenovirus. The oval cell response was compromised in these animals, as evidenced by a decreased number of OV6-positive oval cells. In addition, knockdown of SDF-1 expression caused a dramatic decrease in alpha-fetoprotein expression, implying impaired oval cell activation in these animals. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling assay showed no significant apoptosis related to SDF-1 suppression. Instead, as revealed by Ki67 immunohistochemistry, the suppression of SDF-1 resulted in decrease of hepatic cell proliferation, implying the repair process had been inhibited in these animals. These results indicate that SDF-1 is an essential molecule needed in oval cell activation.

Background & Aims: The ability of the bone marrow cells to differentiate into liver, pancreas, and other tissues led to the speculation that these cells might be the source of adult stem cells found in these organs. The present study analyzed whether the bone marrow cells are a source of hepatic oval cells involved in rat liver regeneration induced by 2-acetylaminofluorene (2-AAF) and 70% partial hepatectomy (PHx). Methods: Three groups of mutant F344 dipeptidyl peptidase IV–deficient (DPPIV−) rats were required for the study. Groups A and B received the mitotic inhibitor monocrotaline, followed by male F344 (DPPIV+) bone marrow transplantation. Next, group A received PHx only, while group B received the 2-AAF/PHx required for the oval cell activation. The last group C was used to analyze the effects of monocrotaline on transplanted bone marrow cells. These rats underwent transplantation with bone marrow cells and were then treated with monocrotaline. Subsequently, the animals were treated with 2-AAF/PHx. Results: In group A, DPPIV+ hepatocytes were found in the liver. Group B showed that approximately 20% of the oval cell population expressed both donor marker (DPPIV) and α-fetoprotein, and some differentiated into hepatocytes. In contrast, animals in group C failed to significantly induce oval cells with the donor DPPIV antigen. In addition, X/Y-chromosome analysis revealed that fusion was not contributing to differentiation of donor-derived oval cells. Conclusions: Our results suggest that under certain physiologic conditions, a portion of hepatic stem cells might arise from the bone marrow and can differentiate into hepatocytes.

Background Lethal viral infections produce widespread inflammation with vascular leakage, clotting and bleeding (DIC), and high mortality, up to 40–70% in disseminated viral infections. Treatment for sepsis and DIC remains very limited. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by a cytokine storm and also induce systemic inflammation. Host regulators of thrombosis and thrombolysis by serine proteases inhibitors (serpins) is lost. Serp-1 is a potent myxomavirus anti-inflammatory protein, which inhibits factor X (fX), tPA and uPA in vitro. Serp-1 reduces arterial inflammation and atherogenesis in many animal models and has been successfully tested in patients with unstable plaque and stent implants. Methods and results: Purified Serp-1 protein treatment significantly improved survival in lethal murine Gammaherpesvirus-68 (MHV68) infection in interferon gamma receptor (IFN γ R) knock-out mice ( N = 72 mice; P N = 30 mice; P = NS). Serp-1 reduced viral antigen, lung hemorrhage, and aortic, lung and colon inflammation in MHV68 infected mice. Neuroserpin suppressed a wide swath of immune cell responses, while Serp-1 selectively increased CD11c + splenocytes (macrophage and dendritic cells) and reduced CD11b + tissue macrophage. Serp-1 significantly reduced fX and increased IL-10 gene expression in the aorta and increased plasma IL-10 protein, whereas neuroserpin decreased IL-10. The fX inhibitor fondaparinux, when given alone, did not improve survival. However improved survival with Serp-1 treatment was lost after treatment with IL-10 receptor antibody immediately post infection ( N = 12 mice; P = 0.014). Conclusions: Serp-1, a unique myxomavirus-derived serpin, improves survival after lethal mouse herpesviral infection, reducing viral load, hemorrhage and arterial inflammation. Serp-1 provides a potential new therapeutic approach for lethal viral sepsis and DIC through modulation of FX and IL-10 activity.

Allogeneic stem cell-based transplants may be limited by allograft rejection, as is seen with conventional organ transplantation. One way to avert such a response is to use autologous stem cells, but that may carry the risk of recurrence of the original disease, particularly in the context of a genetic defect. We investigated the potential for gene modification of autologous stem cells to avoid both problems, using recombinant adenoassociated virus vector expressing human alpha1-antitrypsin in murine liver progenitor cells. We showed that recombinant adenoassociated virus 1 was the most efficient vector for liver progenitor cell transduction among five different serotypes of recombinant adenoassociated virus vectors. Ex vivo infected green fluorescent protein-positive liver progenitor cells from C57BL/6 mice with recombinant adenoassociated virus 1-vector-expressing human alpha1 antitrypsin were transplanted into the liver of monocrotaline-treated and partial-hepatectomized C57BL/6 recipients. Using green fluorescent protein as a donor marker, we were able to determine that at 18 weeks after transplantation, approximately 40% to 50% of the regenerated liver was green fluorescent protein positive. In addition, transgene expression (serum human alpha1-antitrypsin) was sustained for the length of the study (18 weeks after transplantation). Immunostaining revealed approximately 5% to 10% of repopulating liver cells expressing human alpha1-antitrypsin. In conclusion, this study demonstrated the feasibility of long-term engraftment and stability of transgene expression from genetically modified liver progenitor cells with a recombinant adenoassociated virus vector and implies a novel approach to gene therapy for treatment of liver diseases, such as alpha1-antitrypsin deficiency.