RESUMO
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.
RESUMO
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 (IFNγR-/-) 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 IFNγR-/- 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 INFγR-/- 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-1ß), IL-6, tumor necrosis factor alpha (TNFα) and INFγ increased. Treatment of gamma herpesvirus-infected mice with exogenous IL-10 prevents severe GI inflammation and dilatation, suggesting benefit for herpesvirus-induced dilatation.
Assuntos
Dilatação Gástrica/terapia , Dilatação Gástrica/virologia , Infecções por Herpesviridae/complicações , Interleucina-10/uso terapêutico , Receptores de Interferon/genética , Rhadinovirus , Animais , Citocinas/sangue , Citocinas/imunologia , Modelos Animais de Doenças , Dilatação Gástrica/genética , Dilatação Gástrica/patologia , Interleucina-10/genética , Camundongos , Camundongos Knockout , Receptores de Interferon/metabolismo , Estatísticas não Paramétricas , Receptor de Interferon gamaRESUMO
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 < 0.0081). M-T7 lost activity in Ndst1-/- allografts, while M-T7 point mutants with modified GAG-chemokine binding displayed a range of anti-rejection activity. CD3+ T cells (P < 0.0001), HS (P < 0.005) and CXC chemokine staining (P < 0.012), gene expression in NFκB and JAK/STAT pathways, and HS and CS disaccharide content were significantly altered with reduced rejection. Transplant of donor allografts with conditional 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.
Assuntos
Células Alógenas/enzimologia , Aorta/transplante , Células Progenitoras Endoteliais/enzimologia , Rejeição de Enxerto/enzimologia , Células Progenitoras Mieloides/enzimologia , Sulfotransferases , Células Alógenas/patologia , Animais , Aorta/patologia , Células Progenitoras Endoteliais/patologia , Deleção de Genes , Rejeição de Enxerto/genética , Rejeição de Enxerto/patologia , Rejeição de Enxerto/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Células Progenitoras Mieloides/patologia , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
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.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Infecções por Herpesviridae/imunologia , Fatores Imunológicos , Proteínas de Membrana , Peptídeos , Rhadinovirus/imunologia , Vasculite/imunologia , Animais , Coagulação Sanguínea/imunologia , Modelos Animais de Doenças , Infecções por Herpesviridae/tratamento farmacológico , Humanos , Fatores Imunológicos/síntese química , Fatores Imunológicos/química , Fatores Imunológicos/imunologia , Células Jurkat , Proteínas de Membrana/síntese química , Proteínas de Membrana/química , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Knockout , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Vasculite/tratamento farmacológicoRESUMO
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 ß6(-/-) 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 Itgß6(-/-) 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 Itgß6(-/-) 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.
Assuntos
Túnica Adventícia/patologia , Antígenos de Neoplasias/imunologia , Aorta/patologia , Aterosclerose/complicações , Integrinas/imunologia , Periodontite/complicações , Placa Aterosclerótica/complicações , Túnica Adventícia/imunologia , Túnica Adventícia/microbiologia , Animais , Antígenos de Neoplasias/genética , Aorta/imunologia , Aorta/microbiologia , Aterosclerose/imunologia , Aterosclerose/microbiologia , Aterosclerose/patologia , Bacteroidetes/crescimento & desenvolvimento , Bacteroidetes/imunologia , Bacteroidetes/patogenicidade , Reabsorção Óssea , Modelos Animais de Doenças , Fusobacterium nucleatum/crescimento & desenvolvimento , Fusobacterium nucleatum/imunologia , Fusobacterium nucleatum/patogenicidade , Expressão Gênica , Gengiva/imunologia , Gengiva/microbiologia , Gengiva/patologia , Hibridização in Situ Fluorescente , Inflamassomos , Integrinas/deficiência , Integrinas/genética , Lipoproteínas LDL/genética , Lipoproteínas LDL/imunologia , Camundongos , Camundongos Knockout , Consórcios Microbianos , Periodontite/imunologia , Periodontite/microbiologia , Periodontite/patologia , Periodonto/imunologia , Periodonto/microbiologia , Periodonto/patologia , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/microbiologia , Placa Aterosclerótica/patologia , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/patogenicidade , Treponema denticola/crescimento & desenvolvimento , Treponema denticola/imunologia , Treponema denticola/patogenicidadeRESUMO
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-1ß), IL-17, and CD25 and decreased Factor II. Serp-1 decreased IL-1ß 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-1ß.
Assuntos
Arterite de Células Gigantes , Serpinas/farmacologia , Artérias Temporais , Proteínas Virais/farmacologia , Animais , Modelos Animais de Doenças , Feminino , Arterite de Células Gigantes/tratamento farmacológico , Arterite de Células Gigantes/metabolismo , Arterite de Células Gigantes/patologia , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Arterite de Takayasu/tratamento farmacológico , Arterite de Takayasu/metabolismo , Arterite de Takayasu/patologia , Artérias Temporais/metabolismo , Artérias Temporais/patologia , Artérias Temporais/transplanteAssuntos
Modelos Animais de Doenças , Transplante de Rim/métodos , Procedimentos de Cirurgia Plástica/métodos , Procedimentos Cirúrgicos Urológicos/métodos , Animais , Cateterismo , Cistotomia , Hidronefrose/fisiopatologia , Rim/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sistema Urinário/cirurgiaRESUMO
Mouse renal transplantation is a technically challenging procedure. Although the first kidney transplants in mice were performed over 34 years ago and refined some years later, the classical techniques of mouse renal transplantation required clamping both vena cava and aorta simultaneously and carry out suture anastomoses of the renal artery and vein in a heterotopic position. In our laboratory, we have successfully developed mouse orthotopic kidney transplantation for the first time, using a rapid "cuffed" renal vein technique for vessel anastomosis, wherein the donor's renal vein was inserted through an intravenous catheter, folded back and tied. During grafting, the cuffed renal vein was directly inserted into the recipient's renal vein without the need for the clamping vena cava and suturing of renal vein. This technique allowed for the exact transplantation of the kidney into the original position, compared to the classical technique, and has significantly shortened the clamping time due to a quicker and precise anastomosis of renal vein as described. This also allowed for a quicker recovery of the lower extremity activity, reduction in myoglobinuria with resultant kidney graft survival of 88.9%. Thus we believe that the cuffed renal vein technique simplifies microvascular anastomoses and affords important additional benefits.
Assuntos
Transplante de Rim , Veias Renais/cirurgia , Procedimentos Cirúrgicos Urológicos/instrumentação , Procedimentos Cirúrgicos Urológicos/métodos , Anastomose Cirúrgica , Animais , Testes de Função Renal , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Duração da Cirurgia , Proteinúria/fisiopatologiaRESUMO
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.
Assuntos
Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Inibidores de Serina Proteinase/metabolismo , Inibidores de Serina Proteinase/uso terapêutico , Serpinas/metabolismo , Serpinas/uso terapêutico , Animais , Humanos , Modelos Moleculares , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Serpinas/química , Serpinas/farmacologiaRESUMO
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 serine protease inhibitor (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.
Assuntos
Hemorragia/tratamento farmacológico , Doença pelo Vírus Ebola/tratamento farmacológico , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/mortalidade , Proteínas de Membrana/farmacologia , Myxoma virus/química , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Modelos Animais de Doenças , Ebolavirus , Fator X/antagonistas & inibidores , Fator X/metabolismo , Gammaherpesvirinae , Hemorragia/mortalidade , Hemorragia/patologia , Hemorragia/virologia , Doença pelo Vírus Ebola/mortalidade , Doença pelo Vírus Ebola/patologia , Doença pelo Vírus Ebola/virologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Inflamação/tratamento farmacológico , Inflamação/mortalidade , Inflamação/patologia , Inflamação/virologia , Interferon gama/deficiência , Interferon gama/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Myxoma virus/fisiologia , Neuropeptídeos/farmacologia , Serpinas/farmacologia , Análise de Sobrevida , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vasculite/tratamento farmacológico , Vasculite/mortalidade , Vasculite/patologia , Vasculite/virologia , NeuroserpinaRESUMO
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.
RESUMO
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.
Assuntos
Anti-Inflamatórios/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Virais/uso terapêutico , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/metabolismo , Quimiocinas/metabolismo , Humanos , Imunomodulação , Camundongos , Neoplasias/imunologia , Neoplasias/metabolismo , Ligação Proteica , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/uso terapêutico , Serpinas/imunologia , Serpinas/metabolismo , Serpinas/uso terapêutico , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Vírus/imunologia , Vírus/metabolismoRESUMO
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.
Assuntos
Serpinas/metabolismo , Vírus/metabolismo , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Camundongos , Serpinas/genética , Serpinas/farmacologia , Serpinas/uso terapêutico , Vírus/genéticaRESUMO
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.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Neoplasias Pancreáticas/patologia , Receptores de Somatomedina/antagonistas & inibidores , Animais , Sequência de Bases , Primers do DNA , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/metabolismo , RNA Interferente Pequeno , Transplante HeterólogoRESUMO
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.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Neoplasias Pancreáticas/enzimologia , Domínios e Motivos de Interação entre Proteínas , Receptor IGF Tipo 1/metabolismo , Linhagem Celular Tumoral , Quinase 1 de Adesão Focal/química , Quinase 1 de Adesão Focal/genética , Humanos , Modelos Moleculares , Conformação Proteica , Mapeamento de Interação de Proteínas , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/genéticaRESUMO
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.
Assuntos
Compostos de Anilina/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Neoplasias Pancreáticas/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Humanos , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/toxicidade , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
BACKGROUND AND 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 alpha-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.
Assuntos
Células da Medula Óssea/citologia , Transplante de Medula Óssea , Diferenciação Celular , Hepatócitos/patologia , Hepatopatias/patologia , Regeneração Hepática , Fígado/patologia , Células-Tronco/citologia , 2-Acetilaminofluoreno , Animais , Animais Geneticamente Modificados , Antimitóticos/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Proliferação de Células , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Modelos Animais de Doenças , Feminino , Hepatectomia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/transplante , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/fisiopatologia , Fígado/cirurgia , Hepatopatias/metabolismo , Hepatopatias/fisiopatologia , Hepatopatias/cirurgia , Masculino , Monocrotalina/farmacologia , Ratos , Ratos Endogâmicos F344/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Fatores de Tempo , Cromossomo X/metabolismo , Cromossomo Y/metabolismo , alfa-Fetoproteínas/metabolismoRESUMO
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, alpha-melanocyte-stimulating hormone in NTS increased nearly 21-fold, whereas hypothalamic alpha-melanocyte-stimulating hormone remained unchanged. Visceral adiposity decreased by 37%; tissue triglyceride content diminished by 26% and 47% in liver and muscle, respectively; serum triglyceride 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.
Assuntos
Anorexia/prevenção & controle , Melanocortinas/genética , Núcleo Solitário/metabolismo , Taquifilaxia , Redução de Peso/genética , Adiposidade/genética , Animais , Animais Geneticamente Modificados , Anorexia/induzido quimicamente , Regulação do Apetite/genética , Peso Corporal , Metabolismo Energético/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Leptina/sangue , Masculino , Melanocortinas/metabolismo , Obesidade/genética , Obesidade/metabolismo , Obesidade/terapia , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Ratos , Ratos Endogâmicos F344 , Taquifilaxia/genética , Fatores de Tempo , alfa-MSH/genética , alfa-MSH/metabolismoRESUMO
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.
Assuntos
Quimiocinas CXC/genética , Fígado/metabolismo , Receptores CXCR4/fisiologia , 2-Acetilaminofluoreno , Animais , Animais Geneticamente Modificados , Apoptose , Carcinógenos/administração & dosagem , Contagem de Células , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Quimiocinas CXC/fisiologia , Feminino , Hepatectomia , Antígeno Ki-67/análise , Fígado/citologia , RNA Interferente Pequeno , Ratos , Ratos Endogâmicos F344RESUMO
Somatostatin (SST) is a regulatory peptide that activates G protein-coupled receptors comprised of five members (somatostatin receptors (SSTRs) 1-5). Despite the broad use of SST and its analogs in clinical practice, the spectrum of SST activities has been incompletely defined. Recently, it has been demonstrated that SST can be a chemoattractant for hematopoietic precursor cells. Since hepatic oval cells (HOCs) share common characteristics with hematopoietic stem cells, we hypothesized that SST could act as a chemoattractant for HOCs by stimulating SSTRs. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot assay revealed an increased expression of SST in the 2-acetyl-aminofluorene (2AAF)/partial hepatectomy (PHx) HOC induction model. Immunohistochemical staining showed the expression of SST in 2AAF/PHx-treated rat liver, as compared to normal liver. Proliferation and migration assays demonstrated that the increase of SST was related to migration of HOCs, but not their proliferation. RT-PCR and quantitative real-time PCR showed that SSTR4 was preferentially expressed by HOCs. Western blot assay and immunohistochemical staining confirmed the expression of SSTR4 by HOCs. In addition, pretreatment with anti-SSTR4 antibody cultures resulted in a dramatic reduction of cell migration as compared to that of control. Lastly, SST stimulated the rearrangement of actin filaments in HOCs, while HOCs treated with anti-SSTR4 antibody failed to do so. These results suggest a positive role for SST in the migration of HOCs, and that this effect is mediated through SSTR4.