RESUMO
Neutrophil (PMN) infiltration of the intestinal mucosa often leads to severe epithelial injury; however, how this process occurs is unclear. This article describes a novel mechanism whereby membrane-derived microparticles released by tissue infiltrating PMNs (PMN-MPs) serve as shuttles to protect and deliver active mediators to locally modulate cellular function during inflammation. Specifically, myeloperoxidase (MPO), which is abundantly expressed in PMN azurophilic granules and is used for microbial killing, was found to be mobilized to the PMN surface and subsequently released in association with PMN-MPs upon PMN activation and binding to intestinal epithelial cells (IECs). The enzymatic activity of PMN-MP-associated MPO was enhanced compared with soluble protein, leading to potent inhibition of wound closure following PMN-MP binding to IECs. Importantly, localized microinjection of PMN-MPs into wounded colonic mucosa was sufficient to impair epithelial wound healing in vivo. PMN-MP/MPO-dependent inhibition of IEC wound healing was due to impaired IEC migration and proliferation, resulting from impeded actin dynamics, cell spreading, and cell cycle arrest. Thus, our findings provide new insight into mechanisms governing PMN-induced tissue injury and implicate PMN-MPs and MPO as important regulators of cellular function.
Assuntos
Micropartículas Derivadas de Células , Neutrófilos/imunologia , Peroxidase/administração & dosagem , Cicatrização/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Humanos , Peroxidase/imunologia , Peroxidase/metabolismo , Cicatrização/fisiologiaRESUMO
Purpose To test whether MPO-Gd, an activatable molecular magnetic resonance (MR) imaging agent specific for myeloperoxidase (MPO) activity, could detect MPO activity in nonalcoholic steatohepatitis (NASH) mouse models and human liver biopsy samples. Materials and Methods In this study, 20 leptin receptor-deficient and three MPO knockout mice were injected with endotoxin (lipopolysaccharide) or fed a methionine and choline-deficient (MCD) diet to induce experimental NASH and underwent MR imaging with MPO-Gd. Saline-injected and control diet-fed leptin receptor-deficient mice were used as respective controls. MPO protein and activity measurements and histologic analyses were performed. Eleven human liver biopsy samples underwent MPO-Gd-enhanced MR imaging ex vivo and subsequent histologic evaluation. Results were compared with Student t test or Mann-Whitney U test. Results With endotoxin, a significantly increased contrast-to-noise ratio (CNR) was found compared with sham (mean CNR, 1.81 [95% confidence interval {CI}: 1.53, 2.10] vs 1.02 [95% CI: 0.89, 1.14]; P = .03) at MPO-Gd MR imaging. In the diet-induced NASH model, an increased CNR was also found compared with sham mice (mean CNR, 1.33 [95% CI: 1.27, 1.40] vs 0.98 [95% CI: 0.83, 1.12]; P = .008). Conversely, CNR remained at baseline in NASH mice imaged with gadopentetate dimeglumine and in MPO knockout NASH mice with MPO-Gd, which proves specificity of MPO-Gd. Ex vivo molecular MR imaging of liver biopsy samples from NASH and control patients confirmed results from animal studies (mean CNR for NASH vs control patients, 2.61 [95% CI: 1.48, 3.74] vs 1.29 [95% CI: 1.06, 1.52]; P = .004). Conclusion MPO-Gd showed elevated MPO activity in NAFLD mouse models and human liver biopsy samples. © RSNA, 2017 Online supplemental material is available for this article. An earlier incorrect version of this article appeared online. This article was corrected on April 6, 2017.
Assuntos
Fígado Gorduroso/diagnóstico por imagem , Fígado Gorduroso/enzimologia , Imageamento por Ressonância Magnética/métodos , Imagem Molecular/métodos , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/enzimologia , Peroxidase/administração & dosagem , Adulto , Animais , Biópsia , Meios de Contraste/administração & dosagem , Diagnóstico Diferencial , Modelos Animais de Doenças , Feminino , Gadolínio DTPA/administração & dosagem , Humanos , Técnicas Imunoenzimáticas , Camundongos , Pessoa de Meia-Idade , Estresse OxidativoRESUMO
OBJECTIVES: Pentraxin 3 (PTX3) is a multifunctional soluble factor. PTX3 can be involved in the regulation of vasculitis and is expressed in the cytoplasm of neutrophils. As anti-neutrophil cytoplasmic antibody (ANCA) is recognised as a cause of vasculitis, we aimed to discover the role of PTX3 in ANCA production in vivo. METHODS: To this end, we used aluminum salt (alum), which induces neutrophil extracellular traps, as an adjuvant for producing anti-myeloperoxidase-ANCA (MPO-ANCA). Specifically, we intraperitoneally injected alum and recombinant MPO (rMPO) into MPO-deficient mice and then measured the concentration of anti-MPO IgG in their blood. To show the involvement of extracellular PTX3 in this model, we assessed PTX3 protein content and host double-stranded DNA levels in the mice's peritoneal fluid after alum injection. In addition, we simultaneously administered recombinant PTX3, rMPO and alum to MPO-deficient mice to assess the function of PTX3 in producing anti-MPO IgG in vivo. RESULTS: Anti-MPO IgG was produced by the alum + rMPO immunisation model in MPO-deficient but not wildtype mice. Injection of alum induced extracellular PTX3 as well as double-stranded DNA and dead cells in MPO-deficient mice. Simultaneous injection of recombinant PTX3 with rMPO and alum attenuated the production of anti-MPO IgG in MPO-deficient mice. CONCLUSIONS: Our current findings provide evidence that PTX3 attenuates the production of murine MPO-ANCA.
Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen/farmacologia , Anticorpos Anticitoplasma de Neutrófilos/sangue , Proteína C-Reativa/imunologia , Imunoglobulina G/sangue , Erros Inatos do Metabolismo/imunologia , Proteínas do Tecido Nervoso/imunologia , Peroxidase/imunologia , Animais , Líquido Ascítico/imunologia , Líquido Ascítico/metabolismo , Proteína C-Reativa/administração & dosagem , Proteína C-Reativa/metabolismo , DNA/imunologia , DNA/metabolismo , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Feminino , Masculino , Erros Inatos do Metabolismo/sangue , Erros Inatos do Metabolismo/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/metabolismo , Peroxidase/administração & dosagem , Peroxidase/deficiência , Peroxidase/genéticaRESUMO
Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.
Assuntos
Quimiocina CCL5/metabolismo , Células Epiteliais/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Peroxidase/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL5/administração & dosagem , Quimiocina CCL5/genética , Quimiocinas/efeitos dos fármacos , Quimiocinas/genética , Quimiocinas/fisiologia , Citocinas/efeitos dos fármacos , Citocinas/genética , Citocinas/fisiologia , Regulação para Baixo , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Peróxido de Hidrogênio/farmacologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Ativação Linfocitária , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/virologia , Peroxidase/administração & dosagem , RNA Interferente Pequeno , Proteínas Recombinantes , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Atherosclerosis is linked to inflammation. HDL protects against atherosclerotic cardiovascular disease, mainly by mediating cholesterol efflux and reverse cholesterol transport (RCT). The present study aimed to test the impact of acute inflammation as well as selected acute phase proteins on RCT with a macrophage-to-feces in vivo RCT assay using intraperitoneal administration of [(3)H]cholesterol-labeled macrophage foam cells. In patients with acute sepsis, cholesterol efflux toward plasma and HDL were significantly decreased (P < 0.001). In mice, acute inflammation (75 microg/mouse lipopolysaccharide) decreased [(3)H]cholesterol appearance in plasma (P < 0.05) and tracer excretion into feces both within bile acids (-84%) and neutral sterols (-79%, each P < 0.001). In the absence of systemic inflammation, overexpression of serum amyloid A (SAA, adenovirus) reduced overall RCT (P < 0.05), whereas secretory phospholipase A(2) (sPLA(2), transgenic mice) had no effect. Myeloperoxidase injection reduced tracer appearance in plasma (P < 0.05) as well as RCT (-36%, P < 0.05). Hepatic expression of bile acid synthesis genes (P < 0.01) and transporters mediating biliary sterol excretion (P < 0.01) was decreased by inflammation. In conclusion, our data demonstrate that acute inflammation impairs cholesterol efflux in patients and macrophage-to-feces RCT in vivo in mice. Myeloperoxidase and SAA contribute to a certain extent to reduced RCT during inflammation but not sPLA(2). However, reduced bile acid formation and decreased biliary sterol excretion might represent major contributing factors to decreased RCT in inflammation.
Assuntos
Reação de Fase Aguda/fisiopatologia , Colesterol/metabolismo , Fosfolipases A2 do Grupo II/fisiologia , Peroxidase/fisiologia , Proteína Amiloide A Sérica/fisiologia , Reação de Fase Aguda/sangue , Reação de Fase Aguda/induzido quimicamente , Reação de Fase Aguda/metabolismo , Animais , Aterosclerose/fisiopatologia , Aterosclerose/prevenção & controle , Transporte Biológico , Células Cultivadas , Colesterol/sangue , Fezes/química , Células Espumosas/metabolismo , Fosfolipases A2 do Grupo II/biossíntese , Fosfolipases A2 do Grupo II/sangue , Fosfolipases A2 do Grupo II/genética , Humanos , Lipídeos/sangue , Lipoproteínas/sangue , Fígado/enzimologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Peroxidase/administração & dosagem , Peroxidase/sangue , Peroxidase/isolamento & purificação , RNA Mensageiro/metabolismo , Sepse/sangue , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Esteroides/metabolismoRESUMO
Abnormal glycerophospholipid (GPL) metabolism represented by phosphatidylcholine (PC) and phosphatidylethanolamine (PE) has been as a universal metabolic hallmark of cancer, which is involved in tumor progression. Our previous finding showed that peroxidase from foxtail millet bran (FMBP) exhibited significant anticolorectal cancer (CRC) activity in vitro and in nude mice. Presently, the potential of FMBP in clinical application was further evaluated by an azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated carcinogenesis (CAC) mice model, revealed the pivotal role of GPL metabolism in anti-CRC effects of FMBP. Excitedly, FMBP significantly reduced the number and volume of CAC polyps of mice and effectively improved physiological indexes of CAC mice. Meanwhile, the elevated expressions of CRC early markers (cyclooxygenase 2, tumor-proliferating nuclear antigen Ki-67, and EGF module-containing mucin-like receptor 1) in CAC mice were efficiently prevented by FMBP treatment. Metabolomics analysis showed that the elevated abundances of PC and PE involved in GPL metabolism in CAC mice were markedly decreased in FMBP-treated groups, which was also verified in human CRC cells. Further, FMBP reduced the expression levels of PE and PC key metabolic enzymes, resulting in the blockage of GPL metabolism and insufficient adenosine triphosphate to maintain CRC growth. Collectively, FMBP has the potential as a preventive and therapeutic candidate for CRC through the blockage of GPL metabolism.
Assuntos
Colite/complicações , Neoplasias Colorretais/tratamento farmacológico , Glicerofosfolipídeos/metabolismo , Peroxidase/administração & dosagem , Proteínas de Plantas/administração & dosagem , Setaria (Planta)/enzimologia , Animais , Benzofuranos , Carcinogênese , Linhagem Celular Tumoral , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Nus , Quinolinas , Setaria (Planta)/químicaRESUMO
Atherosclerosis is one of the main causes of cardiovascular diseases. Our previous study indicated that a type of peroxidase derived from foxtail millet bran (FMBP) had prominent antitumor activities. In the present study, we found that FMBP had potential antiatherosclerosis effects. The results showed that FMBP treatment strongly suppressed lipid phagocytosis in both HASMCs and THP-1 cells by 52% and 49%, respectively. Further, FMBP significantly inhibited HASMCs migration by promoting transformation of HASMCs from synthetic to contractile, leading to the decrease of lipid phagocytosis. Simultaneously, FMBP repressed lipid uptake by reducing the expression of CD36 in THP-1 cells. In addition, FMBP reduced the secretion of inflammatory factor IL-1ß by inhibiting the expression of STAT3 in THP-1 cells. Interestingly, FMBP also had the same effects in models of atherosclerosis constructed with ApoE-/- mice, including decreased aortic lesion area, repressed aortic sinus CD36 and STAT3 expression, and elevated serum HDL-C concentration. Collectively, these results indicate that FMBP has great potential in preventing the development of atherosclerosis.
Assuntos
Aterosclerose/tratamento farmacológico , Antígenos CD36/genética , Peroxidase/administração & dosagem , Proteínas de Plantas/administração & dosagem , Fator de Transcrição STAT3/genética , Setaria (Planta)/enzimologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Antígenos CD36/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Fator de Transcrição STAT3/metabolismo , Setaria (Planta)/química , Células THP-1RESUMO
Influenza poses a severe threat to human health in the world. However, developing a universal anti-viral strategy has remained challenging due to the presence of diverse subtypes as well as its high mutation rate, resulting in antigenic shift and drift. Here we developed an antiviral strategy using iron oxide nanozymes (IONzymes) to target the lipid envelope of the influenza virus. Methods: We evaluated the antiviral activities of our IONzymes using a hemagglutination assay, together with a 50% tissue culture infectious doses (TCID50) method. Lipid peroxidation of the viral envelope was analyzed using a maleic dialdehyde (MDA) assay and transmission electron microscopy (TEM). The neighboring viral proteins were detected by western blotting. Results: We show that IONzymes induce envelope lipid peroxidation and destroy the integrity of neighboring proteins, including hemagglutinin, neuraminidase, and matrix protein 1, causing the inactivation of influenza A viruses (IAVs). Furthermore, we show that our IONzymes possess a broad-spectrum antiviral activity on 12 subtypes of IAVs (H1~H12). Lastly, we demonstrate that applying IONzymes to a facemask improves the ability of virus protection against 3 important subtypes that pose a threat to human, including H1N1, H5N1, and H7N9 subtype. Conclusion: Together, our results clearly demonstrate that IONzymes can catalyze lipid peroxidation of the viral lipid envelope to inactivate enveloped viruses and provide protection from viral transmission and infection.
Assuntos
Antivirais/administração & dosagem , Compostos Férricos/química , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Lipídeos de Membrana/química , Peroxidase/administração & dosagem , Animais , Antivirais/química , Biocatálise , Feminino , Compostos Férricos/administração & dosagem , Humanos , Vírus da Influenza A/classificação , Vírus da Influenza A/metabolismo , Influenza Humana/virologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Peroxidase/químicaRESUMO
To develop a new strategy for inflamed site-specific drug delivery in the colon for the treatment of ulcerative colitis (UC), we leveraged on the interaction between myeloperoxidase (MPO) and human serum albumin (HSA) and prepared nanoparticles (HSA NPs) conjugated with 5-aminosalicylic acid (5-ASA). The 5-ASA-HSA NPs (nine molecules of 5-ASA per HSA molecule) were uniform particles with an average particle size of 190â¯nm, a zeta potential of --11.8â¯mV, and a polydispersity index of 0.35. This was considered a suitable particle characteristic to pass through the mucus layer and accumulate into the mucosa. The specific interaction between the 5-ASA-HSA NPs and MPO was observed using quartz crystal microbalance analysis in vitro. In addition, the 5-ASA-HSA NPs group containing one thousandth of the dose of the 5-ASA (75⯵g/kg) showed significantly lower disease activity index values and colon weight/length ratios in UC model mice as similar to large amount of neat 5-ASA group (75â¯mg/kg), indicating that the therapeutic effect of the 5-ASA-HSA NP formulation was confirmed in vivo. Microscopic images of tissue sections of colon extracted from UC model mice demonstrated that HSA NPs and MPO were both localized in the colon, and this specific interaction between HSA NPs and MPO would be involved the in the therapeutic effect in vivo. Furthermore, in the 5-ASA and 5-ASA-HSA NPs groups, some inflammatory damage was observed in the colon, but the degree of damage was mild compared with the control and HSA NPs groups, suggesting mucosal repair and replacement with fibrous granulation tissue had occurred. Therefore, these data demonstrated that an HSA NP formulation has the potential to specifically deliver 5-ASA to an inflamed site where MPO is highly expressed.
Assuntos
Colite/tratamento farmacológico , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/administração & dosagem , Peroxidase/administração & dosagem , Albumina Sérica Humana/administração & dosagem , Animais , Colite/metabolismo , Colite/patologia , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/metabolismo , Peroxidase/metabolismo , Distribuição Aleatória , Albumina Sérica Humana/metabolismoRESUMO
Rational:Salmonella Enteritidis (S. Enteritidis) is a globally significant zoonotic foodborne pathogen which has led to large numbers of deaths in humans and caused economic losses in animal husbandry. S. Enteritidis invades host cells and survives within the cells, causing resistance to antibiotic treatment. Effective methods of elimination and eradication of intracellular S. Enteritidis are still very limited. Here we evaluated whether a new intracellular antibacterial strategy using iron oxide nanozymes (IONzymes) exerted highly antibacterial efficacy via its intrinsic peroxidase-like activity in vitro and in vivo.Methods: The antibacterial activities of IONzymes against planktonic S. Enteritidis, intracellular S. Enteritidis in Leghorn Male Hepatoma-derived cells (LMH), and liver from specific pathogen free (SPF) chicks were investigated by spread-plate colony count method and cell viability assay. Changes in levels of microtubule-associated protein light chain 3 (LC3), a widely used marker for autophagosomes, were analyzed by immunoblotting, immunofluorescence, and electron microscopy. Reactive oxygen species (ROS) production was also assessed in vitro. High-throughput RNA sequencing was used to investigate the effects of IONzymes on liver transcriptome of S. Enteritidis-infected chicks. Results: We demonstrated that IONzymes had high biocompatibility with cultured LMH cells and chickens, which significantly inhibited intracellular S. Enteritidis survival in vitro and in vivo. In addition, co-localization of IONzymes with S. Enteritidis were observed in autophagic vacuoles of LMH cells and liver of chickens infected by S. Enteritidis, indicating that IONzymes mediated antibacterial reaction of S. Enteritidis with autophagic pathway. We found ROS level was significantly increased in infected LMH cells treated with IONzymes, which might enhance the autophagic elimination of intracellular S. Enteritidis. Moreover, orally administered IONzymes decreased S. Enteritidis organ invasion of the liver and prevented pathological lesions in a chicken-infection model. Non-target transcriptomic profiling also discovered IONzymes could change hepatic oxidation-reduction and autophagy related gene expressions in the S. Enteritidis infected chickens. Conclusion: These data suggest that IONzymes can increase ROS levels to promote the antibacterial effects of acid autophagic vacuoles, and thus suppress the establishment and survival of invading intracellular S. Enteritidis. As a result, IONzymes may be a novel alternative to current antibiotics for the control of intractable S. Enteritidis infections.
Assuntos
Compostos Férricos/administração & dosagem , Peroxidase/administração & dosagem , Infecções por Salmonella/tratamento farmacológico , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/crescimento & desenvolvimento , Animais , Antibacterianos/administração & dosagem , Antibacterianos/química , Galinhas , Compostos Férricos/química , Humanos , Fígado/microbiologia , Nanoestruturas/química , Peroxidase/química , Espécies Reativas de Oxigênio/metabolismo , Infecções por Salmonella/microbiologia , Salmonella enteritidis/metabolismoRESUMO
Pulmonary arterial hypertension (PAH) remains a disease with limited therapeutic options and dismal prognosis. Despite its etiologic heterogeneity, the underlying unifying pathophysiology is characterized by increased vascular tone and adverse remodeling of the pulmonary circulation. Myeloperoxidase (MPO), an enzyme abundantly expressed in neutrophils, has potent vasoconstrictive and profibrotic properties, thus qualifying as a potential contributor to this disease. Here, we sought to investigate whether MPO is causally linked to the pathophysiology of PAH. Investigation of 2 independent clinical cohorts revealed that MPO plasma levels were elevated in subjects with PAH and predicted adverse outcome. Experimental analyses showed that, upon hypoxia, right ventricular pressure was less increased in Mpo-/- than in WT mice. The hypoxia-induced activation of the Rho-kinase pathway, a critical subcellular signaling pathway yielding vasoconstriction and structural vascular remodeling, was blunted in Mpo-/- mice. Mice subjected to i.v. infusion of MPO revealed activation of Rho-kinase and increased right ventricular pressure, which was prevented by coinfusion of the Rho-kinase inhibitor Y-27632. In the Sugen5416/hypoxia rat model, PAH was attenuated by the MPO inhibitor AZM198. The current data demonstrate a tight mechanistic link between MPO, the activation of Rho-kinase, and adverse pulmonary vascular function, thus pointing toward a potentially novel avenue of treatment.
Assuntos
Hipertensão Pulmonar/patologia , Hipóxia/patologia , Peroxidase/metabolismo , Artéria Pulmonar/patologia , Quinases Associadas a rho/metabolismo , Adulto , Amidas/administração & dosagem , Animais , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Humanos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/mortalidade , Hipertensão Pulmonar/fisiopatologia , Hipóxia/sangue , Hipóxia/etiologia , Infusões Intravenosas , Estimativa de Kaplan-Meier , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Peroxidase/administração & dosagem , Peroxidase/sangue , Artéria Pulmonar/fisiopatologia , Piridinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/sangue , Proteínas Recombinantes/metabolismo , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/fisiologia , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Current biomarkers of renal disease in systemic vasculitis lack predictive value and are insensitive to early damage. To identify novel biomarkers of renal vasculitis flare, we analysed the longitudinal urinary metabolomic profile of a rat model of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Wistar-Kyoto (WKY) rats were immunised with human myeloperoxidase (MPO). Urine was obtained at regular intervals for 181 days, after which relapse was induced by re-challenge with MPO. Urinary metabolites were assessed in an unbiased fashion using nuclear magnetic resonance (NMR) spectroscopy, and analysed using partial least squares discriminant analysis (PLS-DA) and partial least squares regression (PLS-R). At 56 days post-immunisation, we found that rats with vasculitis had a significantly different urinary metabolite profile than control animals; the observed PLS-DA clusters dissipated between 56 and 181 days, and re-emerged with relapse. The metabolites most altered in rats with active or relapsing vasculitis were trimethylamine N-oxide (TMAO), citrate and 2-oxoglutarate. Myo-inositol was also moderately predictive. The key urine metabolites identified in rats were confirmed in a large cohort of patients using liquid chromatography-mass spectrometry (LC-MS). Hypocitraturia and elevated urinary myo-inositol remained associated with active disease, with the urine myo-inositol:citrate ratio being tightly correlated with active renal vasculitis.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/urina , Nefropatias/urina , Metabolômica/métodos , Peroxidase/administração & dosagem , Animais , Ácido Cítrico/urina , Modelos Animais de Doenças , Feminino , Humanos , Imunização , Ácidos Cetoglutáricos/urina , Análise dos Mínimos Quadrados , Masculino , Metilaminas/urina , Peroxidase/imunologia , Ratos , Ratos Endogâmicos WKY , RecidivaRESUMO
Enzyme tablets with butyrylcholine esterase (CHE) and peroxidase (POD) partly lose enzymatic activity during compaction at a pressure of 495 MPa. Compared to solutions of the original enzyme, no changes of ultraviolet absorbance and fluorescence intensity in the tablet solutions were found. Only small changes were observed in the far ultraviolet circular dichroism spectra. Neither missing nor additional bands were detected with polyacrylamide gel electrophoresis. Heated (150 degrees C) solid starting material with CHE and POD showed still part of its original enzymatic activity. The ultraviolet absorbance increased with continued heating until precipitation occurred. The circular dichroism spectra are changed clearly.
Assuntos
Enzimas/análise , Butirilcolinesterase/administração & dosagem , Butirilcolinesterase/análise , Dicroísmo Circular , Composição de Medicamentos , Eletroforese em Gel de Poliacrilamida , Enzimas/administração & dosagem , Peroxidase/administração & dosagem , Peroxidase/análise , Proteínas/administração & dosagem , Proteínas/análise , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , ComprimidosRESUMO
Systemic small vessel vasculitis is associated with antineutrophil cytoplasm antibodies (ANCAs). While there is mounting in vitro evidence to suggest that ANCAs are capable of enhancing leukocyte-endothelial interactions, no in vivo evidence for this has been provided. In this study a novel rat model of ANCA-associated experimental autoimmune vasculitis (EAV), induced by immunization with human myeloperoxidase (MPO), was used to analyze directly the potential effect of ANCAs on leukocyte-venular wall interactions in vivo as observed by intravital microscopy. These rats developed anti-MPO antibodies directed against rat leukocytes, showed pathologic evidence of small vessel vasculitis, and had enhanced leukocyte adhesion and transmigration in response to the chemokine Groalpha (CXCL1 [CXC ligand 1]). Passive transfer of immunoglobulin from rats with EAV to naive rats conferred enhanced adhesion and transmigration responses in the recipients. Furthermore, rats with EAV and recipients of ANCA-positive immunoglobulin developed extensive microvascular injury, as manifested by mesenteric hemorrhage, in response to CXCL1. This study provides the first direct in vivo evidence for the ability of ANCAs to enhance leukocyte-endothelial interactions and cause microvascular hemorrhage, thereby providing a mechanism by which ANCAs could exert pathogenic effects in systemic vasculitis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Endotélio Vascular/citologia , Migração e Rolagem de Leucócitos/imunologia , Peroxidase/imunologia , Vasculite Leucocitoclástica Cutânea/imunologia , Animais , Permeabilidade Capilar/imunologia , Adesão Celular/imunologia , Quimiocina CXCL1 , Quimiocinas CXC/farmacologia , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Humanos , Imunização , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peroxidase/administração & dosagem , Ratos , Ratos Endogâmicos WKY , Circulação Esplâncnica/imunologia , Vasculite Leucocitoclástica Cutânea/patologiaRESUMO
Lyophilized horseradish peroxidase (activity 100 U/mg) administered per os in a dose of 100-200 mg/kg fodder enhanced bactericidal activity of phagocytes in mice experimentally infected with Mycobacterium leprae, which manifested in suppression of M. leprae growth in comparison with untreated controls.
Assuntos
Hanseníase/tratamento farmacológico , Hanseníase/imunologia , Peroxidase/administração & dosagem , Animais , Imunidade/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos CBA , Mycobacterium leprae , Peroxidase/uso terapêuticoRESUMO
Anti-neutrophil cytoplasmic antibodies (ANCA), including anti-myeloperoxidase (MPO) antibodies, are associated with pauci-immune necrotizing small vessel vasculitis or glomerulonephritis. In order to substantiate a pathogenic role for ANCA, an animal model of pauci-immune ANCA-induced glomerulonephritis or vasculitis is required. Brouwer et al. reported pauci-immune glomerulonephritis in rats immunized with human MPO followed by perfusion of kidneys with lysosomal enzyme extract combined with H2O2, and suggested that this could serve as a model of ANCA-induced disease. We repeated these studies in spontaneously hypertensive rats (SHR) and Brown Norway rats (BNR). We immunized rats with human MPO. When circulating anti-MPO antibodies were detectable by indirect immunofluorescence microscopy and ELISA, blood pressure was measured, then perfusion of the left kidney of each rat was done via the renal artery in a closed, blood-free circuit with either MPO + H2O2, MPO, H2O2 alone or MPO + H2O2 + neutral protease. Rats were killed on day 4 or day 10 after perfusion, and specimens were examined by light and immunofluorescence microscopy. Pathological lesions and deposits of IgG, C3, and MPO were found in immunized rats perfused with MPO + H2O2 with or without neutral protease, or MPO alone, in both rat strains and on both day 4 and day 10. The degree of histologic injury was proportional in intensity to the amount of IgG immune deposits. Spontaneously hypertensive rats sustained more damage and higher blood pressure than Brown Norway rats. No lesion was observed in immunized rats perfused with H2O2 or in the non-perfused right kidneys. Some of the non-immunized rats perfused with MPO + H2O2 developed pathological lesions. In conclusion, these rat models are examples of immune complex-mediated glomerulonephritis, and therefore are not similar to human ANCA-associated disease.
Assuntos
Complexo Antígeno-Anticorpo , Glomerulonefrite/imunologia , Peroxidase/imunologia , Animais , Autoanticorpos/imunologia , Pressão Sanguínea , Complemento C3/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Glomerulonefrite/patologia , Humanos , Hipertensão/etiologia , Hipertensão/patologia , Imunização , Imunoglobulina G/imunologia , Rim/imunologia , Rim/patologia , Masculino , Peroxidase/administração & dosagem , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos SHRRESUMO
The capacity of myeloperoxidase which is the product of polymorphonuclear leucocytes to induce the lens opacity was studied in young and old rabbits. It was found that the injection of myeloperoxidase solution into anterior chamber of the eye causes the irreversible lens opacification in old rabbits, not in young ones. Light microscopy of the lens section has shown the following alterations: the local thickening of the anterior capsule, disorderly accumulation of epithelial cells, formation of so-called "bladder cells" under the lens epithelium. Changes in experimental eyes are typical for cataract.
Assuntos
Catarata/etiologia , Neutrófilos/enzimologia , Peroxidase/fisiologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/fisiologia , Animais , Câmara Anterior , Catarata/patologia , Relação Dose-Resposta a Droga , Fibrinogênio/administração & dosagem , Cristalino/efeitos dos fármacos , Cristalino/patologia , Peroxidase/administração & dosagem , Coelhos , Fatores de TempoRESUMO
The strong association of anti-neutrophil cytoplasmic antibodies with various forms of systemic vasculitis suggests a role for these autoantibodies in the pathophysiology of systemic vasculitis. In the present study, we tested the hypothesis that release of neutrophil lysosomal enzymes in the presence of an anti-myeloperoxidase (anti-MPO) immune response may underlie the development of systemic vasculitis. Brown Norway rats were immunized with MPO in complete Freund's adjuvant or complete Freund's adjuvant alone. Two weeks after immunization, rats bad developed antibodies to human and rat MPO as measured by enzyme-linked immunosorbent assay. Next, rats were intravenously infused with 400 micrograms of a human neutrophil lysosomal extract containing 200 micrograms of MPO followed by 0.5 ml of a 1 mmol/L solution of H2O2 through a cannula inserted into the right jugular vein. Rats were sacrificed at 4 hours, 24 hours, 7 days, or 14 days, and several organs (lungs, heart, liver, spleen, gut, and kidneys) were examined for vasculitic lesions and inflammatory cell infiltrates. Macroscopically, patchy hemorrhagic spots were observed in the lungs and gut of MPO-immunized rats at days 7 and 14 after systemic infection of the neutrophil lysosomal extract and H2O2. Such changes were not observed at earlier time points or in control immunized rats. Histologically, the lungs of MPO-immunized rats sacrificed at days 7 and 14 showed patchy inflammatory cell infiltrates associated with vasculitis, granuloma formation, giant cells, and foci of hemorrhage. At 14 days, early signs of fibrosis were found with deposition of collagen and proliferation of fibroblasts. Furthermore, a prominent leukocytoclastic vasculitis was found in the small intestine of these rats characterized by fibrinoid necrosis and an extensive neutrophilic infiltrate. No inflammatory changes were found in the other organs studied (heart, liver, spleen, and kidneys). Control immunized rats, sacrificed at days 7 and 14 showed only some small foci of inflammatory infiltrates in the lungs whereas no inflammatory changes were found in the gastrointestinal tract. These studies show that release of products from activated neutrophils in the presence of anti-MPO autoantibodies may be relevant to the pathogenesis of anti-MPO-associated vasculitides.
Assuntos
Peróxido de Hidrogênio/metabolismo , Intestino Delgado/patologia , Pulmão/patologia , Ativação de Neutrófilo/imunologia , Neutrófilos/metabolismo , Peroxidase/imunologia , Vasculite/induzido quimicamente , Vasculite/patologia , Animais , Humanos , Peróxido de Hidrogênio/administração & dosagem , Infusões Intravenosas , Elastase de Leucócito/administração & dosagem , Lisossomos/enzimologia , Mieloblastina , Necrose , Peroxidase/administração & dosagem , Ratos , Ratos Endogâmicos BN , Serina Endopeptidases/administração & dosagemRESUMO
The occurrence of focal fibrinoid necrosis of capillary loops in the very early stages of ANCA-associated necrotizing crescentic glomerulonephritis (NCGN) and the increased prevalence of this disease at older age suggest that renal ischemia may play an additional role in its pathophysiology. In the present study we investigated the contribution of renal ischemia to the induction of anti-myeloperoxidase (MPO) associated NCGN in a previously described rat model of this disease. The development of renal lesions is dependent on the presence of an anti-MPO immune response and the localization of a lysosomal extract containing lytic enzymes and MPO in combination with hydrogen peroxide (H2O2) along the glomerular basement membrane (GBM). The hypothesis tested whether perfusion of hydrogen peroxide (H2O2) could be replaced by ischemia/reperfusion (I/R) injury, as I/R injury activates endothelial cells to produce oxygen metabolites. I/R was induced by clamping the renal artery for 20 minutes in kidneys in which the circulation had been restored several minutes after perfusion with the lysosomal extract in MPO immunized rats. Rats developed lesions characterized by intra- and extracapillary cell proliferation, periglomerular infiltration, ruptures in Bowman's capsule, ischemic tubuli, and interstitial mononuclear infiltrate. Immune deposits, however, persisted for a longer time along the GBM after perfusion of lytic enzymes followed by I/R injury compared to previous studies in which H2O2 in conjunction with lytic enzymes were perfused in MPO-immunized rats.(ABSTRACT TRUNCATED AT 250 WORDS)