RESUMO
Regulation of autoreactive cells is key for both prevention and amelioration of autoimmune disease. A better understanding of the key cell population(s) responsible for downregulation of autoreactive cells would provide necessary foundational insight for cellular-based therapies in autoimmune disease. Utilizing a mouse model of anti-myeloperoxidase (MPO) glomerulonephritis, we sought to understand which immune cells contribute to downregulation of the anti-MPO autoimmune response. MPO-/- mice were immunized with whole MPO to induce an anti-MPO response. Anti-MPO splenocytes were then transferred into recipient mice (Rag2-/- mice or WT mice). Anti-MPO titers were followed over time. After anti-MPO splenocyte transfer, WT mice are able to downregulate the anti-MPO response while anti-MPO titers persist in Rag2-/- recipients. Reconstitution with WT splenocytes into Rag2-/- recipients prior to anti-MPO splenocyte transfer enabled mice to downregulate the anti-MPO immune response. Therefore, wildtype splenocytes contain a cellular population that is capable of downregulating the autoimmune response. Through splenocyte transfer, antibody depletion experiments, and purified cell population transfers, we confirmed that the regulatory T cell (Treg) population is responsible for the downregulation of the anti-MPO autoimmune response. Further investigation revealed that functional Tregs from WT mice are capable of downregulating anti-MPO antibody production and ameliorate anti-MPO induced glomerulonephritis. These data underscore the importance of functional Tregs for control of autoimmune responses and prevention of end-organ damage due to autoimmunity.
Assuntos
Autoimunidade , Modelos Animais de Doenças , Glomerulonefrite , Camundongos Knockout , Peroxidase , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Glomerulonefrite/imunologia , Glomerulonefrite/terapia , Camundongos , Peroxidase/metabolismo , Peroxidase/imunologia , Autoanticorpos/imunologia , Baço/imunologia , Regulação para Baixo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Transferência Adotiva , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Myeloperoxidase-specific ANCA (MPO-ANCA) are implicated in the pathogenesis of vasculitis and GN. Kinins play a major role during acute inflammation by regulating vasodilatation and vascular permeability and by modulating adhesion and migration of leukocytes. Kinin system activation occurs in patients with ANCA vasculitis. Previous studies in animal models of GN and sclerosing kidney diseases have demonstrated protective effects of bradykinin receptor 1 (B1R) blockade via interference with myeloid cell trafficking. METHODS: To investigate the role of B1R in a murine model of MPO-ANCA GN, we evaluated effects of B1R genetic ablation and pharmacologic blockade. We used bone marrow chimeric mice to determine the role of B1R in bone marrow-derived cells (leukocytes) versus nonbone marrow-derived cells. We elucidated mechanisms of B1R effects using in vitro assays for MPO-ANCA-induced neutrophil activation, endothelial adherence, endothelial transmigration, and neutrophil adhesion molecule surface display. RESULTS: B1R deficiency or blockade prevented or markedly reduced ANCA-induced glomerular crescents, necrosis, and leukocyte influx in mice. B1R was not required for in vitro MPO-ANCA-induced neutrophil activation. Leukocyte B1R deficiency, but not endothelial B1R deficiency, decreased glomerular neutrophil infiltration induced by MPO-ANCA in vivo. B1R enhanced ANCA-induced neutrophil endothelial adhesion and transmigration in vitro. ANCA-activated neutrophils exhibited changes in Mac-1 and LFA-1, important regulators of neutrophil endothelial adhesion and transmigration: ANCA-activated neutrophils increased surface expression of Mac-1 and increased shedding of LFA-1, whereas B1R blockade reduced these effects. CONCLUSIONS: The leukocyte B1R plays a critical role in the pathogenesis of MPO-ANCA-induced GN in a mouse model by modulating neutrophil-endothelial interaction. B1R blockade may have potential as a therapy for ANCA GN and vasculitis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Glomerulonefrite/etiologia , Peroxidase/imunologia , Receptor B1 da Bradicinina/fisiologia , Animais , Antagonistas de Receptor B1 da Bradicinina/uso terapêutico , Adesão Celular , Modelos Animais de Doenças , Células Endoteliais/fisiologia , Glomerulonefrite/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologiaRESUMO
ANCA vasculitis is an autoimmune disease with increased expression of the autoantigen genes, myeloperoxidase (MPO) and proteinase 3 (PRTN3), but the origin and significance of expression is less distinct. To clarify this, we measured MPO and PRTN3 messenger RNA in monocytes, normal-density neutrophils, and in enriched leukocytes from peripheral blood mononuclear cells. Increased autoantigen gene expression was detected in normal-density neutrophils and enriched leukocytes from patients during active disease compared to healthy individuals, with the largest difference in enriched leukocytes. RNA-seq of enriched leukocytes comparing active-remission pairs identified a gene signature for low-density neutrophils. Cell sorting revealed low-density neutrophils contained mature and immature neutrophils depending on the presence or absence of CD10. Both populations contributed to autoantigen expression but the frequency of immature cells in low-density neutrophils did not correlate with low-density neutrophil MPO or PRTN3 expression. Low-density neutrophils were refractory to MPO-ANCA induced oxidative burst, suggesting an alternative role for low-density neutrophils in ANCA vasculitis pathogenesis. In contrast, normal-density neutrophils were activated by MPO-ANCA and monoclonal anti-PR3 antibody. Normal-density neutrophil activation correlated with MPO and PRTN3 mRNA. Increased autoantigen gene expression originating from the mature low-density and normal-density neutrophils suggests transcriptional dysregulation is a hallmark of ANCA vasculitis. Thus, the correlation between autoantigen gene expression and antibody-mediated normal-density neutrophil activation connects autoantigen gene expression with disease pathogenesis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos , Neutrófilos , Autoantígenos/genética , Expressão Gênica , Humanos , Leucócitos Mononucleares , Mieloblastina , Ativação de Neutrófilo , Peroxidase/genéticaRESUMO
Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO-induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO-induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/prevenção & controle , Autoantígenos/imunologia , Glomerulonefrite/prevenção & controle , Peroxidase/imunologia , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Administração Oral , Animais , Complemento C6/imunologia , Via Alternativa do Complemento , Relação Dose-Resposta a Droga , Técnicas de Introdução de Genes , Glomerulonefrite/complicações , Glomerulonefrite/imunologia , Hematúria/etiologia , Hematúria/prevenção & controle , Humanos , Imunização Passiva , Leucócitos , Erros Inatos do Metabolismo/complicações , Erros Inatos do Metabolismo/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidase/deficiência , Proteinúria/etiologia , Proteinúria/prevenção & controle , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/fisiologia , Proteínas Recombinantes de Fusão , Urina/citologiaRESUMO
Myeloperoxidase (MPO) is a target antigen for antineutrophil cytoplasmic autoantibodies (ANCA). There is evidence that MPO-ANCA cause necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. NCGN severity varies among patients with ANCA disease, and genetic factors influence disease severity. The role of genetics in MPO-ANCA NCGN severity was investigated using 13 inbred mouse strains, F1 and F2 hybrids, bone marrow chimeras, and neutrophil function assays. Mouse strains include founders of the Collaborative Cross. Intravenous injection of anti-MPO IgG induced glomerular crescents in >60% of glomeruli in 129S6/SvEv and CAST/EiJ mice, but <1% in A/J, DBA/1J, DBA/2J, NOD/LtJ, and PWK/PhJ mice. C57BL6J, 129S1/SvImJ, LP/J, WSB/EiJ, NZO/HILtJ, and C3H mice had intermediate severity. High-density genotypes at 542,190 single nucleotide polymorphisms were used to identify candidate loci for disease severity by identifying genomic regions that are different between 129S6/SvEv and 129S1/SvImJ mice, which are genetically similar but phenotypically distinct. C57BL/6 × 129S6 F2 mice were genotyped at 76 SNPs to capture quantitative trait loci for disease severity. The absence of a dominant quantitative trait locus suggests that differences in severity are the result of multiple gene interactions. In vivo studies using bone marrow chimeric mice and in vitro studies of neutrophil activation by anti-MPO IgG indicated that severity of NCGN is mediated by genetically determined differences in the function of neutrophils.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Glomerulonefrite/genética , Glomerulonefrite/patologia , Peroxidase/genética , Peroxidase/imunologia , Animais , Células da Medula Óssea/patologia , Quimera , Cruzamentos Genéticos , Feminino , Ligação Genética , Genoma/genética , Genótipo , Glomerulonefrite/imunologia , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Ativação de Neutrófilo/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
As the primary goal of the 17 Sustainable Development Goals (SDGs), poverty eradication is still one of the major challenges faced by countries around the world, and relative poverty is a comprehensive poverty pattern triggered by the superposition of economic, social, and environmental dimensions. Therefore, Therefore, this paper introduces the perspective of coupled coordination to consider the formation of relative poverty, constructs indicators in three major dimensions: economic, social, and environmental, proposes a fast and more accurate method of identifying relative poverty in a region by using machine learning, measures the degree of coupled coordination of China's relatively poor provinces using a coupled coordination model and analyzes the relationship with the level of relative poverty, and puts forward suggestions for poverty management on this basis using typology classification. The results of the study show that: 1) the fusion of data crawlers, remote sensing space, and other multi-source data to construct the dataset and propose a fast and efficient regional relative poverty identification method based on big data with low comprehensive cost and high identification accuracy of 0.914. 2) Currently, 70.83% of the economic-social-environmental systems of the relatively poor regions are in the dysfunctional type and are in a state of disordered development and malignant constraints. The regions showing coupling disorders are mainly clustered in the three southern prefectures of Xinjiang, Qinghai, Gansu, Yunnan, and Sichuan, and their spatial distribution is relatively concentrated. 3) The types of poverty and their coupled and coordinated development in each region show large spatial variability, requiring differentiated poverty eradication countermeasures tailored to local conditions to achieve sustainable regional economic-social-environmental development.
Assuntos
Pobreza , Desenvolvimento Sustentável , China , Humanos , Desenvolvimento Sustentável/economia , Desenvolvimento Econômico , Meio Ambiente , Aprendizado de MáquinaRESUMO
Poverty eradication has always been a major challenge to global development and governance, which received widespread attention from each country. With the completion poverty alleviation task in 2020, relative poverty governance becomes an important issue to be solved in China urgently. Because of a large population, poor infrastructures, insufficient resources, and long-term uneven development raising the living standard of farmers in rural areas is critical to China's success in realizing moderate prosperity. Therefore, identifying the poor farmers, exploring the influence factors to relative poverty, and clarifying its effect mechanism in rural areas are significant for the subsequent poverty governance. Most of the previous studies adopted the method of apriori assuming the factor system and verifying the hypothesis. We innovatively constructed a relative poverty index system consistent with China's actual conditions, selecting all the possible variables that could affect relative poverty based on the existing literature, including individual characteristics, psychological endowment, and geographical environment, and rebuilt an experimental database. Then, through data processing and data analysis, the main factors influencing the relative poverty of farmers were systematically sorted out based on the machine learning method. Finally, 25 chosen influencing factors were discussed in detail. Research findings show that: 1) Machine learning algorithm is proved it could be well applied in relative poverty fields, especially XGBoost, which achieves 81.9% accuracy and the score of ROC_AUC reaches 0.819. 2) This study sheds light on many new research directions in applying machine learning for relative poverty research, besides, the paper offers an integral framework and beneficial reference for target identification using machine learning algorithms. 3) In addition, by utilizing the interpretable tools, the "black-box" of ML become transparent through PDP and SHAP explanation, it also reveals that machine learning models can readily handle the non-linear association relationship.
RESUMO
Based on the extended STIRPAT model, this paper examines social and economic factors regarding PM2.5 concentration intensity in 255 Chinese cities from 2007 to 2016, and includes quantile regressions to analyze the different effects of these factors among cities of various sizes. The results indicate that: (1) during 2007-2016, urban PM2.5 concentration exhibited declining trends in fluctuations concerning the development of the urban economy, accompanied by uncertainty under different city types; (2) population size has a significant effect on propelling PM2.5 concentration; (3) the effect of structure reformation on PM2.5 concentration is evident among cities with different populations and levels of economic development; and (4) foreign investment and scientific technology can significantly reduce PM2.5 emission concentration in cities. Accordingly, local governments not only endeavor to further control population size, but should implement a recycling economy, and devise a viable urban industrial structure. The city governance policies for PM2.5 concentration reduction require re-classification according to different population scales. Cities with large populations (i.e., over 10 million) should consider reducing their energy consumption. Medium population-sized cities (between 1 million and 10 million) should indeed implement effective population (density) control policies, while cities with small populations (less than 1 million) should focus on promoting sustainable urban development to stop environmental pollution from secondary industry sources.
Assuntos
Poluentes Atmosféricos , Poluição do Ar , Poluentes Atmosféricos/análise , Poluição do Ar/análise , China , Cidades , Monitoramento Ambiental/métodos , Poluição Ambiental , Material Particulado/análiseRESUMO
The aim of the study was to investigate the application of algal-bacterial granules in treatment of high ammonia wastewater. Two identical cylindrical reactors, i.e., Rc and Rs was used to develop granular sludge system with synthetic biogas slurry. Rs was run under an artificial solar lamp controlled at 12 h power on and 12 h power off (â¼10,000 lux); Rc was operated as control (no light). Results showed that algal-bacterial granules (ABGS) developed in Rs exhibited better structural stability in the face of high ammonia influent. Compared with aerobic granules (AGS), ABGS possessed high proteins (PN) content (145.3 mg/g-VSS) in extracellular polymeric substances (EPS) and better O2 mass transfer inner granules. Higher phosphorus (P) removal capacity was obtained in Rs even under 400 mg/L NH3-N which resulted in higher P content in ABGS biomass (56.4 mg/g-TSS). Bioavailable P in ABGS was 44 mg P/g-SS on day 160, approximately 1.53-times higher than that in AGS.
RESUMO
Antibodies that specifically bind polyethylene glycol (PEG), i.e. anti-PEG antibodies (APA), are associated with reduced efficacy and increased risk of serious adverse events for several PEGylated therapeutics. Here, we explored the concept of using free PEG molecules to saturate circulating APA. Surprisingly, we found that 40â¯kDa free PEG effectively restored the prolonged circulation of PEGylated liposomes in the presence of high titers of pre-existing APA for at least 48â¯h in mice. In contrast, lower molecular weight free PEG (≤10â¯kDa) failed to restore circulation beyond a few hours. These in vivo results were consistent with estimates from a minimal physiologically based pharmacokinetic model. Importantly, the infusion of free PEG appeared to be safe in mice previously sensitized by injection of PEGylated liposomes, and free PEG did not elicit excess APA production even in mice with pre-existing adaptive immunity against PEG. Our results support further investigation of high molecular weight free PEG as a potential method to control and overcome high titers of APA, restoring the prolonged circulation of PEGylated liposomes and possibly other PEGylated therapeutics.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Anticorpos/imunologia , Doxorrubicina/administração & dosagem , Polietilenoglicóis/administração & dosagem , Administração Intravenosa , Animais , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/farmacocinética , Feminino , Lipossomos , Fígado/metabolismo , Camundongos Endogâmicos BALB C , Peso Molecular , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinéticaRESUMO
OBJECTIVE: There is accumulating evidence that complement activation is important in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) pathogenesis. This study was undertaken to investigate complement activation in AAV with myeloperoxidase (MPO) positivity and AAV with proteinase 3 (PR3) positivity after determining optimal methods for measuring activated complement factors in circulation. METHODS: Participants included 98 patients with AAV (45 MPO-ANCA positive, 53 PR3-ANCA positive) and 35 healthy controls. Plasma was obtained from blood collected using EDTA tubes, with or without 100 µg/ml Futhan. Levels of Bb, C3a, C5a, soluble C5b-9 (sC5b-9), properdin, and C4d were measured by enzyme-linked immunosorbent assay. Group comparisons were made using Wilcoxon's 2-sample test. Paired data were analyzed using a matched pairs signed rank test. RESULTS: Compared to healthy controls, certain complement analyte levels were high in patients with active AAV with MPO positivity, including C3a (P < 0.0001), C5a (P = 0.0004), and sC5b-9 (P = 0.0007). During remission, levels of Bb (P = 0.001), C3a (P < 0.0001), and sC5b-9 (P = 0.003) were higher. Compared to healthy controls, C3a (P < 0.0001), C5a (P = 0.002), sC5b-9 (P = 0.0001), and C4d (P = 0.005) levels were higher in patients with active AAV with PR3 positivity; levels of C3a (P < 0.0001) and C4d (P = 0.007) were also higher duriing remission. There were no significant differences in any complement analyte for either ANCA serotype between patients with active disease and those with disease in remission. Among patients with paired samples, sC5-9 levels were significantly lower during disease remission compared to active disease. C5a was significantly lower among patients with disease in long-term remission who were not receiving therapy. For Bb, C5a, and sC5b-9, median levels and individual values were considerably higher in control and patient samples processed without Futhan compared to those processed with Futhan. CONCLUSION: Complement activation occurs in both MPO-positive AAV and PR3-positive AAV. The complement activation profile differs according to disease activity and possibly ANCA serotype. Futhan reduces in vitro complement activation and provides a more accurate measurement.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Complemento C3a/imunologia , Complemento C5a/imunologia , Fator B do Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Mieloblastina/imunologia , Peroxidase/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Complemento C4/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Properdina/imunologia , Índice de Gravidade de DoençaRESUMO
Antineutrophil cytoplasmic autoantibodies (ANCAs) are identified in the circulation of approximately 80% of patients with pauci-immune necrotizing and crescentic glomerulonephritis and systemic small vessel vasculitis, such as microscopic polyangiitis and Wegener granulomatosis. The most common antigen target for ANCAs is myeloperoxidase (MPO), which is found in neutrophils and monocytes. We report definitive experimental animal evidence that ANCAs are pathogenic. MPO knockout (Mpo(-/-)) mice were immunized with mouse MPO. Splenocytes from these mice or from control mice were injected intravenously into recombinase-activating gene-2-deficient (Rag2(-/-)) mice, which lack functioning B lymphocytes and T lymphocytes. All mice that received splenocytes developed mild to moderate glomerular immune deposits, but only mice that received 1 x 10(8) or 5 x 10(7) anti-MPO splenocytes developed severe necrotizing and crescentic glomerulonephritis, granulomatous inflammation, and systemic necrotizing vasculitis, including necrotizing arteritis and hemorrhagic pulmonary capillaritis. To test the pathogenic potential of antibodies alone, purified anti-MPO IgG or control IgG was injected intravenously into Rag2(-/-) mice and wild-type mice. Mice that received anti-MPO IgG but not mice that received control IgG developed focal necrotizing and crescentic glomerulonephritis with a paucity of glomerular Ig deposition. Thus, anti-MPO IgG alone was able to cause pauci-immune glomerular necrosis and crescent formation in the absence of functional T or B lymphocytes in Rag2(-/-) mice and in the presence of an intact immune system in wild-type C57BL/6J mice. This animal model offers strong support for a direct pathogenic role for ANCA IgG in human glomerulonephritis and vasculitis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Glomerulonefrite/etiologia , Peroxidase/imunologia , Vasculite/etiologia , Transferência Adotiva , Animais , Linhagem Celular , Proteínas de Ligação a DNA/fisiologia , Feminino , Glomerulonefrite/terapia , Imunização Passiva , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo , Soroalbumina Bovina/imunologia , Vasculite/terapiaRESUMO
Anti-neutrophil cytoplasmic antibodies (ANCA) vasculitides are immune-mediated disorders that primarily affect small blood vessels of the airway and kidneys. Lung involvement, one of the hallmarks of microscopic polyangiitis and granulomatosis with polyangiitis, is associated with increased mortality and morbidity. In recent years, several retrospective series and case reports have described the association of interstitial lung disease (ILD) and ANCA vasculitis, particularly those positive for ANCA specific for myeloperoxidase. In the majority of these patients pulmonary fibrosis occurs concurrently or predates the diagnosis of ANCA vasculitis. More importantly, these studies have shown that ILD has an adverse impact on the long-term prognosis of ANCA vasculitis. This review focuses on the main clinical and radiologic features of pulmonary fibrosis associated with anti-neutrophil cytoplasmic antibodies. Major histopathology features, prognosis and therapeutic options are summarized.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Doenças Pulmonares Intersticiais/epidemiologia , Fibrose Pulmonar/epidemiologia , Vasculite/epidemiologia , Animais , Humanos , Pulmão/diagnóstico por imagem , Pulmão/imunologia , Pulmão/patologia , Doenças Pulmonares Intersticiais/diagnóstico por imagem , Doenças Pulmonares Intersticiais/imunologia , Doenças Pulmonares Intersticiais/patologia , Fibrose Pulmonar/diagnóstico por imagem , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/patologia , Tomografia Computadorizada por Raios X , Vasculite/diagnóstico por imagem , Vasculite/imunologia , Vasculite/patologiaRESUMO
BACKGROUND: Antineutrophil cytoplasmic autoantibodies (ANCA) are associated with a spectrum of necrotizing vasculitis including granulomatosis with polyangiitis, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, and renal-limited necrotizing and crescentic glomerulonephritis. Clinical observations and in vitro and in vivo experimental evidence strongly indicate that ANCA are pathogenic. SUMMARY: The etiology and pathogenesis of ANCA-associated vasculitis (AAV) are multifactorial, with contributions from genetic factors, environmental exposures, infections, characteristics of the innate and adaptive immune system, and the intensity and duration of the injury. Acute vascular inflammation is induced when resting neutrophils that have ANCA autoantigens sequestered in cytoplasmic granules are exposed to priming factors - for example, cytokines induced by infection or phlogogenic factors released by complement activation - that cause the release of ANCA antigens on the surface of neutrophils and in the microenvironment around the neutrophils. ANCA bind to these ANCA antigens, which activates neutrophils by Fcγ receptor engagement and F(ab')2 binding at the neutrophil cell surface. ANCA-activated neutrophils release factors that activate the alternative complement pathway, which generates C5a, a chemoattractant for neutrophils; C5a also primes the arriving neutrophils for activation by ANCA. Activated neutrophils adhere to and penetrate vessel walls, and they release toxic oxygen radicals and destructive enzymes that cause apoptosis and necrosis of the neutrophils as well as of the adjacent vessel wall cells and matrix. KEY MESSAGES: Patients with active AAV have ongoing asynchronous onsets of countless acute lesions, with each lesion evolving through stereotypical phases within 1 or 2 weeks. Induction of remission results in termination of new waves of acute lesions and allows all lesions to progress to scarring or resolution.
RESUMO
Because changes in cell-cell adhesion have profound effects on cellular behavior, we hypothesized a link between the adhesion and signaling functions of plakoglobin and beta-catenin. To investigate the existence of adherens-junction-mediated signaling, we used peroxovanadate to tyrosine phosphorylate plakoglobin and beta-catenin and to dissociate adherens junctions. The distribution of plakoglobin and beta-catenin was determined by immunofluorescence, western blot analysis, pulse-chase radiolabeling, and biochemical subcellular fractionation. Coimmunoprecipitation studies from nuclear fractions, gel-shift assays, and transient transfections with T cell factor (TCF)/lymphoid enhancer factor (LEF) optimized promoter reporter constructs were used to investigate the ability of plakoglobin and beta-catenin that had redistributed from the membrane to the nucleus to form functional transcriptional regulatory complexes with TCF/LEF family member transcription factors. Tyrosine phosphorylation of plakoglobin and beta-catenin resulted in their rapid translocation from the cell membrane to the nucleus. Nuclear translocation was associated with increased plakoglobin and decreased beta-catenin binding to nuclear TCF/LEF and downregulation of gene transcription from TCF/LEF reporter constructs. These results are consistent with a signaling pathway initiated by structural changes in the adherens junction in which adherens-junction-derived plakoglobin regulates nuclear transcription by antagonizing the binding of beta-catenin to TCF/LEF proteins.
Assuntos
Junções Aderentes/fisiologia , Núcleo Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Queratinócitos/fisiologia , Transdução de Sinais/fisiologia , Transporte Biológico , Membrana Celular/metabolismo , Técnicas de Cultura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desmoplaquinas , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide , Fosforilação , Regiões Promotoras Genéticas , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Tirosina/metabolismo , Vanadatos/farmacologia , beta Catenina , gama CateninaRESUMO
Antineutrophil cytoplasmic autoantibodies (ANCA) are the likely cause for necrotizing small-vessel vasculitis and crescentic glomerulonephritis. Unlike other forms of crescentic glomerulonephritis induced by immune complexes or anti-glomerular basement membrane antibodies that have conspicuous vessel wall immunoglobulin and complement, there is a paucity, although usually not an absence, of vessel wall immunoglobulin and complement in ANCA-associated glomerulonephritis. Despite this comparatively lower level and more localized distribution of vessel wall complement, experimental and clinical observations strongly incriminate alternative complement pathway activation as critically important in the pathogenesis of ANCA disease. Experimental data in animal models and in vitro experiments has shown that primed neutrophils are activated by ANCA, which generates C5a, which engages C5a receptors on neutrophils. This attracts and in turn primes more neutrophils for activation by ANCA. In patients with ANCA disease, plasma levels of C3a, C5a, soluble C5b-9, and Bb have been reported to be higher in active disease than in remission, whereas no difference was reported in plasma C4d in active versus ANCA disease remission. Thus, experimental and clinical data support the hypothesis that ANCA-induced neutrophil activation activates the alternative complement pathway and generates C5a. C5a not only recruits additional neutrophils through chemotaxis but also primes neutrophils for activation by ANCA. This creates a self-fueling inflammatory amplification loop that results in the extremely destructive necrotizing vascular injury.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/metabolismo , Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Via Alternativa do Complemento/fisiologia , Proteínas do Sistema Complemento/metabolismo , Glomerulonefrite/metabolismo , Imunoglobulinas/metabolismo , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Modelos Animais de Doenças , Glomerulonefrite/imunologia , Humanos , CamundongosRESUMO
Clinical, in vitro, and experimental animal observations indicate that antineutrophil cytoplasmic autoantibodies (ANCA) are pathogenic. The genesis of the ANCA autoimmune response is a multifactorial process that includes genetic predisposition, environmental adjuvant factors, an initiating antigen, and failure of T cell regulation. ANCA activate primed neutrophils (and monocytes) by binding to certain antigens expressed on the surface of neutrophils in specific inflammatory microenvironments. ANCA-activated neutrophils activate the alternative complement pathway, establishing an inflammatory amplification loop. The acute injury elicits an innate inflammatory response that recruits monocytes and T lymphocytes, which replace the neutrophils that have undergone karyorrhexis during acute inflammation. Extravascular granulomatous inflammation may be initiated by ANCA-induced activation of extravascular neutrophils, causing tissue necrosis and fibrin formation, which would elicit an influx of monocytes that transform into macrophages and multinucleated giant cells. Over time, the neutrophil-rich acute necrotizing lesions cause the accumulation of more lymphocytes, monocytes, and macrophages and produce typical granulomatous inflammation.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Autoanticorpos/imunologia , Animais , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Neutrófilos/imunologia , Neutrófilos/patologiaRESUMO
Application of adeno-associated virus (AAV) vector in large animal studies and clinical trials often requires high-titer and high-potency vectors. A number of currently used vector production methods, based on either transient transfection or helper virus infection of cell lines, have their advantages and limitations. We previously developed a 293-cell-based producer cell line method for high-titer and high-potency AAV2 vectors. Similar to several other methods, however, it requires multiple cloning steps for the vector and packaging plasmids and a two-step transfection and selection for stable cell lines. Here we report a simplified method with several key improvements and advantages: (1) a one-step cloning of AAV vector cassette into the serotype-specific packaging plasmid; (2) a single plasmid transfection and selection for stable AAV vector producer cell lines; (3) high vector yields of different serotypes, e.g., AAV2, 8, and 9, upon infection with an E1A/E1B-deleted helper adenovirus; (4) efficient packaging of both single-stranded and double-stranded (self-complementary) AAV vectors; and (5) efficient packaging of large AAV cassettes such as a mini-dystrophin vector (5.0 kb). All cell lines were stable with growth rates identical to the parental 293 cells. The vector yields were consistent among serotypes, with 5 × 10(13) to 8 × 10(13) vector genome particles per Nunc cell factory (equivalent to 40 15-cm plates). The vectors showed high potency for in vitro and in vivo transduction. In conclusion, the simple and versatile AAV producer cell line method can be useful for large scale AAV vector production in preclinical and clinical studies.
Assuntos
Biotecnologia/métodos , Linhagem Celular , Dependovirus , Vetores Genéticos/genética , Técnicas de Cultura de Células , Clonagem Molecular , Humanos , Plasmídeos/genética , Transfecção/métodosRESUMO
O-Glycosylation modifies and regulates a variety of intracellular proteins. Plakoglobin, which functions in both cell-cell adhesion and signal transduction, is modified by O-glycosylation; however, the significance is unknown. To investigate the functional consequence of plakoglobin O-glycosylation, we cloned and overexpressed in keratinocytes murine O-GlcNAc transferase (mOGT). Over expression of mOGT in murine keratinocytes resulted in (i) glycosylation of plakoglobin and (ii) increased levels of plakoglobin due to post-translational stabilization of plakoglobin. Additionally, overexpression of mOGT in keratinocytes correlated with increased staining for cell-cell adhesion proteins and greater cell-cell adhesion. These observations suggest that O-glycosylation functions to regulate the post-translational stability of plakoglobin and keratinocyte cell-cell adhesion.
Assuntos
Queratinócitos/metabolismo , gama Catenina/fisiologia , Animais , Catálise , Adesão Celular , Linhagem Celular , Proteínas do Citoesqueleto/química , Detergentes/farmacologia , Glicosilação , Camundongos , Fosfoproteínas Fosfatases/metabolismo , gama Catenina/químicaRESUMO
Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering skin disease characterized by detachment of keratinocytes (acantholysis). It has been proposed that PV IgG might trigger signaling and that this process may lead to acantholysis. Indeed, we recently identified a rapid and dose-dependent phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and heat shock protein (HSP) 27 after binding of PV antibodies to cultured keratinocytes. In human keratinocyte cultures, inhibitors of p38MAPK prevented PV IgG-induced phosphorylation of HSP27 and, more importantly, prevented the early cytoskeletal changes associated with loss of cell-cell adhesion. This study was undertaken to (i) determine whether p38MAPK and HSP25, the murine HSP27 homolog, were similarly phosphorylated in an in vivo model of PV and (ii) investigate the potential therapeutic use of p38MAPK inhibition to block blister formation in an animal model of PV. We now report that p38MAPK inhibitors prevented PV blistering disease in vivo. Targeting the end-organ by inhibiting keratinocyte desmosome signaling may be effective for treating desmosome autoimmune blistering disorders.