RESUMO
OBJECTIVES: In the post-SARS-CoV-2 pandemic era, "breakthrough infections" are still documented, due to variants of concerns (VoCs) emergence and waning humoral immunity. Despite widespread utilization, the definition of the anti-Spike (S) immunoglobulin-G (IgG) threshold to define protection has unveiled several limitations. Here, we explore the advantages of incorporating T-cell response assessment to enhance the definition of immune memory profile. METHODS: SARS-CoV-2 interferon-gamma release assay test (IGRA) was performed on samples collected longitudinally from immunocompetent healthcare workers throughout their immunization by infection and/or vaccination, anti-receptor-binding domain IgG levels were assessed in parallel. The risk of symptomatic infection according to cellular/humoral immune capacities during Omicron BA.1 wave was then estimated. RESULTS: Close to 40% of our samples were exclusively IGRA-positive, largely due to time elapsed since their last immunization. This suggests that individuals have sustained long-lasting cellular immunity, while they would have been classified as lacking protective immunity based solely on IgG threshold. Moreover, the Cox regression model highlighted that Omicron BA.1 circulation raises the risk of symptomatic infection while increased anti-receptor-binding domain IgG and IGRA levels tended to reduce it. CONCLUSION: The discrepancy between humoral and cellular responses highlights the significance of assessing the overall adaptive immune response. This integrated approach allows the identification of vulnerable subjects and can be of interest to guide antiviral prophylaxis at an individual level.
Assuntos
Anticorpos Antivirais , COVID-19 , Imunidade Humoral , Imunoglobulina G , Memória Imunológica , Testes de Liberação de Interferon-gama , SARS-CoV-2 , Humanos , COVID-19/imunologia , SARS-CoV-2/imunologia , Memória Imunológica/imunologia , Imunidade Humoral/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Testes de Liberação de Interferon-gama/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Linfócitos T/imunologia , Pessoal de Saúde , Vacinas contra COVID-19/imunologiaRESUMO
In sepsis, personalized immunotherapy is being evaluated as a means of restoring immune function in the most severely affected patients. Biomarkers play a crucial role in this process, as there are no clear clinical indicators of immune dysfunction. Functional testing is considered a gold standard for assessing immune function, but this approach faces analytical challenges in terms of clinical implementation. The use of technician-dependent, time-consuming, home-made protocols often leads to poor standardization. This study represents the first beta testing of a fully automated interferon-γ release assay (IGRA) for monitoring the functionality of antigen-independent T lymphocytes. We observed a significant decrease in IFN-γ release capacity, which was associated with typical alterations in immunological cellular parameters (such as low mHLA-DR expression and decreased CD8 T lymphocyte count), in 22 patients with septic shock. Since the test is performed using whole blood and requires no technician intervention, with results available within 4 h, it may offer new possibilities for monitoring patients with immune alterations in routine clinical conditions. Further investigations in larger cohorts of patients are now needed to validate its clinical potential.
Assuntos
Sepse , Choque Séptico , Humanos , Testes de Liberação de Interferon-gama , Estudo de Prova de Conceito , Sepse/metabolismo , Linfócitos T CD8-Positivos/metabolismoRESUMO
SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols.
Assuntos
COVID-19 , Vacinas Virais , Dissulfetos , Células HEK293 , Humanos , Mutação , Isomerases de Dissulfetos de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Antigen-specific T-cells are essential for protective immunity against SARS-CoV-2. We set up a semi-automated whole-blood Interferon-gamma release assay (WB IGRA) to monitor the T-cell response after stimulation with SARS-CoV-2 peptide pools. We report that the WB IGRA is complementary to serological assays to assess SARS-CoV-2 immunity.
Assuntos
COVID-19/imunologia , Interferon gama/metabolismo , Células T de Memória/imunologia , SARS-CoV-2/fisiologia , Adulto , Automação Laboratorial , Células Cultivadas , Estudos de Coortes , Feminino , Humanos , Testes de Liberação de Interferon-gama/normas , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Especificidade do Receptor de Antígeno de Linfócitos T , Imagem Corporal Total , Adulto JovemRESUMO
Rationale: Decreased expression and activity of endothelial nitric oxide synthase (eNOS) in response to inflammatory and metabolic insults is the hallmark of endothelial cell (EC) dysfunction that preludes the development of atherosclerosis and hypertension. We previously reported the atheroprotective properties of the ubiquitin-editing and anti-inflammatory protein A20, also known as TNFAIP3, in part through interrupting nuclear factor-kappa B (NF-κB) and interferon signaling in EC and protecting these cells from apoptosis. However, A20's effect on eNOS expression and function remains unknown. In this study, we evaluated the impact of A20 overexpression or knockdown on eNOS expression in EC, at baseline and after tumor necrosis factor (TNF) treatment, used to mimic inflammation. Methods and Results: A20 overexpression in human coronary artery EC (HCAEC) significantly increased basal eNOS mRNA (qPCR) and protein (western blot) levels and prevented their downregulation by TNF. Conversely, siRNA-induced A20 knockdown decreased eNOS mRNA levels, identifying A20 as a physiologic regulator of eNOS expression. By reporter assays, using deletion and point mutants of the human eNOS promoter, and knockdown of eNOS transcriptional regulators, we demonstrated that A20-mediated increase of eNOS was transcriptional and relied on increased expression of the transcription factor Krüppel-like factor (KLF2), and upstream of KLF2, on activation of extracellular signal-regulated kinase 5 (ERK5). Accordingly, ERK5 knockdown or inhibition significantly abrogated A20's ability to increase KLF2 and eNOS expression. In addition, A20 overexpression in HCAEC increased eNOS phosphorylation at Ser-1177, which is key for the function of this enzyme. Conclusions: This is the first report demonstrating that overexpression of A20 in EC increases eNOS transcription in an ERK5/KLF2-dependent manner and promotes eNOS activating phosphorylation. This effect withstands eNOS downregulation by TNF, preventing EC dysfunction in the face of inflammation. This novel function of A20 further qualifies its therapeutic promise to prevent/treat atherosclerosis.
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The COVID-19 pandemic, caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to spread worldwide. Serological testing for SARS-CoV-2-specific antibodies plays an important role in understanding and controlling the pandemic, notably through epidemiological surveillance. Well-validated and highly specific SARS-CoV-2 serological assays are urgently needed. We describe here the analytical and clinical performance of Vidas SARS-CoV-2 IgM and Vidas SARS-CoV-2 IgG, two CE-marked, emergency use authorization (EUA)-authorized, automated, qualitative assays for the detection of SARS-CoV-2-specific IgM and IgG, respectively. Both assays showed high within-run and within-laboratory precision (coefficients of variation < 11.0%) and very low cross-reactivity toward sera of patients with a past common coronavirus or respiratory virus infection. Clinical specificity determined on up to 989 prepandemic healthy donors was ≥99% with a narrow 95% confidence interval for both IgM and IgG assays. Clinical sensitivity was determined on up to 232 samples from 130 reverse transcriptase PCR (RT-PCR)-confirmed SARS-CoV-2 patients. The positive percent agreement (PPA) with SARS-CoV-2 PCR reached 100% at ≥16 days (Vidas SARS-CoV-2 IgM) and ≥32 days (Vidas SARS-CoV-2 IgG) of symptom onset. Combined IgM/IgG test results improved the PPA compared to each test alone. SARS-CoV-2 IgG seroconversion followed closely that of SARS-CoV-2 IgM and remained stable over time, while SARS-CoV-2 IgM levels rapidly declined. Interestingly, SARS-CoV-2-specific IgM and IgG responses were significantly higher in COVID-19 hospitalized versus nonhospitalized patients. Altogether, the Vidas SARS-CoV-2 IgM and IgG assays are highly specific and sensitive serological tests suitable for the reliable detection of past acute SARS-CoV-2 infections.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Humanos , Imunoglobulina G , Imunoglobulina M , Pandemias , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Several unautomated anti-HEV diagnostic tests are presently available. OBJECTIVE: We have evaluated the performance of the new automated VIDAS® ANTI-HEV IgM and IgG assays. STUDY DESIGN: We assessed the reproducibility and cross-reactivity of both VIDAS assays and the analytical sensitivity and linearity of the VIDAS IgG assay. We also tested the VIDAS and comparator assays Wantai IgG and IgM on immunocompetent and immunocompromised patients. Data were analysed according to the infectious profile, with samples from viremic phase (HEV RNA/IgM positive) and post-viremic phase (HEV RNA negative, IgM positive) infections, and uninfected patients (HEV RNA/IgM negative). RESULTS: Within-run reproducibility was <10% and between-run reproducibility was <12% for both assays. We found no cross-reactivity, except for the VIDAS IgG assay in some patients with HBV (1/10) or malaria (3/23) infections and for the VIDAS IgM assay in some HIV-infected patients (1/10). The VIDAS IgG assay was linear over 0.10-10.0 U/mL. Analytical sensitivity of the IgG assay was 0.71 IU/ml (probit analysis). The clinical sensitivity of the VIDAS IgM assay was 97.65% for viremic samples (83/85) and 59.15% (42/71) for post-viremic samples from immunocompetent patients. It was 78.95% (45/57) for acute phase samples and 77.78% (28/36) for post-viremic samples from immunocompromised patients. Specificity was excellent (>99%) in both populations. CONCLUSION: The analytical and clinical performance of the new VIDAS® ANTI-HEV assays was excellent. These rapid, automated assays for detecting HEV antibodies will strengthen the arsenal for diagnosing HEV infections.
Assuntos
Automação Laboratorial/normas , Anticorpos Anti-Hepatite/sangue , Hepatite E/diagnóstico , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Kit de Reagentes para Diagnóstico/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Reações Cruzadas , Feminino , Vírus da Hepatite E/imunologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Testes Sorológicos/normas , Adulto JovemRESUMO
Dengue is a mosquito-borne disease caused by four genetically and serologically related viruses that affect several millions of people. Envelope domain III (EDIII) of the viral envelope protein contains dengue virus (DENV) type-specific and DENV complex-reactive antigenic sites. Here, we describe the expression in Escherichia coli, the refolding and bio-structural analysis of envelope domain III of the four dengue serotypes as a tetravalent dengue protein (EDIIIT2), generating an attractive diagnostic candidate. In vitro refolding of denatured EDIIIT2 was performed by successive dialysis with decreasing concentrations of chaotropic reagent and in the presence of oxidized glutathione. The efficiency of refolding was demonstrated by protein mobility shifting and fluorescent visualization of labeled cysteine in non-reducing SDS-PAGE. The identity and the fully oxidized state of the protein were verified by mass spectrometry. Analysis of the structure by fluorescence, differential scanning calorimetry and circular dichroism showed a well-formed structural conformation mainly composed of ß-strands. A label-free immunoassay based on biolayer interferometry technology was subsequently used to evaluate antigenic properties of folded EDIIIT2 protein using a panel of dengue IgM positive and negative human sera. Our data collectively support the use of an oxidatively refolded EDIIIT2 recombinant chimeric protein as a promising antigen in the serological diagnosis of dengue virus infections.
Assuntos
Anticorpos Antivirais , Antígenos Virais , Vírus da Dengue/genética , Dengue , Epitopos , Imunoglobulina M , Proteínas Virais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/biossíntese , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , Dengue/sangue , Dengue/diagnóstico , Dengue/imunologia , Vírus da Dengue/imunologia , Vírus da Dengue/metabolismo , Epitopos/biossíntese , Epitopos/genética , Epitopos/imunologia , Epitopos/isolamento & purificação , Humanos , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/isolamento & purificaçãoRESUMO
BACKGROUND: Transplant arteriosclerosis (TA) is the pathognomonic feature of chronic rejection, the primary cause of allograft failure. We have shown that the NF-κB inhibitory protein A20 exerts vasculoprotective effects in endothelial and smooth muscle cells (SMC), and hence is a candidate to prevent TA. We sought direct proof for this hypothesis. METHODS: Fully mismatched, C57BL/6 (H2) into BALB/c (H2), aorta to carotid allografts were preperfused with saline, recombinant A20 adenovirus (rAd.A20) or rAd.ß-galactosidase (ß-gal), implanted, harvested 4 weeks after transplantation, and analyzed by histology, immunohistochemistry, and immunofluorescence staining. We measured indoleamine 2,3-dioxygenase, interleukin-6, and transforming growth factor-ß mRNA and protein levels in nontransduced, and rAd.A20 or rAd.ß-gal-transduced human SMC cultures after cytokine treatment. RESULTS: Vascular overexpression of A20 significantly reduced TA lesions. This correlated with decreased graft inflammation and increased apoptosis of neointimal SMC. Paradoxically, T-cell infiltrates increased in A20-expressing allografts, including the immunoprivileged media, which related to A20 preventing indoleamine 2,3-dioxygenase upregulation in SMC. However, infiltrating T cells were predominantly T-regulatory cells (CD25+/Forkhead Box P3 [FoxP3+]). This agrees with A20 inhibiting interleukin-6 and promoting transforming growth factor-ß production by medial SMC and in SMC cultures exposed to cytokines, which favors differentiation of regulatory over pathogenic T cells. CONCLUSIONS: In summary, A20 prevents immune-mediated remodeling of vascular allografts, therefore reduces TA lesions by affecting apoptotic and inflammatory signals and modifying the local cytokine milieu to promote an immunoregulatory response within the vessel wall. This highlights a novel function for A20 in local immunosurveillance, which added to its vasculoprotective effects, supports its therapeutic promise in TA.
Assuntos
Aorta/transplante , Arteriosclerose/imunologia , Rejeição de Enxerto/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adenoviridae/genética , Animais , Aorta/metabolismo , Aorta/patologia , Apoptose , Arteriosclerose/complicações , Arteriosclerose/metabolismo , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Artérias Carótidas/cirurgia , Células Cultivadas , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Citocinas/metabolismo , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Transplante Homólogo , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
Inflammation induces the NF-κB dependent protein A20 in human renal proximal tubular epithelial cells (RPTEC), which secondarily contains inflammation by shutting down NF-κB activation. We surmised that inducing A20 without engaging the pro-inflammatory arm of NF-κB could improve outcomes in kidney disease. We showed that hepatocyte growth factor (HGF) increases A20 mRNA and protein levels in RPTEC without causing inflammation. Upregulation of A20 by HGF was NF-κB/RelA dependent as it was abolished by overexpressing IκBα or silencing p65/RelA. Unlike TNFα, HGF caused minimal IκBα and p65/RelA phosphorylation, with moderate IκBα degradation. Upstream, HGF led to robust and sustained AKT activation, which was required for p65 phosphorylation and A20 upregulation. While HGF treatment of RPTEC significantly increased A20 mRNA, it failed to induce NF-κB dependent, pro-inflammatory MCP-1, VCAM-1, and ICAM-1 mRNA. This indicates that HGF preferentially upregulates protective (A20) over pro-inflammatory NF-κB dependent genes. Upregulation of A20 supported the anti-inflammatory effects of HGF in RPTEC. HGF pretreatment significantly attenuated TNFα-mediated increase of ICAM-1, a finding partially reversed by silencing A20. In conclusion, this is the first demonstration that HGF activates an AKT-p65/RelA pathway to preferentially induce A20 but not inflammatory molecules. This could be highly desirable in acute and chronic renal injury where A20-based anti-inflammatory therapies are beneficial.
Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Inflamação/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Células Cultivadas , Proteínas de Ligação a DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Túbulos Renais Proximais/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: Neointimal hyperplasia is an inflammatory and proliferative process that occurs as a result of injury to the vessel wall. We have shown that the homeostatic protein A20 prevents neointimal hyperplasia by affecting endothelial cell (EC) and smooth muscle cell (SMC) responses to injury. In this work, we questioned whether A20 impacts other pathogenic effectors of neointimal hyperplasia including homing of monocyte/macrophages and EC/SMC precursors to the site of vascular injury, vascular endothelial growth factor (VEGF) secretion, and adventitial neovascularization. METHODS AND RESULTS: Carotid balloon angioplasty was performed on rat recipients of a bone marrow transplant from green fluorescent rats. Adenoviral delivery of A20 prevented neointimal hyperplasia and decreased macrophage infiltration. This was associated with decreased ICAM-1 and MCP-1 expression in vitro. Additionally, A20 reduced neovascularization in the adventitia of balloon injured carotid arteries, which correlated with fewer VEGF positive cells. CONCLUSIONS: A20 downregulates adhesion markers, chemokine production, and adventitial angiogenesis, all of which are required for macrophage trafficking to sites of vascular injury. This, in turn, diminishes the inflammatory milieu to prevent neointimal hyperplasia.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/citologia , Proteínas Nucleares/metabolismo , Animais , Células da Medula Óssea/citologia , Movimento Celular , Regulação para Baixo , Endotélio Vascular/patologia , Humanos , Hiperplasia/patologia , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Neointima/patologia , Neovascularização Patológica , Ratos , Ratos Sprague-Dawley , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Células U937 , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The nuclear factor-kappaB inhibitory protein A20 demonstrates hepatoprotective abilities through combined antiapoptotic, anti-inflammatory, and pro-proliferative functions. Accordingly, overexpression of A20 in the liver protects mice from toxic hepatitis and lethal radical hepatectomy, whereas A20 knockout mice die prematurely from unfettered liver inflammation. The effect of A20 on oxidative liver damage, as seen in ischemia/reperfusion injury (IRI), is unknown. In this work, we evaluated the effects of A20 upon IRI using a mouse model of total hepatic ischemia. Hepatic overexpression of A20 was achieved by recombinant adenovirus (rAd.)-mediated gene transfer. Although only 10%-25% of control mice injected with saline or the control rAd.beta galactosidase survived IRI, the survival rate reached 67% in mice treated with rAd.A20. This significant survival advantage in rAd.A20-treated mice was associated with improved liver function, pathology, and repair potential. A20-treated mice had significantly lower bilirubin and aminotransferase levels, decreased hemorrhagic necrosis and steatosis, and increased hepatocyte proliferation. A20 protected against liver IRI by increasing hepatic expression of peroxisome proliferator-activated receptor alpha (PPARalpha), a regulator of lipid homeostasis and of oxidative damage. A20-mediated protection of hepatocytes from hypoxia/reoxygenation and H(2)O(2)-mediated necrosis was reverted by pretreatment with the PPARalpha inhibitor MK886. In conclusion, we demonstrate that PPARalpha is a novel target for A20 in hepatocytes, underscoring its novel protective effect against oxidative necrosis. By combining hepatocyte protection from necrosis and promotion of proliferation, A20-based therapies are well-poised to protect livers from IRI, especially in the context of small-for-size and steatotic liver grafts. Liver Transpl 15:1613-1621, 2009. (c) 2009 AASLD.
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Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Cisteína Endopeptidases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transplante de Fígado , PPAR alfa/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Animais , Divisão Celular/fisiologia , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Cisteína Endopeptidases/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Fígado Gorduroso/fisiopatologia , Galactosamina/toxicidade , Expressão Gênica/fisiologia , Hepatectomia , Hepatócitos/citologia , Hepatócitos/fisiologia , Peróxido de Hidrogênio/toxicidade , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipopolissacarídeos/toxicidade , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/patologia , Falência Hepática Aguda/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Necrose , Oxidantes/toxicidade , PPAR alfa/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
The pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is unknown but is most consistent with a primary role for neutrophils in the acute injury. Thus, neutrophils are cardinal cells in the pathophysiological process in AAV because they are both effector cells responsible for endothelial damage and targets of autoimmunity. In addition, because of their capacity to synthesize a wide variety of cytokines and chemokines, neutrophils can be considered as important modulators of the inflammatory and potentially of the autoimmune process. ANCA directed against two main autoantigens, namely proteinase 3 and myeloperoxidase, are likely to play a modulatory role in the inflammatory process. Interestingly, neutrophils are an important source of lymphocyte stimulator (BLy), a cytokine that plays a fundamental role in B-cell physiology, including differentiation, proliferation and immunoglobulin production. The issue of B-cell activation and/or dysregulation in vasculitis will be discussed.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/fisiologia , Linfócitos B/fisiologia , Neutrófilos/fisiologia , Vasculite/etiologia , Anticorpos Anticitoplasma de Neutrófilos/sangue , Apoptose , Fator Ativador de Células B/fisiologia , Humanos , Mieloblastina/imunologiaRESUMO
Because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. Using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. Coronin-1 is an actin-binding protein that can associate with phagosomes and NADPH oxidase, but its involvement in apoptosis was currently unknown. In coronin-1-transfected PLB985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by CD11b labeling. Concerning apoptosis, increased coronin-1 expression in dimethylformamide-differentiated PLB985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. Likewise, coronin-1 significantly decreased TRAIL-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or Bid truncation suggesting that coronin-1 interfered with mitochondria-related events. To validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (CF) patients were studied. Circulating neutrophils from CF patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. This was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. In addition, inflammatory neutrophils from CF patients lungs showed an intense coronin-1 immunolabeling. We concluded that coronin-1 could constitute a potential target in resolving inflammation.
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Apoptose , Proteínas dos Microfilamentos/análise , Neutrófilos/citologia , Sobrevivência Celular , Fibrose Cística/patologia , Citosol/química , Humanos , Hidrólise , Inflamação , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Mitocôndrias/fisiologia , Neutrófilos/patologia , ProteômicaRESUMO
Macrophages are the principal source of TNFalpha, yet they are highly resistant to TNFalpha-mediated cell death. Previously, employing in vitro differentiated human macrophages, we showed that following the inhibition of NF-kappaB, TNFalpha-induced caspase-8 activation contributes to DNA fragmentation but is not necessary for the loss of the inner mitochondrial transmembrane potential (DeltaPsim) or cell death. We here extend these observations to demonstrate that, when NF-kappaB is inhibited in macrophages, TNFalpha alters lysosomal membrane permeability (LMP). This results in the release of cathepsin B with subsequent loss of DeltaPsim and caspase-8 independent cell death. Interestingly, the cytoprotective, NF-kappaB-dependent protein A20 was rapidly induced in macrophages treated with TNFalpha. Ectopic expression of A20 in macrophages preserves LMP following treatment with TNFalpha, and as a result, mitochondrial integrity is safeguarded and macrophages are protected from cell death. These observations demonstrate that TNFalpha triggers both caspase 8-dependent and -independent cell death pathways in macrophages and identify a novel mechanism by which A20 protects these cells against both pathways.
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Apoptose/fisiologia , Caspase 8/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Proteínas Nucleares/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Apoptose/efeitos dos fármacos , Catepsina B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA , Vetores Genéticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Monócitos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Proteínas Nucleares/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transfecção , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Gene silencing achieved through small interfering RNA (siRNA) transfection represents a promising approach to vascular gene therapy. Here we characterize the behavior of RNA interference (RNAi) in vascular biology by comparing the RNAi response to single- and multigene siRNA transfections in vitro in human vascular cells. STUDY DESIGN: The strength and specificity of multigene silencing in cultured human coronary artery smooth muscle and human coronary artery endothelial cells (HCASMC/HCAEC) were assessed by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and Western blot after transfection singly or simultaneously with siRNAs targeting glyceraldehyde-3-phosphate dehydrogenase, the myristoylated alanine-rich C kinase substrate, and cadherin 11. RNAi response to low-dose (0.25 to 10 nM) siRNA transfection was characterized between the two cell types by QRT-PCR and fluorescence-activated cell sorter analysis. RESULTS: Powerful and specific silencing of all targets was observed in both cell types after multigene siRNA transfections, but with a reduction in effect compared with single-gene siRNA transfections. Multigene messenger RNA (mRNA) reductions in HCAECs exceeded those achieved in HCASMCs, and superior mRNA silencing and siRNA delivery were observed in HCAECs after low-dose siRNA transfections. CONCLUSIONS: Multigene silencing by siRNA stands as a promising nonviral approach for manipulating gene expression in human vascular cells. Under our in vitro conditions, endothelial cells were more susceptible to siRNA transfection and gene silencing than vascular smooth muscle cells. RNAi technology could potentially find use in the development of siRNA cocktails for application to vein bypass grafts or for modulating endothelial cell function in other forms of vascular disease.
Assuntos
Endotélio Vascular/citologia , Inativação Gênica , Músculo Liso Vascular/citologia , RNA Interferente Pequeno/genética , Western Blotting , Células Cultivadas , Vasos Coronários/citologia , Terapia Genética/métodos , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção/métodosRESUMO
OBJECTIVE: Intimal hyperplasia is a common cause of vein graft failure in cardiovascular surgery. The molecular basis for intimal hyperplasia remains poorly defined. We have previously identified, by gene chip analysis of vein grafts, increased messenger (mRNA) for the adhesion molecule cadherin 11/osteoblast-cadherin (CDH11). The function of CDH11 in vascular cells is unknown. The aim of the present study is to confirm CDH11 expression in vein grafts and characterize its role in vascular remodeling. METHODS: Cephalic vein interposition grafts were implanted in a canine model and harvested at predetermined time points. CDH11 protein expression was determined by immunohistochemistry. Early passage human coronary artery smooth muscle cells (SMCs) were used for in vitro studies. Real-time polymerase chain reaction was used to assess cellular CDH11 mRNA levels. CDH11 signaling was inhibited by either transfection with silencing RNA targeting CDH11 or with a blocking antibody to CDH11. Cellular migration was evaluated and cellular proliferation was assessed. RESULTS: Expression of CDH11 was increased in medial SMCs of vein grafts recovered at 7, 14, and 30 days after surgery compared with control veins from the same animals. In vitro CDH11 mRNA was up-regulated 1.8 +/- 0.2-fold (P = .003) in SMCs after treatment with tumor necrosis factor-alpha. Cellular migration was attenuated by inhibition of CDH11 both with a blocking antibody (0.67 +/- 0.09; P = .063) and gene knockdown mediated by small interfering RNA (0.67 +/- 0.14; P = .036). SMC proliferation decreased by 3.1-fold (P = .006) in the presence of CDH11-blocking antibody. Knockdown of CDH11 mediated by small interfering RNA resulted in a 1.3-fold (P = .018) decrease in proliferation. CONCLUSIONS: CDH11 is up-regulated in SMC in vivo and in vitro as part of the response to injury. Inhibition of CDH11 decreases SMC migration and proliferation, two pathogenic effectors of intimal hyperplasia.
Assuntos
Caderinas/biossíntese , Movimento Celular , Proliferação de Células , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Anastomose Cirúrgica , Animais , Anticorpos/farmacologia , Caderinas/genética , Caderinas/imunologia , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/metabolismo , Vasos Coronários/fisiopatologia , Cães , Artéria Femoral/cirurgia , Humanos , Hiperplasia , Modelos Animais , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima , Veias/metabolismo , Veias/fisiopatologia , Veias/transplanteRESUMO
A20 is a NF-kappaB-dependent gene that has dual anti-inflammatory and antiapoptotic functions in endothelial cells (EC). The function of A20 in smooth muscle cells (SMC) is unknown. We demonstrate that A20 is induced in SMC in response to inflammatory stimuli and serves an anti-inflammatory function via blockade of NF-kappaB and NF-kappaB-dependent proteins ICAM-1 and MCP-1. A20 inhibits SMC proliferation via increased expression of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Surprisingly, A20 sensitizes SMC to cytokine- and Fas-mediated apoptosis through a novel NO-dependent mechanism. In vivo, adenoviral delivery of A20 to medial rat carotid artery SMC after balloon angioplasty prevents neointimal hyperplasia by blocking SMC proliferation and accelerating re-endothelialization, without causing apoptosis. However, expression of A20 in established neointimal lesions leads to their regression through increased apoptosis. This is the first demonstration that A20 exerts two levels of control of vascular remodeling and healing. A20 prevents neointimal hyperplasia through combined anti-inflammatory and antiproliferative functions in medial SMC. If SMC evade this first barrier and neointima is formed, A20 has a therapeutic potential by uniquely sensitizing neointimal SMC to apoptosis. A20-based therapies hold promise for the prevention and treatment of neointimal disease.
Assuntos
Hiperplasia/prevenção & controle , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , NF-kappa B/antagonistas & inibidores , Proteínas/fisiologia , Proteínas/uso terapêutico , Túnica Íntima/patologia , Adenoviridae , Animais , Aorta , Apoptose , Ciclo Celular , Divisão Celular , Primers do DNA , Proteínas de Ligação a DNA , Endotélio Vascular/fisiologia , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Molécula 1 de Adesão Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Doenças Vasculares/prevenção & controleRESUMO
BACKGROUND: Apoptotic death of renal proximal tubular epithelial cells (RPTECs) is a feature of acute and chronic renal failure. RPTECs are directly damaged by ischemia, inflammatory, and cytotoxic mediators but also contribute to their own demise by up-regulating proinflammatory nuclear factor-kappaB (NF-kappaB)-dependent proteins. In endothelial cells, the Bcl family member A1 and the zinc finger protein A20 have redundant and dual antiapoptotic and anti-inflammatory effects. We studied the function(s) of A1 and A20 in human RPTECs in vitro. METHODS: Expression of A1 [reverse transcription-polymerase chain reaction (RT-PCR) and A20 (Northern and Western blot analysis)] in RPTECs was evaluated. A1 and A20 were overexpressed in RPTECs by recombinant adenoviral-mediated gene transfer. Their effect upon inhibitor of NFkappaB alpha (IkappaBalpha) degradation (Western blot), NF-kappaB nuclear translocation [electrophoretic mobility shift assay (EMSA)], up-regulation of intercellular adhesion molecule-1 (ICAM-1) [fluorescence-activated cell sorter (FACS)] and monocyte chemoattractant protein-1 (MCP-1) (Northern blot) and apoptosis [terminal deoxynucleotiddyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL)] and FACS analysis of DNA content) was determined. RESULTS: A1 and A20 were induced in RPTECs as part of the physiologic response to tumor necrosis factor (TNF). A20, but not A1, inhibited TNF-induced NF-kappaB activation by preventing IkappaBalpha degradation, hence subsequent up-regulation of the proinflammatory molecules ICAM-1 and MCP-1. Unexpectedly, A20 did not protect RPTECs from TNF and Fas-mediated apoptosis while A1 protected against both stimuli. Coexpression of A1 and A20 in RPTECs achieved additive anti-inflammatory and antiapoptotic cytoprotection. CONCLUSION: A1 and A20 exert differential cytoprotective effects in RPTECs. A1 is antiapoptotic. A20 is anti-inflammatory via blockade of NF-kappaB. We propose that A1 and A20 are both required for optimal protection of RPTECs from apoptosis (A1) and inflammation (A20) in conditions leading to renal damage.
Assuntos
Apoptose/fisiologia , Células Epiteliais/fisiologia , Túbulos Renais Proximais/fisiologia , Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Células Cultivadas , Quimiocina CCL2/genética , Proteínas de Ligação a DNA , Células Epiteliais/citologia , Expressão Gênica/fisiologia , Humanos , Proteínas I-kappa B/metabolismo , Molécula 1 de Adesão Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular , Túbulos Renais Proximais/citologia , Antígenos de Histocompatibilidade Menor , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Nefrite/patologia , Nefrite/fisiopatologia , Proteínas Nucleares , RNA Mensageiro/análise , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima , Receptor fas/metabolismoRESUMO
The liver has a remarkable regenerative capacity, allowing recovery following injury. Regeneration after injury is contingent on maintenance of healthy residual liver mass, otherwise fulminant hepatic failure (FHF) may arise. Understanding the protective mechanisms safeguarding hepatocytes and promoting their proliferation is critical for devising therapeutic strategies for FHF. We demonstrate that A20 is part of the physiological response of hepatocytes to injury. In particular, A20 is significantly upregulated in the liver following partial hepatectomy. A20 protects hepatocytes from apoptosis and ongoing inflammation by inhibiting NF-kappaB. Hepatic expression of A20 in BALB/c mice dramatically improves survival following extended and radical lethal hepatectomy. A20 expression in the liver limits hepatocellular damage hence maintains bilirubin clearance and the liver synthetic function. In addition, A20 confers a proliferative advantage to hepatocytes via decreased expression of the cyclin-dependent kinase inhibitor p21(waf1). In conclusion, A20 provides a proliferative advantage to hepatocytes. By combining anti-inflammatory, antiapoptotic and pro-proliferative functions, A20-based therapies could be beneficial in prevention and treatment of FHF.