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In this chapter, indirect enzyme-linked immunosorbent assays (ELISAs) to quantitatively measure autoantibodies directed to human natural and homocysteinylated alpha-1 antitrypsin (anti-AATA and anti-HAATA, respectively) in serum are described. The illustrated ELISA protocols are slightly different, since the two protein forms have different biochemical features and, consequently, different affinity for the matrix (polystyrene microplate wells), so that specific experimental conditions have to be performed for the quantification of the serum antibody recognition.These procedures can be carried out to evaluate the anti-AATA and the anti-HAATA levels, testing serum samples, for research use.
Assuntos
Autoanticorpos , Poliestirenos , Humanos , Ensaio de Imunoadsorção EnzimáticaRESUMO
BACKGROUND: Variant transthyretin-mediated amyloidosis (ATTR-v) is a well-characterized disease affecting the neurologic and cardiovascular systems. Patisiran has been approved for neurologic involvement as it reduces hepatic synthesis of transthyretin (TTR). Eye involvement is a lateonset feature increasing the risk of glaucoma and cataracts in patients. AIMS: The aim of this case series was to assess whether patisiran can effectively reduce TTR synthesis in such a barrier-protected organ as the eye. METHODS: Two patisiran-treated ATTR-v patients underwent serum and aqueous humor sampling to measure TTR levels detected by SDS-PAGE and immunoblotting. Serum samples were compared to healthy control (HC), whereas aqueous humor samples were compared to non-amyloidotic subjects affected by cataracts and glaucoma. RESULTS: Serum TTR levels representative of hepatic synthesis were sharply lower in treated patients if compared to the HC (-87.5% and -93.75%, respectively). Aqueous humor TTR levels showed mild-tono reduction in treated patients compared to non-amyloidotic subjects with cataracts (-34.9% and +8.1%, respectively) and glaucoma (-41.1% and -2.1%). CONCLUSION: Patisiran does not seem to be as effective in inhibiting ocular TTR synthesis as it is in inhibiting hepatic synthesis. Re-engineering the envelope could allow the drug to target RPE cells thus avoiding any ocular involvement.
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Catarata , Glaucoma , Humanos , Pré-Albumina , Projetos Piloto , Catarata/tratamento farmacológico , Glaucoma/tratamento farmacológicoRESUMO
The prostacyclin analogue iloprost is used to treat vascular alterations and digital ulcers, the early derangements manifesting in systemic sclerosis (SSc), an autoimmune disease leading to skin and organ fibrosis. Bioindicator(s) of SSc onset and progress are still lacking and the therapeutic approach remains a challenge. The T helper 1 (Th1) chemokine interferon (IFN)γ-induced protein 10 (IP-10/CXCL10) associates with disease progression and worse prognosis. Endothelial cells and fibroblasts, under Th1-dominance, release CXCL10, further enhancing SSc's detrimental status. We analyzed the effect of iloprost on CXCL10 in endothelial cells, dermal fibroblasts, and in the serum of SSc patients. Human endothelial cells and dermal fibroblasts activated with IFNγ/Tumor Necrosis Factor (TNF)α, with/without iloprost, were investigated for CXCL10 secretion/expression and for intracellular signaling cascade underlying chemokine release (Signal Transducer and Activator of Transcription 1, STAT1; Nuclear Factor kappa-light-chain-enhancer of activated B cells, NF-kB; c-Jun NH2-terminal kinase, JNK: Phosphatidyl-Inositol 3-kinase (PI3K)/protein kinase B, AKT; Extracellular signal-Regulated Kinase 1/2, ERK1/2). CXCL10 was quantified in sera from 25 patients taking iloprost, satisfying the American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) 2013 classification criteria for SSc, and in sera from 20 SSc sex/age-matched subjects without therapy, previously collected. In human endothelial cells and fibroblasts, iloprost targeted CXCL10, almost preventing IFNγ/TNFα-dependent cascade activation in endothelial cells. In SSc subjects taking iloprost, serum CXCL10 was lower. These in vitro and in vivo data suggest a potential role of iloprost to limit CXCL10 at local vascular/dermal and systemic levels in SSc and warrant further translational research aimed to ameliorate SSc understanding/management.
Assuntos
Iloprosta , Escleroderma Sistêmico , Quimiocina CXCL10/metabolismo , Quimiocinas/metabolismo , Células Endoteliais/metabolismo , Epoprostenol/metabolismo , Humanos , Iloprosta/metabolismo , Iloprosta/farmacologia , Iloprosta/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Belimumab (BLM) is a B lymphocyte stimulator (BLyS) inhibitor approved for the treatment of systemic lupus erythematosus (SLE). Autophagy is a cell survival mechanism involved in the pathogenesis of SLE. Citrullination is a post-translational modification catalyzed by peptidylarginine deiminase (PAD) enzymes. Autophagy and citrullination may generate neoepitopes, evoking an autoimmune response. No previous studies have investigated the connection of these processes, and how BLM could affect them, in SLE. Ex vivo autophagy and protein citrullination were analyzed by western blot in lysates from 26 SLE patients' PBMCs at baseline and after 2, 4, and 12 weeks of BLM administration, and from 16 healthy donors' PBMCs. Autophagic PBMCs were identified by the immunofluorescent detection of the autophagy-associated proteins LC3B (LC3 puncta) and LAMP-1. Autophagosome accumulation was evaluated in CD14- (PBLs) and CD14+ (monocytes) SLE cells. The presence of the BLyS receptors BAFF-R, BCMA, and TACI on SLE CD4+, CD8+ T cells and monocytes, as well as serum IL-18 levels, was also assessed. Following BLM administration, we observed a decrease in autophagy and citrullination, with a lowering of LC3-II, citrullinated vimentin, and PAD4 expression levels in PBMCs from SLE patients. LC3-II levels showed a correlation with the SLE Disease Activity Index 2000 (SLEDAI-2K) after 12 weeks of therapy. The LC3B/LAMP-1 analysis confirmed the reduction in autophagy. A lesser autophagosome accumulation occurred in PBLs and monocytes which, in turn, seemed to be the main cellular populations contributing to autophagy. A reduction in patients' serum IL-18 concentrations occurred. CD4+ and CD8+ cells weakly expressed BAFF receptors; monocytes expressed only BAFF-R. BLM could impact on autophagy and citrullination, offering an opportunity for a deeper understanding of these mechanisms in SLE, and a possible tool for the clinical management of SLE.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Autofagia , Citrulinação , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Adulto , Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD/metabolismo , Autofagia/efeitos dos fármacos , Receptor do Fator Ativador de Células B/metabolismo , Antígeno de Maturação de Linfócitos B/metabolismo , Biomarcadores/sangue , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citrulinação/efeitos dos fármacos , Feminino , Humanos , Interleucina-18/sangue , Leucócitos Mononucleares/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/patologia , Proteínas de Membrana Lisossomal/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismoRESUMO
BACKGROUND & AIMS: The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis. METHODS: Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice. RESULTS: Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression. CONCLUSIONS: THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.
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Indutores da Angiogênese/metabolismo , Colangiocarcinoma/etiologia , Linfangiogênese/efeitos dos fármacos , Trombospondina 1/farmacologia , Trombospondinas/farmacologia , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Colangiocarcinoma/fisiopatologia , Modelos Animais de Doenças , Camundongos , Proteômica/métodos , Proteômica/estatística & dados numéricos , Trombospondina 1/administração & dosagem , Trombospondinas/administração & dosagem , Microambiente Tumoral/efeitos dos fármacosRESUMO
OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune systemic inflammatory disease associated with a high prevalence of atherosclerosis. Endothelial dysfunction has emerged as a potentially valuable prognostic tool in predicting the development of atherosclerosis. Tumour necrosis factor (TNF) is the main cytokine involved in RA pathogenesis, exerting a pro-atherogenic role. TNF-inhibitors are effective treatments in RA, also improving endothelial function. Regarding this, no experimental data are known about the involvement of etanercept. We investigated the contribution of TNF to endothelial dysfunction and the effect of in vitro treatment with etanercept, with a special focus on autophagy and apoptosis pathways. METHODS: Autophagy and apoptosis were evaluated by Western blot and flow cytometry in EA.hy926 endothelial cells treated with TNF alone or in combination with etanercept for 24h. RESULTS: Blocking autophagy, TNF was able to induce endothelial cell apoptosis. Co-treatment with etanercept reverted this effect, up-regulating the autophagy pathway. CONCLUSIONS: Our results confirm the protective role of etanercept, by restoring autophagy on TNF-induced endothelial damage.
Assuntos
Células Endoteliais , Fator de Necrose Tumoral alfa , Apoptose , Autofagia , Células Endoteliais/metabolismo , Etanercepte/farmacologia , Humanos , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
BACKGROUND: ICIs have strongly improved the outcome of NSCLC patients. However, primary and secondary resistance occur during treatment in most of the patients, with several of them developing fast progressions. Autoantibodies can be related with a dysfunctional immune system, although their association with immune-based anti-cancer therapies has never been investigated. Moreover, so far no reliable predictive factor is currently available to aid in treatment selection. CD137+T-cells are largely known to be the anti-tumor activated effector cells, but they have never been associated with the response to immunotherapies. METHODS: Forty-two patients with metastatic NSCLC receiving anti-PD-1 ICIs at Sant'Andrea Hospital and Policlinico Umberto I, from June 2016 to September 2018 were enrolled. Circulating levels of IgM-Rheumatoid Factor were evaluated at baseline and correlated with patients clinical response following the anti-PD-1 treatment. IgM-RF interaction and effect on T-cells in vivo and in vitro were investigated. FINDINGS: IgM-RF in NSCLC patient sera strongly predicted the development of early progression to ICIs. Also, a significant reduction of progression-free survival rate in anti-PD-1 treated patients could be identified when patients were stratified based on IgM-RF positivity and titers. IgM-RF bound preferentially circulating naïve and central memory T-cells and a significant reduction of CD137+ anti-tumor T effector cells was found in IgM-RF positive patients. In addition, a higher percentage of CD137+T-cells in peripheral blood of NSCLC patients at baseline resulted as a strong independent prognostic factor for a better outcome in terms of PFS and OS after the anti-PD-1 treatment. Furthermore, T-cells exposed to IgM-RF showed a robust defect in their migratory ability in response to CCL19 chemokine. INTERPRETATION: In this study we showed that serum IgM-RF can be regarded as predictive factor for the development of early progression and prognostic factor of a reduced progression-free survival and overall-survival in anti-PD-1 treated NSCLC patients. The ability of IgM-RF to bind naïve and central memory T-cells and impair their migration could make account for the reduction of the tumor-reactive CD137+ T-cells population that may cause a non-effectiveness of these T-cells targeting drugs. FUNDINGS: AIRC, MIUR and Sapienza University of Rome.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoglobulina M/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Fator Reumatoide/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Idoso , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Progressão da Doença , Feminino , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoglobulina M/sangue , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Prognóstico , Estudos Retrospectivos , Fator Reumatoide/sangueRESUMO
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the novel coronavirus, causing coronavirus disease 2019 (COVID-19). During virus infection, several pro-inflammatory cytokines are produced, leading to the "cytokine storm." Among these, interleukin (IL)-6, tumor necrosis factor-α (TNF-α), and IL-1ß seem to have a central role in the progression and exacerbation of the disease, leading to the recruitment of immune cells to infection sites. Autophagy is an evolutionarily conserved lysosomal degradation pathway involved in different aspects of lymphocytes functionality. The involvement of IL-6, TNF-α, and IL-1ß in autophagy modulation has recently been demonstrated. Moreover, preliminary studies showed that SARS-CoV-2 could infect lymphocytes, playing a role in the modulation of autophagy. Several anti-rheumatic drugs, now proposed for the treatment of COVID-19, could modulate autophagy in lymphocytes, highlighting the therapeutic potential of targeting autophagy in SARS-CoV-2 infection.
RESUMO
BACKGROUND: Circulating endothelial progenitor cells (EPCs) are biologic markers of endothelial function. In patients with systemic lupus erythematosus (SLE), the numerical reduction and functional impairment of EPCs contribute to the endothelial dysfunction. Through ex vivo and in vitro studies, we aimed at evaluating the effects of B lymphocyte stimulator (BLyS) on EPC colonies and endothelial cells and also investigating BLyS receptor expression on these cells. METHODS: EPCs were isolated from peripheral blood mononuclear cells (PBMC). In order to evaluate their ability to form colonies, EPCs were cultured on fibronectin-coated dishes and incubated with BlyS alone or BlyS and belimumab. Apoptosis of EPCs and endothelial cell line EA.hy926 was evaluated after 6, 12, and 24 h of incubation with BLyS and after 6 h with BLyS and belimumab. The expression of B cell activating factor-receptor (BAFF-R), B cell maturation antigen (BCMA), and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI) on EPCs and EA.hy926 was analyzed by cytofluorimetry. RESULTS: The number of EPC colonies was lower in patients than in controls. Moreover, the colonies from SLE patients were poorly organized compared to controls; the addition of belimumab restored the colony structure. Incubation with BLyS induced apoptosis of EPCs and EA.hy926 that was inhibited by the co-incubation with belimumab. BAFF-R and BCMA were expressed on both EPCs and EA.hy926, while TACI was expressed only on EPCs. CONCLUSIONS: EPCs and endothelial cells preferentially express BAFF-R which could be involved in the pro-apoptotic effect of BlyS. Belimumab administration seems to restore the quantitative and qualitative changes of EPC colonies both ex vivo and in vitro.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Fator Ativador de Células B/farmacologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Receptor do Fator Ativador de Células B/metabolismo , Antígeno de Maturação de Linfócitos B/metabolismo , Contagem de Células , Linhagem Celular , Células Cultivadas , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Feminino , Humanos , Imunossupressores/farmacologia , Lúpus Eritematoso Sistêmico/patologia , Pessoa de Meia-Idade , Células-Tronco/efeitos dos fármacos , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismoRESUMO
BACKGROUND: Rheumatoid arthritis (RA) is associated with a high prevalence of atherosclerosis. Recently increased levels of microparticles (MPs) have been reported in patients with RA. MPs could represent a link between autoimmunity and endothelial dysfunction by expressing tumor necrosis factor alpha (TNFα), a key cytokine involved in the pathogenesis of RA, altering endothelial apoptosis and autophagy. The aim of this study was to investigate TNFα expression on MPs and its relationship with endothelial cell fate. METHODS: MPs were purified from peripheral blood from 20 healthy controls (HC) and from 20 patients with RA, before (time (T)0) and after (T4) 4-month treatment with etanercept (ETA). Surface expression of TNFα was performed by flow cytometry analysis. EA.hy926 cells, an immortalized endothelial cell line, were treated with RA-MPs purified at T0 and at T4 and also, with RA-MPs in vitro treated with ETA. Apoptosis and autophagy were then evaluated. RESULTS: RA-MPs purified at T0 expressed TNFα on their surface and this expression significantly decreased at T4. Moreover, at T0 RA-MPs, significantly increased both apoptosis and autophagy levels on endothelial cells, in a dose-dependent manner. RA-MPs did not significantly change these parameters after 4 months of in vivo treatment with ETA. CONCLUSIONS: Our data demonstrate that MPs isolated from patients with RA exert a pathological effect on endothelial cells by TNFα expressed on their surface. In vivo and in vitro treatment with ETA modulates this effect, suggesting anti-TNF therapy protects against endothelial damage in patients with RA.
Assuntos
Artrite Reumatoide/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Endoteliais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Idoso , Antirreumáticos/uso terapêutico , Apoptose/efeitos dos fármacos , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Autofagia/efeitos dos fármacos , Linhagem Celular , Micropartículas Derivadas de Células/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Etanercepte/uso terapêutico , Feminino , Humanos , Masculino , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Psoriatic arthritis (PsA) is a chronic inflammatory arthritis associated with psoriasis. A third of psoriatic patients develop PsA via unknown mechanisms. No reliable diagnostic markers are available for PsA, or prognostic biomarkers for PsA development in psoriasis. We previously uncovered a pro-inflammatory role for cathelicidin LL37 in lesional psoriasis skin. LL37 binds nucleic acids and stimulates plasmacytoid/myeloid dendritic cells (pDC, mDCs) to secrete type I interferon (IFN-I) and pro-inflammatory factors. LL37 becomes an autoantigen for psoriatic Th1-Th17/CD8 T cells. Anti-LL37 antibodies were detected in systemic lupus erythematosus, an autoimmune disease characterized by neutrophil-extracellular-traps release (NETosis) in target organs. LL37 can be substrate of irreversible post-translational modifications, citrullination or carbamylation, linked to neutrophil activity. Here we analyzed inflammatory factors, included LL37, in PsA and psoriasis plasma and PsA synovial fluids (SF)/biopsies. We show that LL37 (as a product of infiltrating neutrophils) and autoantibodies to LL37 are elevated in PsA, but not OA SF. Anti-LL37 antibodies correlate with clinical inflammatory markers. Anti-carbamylated/citrullinated-LL37 antibodies are present in PsA SF/plasma and, at lower extent, in psoriasis plasma, but not in controls. Plasma anti-carbamylated-LL37 antibodies correlate with PsA (DAS44) but not psoriasis (PASI) disease activity. Ectopic lymphoid structures, and deposition of immunoglobulin-(Ig)G-complexes (IC) co-localizing with infiltrating neutrophils, are observed in PsA and not OA synovial tissues (ST). Activated complement (C5a, C9), GM-CSF and IFN-I are up-regulated in PsA and not OA synovia and in PsA and psoriasis plasma but not in HD. C9 and GM-CSF levels in PsA SF correlate with clinical inflammatory markers and DAS44 (C9) and with anti-carbamylated/citrullinated-LL37 antibodies (GM-CSF and IFN-I). Thus, we uncover a role for LL37 as a novel PsA autoantibody target and correlation studies suggest participation of anti-LL37 antibodies to PsA pathogenesis. Notably, plasma antibodies to carbamylated-LL37, which correlate with DAS44, suggest their use as new disease activity markers. GM-CSF and complement C5a and C9 elevation may be responsible for autoantigens release by neutrophils and their modification, fueling inflammation and autoreactivity establishment. Finally, targeting GM-CSF, C5a, C9 can be beneficial in PsA.
Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Artrite Psoriásica/imunologia , Autoanticorpos/imunologia , Líquido Sinovial/imunologia , Adolescente , Adulto , Peptídeos Catiônicos Antimicrobianos/metabolismo , Artrite Psoriásica/metabolismo , Autoanticorpos/metabolismo , Biomarcadores/metabolismo , Feminino , Humanos , Masculino , Carbamilação de Proteínas/imunologia , Líquido Sinovial/metabolismo , CatelicidinasRESUMO
A variety of environmental agents has been found to influence the development of autoimmune diseases; in particular, the studies investigating the potential association of systemic autoimmune rheumatic diseases with environmental micro and nano-particulate matter are very few and contradictory. In this study, the role of diesel exhaust particles (DEPs), one of the most important components of environment particulate matter, emitted from Euro 4 and Euro 5 engines in altering the Normal Human Bronchial Epithelial (NHBE) cell biological activity was evaluated. NHBE cells were exposed in vitro to Euro 4 and Euro 5 particle carbon core, sampled upstream of the typical emission after-treatment systems (diesel oxidation catalyst and diesel particulate filter), whose surfaces have been washed from well-assessed harmful species, as polycyclic aromatic hydrocarbons (PAHs) to: (1) investigate their specific capacity to affect cell viability (flow cytometry); (2) stimulate the production of the pro-inflammatory cytokine IL-18 (Enzyme-Linked ImmunoSorbent Assay -ELISA-); (3) verify their specific ability to induce autophagy and elicit protein citrullination and peptidyl arginine deiminase (PAD) activity (confocal laser scanning microscopy, immunoprecipitation, Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis -SDS-PAGE- and Western blot, ELISA). In this study we demonstrated, for the first time, that both Euro 4 and Euro 5 carbon particles, deprived of PAHs possibly adsorbed on the soot surface, were able to: (1) significantly affect cell viability, inducing autophagy, apoptosis and necrosis; (2) stimulate the release of the pro-inflammatory cytokine IL-18; (3) elicit protein citrullination and PAD activity in NHBE cells. In particular, Euro 5 DEPs seem to have a more marked effect with respect to Euro 4 DEPs.
Assuntos
Autofagia , Brônquios/citologia , Citrulinação , Células Epiteliais/metabolismo , Material Particulado/efeitos adversos , Emissões de Veículos , Poluentes Atmosféricos/efeitos adversos , Apoptose , Doenças Autoimunes/etiologia , Sobrevivência Celular , Células Cultivadas , Humanos , Interleucina-18/metabolismo , Necrose , Tamanho da Partícula , Hidrocarbonetos Policíclicos Aromáticos/efeitos adversos , Desiminases de Arginina em Proteínas/metabolismoRESUMO
OBJECTIVE: Several molecules are involved in the pathogenesis of a new bone formation in ankylosing spondylitis (AS). The aim of this study was to evaluate the serum levels of sclerostin in patients with AS as a possible biomarker and to investigate any correlations with radiographic damage, disease activity, and function. METHODS: AS patients fulfilled the modified New York criteria, and healthy controls were enrolled for this study. BASDAI, ASDAS-CRP, BASMI, BASFI, patient and physician VAS, and C-reactive protein were evaluated at baseline visit. Spinal damage was assessed using the mSASSS on radiographs performed within 3 months from baseline. Serum concentrations of sclerostin were assessed at baseline and after four months of therapy in patients who started an anti-TNF. RESULTS: Twenty healthy subjects and 40 AS patients were enrolled in the study. In our group, serum sclerostin levels (median (25th-75th percentile)) were significantly higher in healthy controls (18.04 (13.6-24) pg/ml) than in AS patients (6.46 (4.5-11.1) pg/ml; P value < 0.01). However, no significant correlations were found between serum sclerostin levels and radiographic damage, assessed by mSASSS, and between serum sclerostin levels and clinical indices of activity and disability or with laboratory parameters. Sclerostin levels did not show significant changes after 4 months of anti-TNF therapy. CONCLUSIONS: The results of our study suggest a possible role of sclerostin in the identification of AS patients. Further studies are needed to prove the role of sclerostin as a disease activity biomarker and progression of disease in AS.
Assuntos
Biomarcadores/sangue , Proteínas Morfogenéticas Ósseas/sangue , Osso e Ossos/patologia , Coluna Vertebral/patologia , Espondilite Anquilosante/diagnóstico , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Antirreumáticos/uso terapêutico , Terapia Biológica , Osso e Ossos/diagnóstico por imagem , Estudos de Casos e Controles , Progressão da Doença , Marcadores Genéticos , Humanos , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Coluna Vertebral/diagnóstico por imagem , Espondilite Anquilosante/terapia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVES: Several molecules are involved in the pathogenesis of new bone formation in ankylosing spondylitis (AS). The aim of the present study was to evaluate serum levels of semaphoring 3A in AS and to investigate any correlations with radiographic damage, disease activity, function and treatment. METHODS: AS patients who fulfilled the modified New York criteria were enrolled for this study. Healthy subjects were also enrolled as control group. BASDAI, ASDAS-CRP, BASMI, BASFI, patients and physician VAS, C-reactive protein and erythrocyte sedimentation rate were evaluated at baseline visit. Radiographs of the spine and pelvis performed within six months from the enrolment in the study were collected in all patients. Spinal damage was assessed using the mSASSS. Serum concentrations of semaphorin3A were assessed at baseline and after four months of therapy in patients who started an anti-TNF. RESULTS: Twenty healthy subjects and forty AS patients were enrolled in the study. Of these patients, 15 started anti-TNF therapy the day of baseline visit. Semaphorin3A serum concentrations [median (25th-75th)] were similar in AS patients [0.26 (0.20-0.31) ng/ml] and controls [0.28 (0.26-0.3) ng/ml; p=ns). No significant correlation was found between semaphorin 3A serum levels and radiographic damage index. Semaphorin 3A serum levels positively correlated with ESR values (rho=0.37, p=0.049) and with disease activity assessed by the physician VAS (rho=0.47, p<0.01). No differences were found in the semaphorin3A serum levels after 4 months, compared to baseline values. CONCLUSIONS: The results of the present study could contribute to the intriguing topic of bone remodelling in AS.
Assuntos
Remodelação Óssea , Ossos Pélvicos/fisiopatologia , Semaforina-3A/sangue , Coluna Vertebral/fisiopatologia , Espondilite Anquilosante/sangue , Espondilite Anquilosante/fisiopatologia , Adulto , Produtos Biológicos/uso terapêutico , Biomarcadores/sangue , Sedimentação Sanguínea , Remodelação Óssea/efeitos dos fármacos , Estudos de Casos e Controles , Estudos Transversais , Avaliação da Deficiência , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ossos Pélvicos/diagnóstico por imagem , Ossos Pélvicos/efeitos dos fármacos , Estudos Prospectivos , Índice de Gravidade de Doença , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/efeitos dos fármacos , Espondilite Anquilosante/diagnóstico , Espondilite Anquilosante/tratamento farmacológico , Fatores de Tempo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
BACKGROUND: Current evidence indicates that estrogens, in particular 17ß-estradiol (E2), play a crucial role in the gender bias of autoimmune diseases although the underlying molecular mechanisms have not yet been fully elucidated. Immune cells have estrogen receptors (ERs), i.e., ERα and ERß, that play pro- and anti-inflammatory functions, respectively, and the presence of one estrogen receptor (ER) subtype over the other might change estrogen effects, promoting or dampening inflammation. In this study, we contributed to define the influences of E2 on T cells from female patients with systemic lupus erythematosus (SLE), a representative autoimmune disease characterized by a higher prevalence in women than in men (female/male ratio 9:1). Particularly, our aim was to evaluate whether alterations of ERα and ERß expression in T cells from female SLE patients may impact lymphocyte sensitivity to E2 and anti-ERα antibody (anti-ERα Ab) stimulation interfering with cell signaling and display a direct clinical effect. METHODS: Sixty-one premenopausal female patients with SLE and 40 age-matched healthy donors were recruited. Patients were divided into two groups based on the SLE Disease Activity Index 2000 (SLEDAI-2K) (i.e., <6 and ≥6). ER expression was evaluated in T lymphocytes by flow cytometry, immunofluorescence, and Western blot analyses. Serum anti-ERα Ab levels were analyzed by enzyme-linked immunosorbent assay (ELISA). ER-dependent signaling pathways were measured by a phosphoprotein detection kit. RESULTS: Intracellular ERß expression was significantly lower in T cells from patients with SLEDAI-2K ≥6 as compared with healthy donors and patients with SLEDAI-2K <6 and negatively correlated with disease activity. The expression of intracellular and membrane-associated-ERα was similar in SLE and control T cells. ER-dependent signaling pathways were activated in T cells from SLE patients with SLEDAI-2K ≥6, but not with SLEDAI-2K <6, when both membrane and intracellular ERs were stimulated by co-treatment with E2 and anti-ERα Abs. CONCLUSIONS: Our results demonstrate an altered ER profile in SLE patients, possibly contributing to SLE pathogenesis and interfering with clinical activity, and highlight the potential exploitation of T cell-associated ERß as a biomarker of disease activity.
RESUMO
Systemic sclerosis (SSc) is a multisystem autoimmune disease of unknown etiology characterized by inflammation, autoantibody production, and fibrosis. It predominantly affects women, this suggesting that female sex hormones such as estrogens may play a role in disease pathogenesis. However, up to date, the role of estrogens in SSc has been scarcely explored. The activity of estrogens is mediated either by transcription activity of the intracellular estrogen receptors (ER), ERα and ERß, or by membrane-associated ER. Since the presence of autoantibodies to ERα and their role as estrogen agonists interfering with T lymphocyte homeostasis were demonstrated in other autoimmune diseases, we wanted to ascertain whether anti-ERα antibodies were detectable in sera from patients with SSc. We detected anti-ERα antibody serum immunoreactivity in 42% of patients with SSc (30 out of 71 analyzed). Importantly, a significant association was found between anti-ERα antibody values and key clinical parameters of disease activity and severity. Fittingly, anti-ERα antibody levels were also significantly associated with alterations of immunological features of SSc patients, including increased T cell apoptotic susceptibility and changes in T regulatory cells (Treg) homeostasis. In particular, the percentage of activated Treg (CD4(+)CD45RA(-) FoxP3(bright)CD25(bright)) was significantly higher in anti-ERα antibody positive patients than in anti-ERα antibody negative patients. Taken together our data clearly indicate that anti-ERα antibodies, probably via the involvement of membrane-associated ER, can represent: i) promising markers for SSc progression but, also, ii) functional modulators of the SSc patients' immune system.
Assuntos
Autoanticorpos/sangue , Progressão da Doença , Receptor alfa de Estrogênio/imunologia , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/patologia , Adulto , Idoso , Autoanticorpos/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Demografia , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Escleroderma Sistêmico/sangue , Adulto JovemRESUMO
ß(2)-glycoprotein I (ß(2)GPI) is the major antigenic target for antiphospholipid Abs. Anti-ß(2)GPI Abs are a heterogeneous population of Igs targeting all domains of the molecule. Abs specific to ß(2)GPI domain I are strongly associated with thrombosis and obstetric complications. In the present study, we sought to understand the possible pathogenic mechanism for this subset of anti-ß(2)GPI Abs, investigating their potential cross-reactivity with other self-proteins involved in inflammatory or coagulant events. We compared the amino acid sequence of the ß(2)GPI domain I with human proteins in a protein databank and identified a peptide sharing 88% identity with an epitope of human TLR4. A high percentage of patients with antiphospholipid syndrome (41%) and systemic lupus erythematosus (50%) presented serum IgG specific to this peptide. Anti-ß(2)GPI peptide Abs binding the TLR4 were able to induce NF-κB activation in HEK293 cells that were stably transfected with the TLR4 gene. Anti-ß(2)GPI peptide Abs induced activation of TLR4 and triggered interleukin-1 receptor-associated kinase phosphorylation and NF-κB translocation, promoting VCAM expression on endothelial cells and TNF-α release by monocytes. In conclusion, our observations suggest a novel pathogenic mechanism in the TLR4 stimulation by anti-ß(2)GPI peptide Abs that links adaptive immune responses with innate immunity in antiphospholipid syndrome and systemic lupus erythematosus.
Assuntos
Autoanticorpos/sangue , Células Endoteliais da Veia Umbilical Humana/imunologia , Mediadores da Inflamação/metabolismo , Monócitos/imunologia , Fragmentos de Peptídeos/imunologia , Receptor 4 Toll-Like/imunologia , beta 2-Glicoproteína I/imunologia , Adolescente , Adulto , Idoso , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/metabolismo , Síndrome Antifosfolipídica/patologia , Estudos de Casos e Controles , Periodontite Crônica/imunologia , Periodontite Crônica/metabolismo , Periodontite Crônica/patologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Monócitos/patologia , NF-kappa B/metabolismo , Transporte Proteico , RNA Interferente Pequeno/genética , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
OBJECTIVE: Estrogens influence many physiologic processes and are also implicated in the development or progression of numerous diseases, including autoimmune disorders. Aberrations of lymphocyte homeostasis that lead to the production of multiple pathogenic autoantibodies, including autoantibodies specific to estrogen receptor (ER), have been detected in the peripheral blood of patients with systemic lupus erythematosus (SLE). This study was undertaken to assess the presence of both anti-ERα and anti-ERß antibodies in sera from patients with SLE, to analyze the effect of these antibodies on peripheral blood T lymphocyte homeostasis, and to evaluate their role as determinants of disease pathogenesis and progression. METHODS: Anti-ER antibody serum immunoreactivity was analyzed by enzyme-linked immunosorbent assay in samples from 86 patients with SLE and 95 healthy donors. Phenotypic and functional analyses were performed by flow cytometry and Western blotting. RESULTS: Anti-ERα antibodies were present in 45% of the patients with SLE, whereas anti-ERß antibodies were undetectable. In healthy donors, anti-ERα antibodies induced cell activation and consequent apoptotic cell death in resting lymphocytes as well as proliferation of anti-CD3-stimulated T lymphocytes. A significant association between anti-ERα antibody values and clinical parameters, i.e., the SLE Disease Activity Index and arthritis, was found. CONCLUSION: Our data suggest that anti-ERα autoantibodies interfere with T lymphocyte homeostasis and are significantly associated with lupus disease activity.
Assuntos
Autoanticorpos/imunologia , Receptor alfa de Estrogênio/imunologia , Homeostase/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Autoantígenos/imunologia , Síndrome de Behçet/sangue , Síndrome de Behçet/imunologia , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Receptor beta de Estrogênio/imunologia , Feminino , Citometria de Fluxo , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/fisiopatologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Adulto JovemRESUMO
AIM: Ral-binding protein 1 (RLIP76) is a cell surface protein that catalyzes the extrusion from the cell of reduced glutathione (GSH) conjugates. We recently demonstrated the presence of serum antibodies to RLIP76 (aaRLIP76) in patients with immune-mediated diseases characterized by vascular dysfunction. The aim of this work was to analyze the possible implication of gender in this issue, investigating the effects of aaRLIP76 in rat vascular smooth muscle cells and human endothelial cells from males and females. RESULTS: We observed that, after aaRLIP76 treatment, vascular cells from females showed a significantly higher susceptibility to the disturbance of intracellular redox balance, in terms of H(2)O(2) and O(2)(*) production, 4-hydroxy-t-2,3-nonenal and GSH levels, C-Jun NH2 kinase signaling activation, and apoptosis in comparison with cells from males. Interestingly, under mild oxidative stress (H(2)O(2) 30 µm for 30 min), these sex-associated differences became significantly more pronounced. Experiments carried out in the presence of sex hormones in the culture medium clearly suggested that estrogens could significantly increase the susceptibility of cells from females to the effects of aaRLIP76, whereas cells from males appeared unaffected. INNOVATION: These results open a new perspective in the gender-dependent pathogenic mechanisms of autoimmune diseases characterized by vascular dysfunction. CONCLUSIONS: Altogether these results suggest that the impairment of RLIP76 by aaRLIP76 can play a role in the damage of vascular cells from females, contributing to the gender-associated pathogenesis of immune-mediated vascular diseases.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apoptose , Autoanticorpos/farmacologia , Proteínas Ativadoras de GTPase/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Músculo Liso Vascular/metabolismo , Estresse Oxidativo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Aldeídos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Autoanticorpos/fisiologia , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Estrogênios/farmacologia , Estrogênios/fisiologia , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/imunologia , Técnicas de Silenciamento de Genes , Glutationa/metabolismo , Histidina/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Músculo Liso Vascular/citologia , Interferência de RNA , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fatores Sexuais , Análise de Célula ÚnicaRESUMO
Estrogens are well-known regulators of the immune responses. Most of their effects are mediated by two receptors: estrogen receptor (ER)alpha and ERbeta. Up to date the presence of intracellular ER in human immune cells represents a controversial issue, while their surface membrane expression has scarcely been explored. In this study we investigated the intracellular and cell surface expression of ERalpha and ERbeta in human peripheral blood lymphocytes (PBL) by flow and static cytometry as well as by Western Blot. To this aim we used five different commercial antibodies recognizing distinct ER epitopes. We observed that CD4(+) and CD8(+) T lymphocytes, B lymphocytes and NK cells contain intracellular ERalpha and ERbeta, being the ERalpha46 isoform the most represented ER. However, significant differences could be observed among the antibodies studied in terms of immunoreactivity and specificity. Importantly, we also found a cell surface expression of ERalpha46 isoform. We also observed that a membrane-impermeant form of E2 induced a rapid phosphorylation of extracellular signal-regulated kinase (ERK), a significant proliferation of T lymphocytes, and IFN-gamma production by NK cells, thus suggesting the expression of a functional mERalpha. In conclusion our data could provide new insights as concerns the estrogen-related mechanisms of immune system modulation. They also suggest the need for a reappraisal of the experimental conditions for the characterization of the ER expression.