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BACKGROUND: Low-density lipoprotein (LDL) levels are increased by proprotein convertase subtilisin kexin 9 (PCSK9) which targets the LDL receptor. We recently reported that PCSK9 ameliorates dendritic cell (DC) activation by oxidized LDL (OxLDL), which is abundant in atherosclerotic plaques and is also associated with cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Here, we investigated the role of PCSK9 in SLE. METHODS: PCSK9 levels were determined by ELISA among SLE patients (N = 109) and age- and sex-matched population-based controls (N = 91). Common carotid intima-media thickness (IMT) and plaque occurrence were determined by B-mode ultrasound. Plaques were graded by echogenicity. Human peripheral blood monocytes from SLE patients or controls were differentiated into DCs. The effects of PCSK9 and its inhibition by silencing were studied. RESULTS: PCSK9 levels were non-significantly higher among SLE-patients compared to controls but significantly associated with SLE disease activity, as determined by the Systemic Lupus Activity Measure (p = 0.020) or the SLE Disease Activity Index (p = 0.0178). There was no association between PCSK9 levels and atherosclerosis as determined by IMT, prevalence of plaques or echolucent (potentially vulnerable) plaques. PCSK9 levels were significantly associated with CVD among SLE patients but not after adjusting for age. OxLDL induced PCSK9 in DCs and DC maturation with increased expression of CD86 and HLA-DR. The effects were significantly stronger in DCs from SLE patients than from controls. Silencing of PCSK9 abolished OxLDL-induced DC maturation. CONCLUSIONS: PCSK9 is associated with disease activity in SLE. One underlying cause could be OxLDL promoting DC activation which depends on PCSK9. OxLDL induces PCSK9 - an effect which is higher among SLE patients. PCSK9 could play an unexpected immunological role in SLE.
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Lúpus Eritematoso Sistêmico/sangue , Pró-Proteína Convertase 9/sangue , Adulto , Espessura Intima-Media Carotídea , Estudos de Casos e Controles , Feminino , Humanos , Lipoproteínas LDL/sangue , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , Placa Aterosclerótica/diagnóstico por imagem , Pró-Proteína Convertase 9/imunologia , Fatores de RiscoRESUMO
OBJECTIVES: Phosphorylcholine (PC) and malondialdehyde (MDA) are generated during lipid peroxidation and form adducts with proteins as albumin as studied herein. Atherosclerosis and cardiovascular disease (CVD) are increased in systemic lupus erythematosus (SLE). We here investigate the role and regulation of IgM antibodies against PC (anti-PC) and MDA (anti-MDA). METHODS: IgM anti-PC and anti-MDA in SLE patients (n=114) were compared with age- and sex-matched population-based controls (n=108). Common carotid intima-media thickness (IMT) and plaque occurrence were determined by B-mode ultrasound. Plaques were graded according to echogenicity (potentially vulnerability). Production of IgM anti-PC and anti-MDA by B cells was determined by ELISA and ELISPOT. The effect of anti-PC and anti-MDA on macrophage uptake of apoptotic cells and oxidative stress was studied by flow cytometry. RESULTS: Above 66rd percentile together, IgM anti-PC and anti-MDA were striking protection markers for plaque prevalence and echolucency in SLE (OR: 0.08, CI: 0.01-0.46 and OR: 0.10, CI: 0.01-0.82), respectively, and risk markers for plaque prevalence when below 33rd percentile: OR: 3.79, CI: (1.10-13.00). In vitro, IgM anti-PC and anti-MDA were much higher when B cells were co-cultured with CD3 T cells. Anti-HLA-, anti-CD40 antibody or CD40 silencing abolished these effects. Uptake of apoptotic cells was increased by IgM anti-PC and anti-MDA. MDA induced increased oxidative stress, which was inhibited by IgM anti-MDA. CONCLUSIONS: Unexpectedly, both IgM anti-MDA and IgM anti-PC are T-cell dependent and especially together, are strong protection markers for atherosclerosis in SLE. Underlying mechanisms include increased phagocytosis of apoptotic cells and decrease of oxidative stress.
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Aterosclerose/imunologia , Imunoglobulina M/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Malondialdeído/imunologia , Fosforilcolina/imunologia , Adulto , Apoptose/imunologia , Aterosclerose/complicações , Aterosclerose/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Espessura Intima-Media Carotídea , Células Cultivadas , Técnicas de Cocultura , Ensaio de Imunoadsorção Enzimática , ELISPOT , Feminino , Humanos , Imunoglobulina M/metabolismo , Células Jurkat , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Malondialdeído/metabolismo , Pessoa de Meia-Idade , Estresse Oxidativo/imunologia , Fagocitose/imunologia , Fosforilcolina/metabolismo , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , UltrassonografiaRESUMO
OBJECTIVE: Atherosclerosis is an inflammatory disease, where activated immunocompetent cells, including dendritic cells (DCs) and T cells are abundant in plaques. Low-density lipoprotein modified either by oxidation (oxLDL) or by human group X-secreted phospholipase A2 (LDLx) and heat shock proteins (HSP), especially HSP60 and 90, have been implicated in atherosclerosis. We previously reported that Annexin A5 inhibits inflammatory effects of phospholipids, decreases vascular inflammation and improves vascular function in apolipoprotein E(-/-) mice. Here, we focus on the LDLx effects on human DCs and T cells. APPROACH AND RESULTS: Human DCs were differentiated from peripheral blood monocytes, stimulated by oxLDL or LDLx. Naive autologous T cells were cocultured with pretreated DCs. oxLDL and LDLx, in contrast to LDL, induced DC-activation and T-cell proliferation. T cells exposed to LDLx-treated DCs produced interferon-γ, interleukin (IL)-17 but not IL-4 and IL-10. Annexin A5 abrogated LDLx effects on DCs and T cells and increased production of transforming growth factor-ß and IL-10. Furthermore, IL-10 producing T cells suppressed primary T-cell activation via soluble IL-10, transforming growth factor-ß, and cell-cell contact. Lentiviral-mediated shRNA knock-down HSP60 and 90 in DCs attenuated the effect of LDLx on DCs and subsequent T-cell proliferation. Experiments on DC and T cells derived from carotid atherosclerotic plaques gave similar results. CONCLUSIONS: Our data show that modified forms of LDL such as LDLx but not native LDL activate human T cells through DCs. HSP60 and 90 contribute to such T-cell activation. Annexin A5 promotes induction of regulatory T cells and is potentially interesting as a therapeutic agent.
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Anexina A5/metabolismo , Comunicação Celular , Chaperonina 60/metabolismo , Células Dendríticas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Lipoproteínas LDL/metabolismo , Ativação Linfocitária , Proteínas Mitocondriais/metabolismo , Subpopulações de Linfócitos T/metabolismo , Doenças das Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Chaperonina 60/genética , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/imunologia , Fosfolipases A2 do Grupo X/metabolismo , Proteínas de Choque Térmico HSP90/genética , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Mitocondriais/genética , Placa Aterosclerótica , Interferência de RNA , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , TransfecçãoRESUMO
Phosphorylcholine (PC) is a classic T-independent Ag that is exposed on apoptotic cells, oxidized phospholipids, and bacterial polysaccharides. Experimental as well as epidemiological studies have over the past decade implicated Abs against PC (anti-PC) as anti-inflammatory and a strong protective factor in cardiovascular disease. Although clinically important, little is known about the development of anti-PC in humans. This study was conceived to dissect the human anti-PC repertoire and generate human mAbs. We designed a PC-specific probe to identify, isolate, and characterize PC-reactive B cells from 10 healthy individuals. The donors had all mounted somatically mutated Abs toward PC using a broad variety of Ig genes. PC-reactive B cells were primarily found in the IgM(+) memory subset, although significant numbers also were detected among naive, IgG(+), and CD27(+)CD43(+) B cells. Abs from these subsets were clonally related, suggesting a common origin. mAbs derived from the same donors exhibited equivalent or higher affinity for PC than the well-characterized murine T-15 clone. These results provide novel insights into the cellular and molecular ontogeny of atheroprotective PC Abs, thereby offering new opportunities for Ab-based therapeutic interventions.
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Anticorpos Antifosfolipídeos/imunologia , Subpopulações de Linfócitos B/imunologia , Imunoglobulina M/imunologia , Memória Imunológica/fisiologia , Fosforilcolina/imunologia , Adulto , Animais , Subpopulações de Linfócitos B/citologia , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , CamundongosRESUMO
PURPOSE: Interferon-γ inhibits cancer cell proliferation and induces re-expression of different tumor suppressor genes. As a candidate, HEPACAM is almost lost in bladder transitional cell carcinoma. To our knowledge whether interferon-γ inhibits BIU-87 proliferation and re-expresses HEPACAM mRNA is still unknown. Thus, we probed the mechanism and examined the correlations between interferon-γ in patient serum and HEPACAM in bladder transitional cell carcinoma. MATERIALS AND METHODS: Using enzyme-linked immunosorbent assay we measured serum interferon-γ in 27 men and 6 women, and 15 volunteers. Disease was Ta-T1 in 12 patients, T2-T4 in 21, low grade in 25, high grade in 8, primary in 13 and recurrent in 20. A total of 33 cancer and 26 adjacent tissues were examined by immunohistochemistry to detect HEPACAM protein and ensure the position. Under interferon-γ stimulation we detected BIU-87 proliferation by MTT assay. Cell cycles were examined by flow cytometry. HEPACAM mRNA expression was determined by reverse transcription-polymerase chain reaction. Western blot was used to detect p21(WAF1). RESULTS: Interferon-γ was remarkably low in patients with bladder transitional cell carcinoma vs volunteers (p <0.01). HEPACAM protein was highly expressed in adjacent tissue, mainly at the cytomembrane, but it was almost absent in bladder transitional cell carcinoma (p <0.01). The interferon-γ decrease in the serum of patients with bladder transitional cell carcinoma and the low HEPACAM expression in tumors correlated linearly (r = 0.899, p <0.01). In vitro interferon-γ inhibited BIU-87 proliferation (p <0.01) and slightly re-expressed HEPACAM mRNA (p <0.05). The cell cycle was arrested at G(0)/G(1) and p21(WAF1) was concurrently increased in response to interferon-γ (p <0.01). CONCLUSIONS: Results suggest an important connection between HEPACAM and interferon-γ, which may inhibit BIU-87 proliferation through HEPACAM re-expression and p21(WAF1) up-regulation to arrest cells at the G(0)/G(1) phase.
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Carcinoma de Células de Transição/sangue , Carcinoma de Células de Transição/genética , Interferon gama/sangue , Interferon gama/fisiologia , Proteínas/genética , Neoplasias da Bexiga Urinária/sangue , Neoplasias da Bexiga Urinária/genética , Carcinoma de Células de Transição/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias da Bexiga Urinária/patologiaRESUMO
BACKGROUND: Gastric cancer (GC) is a common and deadly malignancy in the world. CircRNAs have emerged as important regulators in human diseases, including GC. In this work, we intended to explore the role of circ_CORO1C in GC progression and potential mechanism. METHODS: Quantitative real-time PCR (qRT-PCR) or Western blot assay was performed to examine the expression of circRNA coronin-like actin-binding protein 1C (circ_CORO1C), microRNA (miR)-138-5p and Krueppel-like factor 12 (KLF12) in clinical samples and cells. Cell colony formation ability and viability were measured by colony formation assay and methyl thiazolyl tetrazolium (MTT) assay, respectively. Expression of cell proliferation and epithelia-mesenchymal transition (EMT) biomarker was detected by Western blot analysis. And cell metastasis, including migration and invasion, and apoptosis were analyzed via Transwell assay and flow cytometry, respectively. Target relationship among circ_CORO1C, miR-138-5p and KLF12 was validated by dual-luciferase reporter assay. The in vivo role of circ_CORO1C was investigated by tumor xenograft assay. RESULTS: Circ_CORO1C and KLF12 were upregulated, while miR-138-5p was downregulated in GC tissues and cells. Circ_CORO1C knockdown suppressed colony formation ability, viability, migration, invasion and EMT in GC cells, while promoted cell apoptosis in vitro. Circ_CORO1C targeted miR-138-5p, the inhibition of which could attenuate silenced circ_CORO1C-induced inhibitory effects on GC progression. MiR-138-5p repressed the aggressive malignant behaviors of GC cells by directly targeting KLF12. Circ_CORO1C deficiency inhibited GC tumor growth in vivo. CONCLUSION: Depletion of circ_CORO1C suppressed GC progression by regulating miR-138-5p/KLF12 axis, offering a potential molecular target for GC therapy.
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OBJECTIVE: We study activation of T helper 17 (Th17) and regulatory T (Treg) cells and induction of apoptosis in cells from patients with systemic lupus erythematosus (SLE) compared with controls and effects of atorvastatin and its simulated interactions with other compounds. METHODS: Mononuclear cells from 10 patients with SLE and 10 controls were cultured in conditions that induce Th17 and/or Treg cell polarization and/or apoptosis and were studied by FACScan. Gene expression was determined by quantitative real-time reverse transcription-polymerase chain reaction. Cytokines in plasma were determined by enzyme-linked immunosorbent assay. The Search Tool for Interactions of Chemicals (STITCH) was used to retrieve information regarding the binding properties of atorvastatin. RESULTS: Among patients with SLE, the proportion of Th17 (CD4+ IL17+ ) cells was higher compared with controls after activation, with Th17 or Treg polarizing cytokines, phorbol myristate acetate, and ionomycin. In contrast, Treg cells (CD4+ CD25+ CD127dim/- ) frequencies were lower. CD95 stimulation induced relatively more apoptosis in Treg cells and less in Th17 cells, as compared with controls. Addition of atorvastatin normalized Th17/Treg cell balance and apoptosis induction. Accordingly, the ratio of RORC/FoxP3 decreased in patients with SLE. Interleukin 17 and interleukin 6 (IL-6) levels were increased in patients with SLE. Atorvastatin interacted strongly with C-reactive protein (CRP) and also significantly with IL-6. CONCLUSION: There is a higher proportion of Th17 cells and a lower proportion of Treg cells in patients with SLE after activation. Th17 cells were more resistant than Treg cells to CD95-induced apoptosis in SLE. Atorvastatin normalized these effects. Our findings reveal a novel mechanism behind the imbalance of Th17/Treg cells with implications for treatment in SLE. We determine for the first time simulated interaction between atorvastatin, CRP, and IL-6, implying a novel role of atorvastatin.
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Refractory anaemia with ring sideroblasts (RARS) is characterized by anaemia, erythroid apoptosis, cytochrome c release and mitochondrial ferritin accumulation. Granulocyte-colony-stimulating factor (G-CSF) inhibits the first three of these features in vitro and in vivo. To dissect the molecular mechanisms underlying the RARS phenotype and anti-apoptotic effects of G-CSF, erythroblasts generated from normal (NBM) and RARS marrow CD34(+) cells were cultured +/-G-CSF and subjected to gene expression analysis (GEP). Several erythropoiesis-associated genes that were deregulated in RARS CD34(+) cells showed normal expression in erythroblasts, underscoring the importance of differentiation-specific GEP. RARS erythroblasts showed a marked deregulation of several pathways including apoptosis, DNA damage repair, mitochondrial function and the JAK/Stat pathway. ABCB7, transporting iron from mitochondria to cytosol and associated with inherited ring sideroblast formation was severely suppressed and expression decreased with differentiation, while increasing in NBM cultures. The same pattern was observed for the mitochondrial integrity gene MFN2. Other downregulated key genes included STAT5B, HSPA5, FANCC and the negative apoptosis regulator MAP3K7. Methylation status of key downregulated genes was normal. The mitochondrial pathway including MFN2 was significantly modified by G-CSF, and several heat shock protein genes were upregulated, as evidence of anti-apoptotic protection of erythropoiesis. By contrast, G-CSF had no effect on iron-transport or erythropoiesis-associated genes.
Assuntos
Anemia Refratária/metabolismo , Anemia Sideroblástica/metabolismo , Eritroblastos/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Idoso , Apoptose/genética , Diferenciação Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Eritroblastos/efeitos dos fármacos , Eritroblastos/patologia , Células Precursoras Eritroides/patologia , Eritropoese/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Ferro/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais/genéticaRESUMO
Human dendritic cells were differentiated from blood monocytes and treated with malondialdehyde (MDA) conjugated with human serum albumin (HSA). Autologous T cells from human plaques or blood were co-cultured with the pre-treated dendritic cells or treated directly. MDA modifications were studied by mass spectrometry. MDA-HSA induced a pro-inflammatory DC-mediated T-cell activation and also a strong direct effect on T cells, inhibited by an inhibitor of oxidative stress and antibodies against MDA. Atherogenic heat shock protein-60 was strongly induced in T cells activated by MDA-HSA. Two peptide modifications in atherosclerotic patients' HSA were similar to those present in in vitro MDA-modified HSA.
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Moving in groups is an amazing spectacle of collective behaviour in fish and has attracted considerable interest from many fields, including biology, physics and engineering. Although robotic fish have been well studied, including algorithms to simulate group swimming, experiments that demonstrate multiple robotic fish as a stable group are yet to be achieved. One of the challenges is the lack of a robust bottom-level motion control system for robotic fish platforms. Here we seek to overcome this challenge by focusing on the design and implementation of a motion controller for robotic fish that allows multiple individuals to swim in groups. As direction control is essential in motion control, we first propose a high-accuracy controller which can control a sub-carangiform robotic fish from one arbitrary position/pose (position and direction) to another. We then develop a hydrodynamic-model-based simulation platform to expedite the process of the parameter tuning of the controller. The accuracy of the simulation platform was assessed by comparing the results from experiments on a robotic fish using speeding and turning tests. Subsequently, extensive simulations and experiments with robotic fish were used to verify the accuracy and robustness of the bottom-level motion control. Finally, we demonstrate the efficacy of our controller by implementing group swimming using three robotic fish swimming freely in prescribed trajectories. Although the fluid environment can be complex during group swimming, our bottom-level motion control remained nominally accurate and robust. This motion control strategy lays a solid foundation for further studies of group swimming with multiple robotic fish.
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Peixes/fisiologia , Robótica/instrumentação , Natação/fisiologia , Animais , Fenômenos Biomecânicos , Materiais Biomiméticos , Simulação por Computador , Desenho de Equipamento , Hidrodinâmica , Modelos Biológicos , Tecnologia sem FioRESUMO
EBV specific cellular memory is not transferred from mother to child. By addition of the immunomodulators PSK and Trx80, that act on monocytes we could generate EBV-specific T cell response in cord-blood derived mononuclear cultures infected with the virus. In such cultures EBV infected B lymphocytes activated T and NK cells, and the immunostimulators activated the monocytes. Lymphokines produced by the monocytes induced the T and NK cells to progress into a functional activation state and they inhibited the EBV induced proliferation of B lymphocytes. Leukotrienes have been well studied in allergic and inflammatory responses, but less is known about their contribution to cellular immunity. In these experiments leukotriene LTB, produced by the activated monocytes was involved in the activation of effector cells. LTB4 was detected in the culture supernatants and addition of LTB4 biosynthesis inhibitors abolished the activation. These experiments showed thus that endogenously produced LTB4 can induce effector cell responses. Addition of LTB4 to the infected culture or readdition of LTB4 treated monocytes to the culture of infected monocyte depleted cell population induced also T cell activation and led to inhibition of B cell proliferation. These results demonstrated thus that LTB4 can contribute to the activation of innate immunity.
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Linfócitos B/patologia , Linfócitos B/virologia , Transformação Celular Neoplásica , Sangue Fetal/citologia , Herpesvirus Humano 4/imunologia , Imunidade Inata/imunologia , Leucotrieno B4/imunologia , Linfócitos B/citologia , Células Cultivadas , Sangue Fetal/virologia , Herpesvirus Humano 4/patogenicidade , Humanos , Leucotrieno B4/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
BACKGROUND AND AIMS: IgM antibodies against phosphorylcholine (anti-PC) are negatively associated with atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE), where the risk of CVD and atherosclerosis is high. We here study the effects of IgM anti-PC immune regulation. METHODS: Mononuclear leukocytes were isolated from peripheral blood (PBMC) obtained from healthy blood donors, six SLE patients with age- and sex-matched controls, and symptom-giving human atherosclerotic plaques. The proportion of Th17 (CD4+CCR6+) and Treg (CD4+CD25+CD127dim/-) cells was determined by flow cytometry in CD4+T cells after 6 days of culture with Th17 or Treg-polarizing cytokines, with PMA and Ionomycin stimulation. IgM anti-PC were extracted from total IgM, with flow-through IgM as controls. Dendritic cells (DC) were differentiated from PBMC. Antibody peptide/protein characterization was done by a proteomics de novo sequencing approach. RESULTS: IgM anti-PC increased significantly the proportion of Tregs from healthy donors, SLE patients and atherosclerotic plaque cells while control antibodies did not. T cells from SLE patients had a significantly lower proportion of Tregs and a higher proportion of Th17 cells as compared to matched controls. IgM anti-PC, but not control antibodies, significantly reduced the production of IL-17 and TNF-α in cell cultures from SLE patients and atherosclerotic plaque cells. IgM anti-PC interacted with CD40 and kept DCs in an immature stage, potentially being tolerogenic. We observed differences in the IgM peptide expression levels in anti-PC compared to control antibodies. CONCLUSIONS: IgM anti-PC promote polarization of Tregs, which could represent a novel protective mechanism in atherosclerosis and autoimmune conditions as SLE.
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Aterosclerose/imunologia , Imunoglobulina M/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária , Fosforilcolina/imunologia , Placa Aterosclerótica , Linfócitos T Reguladores/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Autoimunidade , Estudos de Casos e Controles , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Tolerância Imunológica , Interleucina-17/imunologia , Interleucina-17/metabolismo , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/prevenção & controle , Masculino , Pessoa de Meia-Idade , Fenótipo , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Atherosclerosis is characterized by the presence of activated immune-competent cells including dendritic cells (DCs) and T cells, dead cells, and oxidized low-density lipoprotein. HSP60 (Heat shock protein 60) has been implicated in atherosclerosis. A plasma protein, Annexin A5, has atheroprotective properties. METHODS AND RESULTS: Human DCs differentiated from peripheral blood monocytes were treated with human HSP60 or HSP90 and autologous T cells were cocultured with these pretreated DCs (mDCs). HSP60 induced mDCs and T-cell activation as determined by FACScan (Fluorescence associated cell scan), gene-activation, and cytokine production. HSP60-induced T-cell activation was partly major histocompatibility complex class II-dependent. T cells exposed to HSP60-treated mDCs produced interferon-γ, interleukin-17, but not transforming growth factor-ß. HSP60 did not promote expression of Toll-like receptors 2 or 4. HSP90 promoted mDCs maturation but had no effect on T-cell activation. Annexin A5 inhibited HSP60-proinflammatory Th1/Th17 effects on mDCs and T cells, and partly bound HSP60. Further, Annexin A5 inhibited HSP-induced activation of mDCs and also oxidized low-density lipoprotein-induced HSP-production from mDCs. Experiments on mDCs and T cells derived from carotid atherosclerotic plaques from patients with symptomatic carotid disease gave similar results as from blood donors. CONCLUSIONS: HSP60 induces mDCs activation and partly major histocompatibility complex class II-dependent activation of blood- and plaque-derived T cells, which is mostly of Th1/Th17 type. HSP60 could thus be an important T-cell antigen in plaques, and also mediate oxidized low-density lipoproteins immunogenic effects on DC-T-cell activation, promoting plaque rupture and clinical manifestations of cardiovascular disease. Annexin A5 inhibits both oxidized low-density lipoprotein-induced HSP60, and HSP60-mediated immune activation, which suggests a potential therapeutic role.
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Chaperonina 60/farmacologia , Células Dendríticas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Placa Aterosclerótica/imunologia , Células Th17/imunologia , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Humanos , Placa Aterosclerótica/tratamento farmacológico , Placa Aterosclerótica/metabolismo , Células Th17/efeitos dos fármacosRESUMO
Coal tar is one of the oldest and an effective treatment for psoriasis. Coal tar has been directly applied to the skin, or used in combination with UV light as part of the Goeckerman treatment. The use of coal tar has caused long-term remissions in psoriasis, but has fallen out of favor because the treatment requires hospitalization and coal tar is poorly acceptable aesthetically to patients. Thus, determining the active antipsoriatic component of coal tar is of considerable therapeutic interest. We fractionated coal tar into its components, and tested them using the SVR angiogenesis inhibitor assay. Treatment of SVR endothelial cells with coal tar fractions resulted in the isolation of a single fraction with antiangiogenic activity. The active antiangiogenic compound in coal tar is carbazole. In addition to antiangiogenic activity, carbazole inhibited the production of inflammatory IL-15 by human mononuclear cells. IL-15 is elevated in psoriasis and is thought to contribute to psoriatic inflammation. Carbazole treatment also reduced activity of inducible nitric oxide synthase (iNOS), which is proinflammatory and elevated in psoriasis. The effect of carbazole on upstream pathways in human psoriasis was determined, and carbazole was shown to inhibit signal transducer and activator of transcription (stat)3-mediated transcription, which has been shown to be relevant in human psoriasis. IL-15, iNOS, and stat3 activation require the activation of the small GTPase rac for optimal activity. Carbazole was found to inhibit rac activation as a mechanism for its inhibition of downstream inflammatory and angiogenic pathways. Given its antiangiogenic and anti-inflammatory activities, carbazole is likely a major component of the antipsoriatic activity of coal tar. Carbazole and derivatives may be useful in the therapy of human psoriasis.
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Inibidores da Angiogênese/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Carbazóis/farmacologia , Alcatrão/química , Fármacos Dermatológicos/farmacologia , Psoríase/tratamento farmacológico , Inibidores da Angiogênese/isolamento & purificação , Animais , Anti-Inflamatórios não Esteroides/isolamento & purificação , Carbazóis/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Fármacos Dermatológicos/isolamento & purificação , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Humanos , Interleucina-15/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Camundongos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição AP-1/antagonistas & inibidoresRESUMO
BACKGROUND: Activated T cells and dendritic cells (DCs) are colocalized in atherosclerotic plaques in association with plaque rupture. Oxidized low-density lipoprotein (oxLDL) promotes immune activation and inflammation. We studied the effects of statins (atorvastatin and simvastatin) on human DC maturation and T-cell activation. METHODS AND RESULTS: Human peripheral blood monocytes were differentiated to DCs and stimulated with oxLDL. T cells were isolated from carotid endarterectomy specimens from patients undergoing carotid endarterectomy or from healthy individuals. Naïve T cells were cocultured with pretreated DCs. The effects of statin were studied. OxLDL induced DC maturation and T-cell activation. OxLDL induced atherogenic heat shock proteins (HSP) 60 and 90 and decreased potentially atheroprotective heat shock protein 27, effects restored by atorvastatin. T cells exposed to oxLDL-treated DCs produced interferon-γ and interleukin (IL)-17. Atorvastatin and simvastatin suppressed the DC maturation showing lower expression of CD80, CD83, and CD86, and limited their production of tumor necrosis factor-α, IL-1ß and IL-6, and increased transforming growth factor-ß and IL-10 secretion. Statin-treated DCs inhibited Th1 and/or Th17 polarization by downregulation of transcriptional factors T-bet and RORγt expression, and induced T regulatory cells with IL-10 production. OxLDL-induced miRNA let7c and phosphorylation of Akt and ERK were repressed by statins. Let-7c had a pivotal role in mediating effect of oxLDL. Experiments on T cells derived from carotid atherosclerotic plaques or healthy individuals showed similar results. CONCLUSIONS: Statins repress human DC maturation induced by oxLDL, limit T-cell activation, and repress an atherogenic heat shock protein profile and promote induction of T regulatory cells. MicroRNA let-7c is integral to the effects.
Assuntos
Células Dendríticas/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lipoproteínas LDL/farmacologia , Ativação Linfocitária/efeitos dos fármacos , MicroRNAs/efeitos dos fármacos , Placa Aterosclerótica/imunologia , Linfócitos T/efeitos dos fármacos , Atorvastatina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Chaperonina 60/efeitos dos fármacos , Chaperonina 60/imunologia , Células Dendríticas/imunologia , Endarterectomia das Carótidas , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico HSP27/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/imunologia , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/imunologia , Proteínas de Choque Térmico , Humanos , Interferon gama/efeitos dos fármacos , Interferon gama/imunologia , Interleucina-17/imunologia , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Ativação Linfocitária/imunologia , MicroRNAs/imunologia , Proteínas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/imunologia , Chaperonas Moleculares , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/efeitos dos fármacos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinvastatina/farmacologia , Proteínas com Domínio T/efeitos dos fármacos , Proteínas com Domínio T/metabolismo , Linfócitos T/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: Malondialdehyde (MDA) is generated during lipid peroxidation as in oxidized low-density lipoprotein, but antibodies against oxidized low-density lipoprotein show variable results in clinical studies. We therefore studied the risk of cardiovascular disease (CVD) associated with IgM antibodies against MDA conjugated with human albumin (anti-MDA). METHODS AND RESULTS: In a 5- to 7-year follow-up of 60-year-old men and women from Stockholm County previously screened for cardiovascular risk factors (2039 men, 2193 women), 209 incident CVD cases (defined as new events of coronary heart disease, fatal and nonfatal myocardial infarction, ischemic stroke, and hospitalization for angina pectoris) and 620 age- and sex-matched controls were tested for IgM anti-MDA by ELISA. Antibody peptide/protein characterization was done using a proteomics de novo sequencing approach. After adjustment for smoking, body-mass index, type 2 diabetes mellitus, hyperlipidemia, and hypertension, an increased CVD risk was observed in the low IgM anti-MDA percentiles (below 10th and 25th) (odds ratio and 95% CI: 2.0; 1.19-3.36 and 1.67; 1.16-2.41, respectively). Anti-MDA above the 66th percentile was associated with a decreased CVD risk (odds ratio 0.68; CI: 0.48-0.98). After stratification by sex, associations were only present among men. IgM anti-MDA levels were lower among cases (median [interquartile range]: 141.0 [112.7-164.3] versus 147.4 [123.5-169.6]; P=0.0177), even more so among men (130.6 [107.7-155.3] versus 143.0 [120.1-165.2]; P=0.001). The IgM anti-MDA variable region profiles are distinctly different and also more homologous in their content (correlates strongly with fewer peptides) than control antibodies (not binding MDA). CONCLUSIONS: IgM anti-MDA is a protection marker for CVD. This finding could have diagnostic and therapeutic implications.
Assuntos
Autoanticorpos/imunologia , Doenças Cardiovasculares/imunologia , Imunoglobulina M/imunologia , Malondialdeído/imunologia , Albumina Sérica/imunologia , Idoso , Angina Pectoris/epidemiologia , Angina Pectoris/imunologia , Doenças Cardiovasculares/epidemiologia , Estudos de Casos e Controles , Doença das Coronárias/epidemiologia , Doença das Coronárias/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Hospitalização , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/imunologia , Estudos Prospectivos , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/imunologia , Suécia/epidemiologiaRESUMO
We infected umbilical cord blood lymphocytes (CBL) with EBV in vitro. Analysis of the cell population in 3- and 6-day-old cultures showed a relative increase of B cells and outgrowth of B cells after prolonged culture period. The immunomodulator PSK was added to parallel cultures. In these cultures, B cell growth was inhibited and the activation of T and NK cells was potentiated. This was detected by assessment of SH2D1A (also named SAP or DSHP) expression (a molecule which participates in signal transduction and is mutated in X-linked lymphoproliferative disease, XLP). Upon further cultivation, irradiated autologous EBV infected B cells and IL-2 were added to the cultures. After 17 days, the B lymphocytes were removed from the PSK containing cultures. The remaining populations, containing mainly T and NK cells, exerted cytotoxic function which could act on EBV infected autologous B cells, allogeneic LCL and on K562. Since cellular immunity to EBV is not transmitted to the newborn, EBV specific memory is not involved in the activation of effector cells. Our finding of an in vitro response of T and NK cells to EBV infected B lymphocytes in the absence of EBV specific immunological memory is of particular interest, because it may also operate in vivo and participate in the scenario of primary infection. Its potentiation by immunomodulators may have practical significance.
Assuntos
Proteínas de Transporte/metabolismo , Sangue Fetal/imunologia , Herpesvirus Humano 4/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Polissacarídeos Bacterianos/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/virologia , Divisão Celular , Células Cultivadas , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Sangue Fetal/virologia , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Fatores de TempoRESUMO
The unique manifestation of the inherited immunodeficiency, X-linked lymphoproliferative disease (XLP), is the impaired control of EBV infection. The gene, which carries mutations or is deleted in the patients, has been identified (Xq25). The encoded protein (SAP, 128 aa) contains a single SH2 domain and binds to signaling lymphocytic activation molecule (SLAM) and to other related surface molecules that are expressed on activated T, B and NK cells. SAP modifies signal transduction through its association with these molecules. Initially it was assumed that SAP acts passively by interfering and blocking active interactions involving other SH2 carrying molecules. We demonstrated that SAP protein is expressed in activated T and NK, but not in activated B cells. This finding is in line with the fact that in vitro performance of effector cells derived from XLP patients is impaired. However, it is still not known why the severe symptoms (fatal mononucleosis or malignant lymphoproliferation in the survivors of the primary infection) are elicited by EBV. We studied SAP expression in several Burkitt lymphoma (BL) derived lines. In contrast to normal B cells, certain lines expressed SAP. These were all type I cells in the Burkitt line nomenclature: they expressed only one of the EBV encoded proteins (EBNA-1) and their phenotype corresponded to resting B cells. Lymphoblastoid cell lines and type III BLs, whose phenotype resembled activated B cells and expressed all nine EBV encoded proteins, were devoid of SAP. The relationship between cell activation and SAP expression is reciprocal in T and B cells i.e. BL lines, activated T and NK cells express SAP, while BL blasts do not express SAP. This opposite relationship may be exploited for studies about the function of SAP.
Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Antígenos CD , Linfócitos B/imunologia , Linfoma de Burkitt/imunologia , Transformação Celular Viral , Células Cultivadas , Ligação Genética , Glicoproteínas/análise , Herpesvirus Humano 4/patogenicidade , Humanos , Imunoglobulinas/análise , Mononucleose Infecciosa/imunologia , Células Jurkat , Ativação Linfocitária , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/imunologia , Receptores de Superfície Celular , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Triexosilceramidas/análise , Células Tumorais Cultivadas , Cromossomo XRESUMO
Refractory anemia with ring sideroblasts (RARS) is characterized by mitochondrial ferritin (FTMT) accumulation and markedly suppressed expression of the iron transporter ABCB7. To test the hypothesis that ABCB7 is a key mediator of ineffective erythropoiesis of RARS, we modulated its expression in hematopoietic cells. ABCB7 up and downregulation did not influence growth and survival of K562 cells. In normal bone marrow, ABCB7 downregulation reduced erythroid differentiation, growth and colony formation, and resulted in a gene expression pattern similar to that observed in intermediate RARS erythroblasts, and in the accumulation of FTMT. Importantly, forced ABCB7 expression restored erythroid colony growth and decreased FTMT expression level in RARS CD34+ marrow cells. Mutations in the SF3B1 gene, a core component of the RNA splicing machinery, were recently identified in a high proportion of patients with RARS and 11 of the 13 RARS patients in this study carried this mutation. Interestingly, ABCB7 exon usage differed between normal bone marrow and RARS, as well as within the RARS cohort. In addition, SF3B1 silencing resulted in downregulation of ABCB7 in K562 cells undergoing erythroid differentiation. Our findings support that ABCB7 is implicated in the phenotype of acquired RARS and suggest a relation between SF3B1 mutations and ABCB7 downregulation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Anemia Refratária/genética , Anemia Sideroblástica/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Regulação para Baixo , Éxons , Feminino , Citometria de Fluxo , Inativação Gênica , Humanos , Imuno-Histoquímica , Células K562 , Masculino , Pessoa de Meia-Idade , Fenótipo , Splicing de RNA , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Chronic lymphocytic leukemia (CLL) cells express the receptor for Epstein-Barr virus (EBV) and can be infected in vitro. Infected cells do not express the growth-promoting set of EBV-encoded genes and therefore they do not yield LCLs, in most experiments. With exceptional clones, lines were obtained however. We describe a new line, HG3, established by in vitro EBV-infection from an IGHV1-2 unmutated CLL patient clone. All cells expressed EBNA-2 and LMP-1, the EBV-encoded genes pivotal for transformation. The karyotype, FISH cytogenetics and SNP-array profile of the line and the patient's ex vivo clone showed biallelic 13q14 deletions with genomic loss of DLEU7, miR15a/miR16-1, the two micro-RNAs that are deleted in 50% of CLL cases. Further features of CLL cells were: expression of CD5/CD20/CD27/CD43 and release of IgM natural antibodies reacting with oxLDL-like epitopes on apoptotic cells (cf. stereotyped subset-1). Comparison with two LCLs established from normal B cells showed 32 genes expressed at higher levels (> 2-fold). Among these were LHX2 and LILRA. These genes may play a role in the development of the disease. LHX2 expression was shown in self-renewing multipotent hematopoietic stem cells, and LILRA4 codes for a receptor for bone marrow stromal cell antigen-2 that contributes to B cell development. Twenty-four genes were expressed at lower levels, among these PARD3 that is essential for asymmetric cell division. These genes may contribute to establish precursors of CLL clones by regulation of cellular phenotype in the hematopoietic compartment. Expression of CD5/CD20/CD27/CD43 and spontaneous production of natural antibodies may identify the CLL cell as a self-renewing B1 lymphocyte.