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1.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807899

RESUMO

We aimed to evaluate the angiogenic capacity of CXCL2 and IL8 affecting human endothelial cells to clarify their potential role in glioblastoma (GBM) angiogenesis. Human GBM samples and controls were stained for proangiogenic factors. Survival curves and molecule correlations were obtained from the TCGA (The Cancer Genome Atlas) database. Moreover, proliferative, migratory and angiogenic activity of peripheral (HUVEC) and brain specific (HBMEC) primary human endothelial cells were investigated including blockage of CXCR2 signaling with SB225502. Gene expression analyses of angiogenic molecules from endothelial cells were performed. Overexpression of VEGF and CXCL2 was observed in GBM patients and associated with a survival disadvantage. Molecules of the VEGF pathway correlated but no relation for CXCR1/2 and CXCL2/IL8 was found. Interestingly, receptors of endothelial cells were not induced by addition of proangiogenic factors in vitro. Proliferation and migration of HUVEC were increased by VEGF, CXCL2 as well as IL8. Their sprouting was enhanced through VEGF and CXCL2, while IL8 showed no effect. In contrast, brain endothelial cells reacted to all proangiogenic molecules. Additionally, treatment with a CXCR2 antagonist led to reduced chemokinesis and sprouting of endothelial cells. We demonstrate the impact of CXCR2 signaling on endothelial cells supporting an impact of this pathway in angiogenesis of glioblastoma.


Assuntos
Neoplasias Encefálicas , Quimiocina CXCL2/metabolismo , Glioblastoma , Células Endoteliais da Veia Umbilical Humana/metabolismo , Interleucina-8/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Glioblastoma/patologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
J Med Microbiol ; 70(3)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33734952

RESUMO

Introduction. Oral tissues are generally homeostatic despite exposure to many potential inflammatory agents including the resident microbiota. This requires the balancing of inflammation by regulatory mechanisms and/or anti-inflammatory commensal bacteria. Thus, the levels of anti-inflammatory commensal bacteria in resident populations may be critical in maintaining this homeostatic balance.Hypothesis/Gap Statement. The incidence of immunosuppressive streptococci in the oral cavity is not well established. Determining the proportion of these organisms and the mechanisms involved may help to understand host-microbe homeostasis and inform development of probiotics or prebiotics in the maintenance of oral health.Aim. To determine the incidence and potential modes of action of immunosuppressive capacity in resident oral streptococci.Methodology. Supragingival plaque was collected from five healthy participants and supragingival and subgingival plaque from five with gingivitis. Twenty streptococci from each sample were co-cultured with epithelial cells±flagellin or LL-37. CXCL8 secretion was detected by ELISA, induction of cytotoxicity in human epithelial cells by lactate dehydrogenase release and NFκB-activation using a reporter cell line. Bacterial identification was achieved through partial 16S rRNA gene sequencing and next-generation sequencing.Results. CXCL8 secretion was inhibited by 94/300 isolates. Immunosuppressive isolates were detected in supragingival plaque from healthy (4/5) and gingivitis (4/5) samples, and in 2/5 subgingival (gingivitis) plaque samples. Most were Streptococcus mitis/oralis. Seventeen representative immunosuppressive isolates all inhibited NFκB activation. The immunosuppressive mechanism was strain specific, often mediated by ultra-violet light-labile factors, whilst bacterial viability was essential in certain species.Conclusion. Many streptococci isolated from plaque suppressed epithelial cell CXCL8 secretion, via inhibition of NFκB. This phenomenon may play an important role in oral host-microbe homeostasis.


Assuntos
Imunomodulação , Interleucina-8/metabolismo , Microbiota/imunologia , Boca/microbiologia , NF-kappa B/metabolismo , Streptococcus/imunologia , Células A549 , Linhagem Celular , Células Epiteliais/metabolismo , Gengiva/microbiologia , Gengivite/microbiologia , Humanos , Microbiota/genética , Streptococcus/classificação , Streptococcus/genética , Streptococcus/isolamento & purificação
3.
J Enzyme Inhib Med Chem ; 36(1): 659-668, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33641565

RESUMO

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 µM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 µM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 µM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.


Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Peróxido de Hidrogênio/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Ocludina/genética , Ocludina/metabolismo , Oxirredução/efeitos dos fármacos , Fenilalanina/análogos & derivados , Cultura Primária de Células , Serina Endopeptidases/genética
4.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669091

RESUMO

Oxidative stress and inflammation play important roles in the pathophysiology of acute kidney injury (AKI). Transient receptor potential ankyrin 1 (TRPA1) is a Ca2+-permeable ion channel that is sensitive to reactive oxygen species (ROS). The role of TRPA1 in AKI remains unclear. In this study, we used human and animal studies to assess the role of renal TRPA1 in AKI and to explore the regulatory mechanism of renal TRPA1 in inflammation via in vitro experiments. TRPA1 expression increased in the renal tubular epithelia of patients with AKI. The severity of tubular injury correlated well with tubular TRPA1 or 8-hydroxy-2'-deoxyguanosine expression. In an animal model, renal ischemia-reperfusion injury (IR) increased tubular TRPA1 expression in wild-type (WT) mice. Trpa1-/- mice displayed less IR-induced tubular injury, oxidative stress, inflammation, and dysfunction in kidneys compared with WT mice. In the in vitro model, TRPA1 expression increased in renal tubular cells under hypoxia-reoxygenation injury (H/R) conditions. We demonstrated that H/R evoked a ROS-dependent TRPA1 activation, which elevated intracellular Ca2+ level, increased NADPH oxidase activity, activated MAPK/NF-κB signaling, and increased IL-8. Renal tubular TRPA1 may serve as an oxidative stress sensor and a crucial regulator in the activation of signaling pathways and promote the subsequent transcriptional regulation of IL-8. These actions might be evident in mice with IR or patients with AKI.


Assuntos
Lesão Renal Aguda/metabolismo , Desoxiguanosina/metabolismo , Túbulos Renais/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/genética , Traumatismo por Reperfusão/metabolismo , Canal de Cátion TRPA1/metabolismo , Lesão Renal Aguda/enzimologia , Lesão Renal Aguda/genética , Adulto , Animais , Cálcio/metabolismo , Linhagem Celular , Desoxiguanosina/análogos & derivados , Modelos Animais de Doenças , Epitélio/metabolismo , Epitélio/patologia , Humanos , Imuno-Histoquímica , Interleucina-8/metabolismo , Túbulos Renais/citologia , Túbulos Renais/enzimologia , Túbulos Renais/patologia , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADP/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canal de Cátion TRPA1/genética
5.
J Vis Exp ; (168)2021 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-33645590

RESUMO

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due to the lack of causative therapies, there is an urgent need for innovative experimental organ culture models to study the mechanisms involved in the progression of the disease, find therapeutic targets, and reduce the need for animal models. We here present a novel, three-dimensional organ culture model protocol mimicking the proinflammatory and catabolic microenvironment, which is present during IDD. Initially, bovine caudal IVDs were dissected, cleaned, and cultured in the tissue culture medium. Dynamic physiologic or pathologic loading was applied in a custom-made bioreactor for 2 hours per day. IVDs were assigned to a control group (high glucose medium, physiological loading, phosphate-buffered saline injection) and a pathological group (low glucose medium, pathological loading, tumor necrosis factor-alpha injection) for four days. Gene expression analysis from collected nucleus pulposus cells of the IVDs and enzyme-linked immunosorbent assay of the conditioned organ culture media was performed. Our data revealed a higher expression of inflammatory markers and reduced disc heights after loading in the pathological group compared to the control group. This protocol is reliable to simulate IVD inflammation and degeneration and can be further expanded to broaden its application scope.


Assuntos
Inflamação/patologia , Degeneração do Disco Intervertebral/patologia , Deslocamento do Disco Intervertebral/patologia , Modelos Biológicos , Técnicas de Cultura de Órgãos/métodos , Animais , Anel Fibroso/patologia , Bovinos , Meios de Cultivo Condicionados/metabolismo , Regulação da Expressão Gênica , Injeções , Interleucina-8/metabolismo , Núcleo Pulposo/patologia , Fator de Necrose Tumoral alfa/administração & dosagem
6.
Mol Carcinog ; 60(3): 188-200, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33544929

RESUMO

Interaction between a tumor and its microenvironment is important for tumor initiation and progression. Cancer stem cells (CSCs) within the tumor interact with a microenvironmental niche that controls their maintenance and differentiation. We investigated the CSC-promoting effect of factors released from myofibroblasts into the microenvironment of early colorectal cancer tumors and its molecular mechanism. By messenger RNA microarray analysis, expression of HES1, a Notch signaling target, significantly increased in Caco-2 cells cocultured with 18Co cells (pericryptal myofibroblasts), compared to its expression in Caco-2 cells cultured alone. Caco-2 cells cultured in 18Co-conditioned media (CM) showed a significant increase in CD133+CD44+ cells and HES1 expression compared to that in Caco-2 cells cultured in regular media. Significant amounts of interleukin-6 (IL-6) and IL-8 were detected in 18Co-CM compared to levels in regular media. The 18Co-CM-induced increase in CD133+CD44+ cells was attenuated by IL-6- and IL-8-neutralizing antibodies. Furthermore, these neutralizing antibodies and inhibitors of STAT3 and gamma-secretase reduced the expression of HES1 induced in Caco-2 cells cultured in 18Co-CM. Immunohistochemical analysis of human tissues revealed that IL-6, IL-8, and HES1 expression increased from normal to adenoma, and from adenoma to cancer tissues. In addition, IL-6 and HES1 expression was positively correlated in early colorectal cancer tissues. In conclusion, the increase of CSCs by myofibroblasts could be mediated by IL-6/IL-8-induced HES1 activation in the tumor microenvironment. Based on these data, the IL-6/IL-8-mediated Notch/HES1 and STAT3 pathway, through which CSCs interact with their microenvironment, might be a potential target for the prevention and treatment of colorectal tumors.


Assuntos
Neoplasias Colorretais/patologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição HES-1/metabolismo , Células CACO-2 , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Meios de Cultivo Condicionados/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Organoides/patologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição HES-1/genética , Microambiente Tumoral/efeitos dos fármacos
7.
Cell Prolif ; 54(4): e13002, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33615584

RESUMO

OBJECTIVES: Ex vivo engineered production of megakaryocytes (MKs) and platelets (PLTs) from human pluripotent stem cells is an alternative approach to solve shortage of donor-donated PLTs in clinics and to provide induced PLTs for transfusion. However, low production yields are observed and the generation of clinically applicable MKs and PLTs from human pluripotent stem cells without genetic modifications still needs to be improved. MATERIALS AND METHODS: We defined an optimal, stepwise and completely xeno-free culture protocol for the generation of MKs from human embryonic stem cells (hESCs). To generate MKs from hESCs on a large scale, we improved the monolayer induction manner to define three-dimensional (3D) and sphere-like differentiation systems for MKs by using a special polystyrene CellSTACK culture chamber. RESULTS: The 3D manufacturing system could efficiently generate large numbers of MKs from hESCs within 16-18 days of continuous culturing. Each CellSTACK culture chamber could collect on an average 3.4 × 108 CD41+ MKs after a three-stage orderly induction process. MKs obtained from hESCs via 3D induction showed significant secretion of IL-8, thrombospondin-1 and MMP9. The induced cells derived from hESCs in our culture system were shown to have the characteristics of MKs as well as the function to form proPLTs and release PLTs. Furthermore, we generated clinically applicable MKs from clinical-grade hESC lines and confirmed the biosafety of these cells. CONCLUSIONS: We developed a simple, stepwise, 3D and completely xeno-free/feeder-free/transgene-free induction system for the generation of MKs from hESCs. hESC-derived MKs were shown to have typical MK characteristics and PLT formation ability. This study further enhances the clinical applications of MKs or PLTs derived from pluripotent stem cells.


Assuntos
Diferenciação Celular , Meios de Cultura/química , Células-Tronco Embrionárias Humanas/citologia , Megacariócitos/citologia , Técnicas de Cultura de Células , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Interleucina-8/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Megacariócitos/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Trombospondina 1/metabolismo , Fatores de Transcrição/metabolismo
8.
Int J Mol Sci ; 22(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572938

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global public health emergency. Periodontitis, the most prevalent disease that leads to tooth loss, is caused by infection by periodontopathic bacteria. Periodontitis is also a risk factor for pneumonia and the exacerbation of chronic obstructive pulmonary disease, presumably because of the aspiration of saliva contaminated with periodontopathic bacteria into the lower respiratory tract. Patients with these diseases have increased rates of COVID-19 aggravation and mortality. Because periodontopathic bacteria have been isolated from the bronchoalveolar lavage fluid of patients with COVID-19, periodontitis may be a risk factor for COVID-19 aggravation. However, the molecular links between periodontitis and COVID-19 have not been clarified. In this study, we found that the culture supernatant of the periodontopathic bacterium Fusobacterium nucleatum (CSF) upregulated the SARS-CoV-2 receptor angiotensin-converting enzyme 2 in A549 alveolar epithelial cells. In addition, CSF induced interleukin (IL)-6 and IL-8 production by both A549 and primary alveolar epithelial cells. CSF also strongly induced IL-6 and IL-8 expression by BEAS-2B bronchial epithelial cells and Detroit 562 pharyngeal epithelial cells. These results suggest that when patients with mild COVID-19 frequently aspirate periodontopathic bacteria, SARS-CoV-2 infection is promoted, and inflammation in the lower respiratory tract may become severe in the presence of viral pneumonia.


Assuntos
/metabolismo , Meios de Cultivo Condicionados/química , Citocinas/metabolismo , Fusobacterium nucleatum/metabolismo , /genética , /virologia , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Regulação para Cima/efeitos dos fármacos
9.
Nat Commun ; 12(1): 289, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436554

RESUMO

Mechanical ventilation generates injurious forces that exacerbate lung injury. These forces disrupt lung barrier integrity, trigger proinflammatory mediator release, and differentially regulate genes and non-coding oligonucleotides including microRNAs. In this study, we identify miR-146a as a mechanosensitive microRNA in alveolar macrophages that has therapeutic potential to mitigate lung injury during mechanical ventilation. We use humanized in-vitro systems, mouse models, and biospecimens from patients to elucidate the expression dynamics of miR-146a needed to decrease lung injury during mechanical ventilation. We find that the endogenous increase in miR-146a following injurious ventilation is not sufficient to prevent lung injury. However, when miR-146a is highly overexpressed using a nanoparticle delivery platform it is sufficient to prevent injury. These data indicate that the endogenous increase in microRNA-146a during mechanical ventilation is a compensatory response that partially limits injury and that nanoparticle delivery of miR-146a is an effective strategy for mitigating lung injury during mechanical ventilation.


Assuntos
Técnicas de Transferência de Genes , Lesão Pulmonar/genética , Macrófagos Alveolares/metabolismo , Mecanotransdução Celular , Nanopartículas/química , Respiração Artificial/efeitos adversos , Transferência Adotiva , Animais , Lavagem Broncoalveolar , Feminino , Humanos , Inflamação/genética , Inflamação/patologia , Interleucina-8/metabolismo , Masculino , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Células THP-1 , Regulação para Cima/genética
10.
Ecotoxicol Environ Saf ; 208: 111609, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396129

RESUMO

With the wide application of neodymium oxide nanoparticles (NPs-Nd2O3) in various fields, their health hazards have aroused public concern in recent years. However, data regarding the cytotoxicity of NPs-Nd2O3 is limited. In this study, we investigated the function and mechanism of long-chain non-coding RNAs (lncRNAs) in NPs-Nd2O3-induced airway inflammation. Treatment with NPs-Nd2O3 induced an inflammatory response in human bronchial epithelial cells (16HBE) by upregulating the expression of interleukin-6 (IL-6) and interleukin-8 (IL-8). The levels of LDH and intracellular ROS in the cells treated by various doses of NPs-Nd2O3 also increased significantly. After treatment with 10 µg/ml NPs-Nd2O3, RNA microarray and real-time quantitative polymerase chain reaction (qRT-PCR) showed a significant upregulation of lncRNA loc105377478. Functional experiments suggested lncRNA loc105377478 enhanced the expression of IL-6, IL-8 and ROS in NPs-Nd2O3-treated 16HBE cells, and it was further demonstrated that lncRNA loc105377478 promoted the activation of NF-κB by negatively regulating ADIPOR1 expression. Moreover, the expression of IL-6 and IL-8 in NPs-Nd2O3-treated 16HBE cells was regulated by lncRNA loc105377478, which was mediated by the NF-κB signaling pathway. In conclusion, lncRNA loc105377478 promotes NF-κB activation by negatively regulating ADIPOR1 expression, thereby upregulating the expression of IL-6 and IL-8 in 16HBE cells treated with NPs-Nd2O3.


Assuntos
Células Epiteliais/efeitos dos fármacos , NF-kappa B/metabolismo , Nanopartículas/química , Nanopartículas/toxicidade , Neodímio/toxicidade , Óxidos/toxicidade , RNA Longo não Codificante/genética , Receptores de Adiponectina/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Brônquios/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Relação Dose-Resposta a Droga , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Inflamação , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Neodímio/química , Óxidos/química , Transdução de Sinais , Regulação para Cima
11.
Ecotoxicol Environ Saf ; 208: 111693, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396024

RESUMO

Arsenic is a natural chemical element that is strongly associated with bladder cancer. Understanding the underlying mechanisms behind the association between arsenic and bladder cancer as well as identifying effective preventive interventions will help reduce the incidence and mortality of this disease. The epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties play key roles in cancer development and progression. Here, we reported that chronic exposure to arsenic resulted in EMT and increased levels of the CSC marker CD44 in human uroepithelial cells. Furthermore, IL-8 promoted a mesenchymal phenotype and upregulated CD44 by activating the ERK, AKT and STAT3 signaling. Phosphorylation of the human epidermal growth factor receptor 2 (HER2) was key for arsenic-induced IL-8 overexpression and depended on the simultaneous activation of the MAPK, JNK, PI3K/AKT and GSK3ß signaling pathways. We also found that genistein inhibited arsenic-induced HER2 phosphorylation and downregulated its downstream signaling pathways, thereby inhibiting progression of EMT, and reducing CD44 expression levels. These results demonstrate that the HER2/IL-8 axis is related to the acquisition of an EMT phenotype and CSCs in arsenic-treated cells. The inhibitory effects of genistein on EMT and CSCs provide a new perspective for the intervention and potential chemotherapy against arsenic-induced bladder cancer.


Assuntos
Arsênico/toxicidade , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Interleucina-8/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptor ErbB-2/genética , Bexiga Urinária/metabolismo , Arsênico/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/imunologia , Humanos , Receptores de Hialuronatos/genética , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bexiga Urinária/citologia
12.
Nat Commun ; 12(1): 302, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436591

RESUMO

Pemphigoid diseases refer to a group of severe autoimmune skin blistering diseases characterized by subepidermal blistering and loss of dermal-epidermal adhesion induced by autoantibody and immune cell infiltrate at the dermal-epidermal junction and upper dermis. Here, we explore the role of the immune cell-secreted serine protease, granzyme B, in pemphigoid disease pathogenesis using three independent murine models. In all models, granzyme B knockout or topical pharmacological inhibition significantly reduces total blistering area compared to controls. In vivo and in vitro studies show that granzyme B contributes to blistering by degrading key anchoring proteins in the dermal-epidermal junction that are necessary for dermal-epidermal adhesion. Further, granzyme B mediates IL-8/macrophage inflammatory protein-2 secretion, lesional neutrophil infiltration, and lesional neutrophil elastase activity. Clinically, granzyme B is elevated and abundant in human pemphigoid disease blister fluids and lesional skin. Collectively, granzyme B is a potential therapeutic target in pemphigoid diseases.


Assuntos
Doenças Autoimunes/enzimologia , Doenças Autoimunes/patologia , Granzimas/antagonistas & inibidores , Granzimas/metabolismo , Animais , Autoantígenos/metabolismo , Vesícula , Quimiocina CXCL2/metabolismo , Fatores Quimiotáticos/farmacologia , Modelos Animais de Doenças , Epidermólise Bolhosa/enzimologia , Epidermólise Bolhosa/patologia , Humanos , Inflamação/patologia , Integrina alfa6/metabolismo , Interleucina-8/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Colágenos não Fibrilares/metabolismo , Penfigoide Bolhoso/enzimologia , Penfigoide Bolhoso/patologia , Índice de Gravidade de Doença
13.
Anticancer Res ; 41(2): 783-794, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33517283

RESUMO

BACKGROUND/AIM: The inflammatory cytokine IL-8 and its receptor CXCR2 are key signalling pathway molecules in cancer development. We hypothesized that IL-8/CXCR2 signalling promotes tumour progression in oesophageal squamous cell carcinoma (ESCC) patients. MATERIALS AND METHODS: We examined the relationship between IL-8/CXCR2 expression and clinicopathological factors by immunohistochemistry in samples from 63 patients with resectable ESCC. The effects of IL-8/CXCR2 signalling on cell proliferation and gene expression were examined in vitro and in vivo using ESCC cell lines. RESULTS: Increased IL-8/CXCR2 signalling was associated with shorter overall survival (p<0.05) and recurrence-free survival (p<0.05) in ESCC patients. Multivariate analysis identified IL-8/CXCR2 expression as a prognostic factor for surgically treated ESCC (p<0.05). In vitro, IL-8 exposure or over-expression significantly enhanced ESCC cell proliferation. SB225002, a CXCR2-specific antagonist, and IL-8 siRNA significantly suppressed cell proliferation. CONCLUSION: IL-8/CXCR2 expression is an independent prognostic factor for surgically treated ESCC, and IL-8/CXCR2 signalling contributes to ESCC cell proliferation.


Assuntos
Neoplasias Esofágicas/cirurgia , Carcinoma de Células Escamosas do Esôfago/cirurgia , Interleucina-8/metabolismo , Receptores de Interleucina-8B/metabolismo , Regulação para Cima , Idoso , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/antagonistas & inibidores , Interleucina-8/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Compostos de Fenilureia/farmacologia , Prognóstico , RNA Interferente Pequeno/farmacologia , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/genética , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida
14.
Mol Med Rep ; 23(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33215220

RESUMO

In recent years hydrogen sulfide (H2S) has demonstrated vasculoprotective effects against cell death, which suggests its promising therapeutic potential for numerous types of disease. Additionally, a protective effect of exogenous H2S in HG­induced injuries in HUVECs was demonstrated, suggesting a potential protective effect for diabetic vascular complications. The present study aimed to investigate the mechanism accounting for the cytoprotective role of exogenous H2S against high glucose [HG (40 mM glucose)]­induced injury and inflammation in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to HG for 24 h to establish an in vitro model of HG­induced cytotoxicity. The cells were pretreated with sodium hydrosulfide (NaHS), a donor of H2S, or inhibitors of necroptosis and p38 MAPK prior to the exposure to HG. Cell viability, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), IL­1ß, IL­6, IL­8, TNF­α, phosphorylated­(p)38 and receptor­interacting protein 3 (RIP3) expression levels were detected using the indicated methods, including Cell Counting Kit 8, fluorescence detection, western blotting, immunofluorescence assay and ELISAs. The results demonstrated that necroptosis and the p38 MAPK signaling pathway mediated HG­induced injury and inflammation. Notably, NaHS was discovered to significantly ameliorate p38 MAPK/necroptosis­mediated injury and inflammation in response to HG, as evidenced by an increase in cell viability, a decrease in ROS generation and loss of MMP, as well as the reduction in the secretion of proinflammatory cytokines. In addition, the upregulated expression of RIP3 induced by HG was repressed by treatment with SB203580, while the HG­induced upregulation of p­p38 expression levels were significantly downregulated following the treatment of Nec­1 and RIP3­siRNA. In conclusion, the findings of the present study indicated that NaHS may protect HUVECs against HG­induced injury and inflammation by inhibiting necroptosis via the p38 MAPK signaling pathway, which may represent a promising drug for the therapy of diabetic vascular complications.


Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Inflamação/prevenção & controle , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Necroptose/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Sulfetos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/prevenção & controle , Glucose/toxicidade , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/induzido quimicamente , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Metaloproteinases da Matriz/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Front Immunol ; 11: 563699, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33324395

RESUMO

IgA nephropathy (IgAN) is the most common primary glomerular disease. The characteristic pathology involves immune complexes formed by the deposition of IgA1 and underglycosylated IgA1 aggregates in the mesangial area, which may be accompanied by the deposition of IgG and/or IgM and complement components. However, the molecular mechanisms of IgAN remain unclear. In the present study, microarray analysis showed that the expression of microRNA-630 (miR-630) was significantly reduced in palatal tonsils from IgAN patients compared with chronic tonsillitis. Additionally, bioinformatic analysis showed that Toll-like receptor 4 (TLR4) was the predicted target gene of miR-630 and was regulated by miR-630. When miR-630 was overexpressed in palatal tonsil mononuclear cells from IgAN patients, the expression of TLR4 was reduced and the content of IgA1 in the cell culture supernatant was decreased, and the level of galactosylation in the IgA1 hinge region was increased. Moreover, immunohistochemical analysis showed that the expression of TLR4 in IgAN patients was significantly increased. After knocking down the expression of TLR4, both the concentration of IgA1 and the binding force of IgA1 with broad bean lectin were significantly reduced in IgAN. Furthermore, the mechanism study demonstrated that TLR4 might regulate the expression of IL-1ß and IL-8 through NF-κB signaling pathway to modulate the concentration of IgA1 and the glycosylation level of IgA1. This interesting finding may offer new insight into the molecular mechanism of IgAN.


Assuntos
Glomerulonefrite por IGA/imunologia , Imunoglobulina A/biossíntese , MicroRNAs/metabolismo , Tonsila Palatina/imunologia , Receptor 4 Toll-Like/metabolismo , Adolescente , Adulto , Biomarcadores/metabolismo , Células Cultivadas , Criança , Feminino , Técnicas de Silenciamento de Genes , Glicosilação , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Masculino , MicroRNAs/genética , NF-kappa B/metabolismo , Tonsila Palatina/patologia , Transdução de Sinais/genética , Transfecção , Adulto Jovem
16.
PLoS One ; 15(12): e0244720, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382822

RESUMO

OBJECTIVE: We sought to identify novel biomarkers in the amniotic fluid (AF) related to imminent spontaneous preterm delivery (SPTD) (≤ 14 days after sampling) in women with early preterm premature rupture of membranes (PPROM), using a protein microarray. METHOD: This was a retrospective cohort study of a total of 88 singleton pregnant women with PPROM (23+0 to 30+6 weeks) who underwent amniocentesis. A nested case-control study for biomarker discovery was conducted using pooled AF samples from controls (non-imminent delivery, n = 15) and cases (imminent SPTD, n = 15), which were analyzed using an antibody microarray. Quantitative validation of four candidate proteins was performed, using ELISA, in the total cohort (n = 88). IL-8, MMP-9, and Fas levels were additionally measured for the comparison and to examine association of SPTD with the etiologic factors of PPROM. RESULTS: Of all the proteins studied in the protein microarray, four showed significant intergroup differences. Analyses of the total cohort by ELISA confirmed the significantly elevated concentrations of AF lipocalin-2, MMP-9, and S100 A8/A9, but not of endostatin and Fas, in women who delivered within 14 days of sampling. For inflammatory proteins showing a significant association, the odds of SPTD within 14 days increased significantly with an increase in baseline AF levels of the proteins (P for trend <0.05 for each) in each quartile, especially in the 3rd and 4th quartile. CONCLUSIONS: We identified several potential novel biomarkers (i.e., lipocalin-2, MMP-9, and S100 A8/A9) related to SPTD within 14 days of sampling, all of which are inflammation-related molecules. Furthermore, the SPTD risk increased with increasing quartiles of each of these inflammatory proteins, especially the 3rd and 4th quartile of each protein. The present findings may highlight the importance of inflammatory mechanisms and the degree of activated inflammatory response in developing SPTD in early PPROM.


Assuntos
Líquido Amniótico/metabolismo , Ruptura Prematura de Membranas Fetais/metabolismo , Trabalho de Parto Prematuro/metabolismo , Nascimento Prematuro/diagnóstico , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Recém-Nascido , Interleucina-8/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Gravidez , Nascimento Prematuro/metabolismo , Análise Serial de Proteínas , Estudos Retrospectivos , Receptor fas/metabolismo
17.
PLoS One ; 15(12): e0244327, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33373367

RESUMO

Limbal stem cells (LSCs) are a quiescent cell population responsible for the renewal of the corneal epithelium. Their deficiency is responsible for the conjunctivization of the cornea that is seen in different ocular pathologies, both in humans and in the canine species. The canine species represents an interesting preclinical animal model in ocular surface pathologies. However, the role of LSCs in physiological and pathological conditions in canine species is not well understood. Our objective was to characterize for the first time the soluble factors and the proteomic profile of the secretome and exosomes of canine LSCs (cLSCs). In addition, given the important role that fibroblasts play in the repair of the ocular surface, we evaluated the influence of the secretome and exosomes of cLSCs on their proliferation in vitro. Our results demonstrated a secretory profile of cLSCs with high concentrations of MCP-1, IL-8, VEGF-A, and IL-10, as well as significant production of exosomes. Regarding the proteomic profile, 646 total proteins in the secretome and 356 in exosomes were involved in different biological processes. Functionally, the cLSC secretome showed an inhibitory effect on the proliferation of fibroblasts in vitro, which the exosomes did not. These results open the door to new studies on the possible use of the cLSC secretome or some of its components to treat certain pathologies of the ocular surface in canine species.


Assuntos
Biomarcadores/metabolismo , Meios de Cultura/análise , Exossomos/metabolismo , Limbo da Córnea/citologia , Proteômica/métodos , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Quimiocina CCL2/metabolismo , Cães , Interleucina-10/metabolismo , Interleucina-8/metabolismo , Limbo da Córnea/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
18.
Cell Death Dis ; 11(11): 957, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33159040

RESUMO

A global effort is currently undertaken to restrain the COVID-19 pandemic. Host immunity has come out as a determinant for COVID-19 clinical outcomes, and several studies investigated the immune profiling of SARS-CoV-2 infected people to properly direct the clinical management of the disease. Thus, lymphopenia, T-cell exhaustion, and the increased levels of inflammatory mediators have been described in COVID-19 patients, in particular in severe cases1. Age represents a key factor in COVID-19 morbidity and mortality2. Understanding age-associated immune signatures of patients are therefore important to identify preventive and therapeutic strategies. In this study, we investigated the immune profile of COVID-19 hospitalized patients identifying a distinctive age-dependent immune signature associated with disease severity. Indeed, defined circulating factors - CXCL8, IL-10, IL-15, IL-27, and TNF-α - positively correlate with older age, longer hospitalization, and a more severe form of the disease and may thus represent the leading signature in critical COVID-19 patients.


Assuntos
Infecções por Coronavirus/patologia , Citocinas/metabolismo , Pneumonia Viral/patologia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Análise por Conglomerados , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Imunoglobulina G/sangue , Interleucina-10/metabolismo , Interleucina-8/metabolismo , Tempo de Internação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/metabolismo
19.
Folia Biol (Praha) ; 66(3): 104-110, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33069189

RESUMO

Cancer development is a highly complicated process in which tumour growth depends on the development of its vascularization system. To support their own growth, tumour cells significantly modify their microenvironment. One of such modifications inflicted by tumours is stimulation of endothelial cell migration and proliferation. There is accumulating evidence that extracellular vesicles (EVs) secreted by tumour cells (tumour-derived EVs, TEVs) may be regarded as "messengers" with the potential for affecting the biological activities of target cells. Interaction of TEVs with different cell types occurs in an auto- and paracrine manner and may lead to changes in the function of the latter, e.g., promoting motility, proliferation, etc. This study analysed the proangiogenic activity of EVs derived from human pancreatic adenocarcinoma cell line (HPC-4, TEVHPC) in vitro and their effect in vivo on Matrigel matrix vascularization in severe combined immunodeficient (SCID) mice. TEVHPC enhanced proliferation of HPC-4 cells and induced their motility. Moreover, TEVHPC stimulated human umbilical vein endothelial cell (HUVEC) proliferation and migration in vitro. Additionally, TEVHPC influenced secretion of proangiogenic factors (IL-8, VEGF) by HUVEC cells and supported Matrigel matrix haemoglobinization in vivo. These data show that TEVs may support tumour propagation in an autocrine manner and may support vascularization of the tumour. The presented data are in line with the theory that tumour cells themselves are able to modulate the microenvironment via TEVs to maximize their growth potential.


Assuntos
Carcinoma Ductal Pancreático/patologia , Vesículas Extracelulares/patologia , Neoplasias Pancreáticas/patologia , Animais , Comunicação Autócrina , Divisão Celular , Linhagem Celular Tumoral , Quimiotaxia , Colágeno , Combinação de Medicamentos , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Laminina , Camundongos , Camundongos SCID , Transplante de Neoplasias , Neovascularização Patológica/etiologia , Proteoglicanas , RNA Mensageiro/biossíntese , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
PLoS One ; 15(10): e0235803, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031374

RESUMO

Cystic Fibrosis (CF), caused by mutations affecting the CFTR gene, is characterised by viscid secretions in multiple organ systems. CF airways contain thick mucus, creating a gradient of hypoxia, which promotes the establishment of polymicrobial infection. Such inflammation predisposes to further infection, a self-perpetuating cycle in mediated by NF-κB. Anaerobic Gram-negative Prevotella spp. are found in sputum from healthy volunteers and CF patients and in CF lungs correlate with reduced levels of inflammation. Prevotella histicola (P. histicola) can suppress murine lung inflammation, however, no studies have examined the role of P. histicola in modulating infection and inflammation in the CF airways. We investigated innate immune signalling and NF-kB activation in CF epithelial cells CFBE41o- in response to clinical stains of P. histicola and Pseudomonas aeruginosa (P. aeruginosa). Toll-Like Receptor (TLR) expressing HEK-293 cells and siRNA assays for TLRs and IKKα were used to confirm signalling pathways. We show that P. histicola infection activated the alternative NF-kB signalling pathway in CF bronchial epithelial cells inducing HIF-1α protein. TLR5 signalling was responsible for the induction of the alternative NF-kB pathway through phosphorylation of IKKα. The induction of transcription factor HIF-1α was inversely associated with the induction of the alternative NF-kB pathway and knockdown of IKKα partially restored canonical NF-kB activation in response to P. histicola. This study demonstrates that different bacterial species in the respiratory microbiome can contribute differently to inflammation, either by activating inflammatory cascades (P. aeruginosa) or by muting the inflammatory response by modulating similar or related pathways (P. histicola). Further work is required to assess the complex interactions of the lung microbiome in response to mixed bacterial infections and their effects in people with CF.


Assuntos
Brônquios/imunologia , Fibrose Cística/imunologia , NF-kappa B/metabolismo , Prevotella/imunologia , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/imunologia , Receptores Toll-Like/metabolismo , Brônquios/metabolismo , Brônquios/microbiologia , Brônquios/patologia , Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Humanos , Interleucina-8/metabolismo , NF-kappa B/genética , Prevotella/isolamento & purificação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Transdução de Sinais , Receptores Toll-Like/imunologia
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