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1.
J Surg Res ; 245: 1-12, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31394402

RESUMO

BACKGROUND: The process of aortic injury, repair, and remodeling during aortic aneurysm and dissection is poorly understood. We examined the activation of bone marrow (BM)-derived and resident aortic cells in response to aortic injury in a mouse model of sporadic aortic aneurysm and dissection. MATERIALS AND METHODS: Wild-type C57BL/6 mice were transplanted with green fluorescent protein (GFP)+ BM cells. For 4 wk, these mice were either unchallenged with chow diet and saline infusion or challenged with high-fat diet and angiotensin II infusion. We then examined the aortic recruitment of GFP+ BM-derived cells, growth factor production, and the differentiation potential of GFP+ BM-derived and GFP- resident aortic cells. RESULTS: Aortic challenge induced recruitment of GFP+ BM cells and activation of GFP- resident aortic cells, both of which produced growth factors. Although BM cells and resident aortic cells equally contributed to the fibroblast populations, we did not detect the differentiation of BM cells into smooth muscle cells. Interestingly, aortic macrophages were both of BM-derived (45%) and of non-BM-derived (55%) origin. We also observed a significant increase in stem cell antigen-1 (Sca-1)+ stem/progenitor cells and neural/glial antigen 2 (NG2+) cells in the aortic wall of challenged mice. Although some of the Sca-1+ cells and NG2+ cells were BM derived, most of these cells were resident aortic cells. Sca-1+ cells produced growth factors and differentiated into fibroblasts and NG2+ cells. CONCLUSIONS: BM-derived and resident aortic cells are activated in response to aortic injury and contribute to aortic inflammation, repair, and remodeling by producing growth factors and differentiating into fibroblasts and inflammatory cells.


Assuntos
Aneurisma Dissecante/patologia , Aorta/patologia , Aneurisma Aórtico/patologia , Aneurisma Dissecante/etiologia , Aneurisma Dissecante/imunologia , Animais , Aorta/citologia , Aorta/imunologia , Aneurisma Aórtico/complicações , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Fibroblastos/imunologia , Fibroblastos/metabolismo , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo
3.
Allergol. immunopatol ; 47(6): 585-590, nov.-dic. 2019. graf
Artigo em Inglês | IBECS | ID: ibc-186552

RESUMO

Introduction and objectives: Transforming growth factor β1 (TGFβ1) and dysregulated microRNA-21 (miR-21) expression is associated with TGFβ/Smad signaling pathway activation and fibrosis. While calcitriol has been shown to improve airway remodeling in asthmatic mice, its mechanism remains unknown. In this study, the effect of calcitriol on the TGFβ/Smad signaling pathway and miR-21 expression in human bronchial fibroblasts was investigated to explore the mechanism of action of calcitriol and the inhaled glucocorticoid, budesonide, in airway remodeling. Materials and methods: Human bronchial fibroblasts were pretreated with budesonide, calcitriol, or budesonide plus calcitriol, and stimulated with TGFβ1 for 48h. Quantitative real-time PCR was used to determine the expression of miR-21. Western blot was used to determine airway remodeling-related proteins, TGFβ/Smad signaling pathway-related proteins, glucocorticoid receptor, and vitamin D receptor (VDR) expression. Results: Both budesonide and calcitriol down-regulated miR-21 expression in human bronchial fibroblasts, up-regulated Smad7 expression, and inhibited the expression of airway remodeling-related proteins. Both budesonide and calcitriol up-regulated the low expression of VDR induced by TGFβ1 in human bronchial fibroblasts. The expression of VDR in the combined treatment group (budesonide plus calcitriol) was significantly higher than that in the calcitriol treatment group. The expression of collagen type I in the combined treatment group was significantly lower than that in the calcitriol treatment group. Conclusions: Calcitriol can up-regulate the expression of VDR in human bronchial fibroblasts and exert an anti-airway remodeling effect. Budesonide can up-regulate the expression of VDR in human bronchial fibroblasts and enhance the inhibitory effect of calcitriol on airway remodeling


No disponible


Assuntos
Animais , Camundongos , Budesonida/uso terapêutico , Vitamina D/uso terapêutico , Fibroblastos/imunologia , Calcitriol/uso terapêutico , MicroRNAs/imunologia , Asma/imunologia , Fator de Crescimento Transformador beta1/imunologia , Western Blotting , Fator de Crescimento Transformador beta1/metabolismo , Análise de Variância
4.
Isr Med Assoc J ; 21(7): 454-459, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31507120

RESUMO

BACKGROUND: Platelets have the ability to influence the immune system and the inflammatory process and may be strongly involved in the whole pathogenic process of chronic inflammatory joint diseases, such as rheumatoid arthritis. They may play a significant role even before the clinical onset of the disease, contributing to the loss of tolerance of the immune system and the induction of autoimmunity. Subsequently, they can interact with the most important cellular players involved in autoimmunity and inflammation, namely innate immunity cells and T cells and eventually contribute to the building of inflammation in the synovium, thus inducing the activation, migration, and proliferation of fibroblasts that eventually lead to joint damage. Due to their peculiar features, studying the behavior of platelets is a challenging task; however, platelets may prove to be valuable therapeutic targets in the future.


Assuntos
Artrite Reumatoide/imunologia , Plaquetas/imunologia , Sinovite/imunologia , Artrite Reumatoide/patologia , Autoimunidade/imunologia , Fibroblastos/imunologia , Humanos , Imunidade Inata/imunologia , Inflamação/imunologia , Inflamação/patologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Sinovite/patologia , Linfócitos T/imunologia
5.
Mater Sci Eng C Mater Biol Appl ; 105: 110086, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546413

RESUMO

Chitosan (CS) has been reported to have utility for various potential applications in biomedicine, tissue engineering, and cosmetics, as well as in the formulation of antibacterial agents because it exhibits a variety of desirable attributes, including low-toxicity, biodegradability, excellent biocompatibility, and broad-spectrum antibacterial activity. However, the full realization of CS's biomedical applications are practically constrained by its poor solubility. The goal of the present study is to prepare hydroxybutyl chitosan (HBCS) and investigate its impacts on immunocompetence, and its antibacterial activity. In the current study, HBCS was synthesized by modifying the hydroxybutyl group on the chitosan molecule using an etherification method. The physicochemical properties of the synthesized HBCS were characterized by various methods. Results showed that hydro-soluble HBCS exhibited excellent hygroscopicity and moisture retention. It was also found that HBCS exhibited notable cytocompatibility when cultured with mouse embryo fibroblasts. HBCS was able regulate immuno-functionality and promote immunocompetence by improving phagocytosis of macrophages and reinforcing lymphocyte activity in vivo and in vitro. Moreover, HBCS was also found to inhibit L-929 cell migration, indicating the impeded migration and metastasis behaviors of fibrosarcoma cells. Additionally, HBCS displayed favorable antimicrobial functionality against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. This study demonstrated that HBCS could in turn lock moisture, can promote immunocompetence activity, can inhibit fibrosarcoma cell migration, and exhibits anti-bacterial functionality. Taken together, these results indicate that HBCS shows substantial promise for applications in cosmetics, biomedicine, and antibacterial therapies.


Assuntos
Antibacterianos , Quitosana/análogos & derivados , Embrião de Mamíferos/imunologia , Escherichia coli/crescimento & desenvolvimento , Fibroblastos/imunologia , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Quitosana/síntese química , Quitosana/química , Quitosana/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos ICR
6.
Folia Histochem Cytobiol ; 57(3): 116-126, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31388982

RESUMO

INTRODUCTION: Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a selective suppressor of the immune response that has been linked to the evasion of immune surveillance by cancer cells. However, the exact prognostic impact of RCAS1 on epithelial ovarian cancer (EOC) has not been fully elucidated. The main aim of our study was to evaluate the influence of RCAS1 immunoreactivity (RCAS1-Ir) in EOC cells and in tumor stroma cells on patient overall survival. We also focused on RCAS1-Ir and the structure of the tumor stroma. MATERIAL AND METHODS: RCAS1-Ir was evaluated by means of immunohistochemistry in 67 patients with EOC. We distinguished cytoplasmic and membranous immunoreactivity patterns. RESULTS: We found that high cytoplasmic RCAS1-Ir in cancer cells was associated with more than a two-time shortened period of overall survival. Membranous RCAS1-Ir in cancer cells, as well as in tumor stroma macrophages and fibroblasts, did not correlate with patient survival. RCAS1-Ir in the cytoplasm of cancer cells was positively correlated with the degree of tumor stroma infiltration by fibroblasts and macrophages, but not with RCAS1-Ir in these cells. On the other hand, membranous RCAS1-Ir in cancer cells was positively correlated with RCAS1-Ir in fibroblasts and macrophages, but not with their quantity. CONCLUSIONS: Due to their different impacts on patient prognosis and tumor stroma structure, it seems that cytoplasmic and membranous RCAS1-Ir in EOC cells may have different biological functions.


Assuntos
Antígenos de Neoplasias/imunologia , Carcinoma Epitelial do Ovário/diagnóstico , Neoplasias Ovarianas/diagnóstico , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Epitelial do Ovário/imunologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Citoplasma/imunologia , Feminino , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Pessoa de Meia-Idade , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Prognóstico
7.
PLoS Pathog ; 15(8): e1008002, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31404116

RESUMO

The galectin 3 binding protein (LGALS3BP, also known as 90K) is a ubiquitous multifunctional secreted glycoprotein originally identified in cancer progression. It remains unclear how 90K functions in innate immunity during viral infections. In this study, we found that viral infections resulted in elevated levels of 90K. Further studies demonstrated that 90K expression suppressed virus replication by inducing IFN and pro-inflammatory cytokine production. Upon investigating the mechanisms behind this event, we found that 90K functions as a scaffold/adaptor protein to interact with TRAF6, TRAF3, TAK1 and TBK1. Furthermore, 90K enhanced TRAF6 and TRAF3 ubiquitination and served as a specific ubiquitination substrate of TRAF6, leading to transcription factor NF-κB, IRF3 and IRF7 translocation from the cytoplasm to the nucleus. Conclusions: 90K is a virus-induced protein capable of binding with the TRAF6 and TRAF3 complex, leading to IFN and pro-inflammatory production.


Assuntos
Antígenos de Neoplasias/fisiologia , Biomarcadores Tumorais/fisiologia , Glicoproteínas/fisiologia , Fator 3 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Viroses/imunologia , Replicação Viral , Vírus/imunologia , Animais , Células Cultivadas , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Viroses/metabolismo , Viroses/virologia
8.
J Immunol Res ; 2019: 4080735, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428656

RESUMO

Rheumatoid arthritis (RA) and osteoarthritis (OA) are common rheumatic disorders that primarily involve joints. The inflammation of the synovium can be observed in both of the two diseases. Synovial fibroblasts (SFs) play an important role in the inflammatory process of the synovium. The functional states of synovial fibroblasts are heterogeneous, and the detailed transition process of their functional states is still unclear. By using transcriptomic data of SFs at a single-cell level, we found a similar transition process for SFs in RA and OA. We also identified the potential regulatory effects of the WNT signaling pathway, the TGF-ß signaling pathway, the FcεRI signaling pathway, and the ERBB signaling pathway on modifying the SFs' functional state. These findings indicate potentially overlapped pathogenic mechanisms in these two diseases, which may help uncover new therapeutic targets to ameliorate disease progression.


Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Osteoartrite/metabolismo , Membrana Sinovial/citologia , Artrite Reumatoide/imunologia , Progressão da Doença , Fibroblastos/imunologia , Genes erbB , Humanos , Osteoartrite/imunologia , Análise de Célula Única , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt
9.
Gastroenterology ; 157(6): 1572-1583.e8, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31470007

RESUMO

BACKGROUND & AIMS: Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1 beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. METHODS: We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. RESULTS: Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha+ (PDGRFA+) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genes that regulate matrix fibroblasts and inflammation. CONCLUSIONS: In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA+ fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA+ fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.


Assuntos
Pólipos do Colo/imunologia , Receptores ErbB/metabolismo , Interleucina-1beta/metabolismo , Mucosa Intestinal/patologia , Metaloproteinase 3 da Matriz/metabolismo , Animais , Apoptose/imunologia , Ceco/citologia , Ceco/imunologia , Ceco/patologia , Toxina Diftérica/administração & dosagem , Toxina Diftérica/imunologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/patologia , Receptores ErbB/antagonistas & inibidores , Fibroblastos/imunologia , Fibroblastos/metabolismo , Gefitinibe/farmacologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Interleucina-1beta/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Metaloproteinase 3 da Matriz/imunologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Camundongos Transgênicos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Sulfonamidas/farmacologia
10.
Saudi Med J ; 40(7): 657-668, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31287125

RESUMO

OBJECTIVES: To investigate the use of leukocyte-platelet rich fibrin on suppressing the porphyromonas gingivalis (PG-LPS)-induced secretion of proinflammatory cytokines. Methods:This quantitative experimental study was conducted at the School and Hospital of Stomatology, Jilin University, Changchun, China, between September 2017 and January 2019. A modified technique was used to obtain human gingival fibroblast cells (HGFCs). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Cell Counting Kit-8 tests were established to determine the proliferation rate. Human gingival fibroblast cells were treated by PG-LPS at different periods and the isolated mRNA was subjected to reverse transcription polymerase chain reaction and real time quantitative polymerase chain reaction. The release of platelet-derived growth factor and transforming-growth factor-ß1 at various time intervals was observed. Results: We successfully established a modified technique for the production of HGFCs culture. One µg/mL PG-LPS was the recommended concentration to inhibit fibroblast proliferation. The expression of the pro-inflammatory cytokines messenger ribnucleic acid was notably raised at 3 and 6 hours post-PG-LPS treatment. The cumulative release of growth factors peaked during the first 24 hours and the production continued for 10 days. However, the fibroblast expression of cytokines was significantly suppressed after treatment with leucocyte- and platelet-rich fibrin (L-PRF). Conclusion: This study provided a novel way of obtaining HGFCs and greater understanding of the clinical impacts through the assessment of the anti-inflammatory properties of L-PRF in vitro.


Assuntos
Citocinas/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fibrina Rica em Plaquetas , Porphyromonas gingivalis/metabolismo , Estomatite , Animais , Técnicas de Cultura de Células , Proliferação de Células , Citocinas/genética , Citocinas/imunologia , Fibroblastos/imunologia , Gengiva/citologia , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase em Tempo Real
11.
PLoS One ; 14(7): e0218736, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31260471

RESUMO

LL-37 is the only human cathelicidin-family host defense peptide and has been reported to interact with invading pathogens causing inflammation at various body sites. Recent studies showed high levels of LL-37 in the synovial-lining membrane of patients with rheumatoid arthritis, a common type of inflammatory arthritis. The present study aims to investigate the role of LL-37 on mechanisms associated with pathogenesis of inflammatory arthritis. The effects of LL-37 on the expression of proinflammatory cytokines, hyaluronan (HA) metabolism-related genes, cell death-related pathways, and cell invasion were investigated in SW982, a human synovial sarcoma cell line. Time-course measurements of proinflammatory cytokines and mediators showed that LL-37 significantly induced IL6 and IL17A mRNA levels at early time points (3-6 hr). HA-metabolism-related genes (i.e., HA synthase 2 (HAS2), HAS3, hyaluronidase 1 (HYAL1), HYAL2, and CD44) were co-expressed in parallel. In combination, LL-37 and IL17A significantly enhanced PTGS2, TNF, and HAS3 gene expression concomitantly with the elevation of their respective products, PGE2, TNF, and HA. Cell invasion rates and FN1 gene expression were also significantly enhanced. However, LL-37 alone or combined with IL17A did not affect cell mortality or cell cycle. Treatment of SW982 cells with both LL-37 and IL17A significantly enhanced IKK and p65 phosphorylation. These findings suggest that the chronic production of a high level of LL-37 may synchronize with its downstream proinflammatory cytokines, especially IL17A, contributing to the co-operative enhancement of pathogenesis mechanisms of inflammatory arthritis, such as high production of proinflammatory cytokines and mediators together with the activation of HA-metabolism-associated genes and cell invasion.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Hialurônico/metabolismo , Interleucina-17/farmacologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Combinação de Medicamentos , Sinergismo Farmacológico , Fibroblastos/imunologia , Fibroblastos/patologia , Fibronectinas/genética , Fibronectinas/imunologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Hialuronan Sintases/genética , Hialuronan Sintases/imunologia , Ácido Hialurônico/imunologia , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/imunologia , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Inflamação , Interleucina-6/genética , Interleucina-6/imunologia , Transdução de Sinais , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
12.
Nucleic Acids Res ; 47(16): 8838-8859, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31329944

RESUMO

Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem-loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem-loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem-loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1-Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses.


Assuntos
Macrófagos Peritoneais/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Ribonucleases/genética , Transativadores/genética , Animais , Fibroblastos/citologia , Fibroblastos/imunologia , Células HEK293 , Células HeLa , Homeostase/genética , Homeostase/imunologia , Humanos , Imunidade Inata , Inflamação , Sequências Repetidas Invertidas , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Knockout , Mutação , Cultura Primária de Células , Ligação Proteica , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/imunologia , RNA Mensageiro/metabolismo , Ribonucleases/deficiência , Ribonucleases/imunologia , Imagem Individual de Molécula , Transativadores/imunologia
13.
Monoclon Antib Immunodiagn Immunother ; 38(4): 145-156, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31305212

RESUMO

Tumor necrosis factor-α (TNFα), one of the major proinflammatory cytokines, plays a key role in an effective immune response. However, the chronic presence of TNFα can lead to several inflammatory disorders, such as rheumatoid arthritis, psoriasis, Crohn's disease, etc. Inhibition of TNFα by pharmacological inhibitors or antibodies has proven to be effective in palliative treatment to some extent. The aim of this study was to develop an anti-TNFα antibody, which may be used as a therapeutic option to inhibit TNFα-mediated cytotoxicity. We characterized several hybridoma clones secreting monoclonal antibodies (mAbs) to human-TNFα. Four mAbs rescued L929 fibroblast cells from TNFα-triggered cell death and one of these, namely C8, was found to have the highest affinity. To gain insights into the mechanism by which mAb C8 inhibits human TNFα-mediated toxicity, the epitope corresponding to the mAb was delineated. The antigenic determinant was found to comprise of the stretch of amino acids 99-120, of which, 102-104 (glutamine, arginine, glutamic acid) form the core epitope. The observation was supported by bioinformatics analyses of an antigen/antibody complex model. In addition, the binding affinity of mAb C8 to TNFα was found to be comparable with that of infliximab, which is a commercially available anti-TNFα mAb.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Fibroblastos/imunologia , Hibridomas/imunologia , Imunoglobulina G/imunologia , Proteínas Recombinantes/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/biossíntese , Anticorpos Monoclonais Humanizados/farmacologia , Formação de Anticorpos , Células Cultivadas , Feminino , Fibroblastos/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C
14.
PLoS Biol ; 17(7): e3000072, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31306410

RESUMO

Lymphoid T-zone fibroblastic reticular cells (FRCs) actively promote T-cell trafficking, homeostasis, and expansion but can also attenuate excessive T-cell responses via inducible nitric oxide (NO) and constitutive prostanoid release. It remains unclear how these FRC-derived mediators dampen T-cell responses and whether this occurs in vivo. Here, we confirm that murine lymph node (LN) FRCs produce prostaglandin E2 (PGE2) in a cyclooxygenase-2 (COX2)-dependent and inflammation-independent fashion. We show that this COX2/PGE2 pathway is active during both strong and weak T-cell responses, in contrast to NO, which only comes into play during strong T-cell responses. During chronic infections in vivo, PGE2-receptor signaling in virus-specific cluster of differentiation (CD)8 cytotoxic T cells was shown by others to suppress T-cell survival and function. Using COX2flox/flox mice crossed to mice expressing Cre recombinase expression under control of the CC chemokine ligand (CCL19) promoter (CCL19cre), we now identify CCL19+ FRC as the critical source of this COX2-dependent suppressive factor, suggesting PGE2-expressing FRCs within lymphoid tissues are an interesting therapeutic target to improve T-cell-mediated pathogen control during chronic infection.


Assuntos
Ciclo-Oxigenase 2/imunologia , Fibroblastos/imunologia , Linfonodos/imunologia , Prostaglandinas/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Movimento Celular/genética , Movimento Celular/imunologia , Proliferação de Células/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Linfonodos/citologia , Linfonodos/metabolismo , Ativação Linfocitária/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Prostaglandinas/biossíntese , Linfócitos T/virologia
15.
Nat Commun ; 10(1): 2699, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221976

RESUMO

Human cytomegalovirus (CMV) causes a wide array of disease to diverse populations of immune-compromised individuals. Thus, a more comprehensive understanding of how CMV enters numerous host cell types is necessary to further delineate the complex nature of CMV pathogenesis and to develop targeted therapeutics. To that end, we establish a vaccination strategy utilizing membrane vesicles derived from epithelial cells to generate a library of monoclonal antibodies (mAbs) targeting cell surface proteins in their native conformation. A high-throughput inhibition assay is employed to screen these antibodies for their ability to limit infection, and mAbs targeting CD46 are identified. In addition, a significant reduction of viral proliferation in CD46-KO epithelial cells confirms a role for CD46 function in viral dissemination. Further, we demonstrate a CD46-dependent entry pathway of virus infection in trophoblasts, but not in fibroblasts, highlighting the complexity of CMV entry and identifying CD46 as an entry factor in congenital infection.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Proteína Cofatora de Membrana/imunologia , Internalização do Vírus , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/imunologia , Linhagem Celular , Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/virologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Técnicas de Inativação de Genes , Humanos , Proteína Cofatora de Membrana/genética , RNA Interferente Pequeno/metabolismo , Trofoblastos/imunologia , Trofoblastos/virologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
16.
PLoS Negl Trop Dis ; 13(6): e0007537, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31251739

RESUMO

Infection by Zika virus (ZIKV) is linked to microcephaly and other neurological disorders, posing a significant health threat. Innate immunity is the first line of defense against invading pathogens, but relatively little is understood regarding host intrinsic mechanisms that guard against ZIKV. Here, we show that host tripartite motif-containing protein 56 (TRIM56) poses a barrier to ZIKV infection in cells of neural, epithelial and fibroblast origins. Overexpression of TRIM56, but not an E3 ligase-dead mutant or one lacking a short C-terminal portion, inhibited ZIKV RNA replication. Conversely, depletion of TRIM56 increased viral RNA levels. Although the C-terminal region of TRIM56 bears sequence homology to NHL repeat of TRIM-NHL proteins that regulate miRNA activity, knockout of Dicer, which abolishes production of miRNAs, had no demonstrable effect on ZIKV restriction imposed by TRIM56. Rather, we found that TRIM56 is an RNA-binding protein that associates with ZIKV RNA in infected cells. Moreover, a recombinant TRIM56 fragment comprising the C-terminal 392 residues captured ZIKV RNA in cell-free reactions, indicative of direct interaction. Remarkably, deletion of a short C-terminal tail portion abrogated the TRIM56-ZIKV RNA interaction, concomitant with a loss in antiviral activity. Altogether, our study reveals TRIM56 is an RNA binding protein that acts as a ZIKV restriction factor and provides new insights into the antiviral mechanism by which this E3 ligase tackles flavivirus infections.


Assuntos
Fatores Imunológicos/metabolismo , MicroRNAs/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Zika virus/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos , Neurônios/imunologia , Neurônios/virologia , Ligação Proteica , Replicação Viral
17.
Nat Commun ; 10(1): 2387, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160572

RESUMO

Senescent cells accumulate in human tissues during ageing and contribute to age-related pathologies. The mechanisms responsible for their accumulation are unclear. Here we show that senescent dermal fibroblasts express the non-classical MHC molecule HLA-E, which interacts with the inhibitory receptor NKG2A expressed by NK and highly differentiated CD8+ T cells to inhibit immune responses against senescent cells. HLA-E expression is induced by senescence-associated secretary phenotype-related pro-inflammatory cytokines, and is regulated by p38 MAP kinase signalling in vitro. Consistently, HLA-E expression is increased on senescent cells in human skin sections from old individuals, when compared with those from young, and in human melanocytic nevi relative to normal skin. Lastly, blocking the interaction between HLA-E and NKG2A boosts immune responses against senescent cells in vitro. We thus propose that increased HLA-E expression contributes to persistence of senescent cells in tissues, thereby suggesting a new strategy for eliminating senescent cells during ageing.


Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , Senescência Celular/imunologia , Fibroblastos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Adulto , Idoso , Envelhecimento/patologia , Citocinas/imunologia , Derme/citologia , Fibroblastos/patologia , Humanos , Técnicas In Vitro , Nevo Pigmentado/congênito , Nevo Pigmentado/imunologia , Nevo Pigmentado/patologia , Fenótipo , RNA Interferente Pequeno , Transdução de Sinais , Pele/imunologia , Pele/patologia , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia
18.
Arthritis Rheumatol ; 71(11): 1913-1922, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31233292

RESUMO

OBJECTIVE: Chronic periaortitis (CP) is a rare disease characterized by periaortic and periiliac fibroinflammatory tissue. The pathogenic mechanisms leading to tissue accumulation and activation of fibroblasts are unclear. This study was undertaken to explore the role of fibrocytes, circulating precursors of tissue fibroblasts, in patients with CP. METHODS: We studied 44 patients with newly diagnosed CP and 30 healthy controls. Circulating fibrocytes were identified as Col1+CD45+ cells using flow cytometry. Retroperitoneal tissue biopsy samples from 9 CP patients were stained with anti-type I procollagen, anti-CXCR4, and anti-CD45 antibodies and analyzed by confocal microscopy to detect tissue-infiltrating fibrocytes. Circulating levels and tissue expression of CXCL12, a CXCR4 ligand that promotes fibrocyte homing, were investigated using enzyme-linked immunosorbent assay and immunohistochemistry, respectively. We also characterized T helper polarization in biopsy samples from CP patients and measured serum levels of a panel of cytokines that are hallmarks of T helper responses and capable of influencing fibrocyte differentiation. RESULTS: The frequency of circulating Col1+CD45+ fibrocytes was higher in patients than in controls (P = 0.0371). CD45+proCol1+ and CXCR4+proCol1+ cells were detected in all examined biopsy samples from CP patients. Serum levels of CXCL12 were also higher in CP patients than controls (P = 0.0056), and tissue-infiltrating inflammatory cells intensely expressed CXCL12. Increased serum levels of Th2 cytokines (e.g., interleukin-13 [IL-13] and IL-10) were found in patients, and immunohistochemistry revealed a dominant infiltration of GATA-3+ cells, also indicating Th2 polarization; Th2-skewed responses are known to promote fibrocyte differentiation. CONCLUSION: Our findings indicate that fibrocytes are enriched in the peripheral blood of CP patients and infiltrate target lesions. The accumulation of fibrocytes in the pathologic tissue might be driven by CXCL12, and Th2-skewed immune responses are likely to facilitate their differentiation.


Assuntos
Fibroblastos/metabolismo , Fibrose Retroperitoneal/metabolismo , Células Th2/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Quimiocina CXCL12/metabolismo , Colágeno Tipo I/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/citologia , Fibroblastos/imunologia , Fibrose , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Inflamação , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-13/imunologia , Interleucina-13/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Receptores CXCR4/metabolismo , Fibrose Retroperitoneal/imunologia , Fibrose Retroperitoneal/patologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Células Th2/imunologia
19.
Microb Pathog ; 133: 103556, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31128172

RESUMO

To investigate cytokine expression in chicken embryo fibroblast (CEF) cells, a virulent avian avulavirus 1 (AAvV-1) strain called SG10 that rapidly causes 100% mortality in its host, and a vaccine strain (La Sota) were characterized. Real-time quantitative PCR was performed on RNA samples from CEF cells, which were collected at 0, 24, 48 and 72 h post-infection. The dynamic expression patterns of ten cytokines (TNF-α, IFN-α, IFN-ß, IL-1ß, IL-2, IL-6, IL-10, IL-13, IL-15 and IL-18) were investigated. The results showed that infection with lentogenic La Sota induced significantly higher levels of the antiviral cytokines IFN-α and IFN-ß, proinflammatory cytokines IL-2, IL-15 and IL-18, and the anti-inflammatory cytokine IL-10, than did infection with virulent SG10. Furthermore, the SG10 strain induced dramatically higher levels of the inflammatory cytokine IL-6 than those observed in cells infected with La Sota. However, the expression patterns of the other cytokines that were tested did not show any obvious trends or statistically significant differences between cells infected with the virulent and avirulent strains. These data show that infection with lentogenic La Sota induced more effective immune responses and anti-viral effects than did infection with virulent SG10 in CEFs. Our data provide distinct expression patterns of IFNs and proinflammatory and anti-inflammatory cytokines to AAvV-1 by virulence in CEF cells.


Assuntos
Embrião de Galinha/imunologia , Citocinas/metabolismo , Fibroblastos/imunologia , Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/imunologia , Animais , Galinhas/imunologia , Galinhas/metabolismo , Citocinas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Doença de Newcastle/virologia , Vacinas Virais/imunologia
20.
J Orthop Res ; 37(9): 1979-1987, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31062877

RESUMO

The synovium plays a key role in the development of osteoarthritis, as evidenced by pathological changes to the tissue observed in both early and late stages of the disease. One such change is the attachment of cartilage wear particles to the synovial intima. While this phenomenon has been well observed clinically, little is known of the biological effects that such particles have on resident cells in the synovium. The present work investigates the hypothesis that cartilage wear particles elicit a pro-inflammatory response in diseased and healthy human fibroblast-like synoviocytes, like that induced by key cytokines in osteoarthritis. Fibroblast-like synoviocytes from 15 osteoarthritic human donors and a subset of three non-osteoarthritic donors were exposed to cartilage wear particles, interleukin-1α or tumor necrosis factor-α for 6 days and analyzed for proliferation, matrix production, and release of pro-inflammatory mediators and degradative enzymes. Wear particles significantly increased proliferation and release of nitric oxide, interleukin-6 and -8, and matrix metalloproteinase-9, -10, and -13 in osteoarthritic synoviocytes, mirroring the effects of both cytokines, with similar trends in non-osteoarthritic cells. These results suggest that cartilage wear particles are a relevant physical factor in the osteoarthritic environment, perpetuating the pro-inflammatory and pro-degradative cascade by modulating synoviocyte behavior at early and late stages of the disease. Future work points to therapeutic strategies for slowing disease progression that target cell-particle interactions. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1979-1987, 2019.


Assuntos
Cartilagem/fisiologia , Citocinas/farmacologia , Inflamação/etiologia , Sinoviócitos/imunologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Fibroblastos/imunologia , Humanos , Interleucina-1/farmacologia , Masculino , Pessoa de Meia-Idade , Osteoartrite/etiologia , Fator de Necrose Tumoral alfa/farmacologia
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