ABSTRACT
Abstract Background Fibroblast-like synoviocytes (FLS) play a prominent role in rheumatoid synovitis and degradation of the extracellular matrix through the production of inflammatory cytokines and metalloproteinases (MMPs). Since animal models are frequently used for elucidating the disease mechanism and therapeutic development, it is relevant to study the ultrastructural characteristics and functional responses in human and mouse FLS. The objective of the study was to analyze ultrastructural characteristics, Interleukin-6 (IL-6) and Metalloproteinase-3 (MMP-3) production and the activation of intracellular pathways in Fibroblast like synoviocytes (FLS) cultures obtained from patients with rheumatoid arthritis (RA) and from mice with collagen-induced arthritis (CIA). Methods FLSs were obtained from RA patients (RA-FLSs) (n = 8) and mice with CIA (CIA-FLSs) (n = 4). Morphology was assessed by transmission and scanning electron microscopy. IL-6 and MMP-3 production was measured by ELISA, and activation of intracellular signaling pathways (NF-κB and MAPK: p-ERK1/2, p-P38 and p-JNK) was measured by Western blotting in cultures of RA-FLSs and CIA-FLSs stimulated with tumor necrosis factor-alpha (TNF-α) and IL-1β. Results RA-FLS and CIA-FLS cultures exhibited rich cytoplasm, rough endoplasmic reticula and prominent and well- developed Golgi complexes. Transmission electron microscopy demonstrated the presence of lamellar bodies, which are cytoplasmic structures related to surfactant production, in FLSs from both sources. Increased levels of pinocytosis and numbers of pinocytotic vesicles were observed in RA-FLSs (p < 0.05). Basal production of MMP-3 and IL-6 was present in RA-FLSs and CIA-FLSs. Regarding the production of MMP-3 and IL-6 and the activation of signaling pathways, the present study demonstrated a lower response to IL-1β by CIA-FLSs than by RA-FLSs. Conclusion This study provides a comprehensive understanding of the biology of RA-FLS and CIA-FLS. The differences and similarities in ultrastructural morphology and important inflammatory cytokines shown, contribute to future in vitro studies using RA-FLS and CIA-FLS, in addition, they indicate that the adoption of CIA-FLS for studies should take careful and be well designed, since they do not completely resemble human diseases.
ABSTRACT
INTRODUÇÃO: A identificação precoce de citocinas pode favorecer a condução clínica e adoção de medidas preventivas na população neonatal. OBJETIVO: Avaliar os níveis séricos de Interleucinas (IL) 2, IL-4, IL-6, IL-8, IL-10, Interferon gama (IFN-γ), Fator de Necrose Tumoral alfa (TNF-α) e Fator Estimulador de Colônias de Granulócitos e Mastócitos (GM-CSF) nas primeiras três horas de vida de recém-nascidos prematuros através de coleta de sangue em papel filtro. MÉTODOS: Estudo analítico observacional prospectivo desenvolvido com 34 recém-nascidos prematuros com idade gestacional entre 28 e 32 semanas. As dosagens foram feitas com 10, 60 e 180 minutos de vida em papel filtro. Utilizado o reagente Bio-Plex ProT Human Cytokine Standard 8 plex, Group I BIO-RAD e as leituras realizadas com Luminex 100. RESULTADOS: Houve associação entre sepse e a presença de IFN-γ e TNF-α com 10, 60 e 180 minutos de vida. A associação entre sepse e a presença de IL-6 foi observada com 180 minutos de vida. Níveis reduzidos de GM-CSF na primeira hora de vida foram relacionados à hipotermia (p=0,005) à admissão na unidade neonatal. CONCLUSÃO: A detecção dos níveis de citocinas em papel filtro é viável nas primeiras horas de vida de recém-nascidos prematuros. As vantagens são a facilidade de coleta, armazenamento, transporte e utilização de pequenos volumes. O IFN-γ e TNF-α e IL-6 podem ser considerados marcadores para o desenvolvimento de sepse nessa população. A redução dos níveis de GM-CSF em pacientes hipotérmicos favorecendo quadros de neutropenia aponta para a importância da prevenção da hipotermia. (AU)
Introduction: Early identification of cytokines may favor clinical management and the adoption of preventive measures. Objective: This study aims to evaluate the serum levels of IL-2, IL-6, IL-8, IL- 10, Interferon gamma (IFN-γ), Tumor Necrosis Factor alpha (TNF-α) and Stimulating Factor of Granulocyte and Mast Cell Colonies (GMCSF) in the first three hours of life of preterm newborns through blood collection on filter paper. Methods: Prospective observational analytical study developed with 34 preterm infants with gestational age between 28 and 32 weeks. Measurements were made at 10, 60 and 180 minutes of life on filter paper. Used Bio-Plex Pro T Human Cytokine Standard 8 plex reagent, Group I BIO-RAD and readings performed through the Luminex 100. Results: There was an association between sepsis and the presence of IFN-γ and TNF-α at 10, 60 and 180 minutes of life. Association between sepsis and the presence of IL-6 was observed with 180 minutes. Reduced levels of GM-CSF in the first hour of life were related to hypothermia on admission to the neonatal unit (p=0.005). Conclusion: The detection of levels of cytokines in samples collected on filter paper showed to be viable in the first few hours of life with the following advantages: ease of collection, storage / transport and the use of small volumes. IFN-γ and TNF-α and IL-6 may be considered promising markers for the development of sepsis in this population. The reduction of levels of GM-CSF in hypothermic patients favoring neutropenia alerts the need for preventive care of this condition. (AU)