Three-dimensional cell culture for the study of nasal polyps

Abstract Objectives: Three-dimensional (3D) cell cultures have many applications such as stem cell biology research, new drug discovery, cancer, and Chronic Rhinosinusitis with Nasal Polyps (CRSwNP). This disease is characterized by a significant impact on quality of life and productivity. The diversity of factors that act in the progression of CRSwNP point to the creation of a cell culture model that allows the integration of different cell types with extracellular matrix. This work aimed to create a cell culture model in 3 dimensions (spheroids) for the study of Nasal Polyposis. Methods: Nasal polyp tissue from patients diagnosed with CRSwNP was mechanically dissociated using tweezers and a scalpel and the solution containing cells and small aggregates of nasal polyps was transferred to a Petri dish containing 5 mL of culture medium at the concentration of 106 cells/mL. Results: The spheroids were cultivated for 20 days, fixed and analyzed using confocal microscopy. In a 3D culture environment, the spheroids were formed both by clustering cells and from small tissue fragments. In the cultures analyzed, the ciliary beat was present from the dissociation of the cells up to 20 days in culture. Conclusion: Our findings also point to these characteristics showing the environment generated in our study, the cells remained differentiated for a longer time and with ciliary beating. Thus, this work shows that nasal polyp-derived cells can be maintained in a 3D environment, enabling better strategies for understanding CRSwNP in situations similar to those found in vivo. Level of evidence: Laboratory studies.

CD133, a Progenitor Cell Marker, is Reduced in Nasal Polyposis and Showed Significant Correlations with TGF-β1 and IL-8

Abstract Introduction Combination of chronic inflammation and an altered tissue remodeling process are involved in the development of Chronic Rhinosinusitis with Nasal Polyps (CRSwNP). Studies demonstrated that mesenchymal stem cells expressing the progenitor gene CD133 were involved in a significant reduction of the chronic inflammatory process in the polypoid tissue. Objective To evaluate the levels of CD133 (Prominin-1) in nasal polypoid tissue and its correlation with interleukin-8 (IL-8) and transforming growth factor β1 (TGF-β1). Methods A total of 74 subjects were divided in the following groups: control group (n = 35); chronic rhinosinusitis with nasal polyps nonpresenting comorbid asthma and aspirin intolerance (CRSwNPnonAI) group (n = 27); and chronic rhinosinusitis with nasal polyps presenting comorbid asthma and aspirin intolerance (CRSwNPAI) group (n = 12). Histologic analysis and also evaluation of the concentration of CD133, IL-8, and TGF-β1 by enzyme-linked immunosorbent assay (ELISA) kits were performed in nasal tissue obtained from nasal polypectomy or from middle turbinate tissue. Results Higher eosinophilic infiltration was found in both CRSwNP groups by histologic analysis. Lower levels of TGF-β1 and IL-8 were observed in both CRSwNP groups when compared with the control group, whereas the CD133 levels were significantly reduced only in the CRSwNPnonAI group compared with the control group. Conclusion It was demonstrated that the nasal mucosa presenting polyposis showed a significant reduction of CD133 levels, and also that this reduction was significantly correlated with the reduction of TGF-β1 levels, but not with IL-8 levels. Therefore, these findings may be involved in the altered inflammatory and remodeling processes observed in the nasal polyposis.

Inflammatory markers in palatine tonsils of children with obstructive sleep apnea syndrome / Marcadores inflamatórios em tonsilas palatinas de crianças com síndrome da apneia obstrutiva do sono

Abstract Introduction Obstrutive sleep apnea syndrome is characterized by repeated episodes of upper airway obstruction, associated with intermittent hypoxia and hypercapnia, and the main risk factor in childhood is adenotonsillar hypertrophy. The lymphocytes in these structures are responsible for local and systemic immune responses. Objective Verify the levels of the inflammatory markers, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-15, TNF-α, CRP and α1-GP, in the tonsils of children with and without obstructive sleep apnea syndrome. Methods This cross-sectional prospective study included 34 children with complains of snoring, difficulty breathing during sleep or recurrent tonsillitis. Patients underwent to a complete otorhinolaryngological examination, nasal endoscopy and polysomnography and were divided into two groups with 17 children each: obstructive sleep apnea syndrome group and control group. All underwent an adenotonsillectomy. Cytokines were measured in the collected tonsils (ELISA and Multiplex methods). Results Statistically significant increasing were observed between IL-8 and IL-10 cytokines of patients with obstructive sleep apnea when compared to the control group; also between c-reactive protein and α1-GP of the tonsils cortical region in children with obstructive sleep apnea syndrome when compared with the medullary region. There were no statistically significant differences for the remaining inflammatory mediators. Conclusion After the analysis of the levels of pro and anti-inflammatory markers (IL-1β, IL-4, IL-6, IL-8, IL-10, Il-15, TNF-α, CRP, α1-GP) in the tonsils, we observed higher levels of markers IL-8 and IL-10 in pediatric patients with obstructive sleep apnea syndrome.

Resumo Introdução A síndrome da apneia obstrutiva do sono é caracterizada por episódios repetidos de obstrução das vias aéreas superiores, associados a hipóxia intermitente e hipercapnia, e o principal fator de risco na infância é a hipertrofia adenotonsilar. Os linfócitos nessas estruturas são responsáveis por respostas imunes locais e sistêmicas. Objetivo Dosar os marcadores inflamatórios, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-15, TNF-α, PCR e α1-GP, nas tonsilas de crianças com e sem síndrome da apneia obstrutiva do sono. Método Estudamos prospectivamente 34 crianças que se queixavam de ronco, dificuldade para respirar durante o sono ou tonsilites recorrentes. Os pacientes foram submetidos a exame otorrinolaringológico completo, endoscopia nasal e polissonografia e foram divididos em dois grupos com 17 crianças cada: síndrome de apneia obstrutiva do sono e controle. Todos foram submetidos à adenotonsilectomia. As citocinas foram medidas nas tonsilas coletadas (métodos ELISA e Multiplex). Resultados Com diferenças estatisticamente significantes, observou-se aumento das citocinas IL-8 e IL-10 em pacientes com apneia obstrutiva do sono em comparação ao grupo controle, assim como aumento dos níveis de proteína C reativa e de α1-GP na região cortical das tonsilas de crianças portadoras de síndrome da apneia obstrutiva do sono em comparação com a região medular. Não houve diferenças estatisticamente significantes para o restante dos mediadores inflamatórios. Conclusão Após a análise dos níveis de marcadores pró e anti-inflamatórios (IL-1β, IL-4, IL-6, IL-8, IL-10, Il-15, TNF-α, PCR, α1-GP) nas tonsilas, observamos níveis mais altos de marcadores IL-8 e IL-10 em pacientes pediátricos com síndrome da apneia obstrutiva do sono.

Estresse em Estudantes do Quarto Ano de Graduação Expostos à Realização do Trabalho de Conclusão de Curso / Stress Among Fourth-Year Undergraduate Students in the Process of Developing Their Course Term Project

Objetivo: Avaliar o nível de estresse em alunos do quarto (último) ano de graduação, na fase de elaboração do Trabalho de Conclusão de Curso. Material e Métodos: Foram incluídos 51 alunos dos cursos de Graduação em Fisioterapia, Biologia e Sistemas de Informação de uma universidade de São Paulo - SP, que foram avaliados por meio de questionário de coleta de dados e pelo Inventário para Sintomas de Estresse em Adultos de Lipp. Resultados: Observou-se que o grupo Fisioterapia referiu com maior frequência (n= 10, 76,9%) que tem que fazer as atividades com muita rapidez e 69,2% (n= 9) do mesmo grupo afirmou que o trabalho exige frequentemente habilidades ou conhecimentos especializados (p = 0,002 e p = 0,02, respectivamente, Teste de Kruskal-Wallis). O grupo Biologia pareceu ter o pior relacionamento com os chefes e o melhor com os colegas e também referiu o maior número de sintomas associados ao estresse em 24 horas, uma semana e um mês (p < 0,0001, p < 0,0001 e p = 0,009, respectivamente, Teste de KruskalWallis). Conclusão: O grupo Sistemas de Informação apresentou menor número de sintomas associados ao estresse, sugerindo maior adaptabilidade às exigências e problemas do dia-a-dia por esse grupo, comparado à Fisioterapia e Biologia.(AU)

Objective: To evaluate the level of stress among fourthyear undergraduate students in preparation of their course term projects. Materials and Methods: 51 students of Physiotherapy, Biology and Information Systems from a university in São Paulo were included. The student were evaluated using a questionnaire and the Lipp's Stress Symptoms Inventory for adults. Results: The Physiotherapy group reported most frequently having to do the activities very quickly (n= 10, 76.9%). Of these, 69.2% (n=9) reported that the work often requires skill or specialized knowledge (p=0.002 and p=0.02, respectively, Kruskal-Wallis Test). The Biology group seemed to have the worst relationship with the bosses and the best with colleagues and also reported the highest number of symptoms associated with stress in 24 hours, one week and one month (p<0.0001, p<0.0001 and p=0.009 respectively, Kruskal-Wallis Test). Conclusion: The Information Systems group presented a lower number of symptoms related to stress, suggesting more adaptability to the demands and day-by-day problems as compared to Physiotherapy and Biology.(AU)

Microparticles as Potential Biomarkers of Cardiovascular Disease / Micropartículas como Possíveis Biomarcadores da Doença Cardiovascular

Primary prevention of cardiovascular disease is a choice of great relevance because of its impact on health. Some biomarkers, such as microparticles derived from different cell populations, have been considered useful in the assessment of cardiovascular disease. Microparticles are released by the membrane structures of different cell types upon activation or apoptosis, and are present in the plasma of healthy individuals (in levels considered physiological) and in patients with different pathologies. Many studies have suggested an association between microparticles and different pathological conditions, mainly the relationship with the development of cardiovascular diseases. Moreover, the effects of different lipid-lowering therapies have been described in regard to measurement of microparticles. The studies are still controversial regarding the levels of microparticles that can be considered pathological. In addition, the methodologies used still vary, suggesting the need for standardization of the different protocols applied, aiming at using microparticles as biomarkers in clinical practice.

A prevenção primária da doença cardiovascular constitui uma opção de grande relevância pelos seus impactos na saúde. Alguns biomarcadores têm sido considerados úteis na avaliação da doença cardiovascular, dentre eles micropartículas originadas de diferentes populações de células. Micropartículas são estruturas liberadas pela membrana de diferentes tipos celulares após ativação ou apoptose, presentes tanto no plasma de indivíduos saudáveis (níveis considerados fisiológicos) quanto em portadores de diferentes doenças. Muitos estudos têm sugerido uma associação entre micropartículas e diferentes condições patológicas, destacando-se a relação com o desenvolvimento das doenças cardiovasculares. Além disso, têm sido descritos os efeitos de diferentes terapias hipolipemiantes na mensuração de micropartículas. Os estudos ainda são controversos quanto aos níveis de micropartículas que possam ser considerados patológicos, e os métodos utilizados ainda são variados, o que sugere a necessidade da padronização dos diferentes protocolos utilizados, visando à utilização de micropartículas como biomarcadores úteis na prática clínica.

Micropartículas e células progenitoras: novos marcadores da disfunção endotelial / Microparticles and progenitor cells: new markers of endothelial dysfunction

O endotélio vascular compreende uma população altamente dinâmica decélulas com papel na proteção vascular contra inflamação, resposta imune,trombose e doenças cardiovasculares, Células progenitoras endoteliais(CPE) compreendem um grupo celular extremamente raro de células nãohematopoiéticas que podem ser recrutadas da medula óssea por váriosestímulos, como citocinas, fármacos, estrógenos, eritropoietina, atividadefísica,e participam na manutenção da integridade endotelial. Aregeneraçãovascular após dano endotelial ocorre tanto pela proliferação de célulasendoteliais maduras, residentes na parede dos vasos (angiogênese), comopor migração e diferenciação de CPE da medula óssea para áreas deisquemia, com a formação de novos vasos sanguíneos (vasculogênese).As CPE podem ser identificadas por citometria de fluxo por meio daexpressão dos marcadores de superfície celular CD34, CD309 (KDR) eCD133. Micropartículas (MP) são pequenas vesículas liberadas por diferentestipos celulares após ativação ou apoptose, contendo material celularcomo proteínas, mRNA, lipoproteínas e debris. Têm papel relacionado àresposta imune adaptativa, sendo utilizados também como biomarcadoresde disfunção endotelial e de algumas afecções como diabetes, infarto, HIV,além de promoverem a transferência de material e comunicação entrecélulas. CPE associadas às MP podem ser consideradas biomarcadoresúteis da doença cardiovascular por estarem diretamente relacionadas àhomeostase endotelial. O recrutamento de CPE sugere um mecanismocompensatório de reparo vascular que contribui para a restauração daintegridade endotelial. Aumento de MP associa-se à disfunção endoteliale à progressão da aterosclerose. Assim, a relação MP/CPE pode indicar ograu de desequilíbrio entre dano endotelial e capacidade de reparo...

The vascular endothelium comprises a highly dinamic population of cellswith a role in vascular protection against inflammation, imune response,thrombosis and cardiovascular diseases. Endothelial progenitor cells(EPC) are an extremely rare population of non-hematopoietic cells thatcan be recruited from bone marrow by many stimuli, such as cytokines,drugs, estrogens, erithropoetin, physical activity and can participate inthe manteinance of endothelium integrity. Vascular repair after injury ofthe endothelium can occur by both proliferation of mature adjacent cells(angiogenesis), or by EPC migration and proliferation from bone marrowto ischemic areas, forming new vessels (vasculogenesis). EPC can beidentified by flow citometry by using surface markers CD34, CD309(KDR) and CDl33. Microparticles (MP) are small vesicles released bydiferent cell types after acti vation or apoptosis, and contain cell materialsuch as proteins, mRNA, lipoproteins and debris. Their role is related tothe adaptive immune responses, and are also potential biomarkers of endotelialdysfunction in diabetes, myocardial infarction and in HIV infection.Further, MP can promote transfer of cell material and cell communication.EPC and MP can be used as useful biomarkers in cardiovascular diseasefor being associated with endothelial homeostasis. EPC mobilization is acompensatory mechanisrn for vascular repair that contributes to restoreendothelial integrity. Conversely, increase in circulating MP is associatedwith endotelial dysfunction and atherosclerosis progression. Though, theassessment of the MPIEPC ratio can indicate the degree of disbalancebetween endothelial damage and repair...