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1.
Rev. bras. ortop ; 57(5): 863-867, Sept.-Oct. 2022. graf
Article in English | LILACS | ID: biblio-1407705

ABSTRACT

Abstract Objectives The capsuloligamentous structures of the shoulder work as static stabilizers, together with the biceps and rotator cuff muscles, increasing the contact surface of the glenoid cavity. Free nerve endings and mechanoreceptors have been identified in the shoulder; however, there are a few studies that describe the presence of these nerves in the biceps' insertion. The present study aimed to describe the morphology and distribution of nerve endings using immunofluorescence with protein gene product 9.5 (PGP 9.5) and confocal microscopy. Methods Six labrum-biceps complexes from six fresh-frozen cadavers were studied. The specimens were coronally cut and prepared using the immunofluorescence technique. In both hematoxylin and eosin (H&E) and immunofluorescence, the organization of the connective tissue with parallel collagen fibers was described. Results In the H&E study, vascular structures and some nerve structures were visualized, which were identified by the elongated presence of the nerve cell. All specimens analyzed with immunofluorescence and confocal microscopy demonstrated poor occurrence of morphotypes of sensory corpuscles and free nerve endings. We identified free nerve endings located in the labrum and in the bicipital insertion, and sparse nerve endings along the tendon. Corpuscular endings with fusiform, cuneiform, and oval aspect were identified in the tendon. Conclusion These findings support the hypothesis that the generation of pain in the superior labral tear from Anterior to posterior (SLAP) lesions derives from the more proximal part of the long biceps cord and even more from the upper labrum. Future quantitative studies with a larger number of specimens may provide more information on these sensory systems.


Resumo Objetivos As estruturas capsulo-ligamentares do ombro funcionam como estabilizadores estáticos, juntamente com os músculos do bíceps e do manguito rotador, aumentando a superfície de contato da cavidade glenoide. Terminações nervosas livres e mecanorreceptores foram identificados no ombro; no entanto, existem alguns estudos que descrevem a presença desses nervos na inserção do bíceps. Este estudo teve como objetivo descrever a morfologia e distribuição de terminações nervosas utilizando imunofluorescência com protein gene product 9.5 (PGP 9.5) e microscopia confocal. Métodos Foram estudados seis complexos labrum-bíceps de seis cadáveres congelados frescos. Os espécimes foram cortados coronalmente e preparados pelo método de imunofluorescência. Tanto em hematoxilina e eosina (H&E) quanto em imunofluorescência, foi descrita a organização do tecido conjuntivo com fibras paralelas de colágeno. Resultados No estudo de H&E, foram visualizadas estruturas vasculares e algumas estruturas nervosas, que foram identificadas pela presença alongada da célula nervosa. Todas as amostras analisadas com imunofluorescência e microscopia confocal demonstraram baixa ocorrência de morfotipos de corpúsculos sensoriais e terminações nervosas livres. Identificamos terminações nervosas livres localizadas no labrum, inserção bicipital e terminações nervosas esparsas ao longo do tendão. Terminais corpusculares com aspecto fusiforme, cuneiforme e oval foram identificados no tendão. Conclusão Esses achados corroboram a hipótese de que a geração de dor nas lesões labrais superiores de anterior a posterior (SLAP, na sigla em inglês) deriva da parte mais proximal do cabo longo do bíceps e ainda mais do labrum superior. Estudos quantitativos futuros com um número maior de espécimes podem fornecer mais informações sobre esses sistemas sensoriais.


Subject(s)
Humans , Shoulder Joint , Cadaver , Fluorescent Antibody Technique , Hamstring Muscles , Mechanoreceptors , Nerve Endings
2.
Int. j. morphol ; 40(5): 1152-1164, 2022. ilus, tab
Article in English | LILACS-Express | LILACS | ID: biblio-1405284

ABSTRACT

SUMMARY: Coreopsis tinctoria Nutt. (C. tinctoria Nutt.) can protect diabetic kidneys, but the mechanisms are unclear. This work is to investigate the potential mechanisms of C. tinctoria Nutt. in the treatment of diabetic nephropathy based on network pharmacology analysis of its active ingredients. Twelve small molecular compounds of C. tinctoria Nutt. and targets related to diabetic nephropathy were docked by Discovery Studio 3.0. DAVID database was used for GO enrichment and KEGG pathway analysis. Cytoscape 3.6.1 was used to construct active ingredient-target network. Cell viability was detected with MTT. Glucose consumption was analyzed with glucose oxidase method. Protein expression was measured with Western blot and immunofluorescence. Electron microscopy observed autophagosomes. The core active ingredients of C. tinctoria Nutt. included heriguard, flavanomarein, maritimein, and marein. Twenty-one core targets of the 43 potential targets were PYGM, TLR2, RAF1, PRKAA2, GPR119, INS, CSF2, TNF, IAPP, AKR1B1, GSK3B, SYK, NFKB2, ESR2, CDK2, FGFR1, HTRA1, AMY2A, CAMK4, GCK, and ABL2. These 21 core targets were significantly enriched in 50 signaling pathways. Thirty- four signaling pathways were closely related to diabetic nephropathy, of which the top pathways were PI3K/AKT, insulin, and mTOR, and insulin resistance. The enriched GO terms included biological processes of protein phosphorylation, and the positive regulation of PI3K signaling and cytokine secretion; cellular components of cytosol, extracellular region, and extracellular space; and molecular function of protein kinase activity, ATP binding, and non-membrane spanning protein tyrosine kinase activity. In vitro experiments found that marein increased the expression of phosphorylated AKT/AKT in human renal glomerular endothelial cells of an insulin resistance model induced by high glucose, as well as increased and decreased, respectively, the levels of the microtubule-associated proteins, LC3 and P62. C. tinctoria Nutt. has many active ingredients, with main ingredients of heriguard, flavanomarein, maritimein, and marein, and may exert anti-diabetic nephropathy effect through various signaling pathways and targets.


RESUMEN: Coreopsis tinctoria Nutt. (C. tinctoria Nutt.) puede proteger riñones diabéticos, sin embargo los mecanismos son desconocidos. Este trabajo se realizó para investigar los potenciales mecanismos de C. tinctoria Nutt. en el tratamiento de la nefropatía diabética basado en el análisis de farmacología en red de sus principios activos. Doce compuestos moleculares pequeños de C. tinctoria Nutt. y los objetivos relacionados con la nefropatía diabética fueron acoplados por Discovery Studio 3.0. La base de datos DAVID se utilizó para el enriquecimiento GO y el análisis de la vía KEGG. Se usó Cytoscape 3.6.1 para construir una red de ingrediente-objetivo activa. La viabili- dad celular se detectó mediante MTT. El consumo de glucosa se analizó con el método de glucosa oxidasa. La expresión proteica fue determinada mediante Western blot e inmunofluorescencia. En la microscopía electrónica se observó autofagosomas. Los principales ingredientes activos de C. tinctoria Nutt. incluyeron heriguard, flavanomarein, maritimin y marein. Veintiún de los 43 objetivos potenciales fueron PYGM, TLR2, RAF1, PRKAA2, GPR119, INS, CSF2, TNF, IAPP, AKR1B1, GSK3B, SYK, NFKB2, ESR2, CDK2, FGFR1, HTRA1, AMY2A, CAMK4, GCK y ABL2. Estos 21 objetivos principales se enriquecieron significativamente en 50 vías de señalización. Treinta y cuatro vías de señalización estuvieron estrechamente relacionadas con la nefropatía diabética, de las cuales las principales vías fueron PI3K/ AKT, insulina y mTOR, y resistencia a la insulina. Los términos GO enriquecidos incluyeron procesos biológicos de fosforilación proteica, la regulación positiva de la señalización de PI3K y la secreción de citoquinas; componentes celulares del citosol, región extracelular y espacio extracelular; y la función molecular de la actividad de la proteína quinasa, la unión de ATP y la actividad de la proteína tirosina quinasa que no se extiende por la membrana. Los experimentos in vitro encontraron que la mareína aumentaba la expresión de AKT/AKT fosforilada en células endoteliales glomerulares renales humanas en un modelo de resistencia a la insulina inducida por niveles elevados de glucosa, así como aumentaron y disminuyeron respectivamente, los niveles de las proteínas asociadas a los microtúbulos, LC3 y P62. C. tinctoria Nutt. tiene muchos principios activos, con ingredientes principales de heriguard, flavanomarein, maritimain y marein, y puede ejercer un efecto de nefropatía antidiabética a través de distintass vías de señalización y objetivos.


Subject(s)
Coreopsis/chemistry , Diabetic Nephropathies , Network Pharmacology , Microscopy, Electron , Blotting, Western , Fluorescent Antibody Technique , Chalcones
3.
J. bras. nefrol ; 43(4): 586-590, Dec. 2021. tab
Article in English, Portuguese | LILACS | ID: biblio-1350902

ABSTRACT

Abstract Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune inflammatory disease. However, some patients may exhibit a histological pattern of kidney injury, with characteristics indistinguishable from lupus nephritis, but without presenting any extrarenal symptoms or serologies suggestive of SLE. Such involvement has recently been called non-lupus full-house nephropathy. The objective is to report a series of clinical cases referred to the Laboratory of the Federal University of Maranhão that received the diagnosis of "full-house" nephropathy unrelated to lupus, upon immunofluorescence and to discuss its evolution and outcomes. Non-lupus full-house nephropathy represents a diagnostic and therapeutic challenge, because it is a new entity, which still needs further studies and may be the initial manifestation of SLE, isolated manifestation of SLE or a new pathology unrelated to SLE.


Resumo O lúpus eritematoso sistêmico (LES) é uma doença inflamatória crônica autoimune multissistêmica. Alguns pacientes, contudo, podem exibir um padrão histológico de lesão renal, com características indistinguíveis da nefrite lúpica, porém sem apresentar quaisquer sintomas extrarrenais ou sorologias sugestivas de LES. Tal acometimento tem sido recentemente denominado nefropatia "full-house" não relacionada ao lúpus. O objetivo é relatar uma série de casos clínicos encaminhados ao Laboratório da Universidade Federal do Maranhão que receberam o diagnóstico de nefropatia "full-house" não relacionada ao lúpus à imunofluorescência e discutir sua evolução e desfechos. A nefropatia "full-house" não relacionada ao lúpus representa um desafio diagnóstico e terapêutico por ser uma entidade nova, que ainda necessita de maiores estudos e pode ser a manifestação inicial do LES, manifestação isolada do LES ou uma patologia nova não relacionada ao LES.


Subject(s)
Humans , Lupus Nephritis/diagnosis , Kidney Diseases , Lupus Erythematosus, Systemic/complications , Lupus Erythematosus, Systemic/diagnosis , Fluorescent Antibody Technique , Kidney
4.
Electron. j. biotechnol ; 51: 50-57, May. 2021. ilus, graf
Article in English | LILACS | ID: biblio-1343384

ABSTRACT

BACKGROUND: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. RESULTS: In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. CONCLUSIONS: In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.


Subject(s)
Gene Expression Regulation , Optogenetics/methods , Light , Neurons/metabolism , Immunoblotting , Gene Expression , Fluorescent Antibody Technique , Lentivirus
5.
Rev. pediatr. electrón ; 18(1): 33-44, abr. 2021. tab, graf
Article in Spanish | LILACS | ID: biblio-1369717

ABSTRACT

OBJETIVO. Comparar la gravedad de las infecciones respiratorias agudas bajas (IRAb) producidas por Adenovirus (ADV) entre los años 2015 y 2016, en el Hospital de Niños Roberto del Río. MÉTODOS. Se identificó a pacientes hospitalizados por IRAb por ADV, y se registró edad, sexo, días de hospitalización, ingreso a unidad de paciente crítico (UPC), necesidad de ventilación mecánica (VM), entre otros. Se comparó la evolución de los pacientes, y se analizó la relación entre gravedad y presencia de infecciones asociadas a atención de salud (IAAS). RESULTADOS. Se identificó 158 pacientes hospitalizados por IRAb por ADV. La relación hombre: mujer fue 6:4, con una edad promedio de 17.4 meses. La media de días de hospitalización fue de 9.88 el 2015 y 16.06 el 2016 (p=0.01). El promedio de días de oxigenoterapia fue de 5.86 el 2015 y 8.76 el 2016. Un 22.8% (n:36) de los pacientes ingresó a UPC, y el 20.25% (n:32) requirió VM. Un 41.8% de los casos (n:66) correspondió a IAAS. 3 pacientes fallecieron. CONCLUSIONES. Durante el 2016 hubo hospitalizaciones más prolongadas en comparación al 2015, sin diferencias estadísticamente significativas en relación a requerimientos de oxigenoterapia, VM y fallecimiento. La prolongación de la hospitalización se podría asociar a mayor presencia de IAAS.


OBJETIVE. To compare the severity of lower respiratory infections produced by Adenovirus between 2015 and 2016 at the Hospital de Niños Roberto del Río. METHODS. We identified patients hospitalized for lower respiratory tract infection for ADV and we recorded age, sex, days of hospitalization, admission to Intensive Care Unit (ICU), need for mechanical ventilation (MV), among others. The evolution of the patients between the two years were compared. The relationship between severity and the presence of nosocomial infections was also analyzed. RESULTS. We identified 158 hospitalized patients for ADV. The male:female ratio was 6:4, with an average age of 17.4 months. The mean of hospitalization days was 9.88 days in 2015 and 16.06 days in 2016 (p = 0.01). The average number of oxygen therapy days was 5.86 in 2015 and 8.76 in 2016. The 22.8% (n: 36) of cases required admission in the ICU, and 20.25% (n: 32) required MV. The 41.8% (n: 66) of cases corresponded to nosocomial infections. 3 patients died. CONCLUSIONS. During 2016, there were longer hospitalizations for adenovirus infections compared to 2015, without significant differences in relation to oxygen therapy, MV requirements and death. The prolongation of the hospitalization could be associated by the greater presence of nosocomial infections.


Subject(s)
Humans , Male , Female , Infant, Newborn , Infant , Child, Preschool , Respiratory Tract Infections/complications , Cross Infection , Community-Acquired Infections , Adenoviridae Infections/complications , Hospitals, Pediatric/statistics & numerical data , Oxygen Inhalation Therapy , Respiration, Artificial , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/therapy , Respiratory Tract Infections/epidemiology , Vasoconstrictor Agents/therapeutic use , Polymerase Chain Reaction , Acute Disease , Retrospective Studies , Fluorescent Antibody Technique , Adenoviridae Infections/diagnosis , Adenoviridae Infections/therapy , Adenoviridae Infections/epidemiology , Length of Stay
6.
Chinese Journal of Biotechnology ; (12): 4066-4074, 2021.
Article in Chinese | WPRIM | ID: wpr-921487

ABSTRACT

Different fragments of SARS-CoV-2 nucleocapsid (N) protein were expressed and purified, and a fluorescence immunochromatography method for detection of SARS-CoV-2 total antibody was established. The effect of different protein fragments on the performance of the method was evaluated. The N protein sequence was analyzed by bioinformatics technology, expressed in prokaryotic cell and purified by metal ion affinity chromatography column. Different N protein fragments were prepared for comparison. EDC reaction was used to label fluorescence microsphere on the synthesized antigen to construct sandwich fluorescence chromatography antibody detection assay, and the performance was systemically evaluated. Among the 4 prepared N protein fragments, the full-length N protein (N419) was selected as the optimized coating antigen, N412 with 0.5 mol/L NaCl was used as the optimal combination; deleting 91-120 amino acids from the N-terminal of N412 reduced non-specific signal by 87.5%. the linear range of detection was 0.312-80 U/L, the limit of detection was 0.165 U/L, and the accuracy was more than 95%. A fluorescence immunochromatographic detection method for analysis of SARS-CoV-2 total antibody was established by pairing N protein fragments. The detection result achieved 98% concordance with the commercially available Guangzhou Wanfu test strip, which is expected to be used as a supplementary approach for detection of SARS-CoV-2. The assay could also provide experimental reference for improving the performance of COVID-19 antibody detection reagents.


Subject(s)
Antibodies, Viral , COVID-19 , Chromatography, Affinity , Fluorescent Antibody Technique , Humans , Microspheres , SARS-CoV-2 , Sensitivity and Specificity
7.
Rev. bras. parasitol. vet ; 30(2): e026220, 2021. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1288692

ABSTRACT

Abstract Brazilian spotted fever (BSF) is a common tick-borne disease caused by Rickettsia rickettsii. Horses are the primary hosts of the main vector, Amblyomma sculptum, and are considered efficient sentinels for circulation of Rickettsia. Therefore, the aim of this study was to detect antibodies reactive to R. rickettsii antigens in horses from a non-endemic area in the north-central region of Bahia state, Brazil. Blood samples and ticks were collected from 70 horses from the municipalities of Senhor do Bonfim, Antônio Gonçalves, Pindobaçu, and Campo Formoso. The sera obtained were tested by an indirect immunofluorescence assay to detect anti-Rickettsia antibodies. Overall, 5.7% (4/70) of the horses reacted to R. rickettsii antigens. Ticks were collected from 18.6% (13/70) of the horses and were identified as A. sculptum and Dermacentor nitens. Despite being a non-endemic area, seropositive animals were identified in our study, suggesting circulation of rickettsial agents in the region. This is the first serological survey of this agent in horses from the north-central region of Bahia, and further studies are needed to understand the epidemiology of BSF in these locations.


Resumo A Febre Maculosa Brasileira (FMB) é uma importante zoonose causada por Rickettsia rickettsii, sendo seu vetor o Amblyomma sculptum. Cavalos são os principais hospedeiros desse carrapato e considerados sentinelas eficientes para detectar a circulação de Rickettsia. O objetivo deste estudo foi detectar anticorpos reativos a antígenos de R. rickettsii em cavalos de uma área não-endêmica na região Centro-Norte da Bahia, Brasil. Foram coletadas amostras de sangue e carrapatos de 70 cavalos nos municípios de Senhor do Bonfim, Antônio Gonçalves, Pindobaçu e Campo Formoso. Os soros obtidos foram testados pela reação de imunofluorescência indireta para detectar anticorpos anti-Rickettsia. No geral, 5,7% (4/70) dos cavalos reagiram sorologicamente para antígenos de R. rickettsii. Os carrapatos foram coletados em 18,6% (13/70) dos cavalos e foram identificados como A. sculptum e Dermacentor nitens. Embora seja uma área não endêmica, foram observados animais soropositivos, sugerindo a circulação de riquétsias na região estudada. Este é o primeiro estudo de levantamento sorológico desse agente em cavalos do Centro-Norte da Bahia. São necessários mais estudos para compreender a epidemiologia da FMB nesses locais.


Subject(s)
Animals , Rickettsia rickettsii , Ticks/classification , Ticks/microbiology , Rocky Mountain Spotted Fever/veterinary , Horse Diseases/diagnosis , Horses/microbiology , Brazil/epidemiology , Rocky Mountain Spotted Fever/diagnosis , Rocky Mountain Spotted Fever/epidemiology , Fluorescent Antibody Technique , Horse Diseases/microbiology , Horse Diseases/epidemiology
8.
Autops. Case Rep ; 11: e2021267, 2021. graf
Article in English | LILACS | ID: biblio-1249009

ABSTRACT

Pemphigus Vulgaris (PV) is an uncommon autoimmune and blistering mucocutaneous disease. Childhood Pemphigus Vulgaris (CPV) is a pediatric variant of PV, which affects children below 12 years, being very rare among children under 10 years of age. CPV has similar clinical, histological, and immunological features as seen in PV in adults. The mucocutaneous clinical presentation is the most common in both age groups. Vesicles and erosions arising from the disease usually cause pain. A few CPV cases have been reported in the literature. This study reports a case of an 8-year-old male patient with oral lesions since the age of 3 years, and the diagnosis of pemphigus was achieved only 2 years after the appearance of the initial lesions. CPV remains a rare disease, making the diagnosis of this clinical case a challenge due to its age of onset and clinical features presented by the patient. Therefore, dentists and physicians should know how to differentiate CPV from other bullous autoimmune diseases more common in childhood.


Subject(s)
Humans , Male , Child , Pemphigus/complications , Fluorescent Antibody Technique , Rare Diseases
9.
Int. j. morphol ; 38(6): 1742-1750, Dec. 2020. tab, graf
Article in English | LILACS | ID: biblio-1134507

ABSTRACT

SUMMARY: Mesenchymal stem cells are present in adult tissues such as the human dental pulp. They are pluripotent and can differentiate into various specialized cell types in vitro through appropriate stimuli. Ameloblasts produce human tooth enamel only during embryonic development before tooth eruption, so endogenous regeneration is not possible. Various efforts have been aimed at generating natural or artificial substitutes for dental enamel with properties similar to the specific components of said tissue. The purpose of this study was to induce human dental pulp stem cells to produce enamel proteins using extracellular matrix derived from the rat tail tendon and pigskin. Primary cultures of human dental pulp stem cells were established and characterized by RT-PCR and immunofluorescence, using mesenchymal cell markers such as CD14, CD40, CD44, CD105, and STRO-1. The cells were then incubated with the extracellular matrix for fourteen days and labeled with specific antibodies to detect the expression of dental enamel proteins such as amelogenin, ameloblastin, enamelisin, tuftelin, and parvalbumin, characteristics of the phenotype of ameloblasts. This work demonstrated a positive effect of the extracellular matrix to induce the expression of enamel proteins in the stem cells of the human dental pulp.


RESUMEN: Las células madre mesenquimales están presentes en los tejidos adultos como la pulpa dental humana. Son pluripotentes y pueden diferenciarse en varios tipos de células especializadas in vitro a través de estímulos adecuados. Los ameloblastos producen esmalte dental humano sólo durante el desarrollo embrionario antes de la erupción dental, por lo que no es posible su regeneración endógena. Varios esfuerzos se han orientado a generar sustitutos naturales o artificiales de esmalte dental con propiedades similares a los componentes específicos de este tejido. El propósito de este estudio fue inducir células madre de pulpa dental humana para producir proteínas del esmalte dental a través del estímulo de matriz extracelular derivada del tendón de la cola de rata y piel de cerdo. Se establecieron cultivos primarios de células madre de pulpa dental humana y se caracterizaron por RT-PCR e inmunofluorescencia utilizando marcadores de células mesenquimales como CD14, CD40, CD44, CD105 y STRO-1. Posteriormente, las células se incubaron con matriz extracelular durante un período de catorce días y se marcaron con anticuerpos específicos para detectar la expresión de proteínas de esmalte dental como amelogenina, ameloblastina, enamelisina, tuftelina y parvalbúmina, las cuales son características del fenotipo de ameloblastos. Este trabajo demostró el efecto positivo que tiene el empleo de la matriz extracelular para inducir la expresión de proteínas de esmalte en las células pluripotenciales de la pulpa dental humana.


Subject(s)
Humans , Stem Cells , Dental Enamel Proteins , Dental Pulp , Extracellular Matrix , Immunophenotyping , Fluorescent Antibody Technique , Cell Culture Techniques , Tissue Engineering
10.
Int. j. morphol ; 38(5): 1463-1472, oct. 2020. graf
Article in English | LILACS | ID: biblio-1134463

ABSTRACT

SUMMARY: The vomeronasal organ (VNO) is an accessory organ involved on the olfactory pathway, that detects pheromones and emits signals in order to modulate social and reproductive behavior. The VNO stem cells replace neurons throughout life. The aim of this study was to isolate and characterize cells derived from the vomeronasal organ from New Zealand rabbits. Five male rabbits with 120 days were used for cell isolation and culture. Results: VNO-derived cells presented labelling for proliferation (PCNA), undifferentiated profile (Nanog), neuronal (GFAP), mesenchymal stem cells (CD73, CD90 and CD105 and Stro-1). Also, presence of cytoskeletal (Vimentin, b-tubulin and CK-18) and absence of hematopoietic markers (CD34, CD117 and CD45) both by immunofluorescence and flow cytometry. By PCR it was possible to verify the expression of some undifferentiated profile (Oct-4), neuronal (Nestin) and mesenchymal (CD73, CD105 and Vimentin) genes. Functionally, VNO-derived cells differentiate in vitro into adipocytes, osteocytes and chondrocytes, and presented no tumorigenic potential when injected to Balb/c nu/nu mice. In conclusion, the rabbit VNO-derived cells have a profile that could be supportive to VNO olfactory/neuroreceptor epithelium by delivering factors to epithelial turnover or even by differentiation into epithelial cells to replacement of commissural epithelium.


RESUMEN: El órgano vomeronasal (OVN) es un órgano accesorio de la vía olfatoria, que detecta feromonas y emite señales que afectan la modulación del comportamiento social y reproductivo. Las células madre OVN reemplazan las neuronas durante toda la vida. El objetivo de este estudio fue aislar y caracterizar células derivadas del órgano vomeronasal de conejos raza Nueva Zelanda. Para el aislamiento y el cultivo celular se utilizaron cinco conejos machos con una edad de 120 días. Las células del OVN presentaron etiquetado para la proliferación (PCNA), un perfil indiferenciado (Nanog), neuronal (GFAP), células madre mesenquimales (CD73, CD90 y CD105 y Stro-1). Además, se ob- servó presencia de citoesqueleto (Vimentina, β-tubulina y CK-18) y ausencia de marcadores hematopoyéticos (CD34, CD117 y CD45) tanto por inmunofluorescencia como por citometría de flujo. Me- diante PCR fue posible verificar la expresión de algunos genes de perfil indiferenciado (Oct-4), neuronal (Nestin) y mesenquimatoso (CD73, CD105 y Vimentin). Las células derivadas del OVN se diferencian in vitro en adipocitos, osteocitos y condrocitos, y no presentan un potencial tumorigénico al ser infiltrados en ratones Balb / c nu / nu. En conclusión, las células derivadas de OVN de conejo tienen un perfil que podría ser compatible con el epitelio olfatorio / neurorreceptor de OVN transmitiendo factores al recambio epitelial o incluso mediante la diferenciación en células epiteliales para reemplazar el epitelio comisural.


Subject(s)
Animals , Rabbits/anatomy & histology , Vomeronasal Organ/cytology , Mesenchymal Stem Cells/physiology , Olfactory Bulb/cytology , Stem Cells/physiology , Olfactory Mucosa/cytology , Polymerase Chain Reaction , Fluorescent Antibody Technique , Flow Cytometry , Neurons/physiology
11.
Brasília; s.n; maio 2020.
Non-conventional in Portuguese | LILACS, BRISA | ID: biblio-1099659

ABSTRACT

INTRODUÇÃO: O coronavírus da Síndrome Respiratória Aguda Grave 2 (abreviado para SARSCoV-2, do inglês Severe Acute Respiratory Syndrome Coronavirus 2), anteriormente conhecido como novo coronavírus (2019-nCoV), é um agente zoonótico recémemergente que surgiu em dezembro de 2019, em Wuhan, China, causando manifestações respiratórias, digestivas e sistêmicas, que se articulam no quadro clínico da doença denominada COVID-19 (do inglês Coronavirus Disease 2019). Ainda não há informações robustas sobre a história natural da doença, tampouco sobre as medidas de efetividade para o manejo clínico dos casos de infecção pelo COVID19, restando ainda muitos detalhes a serem esclarecidos. No entanto, sabe-se que o vírus tem alta transmissibilidade e provoca uma síndrome respiratória aguda que varia de casos leves ­ cerca de 80% ­ a casos muito graves com insuficiência respiratória - entre 5% e 10% dos casos ­, os quais requerem tratamento especializado em unidades de terapia intensiva (UTI). Sua letalidade varia, principalmente, conforme a faixa etária. TECNOLOGIA: Os testes de diagnóstico para a COVID-19 se destacaram na pandemia de coronavírus em andamento como uma ferramenta essencial para rastrear a propagação da doença. Uma ampla gama de testes diagnósticos para o SARS-CoV-2 está disponível comercialmente, alguns dos quais receberam autorizações para uso por várias agências reguladoras nacionais. Com as informações da sequência genética devidamente identificadas, os testes de diagnóstico baseados na detecção da sequência viral por reação em cadeia da polimerase com transcriptase reversa (RT-PCR) ou plataformas de sequenciamento logo se tornaram disponíveis. Isso permitiu a confirmação do diagnóstico e melhores estimativas da atividade da infecção, que vêm aumentando em velocidades alarmantes. Para a detecção mais sensível de SARS-CoV, MERS-CoV e SARS-CoV-2, recomendavam-se a coleta e o teste de amostras respiratórias superiores e inferiores. O diagnóstico de casos suspeitos era confirmado por testes de RNA com RT-PCR em tempo real ou sequenciamento de próxima geração. Foi demonstrado que o RNA viral poderia ser detectado a partir do swab nasal e faríngeo, lavagem broncoalveolar e plasma sanguíneo usando RT-PCR direcionado ao gene do vírus (5). O padrão-ouro para diagnóstico laboratorial da COVID-19 é a reação da transcriptase reversa, seguida de reação em cadeia da polimerase (RT-PCR) para amostras coletadas no trato respiratório superior ou inferior. OBJETIVO: O objetivo deste relatório é analisar a acurácia dos testes diagnósticos registrados na Agência Nacional de Vigilância Sanitária (ANVISA) até a presente data. METODOLOGIA: foi realizada uma busca por diagnósticos para COVID-19 com registros vigentes na ANVISA. Para tal, foram utilizados os termos "COVID 19", SARS e coronavírus no campo de consulta de registro de produtos para saúde no site da Agência (https://consultas.anvisa.gov.br/#/saude/). Os passos para acesso ao banco de dados de produtos diagnósticos na ANVISA são: 1) consulta produtos; 2) consulta a banco de dados; 3) produtos para a saúde e 4) pesquisa de produtos para a saúde registrados. CONCLUSÕES: A ANVISA já avaliou mais de 120 pedidos de registro de produtos para testagens relacionadas à COVID-19 desde o dia 18 de março. A maior parte das petições aguarda complementação de informações por parte das empresas e outras estão sendo analisadas com prioridade. O tempo médio para avaliação dos registros na ANVISA gira em torno de 15 dias. Atualmente, mais da metade dos registros concedidos diz respeito a testes rápidos para anticorpos. Até a presente data, foram registrados 64 testes para diagnóstico da COVID-19, sendo 15 deles moleculares. O teste de polymerase chain reaction em tempo real (RT-PCR) para identificação de SARS-CoV-2 é um teste de elevada sensibilidade e especificidade, ainda que os doentes com maior carga viral possam ter maior probabilidade de um teste positivo. Os testes moleculares baseados em RNA exigem instalações laboratoriais específicas com níveis restritos de biossegurança e técnica. A sensibilidade e especificidade dos testes sorológicos variaram entre os fabricantes. É importante destacar que uma baixa sensibilidade do teste diagnóstico pode resultar em uma maior probabilidade de detectar falsos-negativos, o que poderia interferir principalmente em casos de indivíduos assintomáticos. Em geral, a sensibilidade dos testes foi superior a 85% e a especificidade, superior a 94%. Os testes sorológicos medem a quantidade de dois anticorpos (IgG e IgM) que o organismo produz quando entra em contato com um invasor. Contudo, o desenvolvimento da resposta de um anticorpo à infecção pode ser dependente do hospedeiro e levar tempo. No caso de SARS-CoV-2, estudos iniciais sugerem que a maioria dos pacientes se converte entre 7 e 11 dias após a exposição ao vírus, embora alguns pacientes possam desenvolver anticorpos mais cedo. Devido a esse atraso natural, o teste de anticorpos pode não ser útil no cenário de uma doença aguda (11). Os testes de anticorpos para SARS-CoV-2 podem facilitar (i) o rastreamento de contatos (os testes baseados em RNA também podem ajudar); (ii) a vigilância sorológica nos níveis local, regional, estadual e nacional; e (iii) a identificação de quem já teve contato com o vírus e, portanto, pode (se houver imunidade protetora) ser imune (11,12). Alguns conjuntos de reagentes para testes sorológicos foram autorizados pela ANVISA em caráter emergencial devido à gravidade da situação e à necessidade de ampliar a testagem da população, mas a validação desses reagentes pelos laboratórios é fundamental, uma vez que poucos trabalhos conseguiram ser publicados até o momento. As aprovações estão de acordo com a Resolução da Diretoria Colegiada (RDC) 348/2020, que define os critérios e os procedimentos extraordinários e temporários para tratamento de petições de registro de medicamentos, produtos biológicos e produtos para diagnóstico in vitro, e mudança pós-registro de medicamentos e produtos biológicos em virtude da emergência de saúde pública internacional decorrente do novo coronavírus. Na RDC, para registro de testes diagnósticos, a ausência de qualquer estudo de desempenho ou restrição de dados deve ser justificada por motivações técnicas que permitam a avalição da confiabilidade dos resultados e da efetividade diagnóstica do produto. Os registros concedidos nas condições dessa Resolução terão a validade de um ano, exceto para situações em que a avaliação da estabilidade seja apresentada por comparação com produtos similares e os demais critérios descritos no Regulamento sejam atendidos. Nesse caso, poderão ter a concessão regular de validade de registro de produtos para saúde por um período de 10 anos. Em resumo, as duas categorias de testes para SARS-CoV-2 podem ser úteis nesse surto, pois, eventualmente, a coleta de múltiplas amostras, regiões e em tempos diferentes durante a evolução da doença pode ser necessária para o diagnóstico da COVID-19.


Subject(s)
Humans , Enzyme-Linked Immunosorbent Assay/instrumentation , Chromatography, Affinity/instrumentation , Fluorescent Antibody Technique/instrumentation , Coronavirus Infections/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Technology Assessment, Biomedical , Health Evaluation
12.
Int. arch. otorhinolaryngol. (Impr.) ; 24(1): 47-52, Jan.-Mar. 2020. graf
Article in English | LILACS | ID: biblio-1090559

ABSTRACT

Abstract Introduction Cisplatin damages the auditory system and is related to the generation of free radicals. Glutathione peroxidase is an endogenous free radicals remover. Objective To investigate the mechanisms involved in otoprotection by N-acetylcys- teine through the expression of glutathione peroxidase in outer hair cells from rats treated with cisplatin. Methods Male Wistar rats were intraperitoneally injected with cisplatin (8 mg/Kg) and/or received oral administration by gavage of N-acetylcysteine (300 mg/Kg) for 3 consecutive days. On the 4th day, the animals were euthanized and beheaded. The tympanic bullae were removed and prepared for scanning electron microscopy and Results Among the groups exposed to ototoxic doses of cisplatin, there was an increase in glutathione peroxidase immunostaining in two groups, the one exposed to cisplatin alone, and the group exposed to both cisplatin and N-acetylcysteine. Conclusion The expression of glutathione peroxidase in the outer hair cells of rats exposed to cisplatin showed the synthesis of this enzyme under cellular toxicity conditions.


Subject(s)
Animals , Male , Acetylcysteine/therapeutic use , Free Radical Scavengers/therapeutic use , Cisplatin/toxicity , Oxidative Stress/drug effects , Antineoplastic Agents/toxicity , Acetylcysteine/metabolism , Acetylcysteine/pharmacology , Microscopy, Electron, Scanning , Evoked Potentials, Auditory, Brain Stem , Free Radical Scavengers/metabolism , Free Radical Scavengers/pharmacology , Fluorescent Antibody Technique , Cisplatin/therapeutic use , Rats, Wistar , Cochlea/anatomy & histology , Cochlea/drug effects , Free Radicals , Glutathione Peroxidase/metabolism , Hearing Loss, Sensorineural/prevention & control
13.
Article in English | WPRIM | ID: wpr-828988

ABSTRACT

Objective@#The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.@*Methods@#We prepared a PrP-specific polyclonal antibody (pAb P54) in a -knockout mouse model immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays.@*Results@#Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP from healthy rodents and humans, and pathological PrP in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP and PrP observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.@*Conclusion@#The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.


Subject(s)
Animals , Antibodies , Allergy and Immunology , Blotting, Western , Fluorescent Antibody Technique , Immunization , Immunohistochemistry , Mice , Mice, Knockout , PrPC Proteins , Allergy and Immunology , PrPSc Proteins , Allergy and Immunology , Prion Proteins , Allergy and Immunology , Recombinant Proteins , Allergy and Immunology
14.
Article in English | WPRIM | ID: wpr-811062

ABSTRACT

PURPOSE: Th17-associated inflammation is increased in chronic rhinosinusitis with nasal polyp (CRSwNP), and is associated with disease severity and steroid resistance. Overexpressed interleukin (IL)-17A affects CRSwNP by tissue remodeling, eosinophilic accumulation, and neutrophilic infiltration. We aimed to identify the role of IL-17A in CRSwNP and to evaluate the effects of anti-IL-17A blocking antibody on nasal polyp (NP) formation using a murine NP model. Moreover, we sought to investigate whether the inhibition of mechanistic target of the rapamycin (mTOR) signal pathway could suppress IL-17A expression and NP formation.METHODS: Human sinonasal tissues from control subjects and patients with chronic rhinosinusitis (CRS) were analyzed using immunohistochemistry (IHC) and immunofluorescence staining. The effects of IL-17A neutralizing antibody and rapamycin were evaluated in a murine NP model. Mouse samples were analyzed using IHC, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay.RESULTS: IL-17A+ inflammatory cells were significantly increased in number in NP from patients with CRSwNP compared to that in uncinate process tissues from control subjects and patients with CRS without NP or CRSwNP. CD68+ M1 macrophages dominantly expressed IL-17A, followed by neutrophils and T helper cells, in NP tissues. Neutralization of IL-17A effectively reduced the number of NPs, inflammatory cytokines, and IL-17A-producing cells, including M1 macrophages. Inhibition of IL-17A via the mTOR pathway using rapamycin also attenuated NP formation and inflammation in the murine NP model.CONCLUSIONS: IL-17A possibly plays a role in the pathogenesis of CRSwNP, the major cellular source being M1 macrophage in NP tissues. Targeting IL-17A directly or indirectly may be an effective therapeutic strategy for CRSwNP.


Subject(s)
Animals , Antibodies, Neutralizing , Cytokines , Enzyme-Linked Immunosorbent Assay , Eosinophils , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Inflammation , Interleukin-17 , Interleukins , Macrophages , Mice , Nasal Polyps , Neutrophils , Real-Time Polymerase Chain Reaction , Signal Transduction , Sinusitis , Sirolimus , T-Lymphocytes, Helper-Inducer
15.
Article in English | WPRIM | ID: wpr-762184

ABSTRACT

PURPOSE: Various immune cells, including eosinophils and neutrophils, are known to contribute to the development of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the current understanding of the role of neutrophils in the development of CRSwNP still remains unclear. Therefore, we investigated risk factors for refractoriness of CRSwNP in an Asian population. METHODS: Protein levels of 17 neutrophil-related mediators in nasal polyps (NPs) were determined by multiplex immunoassay, and exploratory factor analysis using principal component analysis was performed. Immunofluorescence analysis was conducted to detect human neutrophil elastase (HNE) or myeloperoxidase (MPO)-positive cells. Tissue eosinophilic nasal polyp (ENP) and tissue neutrophilia (Neu(high)) were defined as greater than 70 eosinophils and 20 HNE-positive cells, otherwise was classified into non-eosinophilic nasal polyp (NENP) and absence of tissue neutrophilia (Neu(low)). RESULTS: In terms of disease control status, NENP-Neu(low) patients showed the higher rate of disease control than NENP-Neu(high) and ENP-Neu(high) patients. Linear by linear association demonstrated the trend in refractoriness from NENP-Neu(low) to NENP-Neu(high) or ENP-Neu(low) to ENP-Neu(high). When multiple logistic regression was performed, tissue neutrophilia (hazard ratio, 4.38; 95% confidence interval, 1.76-10.85) was found as the strongest risk factor for CRSwNP refractoriness. Additionally, exploratory factor analysis revealed that interleukin (IL)-18, interferon-γ, IL-1Ra, tumor necrosis factor-α, oncostatin M, and MPO were associated with good disease control status, whereas IL-36α and IL-1α were associated with refractory disease control status. In subgroup analysis, HNE-positive cells and IL-36α were significantly upregulated in the refractory group (P = 0.0132 and P = 0.0395, respectively), whereas MPO and IL-18 showed higher expression in the controlled group (P = 0.0002 and P = 0.0009, respectively). Moreover, immunofluorescence analysis revealed that IL-36R⁺HNE⁺-double positive cells were significantly increased in the refractory group compared to the control group. We also found that the ratio of HNE-positive cells to α1 anti-trypsin was increased in the refractory group. CONCLUSIONS: Tissue neutrophilia had an influence on treatment outcomes in the Asian CRSwNP patients. HNE-positive cells and IL-36α may be biomarkers for predicting refractoriness in Asians with CRSwNP. Additionally, imbalances in HNE and α1 anti-trypsin may be associated with pathophysiology of neutrophilic chronic rhinosinusitis.


Subject(s)
Asians , Biomarkers , Eosinophils , Fluorescent Antibody Technique , Humans , Immunoassay , Interleukin 1 Receptor Antagonist Protein , Interleukin-18 , Interleukins , Leukocyte Elastase , Logistic Models , Nasal Polyps , Necrosis , Neutrophils , Oncostatin M , Peroxidase , Principal Component Analysis , Rhinitis , Risk Factors , Sinusitis
16.
Article in English | WPRIM | ID: wpr-762183

ABSTRACT

PURPOSE: The effect of air pollution-related particulate matter (PM) on epithelial barrier function and tight junction (TJ) expression in human nasal mucosa has not been studied to date. This study therefore aimed to assess the direct impact of PM with an aerodynamic diameter less than 2.5 μm (PM2.5) on the barrier function and TJ molecular expression of human nasal epithelial cells. METHODS: Air-liquid interface cultures were established with epithelial cells derived from noninflammatory nasal mucosal tissue collected from patients undergoing paranasal sinus surgery. Confluent cultures were exposed to 50 or 100 µg/mL PM2.5 for up to 72 hours, and assessed for 1) epithelial barrier integrity as measured by transepithelial resistance (TER) and permeability of fluorescein isothiocyanate (FITC) 4 kDa; 2) expression of TJs using real-time quantitative polymerase chain reaction and immunofluorescence staining, and 3) proinflammatory cytokines by luminometric bead array or enzyme-linked immunosorbent assay. RESULTS: Compared to control medium, 50 and/or 100 µg/mL PM2.5-treatment 1) significantly decreased TER and increased FITC permeability, which could not be restored by budesonide pretreatment; 2) significantly decreased the expression of claudin-1 messenger RNA, claudin-1, occludin and ZO-1 protein; and 3) significantly increased production of the cytokines interleukin-8, TIMP metallopeptidase inhibitor 1 and thymic stromal lymphopoietin. CONCLUSIONS: Exposure to PM2.5 may lead to loss of barrier function in human nasal epithelium through decreased expression of TJ proteins and increased release of proinflammatory cytokines. These results suggest an important mechanism of susceptibility to rhinitis and rhinosinusitis in highly PM2.5-polluted areas.


Subject(s)
Asthma , Budesonide , Claudin-1 , Cytokines , Enzyme-Linked Immunosorbent Assay , Epithelial Cells , Fluorescein , Fluorescein-5-isothiocyanate , Fluorescent Antibody Technique , Humans , Interleukin-8 , Mucous Membrane , Nasal Mucosa , Occludin , Particulate Matter , Permeability , Polymerase Chain Reaction , Rhinitis , RNA, Messenger , Tight Junctions
18.
Article in English | WPRIM | ID: wpr-787136

ABSTRACT

Regulator of calcineurin 1 (RCAN1) can be induced by an intracellular calcium increase and oxidative stress, which are characteristic features of temporal lobe epilepsy. Thus, we investigated the spatiotemporal expression and cellular localization of RCAN1 protein and mRNA in the mouse hippocampus after pilocarpine-induced status epilepticus (SE). Male C57BL/6 mice were given pilocarpine hydrochloride (280 mg/kg, i.p.) and allowed to develop 2 h of SE. Then the animals were given diazepam (10 mg/kg, i.p.) to stop the seizures and sacrificed at 1, 3, 7, 14, or 28 day after SE. Cresyl violet staining showed that pilocarpine-induced SE resulted in cell death in the CA1 and CA3 subfields of the hippocampus from 3 day after SE. RCAN1 immunoreactivity showed that RCAN1 was mainly expressed in neurons in the shammanipulated hippocampi. At 1 day after SE, RCAN1 expression became detected in hippocampal neuropils. However, RCAN1 signals were markedly enhanced in cells with stellate morphology at 3 and 7 day after SE, which were confirmed to be reactive astrocytes, but not microglia by double immunofluorescence. In addition, real-time reverse transcriptase–polymerase chain reaction showed a significant upregulation of RCAN1 isoform 4 (RCAN1-4) mRNA in the SE-induced hippocampi. Finally, in situ hybridization with immunohistochemistry revealed astrocytic expression of RCAN1-4 after SE. These results demonstrate astrocytic upregulation of RCAN1 and RCAN1-4 in the mouse hippocampus in the acute and subacute phases of epileptogenesis, providing foundational information for the potential role of RCAN1 in reactive astrocytes during epileptogenesis.


Subject(s)
Animals , Astrocytes , Calcineurin , Calcium , Cell Death , Diazepam , Epilepsy , Epilepsy, Temporal Lobe , Fluorescent Antibody Technique , Hippocampus , Humans , Immunohistochemistry , In Situ Hybridization , Male , Mice , Microglia , Neurons , Neuropil , Oxidative Stress , Pilocarpine , RNA, Messenger , Seizures , Status Epilepticus , Up-Regulation , Viola
19.
Einstein (Säo Paulo) ; 18: eAO5236, 2020. graf
Article in English | LILACS | ID: biblio-1133772

ABSTRACT

ABSTRACT Objective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton's jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.


RESUMO Objetivo Acompanhar a expansão de células-tronco mesenquimais de cordão umbilical por dois marcadores clássicos de senescência, p16 (INK4A) e p21 (CDKN1A), usando métodos práticos, rápidos e com custo menor do que a técnica padrão-ouro de Western blotting, para avaliar sua aplicabilidade em laboratório. Métodos Células-tronco mesenquimais de cordão umbilical foram isoladas da geleia de Wharton e, após controle de qualidade e caracterização morfológica e imunofenotípica por citometria de fluxo, foram expandidas em cultura, até chegarem próximas à parada do ciclo celular (senescência replicativa). Resultados Foi feita a comparação entre células jovens, na passagem 5, e células pré-senescentes, na passagem 10, avaliando a expressão proteica dos marcadores clássicos de senescência celular p16 e p21, comparando os resultados obtidos por Western blotting com os obtidos por citometria de fluxo e imunofluorescência indireta. Conclusão O seguimento de culturas celulares, por meio da imunofluorescência indireta de p16, permite identificar as culturas de células-tronco mesenquimais de cordão umbilical em risco de atingirem a senescência replicativa.


Subject(s)
Humans , Umbilical Cord/physiology , Fluorescent Antibody Technique/methods , Cellular Senescence , Mesenchymal Stem Cells/physiology , Flow Cytometry/methods , Biomarkers/blood , Cells, Cultured , Blotting, Western , Cyclin-Dependent Kinase Inhibitor p16 , Cyclin-Dependent Kinase Inhibitor p21
20.
Braz. oral res. (Online) ; 34: e006, 2020. tab, graf
Article in English | LILACS | ID: biblio-1055522

ABSTRACT

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.


Subject(s)
Signal Transduction/physiology , Smad1 Protein/physiology , Induced Pluripotent Stem Cells/cytology , Ameloblasts/cytology , Phosphorylation , Time Factors , Gene Expression , Cell Differentiation/physiology , Cell Differentiation/genetics , Cells, Cultured , Blotting, Western , Fluorescent Antibody Technique , Culture Media, Serum-Free , Reverse Transcriptase Polymerase Chain Reaction , MAP Kinase Signaling System/physiology , Activin Receptors/analysis , Activin Receptors/physiology , RNA Interference , p38 Mitogen-Activated Protein Kinases/analysis , p38 Mitogen-Activated Protein Kinases/physiology , Bone Morphogenetic Protein Receptors, Type II/analysis , Bone Morphogenetic Protein Receptors, Type II/physiology , Smad1 Protein/analysis
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