RESUMEN

The prevalent pathogens associated with bovine uterine infections are bacteria that appear to increase the host's susceptibility to secondary infections with other bacteria or viruses, among which BoGHV4 is the most frequently found. In this work, the study of the pathways of apoptosis induction was carried out on an experimental model of primary culture of endometrial cells, in order to know the implication of BoGHV4 and the presence of bacterial LPS in the pathogenesis of the bovine reproductive tract. For this, different staining techniques and molecular analysis by RT-PCR were used. The results obtained allowed us to conclude that the level of cell death observed in the proposed primary culture is directly related to the time of viral infection and the presence of LPS in BoGHV4 infection. The apoptosis indices in cells infected with BoGHV4 and BoGHV4 + LPS revealed a maximum that correlated with the appearance of cytopathic effects and the maximum viral titers in the model studied. However, morphological, biochemical, and molecular changes were evident during both early and late stages of apoptosis. These findings provide information on the factors that may influence the pathogenesis of BoGHV4 and help to better understand the mechanisms involved in virus infection.

RESUMEN

Fish cell culture is a common in vitro tool for studies in different fields such as virology, toxicology, pathology and immunology of fish. Fish cell cultures are a promising help to study how to diagnose and control relevant viral and intracellular bacterial infections in aquaculture. They can also be used for developing vaccines and immunostimulants, especially with the ethical demand aiming to reduce and replace the number of fish used in research. This study aimed to isolate head kidney primary cell cultures from three Chilean salmonids: Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss, and characterize the response to bacterial and viral stimuli by evaluating various markers of the innate and adaptive immune response. Specifically, the primary cell cultures of the head kidney from the three salmonids studied were cultured and exposed to two substances that mimic molecular patterns of different pathogens, i.e., Lipopolysaccharide (LPS) (bacterial) and Polyinosinic: polycytidylic acid (POLY I:C). Subsequently, we determined the mRNA expression profiles of the TLR-1, TLR-8, IgM, TLR-5, and MHC II genes. Head kidney primary cell cultures from the three species grown in vitro responded differently to POLY I:C and LPS. This is the first study to demonstrate and characterize the expression of immune genes in head kidney primary cell culture isolated from three salmonid species. It also indicates their potential role in developing immune responses as defense response agents and targets of immunoregulatory factors.

RESUMEN

The neurovascular unit (NVU) is a multicellular structure comprising of neurons, glial cells, and non-neural cells, and it is supported by a specialized extracellular matrix, the basal lamina. Astrocytes, brain microvascular endothelial cells (BMECs), pericytes, and smooth muscle cells constitute the blood-brain barrier (BBB). BMECs have a mesodermal origin and invade the nervous system early in neural tube development, forming the BBB anatomical core. BMECs are connected by adherent junction complexes composed of integral membrane and cytoplasmic proteins. In vivo and in vitro studies have shown that, given the proximity and relationship with neural cells, BMECs acquire a unique gene expression profile, proteome, and specific mechanical and physical properties compared to endothelial cells from the general vasculature. BMECs are fundamental in maintaining brain homeostasis by regulating transcellular and paracellular transport of fluids, molecules, and cells. Therefore, it is essential to gain in-depth knowledge of the dynamic cellular structure of the cells in the NVU and their interactions with health and disease. Here we describe a significantly improved and simplified protocol using C57BL/6 newborn mice at postnatal day 1 (PND1) to isolate, purify, and culture BMECs monolayers in two different substrates (glass coverslips and transwell culture inserts). In vitro characterization and validation of the BMEC primary culture monolayers seeded on glass or insert included light microscopy, immunolabeling, and gene expression profile. Transendothelial electrical resistance (TEER) measurement and diffusion test were used as functional assays for adherent junction complexes and integrity and permeability of BMECs monolayers. The protocol presented here for the isolation and culture of BMECs is more straightforward than previously published protocols and yields a high number of purified cells. Finally, we tested BMECs function using the oxygen-glucose deprivation (OGD) model of hypoxia. This protocol may be suitable as a bioscaffold for secondary cell seeding allowing the study and better understanding of the NVU.

RESUMEN

This chapter presents the protocols for developing of skin equivalents (SE) and reconstructed human epidermis (RHE) models for dermal toxicity evaluation as an alternative method to animal use in research. It provides a detailed protocol for the in vitro reconstruction of human skin from primary keratinocytes, melanocytes, and fibroblasts obtained from foreskin biopsies, including the procedures for reconstruction of a stratified epidermis on a polyester membrane. SE and RHE developed through these methods have been proven suitable not only for dermal toxicity studies, but also for investigating of pathological conditions in the skin, such as diabetes and invasion of melanoma.

Asunto(s)

Epidermis/efectos de los fármacos , Cultivo Primario de Células/métodos , Pruebas de Irritación de la Piel/métodos , Células Cultivadas , Humanos

RESUMEN

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-old's lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V-FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days' culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.

RESUMEN

Abstract The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-old's lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V-FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days' culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.

RESUMEN

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-olds lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V–FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.(AU)

Asunto(s)

Animales , Masculino , Ovinos , Células Madre , Inhibidores de Proteínas Quinasas/análisis , Espermatogonias , Citometría de Flujo

RESUMEN

ABSTRACT Zika virus (ZIKV) infection during pregnancy is associated with a congenital syndrome. Although the virus can be detected in human placental tissue and sexual transmission has been verified, it is not clear how the virus reaches the fetus. Despite the emerging severity caused by ZIKV infection, no specific prophylactic and/or therapeutic treatment is available. The aim of the present study was to evaluate the effectiveness antiviral of nitazoxanide (NTZ) in two important congenital transmission targets: (i) a primary culture of human placental chorionic cells, and (ii) human cervical epithelial cells (C33-A) infected with Brazilian ZIKV strain. Initially, NTZ activity was screened in ZIKV infected Vero cells under different treatment regimens with non-toxic drug concentrations for 48 h. Antiviral effect was found only when the treatment was carried out after the viral inoculum. A strong effect against the dengue virus serotype 2 (DENV-2) was also observed suggesting the possibility of treating other Flaviviruses. Additionally, it was shown that the treatment did not reduce the production of infectious viruses in insect cells (C6/36) infected with ZIKV, indicating that the activity of this drug is also related to host factors. Importantly, we demonstrated that NTZ treatment in chorionic and cervical cells caused a reduction of infected cells in a dose-dependent manner and decreased viral loads in up to 2 logs. Pre-clinical in vitro testing evidenced excellent therapeutic response of infected chorionic and cervical cells and point to future NTZ activity investigation in ZIKV congenital transmission models with the perspective of possible repurposing of NTZ to treat Zika fever, especially in pregnant women.

Asunto(s)

Animales , Femenino , Humanos , Embarazo , Virus Zika , Infección por el Virus Zika , Tiazoles , Replicación Viral , Células Vero , Brasil , Chlorocebus aethiops , Infección por el Virus Zika/tratamiento farmacológico , Nitrocompuestos

RESUMEN

Zika virus (ZIKV) infection during pregnancy is associated with a congenital syndrome. Although the virus can be detected in human placental tissue and sexual transmission has been verified, it is not clear how the virus reaches the fetus. Despite the emerging severity caused by ZIKV infection, no specific prophylactic and/or therapeutic treatment is available. The aim of the present study was to evaluate the effectiveness antiviral of nitazoxanide (NTZ) in two important congenital transmission targets: (i) a primary culture of human placental chorionic cells, and (ii) human cervical epithelial cells (C33-A) infected with Brazilian ZIKV strain. Initially, NTZ activity was screened in ZIKV infected Vero cells under different treatment regimens with non-toxic drug concentrations for 48 h. Antiviral effect was found only when the treatment was carried out after the viral inoculum. A strong effect against the dengue virus serotype 2 (DENV-2) was also observed suggesting the possibility of treating other Flaviviruses. Additionally, it was shown that the treatment did not reduce the production of infectious viruses in insect cells (C6/36) infected with ZIKV, indicating that the activity of this drug is also related to host factors. Importantly, we demonstrated that NTZ treatment in chorionic and cervical cells caused a reduction of infected cells in a dose-dependent manner and decreased viral loads in up to 2 logs. Pre-clinical in vitro testing evidenced excellent therapeutic response of infected chorionic and cervical cells and point to future NTZ activity investigation in ZIKV congenital transmission models with the perspective of possible repurposing of NTZ to treat Zika fever, especially in pregnant women.

Asunto(s)

Infección por el Virus Zika , Virus Zika , Animales , Brasil , Chlorocebus aethiops , Femenino , Humanos , Nitrocompuestos , Embarazo , Tiazoles , Células Vero , Replicación Viral , Infección por el Virus Zika/tratamiento farmacológico

RESUMEN

BACKGROUND: Integrins are highly attractive targets in oncology due to their involvement in angiogenesis in a wide spectrum of cancer entities. Among several integrin inhibitors, cilengitide is suggested to be one of the most promising inhibitors. However, little is known about the cellular processes induced during cilengitide chemotherapy in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: For the current study, 3 HNSCC cell lines, SCC4, SCC15 and SCC25; and 3 primary culture cells, TU53, TU57, and TU63 were used. CD90, cytokeratin, and vimentin were stained immunohistochemically to identify the biological characteristics of these cell lines and primary culture cells and the cytostatic effect of cilengitide was evaluated. Quantitative polymerase chain reaction (qPCR) arrays were applied to evaluate target protein genes ITGAV, ITGB3, and ITGB5 of integrin αvß3 and αvß5 at respective concentrations of 50 and 100 µM cilengitide for 72 h. RESULTS: Cilengitide has significantly inhibited the proliferation of HNSCC cells in a dose-dependent way. At the same concentration, cilengitide suppressed the proliferation of primary culture cells even more strongly than it did that of cell lines, suggesting that primary culture cells retain more of their internal biological characteristics than do cell lines. qPCR assay detected downregulation of ITGAV, ITGB3, and ITGB5 gene expression after exposure to 50 µM of cilengitide. However, after exposure to 100-µM cilengitide, expression of these genes significantly increased both in cell lines and primary culture cells. CONCLUSIONS: RGD-containing small-molecule synthetic peptides might be considered in tumor chemotherapy in the near future. The different reactions of primary culture cells and cell lines demonstrated that individualized chemotherapy plans may be a feasible option. However, research on the role of cilengitide in HNSCC therapy is still in its early stages, and further investigations are required.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/patología , Cadenas beta de Integrinas/química , Integrina beta3/química , Venenos de Serpiente/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Apoptosis/efectos de los fármacos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Cadenas beta de Integrinas/genética , Cadenas beta de Integrinas/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Células Tumorales Cultivadas

RESUMEN

Cytoglobin (Cygb) is a hexacoordinate protein, associated with the transport of oxygen, nitric oxide scavenging, tumor suppression and protection against oxidative stress and inflammation. This protein is expressed in brain areas including the preoptic area (POA) of the anterior hypothalamus, the region responsible for the regulation of body temperature. In this study, we show that Cygb is upregulated in the rat hypothalamus 2.5 h and 5 h after intravenous administration of lipopolysaccharide (LPS). We investigated the effect of treatment with Cygb in POA primary cultures stimulated with LPS for 4 h. The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were measured and the results showed that Cygb reduced the concentrations of both cytokines. We further observed a decrease in immunoreactivity of the inflammatory transcription factor nuclear factor-κB (NF-κB), but not NF-IL6 and STAT3, in the nucleus of Cygb-treated POA cells. These findings suggest that Cygb attenuates the secretion of IL-6 and TNF-α in LPS-stimulated POA primary cultures via inhibition of the NF-κB signaling pathway, indicating that this protein might play an important role in the control of neuroinflammation and fever.

RESUMEN

O Sistema Nervoso Central (SNC) humano é formado por cerca de 86,1 bilhões de neurônios entre o encéfalo e a medula espinhal. O desenvolvimento pré-natal humano (tempo da concepção ao nascimento) possui cerca de 38 semanas, e é dividido na fase embrionária que corresponde ao período das 8 semanas iniciais da gestação, seguido pela fase fetal. A fase embrionária é o período mais vulnerável à ocorrência de anormalidades congênitas. Por ser um órgão com grande período de desenvolvimento, o SNC está sujeito às alterações genéticas, epigenéticas e ambientais. Durante a fase de implantação do embrião, o DNA é mais vulnerável às influências externas, como à fumaça do cigarro, aumentando o risco de retardo do desenvolvimento fetal, o risco de morte súbita pós-natal e de anormalidades do sistema imune. Neste contexto, o objetivo deste trabalho é avaliar os efeitos da exposição à fumaça do cigarro sobre o processo de neuroinflamação da prole de camundongos C57BL/6 expostos à fumaça do cigarro durante a gestação e desafiados ou não com LPS. Para tanto, camundongos C57BL/6 fêmeas prenhes foram expostas à fumaça do cigarro desde o plug vaginal até o nascimento da prole. No 3º dia de vida, os filhotes foram separados para três linhas de trabalho: 1) in vivo: os animais foram desafiados com LPS pelo período de 4h, seguidos de eutanasia e análises de PCR Array do SNC. 2) in vitro: os encéfalos dissecados foram utilizados para a preparação de cultura mista de glia e da cultura enriquecida com neurônio. Após a maturação celular, as células foram estimuladas com LPS 100 ng/mL e, após 24h, foram realizados ensaios de CBA, citometria de fluxo, PCR, dosagem de NO, avaliação de morte celular e metilação global. 3) Encefalomielite Autoimune Experimental (EAE): após o desmame, os animais foram mantidos em suas caixas moradia por 8 semanas sem nenhum estímulo externo, e então foram imunizados com MOG35-55 para o desenvolvimento da EAE. Nos experimentos in vivo observamos o aumento da transcrição de genes relacionados ao processo inflamatório, como interleucinas e quimiocinas. Em relação aos experimentos in vitro observamos maior crescimento de células astrocitárias (astrogliose), e células da microglia com aumento de moléculas co-estimuladoras (CD80 e CD86) bem como da transcrição e concentração de citocinas pró-inflamatórias e produção de NO. Em cultura enriquecida de neurônio, foi observado aumento na porcentagem de células em apoptose no grupo exposto à fumaça do cigarro desafiados ou não com LPS. O bloqueio da atividade da microglia pela minociclina reverteu a apoptose e diminuiu a produção de NO minimizando a morte celular. Em relação aos experimentos de EAE, os animais expostos à fumaça do cigarro no período gestacional, quando imunizados na vida adulta apresentam aumento no grau da doença bem como maior persistência da mesma quando observado escore clínico, além de acompanhados de um grau maior de infiltrado celular e desmielinização. Desta forma podemos concluir que a exposição à fumaça do cigarro durante o período gestacional leva a uma programação fetal com aumento da resposta neuroinflamatória frente a um estimulo sistêmico, trazendo consequências na vida adulta

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The human central nervous system (CNS) is made up of about 86.1 billion neurons between the brain and the spinal cord. The human prenatal development (time from conception to birth) is about 38 weeks, and is divided into the embryonic phase that corresponds to the period of the initial 8 weeks of gestation, followed by the fetal phase. The embryonic stage is the period most vulnerable to the occurrence of congenital abnormalities. Because it is an organ with a long period of development, the CNS is subject to genetic, epigenetic and environmental changes. During the embryo implantation phase, DNA is more vulnerable to external influences such as cigarette smoke, increasing the risk of delay on fetal development, risk of sudden postnatal death, and abnormalities of the immune system. In this context, the aim of this work is to evaluate the effects of exposure to cigarette smoke on the neuroinflammation process of offspring of C57BL/6 mice exposed to cigarette smoke during gestation and challenged or not with LPS. For this, pregnant female C57BL/6 mice were exposed to cigarette smoke from vaginal plug to offspring birth. On the 3rd day of life the offspring were separated into three lines of work: 1) in vivo: the animals were challenged with 1mg/Kg LPS and after 4h they followed to euthanasia; PCR analysis of the CNS was made in this period. 2) in vitro: dissected encephalons were used for the preparation of mixed culture of glia and the culture enriched with neuron. After cell maturation, the cells were stimulated with 100 ng/mL LPS and, after 24 hours, CBA, flow cytometry, PCR, NO assay, cell death and global methylation assays were performed. 3) Experimental Autoimmune Encephalomyelitis (EAE): After weaning, the animals were kept in their housing for 8 weeks without any external stimulus, and then were immunized with MOG35-55 for the development of EAE. In the in vivo experiments we observed increased transcription of genes related to the inflammatory process, such as interleukins and chemokines. In vitro experiments showed higher growth of astrocytes (astrogliosis) and microglia cells with increased stimulatory molecules (CD80 and CD86) as well as the transcription and concentration of proinflammatory cytokines and NO production. In the enriched neuron culture, an increase in the percentage of cells in apoptosis was observed in the group exposed to cigarette smoke challenged or not with LPS. Blocking microglial activity by minocycline reversed apoptosis and decreased NO production by minimizing cell death. The EAE experiments shows that the animals exposed to cigarette smoke in the gestational period, when immunized in adulthood, present an increase in the degree of the disease as well as a greater persistence of the disease; The higher as the clinical score higher is the degree of cellular infiltration and demyelination. In this way we can conclude that the exposure to cigarette smoke during the gestational period leads to a fetal programming with increased neuroinflammatory response to a systemic stimulus and that this is able to last until the adult stage

Asunto(s)

Animales , Femenino , Ratones , Contaminación por Humo de Tabaco/efectos adversos , Tabaquismo/complicaciones , Encefalomielitis Autoinmune Experimental/complicaciones , Atención Prenatal/clasificación , Anomalías Congénitas , Técnicas In Vitro , Sistema Nervioso Central

RESUMEN

BACKGROUND: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. RESULTS: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. CONCLUSIONS: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. METHODS: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

RESUMEN

Background: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. Results: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. Conclusions: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. Methods: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

RESUMEN

Parkinson's disease (PD) is the second most common neurodegenerative disorder and has both unknown etiology and non-curative therapeutic options. Patients begin to present the classic motor symptoms of PD-tremor at rest, bradykinesia and rigidity-once 50-70% of the dopaminergic neurons of the nigrostriatal pathway have degenerated. As a consequence of this, it is difficult to investigate the early-stage events of disease pathogenesis. In vitro experimental models are used extensively in PD research because they present a controlled environment that enables the direct investigation of the early molecular mechanisms that are potentially involved with dopaminergic degeneration, as well as for the screening of potential therapeutic drugs. However, the establishment of PD in vitro models is a controversial issue for neuroscience research not only because it is challenging to mimic, in isolated cell systems, the physiological neuronal environment, but also the pathophysiological conditions experienced by human dopaminergic cells in vivo during the progression of the disease. Since no previous work has attempted to systematically review the literature regarding the establishment of an optimal in vitro model, and/or the features presented by available models used in the PD field, this review aims to summarize the merits and limitations of the most widely used dopaminergic in vitro models in PD research, which may help the PD researcher to choose the most appropriate model for studies directed at the elucidation of the early-stage molecular events underlying PD onset and progression.

Asunto(s)

Dopamina/fisiología , Neuronas Dopaminérgicas/fisiología , Enfermedad de Parkinson , Animales , Antiparkinsonianos/farmacología , Técnicas de Cultivo de Célula , Línea Celular , Células Cultivadas , Cuerpo Estriado/patología , Dopamina/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Humanos , Técnicas In Vitro , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Neurotoxinas/toxicidad , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Cultivo Primario de Células , Ratas , Sustancia Negra/patología

RESUMEN

The epithelium is a highly dynamic system, which plays a crucial role in the homeostasis of the intestinal tract. However, studies on the physiological and pathophysiological functions of intestinal epithelial cells (IECs) have been hampered due to lack of normal epithelial cell models. In the present study, we established a reproducible method for primary culture of mouse IECs, which were isolated from the viable small intestinal crypts of murine fetuses (on embryonic day 19), using type I collagenase and hyaluronidase in a short span of time (≤20 min). With this method, continuously growing mouse IECs, which can be subcultured over a number of passages, were obtained. The obtained cell lines formed a tight cobblestone-like arrangement, displayed long and slender microvilli, expressed characteristic markers (cytokeratin 18 and Notch-1), and generated increasing transepithelial electrical resistance and low paracellular permeability during in vitro culture. The cells also had enzymatic activities of alkaline phosphatase and sucrase-isomaltase, and secreted various cytokines (IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1), responding to the stimulation of Escherichia coli. These results show that the primary-cultured mouse IECs obtained by the method established here had the morphological and immunological characteristics of IECs. This culture system can be a beneficial in vitro model for studies on mucosal immunology and toxicology.

Asunto(s)

Animales , Masculino , Femenino , Técnicas de Cultivo de Célula/métodos , Células Epiteliales/citología , Hialuronoglucosaminidasa , Intestino Delgado/citología , Metaloproteinasa 13 de la Matriz , Proliferación Celular , Células Cultivadas , Colagenasas , Citocinas/metabolismo , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Hematoxilina , Ratones Endogámicos BALB C , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Reproducibilidad de los Resultados , Factores de Tiempo

RESUMEN

During gonad and adrenal development, the POD-1/capsulin/TCF21transcription factor negatively regulates SF-1/NR5A1expression, with higher SF-1 levels being associated with increased adrenal cell proliferation and tumorigenesis. In adrenocortical tumor cells, POD-1 binds to the SF-1 E-box promoter region, decreasing SF-1 expression. However, the modulation of SF-1 expression by POD-1 has not previously been described in normal adrenal cells. Here, we analyzed the basal expression of Pod-1 and Sf-1 in primary cultures of glomerulosa (G) and fasciculata/reticularis (F/R) cells isolated from male Sprague-Dawley rats, and investigated whether POD-1 overexpression modulates the expression of endogenous Sf-1 and its target genes in these cells. POD-1 overexpression, following the transfection of pCMVMycPod-1, significantly decreased the endogenous levels of Sf-1 mRNA and protein in F/R cells, but not in G cells, and also decreased the expression of the SF-1 target StAR in F/R cells. In G cells overexpressing POD-1, no modulation of the expression of SF-1 targets, StAR and CYP11B2, was observed. Our data showing that G and F/R cells respond differently to ectopic POD-1 expression emphasize the functional differences between the outer and inner zones of the adrenal cortex, and support the hypothesis that SF-1 is regulated by POD-1/Tcf21 in normal adrenocortical cells lacking the alterations in cellular physiology found in tumor cells.

Asunto(s)

Animales , Masculino , Corteza Suprarrenal/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Fosfoproteínas/metabolismo , Factor Esteroidogénico 1/metabolismo , Corteza Suprarrenal/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Immunoblotting , Cultivo Primario de Células , Fosfoproteínas/análisis , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Mensajero/análisis , Factor Esteroidogénico 1/análisis , Zona Fascicular/citología , Zona Fascicular/metabolismo , Zona Glomerular/citología , Zona Glomerular/metabolismo , Zona Reticular/citología , Zona Reticular/metabolismo

RESUMEN

The aim of this study was establish a protocol for isolation and primary culture of neurons from tropical freshwater fish species Hoplias malabaricus for assessment of the effects of neurotoxic substances as saxitoxins (STXs). Cells from brain of H. malabaricus were treated with different concentrations of trypsin, dispase and papain for tissue dissociation. Cells type was separated by cellular gradient and basic fibroblast growth factor (bFGF) supplement nutrition media were added. The dissociated cells were plated with medium and different STXs concentrations and the toxic cellular effects such as oxidative stress, neurotoxicity, and genotoxicity and apoptosis process were evaluated. Cultures treated with bFGF showed the greatest adherence, survival and cellular development. STXs increased specific activity of glutathione peroxidase and lipoperoxidation levels, were cytotoxic and genotoxic indicated by the comet assay. Although the STXs effects due the blockage of sodium channels is reported to be reversible, the time exposure and concentration of STXs suggested cellular injuries which can lead to neuropathology. The establishment of primary neuronal culture protocol enables new applications for neurotoxicological assessments.

Asunto(s)

Peces Planos , Neuronas/efectos de los fármacos , Estrés Oxidativo , Saxitoxina/toxicidad , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Técnicas de Cultivo de Célula , Neuronas/citología

RESUMEN

Glioblastomas are the most lethal primary brain tumor that frequently relapse or progress as focal masses after radiation, suggesting that a fraction of tumor cells are responsible for the tumor regrowth. The identification of a brain tumor cell subpopulation with potent tumorigenic activity supports the cancer stem cell hypothesis in solid tumors. The goal of this study is to determine a methodology for the establishment of primary human glioblastoma cell lines. Our aim is achieved by taking the following approaches: (i) the establishment of primary glioblastoma cell culture; (ii) isolation of neurospheres derived from glioblastoma primary cultures; (iii) selection of CD133 cells from neurospheres, (iv) formation of subspheres in the CD133-positive population, (v) study of the expression level of GFAP, CD133, Nestin, Nanog, CD34, Sox2, CD44, and CD90 markers on tumor subspheres. Hence, we described a successful method for isolation of CD133-positive cell population and establishment of glioblastoma neurospheres from this primary culture, which are more robust than the ones derived straight from the tumor. Pointed out that the neurospheres derived from glioblastoma primary culture showed 29% more cells expressing CD133 then the ones straight tumor-derived, denoting a higher concentration of CD133-positive cells in the neurospheres derived from glioblastoma primary culture. These CD133-positive fractions were able to further generate subspheres. The subspheres derived from glioblastoma primary culture presented a well-defined morphology while the ones derived from the fresh tumor were sparce and less robust. And the negative fraction of CD133 cells was unable to generate subspheres. The tumor subspheres expressed GFAP, CD133, Nestin, Nanog, CD44, and CD90. Also, the present study describes an optimization of neurospheres/subspheres isolation from glioblastoma primary culture by selection of CD133-positive adherent stem cell.

RESUMEN

O Mycoplasma é considerado cosmopolita, podendo ser disseminado através do comércio internacional de animais, sêmen industrializado e de produtos de transferência de embriões. A expansão de células do cumulus é utilizada como parâmetro de avaliação de oócitos bovinos cultivados in vitro e suas alterações morfológicas são representativas. O objetivo deste trabalho foi avaliar a interação do Mycoplasma bovigenitalium, exposto experimentalmente à cultura primária de célula do cumulus, após o período de maturação. Complexos oócitos cumulus (COCs) obtidos através de punção folicular de ovários bovinos, provenientes de abatedouro, foram divididos em dois grupos para serem maturados durante 24h em meio de maturação (TCM199 + hormônios) em estufa a 38º C, 5% de CO2, 95% de umidade. Posteriormente, os oócitos foram retirados das placas, permanecendo somente com as células do cumulus aderidas. Com o monoestrato celular formado, um grupo foi infectado com 30 mL de M. bovigenitalium, replicado em meio Hayflick modificado a 37º C em estufa de microaerofilia, enquanto o outro foi mantido como controle. Os resultados mostraram que, com 24h de exposição ao patógeno, as culturas apresentaram um pequeno número de células arredondadas e granulosas, quando comparadas as dos controles. Esse efeito persistiu até o sétimo dia, onde se iniciou um processo de descolamento das células. Pode-se concluir que uma contaminação por micoplasma pode ser imperceptível às manipulações da FIV, pois células infectadas por esse grupo de bactérias não apresentam turvações no meio de cultura e, quando não lisam a célula hospedeira, tornam mais suscetível ao ambiente e outros agentes infecciosos.

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INTERACTION OF MYCOPLASMA BOVIGENITALIUM WITH PRIMARY-CULTURE CUMULUS CELLS AFTER IN VITRO MATURATION PERIOD. The Mycoplasma is considered cosmopolitan and can be disseminated through international trade of animals, industrialized semen and embryo transfer products. The expansion of cumulus cells is used as a parameter for evaluating cattle oocytes cultivated in vitro, and their morphological changes are representative. The aim of the present study was to evaluate the interaction of Mycoplasma bovigenitalium with primary-culture cumulus cells, after the in vitro maturation period. Cumulus complex oocytes (COCs) obtained through follicular puncture of ovaries from a cattle slaughterhouse were divided into two groups to be matured for 24 hours in the maturation medium (TCM 199 + hormones) in a climate controlled chamber at 38° C, 5% CO2, and 95% humidity. Subsequently, the oocytes were removed from the plates, which remained with only the cumulus cells attached. With the monostratum cell formed, one group was infected with 30 mL (5 x 106 cells/mL) M. bovigenitalium, replicated in modified Hayflick medium at 37° C in a mycroaerofilic chamber, while the other was kept as a control. The results showed that with 24 hours of exposure to the pathogen, the cultures showed a small number of rounded and grainy cells, when compared to the controls. This effect persisted until the 7th day, when a process of cell detachment began. It can be concluded that a mycoplasma contamination may be imperceptible to the manipulations of FIV, because cells infected by this group of bacteria present no cloudiness in the culture field, and when they do not lyse the host cell, they make it more susceptible to environment and other infectious agents.

Asunto(s)

Animales , Bovinos/clasificación , Embrión de Mamíferos/microbiología , Oocitos