RESUMEN

Our study highlights the apoptosis, cell cycle, DNA ploidy, and autophagy molecular mechanisms network to identify prostate pathogenesis and its prognostic role. Caspase 3/7 expressions, cell cycle, adhesion glycoproteins, autophagy, nuclear shrinkage, and oxidative stress by flow-cytometry analysis are used to study the BPH microenvironment's heterogeneity. A high late apoptosis expression by caspases 3/7 activity represents an unfavorable prognostic biomarker, a dependent predictor factor for cell adhesion, growth inhibition by arrest in the G2/M phase, and oxidative stress processes network. The heterogeneous aggressive phenotype prostate adenoma primary cell cultures present a high S-phase category (>12%), with an increased risk of death or recurrence due to aneuploid status presence, representing an unfavorable prognostic biomarker, a dependent predictor factor for caspase 3/7 activity (late apoptosis and necrosis), and cell growth inhibition (G2/M arrest)-linked mechanisms. Increased integrin levels in heterogenous BPH cultures suggest epithelial-mesenchymal transition (EMT) that maintains an aggressive phenotype by escaping cell apoptosis, leading to the cell proliferation necessary in prostate cancer (PCa) development. As predictor biomarkers, the biological mechanisms network involved in apoptosis, the cell cycle, and autophagy help to establish patient prognostic survival or target cancer therapy development.

Asunto(s)

Apoptosis , Autofagia , Ciclo Celular , Hiperplasia Prostática , Humanos , Masculino , Hiperplasia Prostática/patología , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/genética , Pronóstico , Cultivo Primario de Células , Transición Epitelial-Mesenquimal/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/genética , Fenotipo , Anciano , Caspasa 3/metabolismo , Proliferación Celular , Caspasa 7/metabolismo , Persona de Mediana Edad , Estrés Oxidativo

RESUMEN

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Asunto(s)

11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2 , Aldosterona , Cortisona , Hidrocortisona , Espectrometría de Masas en Tándem , Humanos , Cortisona/metabolismo , Cortisona/análisis , Hidrocortisona/metabolismo , Aldosterona/metabolismo , Espectrometría de Masas en Tándem/métodos , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Cromatografía Liquida/métodos , Cultivo Primario de Células , Células Cultivadas , Cromatografía Líquida con Espectrometría de Masas

RESUMEN

The likelihood that potential new drugs will successfully navigate the current translational pipeline is poor, with fewer than 10% of drug candidates making this transition successfully, even after their entry into clinical trials. Prior to this stage, candidate drugs are typically evaluated by using models of increasing complexity, beginning with basic in vitro cell culture studies and progressing through to animal studies, where many of these candidates are lost due to lack of efficacy or toxicology concerns. There are many reasons for this poor translation, but interspecies differences in functional and physiological parameters undoubtedly contribute to the problem. Improving the human-relevance of early preclinical in vitro models may help translatability, especially when targeting more nuanced species-specific cell processes. The aim of the current study was to define a set of guidelines for the effective transition of human primary cells of multiple lineages to more physiologically relevant, translatable, animal-free in vitro culture conditions. Animal-derived biomaterials (ADBs) were systematically replaced with non-animal-derived alternatives in the in vitro cell culture systems, and the impact of the substitutions subsequently assessed by comparing the kinetics and phenotypes of the cultured cells. ADBs were successfully eliminated from primary human dermal fibroblast, uterine fibroblast, pulmonary fibroblast, retinal endothelial cell and peripheral blood mononuclear cell culture systems, and the individual requirements of each cell subtype were defined to ensure the successful transition toward growth under animal-free culture conditions. We demonstrate that it is possible to transition ('humanise') a diverse set of human primary cell types by following a set of simple overarching principles that inform the selection, and guide the evaluation of new, improved, human-relevant in vitro culture conditions.

Asunto(s)

Materiales Biocompatibles , Humanos , Animales , Cultivo Primario de Células/métodos , Alternativas a las Pruebas en Animales , Células Cultivadas , Fibroblastos/efectos de los fármacos

RESUMEN

Reproduction by egg-laying (oviparity) or live-bearing (viviparity) is a genetically determined trait fundamental to the biology of amniotes. Squamates are an emerging model for the genetics of reproductive mode yet lack cell culture models valuable for exploring molecular mechanisms. Here, we report a novel primary culture model for reproductive biology: cell cultures derived from the oviduct tissues (infundibulum, uterus and vagina) of oviparous and viviparous common lizards (Lacertidae: Zootoca vivipara). We maintained and expanded these cultures for over 100 days, including repeated subculturing and successful revival of cryopreserved cells. Immunocytochemical investigation suggested expression of both epithelial and fibroblast-like proteins, and RNA sequencing of cultured cells as compared to in vivo oviduct tissue showed changes in gene expression in response to the cell culture environment. Despite this, we confirmed the maintenance of distinct gene expression patterns in viviparous and oviparous cells after 60+ days of cell culture, finding 354 differentially expressed genes between viviparous and oviparous cells. Furthermore, we confirmed the expression of 15 viviparity-associated candidate genes in cells maintained for 60+ days in culture. Our study demonstrates the feasibility and utility of oviduct cell culture for molecular analysis of reproductive mode and provides a tool for future genetic experiments.

Asunto(s)

Lagartos , Oviductos , Oviparidad , Viviparidad de Animales no Mamíferos , Animales , Femenino , Lagartos/genética , Lagartos/fisiología , Oviductos/citología , Oviductos/metabolismo , Viviparidad de Animales no Mamíferos/genética , Oviparidad/genética , Células Cultivadas , Cultivo Primario de Células/métodos

RESUMEN

Primary neuronal cultures allow for in vitro analysis of early developmental processes such as axon pathfinding and growth dynamics. When coupled with methods to visualize and measure microtubule dynamics, this methodology enables an inside look at how the cytoskeleton changes in response to extracellular signaling cues. Here, we describe the culturing conditions and tools required to extract primary cortical neurons from postnatal mouse brains and visualize cytoskeletal components.

Asunto(s)

Corteza Cerebral , Neuronas , Animales , Ratones , Neuronas/citología , Neuronas/metabolismo , Corteza Cerebral/citología , Células Cultivadas , Microtúbulos/metabolismo , Cultivo Primario de Células/métodos , Técnicas de Cultivo de Célula/métodos , Citoesqueleto/metabolismo

RESUMEN

BACKGROUND: Few reports have confirmed whether exosomes derived from fibroblasts can regulate the process of melanogenesis. We wondered whether exosomes derived from fibroblasts could have a potent regulatory effect on melanogenesis and explored the underlying mechanisms. OBJECTIVE: This study aimed to find the role of fibroblasts in melanocytes and revealed the related mechanisms. METHODS: RT-qPCR, Western blot analysis were conducted to measure the RNA and protein expression level of various related genes. miRNA sequencing, mass spectrum analysis and subsequent bioinformatics analysis were employed to find the underlying targets. Zebrafish were employed to measure the melanin synthesis related process in vivo. Furthermore, electron microscopy, ROS measurement and dual-luciferase reporter assay were adopted to investigate the relationship between these processes. RESULTS: We found that exosomes derived from human primary dermal fibroblasts were internalized by human primary melanocytes and MNT1 cells and that the melanin content and the expression of melanin synthesis-related proteins TYR and MITF was inhibited by exosomes derived from UVB-induced human primary dermal fibroblasts. The miRNA expression profile in secreted exosomes changed significantly, with miR-25-5p identified as capable of regulating TSC2 expression via the CDS region. The miR-25-5p-TSC2 axis could affect the melanin content through subsequent cellular organelle dysfunction, such as mitochondrial dysfunction, endoplasmic reticulum stress and dysregulation of lysosomal cysteine proteases. CONCLUSION: We unveiled a novel regulatory role of fibroblasts in melanocytes, facilitated by the secretion of exosomes. miR-25-5p within exosomes plays a pivotal role in regulating melanogenesis via TSC2-induced cellular organelle dysfunction.

Asunto(s)

Exosomas , Fibroblastos , Melaninas , Melanocitos , MicroARNs , Proteína 2 del Complejo de la Esclerosis Tuberosa , Rayos Ultravioleta , Pez Cebra , Humanos , Exosomas/metabolismo , Exosomas/efectos de la radiación , MicroARNs/metabolismo , MicroARNs/genética , Fibroblastos/efectos de la radiación , Fibroblastos/metabolismo , Melaninas/biosíntesis , Melaninas/metabolismo , Melanocitos/efectos de la radiación , Melanocitos/metabolismo , Animales , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Rayos Ultravioleta/efectos adversos , Células Cultivadas , Estrés del Retículo Endoplásmico/efectos de la radiación , Cultivo Primario de Células , Factor de Transcripción Asociado a Microftalmía/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Mitocondrias/efectos de la radiación , Mitocondrias/metabolismo , Melanogénesis

RESUMEN

Primary cell culture is a powerful model system to address fundamental questions about organismal physiology at the cellular level, especially for species that are difficult, or impossible, to study under natural or semi-natural conditions. Due to their ease of use, primary fibroblast cultures are the dominant model system, but studies using both somatic and germ cells are also common. Using these models, genome evolution and phylogenetic relationships, the molecular and biochemical basis of differential longevities among species, and the physiological consequences of life history evolution have been studied in depth. With the advent of new technologies such as gene editing and the generation of induced pluripotent stem cells (iPSC), the field of molecular evolutionary physiology will continue to expand using both descriptive and experimental approaches.

Asunto(s)

Evolución Molecular , Cultivo Primario de Células , Animales , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Filogenia , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/fisiología

RESUMEN

Duck hepatitis B virus (DHBV) is an avian member of the hepatotropic DNA viruses, or hepadnaviridae. It shares with the human hepatitis B virus (HBV) a similar genomic organization and replication strategy via reverse transcription, but is simpler than HBV in lacking the X gene and in expressing just two coterminal envelope proteins: Large (L) and small (S). DHBV has been extensively used as a convenient and valuable animal model for study of the hepadnaviral life cycle, and for drug screening in vitro but also in vivo. Ducks and primary duck hepatocytes (PDHs) are inexpensive, easily accessible, and readily infected with DHBV. The high levels of genome replication and protein expression in duck liver and PDHs also facilitate monitoring of viral life cycle using conventional molecular biology techniques such as Southern blot for replicative DNA and covalently closed circular DNA (cccDNA), Northern blot for viral RNAs, and Western blot for viral proteins.

Asunto(s)

Patos , Infecciones por Hepadnaviridae , Virus de la Hepatitis B del Pato , Hepatocitos , Replicación Viral , Animales , Patos/virología , Hepatocitos/virología , Hepatocitos/metabolismo , Virus de la Hepatitis B del Pato/genética , Infecciones por Hepadnaviridae/virología , Infecciones por Hepadnaviridae/veterinaria , Modelos Animales de Enfermedad , Hepatitis Viral Animal/virología , ADN Viral/genética , Células Cultivadas , Cultivo Primario de Células/métodos , Técnicas de Cultivo de Célula/métodos

RESUMEN

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, development, size, and transparency. Conversely, dysfunctional LECs can lead to cataract formation and posterior capsule opacification (PCO). Consequently, establishing a robust primary LEC culture system is important to researchers engaged in lens development, biochemistry, cataract therapeutics, and PCO prevention. However, cultivating primary LECs has long presented challenges due to their limited availability, slow proliferation rate, and delicate nature. This study addresses these hurdles by presenting a comprehensive protocol for primary LEC culture. The protocol encompasses essential steps such as the formulation of an optimized culture medium, precise isolation of lens capsules, trypsinization techniques, subculture procedures, harvest protocols, and guidelines for storage and shipment. Throughout the culture process, cell morphology was monitored using phase-contrast microscopy. To confirm the authenticity of the cultured LECs, immunofluorescence assays were conducted to detect the presence and subcellular distribution of critical lens proteins, namely αA- and γ-crystallins. This detailed protocol equips researchers with a valuable resource for cultivating and characterizing primary LECs, enabling advancements in our comprehension of lens biology and the development of therapeutic strategies for lens-related disorders.

Asunto(s)

Células Epiteliales , Cristalino , Tripsina , Células Epiteliales/citología , Cristalino/citología , Animales , Ratones , Tripsina/química , Tripsina/metabolismo , Técnicas de Cultivo de Célula/métodos , Cultivo Primario de Células/métodos

RESUMEN

The number of chromosomes varies tremendously across species. It is not clear whether having more or fewer chromosomes could be advantageous. The probability of non-disjunction should theoretically decrease with smaller karyotypes, but too long chromosomes should enforce spatial constraint for their segregation during the mitotic anaphase. Here, we propose a new experimental cell system to acquire novel insights into the mechanisms underlying chromosome segregation. We collected the endemic Australian ant Myrmecia croslandi, the only known species with the simplest possible karyotype of a single chromosome in the haploid males (and one pair of chromosomes in the diploid females), since males are typically haploid in hymenopteran insects. Five colonies, each with a queen and a few hundreds of workers, were collected in the Canberra district (Australia), underwent karyotype analysis to confirm the presence of a single pair of chromosomes in worker pupae, and were subsequently maintained in the laboratory in Paris (France). Starting from dissociated male embryos, we successfully conducted primary cell cultures comprised of single-chromosome cells. This could be developed into a unique model that will be of great interest for future genomic and cell biology studies related to mitosis.

Asunto(s)

Hormigas , Cromosomas de Insectos , Animales , Hormigas/genética , Masculino , Femenino , Cultivo Primario de Células , Cariotipificación , Cariotipo , Haploidia , Segregación Cromosómica

RESUMEN

The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and neurological dysfunctions. The SARS-CoV-2 envelope protein (E protein) is a structural viroporin able to form ion channels in cell membranes, which is critical for viral replication. However, its effects in primary neurons have not been addressed. Here we used fluorescence microscopy and calcium imaging to study SARS-CoV-2 viroporin E localization and the effects on neuron damage and intracellular Ca2+ homeostasis in a model of rat hippocampal neurons aged in vitro. We found that the E protein quickly enters hippocampal neurons and colocalizes with the endoplasmic reticulum (ER) in both short-term (6-8 days in vitro, DIV) and long-term (20-22 DIV) cultures resembling young and aged neurons, respectively. Strikingly, E protein treatment induces apoptosis in aged neurons but not in young neurons. The E protein induces variable increases in cytosolic Ca2+ concentration in hippocampal neurons. Ca2+ responses to the E protein are due to Ca2+ release from intracellular stores at the ER. Moreover, E protein-induced Ca2+ release is very small in young neurons and increases dramatically in aged neurons, consistent with the enhanced Ca2+ store content in aged neurons. We conclude that the SARS-CoV-2 E protein quickly translocates to ER endomembranes of rat hippocampal neurons where it releases Ca2+, probably acting like a viroporin, thus producing Ca2+ store depletion and neuron apoptosis in aged neurons and likely contributing to neurological damage in COVID-19 patients.

Asunto(s)

Calcio , Retículo Endoplásmico , Hipocampo , Neuronas , SARS-CoV-2 , Animales , Ratas , Neuronas/metabolismo , Neuronas/virología , Neuronas/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/citología , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Envoltura de Coronavirus/metabolismo , COVID-19/virología , COVID-19/metabolismo , Células Cultivadas , Apoptosis/efectos de los fármacos , Cultivo Primario de Células , Muerte Celular/efectos de los fármacos , Proteínas Viroporinas/metabolismo

RESUMEN

OBJECTIVE: The endothelium regulates crucial aspects of vascular function, including hemostasis, vasomotor tone, proliferation, immune cell adhesion, and microvascular permeability. Endothelial cells (ECs), especially in arterioles, are pivotal for flow distribution and peripheral resistance regulation. Investigating vascular endothelium physiology, particularly in microvascular ECs, demands precise isolation and culturing techniques. METHODS: Freshly isolated ECs are vital for examining protein expression, ion channel behavior, and calcium dynamics. Establishing primary endothelial cell cultures is crucial for unraveling vascular functions and understanding intact microvessel endothelium roles. Despite the significance, detailed protocols and comparisons with intact vessels are scarce in microvascular research. We developed a reproducible method to isolate microvascular ECs, assessing substrate influence by cultivating cells on fibronectin and gelatin matrix gels. This comparative approach enhances our understanding of microvascular endothelial cell biology. RESULTS: Microvascular mesenteric ECs expressed key markers (VE-cadherin and eNOS) in both matrix gels, confirming cell culture purity. Under uncoated conditions, ECs were undetected, whereas proteins linked to smooth muscle cells and fibroblasts were evident. Examining endothelial cell (EC) physiological dynamics on distinct matrix substrates revealed comparable cell length, shape, and Ca2+ elevations in both male and female ECs on gelatin and fibronectin matrix gels. Gelatin-cultured ECs exhibited analogous membrane potential responses to acetylcholine (ACh) or adenosine triphosphate (ATP), contrasting with their fibronectin-cultured counterparts. In the absence of stimulation, fibronectin-cultured ECs displayed a more depolarized resting membrane potential than gelatin-cultured ECs. CONCLUSIONS: Gelatin-cultured ECs demonstrated electrical behaviors akin to intact endothelium from mouse mesenteric arteries, thus advancing our understanding of endothelial cell behavior within diverse microenvironments.

Asunto(s)

Células Endoteliales , Gelatina , Microvasos , Óxido Nítrico Sintasa de Tipo III , Animales , Células Endoteliales/metabolismo , Células Endoteliales/citología , Ratones , Femenino , Masculino , Microvasos/citología , Microvasos/metabolismo , Microvasos/fisiología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Células Cultivadas , Fibronectinas/metabolismo , Fibronectinas/farmacología , Geles , Antígenos CD/metabolismo , Cadherinas/metabolismo , Cultivo Primario de Células , Endotelio Vascular/metabolismo , Endotelio Vascular/citología

RESUMEN

Domestic cats are the natural host of feline morbilliviruses (FeMV). Although other species can also be infected (such as dogs and opossums), no laboratory animal infection model is established so far. In vitro models for studying the molecular pathogenesis are therefore needed. For this purpose, propagation and titration of FeMV are key techniques. Unlike other morbilliviruses, such as canine distemper virus (CDV) or measles virus (MV), FeMV is a slow growing virus in cell culture and is difficult to titrate using classical plaque techniques. Here we describe methods for the efficient isolation of FeMV from natural sources (e.g., urine), the propagation of viral stocks, and their titration. In addition, we establish the generation of a three-dimensional infection model mimicking the feline tubular epithelium.

Asunto(s)

Infecciones por Morbillivirus , Morbillivirus , Animales , Gatos , Morbillivirus/patogenicidad , Morbillivirus/genética , Morbillivirus/fisiología , Infecciones por Morbillivirus/veterinaria , Infecciones por Morbillivirus/virología , Riñón/virología , Riñón/citología , Enfermedades de los Gatos/virología , Células Cultivadas , Cultivo de Virus/métodos , Modelos Animales de Enfermedad , Cultivo Primario de Células/métodos

RESUMEN

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

Asunto(s)

Hipocampo , Neuronas , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Hipocampo/metabolismo , Hipocampo/citología , Neuronas/metabolismo , Animales , Subunidades de Proteína/metabolismo , Subunidades de Proteína/genética , Células Cultivadas , Ratas , Humanos , Lentivirus/genética , Cultivo Primario de Células/métodos , Expresión Génica

RESUMEN

Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.

Asunto(s)

Encéfalo , Animales , Caballos/virología , Encéfalo/virología , Encéfalo/embriología , Encéfalo/citología , Cultivo Primario de Células/métodos , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/fisiología , Línea Celular , Neuronas/virología , Cultivo de Virus/métodos , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/veterinaria , Células Cultivadas , Replicación Viral

RESUMEN

The ability of the immune system to combat pathogens relies on processes like antigen sampling by dendritic cells and macrophages migrating through endo- and epithelia or penetrating them with their dendrites. In addition, other immune cell subtypes also migrate through the epithelium after activation. For paracellular migration, interactions with tight junctions (TJs) are necessary, and previous studies reported TJ protein expression in several immune cells. Our investigation aimed to characterize, in more detail, the expression profiles of TJ proteins in different immune cells in both naïve and activated states. The mRNA expression analysis revealed distinct expression patterns for TJ proteins, with notable changes, mainly increases, upon activation. At the protein level, LSR appeared predominant, being constitutively present in naïve cell membranes, suggesting roles as a crucial interaction partner. Binding experiments suggested the presence of claudins in the membrane only after stimulation, and claudin-8 translocation to the membrane occurred after stimulation. Our findings suggest a dynamic TJ protein expression in immune cells, implicating diverse functions in response to stimulation, like interaction with TJ proteins or regulatory roles. While further analysis is needed to elucidate the precise roles of TJ proteins, our findings indicate important non-canonical functions of TJ proteins in immune response.

Asunto(s)

Granulocitos , Sistema Inmunológico , Macrófagos , Receptores de Lipoproteína , Proteínas de Uniones Estrechas , Factores de Transcripción , Proteínas de Uniones Estrechas/metabolismo , Humanos , Colon , Organoides , Células HT29 , Granulocitos/metabolismo , Macrófagos/metabolismo , Sistema Inmunológico/metabolismo , Cultivo Primario de Células

RESUMEN

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

Asunto(s)

Neuronas , Receptores de N-Metil-D-Aspartato , Transfección , Neuronas/metabolismo , Transfección/métodos , Cultivo Primario de Células , Receptores de N-Metil-D-Aspartato/genética , ADN Complementario/química , ADN Complementario/genética , Animales , Roedores , Lípidos

RESUMEN

BACKGROUND: EcoHIV is a chimeric HIV that replicates in mice in CD4+ T cells, macrophages, and microglia (but not in neurons), causing lasting neurocognitive impairment resembling neurocognitive disease in people living with HIV. The present study was designed to develop EcoHIV-susceptible primary mouse brain cultures to investigate the indirect effects of HIV infection on neuronal integrity. RESULTS: We used two EcoHIV clones encoding EGFP and mouse bone marrow-derived macrophages (BMM), mixed mouse brain cells, or enriched mouse glial cells from two wild-type mouse strains to test EcoHIV replication efficiency, the identity of productively infected cells, and neuronal apoptosis and integrity. EcoHIV replicated efficiently in BMM. In mixed brain cell cultures, EcoHIV targeted microglia but did not cause neuronal apoptosis. Instead, the productive infection of the microglia activated them and impaired synaptophysin expression, dendritic density, and axonal structure in the neurons. EcoHIV replication in the microglia and neuronal structural changes during infection were prevented by culture with an antiretroviral. CONCLUSIONS: In murine brain cell cultures, EcoHIV replication in the microglia is largely responsible for the aspects of neuronal dysfunction relevant to cognitive disease in infected mice and people living with HIV. These cultures provide a tool for further study of HIV neuropathogenesis and its control.

Asunto(s)

Encéfalo , Microglía , Neuronas , Replicación Viral , Animales , Ratones , Encéfalo/virología , Encéfalo/patología , Neuronas/virología , Neuronas/patología , Microglía/virología , Células Cultivadas , Infecciones por VIH/virología , Macrófagos/virología , Modelos Animales de Enfermedad , Apoptosis , Humanos , VIH-1/fisiología , Cultivo Primario de Células , Ratones Endogámicos C57BL

RESUMEN

A 2D primary gill cell culture system of the sevenband grouper (Hyporthodus septemfasciatus) was established to validate the pathogenesis of nervous necrosis virus (NNV) as observed in previous studies. This system, developed using the double-seeded insert (DSI) technique, yielded confluent cell layers. Upon challenge with NNV in a setup containing both autoclaved salt water and L15 media in the apical compartment, viral replication akin to that anticipated based on previous studies was observed. Consequently, we advocate for the utilization of primary gill cell culture as a viable alternative to conventional methodologies for investigating host pathogen interactions.

Asunto(s)

Branquias , Nodaviridae , Replicación Viral , Animales , Branquias/virología , Branquias/citología , Nodaviridae/fisiología , Cultivo Primario de Células/métodos , Lubina/virología , Enfermedades de los Peces/virología , Técnicas de Cultivo de Célula/métodos , Infecciones por Virus ARN/veterinaria , Infecciones por Virus ARN/virología , Células Cultivadas , Interacciones Huésped-Patógeno

RESUMEN

BACKGROUND: The aberrant expression of tight junction (TJ) proteins play an important role in several diseases with impaired skin barriers, including atopic dermatitis, psoriasis, and chronic wounds. The evidence provided thus far suggests an important role of calcitriol in skin homeostasis. However, it is not known whether calcitriol improves the impaired skin barrier. OBJECTIVE: To investigate the effect of calcitriol on TJ barrier function in human primary keratinocytes. METHODS: Normal human primary keratinocytes were stimulated with calcitriol, and the expression of TJ-related proteins was measured by real-time PCR and Western blotting. Immunofluorescence was used to examine the intercellular distribution of TJ-related proteins. TJ barrier function was assessed by the transepithelial electrical resistance (TER) assay. RESULTS: We demonstrated that calcitriol increased the expression levels of TJ-related proteins, including claudin-4, claudin-7, occludin, and zonula occludens (ZO)- 1. Calcitriol enhanced the distribution of TJ-related proteins at cellcell borders and induced the phosphorylation of pathways involved in the regulation of TJ barrier function, such as atypical protein kinase C (aPKC), Ras-related C3 botulinum toxin substrate 1 (Rac1), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt), as evidenced by the effects of specific inhibitors on the above pathways. Indeed, we confirmed that calcitriol enhanced TER in keratinocyte monolayers. CONCLUSION: These findings showed that calcitriol could modify the expression of keratinocyte TJ proteins, contributing to the maintenance of homeostatic barrier function.

Asunto(s)

Calcitriol , Epidermis , Queratinocitos , Uniones Estrechas , Humanos , Calcitriol/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Células Cultivadas , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Transducción de Señal/efectos de los fármacos , Fosforilación/efectos de los fármacos , Ocludina/metabolismo , Cultivo Primario de Células , Proteína de la Zonula Occludens-1/metabolismo , Claudinas/metabolismo , Claudinas/genética , Impedancia Eléctrica