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Mexico City has one of the highest incidences of acute lymphoblastic leukemia (ALL) globally, with patients showing low survival, and high relapse rates. To gain more insight into the molecular features of B-ALL in Mexican children, we isolated CD10 + /CD19 + precursor B lymphoblasts from four bone marrow and nine peripheral blood samples of B-ALL patients using a fluorescence-activated cell sorting protocol. The global gene expression profile (BM vs PB) revealed 136 differentially expressed genes; 62 were upregulated (45.6%) and 74 were downregulated (54.4%). Pearson's correlation coefficient was calculated to determine the similarity between pre-B lymphoblast populations. We selected 26 highly significant genes and validated 21 by RT-qPCR (CNN3, STON2, CALN1, RUNX2, GADD45A, CDC45, CDC20, PLK1, AIDA, HCK, LY86, GPR65, PIK3CG, LILRB2, IL7R, TCL1A, DOCK1, HIST1H3G, PTPN14, CD72, and NT5E). The gene set enrichment analysis of the total expression matrix and the ingenuity pathway analysis of the 136 differentially expressed genes showed that the cell cycle was altered in the bone marrow with four overexpressed genes (PLK1, CDC20, CDC45, and GADD45A) and a low expression of IL7R and PIK3CG, which are involved in B cell differentiation. A comparative bioinformatics analysis of 15 bone marrow and 10 peripheral blood samples from Hispanic B-ALL patients collected by the TARGET program, corroborated the genes observed, except for PIK3CG. We conclude the Mexican and the Hispanic B-ALL patients studied present common driver alterations and histotype-specific mutations that could facilitate risk stratification and diagnostic accuracy and serve as potential therapeutic targets.
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Yellow fever (YF) is a life-threatening viral disease endemic in parts of Africa and Latin America. Although there is a very efficacious vaccine since the 1930s, YF still causes 29,000-60,000 annual deaths. During recent YF outbreaks there were issues of vaccine shortage of the current egg-derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the circulating mosquito vector could potentially start local transmission. Here we investigated the production of YF virus-like particles (VLPs) using stably transfected HEK293 cells. Process intensification was achieved by combining sequential FACS (fluorescence-activated cell sorting) rounds to enrich the stable cell pool in terms of high producers and the use of perfusion processes. At shaken-tube scale, FACS enrichment of cells allowed doubling VLP production, and pseudoperfusion cultivation (with daily medium exchange) further increased VLP production by 9.3-fold as compared to batch operation mode. At perfusion bioreactor scale, the use of an inclined settler as cell retention device showed operational advantages over an ATF system. A one-step steric exclusion chromatography purification allowed significant removal of impurities and is a promising technique for future integration of upstream and downstream operations. Characterization by different techniques confirmed the identity and 3D-structure of the purified VLPs.
Assuntos
Vacinas de Partículas Semelhantes a Vírus , Vacina contra Febre Amarela , Vírus da Febre Amarela/química , Células HEK293 , Humanos , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacina contra Febre Amarela/química , Vacina contra Febre Amarela/isolamento & purificaçãoRESUMO
Mammalian testes are very heterogeneous organs, with a high number of different cell types. Testicular heterogeneity, together with the lack of reliable in vitro culture systems of spermatogenic cells, have been an obstacle for the characterization of the molecular bases of the unique events that take place along the different spermatogenic stages. In this context, flow cytometry has become an invaluable tool for the analysis of testicular heterogeneity, and for the purification of stage-specific spermatogenic cell populations, both for basic research and for clinical applications. In this review, we highlight the importance of flow cytometry for the advances on the knowledge of the molecular groundwork of spermatogenesis in mammals. Moreover, we provide examples of different approaches to the study of spermatogenesis that have benefited from flow cytometry, including the characterization of mutant phenotypes, transcriptomics, epigenetic and genome-wide chromatin studies, and the attempts to establish cell culture systems for research and/or clinical aims such as infertility treatment.
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Citometria de Fluxo , Mamíferos , Espermatogênese/fisiologia , Animais , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular/genética , Suscetibilidade a Doenças , Citometria de Fluxo/métodos , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , MasculinoRESUMO
Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins ßIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.
Assuntos
Transplante de Tecido Encefálico/métodos , Queratinócitos/metabolismo , Animais , Humanos , RatosRESUMO
The Duchenne smile hypothesis is that smiles that include eye constriction (AU6) are the product of genuine positive emotion, whereas smiles that do not are either falsified or related to negative emotion. This hypothesis has become very influential and is often used in scientific and applied settings to justify the inference that a smile is either true or false. However, empirical support for this hypothesis has been equivocal and some researchers have proposed that, rather than being a reliable indicator of positive emotion, AU6 may just be an artifact produced by intense smiles. Initial support for this proposal has been found when comparing smiles related to genuine and feigned positive emotion; however, it has not yet been examined when comparing smiles related to genuine positive and negative emotion. The current study addressed this gap in the literature by examining spontaneous smiles from 136 participants during the elicitation of amusement, embarrassment, fear, and pain (from the BP4D+ dataset). Bayesian multilevel regression models were used to quantify the associations between AU6 and self-reported amusement while controlling for smile intensity. Models were estimated to infer amusement from AU6 and to explain the intensity of AU6 using amusement. In both cases, controlling for smile intensity substantially reduced the hypothesized association, whereas the effect of smile intensity itself was quite large and reliable. These results provide further evidence that the Duchenne smile is likely an artifact of smile intensity rather than a reliable and unique indicator of genuine positive emotion.
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Extracellular matrix (ECM) gives structure, support, and is the niche for several cells found in skeletal muscle. ECM is mainly produced by muscle connective tissue (CT) fibroblasts during development and regeneration. Stromal fibroadipogenic progenitors (FAPs) are CT fibroblasts-like mesenchymal progenitors (MPs) with important roles in regeneration and degeneration. Chronic damage restrains the normal regenerative behavior of muscle fibroblasts/FAPs. Thus, the isolation and study of these mesenchymal progenitors are of crucial importance for understanding their behavior and biology. We investigated whether adult muscle CT fibroblasts (hereafter referred to as adherent fibroblasts [aFbs]) cultured via pre-plating strategy belong to a heterogeneous population of FAPs. By combining microscopy, western blot analyses, flow cytometry, and FACS we determined that aFbs isolated from skeletal muscle largely overlap with FAPs. In addition, we used the PDGFRαEGFP mice in order to corroborate our results with EGFP+ FAPs. Moreover, our strategy allows the isolation of activated EGFP+ FAPs from the murine DMD model PDGFRαEGFP; mdx and PDGFRαEGFP denervated mice. Here we report that 1â¯h 30â¯min of pre-plating strategy allows the isolation and culture of a highly enriched population of aFbs. These cells are phenotypically and biochemically a FAPs-like population of adherent cells. In addition, aFbs respond in the same fashion as FAPs to Nilotinib, an inducer of FAPs apoptosis. Moreover, flow cytometry characterization of these aFbs suggests that 85% of them express the MP marker PDGFRα, and isolation of aFbs from the PDGFRαEGFP mice suggests that 75% of them show high EGFP expression. Furthermore, TGF-ß1 induces aFbs proliferation, myofibroblast differentiation, and ECM production. We were also able to isolate activated aFbs from skeletal muscle of the DMD mice and from the PDGFRαEGFP mice 2-days after denervation. Our findings suggest that the in vitro pre-plating strategy allows the isolation and culture of a relatively pure aFbs population, which resembles FAPs in vitro.
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Neurogenesis is a plastic event modulated by external cues. Systemic inflammation decreases neurogenesis in the dentate gyrus (DG) in part through the proliferative restrain of neural precursor cells (NPCs). To evaluate if inflammation affects the cell cycle progression of particular populations of NPCs, we treated young-adult mice with a single i.p. injection of saline or 1 mg/kg LPS. After 7 days, we analysed proliferation of new BrdU+/DCX+ cells through immunohistochemistry. We extracted the hippocampus and performed a neurosphere assay and a flow cytometric analysis to evaluate proliferation and to identify the phase of the cell cycle in specific populations of DG-derived NPCs. We show that the number of BrdU+/DCX+ cells diminishes in the LPS-treated group and that the number of primary neurospheres derived from LPS-injected animals is significantly reduced compared to the saline-injected group. Flow cytometry revealed that inflammation does not affect the total number of Type 1 BLBP+/TBR2- cells, while the total number of Type 2 intermediate precursor cells (IPCs) (TBR2+) from the LPS-treated group was increased. Cell cycle analysis shows a decrease in the total rate of NPCs in phases S, G2 and M in the LPS-treated group. The percentage of Type 1 BLBP+/TBR2- cells in each cell cycle phase was not different between groups, while there was a fewer number of Type 2 TBR2+ cells in S/G2/M phase. These results show that inflammation alters the appropriate cell cycle progression of Type 2 IPCs, which may contribute to the decrease in the birth rate of DG neurons.
Assuntos
Hipocampo/patologia , Inflamação/patologia , Células-Tronco Neurais/patologia , Animais , Comportamento Animal/efeitos dos fármacos , Contagem de Células , Ciclo Celular , Proliferação de Células , Tamanho Celular , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/administração & dosagem , Masculino , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Atividade Motora/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neuropeptídeos/metabolismo , Esferoides Celulares/metabolismo , Redução de Peso/efeitos dos fármacosRESUMO
The mouse olfactory epithelium is composed of a heterogeneous population of olfactory sensory neurons, where each neuron expresses one single type of odorant receptor gene, out of a repertoire of ~1000 different genes. Fluorescent-activated cell sorting (FACS) is a powerful technique, which can be used to isolate a cellular subpopulation from a heterogeneous tissue. The sorted neurons can then be used in gene expression studies, or analyzed for the presence of different DNA epigenetic modification marks. Here we describe a method to separate a subpopulation of olfactory sensory neurons expressing the odorant receptor Olfr17. In this method, the main olfactory epithelium from transgenic Olfr17-IRES-GFP mice is dissociated into single cells, followed by separation of the GFP positive cells by FACS.
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Citometria de Fluxo/métodos , Mucosa Olfatória/citologia , Receptores Odorantes , Células Receptoras Sensoriais/citologia , Animais , Camundongos , Camundongos Transgênicos , Mucosa Olfatória/metabolismo , Células Receptoras Sensoriais/metabolismoRESUMO
BACKGROUND: The growth-promoting effects of IGF-I is mediated through the IGF-I receptor (IGF1R), a widely expressed cell-surface tyrosine kinase receptor. IGF1R copy number variants (CNV) can cause pre- and postnatal growth restriction or overgrowth. METHODS: Whole exome sequence (WES), chromosomal microarray, and targeted IGF1R gene analyses were performed on 3 unrelated children who share features of small for gestational age, short stature, and elevated serum IGF-I, but otherwise had clinical heterogeneity. Fluorescence-activated cell sorting (FACS) analysis of cell-surface IGF1R was performed on live primary cells derived from the patients. RESULTS: Two novel IGF1R CNV and a heterozygous IGF1R nonsense variant were identified in the 3 patients. One CNV (4.492 Mb) was successfully called from WES, utilizing eXome-Hidden Markov Model (XHMM) analysis. FACS analysis of cell-surface IGF1R on live primary cells derived from the patients demonstrated a â¼50% reduction in IGF1R availability associated with the haploinsufficiency state. CONCLUSION: In addition to conventional methods, IGF1R CNV can be identified from WES data. FACS analysis of live primary cells is a promising method for efficiently evaluating and screening for IGF1R haploinsufficiency. Further investigations are necessary to delineate how comparable IGF1R availability leads to the wide spectrum of clinical phenotypes and variable responsiveness to rhGH therapy.
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Transtornos do Crescimento/genética , Haploinsuficiência , Receptores de Somatomedina/genética , Criança , Exoma , Feminino , Transtornos do Crescimento/diagnóstico , Humanos , Receptor IGF Tipo 1RESUMO
Microglia are the innate immune cells of the central nervous system (CNS) and play an important role in the maintenance of tissue homeostasis, providing neural support and neuroprotection. Microglia constantly survey their environment and quickly respond to homeostatic perturbations. Microglia are increasingly implicated in neuropathological and neurodegenerative conditions, such as Alzheimer's disease, Parkinson's disease, and glioma progression. Here, we describe a detailed isolation protocol for microglia and immune infiltrates, optimized for large amounts of post mortem tissue from human and rhesus macaque, as well as smaller tissue amounts from mouse brain and spinal cord, that yield a highly purified microglia population (up to 98 % purity). This acute isolation protocol is based on mechanical dissociation and a two-step density gradient purification, followed by fluorescence-activated cell sorting (FACS) to obtain pure microglia and immune infiltrate populations.
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Neoplasias Encefálicas/patologia , Separação Celular/métodos , Citometria de Fluxo/métodos , Glioma/patologia , Microglia/patologia , Doenças Neurodegenerativas/patologia , Animais , Anticorpos/química , Antígenos CD/genética , Antígenos CD/imunologia , Biomarcadores/metabolismo , Encéfalo/imunologia , Encéfalo/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Separação Celular/instrumentação , Centrifugação com Gradiente de Concentração/métodos , Citometria de Fluxo/instrumentação , Expressão Gênica , Glioma/genética , Glioma/imunologia , Humanos , Macaca mulatta , Camundongos , Microglia/imunologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/imunologia , Medula Espinal/imunologia , Medula Espinal/patologiaRESUMO
Pictures of facial expressions of emotion are used in a wide range of experiments. The last decade has seen an increase in the number of studies presenting local sets of emotion stimuli. However, only a few existing sets contain pictures of Latin Americans, despite the growing attention emotion research is receiving in this region. Here we present the development and validation of the Universidad Nacional de Cordoba, Expresiones de Emociones Faciales (UNCEEF), a Facial Action Coding System (FACS)-verified set of pictures of Argentineans expressing the six basic emotions, plus neutral expressions. FACS scores, recognition rates, Hu scores, and discrimination indices are reported. Evidence of convergent validity was obtained using the Pictures of Facial Affect in an Argentine sample. However, recognition accuracy was greater for UNCEEF. The importance of local sets of emotion pictures is discussed.
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Bases de Dados Factuais , Emoções , Expressão Facial , Estimulação Luminosa/métodos , Adolescente , Adulto , Argentina , Feminino , Humanos , Masculino , Reconhecimento Psicológico , Adulto JovemRESUMO
T-cell hybridoma assays have been widely used for the in vitro study of antigen processing and presentation because they represent an unlimited source of cells and they bypass the difficulty of maintaining T-cell clones in culture. One of the most widely used methods to assess hybridoma activation is measurement of CTLL-2 cell proliferation, which is dependent on IL-2. However, continuous culture of this cell line results in a loss of sensitivity, and significant interassay variability can occur. Therefore, our goal was to develop a method to assess T-cell hybridoma activation that was fast and sensitive with low variability based on the IL-2 secretion assay. The assay used flow cytometry detection and employed the hen egg lysozyme (HEL)-specific 3A9 hybridoma as a model. The original murine IL-2 secretion assay protocol from Miltenyi Biotec® was tested and modified; the conjugated capture antibody (anti-CD45-anti-IL-2) was added together with the stimulus at the beginning of the antigen presentation assay instead of after antigenic stimulation. With this modification, the percentage of detectable CD4+IL-2+ cells following HEL stimulation rose from 4.5% with the original protocol (0.8% without stimulus) to 94.1% (0.8% without stimulus) with the newly proposed method under the conditions evaluated in this study. This modification allowed us to evaluate the activation of hybridomas directly and more rapidly (~18h) than the reference method that assayed CTLL-2 cell proliferation using the MTT reduction assay (~48h). In conclusion, the proposed method offered a rapid alternative for screening T-cell hybridomas and evaluating their antigen-specific activation.
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Biomarcadores/análise , Imunoensaio/métodos , Interleucina-2/metabolismo , Ativação Linfocitária , Linfócitos T/citologia , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/citologia , Linhagem Celular , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/imunologia , Hibridomas , Camundongos , Camundongos Endogâmicos C3H , Muramidase/imunologiaRESUMO
Research on the postnatal development of pancreatic beta-cells has become an important subject in recent years. Understanding the mechanisms that govern beta-cell postnatal maturation could bring new opportunities to therapeutic approaches for diabetes. The weaning period consists of a critical postnatal window for structural and physiologic maturation of rat beta-cells. To investigate transcriptome changes involved in the maturation of beta-cells neighboring this period, we performed microarray analysis in fluorescence-activated cell-sorted (FACS) beta-cell-enriched populations. Our results showed a variety of gene sets including those involved in the integration of metabolism, modulation of electrical activity, and regulation of the cell cycle that play important roles in the maturation process. These observations were validated using reverse hemolytic plaque assay, electrophysiological recordings, and flow cytometry analysis. Moreover, we suggest some unexplored pathways such as sphingolipid metabolism, insulin-vesicle trafficking, regulation of transcription/transduction by miRNA-30, trafficking proteins, and cell cycle proteins that could play important roles in the process mentioned above for further investigation.
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Células Secretoras de Insulina/metabolismo , Lactação/genética , Transcriptoma , Animais , Biomarcadores , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Ciclo Celular/genética , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Imunofenotipagem , Insulina/metabolismo , Masculino , Fenótipo , RatosRESUMO
Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4(+) and CD8(+) T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4(+) T lymphocytes from HIV chronic infection and in CD8(+) T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes.
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Linfócitos B/enzimologia , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD8-Positivos/enzimologia , Peroxidase/biossíntese , Linfócitos B/imunologia , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Separação Celular , Citometria de Fluxo , Infecções por HIV/enzimologia , Infecções por HIV/imunologia , Hepatite C/enzimologia , Hepatite C/imunologia , Humanos , Imunofenotipagem , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Peroxidase/imunologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
CIGB-247 is a cancer vaccine that is a formulation of a recombinant protein antigen representative of the human vascular endothelial growth factor (VEGF) with a bacterially-derived adjuvant (VSSP). The vaccine has shown an excellent safety profile in mice, rats, rabbits, not-human primates and in recent clinical trials in cancer patients. Response to the vaccine is characterized by specific antibody titers that neutralize VEGF/VEGFR2 binding and a cytotoxic tumor-specific response. To expand our present anti-VEGF active immunotherapy strategies, we have now studied in mice and non-human primates the effects of vaccination with a formulation of our recombinant VEGF antigen and aluminum phosphate adjuvant (hereafter denominated CIGB-247-A). Administered bi-weekly, CIGB-247-A produces high titers of anti-VEGF IgG blocking antibodies in 2 mice strains. Particularly in BALB/c, the treatment impaired subcutaneous F3II mammary tumor growth and reduced the number of spontaneous lung macro metastases, increasing animals' survival. Spleen cells from specifically immunized mice directly killed F3II tumor cells in vitro. CIGB-247-A also showed to be immunogenic in non-human primates, which developed anti-VEGF blocking antibodies and the ability for specific direct cell cytotoxic responses, all without impairing the healing of deep skin wounds or other side effect. Our results support consideration of aluminum phosphate as a suitable adjuvant for the development of new vaccine formulations using VEGF as antigen.
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Adjuvantes Imunológicos/administração & dosagem , Compostos de Alumínio/administração & dosagem , Vacinas Anticâncer/imunologia , Química Farmacêutica , Fosfatos/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Neutralizantes/sangue , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Chlorocebus aethiops , Citotoxicidade Imunológica , Feminino , Esquemas de Imunização , Leucócitos Mononucleares/imunologia , Masculino , Neoplasias Mamárias Animais/terapia , Neoplasias Mamárias Experimentais/terapia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Metástase Neoplásica/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Tuberculosis (TB) remains the world's leading cause of morbidity and mortality. Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine currently in use, its efficacy is highly variable. It has been suggested that early antigenic presentation is a pivotal event leading to a better immune response in TB vaccine models. To investigate this further, we compared in vitro cell-mediated immune responses in the context of early sensitization with TB (i.e. healthy adults vaccinated with BCG when they were young, HD; n = 25) to those in its absence (i.e., newborns with naïve immunity to TB, UV; n = 10) by challenging mononuclear cells with BCG Moreau. After 48 hours, CD4+ and CD8+ T cells were harvested from both groups and stained for PD-1/CD25/ FOXP3. In addition, supernatants were assayed for a broad range of cytokines using an array system. The HD group showed robust reactivity to Protein Purified Derivative and BCG while the naïve, UV group did not. Similarly, in terms of PD-1 expression and Treg cells (CD4+/CD25high(+)/FOXP3+), only the HD group showed higher levels in CD4 lymphocytes. Otherwise, only the UV group showed expression of CD25dim+ as an activation marker dependent on BCG infection. In terms of cytokines, the HD group showed higher levels of Th1 (IL-2/TNF-α/IFN-γ) and regulatory (IL-10) profiles, with monocytes, but not Tr1 cells, acting as the main source of IL-10. Taken together, our results highlight critical roles of early sensitization with TB in mounting cell-mediated immune responses.
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Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Leucócitos Mononucleares/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Brasil , Células Cultivadas , Meios de Cultura/química , Citocinas/análise , Fatores de Transcrição Forkhead/análise , Voluntários Saudáveis , Humanos , Subunidade alfa de Receptor de Interleucina-2/análise , Leucócitos Mononucleares/química , Receptor de Morte Celular Programada 1/análise , Subpopulações de Linfócitos T/química , Adulto JovemRESUMO
Cestodes show a remarkable proliferative capability that sustains the constant growth and differentiation of proglottids essential for their lifestyle. It is believed that a separate population of undifferentiated stem cells (the so-called germinative cells) are the only cells capable of proliferation during growth and development. The study of this particular cell subpopulation is hampered by the current lack of methods to isolate it. In this work, we developed a reproducible flow cytometry and cell sorting method to quantify and isolate the proliferating cells in the tetrathyridia larvae of the model cestode Mesocestoides corti, based on the DNA content of the cells. The isolated cells display the typical germinative cell morphology, and can be used for RNA isolation with a yield in the ng to µg range. We expect that this approach may facilitate the characterization of the germinative cells in M. corti and other model tapeworms.
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Separação Celular/métodos , Citometria de Fluxo/métodos , Mesocestoides/citologia , Animais , Benzimidazóis , Ciclo Celular , Proliferação de Células , Fluoresceínas , Corantes Fluorescentes , Indicadores e Reagentes , Larva/citologia , Mesocestoides/crescimento & desenvolvimento , Camundongos , Modelos Animais , Propídio , Reprodutibilidade dos Testes , Tripsina/metabolismoRESUMO
Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependence of the first step of glycolysis on mitochondrial ATP. The oxidative phosphorylation (OXPHOS) system is responsible for the synthesis of approximately 90% of the ATP in normal cells and up to 50% in most glycolytic cancers; therefore, inhibition of the electron transport chain (ETC) emerges as an attractive therapeutic target. We studied the effect of a lipophilic isoprenylated catechol, 3-hydroxybakuchiol (3-OHbk), a putative ETC inhibitor isolated from Psoralea glandulosa. 3-OHbk exerted cytotoxic and anti-proliferative effects on the TA3/Ha mouse mammary adenocarcinoma cell line and induced a decrease in the mitochondrial transmembrane potential, the activation of caspase-3, the opening of the mitochondrial permeability transport pore (MPTP) and nuclear DNA fragmentation. Additionally, 3-OHbk inhibited oxygen consumption, an effect that was completely reversed by succinate (an electron donor for Complex II) and duroquinol (electron donor for Complex III), suggesting that 3-OHbk disrupted the electron flow at the level of Complex I. The inhibition of OXPHOS did not increase the level of reactive oxygen species (ROS) but caused a large decrease in the intracellular ATP level. ETC inhibitors have been shown to induce cell death through necrosis and apoptosis by increasing ROS generation. Nevertheless, we demonstrated that 3-OHbk inhibited the ETC and induced apoptosis through an interaction with Complex I. By delivering electrons directly to Complex III with duroquinol, cell death was almost completely abrogated. These results suggest that 3-OHbk has antitumor activity resulting from interactions with the ETC, a system that is already deficient in cancer cells.