RESUMEN

The protein p32 (C1QBP) is a multifunctional and multicompartmental homotrimer that is overexpressed in many cancer types, including colon cancer. High expression levels of C1QBP are negatively correlated with the survival of patients. Previously, we demonstrated that C1QBP is an essential promoter of migration, chemoresistance, clonogenic, and tumorigenic capacity in colon cancer cells. However, the mechanisms underlying these functions and the effects of specific C1QBP protein inhibitors remain unexplored. Here, we show that the specific pharmacological inhibition of C1QBP with the small molecule M36 significantly decreased the viability rate, clonogenic capacity, and proliferation rate of different colon cancer cell lines in a dose-dependent manner. The effects of the inhibitor of C1QBP were cytostatic and non-cytotoxic, inducing a decreased activation rate of critical pro-malignant and mitogenic cellular pathways such as Akt-mTOR and MAPK in RKO colon cancer cells. Additionally, treatment with M36 significantly affected the mitochondrial integrity and dynamics of malignant cells, indicating that p32/C1QBP plays an essential role in maintaining mitochondrial homeostasis. Altogether, our results reinforce that C1QBP is an important oncogene target and that M36 may be a promising therapeutic drug for the treatment of colon cancer.

Asunto(s)

Neoplasias del Colon , Citostáticos , Humanos , Citostáticos/farmacología , Mitógenos/farmacología , Transducción de Señal , Proteínas Mitocondriales/metabolismo , Proliferación Celular , Proteínas Portadoras/metabolismo

RESUMEN

Intense stimulation of most living cells triggers the activation of immediate early genes, such as Fos and Jun families. These genes are important in cellular and biochemical processes, such as mitosis and cell death. The present study focused on determining the temporal expression pattern of Fos and Jun families in fibroblasts and neural stem cells of cerebellum, hippocampus, and subventricular zone (SVZ) of rats of different ages at 0, 0.5, 1, 3, and 6 h after stimulation with fibroblast growth factor (FGF)-2. In neonates, a similar expression pattern was observed in all cells analyzed, with lower expression in basal condition, peak expression at 0.5 h after stimulation, returning to baseline values between 1 and 3 h after stimulation. On the other hand, cells from adult animals only showed Fra1 and JunD expression after stimulation. In fibroblasts and hippocampus, Fra1 reached peak expression at 0.5 h after stimulation, while in the SVZ, peak level was observed at 6 h after stimulation. JunD in fibroblasts presented two peak expressions, at 0.5 and 6 h after stimulation. Between these periods, the expression observed was at a basal level. Nevertheless, JunD expression in SVZ and hippocampus was low and without significant changes after stimulation. Differences in mRNA expression in neonate and adult animals characterize the significant differences in neurogenesis and cell response to stimulation at different stages of development. Characterizing these differences might be important for the development of cell cultures, replacement therapy, and the understanding of the physiological response profile of different cell types.

Asunto(s)

Factor 2 de Crecimiento de Fibroblastos , Células-Madre Neurales , Animales , Ratas , Mitógenos , Proliferación Celular , Fibroblastos

RESUMEN

The effects on the ontogeny of serum cytokines and immune cells caused by feeding suckling piglets with sow/gilt colostrum and milk replacer was assessed in the present study. After farrowing, the piglets born were randomized into six groups: GG and SS (n = 10/group): piglets were kept with their dam; GS (n = 10): piglets were changed from gilts to sows; SG (n = 10): piglets were changed from sows to gilts; GMR (n = 6) and SMR (n = 8): piglets from either gilts or sows were isolated from the dams and were bottle-fed ad libitum with commercial formula milk replacer. The piglets remained in the groups during the first 24 h of life and were later returned to their respective mothers. Serum immunoglobulin concentration and lymphocyte proliferation from the blood, spleen, thymus, and mesenteric lymph node of the piglets were assessed at 24 h and at 28 days of age. Serum cytokine concentrations were measured through a cytokine multiplex assay at 24 h. Overall, piglets suckling on sows (SS and GS) had a higher concentration of serum immunoglobulin at 24 h, which was also associated with a rise in plasma cytokine concentration and greater ability of B and T cells from lymphatic organs and blood mononuclear cells to respond to mitogens. We suggest a bias towards Th1-, Th2-, and Th17-cell polarizing and cytokines during the suckling period, which may be influenced by maternal immunological factors in the colostrum, such as dam parity. All findings suggest sow parity having a possible role, which may contribute to exerting a modulating action on immune response development.

Asunto(s)

Calostro , Mitógenos , Animales , Citocinas , Femenino , Leche , Embarazo , Sus scrofa , Porcinos

RESUMEN

Relationship between lymphocyte function and cardiorespiratory fitness (CRF) is well-documented at rest; however, upon mitogen stimulation the proliferation and cytokine production alters, but knowledge is incipient about lymphocyte responses after mitogen stimulus according to CRF. So, the purpose of the present study was to analyze the lymphocyte function according to the physical fitness status of healthy young men. The study is divided in two experiments being the first analyzing the lymphocyte phenotypes profile and the inflammatory responses, according to CRF, in lymphocyte cell cultures treated for 48 h with concanavalin A (ConA). The second experiment analyzed the proliferation, reactive oxygen species production, viability, and mitochondrial polarization state in lymphocytes treated with ConA in different concentrations, considering the CRF levels. The results showed a difference in the percentage of total lymphocytes expression between groups (P = 0.011) observing a lower lymphocytes T expression in the group with high maximal oxygen consumption (V̇o2max) when compared with the moderate V̇o2max group. When treated with ConA, the lymphocytes of the low V̇o2max group released higher TNF-α concentration (P = 0.032), reflecting an elevated TNF-α/IL-10 ratio (P = 0.055), parallel with lower IL-6 production (P = 0.027), mainly when compared with the moderate V̇o2max group. In addition, there is a positive relationship between V̇o2max and IL-6 production (r = 0.507; P = 0.016), whereas the percentage of total lymphocytes (LyT%) shows a negative trend with V̇o2max (r = -0.497; P = 0.060). Also, individuals with lower V̇o2max showed reduced absolute and relative ROS production, lower cell proliferation, and higher mitochondrial membrane depolarization. In conclusion, cardiorespiratory fitness degree exerts a strong impact on lymphocyte function after mitogen stimulation.NEW & NOTEWORTHY The innovation of the research is to elucidate the impact of different physical fitness status on metabolism, cell proliferation, and lymphocyte activity and, consequently, on the specific inflammatory response against a mitogen.

Asunto(s)

Capacidad Cardiovascular , Humanos , Linfocitos , Masculino , Mitógenos , Consumo de Oxígeno , Aptitud Física

RESUMEN

INTRODUCTION: Nitric oxide is a gaseous radical produced by the nitric oxide endothelial synthase (eNOS) whose most studied physiological action is the vasodilation. However, it also acts in the defense of the organism through the formation of cytotoxic radicals, which can potentiate the inflammatory lesion of the cells. The Glu298Asp is a single nucleotide polymorphism (SNP) in the eNOS gene related to the risk of cardiovascular disease. Blacks present a higher prevalence of hypertension and cardiovascular mortality. Then, we aimed to evaluate the influence of Glu298Asp polymorphism on inflammatory response in vitro and gene expression in blacks. MATERIAL AND METHODS: Peripheral blood mononuclear cells (PBMC) from blacks with different Glu298Asp genotypes were treated with phytohemagglutinin (PHA), a mitogen and activator of T cells. Oxidative, inflammatory markers, and expression of inflammation genes were evaluated. RESULTS: The genotype frequencies were TT 6.7%; TG 29.3% and GG 64.0%. Activation of PBMCs with 125 µg of PHA modulated the expression of inflammatory genes and increased levels of inflammatory cytokines. The T allele showed increased susceptibility to inflammation (higher levels of interleukin 1, interleukin 6 and tumor necrosis factor alpha; p < 0.001). The G allele exhibited protection through higher levels of nitric oxide (p < 0.001) and fewer inflammatory cytokines. CONCLUSION: Despite methodological limitations related to in vitro assays, the whole of results suggested that Glu298Asp modulates inflammatory genes, the T allele is more susceptible to inflammation and the G allele is protective.

Asunto(s)

Citocinas/metabolismo , Leucocitos Mononucleares/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Alelos , Población Negra/genética , Estudios de Asociación Genética , Genotipo , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Mitógenos/farmacología , Óxido Nítrico/metabolismo , Fenotipo , Fitohemaglutininas/farmacología , Polimorfismo de Nucleótido Simple , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

BACKGROUND: Lectins have been studied in recent years due to their immunomodulatory activities. OBJECTIVE: We purified a lectin named OniL from tilapia fish (Oreochromis niloticus) and here we analyzed the cell proliferation and cytokine production in Balb/c mice splenocytes. METHODS: Cells were stimulated in vitro in 24, 48, 72 hours and 6 days with different concentrations of OniL and Con A. Evaluation of cell proliferation was performed through [3H]-thymidine incorporation, cytokines were investigated using ELISA assay and cell viability assay was performed by investigation of damage through signals of apoptosis and necrosis. RESULTS: OniL did not promote significant cell death, induced high mitogenic activity in relation to control and Con A and stimulated the cells to release high IL-2 and IL-6 cytokines. CONCLUSION: These findings suggest that, like Con A, OniL lectin can be used as a mitogenic agent in immunostimulatory assays.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Lectinas de Unión a Manosa/farmacología , Mitógenos/farmacología , Bazo/citología , Animales , Muerte Celular , Línea Celular , Supervivencia Celular , Concanavalina A/farmacología , Citocinas/biosíntesis , Masculino , Ratones Endogámicos BALB C , Tilapia

RESUMEN

Allergic conjunctivitis (AC) is one of the most common ophthalmological disorders seen in clinical practice. Growing evidence from recent years suggests that a subset of IL-10-expressing B cells is involved in inflammatory allergic diseases. In this study, we aimed to evaluate the potential involvement of blood Bregs cells in perennial allergic conjunctivitis (PAC), and interleukins (IL)-1ß, IL-6, IL-8, IL-10, and IL-12, and tumor necrosis factor (TNF)-α, were measured in tear samples and compared with healthy controls (HC) using flow cytometry. Non-significant differences in CD19⁺IL-10⁺ cell frequency between PAC patients and healthy controls (HC) were observed. Nevertheless, when we analyzed the mean fluorescence intensity (MFI) of IL-10 on CD19⁺CD38Lo/Med/Hi-gated cells, we observed a significant decrease in MFI in all Bregs subsets in PAC patients. Additionally, tear cytokines showed 2.8 times lower levels of IL-10 than TNF-α in PAC patients when compared to HC. Our findings demonstrate an immunological dysregulation in patients with allergic conjunctivitis, characterized by the low expression of IL-10 in circulating CD19⁺CD38⁺ Bregs subsets and an inverted tear IL-10/TNF-α ratio, promoting a local pro-inflammatory microenvironment. These findings highlight the novel pathologic changes involved in ocular allergic diseases. Understanding systemic and local mechanisms will aid the design of immunomodulating therapeutics at different levels.

Asunto(s)

Linfocitos B Reguladores/metabolismo , Conjuntivitis Alérgica/inmunología , Conjuntivitis Alérgica/metabolismo , Interleucina-10/metabolismo , Lágrimas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adolescente , Estudios de Casos y Controles , Niño , Femenino , Humanos , Subgrupos Linfocitarios/metabolismo , Masculino , Mitógenos/farmacología

RESUMEN

Lectins are carbohydrate binding proteins that can stimulate cell proliferation. This property makes these biomolecules capable of being used as mitogen reagents to study the interaction with lymphocytes allowing evaluation of immunomodulatory action, since B and T lymphocytes are related to humoral and innate immunity, respectively. Isolated cells from spleen, which include lymphocytes, are widely applied as a model in screening lectin mitogenic capacity. This mitotic stimulus is initiated by interaction of the lectin with T-cell receptor on cell surface. This brief review article aims to explain how cell proliferation, especially lymphocytes, can be achieved through lectin induction. Additionally, this work intends to highlight the main colorimetric and radiographic techniques to encourage the scientific community in searching for new mitogenic lectins.

Asunto(s)

Lectinas/metabolismo , Activación de Linfocitos/efectos de los fármacos , Mitosis , Animales , Linfocitos B/citología , Proliferación Celular , Colorimetría , Humanos , Inmunidad Humoral , Inmunidad Innata , Ratones , Mitógenos , Lectinas de Plantas , Ratas , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Bazo/citología , Linfocitos T/citología

RESUMEN

Aflatoxin B1 (AFB1), a mycotoxin found in food and feed, exerts harmful effects on humans and animals. The liver is the earliest target of AFB1, and its effects have been evaluated in animal models exposed to acute or chronic doses. Considering the possibility of sporadic ingestion of AFB1-contaminated food, this study investigated the impact of a single oral dose of AFB1 on liver function/cytokines and the lymphoproliferative response in mice. C57BL/6 mice were treated with a single oral AFB1 dose (44, 442 or 663 µg AFB1/kg of body weight) on the first day. Liver function (ALT, γ-GT, and total protein), cytokines (IL-4, IFN-γ, and IL-17), histopathology, and the spleen lymphoproliferative response to mitogens were evaluated on the 5th day. Although AFB1 did not produce any significant changes in the biochemical parameters, 663 µg AFB1/kg-induced hepatic upregulation of IL-4 and IFN-γ, along with liver tissue injury and suppression of the lymphoproliferative response to ConA (p < 0.05). In conclusion, a single oral dose of AFB1 exposure can induce liver tissue lesions, liver cytokine modulation, and immune suppression in C57BL/6 mice.

Asunto(s)

Aflatoxina B1/toxicidad , Citocinas/metabolismo , Hígado/efectos de los fármacos , Linfocitos/efectos de los fármacos , Administración Oral , Alanina Transaminasa/sangre , Animales , Proliferación Celular/efectos de los fármacos , Concanavalina A/farmacología , Lipopolisacáridos/farmacología , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Mitógenos/farmacología , Bazo/citología , gamma-Glutamiltransferasa/sangre

RESUMEN

OBJECTIVE: Iron deficiency is likely the most common nutritional deficiency worldwide; low iron concentrations have been related to alterations in immune system functions; therefore, the aim of this study was to determine the effect of low serum iron (LSI) concentrations on the production of proinflammatory cytokines by peripheral blood leukocytes in 8- to 12-y-old children from a local community. METHODS: We obtained 120 blood samples and determined full blood counts and serum iron concentrations. An LSI and a control group, paired by age and sex were established using serum iron <60 µg/dL as the cutoff point. Ferritin and C-reactive protein concentrations were quantified. Serum interferon (IFN)-γ and tumor necrosis factor (TNF)-α concentrations were measured in these groups by enzyme-linked immunosorbent assay. A second blood sample was taken from children in both groups to isolate peripheral blood mononuclear cells (PBMCs) and measure IFN-γ and TNF-α production by unstimulated and lipopolysaccharide/phorbol myristate acetate/ionomycin-stimulated leukocytes in vitro. RESULTS: Of the participants in the present study, 17.5% (21 children) presented LSI, as well as decreased ferritin concentrations. Differential counts from total blood samples showed a significant increase in leukocyte numbers in the LSI group, along with increased neutrophil frequencies and numbers but decreased lymphocyte frequencies. Decreased serum IFN-γ concentrations and decreased in vitro production of IFN-γ by PBMCs were found in the LSI group. CONCLUSIONS: The results of the present study suggest that low iron levels alter leukocyte subpopulations in circulation and have a detrimental effect on leukocyte production of proinflammatory cytokines after an antigenic challenge.

Asunto(s)

Anemia Ferropénica/fisiopatología , Enfermedades Asintomáticas , Fenómenos Fisiológicos Nutricionales Infantiles , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitosis/etiología , Factor de Necrosis Tumoral alfa/metabolismo , Anemia Ferropénica/sangre , Anemia Ferropénica/inmunología , Anemia Ferropénica/metabolismo , Recuento de Células Sanguíneas , Proteína C-Reactiva/análisis , Ionóforos de Calcio/farmacología , Niño , Femenino , Ferritinas/sangre , Humanos , Interferón gamma/biosíntesis , Interferón gamma/sangre , Ionomicina/farmacología , Hierro/sangre , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Masculino , México , Mitógenos/farmacología , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/sangre

RESUMEN

BACKGROUND: Metabolic syndrome (MetS), a cluster of various metabolic conditions, has become epidemic and causes increased morbidity and mortality. PURPOSE: The aim of this study was to compare lymphocyte proliferation under two different stimuli, Concanavalin A (ConA) and insulin, in a group of patients with MetS (Group 1) and a healthy group (Group 2). METHODS: Group 1 consisted of 53 patients who met the diagnostic criteria for MetS. Group 2 consisted of 63 patients without MetS. All individuals were evaluated for lipid profile and glycemia. Lymphocyte extraction and culture were performed for each subject and lymphocyte proliferation was assessed using the Alamar blue technique. RESULTS: There was no gender difference between both groups, but in terms of age, there was a significant difference. The use of Con A at concentrations of 1 and 5 µg/mL induced a high lymphocyte proliferation in both groups. In contrast, when different concentrations of insulin were added, no significant changes in lymphocyte proliferation were observed. However, the proliferation of lymphocytes was significantly higher in Group 1 compared to Group 2 under insulin stimulus, which did not happen under ConA stimulation. Even after age and gender correction, this difference was maintained. CONCLUSIONS: The increased lymphocyte proliferative response to insulin in patients with MetS found in this study suggests a role of the lymphocyte response to insulin in the pathophysiology of MetS. This response may be used as an immuno-biological marker for MetS, although further studies to evaluate its clinical usefulness need to be conducted.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Concanavalina A/farmacología , Hipoglucemiantes/farmacología , Insulina/farmacología , Síndrome Metabólico/tratamiento farmacológico , Mitógenos/farmacología , Linfocitos T/efectos de los fármacos , Adulto , Anciano , Concanavalina A/administración & dosificación , Femenino , Humanos , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Masculino , Síndrome Metabólico/sangre , Persona de Mediana Edad , Mitógenos/administración & dosificación

RESUMEN

Mitogen-activated protein kinase (MAPK) pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes (IEGs) of the activator protein 1 (AP-1) family, such as fos, achieve peak expression levels shortly after cells are stimulated with growth factors and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR (Hu-antigen R, Elav-like protein 1, ELAVL1) and KSRP (far upstream element-binding protein 2, KHSRP) bind to a fos AU-rich element (ARE) present in the 3'-UTR (untranslated region) of fos mRNA regulating its stability by a still poorly defined mechanism. We show in the present study that, whereas HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3'-UTR, KSRP counteracts this effect. Furthermore, we found that fos mRNA stability and HuR phosphorylation status are dependent on the activity of p38 MAPK in both epithelial cells and fibroblasts upon proliferative stimulation. Analysing PPI (protein-protein interaction) networks, we performed a thorough query of interacting proteins for p38 MAPKs, HuR and other AUBPs upon growth factor stimulation. This revealed novel HuR interactors including inhibitors of protein phosphatase 2 (PP2A) activity. Over-expression of two of these interactors, pp32 and APRIL (acidic leucine-rich nuclear phosphoprotein 32 family member B, ANP32B) and pharmacological inhibition of PP2A stabilized a fos reporter mRNA. Our results indicate that p38 MAPK regulates fos mRNA decay by affecting the state of phosphorylation of HuR while controlling yet to be fully elucidated PP regulatory networks.

Asunto(s)

Proteínas ELAV/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mitógenos/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Regiones no Traducidas 3'/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Proteínas ELAV/genética , Proteína 1 Similar a ELAV , Células HEK293 , Células HeLa , Humanos , Ratones , Mutación , Células 3T3 NIH , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/química , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo

Asunto(s)

Linfocitos B , Aberraciones Cromosómicas , Mitógenos , Neoplasias

RESUMEN

Immune response plays an important role in the development of hepatic fibrosis. In the present study, we investigated the effects of quercetin on hepatitis and hepatic fibrosis induced by immunological mechanism. In the acute hepatitis model, quercetin (2.5 mg/kg) was injected iv into mice 30 min after concanavalin A (Con A) challenge. Mice were sacrificed 4 or 24 h after Con A injection, and aminotransferase tests and histopathological sections were performed. Treatment with quercetin significantly decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Consistent with this observation, treatment with quercetin markedly attenuated the pathologic changes in the liver. A hepatic fibrosis model was also generated in mice by Con A challenge once a week for 6 consecutive weeks. Mice in the experimental group were treated with daily iv injections of quercetin (0.5 mg/kg). Histopathological analyses revealed that treatment with quercetin markedly decreased collagen deposition, pseudolobuli development, and hepatic stellate cells activation. We also examined the effects of quercetin on the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transforming growth factor beta (TGF-ß) pathways by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). NF-κB and TGF-ß production was decreased after treatment with quercetin, indicating that the antifibrotic effect of quercetin is associated with its ability to modulate NF-κB and TGF-ß production. These results suggest that quercetin may be an effective therapeutic strategy in the treatment of patients with liver damage and fibrosis.

Asunto(s)

Antioxidantes/administración & dosificación , Hepatitis/tratamiento farmacológico , Cirrosis Hepática/tratamiento farmacológico , Quercetina/farmacología , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Colágeno/análisis , Concanavalina A , Modelos Animales de Enfermedad , Femenino , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Liposomas , Cirrosis Hepática/inducido químicamente , Ratones Endogámicos BALB C , Mitógenos , FN-kappa B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Immune response plays an important role in the development of hepatic fibrosis. In the present study, we investigated the effects of quercetin on hepatitis and hepatic fibrosis induced by immunological mechanism. In the acute hepatitis model, quercetin (2.5 mg/kg) was injected iv into mice 30 min after concanavalin A (Con A) challenge. Mice were sacrificed 4 or 24 h after Con A injection, and aminotransferase tests and histopathological sections were performed. Treatment with quercetin significantly decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Consistent with this observation, treatment with quercetin markedly attenuated the pathologic changes in the liver. A hepatic fibrosis model was also generated in mice by Con A challenge once a week for 6 consecutive weeks. Mice in the experimental group were treated with daily iv injections of quercetin (0.5 mg/kg). Histopathological analyses revealed that treatment with quercetin markedly decreased collagen deposition, pseudolobuli development, and hepatic stellate cells activation. We also examined the effects of quercetin on the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transforming growth factor beta (TGF-β) pathways by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). NF-κB and TGF-β production was decreased after treatment with quercetin, indicating that the antifibrotic effect of quercetin is associated with its ability to modulate NF-κB and TGF-β production. These results suggest that quercetin may be an effective therapeutic strategy in the treatment of patients with liver damage and fibrosis.

Asunto(s)

Animales , Femenino , Antioxidantes/administración & dosificación , Hepatitis/tratamiento farmacológico , Cirrosis Hepática/tratamiento farmacológico , Quercetina/farmacología , Alanina Transaminasa/sangre , Aspartato Aminotransferasas/sangre , Concanavalina A , Colágeno/análisis , Modelos Animales de Enfermedad , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Liposomas , Cirrosis Hepática/inducido químicamente , Ratones Endogámicos BALB C , Mitógenos , FN-kappa B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Lecithins, mainly composed of the phospholipids phosphatidylcholines (PC), have many different uses in the pharmaceutical and clinical field. PC are involved in structural and biological functions as membrane trafficking processes and cellular signaling. Considering the increasing applications of lecithin-based nanosystems for the delivery of therapeutic agents, the aim of the present work was to determine the effects of phosphatidylcholine nanoparticles over breast cancer cellular proliferation and signaling. PC dispersions at 0.01 and 0.1% (w/v) prepared in buffer pH 7.0 and 5.0 were studied in the MCF-7 breast cancer cell line. Neutral 0.1% PC-derived nanoparticles induced the activation of the MEK-ERK1/2 pathway, increased cell viability and induced a 1.2 fold raise in proliferation. These biological effects correlated with the increase of epidermal growth factor receptor (EGFR) content and its altered cellular localization. Results suggest that nanoparticles derived from PC dispersion prepared in buffer pH 7.0 may induce physicochemical changes in the plasma membrane of cancer cells which may affect EGFR cellular localization and/or activity, increasing activation of the MEK-ERK1/2 pathway and inducing proliferation. Results from the present study suggest that possible biological effects of delivery systems based on lecithin nanoparticles should be taken into account in pharmaceutical formulation design.

Asunto(s)

Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mitógenos , Nanopartículas/química , Fosfatidilcolinas , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Receptores ErbB/metabolismo , Femenino , Humanos , Mitógenos/química , Mitógenos/farmacología , Proteínas de Neoplasias/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacología

RESUMEN

OBJECTIVES: Serotonin (5-HT)7 receptors in lymphocytes play a relevant role as modulators of T cell functions and might be modified by stress protocols. The aims of this work were to evaluate: (i) the presence of 5-HT7 receptors in specific lymphocyte populations, (ii) the probable modifications of them by inflammatory stress with mitogen and (iii) the effects of physical and pharmacological stress. METHODS: Blood lymphocytes were isolated by density gradients and differential adhesion to plastic. Concanavalin A (Con A) was systemically administered (500 µg/kg) or added to lymphocyte cultures (2.5 µg/ml, final volume 200 µl). Physical restraint was performed in Plexiglass boxes for 5 h per day for 5 days. Reserpine administration was 2.5 mg/kg for 3 days. Immunocytochemical labeling of CD4+, CD8+ and 5-HT7 receptors, and also tryptophan hydroxylase cells was performed. mRNA of 5-HT7 receptors was evaluated by RT-PCR. Controls were included for each protocol. RESULTS: Con A treatment or culture exposure increased the number of lymphocytes expressing 5-HT7 receptors or tryptophan hydroxylase, as compared to absence of the mitogen. Receptors were present in 12-16% of total rat lymphocytes, in ∼10% of CD4+ and in ∼5% of CD8+ cells from control rats. CD4+ decreased, and CD8+ and 5-HT7 cells increased after physical restraint. Reserpine treatment elevated CD8+ and 5-HT7 cells. Con A and physical restraint, but not reserpine treatment, significantly augmented 5-HT7 receptor mRNA in lymphocytes. CONCLUSIONS: Rat lymphocytes, expressing tryptophan hydroxylase, could synthesize 5-HT, functioning as a direct autocrine modulator. The modifications of CD4+, CD8+ and 5-HT7 receptors in lymphocytes by three stress protocols could have an impact on immune responses. In addition, the differential distribution of 5-HT7 receptors indicates potential specific physiopathological roles.

Asunto(s)

Linfocitos/inmunología , Linfocitos/metabolismo , Receptores de Serotonina/biosíntesis , Triptófano Hidroxilasa/biosíntesis , Animales , Antipsicóticos/farmacología , Masculino , Mitógenos/farmacología , Ratas , Ratas Sprague-Dawley , Reserpina/farmacología , Restricción Física/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Psicológico/inmunología , Estrés Psicológico/metabolismo

RESUMEN

A device's instrumentation for magnetic stimulation on human lymphocytes is presented. This is a new procedure to stimulate growing cells with ferrofluid in vortices of magnetic field. The stimulation of magnetic vortices was provided at five different frequencies, from 100 to 2500 Hz and intensities from 1.13 to 4.13 mT. To improve the stimulation effects, a paramagnetic ferrofluid was added on the cell culture medium. The results suggest that the frequency changes and the magnetic field variation produce an important increase in the number of proliferating cells as well as in the cellular viability. This new magnetic stimulation modality could trigger an intracellular mechanism to induce cell proliferation and cellular survival only on mitogen stimulated cells.

Asunto(s)

Proliferación Celular , Medios de Cultivo/farmacología , Linfocitos/metabolismo , Campos Magnéticos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Linfocitos/citología , Mitógenos/farmacología

RESUMEN

BACKGROUND: Intrauterine infection during pregnancy can trigger a local inflammatory response leading to several complications, such as preterm labor. Many studies have used in vitro and in vivo models employing mitogens to induce the expression of the characteristic proinflammatory mediators triggered by infection. However, relative expression assays depend on the stability of housekeeping gene expression, which can vary depending on certain stimuli. In this study, we analyzed the stability and pairwise variation in the expression of GAPDH, ACTB and RNA18S1 in cultured reproductive tissues under mitogen stimulation. We used fetal membranes, placental villous and umbilical cord explants from patients with normal term pregnancies (>37 weeks of gestation), as well as myometrium and cervix explants from patients undergoing hysterectomies. Tissues were stimulated with lipopolysaccharide or phytohemagglutinin for 24 hours. We then analyzed the expression stability and the pairwise variation of GAPDH, ACTB and RNA18S1 from real time quantitative RT-PCR absolute threshold cycles (Cp) using geNorm software. RESULTS: In all of the tissues, the three housekeeping genes showed great stability under our experimental conditions. Pairwise variation analyses showed that only two reference genes are required for adequate normalization, GAPDH and ACTB being optimal in the cervix, fetal membranes and umbilical cord, while GAPDH and RNA18S1 are best for normalization in the placenta and myometrium. CONCLUSION: Our results show that GAPDH, ACTB and RNA18S1 are adequate references for gene expression normalization in reproductive tissues stimulated with mitogens in culture.

Asunto(s)

Expresión Génica , Genes Esenciales , Genitales Femeninos/metabolismo , Mitógenos/farmacología , Secuencia de Bases , Cartilla de ADN , Femenino , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

Asunto(s)

Hormona Folículo Estimulante/metabolismo , Mitógenos/farmacología , Folículo Ovárico/efectos de los fármacos , Fitohemaglutininas/farmacología , Animales , Femenino , Hormona Folículo Estimulante/genética , Cabras , Técnicas In Vitro , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/ultraestructura , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Mensajero/metabolismo