RESUMEN
BACKGROUND: Cell responses to different stress inducers are efficient mechanisms that prevent and fight the accumulation of harmful macromolecules in the cells and also reinforce the defenses of the host against pathogens. Vaccinia virus (VACV) is an enveloped, DNA virus, belonging to the Poxviridae family. Members of this family have evolved numerous strategies to manipulate host responses to stress controlling cell survival and enhancing their replicative success. In this study, we investigated the activation of the response signaling to malformed proteins (UPR) by the VACV virulent strain-Western Reserve (WR)-or the non-virulent strain-Modified Vaccinia Ankara (MVA). METHODS: Through RT-PCR RFLP and qPCR assays, we detected negative regulation of XBP1 mRNA processing in VACV-infected cells. On the other hand, through assays of reporter genes for the ATF6 component, we observed its translocation to the nucleus of infected cells and a robust increase in its transcriptional activity, which seems to be important for virus replication. WR strain single-cycle viral multiplication curves in ATF6α-knockout MEFs showed reduced viral yield. RESULTS: We observed that VACV WR and MVA strains modulate the UPR pathway, triggering the expression of endoplasmic reticulum chaperones through ATF6α signaling while preventing IRE1α-XBP1 activation. CONCLUSIONS: The ATF6α sensor is robustly activated during infection while the IRE1α-XBP1 branch is down-regulated.
Asunto(s)
Factores de Transcripción , Virus Vaccinia , Factores de Transcripción/genética , Virus Vaccinia/genética , Endorribonucleasas , Proteínas Serina-Treonina Quinasas , Estrés del Retículo Endoplásmico , Respuesta de Proteína DesplegadaRESUMEN
BACKGROUND/OBJECTIVES: Platelet-activating factor receptor (PAFR) activation controls adipose tissue (AT) expansion in animal models. Our objective was twofold: (i) to check whether PAFR signaling is involved in human obesity and (ii) investigate the PAF pathway role in hematopoietic or non-hematopoietic cells to control adipocyte size. MATERIALS/SUBJECTS AND METHODS: Clinical parameters and adipose tissue gene expression were evaluated in subjects with obesity. Bone marrow (BM) transplantation from wild-type (WT) or PAFR-/- mice was performed to obtain chimeric PAFR-deficient mice predominantly in hematopoietic or non-hematopoietic-derived cells. A high carbohydrate diet (HC) was used to induce AT remodeling and evaluate in which cell compartment PAFR signaling modulates it. Also, 3T3-L1 cells were treated with PAF to evaluate fat accumulation and the expression of genes related to it. RESULTS: PAFR expression in omental AT from humans with obesity was negatively correlated to different corpulence parameters and more expressed in the stromal vascular fraction than adipocytes. Total PAFR-/- increased adiposity compared with WT independent of diet-induced obesity. Differently, WT mice receiving PAFR-/--BM exhibited similar adiposity gain as WT chimeras. PAFR-/- mice receiving WT-BM showed comparable augmentation in adiposity as total PAFR-/- mice, demonstrating that PAFR signaling modulates adipose tissue expansion through non-hematopoietic cells. Indeed, the PAF treatment in 3T3-L1 adipocytes reduced fat accumulation and expression of adipogenic genes. CONCLUSIONS: Therefore, decreased PAFR signaling may favor an AT accumulation in humans and animal models. Importantly, PAFR signaling, mainly in non-hematopoietic cells, especially in adipocytes, appears to play a significant role in regulating diet-induced AT expansion.
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Tejido Adiposo/fisiopatología , Obesidad/complicaciones , Glicoproteínas de Membrana Plaquetaria/farmacología , Tejido Adiposo/metabolismo , Adulto , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Obesidad/fisiopatología , Paris , Receptores Acoplados a Proteínas G , Transducción de Señal/fisiologíaRESUMEN
The innate and adaptive immune systems play an important role in the development of cardiac diseases. Therefore, it has become critical to identify molecules that can modulate inflammation in the injured heart. In this regard, activation of the cholinergic system in animal models of heart disease has been shown to exert protective actions that include immunomodulation of cardiac inflammation. In this mini-review, we briefly present our current understanding on the cardiac cellular sources of acetylcholine (ACh) (neuronal vs. nonneuronal), followed by a discussion on its contribution to the regulation of inflammatory cells. Although the mechanism behind ACh-mediated protection still remains to be fully elucidated, the beneficial immunomodulatory role of the cholinergic signaling emerges as a potential key regulator of cardiac inflammation.
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Acetilcolina/metabolismo , Antiinflamatorios/uso terapéutico , Cardiotónicos/uso terapéutico , Cardiopatías/metabolismo , Cardiopatías/prevención & control , Corazón/efectos de los fármacos , Acetilcolina/administración & dosificación , Animales , Antiinflamatorios/farmacología , Cardiotónicos/farmacología , Humanos , Inflamación/metabolismo , Inflamación/prevención & control , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
Cryptococcus gattii (Cg) is one of the agents of cryptococcosis, a severe systemic mycosis with a higher prevalence in men than women, but the influence of the female sex hormone, 17-ß-estradiol (E2), on cryptococcosis remains unclear. Our study shows that female mice presented delayed mortality, increased neutrophil recruitment in bronchoalveolar lavage fluid, and reduced fungal load after 24 hr of infection compared to male and ovariectomised female mice (OVX). E2 replacement restored OVX female survival. Female macrophages have more efficient fungicidal activity, which was increased by E2 and reversed by the antagonist of G-protein-coupled oestrogen receptor (GPER), which negatively modulates PI3K activation. Furthermore, E2 induces a reduction in Cg cell diameter, cell charge, and antioxidant peroxidase activity. In conclusion, female mice present improved control of Cg infection, and GPER is important for E2 modulation of the female response.
Asunto(s)
Criptococosis/tratamiento farmacológico , Cryptococcus gattii/efectos de los fármacos , Estradiol/farmacología , Proteínas de Unión al GTP/metabolismo , Macrófagos/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Animales , Antifúngicos/farmacología , Antioxidantes , Criptococosis/inmunología , Modelos Animales de Enfermedad , Femenino , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Lactobacilli are the dominant bacteria of the vaginal tract of healthy women and they play a major role in the maintenance of mucosal homeostasis, preventing genital infections, such as bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). It is now known that one mechanism of this protection is the influence that lactobacilli can exert on host immune responses. In this context, we evaluated two Lactobacillus strains (L. plantarum 59 and L. fermentum 137) for their immunomodulatory properties in response to Gardnerella vaginalis (BV) or Candida albicans (VVC) infections in a HeLa cell infection model. G. vaginalis and C. albicans triggered the secretion of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-8) and the activation of NF-κB in HeLa cells, in contrast to L. plantarum 59 and L. fermentum 137. Treatments with the Lactobacillus strains or their cell-free supernatants before (pre-treatment) or after (post-treatment) the challenge with the pathogens resulted in decreased secretion of pro-inflammatory cytokines and decreased activation of NF-κB. The treatments with Lactobacillus strains not only decreased the secretion of IL-8, but also its expression, as confirmed by gene reporter luciferase assay, suggesting transcription-level control by lactobacilli. In conclusion, L. plantarum 59 and L. fermentum 137 were confirmed to have an anti-inflammatory effect against G. vaginalis and C. albicans and they were able to influence signalling in NF-κB pathway, making them interesting candidates as probiotics for the prevention or treatment of BV and VVC.
Asunto(s)
Antiinflamatorios/farmacología , Candida albicans/efectos de los fármacos , Gardnerella vaginalis/efectos de los fármacos , Lactobacillus plantarum/fisiología , Limosilactobacillus fermentum/fisiología , Probióticos/farmacología , Candida albicans/crecimiento & desarrollo , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Citocinas/genética , Citocinas/metabolismo , Femenino , Gardnerella vaginalis/crecimiento & desarrollo , Células HeLa , Humanos , Factor de Transcripción ReIA/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Malignant gliomas are a lethal type of brain tumors that poorly respond to chemotherapeutic drugs. Several therapy resistance mechanisms have been characterized. However, the response to stress through mRNA translational control has not been evaluated for this type of tumor. A potential target would involve the alpha subunit of eukaryotic translation initiation factor (eIF2α) that leads to assembly of stress granules (SG) which are cytoplasmic granules mainly composed by RNA binding proteins and untranslated mRNAs. We assessed whether glioma cells are capable of assembling SG after exposure to different classes of chemotherapeutic agents through evaluation of the effects of interfering in this process by impairing the eIF2α signaling. C6 and U87MG cells were exposed to bortezomib, cisplatin, or etoposide. Forced expression of a dominant negative mutant of eIF2α (eIF2α(DN)) was employed to block this pathway. We observed that exposure to drugs stimulated SG assembly. This was reduced in eIF2α(DN)-transfected cells and this strategy enhanced chemotherapeutically-induced cell death for all drugs. Our data suggest that SG assembly occurs in glioma cells in response to chemotherapeutic drugs in an eIF2α-dependent manner and this response is relevant for drug resistance. Interfering with eIF2α signaling pathway may be a potential strategy for new co-adjuvant therapies to treat gliomas.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Gránulos Citoplasmáticos/fisiología , Factor 2 Eucariótico de Iniciación/antagonistas & inhibidores , Glioma/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proliferación Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Técnica del Anticuerpo Fluorescente , Glioma/metabolismo , Glioma/patología , Humanos , Fosforilación/efectos de los fármacos , Ratas , Células Tumorales CultivadasRESUMEN
BACKGROUND: Gastric adenocarcinoma is associated with chronic infection by Helicobacter pylori and with the host inflammatory response triggered by it, with substantial inter-person variation in the immune response profile due to host genetic factors. AIM: To investigate the diversity of the proinflammatory genes IL8, its receptors and PTGS2 in Amerindians; to test whether candidate SNPs in these genes are associated with gastric cancer in an admixed population with high Amerindian ancestry from Lima, Peru; and to assess whether an IL8RB promoter-derived haplotype affects gene expression. METHODS: We performed a Sanger-resequencing population survey, a candidate-gene association study (220 cases, 288 controls) and meta-analyses. We also performed an in vitro validation by a reporter gene assay of IL8RB promoter. RESULTS: The diversity of the promoter of studied genes in Native Americans is similar to Europeans. Although an association between candidate SNPs and gastric cancer was not found in Peruvians, trend in our data is consistent with meta-analyses results that suggest PTGS2-rs689466-A is associated with H. pylori-associated gastric cancer in East Asia. IL8RB promoter-derived haplotype (rs3890158-A/rs4674258-T), common in Peruvians, was up-regulated by TNF-α unlike the ancestral haplotype (rs3890158-G/rs4674258-C). Bioinformatics analysis suggests that this effect stemmed from creation of a binding site for the FOXO3 transcription factor by rs3890158G>A. CONCLUSIONS: Our updated meta-analysis reinforces the role of PTGS2-rs689466-A in gastric cancer in Asians, although more studies that control for ancestry are necessary to clarify its role in Latin Americans. Finally, we suggest that IL8RB-rs3890158G>A is a cis-regulatory SNP.
Asunto(s)
Adenocarcinoma/etnología , Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Ciclooxigenasa 2/genética , Indígenas Sudamericanos/genética , Interleucina-8/genética , Polimorfismo de Nucleótido Simple , Neoplasias Gástricas/etnología , Neoplasias Gástricas/genética , Adenocarcinoma/metabolismo , Pueblo Asiatico/genética , Sitios de Unión , Población Negra/genética , Estudios de Casos y Controles , Biología Computacional , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Células HEK293 , Haplotipos , Humanos , Perú/epidemiología , Fenotipo , Regiones Promotoras Genéticas , Factores de Riesgo , Neoplasias Gástricas/metabolismo , Transfección , Población Blanca/genéticaRESUMEN
The factor RasGEF1b is a Ras guanine exchange factor involved in immune responses. Studies have also implicated RasGEF1b in the CNS development. It is still limited the understanding of the role of RasGEF1b in CNS functioning. Using RasGEF1b deficient mice (RasGEF1b-cKO), we investigated the impact of this gene deletion in behavior, cognition, brain neurochemistry and microglia morphology. We showed that RasGEF1b-cKO mice display spontaneous hyperlocomotion and anhedonia. RasGEF1b-cKO mice also exhibited compulsive-like behavior that was restored after acute treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine (5 mg/kg). A down-regulation of mRNA of dopamine receptor (Drd1, Drd2, Drd4 and Drd5) and serotonin receptor genes (5Htr1a, 5Htr1b and 5Htr1d) was observed in hippocampus of RasGEF1b-cKO mice. These mice also had reduction of Drd1 and Drd2 in prefrontal cortex and 5Htr1d in striatum. In addition, morphological alterations were observed in RasGEF1b deficient microglia along with decreased levels of hippocampal BDNF. We provided original evidence that the deletion of RasGEF1b leads to unique behavioral features, implicating this factor in CNS functioning.
Asunto(s)
Encéfalo , Inhibidores Selectivos de la Recaptación de Serotonina , Animales , Ratones , Cognición , Fluoxetina/farmacología , Corteza Prefrontal , Receptores de Dopamina D5RESUMEN
Brucella abortus is recognized by several Toll-like receptor (TLR)-associated pathways triggering proinflammatory responses that affect both the nature and intensity of the immune response. Previously, we demonstrated that B. abortus-mediated dendritic cell (DC) maturation and control of infection are dependent on the adaptor molecule MyD88. However, the involvement of all TLRs in response to B. abortus infection is not completely understood. Therefore, we decided to evaluate the requirement for TLR6 in host resistance to B. abortus. Here, we demonstrated that TLR6 is an important component for triggering an innate immune response against B. abortus. An in vitro luciferase assay indicated that TLR6 cooperates with TLR2 to sense Brucella and further activates NF-κB signaling. However, in vivo analysis showed that TLR6, not TLR2, is required for the efficient control of B. abortus infection. Additionally, B. abortus-infected dendritic cells require TLR6 to induce tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12). Furthermore, our findings demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway is impaired in TLR2, TLR6, and TLR2/6 knockout (KO) DCs when infected with B. abortus, which may account for the lower proinflammatory cytokine production observed in TLR6 KO mouse dendritic cells. In summary, the results presented here indicate that TLR6 is required to trigger innate immune responses against B. abortus in vivo and is required for the full activation of DCs to induce robust proinflammatory cytokine production.
Asunto(s)
Brucella abortus/inmunología , Brucelosis/inmunología , Inmunidad Innata/fisiología , Receptor Toll-Like 6/fisiología , Análisis de Varianza , Animales , Citocinas/metabolismo , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Bazo/microbiología , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 6/deficienciaRESUMEN
Ras guanine nucleotide exchange factor member 1b (RasGEF1b) of the RasGEF/CDC25 domain-containing family is preferentially expressed by macrophages. However, information is lacking about its role in macrophage function. In this study, we generated mice with ubiquitous deletion of Rasgef1b and used RNA-seq-based transcriptomics to compare the global gene expression in wild-type and knock-out primary bone-marrow-derived macrophages under basal conditions and after lipopolysaccharide (LPS) treatment. Transcriptional filtering identified several genes with significantly different transcript levels between wild-type and knock-out macrophages. In total, 49 and 37 differentially expressed genes were identified at baseline and in LPS-activated macrophages, respectively. Distinct biological processes were significantly linked to down-regulated genes at the basal condition only, and largely included chemotaxis, response to cytokines, and positive regulation of GTPase activity. Importantly, validation by RT-qPCR revealed that the expression of genes identified as down-regulated after LPS stimulation was also decreased in the knock-out cells under basal conditions. We used a luciferase-based reporter assay to showcase the capability of RasGEF1b in activating the Serpinb2 promoter. Notably, knockdown of RasGEF1b in RAW264.7 macrophages resulted in impaired transcriptional activation of the Serpinb2 promoter, both in constitutive and LPS-stimulated conditions. This study provides a small collection of genes that shows relative expression changes effected by the absence of RasGEF1b in macrophages. Thus, we present the first evidence that RasGEF1b mediates the regulation of both steady-state and signal-dependent expression of genes and propose that this GEF plays a role in the maintenance of the basal transcriptional level in macrophages.
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Citocinas , Lipopolisacáridos , Animales , Ratones , Quimiotaxis , Citocinas/genética , Citocinas/metabolismo , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , TranscriptomaRESUMEN
We investigated the type I interferon (IFN-1)/PKR axis in the outcome of the Leishmania (Leishmania) amazonensis infection, along with the underlying mechanisms that trigger and sustain this signaling pathway. Reporter assays of cell extracts from RAW-264.7 macrophages infected with L. (L.) amazonensis or HEK-293T cells cotransfected with TLR2 and PKR promoter constructions were employed. Primary macrophages of TLR2-knockout (KO) or IFNR-KO mice were infected, and the levels of PKR, IFN-1, and superoxide dismutase 1 (SOD1) transcript levels were investigated and compared. Immunohistochemical analysis of human biopsy lesions was evaluated for IFN-1 and PKR-positive cells. Leishmania infection increased the expression of PKR and IFN-ß on induction of PKR-promoter activity. The observed effects required the engagement of TLR2. TLR2-KO macrophages expressed low IFN-ß and PKR levels postinfection with a reduced parasite load. We also revealed the requirement of PKR signaling for Leishmania-induced IFN-1 expression, responsible for sustaining PKR expression and enhancing infection. Moreover, during infection, SOD1 transcripts increased and were also enhanced when IFN-1 was added to the cultures. Remarkably, SOD1 expression was abrogated in infected, dominant-negative PKR-expressing cells. Finally, lesions of patients with anergic diffuse cutaneous leishmaniasis exhibited higher levels of PKR/IFN-1-expressing cells compared to those with single cutaneous leishmaniasis. In summary, we demonstrated the mechanisms and relevance of the IFN-1/PKR axis in the Leishmania infection.
Asunto(s)
Interferón Tipo I/metabolismo , Leishmania mexicana , Leishmaniasis Cutánea/enzimología , Leishmaniasis Cutánea/inmunología , Receptor Toll-Like 2/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Glicoesfingolípidos/inmunología , Interacciones Huésped-Parásitos , Humanos , Leishmania mexicana/inmunología , Leishmania mexicana/patogenicidad , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea Difusa/enzimología , Leishmaniasis Cutánea Difusa/genética , Leishmaniasis Cutánea Difusa/inmunología , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Regiones Promotoras Genéticas , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética , Transfección , eIF-2 Quinasa/genéticaRESUMEN
At the site of infection, pro-inflammatory cytokines locally produced by macrophages infected with Trypanosoma cruzi can activate surrounding non-professional phagocytes such as fibroblasts, epithelial and endothelial cells, which can be further invaded by the parasite. The effect of secreted soluble factors on the invasion of these cells remains, however, to be established. We show here that two epithelial cell lines become significantly susceptible to the infection by the Y strain of T. cruzi after tumour necrosis factor (TNF) treatment. The increase in the invasion was correlated with the increasing concentration of recombinant TNF added to cultures of HEK293T or LLC-MK2 cells. Supernatants taken from PMA-differentiated human monocytes infected with T. cruzi also increased the permissiveness of epithelial cells to subsequent infection with the parasite, which was inhibited by a TNF monoclonal antibody. Furthermore, the permissiveness induced by TNF was inhibited by TPCK, and led to significant decrease in the number of intracellular parasites, providing evidence that activation of NF-κB induced by TNF favours the invasion of the epithelial cell lines by T. cruzi through yet an unidentified mechanism. Our data indicate that soluble factors released from macrophages early in the infection favours T. cruzi invasion of non-professional phagocytic cells.
Asunto(s)
Células Epiteliales/inmunología , Células Epiteliales/parasitología , FN-kappa B/metabolismo , Fagocitosis , Trypanosoma cruzi/patogenicidad , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Haplorrinos , HumanosRESUMEN
The evolution of Leishmania infection depends on the balance between microbicidal and suppressor macrophage functions. Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a classic antiviral protein, is able to regulate a number of signaling pathways and macrophage functions. We investigated the possible role of PKR in the modulation of Leishmania infection. Our data demonstrated that Leishmania amazonensis infection led to PKR activation and increased PKR levels. Consistently, in macrophages from PKR knockout 129Sv/Ev mice and RAW-264.7 cells stably expressing a dominant-negative (DN) construct of PKR (DN-PKR), L. amazonensis infection was strongly reduced. The treatment of infected macrophages with the synthetic double-stranded RNA poly(I:C), a potent PKR inductor, increased L. amazonensis intracellular proliferation. This effect was reversed by 2-aminopurine (2-AP), a pharmacological inhibitor of PKR, as well as by the expression of DN-PKR. NO release induced by dsRNA treatment was inhibited by L. amazonensis through NF-kappaB modulation. PKR activation induced by dsRNA also resulted in IL-10 production, whose neutralization with specific antibody completely abrogated L. amazonensis proliferation. Our data demonstrated a new role of PKR in protozoan parasitic infection through IL-10 modulation.
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Leishmania/patogenicidad , Macrófagos/parasitología , eIF-2 Quinasa/metabolismo , 2-Aminopurina/farmacología , Animales , Activación Enzimática , Humanos , Interleucina-10/metabolismo , Leishmania/genética , Ratones , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacología , Poli I-C/farmacología , ARN Bicatenario/genéticaRESUMEN
Toll-like receptor (TLR) signaling induces substantial changes in the phosphoproteome of innate immune cells, mainly in the form of increased phosphorylation of signaling intermediaries. Loss of constitutive phosphorylation occurs simultaneously, but these transitions from a stable, phosphorylated state in resting cells to a sustained underphosphorylated state in activated cells have received far less attention. This review provides an overview of phosphorylation sites downregulated during TLR-mediated signaling, with a particular focus on TLR4 activation by lipopolysaccharide (LPS). Energy homeostasis, the cell cycle, mitochondrial fission, and gene regulation are among the biological events in macrophages that are regulated through the downregulation of phosphoproteins as part of intracellular signaling events. Phosphoproteomics studies on innate immune cells have identified hundreds of hitherto uncharacterized phosphorylation sites that are lost upon stimulation, indicating that protein hypophosphorylation is a significant, largely unexplored layer of complexity in the TLR4 pathway.
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Enfermedad , Salud , Macrófagos/metabolismo , Transducción de Señal , Receptores Toll-Like/metabolismo , Animales , Humanos , FosforilaciónRESUMEN
Brazilian Cerrado is the second largest biome in South America and contains many unstudied valuable plant species rich in bioactive substances. In this study we investigated the phenolic content and proliferative effects on cultured fibroblasts of 32 extracts of different polarities prepared from 11 plants found in Cerrado regions. Eight extracts from six species increased cell proliferation and significantly induced ATP production by the cells. Four of these extracts were obtained from plants used as food, specifically from its fruits or seeds. A high phenolic content for these eight extracts, which directly correlated with the induction of cell proliferation, was corroborated by mass spectrometry analysis. We suggest that the bioactive substance content of these species shows an interesting potential use in cosmetic and food industry, which can contribute to the conservation and sustainable development of this region.
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Frutas , Fenoles , Brasil , Fibroblastos , Frutas/química , Fenoles/análisis , Extractos Vegetales/farmacología , Plantas ComestiblesRESUMEN
Different data suggest that microglia may participate in the drug addiction process as these cells respond to neurochemical changes induced by the administration of these substances. In order to study the role of microglia in drug abuse, Swiss mice aged 8-9 weeks were treated with the CSF1R inhibitor PLX3397 (40 mg/kg, p.o.) and submitted to behavioral sensitization or conditioned place preference (CPP) induced by cocaine (15 mg/kg, i.p.). Thereafter, brains were used to evaluate the effects of CSF1R inhibition and cocaine administration on morphological, biochemical and molecular changes. CSF1R inhibition attenuated behavioral sensitization, reduced the number of Iba-1+ cells and increased ramification and lengths of the branches in the remaining microglia. Additionally, both cocaine and PLX3397 increased the cell body to total cell size ratio of Iba-1+ cells, as well as CD68+ and GFAP+ stained areas, suggesting an activated pattern of the glial cells. Besides, CSF1R inhibition increased CX3CL1 levels in the striatum, prefrontal cortex and hippocampus, as well as reduced CX3CR1 expression in the hippocampus. In this region, cocaine also reduced BDNF levels, an effect that was enhanced by CSF1R inhibition. In summary, our results suggest that microglia participate in the behavioral and molecular changes induced by cocaine. This study contributes to the understanding of the role of microglia in cocaine addiction.
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Aminopiridinas/farmacología , Conducta Animal/efectos de los fármacos , Trastornos Relacionados con Cocaína/prevención & control , Cocaína/toxicidad , Microglía/efectos de los fármacos , Pirroles/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Trastornos Relacionados con Cocaína/etiología , Trastornos Relacionados con Cocaína/patología , Condicionamiento Clásico , Inhibidores de Captación de Dopamina/toxicidad , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Inhibición Psicológica , Masculino , Ratones , Microglía/metabolismo , Microglía/patologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Cecropia pachystachya Trécul (Urticaceae), known as embaúba, are used as hypoglycemic and for weight reduction in Brazilian traditional medicine. AIM OF THE STUDY: This study investigated the effects of a pharmaceutical formulation (ECP20) containing C. pachystachya extract on some metabolic alterations caused by a hypercaloric diet in mice. MATERIAL AND METHODS: Mice were randomly fed with a standard or hypercaloric diet and orally treated with ECP20 or vehicle for 13 weeks. Subsequently, adiposity, glucose intolerance, and the presence of nonalcoholic fatty liver disease were assessed. Adipose tissue and liver were collected after euthanasia and frozen at -80 °C for histological and antioxidant analyzes. The effect of ECP20 on the differentiation of 3T3-L1 pre-adipocytes was also investigated. RESULTS: Animals treated with ECP20 showed less weight gain, reduced glycemia, glucose tolerance restored, and hepatoprotective effect. Also, ECP20 presented significant in vivo antioxidant activity. Treatment of 3T3-L1 preadipocytes with ECP20 did not inhibit cellular differencing. CONCLUSIONS: Therefore, ECP20 presented promising effects in the control of obesity and related disorders. Considering that glucose intolerance and hyperglycemia are strong evidence for the development of type 2 diabetes, the findings corroborated the traditional use of C. pachystachya to treat this disease. The chlorogenic acid and the flavonoids orientin and iso-orientin, present in the extract, might be involved in the activities found.
Asunto(s)
Fármacos Antiobesidad/farmacología , Cecropia/química , Dieta/efectos adversos , Ingestión de Energía/efectos de los fármacos , Hepatopatías/prevención & control , Extractos Vegetales/farmacología , Animales , Fármacos Antiobesidad/química , Glucemia/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Masculino , Medicina Tradicional , Ratones , Obesidad/inducido químicamente , Obesidad/prevención & control , Fitoterapia , Extractos Vegetales/química , Plantas MedicinalesRESUMEN
BACKGROUND: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. RESULTS: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 µg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). CONCLUSIONS: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.
Asunto(s)
Proteínas Bacterianas/administración & dosificación , Chaperonina 60/administración & dosificación , Terapia Genética , ARN Mensajero/administración & dosificación , Vacunas contra la Tuberculosis/administración & dosificación , Tuberculosis/prevención & control , Administración Intranasal , Animales , Células Presentadoras de Antígenos/inmunología , Proteínas Bacterianas/inmunología , Línea Celular , Chaperonina 60/inmunología , Femenino , Células HEK293 , Humanos , Interleucina-10/inmunología , Pulmón/citología , Pulmón/inmunología , Ratones , Ratones Endogámicos BALB C , Mycobacterium leprae/inmunología , Mycobacterium tuberculosis/patogenicidad , ARN Mensajero/inmunología , Tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The aim of this work was to establish a modified pre-diagnostic polymerase chain reaction (PCR) protocol using a single primer set that enables successful amplification of a highly conserved mammalian sequence in order to determine overall sample DNA quality for multiple mammalian species that inhabit areas endemic for leishmaniasis. The gene encoding interphotoreceptor retinoid-binding protein (IRBP), but not other conserved genes, was efficiently amplified in DNA samples from tail skin, ear skin, bone marrow, liver and spleen from all of the species tested. In tissue samples that were PCR-positive for Leishmania, we found that DNA from 100%, 55% and 22% of the samples tested resulted in a positive PCR reaction for the IRBP, beta-actin and beta-globin genes, respectively. Nucleotide sequencing of an IRBP amplicon resolved any questions regarding the taxonomical classification of a rodent, which was previously based simply on the morphological features of the animal. Therefore, PCR amplification and analysis of the IRBP amplicon are suitable for pre-diagnostically assessing DNA quality and identifying mammalian species living in areas endemic to leishmaniasis and other diseases.
Asunto(s)
Actinas/genética , ADN Protozoario/genética , Proteínas del Ojo/genética , Leishmaniasis/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Proteínas de Unión al Retinol/genética , Globinas beta/genética , Actinas/análisis , Animales , Cartilla de ADN/genética , Perros , Enfermedades Endémicas , Proteínas del Ojo/análisis , Leishmaniasis/parasitología , Marsupiales , Reacción en Cadena de la Polimerasa/métodos , Proteínas de Unión al Retinol/análisis , Roedores , Globinas beta/análisisRESUMEN
Ras Guanine Exchange Factor (RasGEF) domain family member 1b is encoded by a Toll-like receptor (TLR)-inducible gene expressed in macrophages, but transcriptional mechanisms that govern its expression are still unknown. Here, we have functionally characterized the 5' flanking Rasgef1b sequence and analyzed its transcriptional activation. We have identified that the inflammation-responsive promoter is contained within a short sequence (-183 to +119) surrounding the transcriptional start site. The promoter sequence is evolutionarily conserved and harbors a cluster of five NF-κB binding sites. Luciferase reporter gene assay showed that the promoter is responsive to TLR activation and RelA or cRel, but not RelB, transcription factors. Besides, site-directed mutagenesis showed that the κB binding sites are required for maximal promoter activation induced by LPS. Analysis by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that the promoter is located in an accessible chromatin region. More important, Chromatin Immunoprecipitation sequencing (ChIP-seq) showed that RelA is recruited to the promoter region upon LPS stimulation of bone marrow-derived macrophages. Finally, studies with Rela-deficient macrophages or pharmacological inhibition by Bay11-7082 showed that NF-κB is required for optimal Rasgef1b expression induced by TLR agonists. Our data provide evidence of the regulatory mechanism mediated by NF-κB that facilitates Rasgef1b expression after TLR activation in macrophages.