RESUMEN
BACKGROUND: Standard cancer treatments show a lack of selectivity that has led to the search for new strategies against cancer. The selective elimination of cancer cells modulating the redox environment, known as "selective oxycution", has emerged as a viable alternative. This research focuses on characterizing the unexplored Escallonia genus plant extracts and evaluating their potential effects on cancer's redox balance, cytotoxicity, and activation of death pathways. METHODS: 36 plant extracts were obtained from 4 different species of the Escallonia genus (E. illinita C. Presl, E. rubra (Ruiz & Pav.) Pers., E. revoluta (Ruiz & Pav.) Pers., and E. pulverulenta (Ruiz & Pav.) Pers.), which were posteriorly analyzed by their phytoconstituents, antioxidant capacity, and GC-MS. Further, redox balance assays (antioxidant enzymes, oxidative damage, and transcription factors) and cytotoxic effects (SRB, ∆Ψmt, and caspases actives) of those plant extracts were analyzed on four cell lines (HEK-293T, MCF-7, HT-29, and PC-3). RESULTS: 36 plant extracts were obtained, and their phytoconstituents and antioxidant capacity were established. Further, only six extracts had EC50 values < 10 µg*mL- 1, indicating high toxicity against the tested cells. From those, two plant extracts were selective against different cancer cell lines: the hexane extract of E. pulverulenta´s stem was selective for HT-29, and the ethyl acetate extract of E. rubra´s stem was selective for PC-3. Both extracts showed unbalanced redox effects and promoted selective cell death. CONCLUSIONS: This is the first study proving "selective oxycution" induced by Chilean native plant extracts.
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Magnoliopsida , Neoplasias , Humanos , Antioxidantes/farmacología , Estrés Oxidativo , Extractos Vegetales/farmacología , Oxidación-Reducción , Células HT29 , Neoplasias/tratamiento farmacológicoRESUMEN
Compelling evidence has shown that interferon (IFN)-γ has dual effects in multiple sclerosis and in its animal model of experimental autoimmune encephalomyelitis (EAE), with results supporting both a pathogenic and beneficial function. However, the mechanisms whereby IFN-γ may promote neuroprotection in EAE and its effects on central nervous system (CNS)-resident cells have remained an enigma for more than 30 years. In this study, the impact of IFN-γ at the peak of EAE, its effects on CNS infiltrating myeloid cells (MC) and microglia (MG), and the underlying cellular and molecular mechanisms were investigated. IFN-γ administration resulted in disease amelioration and attenuation of neuroinflammation associated with significantly lower frequencies of CNS CD11b+ myeloid cells and less infiltration of inflammatory cells and demyelination. A significant reduction in activated MG and enhanced resting MG was determined by flow cytometry and immunohistrochemistry. Primary MC/MG cultures obtained from the spinal cord of IFN-γ-treated EAE mice that were ex vivo re-stimulated with a low dose (1 ng/ml) of IFN-γ and neuroantigen, promoted a significantly higher induction of CD4+ regulatory T (Treg) cells associated with increased transforming growth factor (TGF)-ß secretion. Additionally, IFN-γ-treated primary MC/MG cultures produced significantly lower nitrite in response to LPS challenge than control MC/MG. IFN-γ-treated EAE mice had a significantly higher frequency of CX3CR1high MC/MG and expressed lower levels of program death ligand 1 (PD-L1) than PBS-treated mice. Most CX3CR1highPD-L1lowCD11b+Ly6G- cells expressed MG markers (Tmem119, Sall2, and P2ry12), indicating that they represented an enriched MG subset (CX3CR1highPD-L1low MG). Amelioration of clinical symptoms and induction of CX3CR1highPD-L1low MG by IFN-γ were dependent on STAT-1. RNA-seq analyses revealed that in vivo treatment with IFN-γ promoted the induction of homeostatic CX3CR1highPD-L1low MG, upregulating the expression of genes associated with tolerogenic and anti-inflammatory roles and down-regulating pro-inflammatory genes. These analyses highlight the master role that IFN-γ plays in regulating microglial activity and provide new insights into the cellular and molecular mechanisms involved in the therapeutic activity of IFN-γ in EAE.
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Encefalomielitis Autoinmune Experimental , Ratones , Animales , Microglía/metabolismo , Interferón gamma/metabolismo , Antígeno B7-H1/metabolismo , Sistema Nervioso CentralRESUMEN
BACKGROUND: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare inherited skin disease caused by variants in the COL7A1 gene, coding for type VII collagen (C7), an important component of anchoring fibrils in the basement membrane of the epidermis. RDEB patients suffer from skin fragility starting with blister formation and evolving into chronic wounds, inflammation and skin fibrosis, with a high risk of developing aggressive skin carcinomas. Restricted therapeutic options are limited by the lack of in vitro models of defective wound healing in RDEB patients. RESULTS: In order to explore a more efficient, non-invasive in vitro model for RDEB studies, we obtained patient fibroblasts derived from discarded dressings) and examined their phenotypic features compared with fibroblasts derived from non-injured skin of RDEB and healthy-donor skin biopsies. Our results demonstrate that fibroblasts derived from RDEB chronic wounds (RDEB-CW) displayed characteristics of senescent cells, increased myofibroblast differentiation, and augmented levels of TGF-ß1 signaling components compared to fibroblasts derived from RDEB acute wounds and unaffected RDEB skin as well as skin from healthy-donors. Furthermore, RDEB-CW fibroblasts exhibited an increased pattern of inflammatory cytokine secretion (IL-1ß and IL-6) when compared with RDEB and control fibroblasts. Interestingly, these aberrant patterns were found specifically in RDEB-CW fibroblasts independent of the culturing method, since fibroblasts obtained from dressing of acute wounds displayed a phenotype more similar to fibroblasts obtained from RDEB normal skin biopsies. CONCLUSIONS: Our results show that in vitro cultured RDEB-CW fibroblasts maintain distinctive cellular and molecular characteristics resembling the inflammatory and fibrotic microenvironment observed in RDEB patients' chronic wounds. This work describes a novel, non-invasive and painless strategy to obtain human fibroblasts chronically subjected to an inflammatory and fibrotic environment, supporting their use as an accessible model for in vitro studies of RDEB wound healing pathogenesis. As such, this approach is well suited to testing new therapeutic strategies under controlled laboratory conditions.
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Epidermólisis Ampollosa Distrófica , Humanos , Epidermólisis Ampollosa Distrófica/genética , Fibroblastos , Vendajes , Diferenciación Celular , Colágeno Tipo VII/genéticaRESUMEN
Epidemiological studies show that having a history of cancer protects from the development of Alzheimer's Disease (AD), and vice versa, AD protects from cancer. The mechanism of this mutual protection is unknown. We have reported that the peripheral blood mononuclear cells (PBMC) of amnestic cognitive impairment (aMCI) and Alzheimer's Disease (AD) patients have increased susceptibility to oxidative cell death compared to control subjects, and from the opposite standpoint a cancer history is associated with increased resistance to oxidative stress cell death in PBMCs, even in those subjects who have cancer history and aMCI (Ca + aMCI). Cellular senescence is a regulator of susceptibility to cell death and has been related to the pathophysiology of AD and cancer. Recently, we showed that cellular senescence markers can be tracked in PBMCs of aMCI patients, so we here investigated whether these senescence markers are dependent on having a history of cancer. Senescence-associated ßeta-galactosidase (SA-ß-Gal) activity, G0-G1 phase cell-cycle arrest, p16 and p53 were analyzed by flow cytometry; phosphorylated H2A histone family member X (γH2AX) by immunofluorescence; IL-6 and IL-8 mRNA by qPCR; and plasmatic levels by ELISA. Senescence markers that were elevated in PBMCs of aMCI patients, such as SA-ß-Gal, Go-G1 arrested cells, IL-6 and IL-8 mRNA expression, and IL-8 plasmatic levels, were decreased in PBMCs of Ca + aMCI patients to levels similar to those of controls or of cancer survivors without cognitive impairment, suggesting that cancer in the past leaves a fingerprint that can be peripherally traceable in PBMC samples. These results support the hypothesis that the senescence process might be involved in the inverse association between cancer and AD.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Neoplasias , Humanos , Leucocitos Mononucleares , Enfermedad de Alzheimer/genética , Interleucina-6 , Interleucina-8 , Pruebas Neuropsicológicas , Disfunción Cognitiva/genética , Cognición , ARN MensajeroRESUMEN
BACKGROUND: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare inherited skin disease caused by variants in the COL7A1 gene, coding for type VII collagen (C7), an important component of anchoring fibrils in the basement membrane of the epidermis. RDEB patients suffer from skin fragility starting with blister formation and evolving into chronic wounds, inflammation and skin fibrosis, with a high risk of developing aggressive skin carcinomas. Restricted therapeutic options are limited by the lack of in vitro models of defective wound healing in RDEB patients. RESULTS: In order to explore a more efficient, non-invasive in vitro model for RDEB studies, we obtained patient fibroblasts derived from discarded dressings) and examined their phenotypic features compared with fibroblasts derived from non-injured skin of RDEB and healthy-donor skin biopsies. Our results demonstrate that fibroblasts derived from RDEB chronic wounds (RDEB-CW) displayed characteristics of senescent cells, increased myofibroblast differentiation, and augmented levels of TGF-ß1 signaling components compared to fibroblasts derived from RDEB acute wounds and unaffected RDEB skin as well as skin from healthy-donors. Furthermore, RDEB-CW fibroblasts exhibited an increased pattern of inflammatory cytokine secretion (IL-1ß and IL-6) when compared with RDEB and control fibroblasts. Interestingly, these aberrant patterns were found specifically in RDEB-CW fibroblasts independent of the culturing method, since fibroblasts obtained from dressing of acute wounds displayed a phenotype more similar to fibroblasts obtained from RDEB normal skin biopsies. CONCLUSIONS: Our results show that in vitro cultured RDEB-CW fibroblasts maintain distinctive cellular and molecular characteristics resembling the inflammatory and fibrotic microenvironment observed in RDEB patients' chronic wounds. This work describes a novel, non-invasive and painless strategy to obtain human fibroblasts chronically subjected to an inflammatory and fibrotic environment, supporting their use as an accessible model for in vitro studies of RDEB wound healing pathogenesis. As such, this approach is well suited to testing new therapeutic strategies under controlled laboratory conditions.
Asunto(s)
Humanos , Epidermólisis Ampollosa Distrófica/genética , Vendajes , Diferenciación Celular , Colágeno Tipo VII/genética , FibroblastosRESUMEN
During Inflammaging, a dysregulation of the immune cell functions is generated, and these cells acquire a senescent phenotype with an increase in pro-inflammatory cytokines and ROS. This increase in pro-inflammatory molecules contributes to the chronic inflammation and oxidative damage of biomolecules, classically observed in the Inflammaging process. One of the most critical oxidative damages is generated to the host DNA. Damaged DNA is located out of the natural compartments, such as the nucleus and mitochondria, and is present in the cell's cytoplasm. This DNA localization activates some DNA sensors, such as the cGAS/STING signaling pathway, that induce transcriptional factors involved in increasing inflammatory molecules. Some of the targets of this signaling pathway are the SASPs. SASPs are secreted pro-inflammatory molecules characteristic of the senescent cells and inducers of ROS production. It has been suggested that oxidative damage to nuclear and mitochondrial DNA generates activation of the cGAS/STING pathway, increasing ROS levels induced by SASPs. These additional ROS increase oxidative DNA damage, causing a loop during the Inflammaging. However, the relationship between the cGAS/STING pathway and the increase in ROS during Inflammaging has not been clarified. This review attempt to describe the potential connection between the cGAS/STING pathway and ROS during the Inflammaging process, based on the current literature, as a contribution to the knowledge of the molecular mechanisms that occur and contribute to the development of the considered adaptative Inflammaging process during aging.
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Proteínas de la Membrana , Nucleotidiltransferasas , Humanos , Especies Reactivas de Oxígeno , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Transducción de Señal/fisiología , Inflamación , ADN Mitocondrial/genéticaRESUMEN
Recent studies suggest that cellular senescence plays a role in Alzheimer's Disease (AD) pathogenesis. We hypothesize that cellular senescence markers might be tracked in the peripheral tissues of AD patients. Senescence hallmarks, including altered metabolism, cell-cycle arrest, DNA damage response (DDR) and senescence secretory associated phenotype (SASP), were measured in peripheral blood mononuclear cells (PBMCs) of healthy controls (HC), amnestic mild cognitive impairment (aMCI) and AD patients. Senescence-associated ßeta-galactosidase (SA-ß-Gal) activity, G0-G1 phase cell-cycle arrest, p16 and p53 were analyzed by flow cytometry, while IL-6 and IL-8 mRNA were analyzed by qPCR, and phosphorylated H2A histone family member X (γH2AX) was analyzed by immunofluorescence. Senescent cells in the brain tissue were determined with lipofuscin staining. An increase in the number of senescent cells was observed in the frontal cortex and hippocampus of advanced AD patients. PBMCs of aMCI patients, but not in AD, showed increased SA-ß-Gal compared with HCs. aMCI PBMCs also had increased IL-6 and IL8 mRNA expression and number of cells arrested at G0-G1, which were absent in AD. Instead, AD PBMCs had significantly increased p16 and p53 expression and decreased γH2Ax activity compared with HC. This study reports that several markers of cellular senescence can be measured in PBMCs of aMCI and AD patients.
Asunto(s)
Enfermedad de Alzheimer , Disfunción Cognitiva , Enfermedad de Alzheimer/patología , Biomarcadores , Senescencia Celular , Disfunción Cognitiva/patología , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Leucocitos Mononucleares/metabolismo , ARN Mensajero , Proteína p53 Supresora de TumorRESUMEN
Mesenchymal stem cells (MSC) have emerged as a promising tool to treat inflammatory diseases, such as inflammatory bowel disease (IBD), due to their immunoregulatory properties. Frequently, IBD is modeled in mice by using dextran sulfate sodium (DSS)-induced colitis. Recently, the modulation of autophagy in MSC has been suggested as a novel strategy to improve MSC-based immunotherapy. Hence, we investigated a possible role of Pacer, a novel autophagy enhancer, in regulating the immunosuppressive function of MSC in the context of DSS-induced colitis. We found that Pacer is upregulated upon stimulation with the pro-inflammatory cytokine TNFα, the main cytokine released in the inflammatory environment of IBD. By modulating Pacer expression in MSC, we found that Pacer plays an important role in regulating the autophagy pathway in this cell type in response to TNFα stimulation, as well as in regulating the immunosuppressive ability of MSC toward T-cell proliferation. Furthermore, increased expression of Pacer in MSC enhanced their ability to ameliorate the symptoms of DSS-induced colitis in mice. Our results support previous findings that autophagy regulates the therapeutic potential of MSC and suggest that the augmentation of autophagic capacity in MSC by increasing Pacer levels may have therapeutic implications for IBD.
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Colitis , Enfermedades Inflamatorias del Intestino , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Animales , Autofagia , Colitis/tratamiento farmacológico , Colitis/terapia , Citocinas/metabolismo , Sulfato de Dextran/farmacología , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Ratones , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Infections during pregnancy can seriously damage fetal neurodevelopment by aberrantly activating the maternal immune system, directly impacting fetal neural cells. Increasing evidence suggests that these adverse impacts involve alterations in neural stem cell biology with long-term consequences for offspring, including neurodevelopmental disorders such as autism spectrum disorder, schizophrenia, and cognitive impairment. Here we review how maternal infection with viruses such as Influenza A, Cytomegalovirus, and Zika during pregnancy can affect the brain development of offspring by promoting the release of maternal pro-inflammatory cytokines, triggering neuroinflammation of the fetal brain, and/or directly infecting fetal neural cells. In addition, we review insights into how these infections impact human brain development from studies with animal models and brain organoids. Finally, we discuss how maternal infection with SARS-CoV-2 may have consequences for neurodevelopment of the offspring.
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Trastorno del Espectro Autista , COVID-19 , Virosis , Infección por el Virus Zika , Virus Zika , Animales , Trastorno del Espectro Autista/etiología , Encéfalo , Citocinas , Femenino , Embarazo , SARS-CoV-2 , Virosis/complicacionesRESUMEN
Several drugs are chemically unstable in the gastric environment and have low bioavailability restricted by intestinal absorption, which motivates the development of alternative routes for drug release, such as transdermal drug carriers for drug delivery to specific areas of the skin. Herein, novel polyelectrolyte complexes (PEC) consisting of carboxymethylagarose (CMA) and chitosan (CS) were prepared. pH-responsive CMA/CS hydrogels were obtained by mixing CMA and CS at various weight ratios. Swelling ratio was modulated by varying the CMA and CS weight ratio, and the highest swelling values were achieved for 2:1 wt% hydrogels at 25 °C and pH 6.0. PEC films were characterized by ATR-FTIR spectroscopy, TGA, DSC, and SEM. Results indicated that CMA and CS were successfully crosslinked by ionic complexation. As a model drug, diclofenac sodium (DS) was loaded in CMA/CS PECs. Association efficiency and loading capacity were ca. 69% and 79%, respectively, exhibiting 67% cumulative release after 72 h at 37 °C and pH 6.0 through Fickian diffusion mechanism. Viability assay of immortalized human keratinocyte (HaCat) cells showed ca. 100% survival in the presence of hydrogels and DS. Therefore, this work suggests that CMA/CS PECs can be applied as pH-responsive carriers for dermal drug delivery.
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Quitosano , Quitosano/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Liberación de Fármacos , Humanos , Hidrogeles/química , Concentración de Iones de Hidrógeno , Polielectrolitos/químicaRESUMEN
BACKGROUND: Disturbances in the autophagy/endolysosomal systems are proposed as early signatures of Alzheimer's disease (AD). However, few studies are available concerning autophagy gene expression in AD patients. OBJECTIVE: To explore the differential expression of classical genes involved in the autophagy pathway, among them a less characterized one, DEF8 (Differentially expressed in FDCP 8), initially considered a Rubicon family member, in peripheralblood mononuclear cells (PBMCs) from individuals with mild cognitive impairment (MCI) and probable AD (pAD) and correlate the results with the expression of DEF8 in the brain of 5xFAD mice. METHOD: By real-time PCR and flow cytometry, we evaluated autophagy genes levels in PBMCs from MCI and pAD patients. We evaluated DEF8 levels and its localization in brain samples of the 5xFAD mice by real-time PCR, western blot, and immunofluorescence. RESULTS: Transcriptional levels of DEF8 were significantly reduced in PBMCs of MCI and pAD patients compared with healthy donors, correlating with the MoCA and MoCA-MIS cognitive tests scores. DEF8 protein levels were increased in lymphocytes from MCI but not pAD, compared to controls. In the case of brain samples from 5xFAD mice, we observed a reduced mRNA expression and augmented protein levels in 5xFAD compared to age-matched wild-type mice. DEF8 presented a neuronal localization. CONCLUSION: DEF8, a protein proposed to act at the final step of the autophagy/endolysosomal pathway, is differentially expressed in PBMCs of MCI and pAD and neurons of 5xFAD mice. These results suggest a potential role for DEF8 in the pathophysiology of AD.
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Enfermedad de Alzheimer/metabolismo , Autofagia/fisiología , Encéfalo/metabolismo , Disfunción Cognitiva/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Biomarcadores/metabolismo , Encéfalo/patología , Disfunción Cognitiva/genética , Disfunción Cognitiva/patología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana EdadRESUMEN
Active immunotherapy against cancer is based on immune system stimulation, triggering efficient and long-lasting antigen-specific immune responses. Immunization strategies using whole dead cells from tumor tissue, containing specific antigens inside, have become a promising approach, providing efficient lymphocyte activation through dendritic cells (DCs). In this work, we generate whole dead tumor cells from CT26, E.G7, and EL4 live tumor cells as antigen sources, which termed immunogenic cell bodies (ICBs), generated by a simple and cost-efficient starvation-protocol, in order to determine whether are capable of inducing a transversal anticancer response regardless of the tumor type, in a similar way to what we describe previously with B16 melanoma. We evaluated the anticancer effects of immunization with doses of ICBs in syngeneic murine tumor models. Our results showed that mice's immunization with ICBs-E.G7 and ICBs-CT26 generate 18% and 25% of tumor-free animals, respectively. On the other hand, all carrying tumor-animals and immunized with ICBs, including ICBs-EL4, showed a significant delay in their growth compared to not immunized animals. These effects relate to DCs maturation, cytokine production, increase in CD4+T-bet+ and CD4+ROR-γt+ population, and decrease of T regulatory lymphocytes in the spleen. Altogether, our data suggest that whole dead tumor cell-based cancer immunotherapy generated by a simple starvation protocol is a promising way to develop complementary, innovative, and affordable antitumor therapies in a broad spectrum of tumors.
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Antígenos de Neoplasias , Neoplasias del Colon/inmunología , Inmunoterapia , Linfoma/inmunología , Células Tumorales Cultivadas/inmunología , Animales , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Autofagia , Técnicas de Cultivo de Célula , Citocinas/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Bazo/citologíaRESUMEN
Spontaneous Ca2+ signaling from the InsP3R intracellular Ca2+ release channel to mitochondria is essential for optimal oxidative phosphorylation (OXPHOS) and ATP production. In cells with defective OXPHOS, reductive carboxylation replaces oxidative metabolism to maintain amounts of reducing equivalents and metabolic precursors. To investigate the role of mitochondrial Ca2+ uptake in regulating bioenergetics in these cells, we used OXPHOS-competent and OXPHOS-defective cells. Inhibition of InsP3R activity or mitochondrial Ca2+ uptake increased α-ketoglutarate (αKG) abundance and the NAD+/NADH ratio, indicating that constitutive endoplasmic reticulum (ER)-to-mitochondria Ca2+ transfer promoted optimal αKG dehydrogenase (αKGDH) activity. Reducing mitochondrial Ca2+ inhibited αKGDH activity and increased NAD+, which induced SIRT1-dependent autophagy in both OXPHOS-competent and OXPHOS-defective cells. Whereas autophagic flux in OXPHOS-competent cells promoted cell survival, it was impaired in OXPHOS-defective cells because of inhibition of autophagosome-lysosome fusion. Inhibition of αKGDH and impaired autophagic flux in OXPHOS-defective cells resulted in pronounced cell death in response to interruption of constitutive flux of Ca2+ from ER to mitochondria. These results demonstrate that mitochondria play a fundamental role in maintaining bioenergetic homeostasis of both OXPHOS-competent and OXPHOS-defective cells, with Ca2+ regulation of αKGDH activity playing a pivotal role. Inhibition of ER-to-mitochondria Ca2+ transfer may represent a general therapeutic strategy against cancer cells regardless of their OXPHOS status.
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Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Neoplasias/metabolismo , Fosforilación Oxidativa , Línea Celular Tumoral , Supervivencia Celular , Retículo Endoplásmico/genética , Retículo Endoplásmico/patología , Humanos , Mitocondrias/genética , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologíaRESUMEN
The adverse environmental conditions found in the periodontium during periodontitis pathogenesis stimulate local autophagy responses, mainly due to a continuous inflammatory response against the dysbiotic subgingival microbiome. The junctional epithelium represents the main site of the initial interaction between the host and the dysbiotic biofilm. Here, we investigated the role of autophagy in junctional epithelium keratinocytes (JEKs) in response to Aggregatibacter actinomycetemcomitans or its purified lipopolysaccharides (LPS). Immunofluorescence confocal analysis revealed an extensive nuclear translocation of transcription factor EB (TFEB) and consequently, an increase in autophagy markers and LC3-turnover assessed by immunoblotting and qRT-PCR. Correspondingly, challenged JEKs showed a punctuate cytosolic profile of LC3 protein contrasting with the diffuse distribution observed in untreated controls. Three-dimensional reconstructions of confocal images displayed a close association between intracellular bacteria and LC3-positive vesicles. Similarly, a close association between autophagic vesicles and the protein p62 was observed in challenged JEKs, indicating that p62 is the main adapter protein recruited during A. actinomycetemcomitans infection. Finally, the pharmacological inhibition of autophagy significantly increased the number of bacteria-infected cells as well as their death, similar to treatment with LPS. Our results indicate that A. actinomycetemcomitans infection induces autophagy in JEKs, and this homeostatic process has a cytoprotective effect on the host cells during the early stages of infection.
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Aggregatibacter actinomycetemcomitans/fisiología , Autofagia , Inserción Epitelial/patología , Queratinocitos/microbiología , Queratinocitos/patología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Biomarcadores/metabolismo , Recuento de Células , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular , Humanos , Imagenología Tridimensional , Lipopolisacáridos/aislamiento & purificación , Modelos Biológicos , Transporte de Proteínas , Proteína Sequestosoma-1/metabolismoRESUMEN
Mollusk hemocyanins have been used for decades in immunological and clinical applications as natural, nontoxic, nonpathogenic, and nonspecific immunostimulants for the treatment of superficial bladder cancer, as carriers/adjuvants of tumor-associated antigens in cancer vaccine development and as adjuvants to dendritic cell-based immunotherapy, because these glycoproteins induce a bias towards Th1 immunity. Here, we analyzed the preclinical therapeutic potential of the traditional keyhole limpet hemocyanin (KLH) and two new hemocyanins from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH) in mouse models of oral squamous cell carcinoma. Due to the aggressiveness and deadly malignant potential of this cancer, the hemocyanins were applied in combination with adjuvants, such as alum, AddaVax, and QS-21, which have been shown to be safe and effective in human vaccines, to potentiate their antitumor activity. The immunogenic performance of the hemocyanins in combination with the adjuvants was compared, and the best formulation was evaluated for its antitumor effects in two murine models of oral cancer: MOC7 cells implanted in the flank (heterotopic) and bioluminescent AT-84 E7 Luc cells implanted in the floor of the mouth (orthotopic). The results demonstrated that the hemocyanins in combination with QS-21 showed the greatest immunogenicity, as reflected by a robust, specific humoral response predominantly characterized by IgG2a antibodies and a sustained cellular response manifesting as a delayed hypersensitivity reaction. The KLH- and FLH-QS-21 formulations showed reduced tumor development and greater overall survival. Hemocyanins, as opposed to QS-21, had no cytotoxic effect on either oral cancer cell line cultured in vitro, supporting the idea that the antitumor effects of hemocyanins are associated with their modulation of the immune response. Therefore, hemocyanin utilization would allow a lower QS-21 dosage to achieve therapeutic results. Overall, our study opens a new door to further investigation of the use of hemocyanins plus adjuvants for the development of immunotherapies against oral carcinoma.
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Adyuvantes Inmunológicos/uso terapéutico , Hemocianinas/uso terapéutico , Inmunoterapia , Neoplasias de la Boca/tratamiento farmacológico , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Hemocianinas/química , Inmunidad Celular , Inmunidad Humoral , Ratones , Ratones Endogámicos C57BL , Moluscos/química , Polisorbatos/administración & dosificación , Saponinas/administración & dosificación , Escualeno/administración & dosificaciónRESUMEN
The insular cortex (IC), among other brain regions, becomes active when humans experience fear or anxiety. However, few experimental studies in rats have implicated the IC in threat responses. We have recently reported that inactivation of the primary interoceptive cortex (pIC) during pre-training, or the intra-pIC blockade of protein synthesis immediately after training, impaired the consolidation of auditory fear conditioning. The present study was designed to investigate the role of the pIC in innate and learned defensive responses to predator odor. Freezing behavior was elicited by single or repetitive exposures to a collar that had been worn by a domestic cat. Sessions were video-recorded and later scored by video observation. We found that muscimol inactivation of the pIC reduced the expression of freezing reaction in response to a single or repeated exposure to cat odor. We also found that pIC inactivation with muscimol impaired conditioning of fear to the context in which rats were exposed to cat odor. Furthermore, neosaxitoxin inactivation of the pIC resulted in a prolonged and robust reduction in freezing response in subsequent re-exposures to cat odor. In addition, freezing behavior significantly correlated with the neural activity of the IC. The present results suggest that the IC is involved in the expression of both innate and learned fear responses to predator odor.
RESUMEN
Colorectal cancer is a deadly disease, which is frequently diagnosed at advanced stages, where conventional treatments are no longer effective. Cancer immunotherapy has emerged as a new form to treat different malignancies by turning-on the immune system against tumors. However, tumors are able to evade antitumor immune responses by promoting an immunosuppressive microenvironment. Single-stranded DNA containing M13 bacteriophages are highly immunogenic and can be specifically targeted to the surface of tumor cells to trigger inflammation and infiltration of activated innate immune cells, overcoming tumor-associated immunosuppression and promoting antitumor immunity. Carcinoembryonic antigen (CEA) is highly expressed in colorectal cancers and has been shown to promote several malignant features of colorectal cancer cells. In this work, we targeted M13 bacteriophage to CEA, a tumor-associated antigen over-expressed in a high proportion of colorectal cancers but largely absent in normal cells. The CEA-targeted M13 bacteriophage was shown to specifically bind to purified CEA and CEA-expressing tumor cells in vitro. Both intratumoral and systemic administration of CEA-specific bacteriophages significantly reduced tumor growth of mouse models of colorectal cancer, as compared to PBS and control bacteriophage administration. CEA-specific bacteriophages promoted tumor infiltration of neutrophils and macrophages, as well as maturation dendritic cells in tumor-draining lymph nodes, suggesting that antitumor T-cell responses were elicited. Finally, we demonstrated that tumor protection provided by CEA-specific bacteriophage particles is mediated by CD8+ T cells, as depletion of circulating CD8+ T cells completely abrogated antitumor protection. In summary, we demonstrated that CEA-specific M13 bacteriophages represent a potential immunotherapy against colorectal cancer.
Asunto(s)
Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/inmunología , Inovirus/inmunología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
DNA vaccination is an attractive approach to elicit tumor-specific cytotoxic CD8+ T lymphocytes (CTL), which can mediate protective immunity against tumors. To initiate CTL responses, antigen-encoding plasmids employed for DNA vaccination need to activate dendritic cells (DC) through the stimulation of DNA-sensing innate immune receptors that converge in the activation of the master transcription factor NF-κB. To this end, NF-κB repressor IκBα needs to be degraded, allowing NF-κB to translocate to the nucleus and transcribe proinflammatory target genes, as well as its repressor IκBα. Therefore, NF-κB activation is self-limited by de novo synthesis of IκBa, which sequesters NF-κB in the cytosol. Hence, we tested whether co-delivering a shRNA-based adjuvant able to silence IκBα expression would further promote DNA-induced NFκB activation, DC activation and tumor-protective CTL responses induced by DNA vaccination in a preclinical model. First, an IκBα-targeting shRNA plasmid (shIκBα) was shown to reduce IκBα expression and promote NFκB-driven transcription in vitro, as well as up-regulate inflammatory target genes in vivo. Then, we showed that intradermal DNA electroporation induced the migration of skin migratory dendritic cells to draining lymph nodes and maturation of dermal dendritic cells (dDC). Interestingly, shIκBα further promoted the migration of mature skin migratory dendritic cells, in particular dDC, which are specialized in antigen cross-presentation and activation of CD8+ T cells. Consistently, mice vaccinated with a plasmid encoding the melanoma-associated antigen tyrosinase-related protein 2 (TRP2) in combination with shIκBα enhanced TRP2-specific CTL responses and reduced the number of lung melanoma foci in mice challenged with intravenous injection of B16F10 cells. Moreover, therapeutic vaccination with pTRP2 and shIκBα delayed the growth of B16F10 melanoma subcutaneous tumors. Our data suggest that adjuvants promoting NF-κB activation represent an attractive strategy to boost DC activation and promote the generation of tumor-protective CTL responses elicited by DNA vaccines.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Células de Langerhans/inmunología , Ganglios Linfáticos/inmunología , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , ARN Interferente Pequeño/metabolismo , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/metabolismo , Animales , Vacunas contra el Cáncer/administración & dosificación , Movimiento Celular , Modelos Animales de Enfermedad , Células de Langerhans/fisiología , Pulmón/patología , Melanoma/patología , Melanoma/terapia , Ratones Endogámicos C57BL , Resultado del Tratamiento , Vacunación , Vacunas de ADN/administración & dosificaciónRESUMEN
The pathological hallmarks of Alzheimer's disease (AD) are amyloid-ß (Aß) plaques, neurofibrillary tangles, and glia activation. The pathology also includes vascular amyloidosis and cerebrovascular disease. Vascular compromise can result in hypoperfusion, local tissue hypoxia, and acidosis. Activated microglia and astrocytes can phagocytose Aß through membrane receptors that include scavenger receptors. Changes in glial cells induced by extracellular acidosis could play a role in the development of AD. Here, we assess whether extracellular acidosis changes glial cell properties relevant for Aß clearance capacity. Incubation of glial cells on acidified culture medium (pH 6.9 or 6.5) for 24-48âh resulted in decreased cell diameter, with thinner branches in astrocytes, slight reduction in cell body size in microglia, a transient decrease in astrocyte adhesion to substrates, and a persistent decrease in microglia adhesion compared with control media (pH 7.4). Astrocyte Aß phagocytosis decreased at pH 6.9 and 6.5, whereas microglia phagocytosis only transiently decreased in acidified media. Scavenger receptors class B member I (SR-BI) increased and scavenger receptors-macrophage receptors with collagenous structures (SR-MARCO) decreased in astrocytes cultured at pH 6.5. In contrast, in microglia exposed to pH 6.5, expression of SR-BI and SR-MARCO increased and fatty acid translocase (CD-36) decreased. In conclusion, the acidic environment changed the adhesiveness and morphology of both microglia and astrocytes, but only astrocytes showed a persistent decrease in Aß clearance activity. Expression of scavenger receptors was affected differentially in microglia and astrocytes by acidosis. These changes in scavenger receptor patterns can affect the activation of glia and their contribution to neurodegeneration.
Asunto(s)
Acidosis/fisiopatología , Péptidos beta-Amiloides/metabolismo , Astrocitos/metabolismo , Regulación de la Expresión Génica/fisiología , Microglía/metabolismo , Fagocitosis/fisiología , Receptores Depuradores/genética , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Medios de Cultivo Condicionados/farmacología , Medios de Cultivo Condicionados/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Microglía/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fagocitosis/efectos de los fármacos , Ratas , Receptores Depuradores/metabolismo , Factores de Tiempo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Interleukin-3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) are two of the best-characterized cell survival factors in hematopoietic cells; these factors induce an increase in Akt activity in multiple cell lines, a process thought to be involved in cellular survival. It is known that growth factors require sustained glucose metabolism to promote cell survival. It has been determined that IL-3 and GM-CSF signal for increased glucose uptake in hematopoietic cells. Interestingly, receptors for IL-3 and GM-CSF are present in several non-hematopoietic cell types but their roles in these cells have been poorly described. In this study, we demonstrated the expression of IL-3 and GM-CSF receptors in HEK293 cells and analyzed their effect on glucose uptake. In these cells, both IL-3 and GM-CSF, increased glucose uptake. The results indicated that this increase involves the subcellular redistribution of GLUT1, affecting glucose transporter levels at the cell surface in HEK293 cells. Also the data directly demonstrates that the PI 3-kinase/Akt pathway is an important mediator of this process. Altogether these results show a role for non-insulin growth factors in the regulation of GLUT1 trafficking that has not yet been directly determined in non-hematopoietic cells.