RESUMO
Removal of the assembly factor eukaryotic initiation factor 6 (eIF6) is critical for late cytoplasmic maturation of 60S ribosomal subunits. In mammalian cells, the current model posits that eIF6 release is triggered following phosphorylation of Ser 235 by activated protein kinase C. In contrast, genetic studies in yeast indicate a requirement for the ortholog of the SBDS (Shwachman-Bodian-Diamond syndrome) gene that is mutated in the inherited leukemia predisposition disorder Shwachman-Diamond syndrome (SDS). Here, by isolating late cytoplasmic 60S ribosomal subunits from Sbds-deleted mice, we show that SBDS and the GTPase elongation factor-like 1 (EFL1) directly catalyze eIF6 removal in mammalian cells by a mechanism that requires GTP binding and hydrolysis by EFL1 but not phosphorylation of eIF6 Ser 235. Functional analysis of disease-associated missense variants reveals that the essential role of SBDS is to tightly couple GTP hydrolysis by EFL1 on the ribosome to eIF6 release. Furthermore, complementary NMR spectroscopic studies suggest unanticipated mechanistic parallels between this late step in 60S maturation and aspects of bacterial ribosome disassembly. Our findings establish a direct role for SBDS and EFL1 in catalyzing the translational activation of ribosomes in all eukaryotes, and define SDS as a ribosomopathy caused by uncoupling GTP hydrolysis from eIF6 release.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Guanosina Trifosfato/metabolismo , Ribossomos/patologia , Animais , Doenças da Medula Óssea/genética , Doenças da Medula Óssea/fisiopatologia , Catálise , Células Cultivadas , Modelos Animais de Doenças , Fatores de Iniciação em Eucariotos/genética , Insuficiência Pancreática Exócrina/genética , Insuficiência Pancreática Exócrina/fisiopatologia , Humanos , Hidrólise , Lipomatose , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutação , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos , Síndrome de Shwachman-DiamondRESUMO
Controlling the outer surface of nanometric metal-organic frameworks (nanoMOFs) and further understanding the in vivo effect of the coated material are crucial for the convenient biomedical applications of MOFs. However, in most studies, the surface modification protocol is often associated with significant toxicity and/or lack of selectivity. As an alternative, how the highly selective and general grafting GraftFast method leads, through a green and simple process, to the successful attachment of multifunctional biopolymers (polyethylene glycol (PEG) and hyaluronic acid) on the external surface of nanoMOFs is reported. In particular, effectively PEGylated iron trimesate MIL-100(Fe) nanoparticles (NPs) exhibit suitable grafting stability and superior chemical and colloidal stability in different biofluids, while conserving full porosity and allowing the adsorption of bioactive molecules (cosmetic and antitumor agents). Furthermore, the nature of the MOF-PEG interaction is deeply investigated using high-resolution soft X-ray spectroscopy. Finally, a cell penetration study using the radio-labeled antitumor agent gemcitabine monophosphate (3 H-GMP)-loaded MIL-100(Fe)@PEG NPs shows reduced macrophage phagocytosis, confirming a significant in vitro PEG furtiveness.
RESUMO
One of the main challenges in the development of vaccine has been to improve their stability at room temperature and eliminate the limitations associated with the cold chain storage. In this paper, we describe the development and optimization of thermostable nanocarriers consisting of an oily core with immunostimulating activity, containing squalene or α tocopherol surrounded by a protamine shell. The results showed that these nanocapsules can efficiently associate the recombinant hepatitis B surface antigen (rHBsAg) without compromising its antigenicity. Furthermore, the freeze-dried protamine nanocapsules were able to preserve the integrity and bioactivity of the associated antigen upon storage for at least 12 months at room temperature. In vitro studies evidenced the high internalization of the nanocapsules by immunocompetent cells, followed by cytokine secretion and complement activation. In vivo studies showed the capacity of rHBsAg-loaded nanocapsules to elicit protective levels upon intramuscular or intranasal administration to mice. Overall, our data indicate that protamine nanocapsules are an innovative thermostable nanovaccine platform for improved antigen delivery.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos de Superfície da Hepatite B/administração & dosagem , Vacinas contra Hepatite B/administração & dosagem , Nanocápsulas/química , Animais , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Feminino , Liofilização , Voluntários Saudáveis , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Humanos , Imunogenicidade da Vacina , Leucócitos Mononucleares , Camundongos , Camundongos Endogâmicos BALB C , Protaminas/química , Células RAW 264.7 , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Temperatura , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
PURPOSE: Diagnosis of tuberculous uveitis (TBU) is often challenging and is usually made after excluding other causes of uveitis. We analysed the characteristics of TBU and variables associated with visual outcome. METHODS: A retrospective, observational analysis was performed in patients with presumptive TBU who were started on specific TB treatment between January 2006 and June 2016. Demographic, clinical, radiological, analytical and ophthalmic examination variables were studied. After completing TB treatment, a follow-up of at least 9 months was performed. A univariate and logistic regression analysis was applied to identify the variables associated with visual acuity and recurrences of uveitis. RESULTS: Forty affected eyes of 24 individuals were identified; 79% of patients were diagnosed during the last 3 years of the study period. Median delay from onset of symptoms to diagnosis was 12 weeks. Loss of visual acuity was the most frequent symptom (87.5%). Posterior uveitis was the most frequent localization (72.9%); 19 patients (79.2%) presented at least one of the Gupta signs predictive of TBU, but there were no confirmed diagnoses. OUTCOME: There was improvement in visual acuity in 74.4% of the eyes, but a complete response was achieved only in 56.4%. There was recurrence in two patients. The initiation of treatment ≥ 24 weeks after onset of symptoms was significantly associated with no improvement (p = 0.026). CONCLUSION: TBU can cause permanent damage to visual acuity, particularly in patients with delayed diagnosis. A prompt initiation of systemic TB treatment is essential to improve visual prognosis.
Assuntos
Tuberculose Ocular/epidemiologia , Uveíte/epidemiologia , Uveíte/microbiologia , Adulto , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Biomarcadores , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Vasculite Retiniana/diagnóstico , Vasculite Retiniana/tratamento farmacológico , Vasculite Retiniana/microbiologia , Estudos Retrospectivos , Tempo para o Tratamento , Resultado do Tratamento , Tuberculose Ocular/diagnóstico , Tuberculose Ocular/tratamento farmacológico , Uveíte/diagnóstico , Uveíte/tratamento farmacológico , Testes VisuaisRESUMO
In recent years, there has been an increase in the production of several types of nanoparticles (Nps) for different purposes. Several studies have been performed to analyse the toxicity induced by some of these individual Nps, but data are scarce on the potential hazards or beneficial effects induced by a range of nanomaterials in the same environment. The purpose of the study described here was to evaluate the toxicological effects induced by in vitro exposure of human cells to ZnO Nps in combination with different concentrations of other metal oxide Nps (Al2O3, CeO2, TiO2 and Y2O3). The results indicate that the presence of these Nps has synergistic or antagonistic effects on the cell death induced by ZnO Nps, with a quite marked beneficial effect observed when high concentrations of Nps were tested. Moreover, analysis by Western blot of the main components of the intracellular activation routes (MAPKs and NFκB) again showed that the presence of other Nps can affect cell activation. In conclusion, the presence of several Nps in the same environment modifies the functional activity of one individual Np. Further studies are required in order to elucidate the effects induced by combinations of nanomaterials.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Nanopartículas Metálicas , Metais , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Óxidos , Óxido de Alumínio/química , Óxido de Alumínio/farmacologia , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Nanopartículas Metálicas/química , Metais/química , Metais/farmacologia , Óxidos/química , Óxidos/farmacologia , Solubilidade , Óxido de Zinco/química , Óxido de Zinco/farmacologiaRESUMO
Tissue membranes are boundaries that isolate organs or cavities in the body. These semi-permeable membranes are responsible for passive protection that acts through the regulation of nutrient absorption, secretion and filtration of small molecules. These functions could be altered as a consequence of inflammation or trauma, which in turn could lead to changes in permeability, allowing the entrance of toxins, antigens, proteins or facilitating the spread of tumors. Membrane permeability therefore plays an important role in numerous diseases. However, current experimental techniques that are available to quantify membrane permeability in small animals have limited precision and temporal specificity. Improvements in such measurements would lead to a deeper understanding of disease pathogenesis and this may accelerate the development of specific therapies. The study reported here concerns the efficacy of a novel, non-invasive imaging analysis-based measurement method that significantly improves the quantification of tissue membrane permeability in small animals, while at the same time mitigating the adverse effects experienced by the animals under study.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Modelos Animais de Doenças , Imagem Óptica/métodos , Peritônio , Animais , Dextranos/análise , Soluções para Diálise/efeitos adversos , Feminino , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/análise , Camundongos , Camundongos Endogâmicos C57BL , Diálise Peritoneal , Peritônio/diagnóstico por imagem , Peritônio/metabolismo , Peritonite/diagnóstico por imagemRESUMO
Selol is a semi-synthetic compound containing selenite that is effective against cancerous cells and safer for clinical applications in comparison with other inorganic forms of selenite. Recently, we have developed a formulation of poly(methyl vinyl ether-co-maleic anhydride)-shelled selol nanocapsules (SPN), which reduced the proliferative activity of lung adenocarcinoma cells and presented little deleterious effects on normal cells in in vitro studies. In this study, we report on the antitumor activity and systemic effects induced by this formulation in chemically induced lung adenocarcinoma-bearing mice. The in vivo antitumor activity of the SPN was verified by macroscopic quantification, immunohistochemistry and morphological analyses. Toxicity analyses were performed by evaluations of the kidney, liver, and spleen; analyses of hemogram and plasma levels of alanine aminotransferase, aspartate transaminase, urea, and creatinine; and DNA fragmentation and cell cycle activity of the bone marrow cells. Furthermore, we investigated the potential of the SPN formulation to cause hemolysis, activate the complement system, provoke an inflammatory response and change the conformation of the plasma proteins. Our results showed that the SPN reduced the area of the surface tumor nodules but not the total number of tumor nodules. The biochemical and hematological findings were suggestive of the low systemic toxicity of the SPN formulation. The surface properties of the selol nanocapsules point to characteristics that are consistent with the treatment of the tumors in vivo: low hemolytic activity, weak inflammatory reaction with no activation of the complement system, and mild or absent conformational changes of the plasma proteins. In conclusion, this report suggests that the SPN formulation investigated herein exhibits anti-tumoral effects against lung adenocarcinoma in vivo and is associated with low systemic toxicity and high biocompatibility.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Maleatos/administração & dosagem , Nanocápsulas/administração & dosagem , Polietilenos/administração & dosagem , Compostos de Selênio/administração & dosagem , Adenocarcinoma/ultraestrutura , Adenocarcinoma de Pulmão , Animais , Antineoplásicos/química , Antineoplásicos/toxicidade , Peso Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas do Sistema Complemento/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Feminino , Inflamação/induzido quimicamente , Neoplasias Pulmonares/ultraestrutura , Maleatos/química , Maleatos/toxicidade , Camundongos , Nanocápsulas/química , Nanocápsulas/toxicidade , Tamanho do Órgão/efeitos dos fármacos , Polietilenos/química , Polietilenos/toxicidade , Compostos de Selênio/química , Compostos de Selênio/toxicidadeRESUMO
BACKGROUND: Selol is an oily mixture of selenitetriacylglycerides that was obtained as a semi-synthetic compound containing selenite. Selol is effective against cancerous cells and less toxic to normal cells compared with inorganic forms of selenite. However, Selol's hydrophobicity hinders its administration in vivo. Therefore, the present study aimed to produce a formulation of Selol nanocapsules (SPN) and to test its effectiveness against pulmonary adenocarcinoma cells (A549). RESULTS: Nanocapsules were produced through an interfacial nanoprecipitation method. The polymer shell was composed of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer. The obtained nanocapsules were monodisperse and stable. Both free Selol (S) and SPN reduced the viability of A549 cells, whereas S induced a greater reduction in non-tumor cell viability than SPN. The suppressor effect of SPN was primarily associated to the G2/M arrest of the cell cycle, as was corroborated by the down-regulations of the CCNB1 and CDC25C genes. Apoptosis and necrosis were induced by Selol in a discrete percentage of A549 cells. SPN also increased the production of reactive oxygen species, leading to oxidative cellular damage and to the overexpression of the GPX1, CYP1A1, BAX and BCL2 genes. CONCLUSIONS: This study presents a stable formulation of PVM/MA-shelled Selol nanocapsules and provides the first demonstration that Selol promotes G2/M arrest in cancerous cells.
Assuntos
Adenocarcinoma/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Maleatos/química , Nanocápsulas/química , Polietilenos/química , Compostos de Selênio/farmacologia , Adenocarcinoma/patologia , Adenocarcinoma/ultraestrutura , Adenocarcinoma de Pulmão , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/ultraestrutura , Ciclina B1/genética , Relação Dose-Resposta a Droga , Glutationa Peroxidase/genética , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/ultraestrutura , Nanoconchas/química , Espécies Reativas de Oxigênio/metabolismo , Compostos de Selênio/administração & dosagem , Compostos de Selênio/química , Termodinâmica , Fosfatases cdc25/genética , Glutationa Peroxidase GPX1RESUMO
The impact of metal oxide nanoparticles (NPs) on the immune system has been studied in vitro using human peripheral blood lymphocytes (PBLs). Metal oxide NPs (ZnO, CeO2, TiO2 and Al2O3) induced changes in the expression levels of adhesion molecules and the C-X-C chemokine receptor type 4 (CXCR4) in these cells. Proliferation studies were carried out with CFSE in response to PHA, finding an increase in T-cell proliferation upon cell exposure to TiO2 and Al2O3 NPs. For ZnO NPs, a decrease in the chemotactic response to SDF-1α was observed. No changes were found in basophil activation and leukocyte oxidative burst after phagocytosis. Despite the absence of cytotoxicity, metal oxide NPs are not inert; they alter the expression levels of adhesion molecules and chemokine receptors, key actors in the immune response, and affect important cell functions such as T-cell proliferative response to mitogens and chemotaxis. FROM THE CLINICAL EDITOR: This study demonstrates the immune-modulating effects of four different metal nanoparticles in a human peripheral blood lymphocyte model system. These effects were clearly present even though these nanoparticles did not display cytotocity in ex vivo experiments.
Assuntos
Linfócitos/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Óxidos/toxicidade , Receptores CXCR4/imunologia , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Humanos , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/metabolismo , Explosão Respiratória/efeitos dos fármacosRESUMO
A series of experiments was undertaken on three different marine microalgae to compare the effect of two metal oxide nanoparticles (NPs) on different physiological responses to stress: zinc oxide (ZnO), a known toxic compound for microalgae, and the never before tested yttrium oxide (Y2O3). The effect of these potential pollutants was estimated for different physiological variables and temporal scales: Growth, carbon content, carbon-to-nitrogen (C:N) ratio, and chlorophyll fluorescence were evaluated in long-term assays, and reactive oxygen species (ROS) production was evaluated in a short-term assay. Population growth was the most susceptible variable to the acute toxic effects of both NPs as measured in terms of number of cells and of biomass. Although Phaeodactylum tricornutum and Alexandrium minutum were negatively affected by ZnO NPs, this effect was not detected in Tetraselmis suecica, in which cell growth was significantly decreased by Y2O3 NPs. Biomass per cell was negatively affected in the most toxic treatments in T. suecica but was positively affected in A. minutum. ZnO treatments induced a sharper decrease in chlorophyll fluorescence and higher ROS than did Y2O3 treatments. The pronounced differences observed in the responses between the species and the physiological variables tested highlight the importance of analyzing diverse groups of microalgae and various physiological levels to determine the potential effects of environmental pollutants.
Assuntos
Nanopartículas Metálicas/toxicidade , Fitoplâncton/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Clorófitas/efeitos dos fármacos , Diatomáceas/efeitos dos fármacos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Óxido de Zinco/toxicidadeRESUMO
Purpose: The growing incidence and lethality of pancreatic cancer urges the development of new therapeutic approaches. Anti-tumoral vaccines can potentiate the immune response against the tumor, targeting specific antigens expressed only on tumor cells. In this work, we designed new vaccines for pancreatic cancer, composed by chitosan nanocapsules (CS NCs) containing imiquimod (IMQ) as adjuvant, and targeting the K-Ras mutation G12V. Experimental design: We tested the immunogenicity of our vaccines in mice, carrying different combinations of K-Ras mutated peptides. Then, we analyzed their prophylactic and therapeutic efficacy in mice bearing heterotopic pancreatic cancer. Results: Unexpectedly, although good results were observed at short time points, the different combinations of our CS NCs vaccines seemed to potentiate tumor growth and reduce survival rate. We propose that this effect could be due to an inadequate immune response, partially because of the induction of a regulatory tolerogenic response. Conclusion: Our results call for caution in the use of some NCs containing IMQ in the immunotherapy against pancreatic cancer.
Assuntos
Vacinas Anticâncer , Nanocápsulas , Neoplasias Pancreáticas , Animais , Camundongos , Imiquimode/farmacologia , Imunização , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Peptídeos , Neoplasias PancreáticasRESUMO
Heparin is the most common anticoagulant used in clinical practice but shows some downsides such as short half-life (for the high molecular weight heparin) and secondary effects. On the other hand, its low molecular weight analogue cannot be neutralized with protamine, and therefore cannot be used in some treatments. To address these issues, we conjugated polyethylene glycol (PEG) to heparin reducing end (end-on) via oxime ligation and studied the interactions of the conjugate (Hep-b-PEG) with antithrombin III (AT) and protamine. Isothermal titration calorimetry showed that Hep-b-PEG maintains the affinity to AT. Dynamic light scattering demonstrated that the Hep-b-PEG formed colloidal stable nanocomplexes with protamine instead of large multi-molecular aggregates, associated with heparin side effects. The in vitro (human plasma) and in vivo experiments (Sprague Dawley rats) evidenced an extended half-life and higher anticoagulant activity of the conjugate when compared to unmodified heparin.
Assuntos
Heparina , Protaminas , Animais , Ratos , Humanos , Heparina/efeitos adversos , Protaminas/química , Ratos Sprague-Dawley , Anticoagulantes/farmacologia , Anticoagulantes/químicaRESUMO
Bordetella spp. are respiratory pathogens equipped with immune evasion mechanisms. We previously characterized a Bordetella bronchiseptica mutant (RB50ΔbtrS) that fails to suppress host responses, leading to rapid clearance and long-lasting immunity against reinfection. This work revealed eosinophils as an exclusive requirement for RB50ΔbtrS clearance. We also show that RB50ΔbtrS promotes eosinophil-mediated B/T cell recruitment and inducible bronchus-associated lymphoid tissue (iBALT) formation, with eosinophils being present throughout iBALT for Th17 and immunoglobulin A (IgA) responses. Finally, we provide evidence that XCL1 is critical for iBALT formation but not maintenance, proposing a novel role for eosinophils as facilitators of adaptive immunity against B. bronchiseptica. RB50ΔbtrS being incapable of suppressing eosinophil effector functions illuminates active, bacterial targeting of eosinophils to achieve successful persistence and reinfection. Overall, our discoveries contribute to understanding cellular mechanisms for use in future vaccines and therapies against Bordetella spp. and extension to other mucosal pathogens.
Assuntos
Infecções por Bordetella , Bordetella bronchiseptica , Bordetella , Humanos , Eosinófilos , Infecções por Bordetella/microbiologia , Infecções por Bordetella/prevenção & controle , ReinfecçãoRESUMO
BACKGROUND: Patients with end-stage renal disease (ESRD) and Mycobacterium tuberculosis infection pose a high risk of developing active TB disease. It is therefore important to detect latent TB infection (LTBI) to be able to offer treatment and prevent progression to TB disease. We assessed the value of the tuberculin skin test (TST) and of an interferon-gamma release assay (Quantiferon®-TB Gold in-Tube, QFT) for diagnosing LTBI in ESRD patients, after prolonged exposure to a highly contagious TB case in a haemodialysis unit. As a high number of patients presented erythema without induration in the TST response, this type of reaction was also analysed. METHOD: The TST and QFT were simultaneously performed twelve weeks after the last possible exposure to a bacilliferous TB patient. If the first TST (TST-1) was negative, a second TST (TST-2) was performed 15 days later to detect a booster response. A comparison was made between the TST responses (including those cases with erythema without induration) and those for the QFT. The correlation with risk of infection and the concordance between tests were both analysed. RESULTS: A total of 52 patients fulfilled the inclusion criteria. Overall, 11 patients (21.2%) had a positive TST response: 3 for TST-1 and 8 for TST-2, and 18 patients (34.6%) showed a positive QFT response (p = 0.065). Erythema without induration was found in 3 patients at TST-1 and in a further 9 patients at TST-2. The three patients with erythema without induration in TST-1 had a positive TST-2 response. Concordance between TST and QFT was weak for TST-1 (κ = 0.21); it was moderate for overall TST (κ = 0.49); and it was strong if both induration and erythema (κ = 0.67) were considered. CONCLUSIONS: In patients with ESRD, erythema without induration in the TST response could potentially be an indicator of M. tuberculosis infection. The QFT shows better accuracy for LTBI diagnosis than the TST.
Assuntos
Técnicas de Laboratório Clínico/métodos , Testes de Liberação de Interferon-gama/métodos , Falência Renal Crônica/complicações , Falência Renal Crônica/terapia , Mycobacterium tuberculosis/imunologia , Teste Tuberculínico/métodos , Tuberculose/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Diálise RenalRESUMO
While Tuberculosis (TB) infection remains a serious challenge worldwide, big data and "omic" approaches have greatly contributed to the understanding of the disease. Transcriptomics have been used to tackle a wide variety of queries including diagnosis, treatment evolution, latency and reactivation, novel target discovery, vaccine response or biomarkers of protection. Although a powerful tool, the elevated cost and difficulties in data interpretation may hinder transcriptomics complete potential. Technology evolution and collaborative efforts among multidisciplinary groups might be key in its exploitation. Here, we discuss the main fields explored in TB using transcriptomics, and identify the challenges that need to be addressed for a real implementation in TB diagnosis, prevention and therapy.
RESUMO
Targeted delivery of dexamethasone to inflamed tissues using nanoparticles is much-needed to improve its efficacy while reducing side effects. To drastically improve dexamethasone loading and prevent burst release once injected intravenously, a lipophilic prodrug dexamethasone palmitate (DXP) was encapsulated into poly(DL-lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) nanoparticles (NPs). DXP-loaded PLGA-PEG NPs (DXP-NPs) of about 150 nm with a drug loading as high as 7.5% exhibited low hemolytic profile and cytotoxicity. DXP-NPs were able to inhibit the LPS-induced release of inflammatory cytokines in macrophages. After an intravenous injection to mice, dexamethasone (DXM) pharmacokinetic profile was also significantly improved. The concentration of DXM in the plasma of healthy mice remained high up to 18 h, much longer than the commercial soluble drug dexamethasone phosphate (DSP). Biodistribution studies showed lower DXM concentrations in the liver, kidneys, and lungs when DXP-NPs were administered as compared with the soluble drug. Histology analysis revealed an improvement in the knee structure and reduction of cell infiltration in animals treated with the encapsulated DXP compared with the soluble DSP or non-treated animals. In summary, the encapsulation of a lipidic prodrug of dexamethasone into PLGA-PEG NPs appears as a promising strategy to improve the pharmacological profile and reduce joint inflammation in a murine model of rheumatoid arthritis.
Assuntos
Artrite , Nanopartículas , Pró-Fármacos , Animais , Dexametasona , Portadores de Fármacos/química , Camundongos , Nanopartículas/química , Tamanho da Partícula , Polietilenoglicóis/química , Distribuição TecidualRESUMO
Epigenetic modifications could drive some of the molecular events implicated in proliferation, drug resistance and metastasis of pancreatic ductal adenocarcinoma (PDAC). Thus, epigenetic enzyme inhibitors could be the key to revert those events and transform PDAC into a drug-sensitive tumor. We performed a systematic study with five different epigenetic enzyme inhibitors (1, UVI5008, MS275, psammaplin A, and BIX01294) targeting either Histone Deacetylase (HDAC) 1 or 1/4, DNA methyltransferase 3a (DNMT3a), Euchromatic histone lysine methyltransferase 2 (EHMT2), or Sirtuin 1 (SIRT1), as well as one drug that restores the p53 function (P53R3), in three different human PDAC cell lines (SKPC-1, MIA PaCa-2, and BxPC-3) using 2D and 3D cell cultures. The synergistic effect of these antitumoral drugs with gemcitabine was tested and the most efficient combinations were characterized by RNA-seq. The inhibition of HDAC1/4 (MS275), HDAC1/4/SIRT1/DNMT3a (UVI5008) or EHMT2 (BIX01294) induced a significant reduction on the cell viability, even in gemcitabine-resistance cells. The combination of UVI5008 or MS275 with gemcitabine induced a synergistic effect at low concentration and the RNA-Seq analysis revealed some synergy candidate genes as potential biomarkers. Reverting aberrant epigenetic modifications in combination with gemcitabine offers an alternative treatment for PDAC patients, with an important reduction of the therapeutic dose.
RESUMO
Pancreatic cancer evades most of the current therapies and there is an urgent need for new treatments that could efficiently eliminate this aggressive tumor, such as the blocking of routes driving cell proliferation. In this work, we propose the use of small interfering RNA (siRNA) to inhibit the combined expression of FOSL-1 and YAP, two signaling proteins related with tumor cell proliferation and survival. To improve the efficacy of cell transfection, DODAB:MO (1:2) liposomes were used as siRNA nanocarriers, forming a complex denominated siRNA-lipoplexes. Liposomes and lipoplexes (carrying two siRNA for each targeted protein, or the combination of four siRNAs) were physico-chemically and biologically characterized. They showed very good biocompatibility and stability. The efficient targeting of FOSL-1 and YAP expression at both mRNA and protein levels was first proved in vitro using mouse pancreatic tumoral cell lines (KRASG12V and p53 knockout), followed by in vivo studies using subcutaneous allografts on mice. The peri-tumoral injection of lipoplexes lead to a significant decrease in the tumor growth in both Athymic Nude-Foxn1nu and C57BL/6 mice, mainly in those receiving the combination of four siRNAs, targeting both YAP and FOSL-1. These results open a new perspective to overcome the fast tumor progression in pancreatic cancer.
RESUMO
OBJECTIVE: Semaphorin 3B (Sema3B) decreases the migratory and invasive capacities of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) and suppresses expression of matrix metalloproteinases. We undertook this study to examine the role of Sema3B in a mouse model of arthritis and its expression in RA patients. METHODS: Clinical responses, histologic features, and FLS function were examined in wild-type (WT) and Sema3B-/- mice in a K/BxN serum transfer model of arthritis. Protein and messenger RNA expression of Sema3B in mouse joints and murine FLS, as well as in serum and synovial tissue from patients with arthralgia and patients with RA, was determined using enzyme-linked immunosorbent assay, immunoblotting, quantitative polymerase chain reaction, and RNA sequencing. FLS migration was determined using a wound closure assay. RESULTS: The clinical severity of serum-induced arthritis was significantly higher in Sema3B-/- mice compared to WT mice. This was associated with increased expression of inflammatory mediators and increased migratory capacity of murine FLS. Administration of recombinant mouse Sema3B reduced the clinical severity of serum-induced arthritis and the expression of inflammatory mediators. Sema3B expression was significantly lower in the synovial tissue and serum of patients with established RA compared to patients with arthralgia. Serum Sema3B levels were elevated in patients with arthralgia that later progressed to RA, but not in those who did not develop RA; however, these levels drastically decreased 1 and 2 years after RA development. CONCLUSION: Sema3B expression plays a protective role in a mouse model of arthritis. In RA patients, expression levels of Sema3B in the serum depend on the disease stage, suggesting different regulatory roles in disease onset and progression.