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1.
Rev Alerg Mex ; 70(4): 206, 2023 Sep.
Artigo em Espanhol | MEDLINE | ID: mdl-37933947

RESUMO

Background: Upper respiratory tract infections (URIs) are very common in the pediatric population. Most of these infections are mild, but due to their chronicity they affect quality of life (QoL), in addition to high costs for medical care. The use of bacterial extracts (BE) that stimulate general immunity can reduce its frequency and improve the QoL of the patient. Objective: Evaluate the effectiveness of a BE in the prevention of ARVI in children from 1 to 6 years of age. Methods: Children between the ages of 1 and 6 years, with a diagnosis of RAVI, were randomized into 3 different groups, with medical follow-up at 6 and 12 weeks after the start. The EB was administered with different doses to each group. An ANOVA test with a Tukey post hoc is used for multiple comparisons (maximum type I error of 0.05). Results: 33 children (12 girls) with a mean age of 3.11 years were included. The average frequency of RAVI prior to treatment was 2.2 events/month and 0.9 and 0.4 events/month at 6 and 12 weeks, respectively. The IVARS were reduced by 76.9% at 3 months of treatment. (Graph). No adverse effects were reported. Conclusions: BE is safe and effective in reducing the frequency of RAVI in children, in agreement with the literature. There is not enough published scientific evidence, but the BE seems to have an application in the prevention and treatment of RAVI. Sublingual administration is comfortable in this age group.


Antecedentes: Las infecciones de vías aéreas superiores (IVASR) son muy frecuentes en la población pediátrica. La mayoría de estas infecciones son leves, pero por la cronicidad afectan la calidad de vida (CdV), además de elevados costos por la atención médica. El uso de extractos bacterianos (EB) que estimulen la inmunidad general pueden reducir su frecuencia y mejorar la CdV del paciente. Objetivo: Evaluar la efectividad de un EB en la prevención de IVASR en niños de 1 a 6 años. Métodos: Se aleatorizaron niños entre 1 y 6 años, con diagnóstico IVASR en 3 grupos distintos, seguimiento médico a las 6 y 12 semanas tras el inicio. El EB se administró con dosis distintas a cada grupo. Se utiliza una prueba de ANOVA con un post hoc Tukey para comparaciones múltiples (error tipo I máximo de 0.05). Resultados: Se incluyeron 33 niños (12 niñas) con una media de edad de 3.11 años. La frecuencia de IVASR previo al tratamiento en promedio fue de 2.2 eventos/mes y de 0.9 y de 0.4 eventos/mes a las 6 y 12 semanas respectivamente. La IVARS se redujeron un 76.9% a los 3 meses de tratamiento. (Gráfica). No se reportaron efectos adversos. Conclusiones: El EB es seguro y efectivo en disminuir la frecuencia de IVASR en niños en concordancia con la literatura. No hay suficiente evidencia científica publicada pero el EB parece tener aplicación en la prevención y tratamiento de las IVASR. La administración sublingual es cómoda en este grupo etario.


Assuntos
Metenamina , Qualidade de Vida , Feminino , Humanos , Criança , Recém-Nascido , Lactente , Pré-Escolar , Administração Sublingual , Azul de Metileno , Estudos Retrospectivos
2.
J Leukoc Biol ; 113(6): 588-603, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-36987875

RESUMO

Tuberculosis remains one of the leading public health problems in the world. The mechanisms that lead to the activation of the immune response against Mycobacterium tuberculosis have been extensively studied, with a focus on the role of cytokines as the main signals for immune cell communication. However, less is known about the role of other signals, such as extracellular vesicles, in the communication between immune cells, particularly during the activation of the adaptive immune response. In this study, we determined that extracellular vesicles released by human neutrophils infected with M. tuberculosis contained several host proteins that are ectosome markers. In addition, we demonstrated that extracellular vesicles released by human neutrophils infected with M. tuberculosis released after only 30 min of infection carried mycobacterial antigens and pathogen-associated molecular patterns, and we identified 15 mycobacterial proteins that were consistently found in high concentrations in extracellular vesicles released by human neutrophils infected with M. tuberculosis; these proteins contain epitopes for CD4 T-cell activation. We found that extracellular vesicles released by human neutrophils infected with M. tuberculosis increased the expression of the costimulatory molecule CD80 and of the coinhibitory molecule PD-L1 on immature monocyte-derived dendritic cells. We also found that immature and mature dendritic cells treated with extracellular vesicles released by human neutrophils infected with M. tuberculosis were able to induce IFN-γ production by autologous M. tuberculosis antigen-specific CD4 T cells, indicating that these extracellular vesicles acted as antigen carriers and transferred mycobacterial proteins to the antigen-presenting cells. Our results provide evidence that extracellular vesicles released by human neutrophils infected with M. tuberculosis participate in the activation of the adaptive immune response against M. tuberculosis.


Assuntos
Vesículas Extracelulares , Mycobacterium tuberculosis , Tuberculose , Humanos , Células Th1 , Neutrófilos , Monócitos , Células Dendríticas
3.
Microbiol Immunol ; 66(10): 477-490, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35856253

RESUMO

Most individuals infected with Mycobacterium tuberculosis (Mtb) have latent tuberculosis (TB), which can be diagnosed with tests (such as the QuantiFERON-TB Gold test [QFT]) that detect the production of IFN-γ by memory T cells in response to the Mtb-specific antigens 6 kDa early secretory antigenic target EsxA (Rv3875) (ESAT-6), 10 kDa culture filtrate antigen EsxB (Rv3874) (CFP-10), and Mtb antigen of 7.7 kDa (Rv2654c) (TB7.7). However, the immunological mechanisms that determine if an individual will develop latent or active TB remain incompletely understood. Here we compared the response of innate and adaptive peripheral blood lymphocytes from healthy individuals without Mtb infection (QFT negative) and from individuals with latent (QFT positive) or active TB infection, to determine the characteristics of these cells that correlate with each condition. In active TB patients, the levels of IFN-γ that were produced in response to Mtb-specific antigens had high positive correlations with IL-1ß, TNF-α, MCP-1, IL-6, IL-12p70, and IL-23, while the proinflammatory cytokines had high positive correlations between themselves and with IL-12p70 and IL-23. These correlations were not observed in QFT-negative or QFT-positive healthy volunteers. Activation with Mtb-soluble extract (a mixture of Mtb antigens and pathogen-associated molecular patterns [PAMPs]) increased the percentage of IFN-γ-/IL-17-producing NK cells and of IL-17-producing innate lymphoid cell 3 (ILC3) in the peripheral blood of active TB patients, but not of QFT-negative or QFT-positive healthy volunteers. Thus, active TB patients have both adaptive and innate lymphocyte subsets that produce characteristic cytokine profiles in response to Mtb-specific antigens or PAMPs. These profiles are not observed in uninfected individuals or in individuals with latent TB, suggesting that they are a response to active TB infection.


Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose , Antígenos de Bactérias , Citocinas , Humanos , Imunidade Inata , Interleucina-17 , Interleucina-23 , Interleucina-6 , Linfócitos , Moléculas com Motivos Associados a Patógenos , Fator de Necrose Tumoral alfa
4.
Open Biol ; 11(8): 200415, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34343464

RESUMO

Protein S-acylation or palmitoylation is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of proteins through a thioester bond. Palmitoylation and palmitoyltransferases (PATs) have been linked to several types of cancers, diseases of the central nervous system and many infectious diseases where pathogens use the host cell machinery to palmitoylate their effectors. Despite the central importance of palmitoylation in cell physiology and disease, progress in the field has been hampered by the lack of potent-specific inhibitors of palmitoylation in general, and of individual PATs in particular. Herein, we present a yeast-based method for the high-throughput identification of small molecules that inhibit protein palmitoylation. The system is based on a reporter gene that responds to the acylation status of a palmitoylation substrate fused to a transcription factor. The method can be applied to heterologous PATs such as human DHHC20, mouse DHHC21 and also a PAT from the parasite Giardia lamblia. As a proof-of-principle, we screened for molecules that inhibit the palmitoylation of Yck2, a substrate of the yeast PAT Akr1. We tested 3200 compounds and were able to identify a candidate molecule, supporting the validity of our method.


Assuntos
Aciltransferases/antagonistas & inibidores , Lipoilação , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Giardia lamblia/efeitos dos fármacos , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Especificidade por Substrato
6.
PLoS One ; 16(4): e0245703, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33819265

RESUMO

The queen conch fishery in Jamaica is sustained by Pedro Bank, which is the main harvesting site located approximately 80 km south-west from Kingston. Due to its relative size, Pedro Bank has been subdivided into zones for management purposes by the Fisheries Division and the Veterinary Services Division. Understanding whether these sub-divisions reflect different sub-populations is critical for managing exploitation levels because fisheries management must demonstrate that harvesting does not endanger the future viability of the population as queen conch are on Appendix II of the Convention in Trade in Endangered Species of Wild Fauna and Flora (CITES). This determination is essential for the continued export to international markets such as the European Union. Two hundred and eight samples were collected across the entire Pedro Bank and were genetically characterized using nine polymorphic microsatellite loci. Population structure analysis for Lobatus gigas from Pedro Bank yielded low but significant values (FST = 0.009: p = 0.006) and suggested a high magnitude of gene flow indicative of a fit and viable population throughout the bank. Analysis of molecular variance (AMOVA) indicated a 100% variation within individual samples with little variation (0.9%) between populations. In contrast pairwise genetic comparisons identified significant differences between populations located to the south eastern and eastern region of the bank to those in the central and western locations. Bayesian clustering analysis also indicated the likelihood of two population sub-divisions (K = 2) on Pedro Bank. The results provided evidence of a weak but significant population structure which has crucial implications for the fishing industry as it suggests the use of ecosystem based management (EBM) in setting quotas to promote sustainable harvesting of L. gigas within each monitoring zone on Pedro Bank.


Assuntos
Gastrópodes/genética , Animais , Espécies em Perigo de Extinção , Pesqueiros , Fluxo Gênico , Variação Genética , Jamaica , Repetições de Microssatélites , Polimorfismo Genético
7.
Sci Rep ; 10(1): 17802, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33082490

RESUMO

Valproic acid (VPA) is a drug commonly used for epileptic seizure control. Recently, it has been shown that VPA alters the activation of several immune cells, including Natural Killer (NK) cells, which play an important role in the containment of viruses and intracellular bacteria. Although VPA can increase susceptibility to extracellular pathogens, it is unknown whether the suppressor effect of VPA could affect the course of intracellular bacterial infection. This study aimed to evaluate the role of VPA during Listeria monocytogenes (L.m) infection, and whether NK cell activation was affected. We found that VPA significantly augmented mortality in L.m infected mice. This effect was associated with increased bacterial load in the spleen, liver, and blood. Concurrently, decreased levels of IFN-γ in serum and lower splenic indexes were observed. Moreover, in vitro analysis showed that VPA treatment decreased the frequency of IFN-γ-producing NK cells within L.m infected splenocytes. Similarly, VPA inhibited the production of IFN-γ by NK cells stimulated with IL-12 and IL-18, which is a crucial system for early IFN-γ production in listeriosis. Finally, VPA decreased the phosphorylation of STAT4, p65, and p38, without affecting the expression of IL-12 and IL-18 receptors. Altogether, our results indicate that VPA increases the susceptibility to Listeria monocytogenes infection and suggest that NK cell is one of the main targets of VPA, but further work is needed to ascertain this effect.


Assuntos
Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Ácido Valproico/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Humanos , Imunomodulação , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Fator de Transcrição STAT4/metabolismo , Transdução de Sinais , Ácido Valproico/imunologia
8.
J Leukoc Biol ; 108(3): 859-866, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32480423

RESUMO

Mast cell activation through the high-affinity IgE receptor (FcεRI) plays a central role in allergic reactions. FcεRI-mediated activation triggers multiple signaling pathways leading to degranulation and synthesis of different inflammatory mediators. IgE-mediated mast cell activation can be modulated by different molecules, including several drugs. Herein, we investigated the immunomodulatory activity of the histone deacetylase inhibitor valproic acid (VPA) on IgE-mediated mast cell activation. To this end, bone marrow-derived mast cells (BMMC) were sensitized with IgE and treated with VPA followed by FcεRI cross-linking. The results indicated that VPA reduced mast cell IgE-dependent degranulation and cytokine release. VPA also induced a significant reduction in the cell surface expression of FcεRI and CD117, but not other mast cell surface molecules. Interestingly, VPA treatment inhibited the phosphorylation of PLCγ2, a key signaling molecule involved in IgE-mediated degranulation and cytokine secretion. However, VPA did not affect the phosphorylation of other key components of the FcεRI signaling pathway, such as Syk, Akt, ERK1/2, or p38. Altogether, our data demonstrate that VPA affects PLCγ2 phosphorylation, which in turn decreases IgE-mediated mast cell activation. These results suggest that VPA might be a key modulator of allergic reactions and might be a promising therapeutic candidate.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Imunoglobulina E/imunologia , Mastócitos/efeitos dos fármacos , Fosfolipase C gama/antagonistas & inibidores , Receptores de IgE/efeitos dos fármacos , Ácido Valproico/farmacologia , Animais , Degranulação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Interleucina-13/metabolismo , Interleucina-6/metabolismo , Mastócitos/citologia , Camundongos , Fosfolipase C gama/fisiologia , Receptores de IgE/biossíntese , Receptores de IgE/genética , Fator de Necrose Tumoral alfa/metabolismo
9.
Mutat Res ; 821: 111693, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32172132

RESUMO

Polo-Like Kinases (PLKs) are central players of mitotic progression in Eukaryotes. Given the intimate relationship between cell cycle progression and cancer development, PLKs in general and PLK1 in particular have been thoroughly studied as biomarkers and potential therapeutic targets in oncology. The oncogenic properties of PLK1 overexpression across different types of human cancers are attributed to its roles in promoting mitotic entry, centrosome maturation, spindle assembly and cytokinesis. While several academic labs and pharmaceutical companies were able to develop potent and selective inhibitors of PLK1 (PLK1i) for preclinical research, such compounds have reached only limited success in clinical trials despite their great pharmacokinetics. Even though this could be attributed to multiple causes, the housekeeping roles of PLK1 in both normal and cancer cells are most likely the main reason for clinical trials failure and withdraw due to toxicities issues. Therefore, great efforts are being invested to position PLK1i in the treatment of specific types of cancers with revised dosages schemes. In this mini review we focus on two potential niches for PLK1i that are supported by recent evidence: triple negative breast cancers (TNBCs) and BRCA1-deficient cancers. On the one hand, we recollect several lines of strong evidence indicating that TNBCs are among the cancers with highest PLK1 expression and sensitivity to PLK1i. These findings are encouraging because of the limited therapeutics options available for TNBC patients, which rely mainly on classic chemotherapy. On the other hand, we discuss recent evidence that unveils synthetic lethality induction by PLK1 inhibition in BRCA1-deficient cancers cells. This previously unforeseen therapeutic link between PLK1 and BRCA1 is promising because it defines novel therapeutic opportunities for PLK1i not only for breast cancer (i.e. TNBCs with BRCA1 deficiencies), but also for other types of cancers with BRCA1-deficiencies, such as pancreatic and prostate cancers.


Assuntos
Antineoplásicos/uso terapêutico , Proteína BRCA1/deficiência , Proteínas de Ciclo Celular/antagonistas & inibidores , Terapia de Alvo Molecular , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Humanos , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Quinase 1 Polo-Like
10.
Clin Infect Dis ; 71(8): e262-e269, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31732729

RESUMO

BACKGROUND: Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL). METHODS: We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos. RESULTS: The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%-2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%-2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative. CONCLUSIONS: The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.


Assuntos
Mycobacterium leprae , Mycobacterium , Animais , Humanos , México , Camundongos , Mycobacterium leprae/genética , Patologia Molecular
11.
Cells ; 10(1)2020 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-33396205

RESUMO

Studying tissue-independent components of cancer and defining pan-cancer subtypes could be addressed using tissue-specific molecular signatures if classification errors are controlled. Since PAM50 is a well-known, United States Food and Drug Administration (FDA)-approved and commercially available breast cancer signature, we applied it with uncertainty assessment to classify tumor samples from over 33 cancer types, discarded unassigned samples, and studied the emerging tumor-agnostic molecular patterns. The percentage of unassigned samples ranged between 55.5% and 86.9% in non-breast tissues, and gene set analysis suggested that the remaining samples could be grouped into two classes (named C1 and C2) regardless of the tissue. The C2 class was more dedifferentiated, more proliferative, with higher centrosome amplification, and potentially more TP53 and RB1 mutations. We identified 28 gene sets and 95 genes mainly associated with cell-cycle progression, cell-cycle checkpoints, and DNA damage that were consistently exacerbated in the C2 class. In some cancer types, the C1/C2 classification was associated with survival and drug sensitivity, and modulated the prognostic meaning of the immune infiltrate. Our results suggest that PAM50 could be repurposed for a pan-cancer context when paired with uncertainty assessment, resulting in two classes with molecular, biological, and clinical implications.


Assuntos
Biomarcadores Tumorais/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Dano ao DNA/genética , Células-Tronco Embrionárias/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/classificação , Neoplasias/metabolismo , Algoritmos , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Centrossomo/metabolismo , Estudos de Coortes , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Humanos , Concentração Inibidora 50 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Neoplasias/genética , Neoplasias/mortalidade , Prognóstico , Modelos de Riscos Proporcionais , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética
12.
Front Pharmacol ; 11: 593845, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33424604

RESUMO

Several plants from South America show strong antitumoral properties based on anti-proliferative and/or pro-apoptotic activities. In this work we aimed to identify selective cytotoxic compounds that target BRCA1-deficient cancer cells by Synthetic Lethality (SL) induction. Using a high-throughput screening technology developed in our laboratory, we analyzed a collection of extracts from 46 native plant species from Argentina using a wide dose-response scheme. A highly selective SL-induction capacity was found in an alkaloidal extract from Zanthoxylum coco (Fam. Rutaceae). Bio-guided fractionation coupled to HPLC led to the identification of active benzophenanthridine alkaloids. The most potent SL activity was found with the compound oxynitidine, which showed a remarkably low relative abundance in the active fractions. Further validation experiments were performed using the commercially available and closely related analog nitidine, which showed SL-induction activity against various BRCA1-deficient cell lines with different genetic backgrounds, even in the nanomolar range. Exploration of the underlying mechanism of action using BRCA1-KO cells revealed AKT and topoisomerases as the potential targets responsible of nitidine-triggered SL-induction. Taken together, our findings expose an unforeseen therapeutic activity of alkaloids from Zanthoxylum-spp. that position them as novel lead molecules for drug discovery.

13.
Front Oncol ; 9: 1323, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31828042

RESUMO

ZEB1 is a master regulator of the Epithelial-to-Mesenchymal Transition (EMT) program. While extensive evidence confirmed the importance of ZEB1 as an EMT transcription factor that promotes tumor invasiveness and metastasis, little is known about its regulation. In this work, we screened for potential regulatory links between ZEB1 and multiple cellular kinases. Exploratory in silico analysis aided by phospho-substrate antibodies and ZEB1 deletion mutants led us to identify several potential phospho-sites for the family of PKC kinases in the N-terminus of ZEB1. The analysis of breast cancer cell lines panels with different degrees of aggressiveness, together with the evaluation of a battery of kinase inhibitors, allowed us to expose a robust correlation between ZEB1 and PKCα both at mRNA and protein levels. Subsequent validation experiments using siRNAs against PKCα revealed that its knockdown leads to a concomitant decrease in ZEB1 levels, while ZEB1 knockdown had no impact on PKCα levels. Remarkably, PKCα-mediated downregulation of ZEB1 recapitulates the inhibition of mesenchymal phenotypes, including inhibition in cell migration and invasiveness. These findings were extended to an in vivo model, by demonstrating that the stable knockdown of PKCα using lentiviral shRNAs markedly impaired the metastatic potential of MDA-MB-231 breast cancer cells. Taken together, our findings unveil an unforeseen regulatory pathway comprising PKCα and ZEB1 that promotes the activation of the EMT in breast cancer cells.

14.
Int J Nanomedicine ; 14: 6707-6719, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31692512

RESUMO

BACKGROUND: Tuberculosis is the leading cause of death by an infectious microorganism worldwide. Conventional treatment lasts at least six months and has adverse effects; therefore, it is important to find therapeutic alternatives that reduce the bacterial load and may reduce the treatment duration. The immune response against tuberculosis can be modulated by several mechanisms, including extracellular vesicles (EVs), which are nano-sized membrane-bound structures that constitute an efficient communication mechanism among immune cells. METHODS: The EVs released by the J774A.1 mouse macrophage cell line, both spontaneously (S-EV) and after infection with Mycobacterium tuberculosis H37Rv (Mtb-EV), were purified by ultra-centrifugation and size-exclusion chromatography. The size distribution and chemical composition of these EVs were evaluated, and their effect on the bacterial load and the production of cytokines was determined in both in vitro and in vivo models of M. tuberculosis infection. RESULTS: Mtb-EV are larger than S-EV, they contain M. tuberculosis-specific antigens (not detected in EVs released from M. fortuitum-infected J774A.1 cells) and are rich in phosphatidylserine, present in their outer membrane layer. S-EV, but not Mtb-EV, reduced the bacterial load and the production of MCP-1 and TNF-α in M. tuberculosis-infected macrophages, and these effects were reversed when phosphatidylserine was blocked with annexin V. Both S-EV and Mtb-EV significantly reduced the lung bacterial load in mice infected with M. tuberculosis after 60 days of treatment, but they had no effect on survival or on the lung pneumonic area of these mice. CONCLUSION: J774A.1 macrophages infected with M. tuberculosis H37Rv released EVs that differed in size and phosphatidylserine content from spontaneously released EVs, and these EVs also had different biological effects: S-EV reduced the mycobacterial load and the cytokine production in vitro (through a phosphatidylserine-dependent mechanism), while both EVs reduced the lung bacterial load in vivo. These results are the basis for further experiments to evaluate whether EVs improve the efficiency of the conventional treatment for tuberculosis.


Assuntos
Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Tuberculose/terapia , Animais , Carga Bacteriana , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Vesículas Extracelulares/química , Vesículas Extracelulares/transplante , Masculino , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologia
15.
Front Microbiol ; 10: 2097, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31616387

RESUMO

Tuberculosis remains a serious threat worldwide. For this reason, it is necessary to identify agents that shorten the duration of treatment, strengthen the host immune system, and/or decrease the damage caused by the infection. Statins are drugs that reduce plasma cholesterol levels and have immunomodulatory, anti-inflammatory and antimicrobial effects. Although there is evidence that statins may contribute to the containment of Mycobacterium tuberculosis infection, their effects on peripheral blood mononuclear cells (PBMCs) involved in the immune response have not been previously described. Using PBMCs from 10 healthy subjects infected with M. tuberculosis H37Rv, we analyzed the effects of simvastatin on the treatment of the infections in an in vitro experimental model. Direct quantification of M. tuberculosis growth (in CFU/mL) was performed. Phenotypes and cell activation were assessed via multi-color flow cytometry. Culture supernatant cytokine levels were determined via cytokine bead arrays. The induction of apoptosis and autophagy was evaluated via flow cytometry and confocal microscopy. Simvastatin decreased the growth of M. tuberculosis in PBMCs, increased the proportion of NKT cells in culture, increased the expression of co-stimulatory molecules in monocytes, promoted the secretion of the cytokines IL-1ß and IL-12p70, and activated apoptosis and autophagy in monocytes, resulting in a significant reduction in bacterial load. We also observed an increase in IL-10 production. We did not observe any direct antimycobacterial activity. This study provides new insight into the mechanism through which simvastatin reduces the mycobacterial load in infected PBMCs. These results demonstrate that simvastatin activates several immune mechanisms that favor the containment of M. tuberculosis infection, providing relevant evidence to consider statins as candidates for host-directed therapy. They also suggest that future studies are needed to define the roles of statin-induced anti-inflammatory mechanisms in tuberculosis treatment.

16.
Molecules ; 24(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470504

RESUMO

Brucellosis, also known as "undulant fever" is a zoonotic disease caused by Brucella, which is a facultative intracellular bacterium. Despite efforts to eradicate this disease, infection in uncontrolled domestic animals persists in several countries and therefore transmission to humans is common. Brucella evasion of the innate immune system depends on its ability to evade the mechanisms of intracellular death in phagocytic cells. The BvrR-BvrS two-component system allows the bacterium to detect adverse conditions in the environment. The BvrS protein has been associated with genes of virulence factors, metabolism, and membrane transport. In this study, we predicted the DNA sequence recognized by BvrR with Gibbs Recursive Sampling and identified the three-dimensional structure of BvrR using I-TASSER suite, and the interaction mechanism between BvrR and DNA with Protein-DNA docking and molecular dynamics (MD) simulation. Based on the Gibbs recursive Sampling analysis, we found the motif AAHTGC (H represents A, C, and T nucleotides) as a possible sequence recognized by BvrR. The docking and EMD simulation results showed that C-terminal effector domain of BvrR protein is likely to interact with AAHTGC sequence. In conclusion, we predicted the structure, recognition motif, and interaction of BvrR with DNA.


Assuntos
Proteínas de Bactérias/química , Brucella/química , DNA/química , Fatores de Virulência/química , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Brucella/patogenicidade , DNA/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Motivos de Nucleotídeos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Homologia Estrutural de Proteína , Termodinâmica , Fatores de Virulência/metabolismo
17.
Molecules ; 24(19)2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547522

RESUMO

Ursolic and oleanolic acids are natural isomeric triterpenes known for their anticancer activity. Here, we investigated the effect of triterpenes on the viability of A549 human lung cancer cells and the role of autophagy in their activity. The induction of autophagy, the mitochondrial changes and signaling pathway stimulated by triterpenes were systematically explored by confocal microscopy and western blotting. Ursolic and oleanolic acids induce autophagy in A549 cells. Ursolic acid activates AKT/mTOR pathways and oleanolic acid triggers a pathway independent on AKT. Both acids promote many mitochondrial changes, suggesting that mitochondria are targets of autophagy in a process known as mitophagy. The PINK1/Parkin axis is a pathway usually associated with mitophagy, however, the mitophagy induced by ursolic or oleanolic acid is just dependent on PINK1. Moreover, both acids induce an ROS production. The blockage of autophagy with wortmannin is responsible for a decrease of mitochondrial membrane potential (Δψ) and cell death. The wortmannin treatment causes an over-increase of p62 and Nrf2 proteins promote a detoxifying effect to rescue cells from the death conducted by ROS. In conclusion, the mitophagy and p62 protein play an important function as a survival mechanism in A549 cells and could be target to therapeutic control.


Assuntos
Mitofagia/efeitos dos fármacos , Ácido Oleanólico/farmacologia , Triterpenos/farmacologia , Células A549 , Humanos , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ácido Ursólico
18.
J Immunol Res ; 2019: 9678098, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31001564

RESUMO

Valproic acid (VPA) is widely recognized for its use in the control of epilepsy and other neurological disorders in the past 50 years. Recent evidence has shown the potential of VPA in the control of certain cancers, owed in part to its role in modulating epigenetic changes through the inhibition of histone deacetylases, affecting the expression of genes involved in the cell cycle, differentiation, and apoptosis. The direct impact of VPA in cells of the immune system has only been explored recently. In this review, we discuss the effects of VPA in the suppression of some activation mechanisms in several immune cells that lead to an anti-inflammatory response. As expected, immune cells are not exempt from the effect of VPA, as it also affects the expression of genes of the cell cycle and apoptosis through epigenetic modifications. In addition to inhibiting histone deacetylases, VPA promotes RNA interference, activates histone methyltransferases, or represses the activation of transcription factors. However, during the infectious process, the effectiveness of VPA is subject to the biological nature of the pathogen and the associated immune response; this is because VPA can promote the control or the progression of the infection. Due to its various effects, VPA is a promising alternative for the control of autoimmune diseases and hypersensitivity and needs to be further explored.


Assuntos
Imunidade Adaptativa , Reposicionamento de Medicamentos , Imunidade Inata , Neoplasias/tratamento farmacológico , Ácido Valproico/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Epigênese Genética , Inibidores de Histona Desacetilases/administração & dosagem , Histona Desacetilases/metabolismo , Humanos , Camundongos , Interferência de RNA
19.
Clin Cancer Res ; 25(13): 4049-4062, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30890549

RESUMO

PURPOSE: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds. EXPERIMENTAL DESIGN: We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1- and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dual-tumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database. RESULTS: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells. CONCLUSIONS: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.


Assuntos
Proteína BRCA1/deficiência , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Mutações Sintéticas Letais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Aberrações Cromossômicas , Dano ao DNA , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-Like
20.
Tuberculosis (Edinb) ; 114: 123-126, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711151

RESUMO

Tuberculosis is one of the leading causes of mortality worldwide, it is caused by Mycobacterium tuberculosis (Mtb), a bacteria that employs several strategies to evade the host immune response. For instance, Mtb interferes with the overexpression of class II transactivator (CIITA) in macrophages exposed to IFN-γ by inhibiting histone acetylation at its promoter, which can be reverted by the histone deacetylase inhibitor (HDACi) sodium butyrate. In this work, we evaluated whether a different HDACi, valproic acid (VPA), could revert the inhibition of gene expression induced by Mtb. J774 macrophages treated with VPA and IFN-γ unexpectedly induced a higher expression of the inducible nitric oxide synthase and a higher production of nitric oxide when exposed to the 19 kDa lipoprotein of Mtb or the whole bacteria. However, VPA was unable to revert the inhibition of CIITA expression induced by the 19 kDa lipoprotein of Mtb. Finally, macrophages infected with Mtb and treated with VPA and IFN-γ showed a significant reduction in intracellular bacteria. Our findings suggest a new therapeutic potential of VPA for the treatment of tuberculosis.


Assuntos
Interferon gama/imunologia , Macrófagos/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Óxido Nítrico/biossíntese , Ácido Valproico/farmacologia , Animais , Antituberculosos/farmacologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética
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