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1.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198513

RESUMO

BACKGROUND: Pulmonary disease caused by Mycobacterium abscessus (M. abscessus) spreads around the world, and this disease is extremely difficult to treat due to intrinsic and acquired resistance of the pathogen to many approved antibiotics. M. abscessus is regarded as one of the most drug-resistant mycobacteria, with very limited therapeutic options. METHODS: Whole-cell growth inhibition assays was performed to screen and identify novel inhibitors. The IC50 of the target compounds were tested against THP-1 cells was determined to calculate the selectivity index, and then time-kill kinetics assay was performed against M. abscessus. Subsequently, the synergy of oritavancin with other antibiotics was evaluated by using checkerboard method. Finally, in vivo efficacy was determined in an immunosuppressive murine model simulating M. abscessus infection. RESULTS: We have identified oritavancin as a potential agent against M. abscessus. Oritavancin exhibited time-concentration dependent bactericidal activity against M. abscessus and it also displayed synergy with clarithromycin, tigecycline, cefoxitin, moxifloxacin, and meropenem in vitro. Additionally, oritavancin had bactericidal effect on intracellular M. abscessus. Oritavancin significantly reduced bacterial load in lung when it was used alone or in combination with cefoxitin and meropenem. CONCLUSIONS: Our in vitro and in vivo assay results indicated that oritavancin may be a viable treatment option against M. abscessus infection.


Assuntos
Antibacterianos/uso terapêutico , Lipoglicopeptídeos/uso terapêutico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/fisiologia , Animais , Antibacterianos/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Imunossupressão , Espaço Intracelular/microbiologia , Lipoglicopeptídeos/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Células THP-1
2.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199262

RESUMO

As the number of manned space flights increase, studies on the effects of microgravity on the human body are becoming more important. Due to the high expense and complexity of sending samples into space, simulated microgravity platforms have become a popular way to study these effects on earth. In addition, simulated microgravity has recently drawn the attention of regenerative medicine by increasing cell differentiation capability. These platforms come with many advantages as well as limitations. A main limitation for usage of these platforms is the lack of high-throughput capability due to the use of large cell culture vessels. Therefore, there is a requirement for microvessels for microgravity platforms that limit waste and increase throughput. In this work, a microvessel for commercial cell culture plates was designed. Four 3D printable (polycarbonate (PC), polylactic acid (PLA) and resin) and castable (polydimethylsiloxane (PDMS)) materials were assessed for biocompatibility with adherent and suspension cell types. PDMS was found to be the most suitable material for microvessel fabrication, long-term cell viability and proliferation. It also allows for efficient gas exchange, has no effect on cell culture media pH and does not induce hypoxic conditions. Overall, the designed microvessel can be used on simulated microgravity platforms as a method for long-term high-throughput biomedical studies.


Assuntos
Técnicas de Cultura de Células/métodos , Microvasos/fisiologia , Engenharia Tecidual/métodos , Simulação de Ausência de Peso , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Células Jurkat , Teste de Materiais , Microvasos/efeitos dos fármacos , Células THP-1
3.
Molecules ; 26(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207168

RESUMO

Xanthone derivatives have shown promising antitumor properties, and 1-carbaldehyde-3,4-dimethoxyxanthone (1) has recently emerged as a potent tumor cell growth inhibitor. In this study, its effect was evaluated (MTT viability assay) against a new panel of cancer cells, namely cervical cancer (HeLa), androgen-sensitive (LNCaP) and androgen-independent (PC-3) prostate cancer, and nonsolid tumor derived cancer (Jurkat) cell lines. The effect of xanthone 1 on macrophage functions was also evaluated. The effect of xanthone 1-conditioned THP-1 human macrophage supernatants on the metabolic viability of cervical and prostate cancer cell lines was determined along with its interference with cytokine expression characteristic of M1 profile (IL-1 ≤ ß; TNF-α) or M2 profile (IL-10; TGF-ß) (PCR and ELISA). Nitric oxide (NO) production by murine RAW264.7 macrophages was quantified by Griess reaction. Xanthone 1 (20 µM) strongly inhibited the metabolic activity of the cell lines and was significantly more active against prostate cell lines compared to HeLa (p < 0.05). Jurkat was the cell most sensitive to the effect of xanthone 1. Compound 1-conditioned IL-4-stimulated THP-1 macrophage supernatants significantly (p < 0.05) inhibited the metabolic activity of HeLa, LNCaP, and PC-3. Xanthone 1 did not significantly affect the expression of cytokines by THP-1 macrophages. The inhibiting effect of compound 1 observed on the production of NO by RAW 264.7 macrophages was moderate. In conclusion, 1-carbaldehyde-3,4-dimethoxyxanthone (1) decreases the metabolic activity of cancer cells and seems to be able to modulate macrophage functions.


Assuntos
Antineoplásicos/farmacologia , Macrófagos/efeitos dos fármacos , Próstata/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias do Colo do Útero/tratamento farmacológico , Xantonas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Células HeLa , Papillomavirus Humano 18/patogenicidade , Humanos , Células Jurkat , Macrófagos/metabolismo , Masculino , Camundongos , Óxido Nítrico/metabolismo , Células PC-3 , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Células RAW 264.7 , Células THP-1 , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia
4.
Nat Commun ; 12(1): 4136, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230486

RESUMO

Acute pancreatitis is a disease associated with suffering and high lethality. Although the disease mechanism is unclear, phospholipase A2 (PLA2) produced by pancreatic acinar cells is a known pathogenic trigger. Here, we show macrophage membrane-coated nanoparticles with a built-in 'lure and kill' mechanism (denoted 'MΦ-NP(L&K)') for the treatment of acute pancreatitis. MΦ-NP(L&K) are made with polymeric cores wrapped with natural macrophage membrane doped with melittin and MJ-33. The membrane incorporated melittin and MJ-33 function as a PLA2 attractant and a PLA2 inhibitor, respectively. These molecules, together with membrane lipids, work synergistically to lure and kill PLA2 enzymes. These nanoparticles can neutralize PLA2 activity in the sera of mice and human patients with acute pancreatitis in a dose-dependent manner and suppress PLA2-induced inflammatory response accordingly. In mouse models of both mild and severe acute pancreatitis, MΦ-NP(L&K) confer effective protection against disease-associated inflammation, tissue damage and lethality. Overall, this biomimetic nanotherapeutic strategy offers an anti-PLA2 treatment option that might be applicable to a wide range of PLA2-mediated inflammatory disorders.


Assuntos
Doença Aguda/terapia , Macrófagos , Nanopartículas/uso terapêutico , Pancreatite/terapia , Animais , Citocinas , Modelos Animais de Doenças , Feminino , Humanos , Inflamação , Meliteno , Camundongos , Fosfolipases A2/sangue , Células THP-1
5.
Molecules ; 26(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209843

RESUMO

In the search for new chemical scaffolds able to afford NLRP3 inflammasome inhibitors, we used a pharmacophore-hybridization strategy by combining the structure of the acrylic acid derivative INF39 with the 1-(piperidin-4-yl)1,3-dihydro-2H-benzo[d]imidazole-2-one substructure present in HS203873, a recently identified NLRP3 binder. A series of differently modulated benzo[d]imidazole-2-one derivatives were designed and synthesised. The obtained compounds were screened in vitro to test their ability to inhibit NLRP3-dependent pyroptosis and IL-1ß release in PMA-differentiated THP-1 cells stimulated with LPS/ATP. The selected compounds were evaluated for their ability to reduce the ATPase activity of human recombinant NLRP3 using a newly developed assay. From this screening, compounds 9, 13 and 18, able to concentration-dependently inhibit IL-1ß release in LPS/ATP-stimulated human macrophages, emerged as the most promising NLRP3 inhibitors of the series. Computational simulations were applied for building the first complete model of the NLRP3 inactive state and for identifying possible binding sites available to the tested compounds. The analyses led us to suggest a mechanism of protein-ligand binding that might explain the activity of the compounds.


Assuntos
Imidazóis , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células THP-1
6.
Sci Rep ; 11(1): 13300, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172780

RESUMO

Nucleotide-binding domain and leucine-rich repeat (LRR)-containing family protein 3 (NLRP3) regulated the maturation of inflammation-related cytokines by forming NLRP3 inflammasome, which plays pivotal roles in sepsis pathogenesis. In this study, we evaluated the genetic association of NLRP3 polymorphisms with sepsis (640 patients and 769 controls) and characterized the impact of NLRP3 polymorphisms on NLRP3 expression and inflammatory responses. No significant differences were observed in genotype/allelic frequencies of NLRP3 29940G>C between sepsis cases and controls. The G allele was significantly overrepresented in patients with septic shock than those in sepsis subgroup, and the GC/GG genetypes were related to the 28-day mortality of sepsis. Lipopolysaccharide challenge to peripheral blood mononuclear cells showed a significant suppression of NLRP3 mRNA expression and release of IL-1ß and TNF-α in CC compared with the GC/GG genotype category. Functional experiments with luciferase reporter vectors containing the NLRP3 3'-UTR with the 29940 G-to-C variation in HUVECs and THP-1 cells showed a potential suppressive effect of miR-146a on NLRP3 transcription in the presence of the C allele. Taken together, these results demonstrated that the 29940 G-to-C mutation within the NLRP3 3'-UTR was a gain-of-function alteration that caused the suppression of NLRP3 expression and downstream inflammatory cytokine production via binding with miR-146a, which ultimately protected patients against susceptibility to sepsis progression and poor clinical outcome.


Assuntos
MicroRNAs , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Sepse , China/epidemiologia , Citocinas/metabolismo , Progressão da Doença , Suscetibilidade a Doenças , Feminino , Mutação com Ganho de Função , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamassomos/metabolismo , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Polimorfismo Genético , Sepse/epidemiologia , Sepse/genética , Células THP-1
7.
Molecules ; 26(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072265

RESUMO

Though siRNA-based therapy has achieved great progress, efficient siRNA delivery remains a challenge. Here, we synthesized a copolymer PAsp(-N=C-PEG)-PCys-PAsp(DETA) consisting of a poly(aspartate) block grafted with comb-like PEG side chains via a pH-sensitive imine bond (PAsp(-N=C-PEG) block), a poly(l-cysteine) block with a thiol group (PCys block), and a cationic poly(aspartate) block grafted with diethylenetriamine (PAsp(DETA) block). The cationic polymers efficiently complexed siRNA into polyplexes, showing a sandwich-like structure with a PAsp(-N=C-PEG) out-layer, a crosslinked PCys interlayer, and a complexing core of siRNA and PAsp(DETA). Low pH-triggered breakage of pH-sensitive imine bonds caused PEG shedding. The disulfide bond-crosslinking and pH-triggered PEG shedding synergistically decreased the polyplexes' size from 75 nm to 26 nm. To neutralize excessive positive charges and introduce the targeting ligand, the polyplexes without a PEG layer were coated with an anionic copolymer modified with the targeting ligand lauric acid. The resulting polyplexes exhibited high transfection efficiency and lysosomal escape capacity. This study provides a promising strategy to engineer the size and surface of polyplexes, allowing long blood circulation and targeted delivery of siRNA.


Assuntos
Polímeros/química , RNA Interferente Pequeno/metabolismo , Ânions , Cátions , Sobrevivência Celular , Reagentes para Ligações Cruzadas/química , Citoplasma/metabolismo , Dissulfetos , Sistemas de Liberação de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Ácidos Láuricos/química , Ligantes , Espectroscopia de Ressonância Magnética , Oxigênio/química , Tamanho da Partícula , Polietilenoglicóis/química , Espectroscopia de Infravermelho com Transformada de Fourier , Células THP-1
8.
Toxicol Lett ; 349: 134-144, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34153406

RESUMO

Recent epidemiological studies reported cases of cholangiocarcinoma in workers exposed to 1,2-dichloropropane (1,2-DCP) in an offset proof printing factory in Japan. The present study investigated the effects of 1,2-DCP on the expression of histone family member X (H2AX) phosphorylated on Ser 139 (γ-H2AX), a marker of DNA double strand break, in human immortalized cholangiocytes MMNK-1 cells. Mono-cultures of MMNK-1 cells and co-cultures of MMNK-1 cells with THP-1 macrophages were exposed to 1,2-DCP at concentrations of 100 and 500 µM for 24 h. Expression of γ-H2AX was visualized by immunofluorescence staining. Exposure to 1,2-DCP had no effect on the expression of γ-H2AX in mono-cultured MMNK-1 cells, but significantly increased the number of nuclear foci stained by γ-H2AX in MMNK-1 cells co-cultured with THP-1 macrophages. Exposure to 1,2-DCP also significantly increased the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in co-cultured MMNK-1 cells. The results suggest that macrophages play a critical role by producing cytokines in 1,2-DCP-induced DNA double strand break in MMNK-1 cells.


Assuntos
Ductos Biliares/efeitos dos fármacos , Histonas/metabolismo , Macrófagos/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Propano/análogos & derivados , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Técnicas de Cocultura , Quebras de DNA de Cadeia Dupla , Humanos , Interleucina-6/metabolismo , Macrófagos/metabolismo , Propano/toxicidade , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
9.
Theranostics ; 11(15): 7379-7390, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34158856

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel strain of highly contagious coronaviruses that infects humans. Prolonged fever, particularly that above 39.5 °C, is associated with SARS-CoV-2 infection. However, little is known about the pathological effects of fever caused by SARS-CoV-2. Methods: Primary bovine alveolar macrophages (PBAMs), RAW264.7 mouse macrophages, and THP-1 human cells were transfected with plasmids carrying the genes encoding the SARS-CoV-2 spike (S) protein or receptor-binding domain (RBD). Proteins in the macrophages interacting with S-RBD at 39.5 °C or 37 °C were identified by immunoprecipitation-mass spectrometry. Glutathione S-transferase pulldown, surface plasmon resonance, and immunofluorescence were performed to evaluate the transient receptor potential vanilloid 2 (TRPV2) interaction with SARS-CoV-2-S-RBD at 39.5 °C. Using an RNA sequencing-based approach, cytokine gene expression induced by SARS-CoV-2 S transfection at 39.5 °C and 37.5 °C in primary alveolar macrophages was measured. Fluo-4 staining and enzyme-linked immunosorbent assays were used to assess the regulatory function of TRPV2 in intracellular Ca 2+ and cytokines under SARS-CoV-2-S-RBD at 39.5 °C. Additionally, cytokine release was examined after TRPV2 knockdown with shRNA oligonucleotides or inhibition using the SKF-96365 antagonist. Results: We identified an interaction between the primary alveolar macrophage receptor TRPV2 and S-RBD under febrile conditions. Febrile temperature promotes Ca2+ influx through SARS-CoV-2 infection in PBAMs, further activates the NF-κB p65 signaling pathway, and enhances the secretion of cytokines. Furthermore, knockdown or antagonist (with SKF-96365) of TRPV2 significantly decreased the release of cytokines that drive the inflammatory response. Conclusion: Collectively, our findings identified TRPV2 as a receptor of SARS-CoV-2 in conditions of febrile temperature, providing insight into critical interactions of SARS-CoV-2 with macrophages, as well as a useful resource and potential drug target for coronavirus disease 2019.


Assuntos
COVID-19/virologia , Febre/virologia , Macrófagos/metabolismo , Macrófagos/virologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Canais de Cátion TRPV/metabolismo , Internalização do Vírus , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Citocinas/metabolismo , Humanos , Imidazóis/farmacologia , Cinética , Macrófagos/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , Células RAW 264.7 , SARS-CoV-2/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células THP-1 , Temperatura , Internalização do Vírus/efeitos dos fármacos
10.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070369

RESUMO

Folate receptor beta (FRß) is a folate binding receptor expressed on myeloid lineage hematopoietic cells. FRß is commonly expressed at high levels on malignant blasts in patients with acute myeloid leukemia (AML), as well as on M2 polarized tumor-associated macrophages (TAMs) in the tumor microenvironment of many solid tumors. Therefore, FRß is a potential target for both direct and indirect cancer therapy. We demonstrate that FRß is expressed in both AML cell lines and patient-derived AML samples and that a high-affinity monoclonal antibody against FRß (m909) has the ability to cause dose- and expression-dependent ADCC against these cells in vitro. Importantly, we find that administration of m909 has a significant impact on tumor growth in a humanized mouse model of AML. Surprisingly, m909 functions in vivo with and without the infusion of human NK cells as mediators of ADCC, suggesting potential involvement of mouse macrophages as effector cells. We also found that TAMs from primary ovarian ascites samples expressed appreciable levels of FRß and that m909 has the ability to cause ADCC in these samples. These results indicate that the targeting of FRß using m909 has the potential to limit the outgrowth of AML in vitro and in vivo. Additionally, m909 causes cytotoxicity to TAMs in the tumor microenvironment of ovarian cancer warranting further investigation of m909 and its derivatives as therapeutic agents in patients with FRß-expressing cancers.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Receptor 2 de Folato , Imunoterapia , Leucemia Mieloide Aguda , Proteínas de Neoplasias , Neoplasias Ovarianas , Animais , Células CHO , Cricetulus , Feminino , Receptor 2 de Folato/antagonistas & inibidores , Receptor 2 de Folato/imunologia , Células HL-60 , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Células THP-1 , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070558

RESUMO

Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with cardiovascular and metabolic dysfunction. However, the mechanisms underlying these morbidities remain poorly delineated. Extracellular vesicles (EVs) mediate intercellular communications, play pivotal roles in a multitude of physiological and pathological processes, and could mediate IH-induced cellular effects. Here, the effects of IH on human primary cells and the release of EVs were examined. Microvascular endothelial cells (HMVEC-d), THP1 monocytes, THP1 macrophages M0, THP1 macrophages M1, THP1 macrophages M2, pre-adipocytes, and differentiated adipocytes (HAd) were exposed to either room air (RA) or IH for 24 h. Secreted EVs were isolated and characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. The effects of each of the cell-derived EVs on endothelial cell (EC) monolayer barrier integrity, on naïve THP1 macrophage polarity, and on adipocyte insulin sensitivity were also evaluated. IH did not alter EVs cell quantal release, but IH-EVs derived from HMVEC-d (p < 0.01), THP1 M0 (p < 0.01) and HAd (p < 0.05) significantly disrupted HMVEC-d monolayer integrity, particularly after H2O2 pre-conditioning. IH-EVs from HMVEC-d and THP1 M0 elicited M2-polarity changes did not alter insulin sensitivity responses. IH induces cell-selective changes in EVs cargo, which primarily seem to target the emergence of endothelial dysfunction. Thus, changes in EVs cargo from selected cell sources in vivo may play causal roles in some of the adverse outcomes associated with OSA.


Assuntos
Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Hipóxia/metabolismo , Apneia Obstrutiva do Sono/metabolismo , Células Endoteliais/patologia , Vesículas Extracelulares/patologia , Humanos , Hipóxia/patologia , Apneia Obstrutiva do Sono/patologia , Células THP-1
13.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064458

RESUMO

Vitamin D and beta-glucans are both immunostimulants. Vitamin D exerts its beneficial effects on many components of the immune system. In macrophages, the hormone modulates both phagocytic activity and cytokine production; therefore, it plays an important role in mediating the innate immune response to infection. The immunomodulatory properties of beta-glucans are attributed to the ability of these fungal cell wall polysaccharides to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes and macrophages. The intracellular signaling pathways activated by beta-glucans lead to enhanced phagocytosis and cytokine response. In this study we investigated the possible potentiation of immunomodulatory properties of the combined treatment with vitamin D and beta-glucans. The effects of 100 nM 1,25-dihydroxyvitamin D3 or 100 µg/mL beta-glucans were evaluated in human macrophages in terms of cytokine production, intracellular vesicle acidification and changes in energy metabolism, three hallmarks of macrophage antimicrobial activation. We found that all the analyzed parameters were enhanced by the co-treatment compared to the response to single molecules. The results of this study support the validity of a novel therapeutic approach that could boost the immune response, taking advantage of the synergy between two natural compounds.


Assuntos
Adjuvantes Imunológicos/farmacologia , Calcitriol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , beta-Glucanas/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Sinergismo Farmacológico , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-8/genética , Interleucina-8/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/imunologia , Transdução de Sinais , Células THP-1 , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/imunologia
14.
AAPS PharmSciTech ; 22(5): 171, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34100170

RESUMO

Macrophages act as a cellular reservoir in HIV infection. Elimination of HIV from macrophages has been an unfulfilled dream due to the failure of drugs to reach them. To address this, we developed CD44 receptor-targeted, novel hyaluronic acid (HA)-coated nanostructured lipid carriers (NLCs) of efavirenz via washless layer-by-layer (LbL) assembly of HA and polyallylamine hydrochloride (PAH). NLCs were subjected to TEM analysis, size and zeta potential, in vitro release and encapsulation efficiency studies. The uptake of NLCs in THP-1 cells was studied using fluorescence microscopy and flow cytometry. The anti-HIV efficacy was evaluated using p24 antigen inhibition assay. NLCs were found to be spherical in shape with anionic zeta potential (-23.66 ± 0.87 mV) and 241.83 ± 5.38 nm particle size. NLCs exhibited prolonged release of efavirenz during in vitro drug release studies. Flow cytometry revealed 1.73-fold higher uptake of HA-coated NLCs in THP-1 cells. Cytotoxicity studies showed no significant change in cell viability in presence of NLCs as compared with the control. HA-coated NLCs distributed throughout the cell including cytoplasm, plasma membrane and nucleus, as observed during fluorescence microscopy. HA-coated NLCs demonstrated consistent and significantly higher inhibition (81.26 ± 1.70%) of p24 antigen which was 2.08-fold higher than plain NLCs. The obtained results suggested preferential uptake of HA-coated NLCs via CD44-mediated uptake. The present finding demonstrates that HA-based CD44 receptor targeting in HIV infection is an attractive strategy for maximising the drug delivery to macrophages and achieve effective viral inhibition.


Assuntos
Portadores de Fármacos/administração & dosagem , HIV-1/efeitos dos fármacos , Receptores de Hialuronatos , Macrófagos/efeitos dos fármacos , Nanoestruturas/administração & dosagem , Inibidores da Transcriptase Reversa/administração & dosagem , Alcinos/administração & dosagem , Alcinos/síntese química , Alcinos/metabolismo , Benzoxazinas/administração & dosagem , Benzoxazinas/síntese química , Benzoxazinas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ciclopropanos/administração & dosagem , Ciclopropanos/síntese química , Ciclopropanos/metabolismo , Relação Dose-Resposta a Droga , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , HIV-1/fisiologia , Humanos , Receptores de Hialuronatos/metabolismo , Lipídeos/administração & dosagem , Lipídeos/síntese química , Macrófagos/metabolismo , Nanoestruturas/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/metabolismo , Células THP-1
15.
Molecules ; 26(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068519

RESUMO

Malaria remains one of the leading causes of death in sub-Saharan Africa, ranked in the top three infectious diseases in the world. Plants of the Eriosema genus have been reported to be used for the treatment of this disease, but scientific evidence is still missing for some of them. In the present study, the in vitro antiplasmodial activity of the crude extract and compounds from Eriosema montanum Baker f. roots were tested against the 3D7 strain of Plasmodium falciparum and revealed using the SYBR Green, a DNA intercalating compound. The cytotoxicity effect of the compounds on a human cancer cell line (THP-1) was assessed to determine their selectivity index. It was found that the crude extract of the plant displayed a significant antiplasmodial activity with an IC50 (µg/mL) = 17.68 ± 4.030 and a cytotoxic activity with a CC50 (µg/mL) = 101.5 ± 12.6, corresponding to a selective antiplasmodial activity of 5.7. Bioactivity-guided isolation of the major compounds of the roots' crude extract afforded seven compounds, including genistein, genistin and eucomic acid. Under our experimental conditions, using Artemisinin as a positive control, eucomic acid showed the best inhibitory activity against the P. falciparum 3D7, a well-known chloroquine-sensitive strain. The present results provide a referential basis to support the traditional use of Eriosema species in the treatment of malaria.


Assuntos
Antimaláricos/farmacologia , Fabaceae/química , Raízes de Plantas/química , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/química , Antimaláricos/isolamento & purificação , Morte Celular/efeitos dos fármacos , Cloroquina/farmacologia , Misturas Complexas , Humanos , Células THP-1
16.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070749

RESUMO

Atherosclerosis and nonalcoholic fatty liver disease are leading causes of morbidity and mortality in the Western countries. The renin-angiotensin system (RAS) with its two main opposing effectors, i.e., angiotensin II (Ang II) and Ang-(1-7), is widely recognized as a major regulator of cardiovascular function and body metabolic processes. Angiotensin-converting enzyme 2 (ACE2) by breaking-down Ang II forms Ang-(1-7) and thus favors Ang-(1-7) actions. Therefore, the aim of our study was to comprehensively evaluate the influence of prolonged treatment with ACE2 activator, diminazene aceturate (DIZE) on the development of atherosclerotic lesions and hepatic steatosis in apoE-/- mice fed a high-fat diet (HFD). We have shown that DIZE stabilized atherosclerotic lesions and attenuated hepatic steatosis in apoE-/- mice fed an HFD. Such effects were associated with decreased total macrophages content and increased α-smooth muscle actin levels in atherosclerotic plaques. Moreover, DIZE changed polarization of macrophages towards increased amount of anti-inflammatory M2 macrophages in the atherosclerotic lesions. Interestingly, the anti-steatotic action of DIZE in the liver was related to the elevated levels of HDL in the plasma, decreased levels of triglycerides, and increased biosynthesis and concentration of taurine in the liver of apoE-/- mice. However, exact molecular mechanisms of both anti-atherosclerotic and anti-steatotic actions of DIZE require further investigations.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Aterosclerose/tratamento farmacológico , Diminazena/análogos & derivados , Fígado Gorduroso/tratamento farmacológico , Placa Aterosclerótica/tratamento farmacológico , Taurina/biossíntese , Angiotensina I/genética , Angiotensina I/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/etiologia , Aterosclerose/genética , Aterosclerose/patologia , Dieta Hiperlipídica , Diminazena/farmacologia , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , Células THP-1 , Taurina/agonistas
17.
Molecules ; 26(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064423

RESUMO

In the present study, we evaluated for the first time the photoprotective effect of fish bone bioactive peptides (FBBP) preparation isolated from silver carp (Hypophthalmichthys molitrix) discarded tissue using in vitro experimental models of skin cells exposed to ultraviolet B (UVB) irradiation and stressing agents. FBBP preparation was obtained by papain treatment of minced bones and centrifugal ultrafiltration, and the molecular weight (MW) distribution was characterized by size exclusion and reversed-phase high performance liquid chromatography (RP-HPLC). In vitro assessment of the effect of FBBP pretreatment in UVB-irradiated L929 fibroblasts and HaCaT keratinocytes revealed their cytoprotective activity. Their capacity to efficiently reduce reactive oxygen species (ROS) production and lipid peroxidation varied in a dose-dependent manner, and it was greater in fibroblasts. A decrease of proinflammatory cytokines secretion, in particular of tumor necrosis factor alpha (TNF-α), was found after FBBP pretreatment of THP-1-derived inflamed macrophages. Melanin production and tyrosinase activity investigated in UVB-irradiated Mel-Juso cells were lowered in direct relation to FBBP concentrations. FBBP fractions with high radical scavenging activity were separated by ion exchange chromatography, and two collagenic sequences were identified. All these results offer new scientific data on aquaculture fish bone-derived peptides confirming their ability to control the antioxidant, anti-inflammatory and pigmentation processes developed during UV irradiation of skin cells and recommend their use as valuable natural ingredients of photoprotective cosmeceutical products.


Assuntos
Osso e Ossos/efeitos dos fármacos , Inflamação/patologia , Peptídeos/farmacologia , Pigmentação , Pele/efeitos da radiação , Raios Ultravioleta , Animais , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Citoproteção/efeitos dos fármacos , Citoproteção/efeitos da radiação , Peixes , Células HaCaT/efeitos dos fármacos , Células HaCaT/efeitos da radiação , Humanos , Mediadores da Inflamação/metabolismo , Espaço Intracelular/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Malondialdeído/metabolismo , Melaninas/biossíntese , Camundongos , Peso Molecular , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Peptídeos/isolamento & purificação , Pigmentação/efeitos dos fármacos , Pigmentação/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Espectrofotometria Ultravioleta , Células THP-1
18.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065977

RESUMO

BACKGROUND: Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor, and macrophages account for 30-40% of its composition. Most of these macrophages derive from bone marrow monocytes playing a crucial role in tumor progression. Unraveling the mechanisms of macrophages-GBM crosstalk in an appropriate model will contribute to the development of specific and more successful therapies. We investigated the interaction of U87MG human GBM cells with primary human CD14+ monocytes or the THP-1 cell line with the aim of establishing a physiologically relevant heterotypic culture model. METHODS: primary monocytes and THP-1 cells were cultured in the presence of U87MG conditioned media or co-cultured together with previously formed GBM spheroids. Monocyte differentiation was determined by flow cytometry. RESULTS: primary monocytes differentiate to M2 macrophages when incubated with U87MG conditioned media in 2-dimensional culture, as determined by the increased percentage of CD14+CD206+ and CD64+CD206+ populations in CD11b+ cells. Moreover, the mitochondrial protein p32/gC1qR is expressed in monocytes exposed to U87MG conditioned media. When primary CD14+ monocytes or THP-1 cells are added to previously formed GBM spheroids, both invade and establish within them. However, only primary monocytes differentiate and acquire a clear M2 phenotype characterized by the upregulation of CD206, CD163, and MERTK surface markers on the CD11b+CD14+ population and induce alterations in the sphericity of the cell cultures. CONCLUSION: our results present a new physiologically relevant model to study GBM/macrophage interactions in a human setting and suggest that both soluble GBM factors, as well as cell-contact dependent signals, are strong inducers of anti-inflammatory macrophages within the tumor niche.


Assuntos
Neoplasias Encefálicas/metabolismo , Técnicas de Cocultura/métodos , Glioblastoma/metabolismo , Macrófagos/citologia , Monócitos/citologia , Biomarcadores/metabolismo , Proteínas de Transporte/metabolismo , Comunicação Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas Mitocondriais/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Cultura Primária de Células , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Células THP-1
19.
Microb Pathog ; 157: 104963, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34022361

RESUMO

Emerging evidence suggests that long noncoding RNAs (lncRNAs) play important roles in disease development. However, the roles of lncRNAs in the pathogenesis of Candida albicans (C. albicans) remain unclear. Our study aimed to investigate and characterize the mRNA and lncRNA transcriptomes of CD14+ monocytes and THP-1 cells stimulated with insoluble ß-glucan by RNA-seq. We identified a total of 10788 differentially expressed (DE) mRNAs and 2021 DE lncRNAs in CD14+ monocytes, while 3349 DE mRNAs and 291 DE lncRNAs were observed in THP-1 cells. A total of 808 DE mRNAs and 51 DE lncRNAs overlapped between the two groups. We examined five collectively DE mRNAs and lncRNAs in both cells using quantitative real-time PCR, validating the reliability of the RNA-seq results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the 808 DE mRNAs were mostly enriched in the inflammatory response and NF-kappa B signaling pathway, respectively. Next, lncRNA-mRNA coexpression analysis was performed for the 51 DE lncRNAs and the 808 DE mRNAs in the two groups. We chose the common network pairs of the two groups to construct the coexpression network and revealed 97 network pairs comprising 8 dysregulated lncRNAs and 60 dysregulated mRNAs. We found that lncRNA lnc-CCL3L3-1:1 might be involved in the NF-kappa B signaling pathway in C. albicans infection. In conclusion, the aberrantly expressed lncRNAs might play a role in the pathogenesis of C. albicans infection and could be used as therapeutic targets in the future.


Assuntos
Monócitos , RNA Longo não Codificante , beta-Glucanas , Candida albicans/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , RNA Longo não Codificante/genética , Reprodutibilidade dos Testes , Células THP-1 , Transcriptoma
20.
Front Immunol ; 12: 562244, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33981296

RESUMO

Polyvalent bacterial lysates have been in use for decades for prevention and treatment of respiratory infections with reported clinical benefits. However, besides claims of broad immune activation, the mode of action is still a matter of debate. The lysates, formulated with the main bacterial species involved in respiratory infections, are commonly prepared by chemical or mechanical disruption of bacterial cells, what is believed influences the biological activity of the product. Here, we prepared two polyvalent lysates with the same composition but different method of bacterial cell disruption and evaluated their biological activity in a comparative fashion. We found that both bacterial lysates induce NF-kB activation in a MyD88 dependent manner, suggesting they work as TLR agonists. Further, we found that a single intranasal dose of any of the two lysates, is sufficient to protect against pneumococcal pneumonia, suggesting that they exert similar biological activity. We have previously shown that protection against pneumococcal pneumonia can also be induced by prior S. pneumoniae sub lethal infection or therapeutic treatment with a TLR5 agonist. Protection in those cases depends on neutrophil recruitment to the lungs, and can be associated with increased local expression of IL-17A. Here, we show that bacterial lysates exert protection against pneumococcal pneumonia independently of neutrophils, IL-17A or Caspase-1/11 activation, suggesting the existence of redundant mechanisms of protection. Trypsin-treated lysates afford protection to the same extent, suggesting that just small peptides suffice to exert the protective effect or that the molecules responsible for the protective effect are not proteins. Understanding the mechanism of action of bacterial lysates and deciphering the active components shall allow redesigning them with more precisely defined formulations and expanding their range of action.


Assuntos
Bactérias/química , Fatores Biológicos/farmacologia , Caspase 1/metabolismo , Interleucina-17/metabolismo , Neutrófilos/metabolismo , Pneumonia Pneumocócica/prevenção & controle , Streptococcus pneumoniae/efeitos dos fármacos , Células A549 , Animais , Fatores Biológicos/química , Ativação Enzimática , Humanos , Camundongos , Pneumonia Pneumocócica/metabolismo , Pneumonia Pneumocócica/microbiologia , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Streptococcus pneumoniae/fisiologia , Análise de Sobrevida , Células THP-1
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