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1.
Mol Biol (Mosk) ; 55(3): 478-490, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34097682

RESUMO

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect almost all organs and tissues, cause genital herpes-the most common sexually transmitted disease-disorders of the central nervous system (CNS), and lead to severe complications in children. Despite the available drugs, the incidence of HSV-1/2 continues to rise. None of the prophylactic vaccine candidates have shown a protective effect in trials nor approval for use in clinical practice. We have investigated the protective properties of mesenchymal stem cells (MSC) isolated from the bone marrow of mice. A comparative analysis of the protective response to the introduction of primary and modified MSCs (mMSC) was carried out using the plasmid containing gene of the HSV and an inactivated virus in a model of lethal HSV-1 infection in mice. mMSCs were obtained by transfection of the Us6 gene encoding glycoprotein D (gD) of the HSV, the plasmid contained the same gene. After twofold immunization with primary MSCs, the formation of antibodies interacting with the viral antigen (according to enzyme immunoassay data) and neutralizing the infectious activity of HSV-1 in the reaction of biological neutralization was observed in the peripheral blood of mice. In addition, the introduction of primary MSCs induced the production of interferon gamma (INF-γ) which is detected in the peripheral blood of mice. After infection with HSV-1, the immunized mice showed significantly increased titers of virus-specific antibodies, an increased level of IFNγ, and were completely protected from lethal HSV-1 infection. The protective effect of the other three immunogens was lower and did not exceed 50-65%. Considering the wide availability of MSCs, the proven safety of intravenous administration, and the results obtained in this work on the ability to induce innate, adaptive and protective immunity to HSV-1, MSCs can be considered a promising basis for the development of new cellular vaccines for the prevention of herpesvirus and other viral infections.


Assuntos
Herpesvirus Humano 1 , Células-Tronco Mesenquimais , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Proteínas do Envelope Viral
2.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068436

RESUMO

Extracellular vesicles (EVs) are small lipid vesicles released by either any prokaryotic or eukaryotic cell, or both, with a biological role in cell-to-cell communication. In this work, we characterize the proteomes and nanomechanical properties of EVs released by tissue-culture cell-derived trypomastigotes (mammalian infective stage; (TCT)) and epimastigotes (insect stage; (E)) of Trypanosoma cruzi, the etiologic agent of Chagas disease. EVs of each stage were isolated by differential centrifugation and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy and atomic force microscopy (AFM). Measurements of zeta-potential were also included. Results show marked differences in the surface molecular cargos of EVs between both stages, with a noteworthy expansion of all groups of trans-sialidase proteins in trypomastigote's EVs. In contrast, chromosomal locations of trans-sialidases of EVs of epimastigotes were dramatically reduced and restricted to subtelomeric regions, indicating a possible regulatable expression of these proteins between both stages of the parasite. Regarding mechanical properties, EVs of trypomastigotes showed higher adhesion compared to the EVs of epimastigotes. These findings demonstrate the remarkable surface remodeling throughout the life cycle of T. cruzi, which shapes the physicochemical composition of the extracellular vesicles and could have an impact in the ability of these vesicles to participate in cell communication in completely different niches of infection.


Assuntos
Doença de Chagas/metabolismo , Vesículas Extracelulares/metabolismo , Estágios do Ciclo de Vida , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Doença de Chagas/parasitologia , Chlorocebus aethiops , Vesículas Extracelulares/parasitologia , Interações Hospedeiro-Parasita , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteoma/análise , Células Vero
3.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068922

RESUMO

INTRODUCTION: AQP4 (aquaporin-4)-immunoglobulin G (IgG)-mediated neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease that affects the central nervous system, particularly the spinal cord and optic nerve; remyelination capacity in neuromyelitis optica is yet to be determined, as is the role of AQP4-IgG in cell differentiation. MATERIAL AND METHODS: We included three groups-a group of patients with AQP4-IgG-positive neuromyelitis optica, a healthy group, and a sham group. We analyzed differentiation capacity in cultures of neurospheres from the subventricular zone of mice by adding serum at two different times: early and advanced stages of differentiation. We also analyzed differentiation into different cell lines. RESULTS AND CONCLUSIONS: The effect of sera from patients with NMOSD on precursor cells differs according to the degree of differentiation, and probably affects oligodendrocyte progenitor cells from NG2 cells to a lesser extent than cells from the subventricular zone; however, the resulting oligodendrocytes may be compromised in terms of maturation and possibly limited in their ability to generate myelin. Furthermore, these cells decrease in number with age. It is very unlikely that the use of drugs favoring the migration and differentiation of oligodendrocyte progenitor cells in multiple sclerosis would be effective in the context of neuromyelitis optica, but cell therapy with oligodendrocyte progenitor cells seems to be a potential alternative.


Assuntos
Aquaporina 4/imunologia , Autoanticorpos/imunologia , Diferenciação Celular , Sistema Nervoso Central/patologia , Imunoglobulina G/imunologia , Neuromielite Óptica/imunologia , Células Precursoras de Oligodendrócitos/patologia , Animais , Autoanticorpos/sangue , Estudos de Casos e Controles , Sistema Nervoso Central/imunologia , Cerebelo/imunologia , Cerebelo/patologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Neuromielite Óptica/sangue , Neuromielite Óptica/patologia , Células Precursoras de Oligodendrócitos/imunologia
4.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 33(5): 523-528, 2021 May.
Artigo em Chinês | MEDLINE | ID: mdl-34112286

RESUMO

OBJECTIVE: To investigate the roles and underlying mechanisms of mixed lineage kinase domain like (MLKL)-mediated inflammatory response induced by NOD-like receptor protein 3 (NLRP3) inflammatory corpuscles in the acute lung injury (ALI) after sepsis. METHODS: Eighteen BALB/c mice were randomly divided into sham operation group (Sham group), cecal ligation and perforation (CLP)-induced sepsis model group (CLP group) and specific inhibitor Necrostatin-1 intervention group [CLP+Nec-1 group, Necrostatin-1 solution (20 mg/kg) was injected intravenously 10 minutes before modeling], with 6 mice in each group. The mice were sacrificed by neck amputation at the 2nd day after operation, and the serum and lung tissue samples were collected. The morphological changes of lung tissue were observed by hematoxylin-eosin (HE) staining. The water content of lung tissue was detected by dry-wet weight method. The pulmonary vascular permeability was measured by Evans blue (EB) staining. The protein expressions of MLKL and NLRP3 in the lung tissue were detected by Western blotting, and the level of serum interleukin-1ß (IL-1ß) was detected by enzyme linked immunosorbent assay (ELISA). RESULTS: HE staining showed that the lung morphological structure in Sham group was normal. In CLP group, congestion and edema in the alveolar cavity and interstitium, infiltration of neutrophils and thickening of alveolar wall were observed. The histopathological changes of lung tissue in CLP+Nec-1 group were better than those in CLP group. Compared with Sham group, the water content of lung tissue [(88.00±0.00)% vs. (78.00±0.01)%], pulmonary vascular permeability [EB content (mg/L): 11.82±1.15 vs. 4.00±0.71], the protein expressions of phosphorylated MLKL (p-MLKL) and NLRP3 in lung tissue (p-MLKL/GAPDH: 0.34±0.04 vs. 0.12±0.01,NLRP3/GAPDH: 0.47±0.07 vs. 0.16±0.04), and the level of serum IL-1ß (ng/L: 183.56±9.61 vs. 44.14±6.95) in CLP group were all significantly increased (all P < 0.01). Compared with CLP group, the water content of lung tissue [(81.00±0.01)% vs. (88.00±0.00)%], pulmonary vascular permeability [EB content (mg/L): 7.90±0.00 vs. 11.82±1.15], protein expressions of p-MLKL and NLRP3 in lung tissue (p-MLKL/GAPDH: 0.13±0.03 vs. 0.34±0.04, NLRP3/GAPDH: 0.18±0.04 vs. 0.47±0.07), and the level of serum IL-1ß (ng/L: 113.81±6.62 vs. 183.56±9.61) were all significantly decreased (all P < 0.01). CONCLUSIONS: MLKL-NLRP3-mediated necroinflammation was significantly up-regulatedin the lung tissue of septic mice, which could be attenuated by specific inhibitor Necrostatin-1.


Assuntos
Lesão Pulmonar Aguda , Sepse , Lesão Pulmonar Aguda/etiologia , Animais , Pulmão , Camundongos , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas NLR , Proteínas Quinases , Sepse/complicações , Fator de Necrose Tumoral alfa
5.
Int J Nanomedicine ; 16: 3755-3773, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34103914

RESUMO

Purpose: Acinetobacter baumannii antibiotic resistant infections in high-risk patients are a great challenge for researchers and clinicians worldwide. In an effort to achieve potent bactericidal outcomes, a novel chitosan-mastoparan nanoconstruct (Mast-Cs NC) was designed and assessed for its therapeutic potential through in silico, in vitro and in vivo experimentation against clinical multidrug-resistant (MDR) A. baumannii. Methods: Optimized 3D structures of mastoparan and chitosan were coupled computationally through an ionic cross-linker to generate a circular ring of chitosan encasing mastoparan. The complex was assessed for interactions and stability through molecular dynamic simulation (MDS). Binding pocket analysis was used to assess the protease-peptide interface. Mast-Cs NC were prepared by the ionic gelation method. Mast-Cs NC were evaluated in vitro and in vivo for their therapeutic efficacy against drug-resistant clinical A. baumannii. Results: MDS for 100 ns showed stable bonds between chitosan and mastoparan; the first at chitosan oxygen atom-46 and mastoparan isoleucine carbon atom with a distance of 2.77 Å, and the second between oxygen atom-23 and mastoparan lysine nitrogen atom with a distance of 2.80 Å, and binding energies of -3.6 and -7.4 kcal/mol, respectively. Mast-Cs complexes approximately 156 nm in size, with +54.9 mV zeta potential and 22.63% loading capacity, offered >90% encapsulation efficiency and were found to be geometrically incompatible with binding pockets of various proteases. The MIC90 of Mast-Cs NC was significantly lower than that of chitosan (4 vs 512 µg/mL, respectively, p<0.05), with noticeable bacterial damage upon morphological analysis. In a BALB/c mouse sepsis model, a significant reduction in bacterial colony count in the Mast-Cs treated group was observed compared with chitosan and mastoparan alone (p<0.005). Mast-Cs maintained good biocompatibility and cytocompatibility. Conclusion: Novel mastoparan-loaded chitosan nanoconstructs signify a successful strategy for achieving a synergistic bactericidal effect and higher therapeutic efficacy against MDR clinical A. baumannii isolates. The Mast-Cs nano-drug delivery system could work as an alternative promising treatment option against MDR A. baumannii.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Quitosana/química , Simulação por Computador , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Nanopartículas/química , Venenos de Vespas/farmacologia , Acinetobacter baumannii/crescimento & desenvolvimento , Acinetobacter baumannii/isolamento & purificação , Adolescente , Adulto , Animais , Antibacterianos/farmacologia , Criança , Pré-Escolar , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Nanopartículas/ultraestrutura , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Adulto Jovem
6.
J Med Microbiol ; 70(6)2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34115583

RESUMO

Introduction. Leishmaniasis is a neglected tropical and subtropical disease caused by over 20 protozoan species.Hypothesis. Treatment of this complex disease with traditional synthetic drugs is a major challenge worldwide. Natural constituents are unique candidates for future therapeutic development.Aim. This study aimed to assess the in vivo anti-leishmanial effect of the Gossypium hirsutum extract, and its fractions compared to the standard drug (Glucantime, MA) in a murine model and explore the mechanism of action.Methodology. Footpads of BALB/c mice were infected with stationary phase promastigotes and treated topically and intraperitoneally with G. hirsutum extract, its fractions, or Glucantime, 4 weeks post-infection. The extract and fractions were prepared using the Soxhlet apparatus with chloroform followed by the column procedure.Results. The crude extract significantly decreased the footpad parasite load and lesion size compared to the untreated control group (P<0.05), as revealed by dilution assay, quantitative real-time PCR, and histopathological analyses. The primary mode of action involved an immunomodulatory role towards the Th1 response in the up-regulation of IFN-γ and IL-12 and the suppression of IL-10 gene expression profiling against cutaneous leishmaniasis caused by Leishmania major.Conclusion. This finding suggests that the extract possesses multiple combinatory effects of diverse bioactive phytochemical compositions that exert its mechanisms of action through agonistic-synergistic interactions. The topical extract formulation could be a suitable and unique candidate for future investigation and pharmacological development. Further studies are crucial to evaluate the therapeutic potentials of the extract alone and in combination with conventional drugs using clinical settings.


Assuntos
Antiprotozoários/uso terapêutico , Gossypium , Leishmania major/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Fitoterapia , Extratos Vegetais/uso terapêutico , Administração Tópica , Animais , Antiprotozoários/farmacologia , Feminino , Injeções Intraperitoneais , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-10/genética , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Leishmania major/fisiologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Leishmaniose Cutânea/fisiopatologia , Linfonodos/patologia , Antimoniato de Meglumina/administração & dosagem , Antimoniato de Meglumina/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Baço/parasitologia , Baço/patologia , Células Th1/imunologia , Transcriptoma
7.
Nan Fang Yi Ke Da Xue Xue Bao ; 41(5): 649-656, 2021 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-34134950

RESUMO

OBJECTIVE: To investigate the effect and safety of Escherichia coli (E.coli) expressing gas vesicle (GVs) for enhancing the efficacy of tumor ablation by high intensity focused ultrasound (HIFU) in tumor-bearing mice. OBJECTIVE: Thirty-two female BALB/c mice were used to establish mouse models bearing 4T1 tumor, which were randomized into GVs group [E.coli BL21 (AI)-PET28a-Arg1] and control group (PBS), and the efficacy of HIFU ablation was evaluated by examining coagulative necrotic volume and pathology of the tumors. Another 104 BALB/c mice were also randomly divided into GVs group and control group, and body weight changes of the mice were recorded on days 1, 4 and 15 after intravenous injection of E.coli containing GVs or PBS. White blood cells, red blood cells, hemoglobin and platelet counts and liver and renal function parameters of the mice were detected, and serum levels of TNF-α and IL-1ß were examined using ELISA. The pathological changes in the liver and spleen were evaluated using HE staining to assess the safety of the treatments. OBJECTIVE: HIFU ablation resulted in a significantly greater volume of coagulative necrosis and severer tissue damage in GVs group than in the control group (P < 0.001). In the 104 BALB/c mice without tumor cell inoculation, intravenous injection of E.coli expressing GVs, as compared with PBS, did not significantly affect body weight or cause changes in white blood cell, red blood cell and platelet counts or hemoglobin level (P1=0.59, P2=0.27, P3=0.76, P4=0.81). The liver and kidney function parameters (P1=0.12, P2=0.46, P3=0.62, P4=0.86) and serum levels of TNF-α and IL-1ß (P1=0.48, P2=0.56) were all comparable between GVs group and control group. No obvious pathological changes were detected in the liver and spleen tissues in either GVs group or the control group. OBJECTIVE: E.coli expressing GVs is safe for enhancing the ablation effect of HIFU in tumor-bearing mice.


Assuntos
Ablação por Ultrassom Focalizado de Alta Intensidade , Neoplasias , Animais , Escherichia coli , Feminino , Ablação por Ultrassom Focalizado de Alta Intensidade/efeitos adversos , Camundongos , Camundongos Endogâmicos BALB C , Ultrassonografia
8.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064275

RESUMO

Sigma-1 receptor (chaperone Sigma1R) is an intracellular protein with chaperone functions, which is expressed in various organs, including the brain. Sigma1R participates in the regulation of physiological mechanisms of anxiety (Su, T. P. et al., 2016) and reactions to emotional stress (Hayashi, T., 2015). In 2006, fabomotizole (ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihydrochloride) was registered in Russia as an anxiolytic (Seredenin S. and Voronin M., 2009). The molecular targets of fabomotizole are Sigma1R, NRH: quinone reductase 2 (NQO2), and monoamine oxidase A (MAO-A) (Seredenin S. and Voronin M., 2009). The current study aimed to clarify the dependence of fabomotizole anxiolytic action on its interaction with Sigma1R and perform a docking analysis of fabomotizole interaction with Sigma1R. An elevated plus maze (EPM) test revealed that the anxiolytic-like effect of fabomotizole (2.5 mg/kg i.p.) administered to male BALB/c mice 30 min prior EPM exposition was blocked by Sigma1R antagonists BD-1047 (1.0 mg/kg i.p.) and NE-100 (1.0 mg/kg i.p.) pretreatment. Results of initial in silico study showed that fabomotizole locates in the active center of Sigma1R, reproducing the interactions with the site's amino acids common for established Sigma1R ligands, with the ΔGbind value closer to that of agonist (+)-pentazocine in the 6DK1 binding site.


Assuntos
Ansiolíticos/farmacologia , Ansiedade/tratamento farmacológico , Benzimidazóis/farmacologia , Chaperonas Moleculares/metabolismo , Morfolinas/farmacologia , Receptores sigma/metabolismo , Animais , Anisóis/farmacologia , Sítios de Ligação/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Etilenodiaminas/farmacologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Propilaminas/farmacologia , Federação Russa
9.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068008

RESUMO

A major contributing factor in triple-negative breast cancer progression is its ability to evade immune surveillance. One mechanism for this immunosuppression is through ribosomal protein S19 (RPS19), which facilitates myeloid-derived suppressor cells (MDSCs) recruitment in tumors, which generate cytokines TGF-ß and IL-10 and induce regulatory T cells (Tregs), all of which are immunosuppressive and enhance tumor progression. Hence, enhancing the immune system in breast tumors could be a strategy for anticancer therapeutics. The present study evaluated the immune response of atovaquone, an antiprotozoal drug, in three independent breast-tumor models. Our results demonstrated that oral administration of atovaquone reduced HCC1806, CI66 and 4T1 paclitaxel-resistant (4T1-PR) breast-tumor growth by 45%, 70% and 42%, respectively. MDSCs, TGF-ß, IL-10 and Tregs of blood and tumors were analyzed from all of these in vivo models. Our results demonstrated that atovaquone treatment in mice bearing HCC1806 tumors reduced MDSCs from tumor and blood by 70% and 30%, respectively. We also observed a 25% reduction in tumor MDSCs in atovaquone-treated mice bearing CI66 and 4T1-PR tumors. In addition, a decrease in TGF-ß and IL-10 in tumor lysates was observed in atovaquone-treated mice with a reduction in tumor Tregs. Moreover, a significant reduction in the expression of RPS19 was found in tumors treated with atovaquone.


Assuntos
Anti-Infecciosos/farmacologia , Apresentação do Antígeno/imunologia , Atovaquona/farmacologia , Imunossupressão , Células Supressoras Mieloides/imunologia , Linfócitos T Reguladores/imunologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Apoptose , Proliferação de Células , Citocinas/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Células Supressoras Mieloides/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Nat Commun ; 12(1): 3279, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078883

RESUMO

Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial-mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart-irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity.


Assuntos
Anticorpos Neutralizantes/farmacologia , Cardiomiopatias/prevenção & controle , Cardiotoxicidade/prevenção & controle , Doxorrubicina/toxicidade , Raios gama/efeitos adversos , Molécula L1 de Adesão de Célula Nervosa/genética , Animais , Antibióticos Antineoplásicos/toxicidade , Cardiomiopatias/etiologia , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiotoxicidade/etiologia , Cardiotoxicidade/genética , Cardiotoxicidade/metabolismo , Estudos de Casos e Controles , Técnicas de Cocultura , Dano ao DNA , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/efeitos da radiação , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
11.
Nat Commun ; 12(1): 3299, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083537

RESUMO

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using ß-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Fenformin/farmacologia , Fator de Transcrição STAT1/metabolismo , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sinergismo Farmacológico , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Feminino , Glutationa/antagonistas & inibidores , Glutationa/biossíntese , Humanos , Interferon gama/administração & dosagem , Interferon gama/deficiência , Interferon gama/metabolismo , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Fenformin/administração & dosagem , Poli I-C/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT1/agonistas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(3): 931-936, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34105496

RESUMO

OBJECTIVE: To explore the kinetics of infiltrated T cell in murine acute graft-versus-host disease (aGVHD) target organs after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and its relationship with tissue pathological damage and aGVHD progress. METHODS: Male C57BL/6 (H-2Kb) mice at age of 8-10 weeks were selected as donors, from which splenic cells and bone marrow cells were isolated. And 10-12 weeks of BALB/c (H-2Kd) male mice which received 7.5 Gy total body irradiation (TBI) were recipients to transplant. Recipients were randomly divided into allogeneic bone marrow transplantation (BMT) group and BMT+T group, which were transplanted bone marrow cells with or without splenic cells, respectively. All recipients were daily monitored and the dynamic changes of the body weights along with clinical scores of aGVHD were detected. HE staining was used to investigate the pathological damage and score of aGVHD target organs. The number of infiltrated CD3+ T cells in target organs was numerated and statistically analyzed after immunohistochemistry staining on day 7, 14, 28, 40 and 47 after transplantation. RESULTS: Compared with BMT group, the number of infiltrated T cells in aGVHD target organs including liver, lung and gut increased since day 7 in BMT+T group (P<0.05). On day 14, 28, 40 and 47 after transplantation, more infiltrated CD3+ T cells were detected in target tissues of mice in BMT+T group than those in BMT group (P<0.05). Higher clinical score and histopathological score of target organs in aGVHD mice were detected (P<0.05). Positive correlation was found in the number of liver infiltrated T cells and pathological damage, and the numbers of infiltrated CD3+ T cells in gut were positively related to aGVHD clinical scores. CONCLUSION: Pathological damage of aGVHD target organs is induced by CD3+ T cell infiltration, and the number of infiltrated T cell may be an important evaluated index of aGVHD severity.


Assuntos
Doença Enxerto-Hospedeiro , Animais , Transplante de Medula Óssea , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T , Transplante Homólogo
13.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(3): 937-943, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34105497

RESUMO

OBJECTIVE: To establish the aGVHD mouse model,and investigate the regulatory effect and its mechanism of low-dose GSI combined with BMSC on aGVHD mice. METHODS: C57BL/6 (H-2b) and BALB/c (H-2d) were selected as donor and recipient of allogeneic transplantation to establish the aGVHD mouse model. BALB/c mice were randomly divided into 6 groups, which were the bone marrow cell infusion after irradiation (BM) group; the bone marrow cells + spleen cells after irradiation (BM+SC) group; the bone marrow cells + spleen cells + DMSO (BM+SC+DMSO) (transplant control) group; bone marrow cells + splenocytes +GSI after irradiation (BM+SC+GSI) group; bone marrow cells + spleen cells + bone marrow mesenchymal stromal infusion after irradiation cell (BM+SC+BMSC) group; bone marrow cells + spleen cells + bone marrow mesenchymal stromal cells +GSI infused after irradiation (BM+SC+BMSC+GSI) group. The mice in the two groups containing GSI were intraperitoneally injected with GSI at 5 µmol/kg on day 1, 2, and 3 after transplantation with DMSO as a control. The general conditions, survival time and hematopoietic recovery of mice were observed, cytokines were detected by ELISA, and histopathological changes were detected by immunohistochemistry. The effects of low-dose GSI combined with BMSC on hematopoietic reconstruction and aGVHD development after allo-BMT were investigated. RESULTS: The survival rate of the mice in BM+SC+BMSC+GSI combination group was 80% during the observation period, which was significantly higher than that in the other groups; the incidence of aGVHD was reduced in the BMSC GSI or their combination groups after 21 days of transplantation. GSI could partly promote the recovery of leukocytes, and show no significant delayed effect on the recovery platelets. Moreover, the level of Th1 cytokines (IFN-γ) in BM+SC+BMSC+GSI combined group was lower than that in BM+SC+GSI group (P<0.01), the level of Th2 cytokines (IL-4) in the combination group was higher than that in BM+SC+GSI group (P<0.01), also the level of IL-17 was significantly lower than that in the corresponding control group (P<0.001). CONCLUSION: Low dose GSI combined with BMSC can promote hematopoietic reconstruction and regulate cytokines secretion including IFN-γ, IL-4 and IL-17. GSI combined with BMSC achieve the goal of synergistically inhibiting the occurrence and progression of aGVHD.


Assuntos
Doença Enxerto-Hospedeiro , Secretases da Proteína Precursora do Amiloide , Animais , Transplante de Medula Óssea , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
14.
Klin Lab Diagn ; 66(6): 353-357, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34105911

RESUMO

Tularemia is an especially dangerous infection caused by the gram-negative bacterium Francisella tularensis. It belongs to natural focal infections, and therefore is under continuous control by quarantine services. When carrying out their activities they use a whole range of diagnostic tools. The objective of this research is to develop an enzyme immunoassay based on highly specific monoclonal antibodies and immunomagnetic particles for monitoring the tularemia pathogen. To produce hybridomas mice were immunized with cells of the vaccine strain F. tularensis subsp. holarctica 15 NIIEG. After cell fusion hybridomas were selected by a solid-phase enzyme immunoassay (ELISA) using lipopolysaccharide (LPS) of the tularemia microbe. As a result, two hybridomas, 1C2 and 3F5, were produced. MABs of the hybridomas were obtained by using BALB / c mice. The MABs were purified by sepharose A affinity chromatography and used for conjugation with magnetic particles, and for biotinylation followed by matching a pair for ELISA. The pair of IMPs and MABs 3F5 as well as biotinylated FB11-x MABs was the best in detecting tularemia cells. The use of this MAB pair in ELISA allowed the identification of 105 microbial cells/ml in a 4 ml sample and 5×103 microbial cells/ml in a 45ml sample. Interaction with F. tularensis subsp. novicida Utah112 cells was absent.


Assuntos
Francisella tularensis , Tularemia , Animais , Imunoensaio , Testes Imunológicos , Camundongos , Camundongos Endogâmicos BALB C , Tularemia/diagnóstico
15.
Nat Commun ; 12(1): 3453, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103484

RESUMO

A once every eight-week cabotegravir (CAB) long-acting parenteral is more effective than daily oral emtricitabine and tenofovir disoproxil fumarate in preventing human immunodeficiency virus type one (HIV-1) transmission. Extending CAB dosing to a yearly injectable advances efforts for the elimination of viral transmission. Here we report rigor, reproducibility and mechanistic insights for a year-long CAB injectable. Pharmacokinetic (PK) profiles of this nanoformulated CAB prodrug (NM2CAB) are affirmed at three independent research laboratories. PK profiles in mice and rats show plasma CAB levels at or above the protein-adjusted 90% inhibitory concentration for a year after a single dose. Sustained native and prodrug concentrations are at the muscle injection site and in lymphoid tissues. The results parallel NM2CAB uptake and retention in human macrophages. NM2CAB nanocrystals are stable in blood and tissue homogenates. The long apparent drug half-life follows pH-dependent prodrug hydrolysis upon slow prodrug nanocrystal dissolution and absorption. In contrast, solubilized prodrug is hydrolyzed in hours in plasma and tissues from multiple mammalian species. No toxicities are observed in animals. These results affirm the pharmacological properties and extended apparent half-life for a nanoformulated CAB prodrug. The report serves to support the mechanistic design for drug formulation safety, rigor and reproducibility.


Assuntos
Liberação Controlada de Fármacos , Lipídeos/química , Nanopartículas/química , Pró-Fármacos/farmacologia , Piridonas/farmacocinética , Animais , Composição de Medicamentos , Endocitose , Humanos , Cinética , Masculino , Camundongos Endogâmicos BALB C , Piridonas/administração & dosagem , Piridonas/sangue , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Distribuição Tecidual
16.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 35(5): 620-626, 2021 May 15.
Artigo em Chinês | MEDLINE | ID: mdl-33998217

RESUMO

Objective: To investigate the correlation between the content of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) and its osteogenic activity in vitro and in vivo, in order to choose a simple and convenient method to evaluate the osteogenic activity of DBM. Methods: The left mid-femoral tissues of 9 donors were taken, and DBMs (S1-S9) were prepared by dynamic decalcification process, and inactivated DBM (control group) was prepared at the same time. Protease inhibitor method, collagenase method, guanidine hydrochloride/ethylene diamine tetraacetic acid (EDTA) method, and RIPA lysate method were used to extract BMP-2 in S1-S9 and inactivated DBMs. The BMP-2 content was measured and the differences between DBMs were compared. Then the S1-S9 and inactivated DBMs were co-cultured with mouse embryonic osteoblasts MC3T3-E1, respectively. The cell proliferation was detected by MTT method and fluorescence staining, and alkaline phosphatase (ALP) activity was detected at the same time. Thirty BALB/c male nude mice were divided into 10 groups, namely S1-S9 DBM groups (S1-S9 groups) and inactivated DBM group (control group), with 3 mice in each group. Muscle pockets of the middle thighs were prepared on both hindlimbs of mice in each group, and implanted corresponding DBM materials. At 4 weeks after operation, the samples were taken for HE staining observation and semi-quantitative evaluation, and the new bone formation score was calculated. Results: The BMP-2 content of DBM derived from different donor bones was distinct. The BMP-2 content obtained by different extraction methods for DBM prepared from the same donor bone was also different, and the extraction efficiency of the guanidine hydrochloride/EDTA method was the highest. In vitro cell experiments, MTT test displayed that cell proliferations and ALP activity were significantly higher in S4 and S6 groups than in other groups at each time point after co-cultivation ( P<0.05). Moreover, the cell proliferation of S4 group was the most significant at 7 days ( P<0.05); fluorescence staining demonstrated that the osteoblasts of each group was in good condition, but the osteoblasts of S1, S2, S3, S4, and S6 groups were significantly more than other groups. In vivo ectopic osteogenesis experiments, the cartilage and new bone formation could be seen in the bone graft area of S1-S6 groups at 4 weeks after operation, and with the increase of BMP-2 content, the more new bone formation induced by the material, the higher the score of new bone formation of the material ( P<0.05). Among them, S4 and S6 groups contained a large number of chondrocytes and osteoblasts in the osteogenesis area. Conclusion: The osteogenic activity of DBM can be evaluated through BMP-2 quantitative detection combined with in vitro osteoblast proliferation and differentiation experiments.


Assuntos
Proteína Morfogenética Óssea 2 , Osteogênese , Animais , Matriz Óssea , Diferenciação Celular , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus
17.
Stem Cell Reports ; 16(5): 1165-1181, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33979601

RESUMO

SARS-CoV-2 infection is associated with lower blood oxygen levels, even in patients without hypoxia requiring hospitalization. This discordance illustrates the need for a more unifying explanation as to whether SARS-CoV-2 directly or indirectly affects erythropoiesis. Here, we show significantly enriched CD71+ erythroid precursors/progenitors in the blood circulation of COVID-19 patients. We found that these cells have distinctive immunosuppressive properties. In agreement, we observed a strong negative correlation between the frequency of these cells with T and B cell proportions in COVID-19 patients. The expansion of these CD71+ erythroid precursors/progenitors was negatively correlated with the hemoglobin levels. A subpopulation of abundant erythroid cells, CD45+ CD71+ cells, co-express ACE2, TMPRSS2, CD147, and CD26, and these can be infected with SARS-CoV-2. In turn, pre-treatment of erythroid cells with dexamethasone significantly diminished ACE2/TMPRSS2 expression and subsequently reduced their infectivity with SARS-CoV-2. This provides a novel insight into the impact of SARS-CoV-2 on erythropoiesis and hypoxia seen in COVID-19 patients.


Assuntos
Imunidade Adaptativa/imunologia , COVID-19/patologia , Células Precursoras Eritroides/virologia , Eritropoese/fisiologia , Hemoglobinas/análise , Oxigênio/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , COVID-19/imunologia , Dexametasona/farmacologia , Células Precursoras Eritroides/imunologia , Feminino , Humanos , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Serina Endopeptidases/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Adulto Jovem
18.
Microb Cell Fact ; 20(1): 95, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33952256

RESUMO

BACKGROUND: The global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the need to develop safe and effective vaccines with a top priority. Multiple vaccine candidates are under development, and several vaccines are currently available. Efforts need to be undertaken to counter the threat of the global COVID-19 pandemic. RESULTS: We generated a Saccharomyces cerevisiae (S. cerevisiae)-based SARS-CoV-2 vaccine, EBY100/pYD1-RBD, in which the full-length receptor binding domain (RBD) of the spike protein of SARS-CoV-2 was expressed on the surface of yeast. Mice vaccinated orally with unadjuvanted EBY100/pYD1-RBD could produce significant humoral and mucosal responses as well as robust cellular immune responses. Notably, EBY100/pYD1-RBD elicited a mixed Th1/Th2-type cellular immune response with a Th1-biased immune response in a mouse model. CONCLUSIONS: Our findings highlight the importance of the RBD as a key target to design and develop vaccines against SARS-CoV-2 and provide evidence of oral administration of a S. cerevisiae-based SARS-CoV-2 vaccine eliciting significant immune responses. Most importantly, the S. cerevisiae surface display system can serve as a universal technology platform and be applied to develop other oral viral or bacterial vaccines.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Imunidade Celular , Saccharomyces cerevisiae , Glicoproteína da Espícula de Coronavírus/imunologia , Administração Oral , Animais , Sítios de Ligação , COVID-19/prevenção & controle , Vacinas contra COVID-19/administração & dosagem , Feminino , Imunidade Humoral , Imunidade nas Mucosas , Camundongos , Camundongos Endogâmicos BALB C
19.
Int J Nanomedicine ; 16: 3073-3089, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33953556

RESUMO

Introduction: Hyperoside (HYP), a flavonol glycoside compound, has been shown to significantly inhibit the proliferation of malignant tumors. Mitochondria serve as both "energy factories" and "suicide weapon stores" of cells. Targeted delivery of cytotoxic drugs to the mitochondria of tumor cells and tumor vascular cells is a promising strategy to improve the efficacy of chemotherapy. Objective: We report a novel dual-functional liposome system possessing both extracellular charge reversal and mitochondrial targeting properties to enhance drug accumulation in mitochondria and trigger apoptosis of cancer cells. Methods: L-lysine was used as a linker to connect 2,3-dimethylmaleic anhydride (DMA) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) to yield a new compound, DSPE-Lys-DMA (DLD). Then, DLD was mixed with other commercially available lipids to form charge reversed and mitochondria-targeted liposomes (DLD-Lip). The size, morphology, zeta potential, serum stability, and protein adsorption of the HYP loaded DLD-Lip (HYP/DLD-Lip) were measured. The release profile, cellular uptake, in vitro and in vivo toxicity, and anticancer activity of HYP/DLD-Lip were investigated. Results: The results showed that the mean diameter of the liposomes was less than 200 nm. The zeta potential of the liposomes was negative at pH 7.4. However, the zeta potential was positive at weak acidic pH values with the cleavage of the DMA amide. The charge reversion of HYP/DLD-Lip facilitated the cellular internalization and mitochondrial accumulation for enhanced antitumor effect. The strongest tumor growth inhibition (TGI 88.79%) without systemic toxicity was observed in DLD/HYP-Lips-treated CBRH-7919 tumor xenograft BALB/C mice. Conclusion: The charge reversed and mitochondria-targeted liposomes represented a promising anticancer drug delivery system for enhanced anticancer therapeutic efficacy.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Lipossomos/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Quercetina/análogos & derivados , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Lipossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidiletanolaminas/química , Quercetina/administração & dosagem , Quercetina/química , Quercetina/farmacologia
20.
mBio ; 12(3)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33975938

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Vacinas contra COVID-19/imunologia , Imunogenicidade da Vacina , Receptores de Coronavírus/genética , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Sítios de Ligação , Vacinas contra COVID-19/química , Feminino , Engenharia Genética , Glicosilação , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Domínios Proteicos , Ratos , Ratos Sprague-Dawley , Receptores de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Conjugadas/genética , Vacinas Conjugadas/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia
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