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1.
Cell Physiol Biochem ; 54(2): 161-179, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-32045141

RESUMO

BACKGROUND/AIMS: We performed co-culture experiments between human RPE cells (ARPE-19) and human umbilical vascular endothelial cells (HUVEC) in order to evaluate how anti-VEGF drugs could affect NO release, mitochondrial function, the oxidative status, proliferation and migration of RPE cells through modulation of their cross talk with vascular endothelial cells. METHODS: The co-culture HUVEC/RPE, was exposed to Ranibizumab/Aflibercept in the absence/presence of the NO synthase (NOS) inhibitor, the phosphatidylinositol 3'-kinase (PI3K), the extracellular-signal-regulated kinases 1/2 (ERK1/2) and the p38 mitogen-activated protein kinase (p38 MAPK) blockers. Specific kits were used for cell viability, mitochondrial membrane potential, NO, ROS and GSH production. Western blot was performed for apoptosis markers, NOS isoforms, and others kinases detection. Cell migration was analyzed by scratch assay, whereas cell proliferation and cell cycle through xCELLigence and flow cytometry. RESULTS: In RPE cells co-cultured with HUVEC in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in peroxidative conditions. Both anti-VEGF agents were able to prevent the fall of cell viability and mitochondrial membrane potential, an effect which was reduced by various inhibitors, and increased cell migration. Aflibercept/Ranibizumab counteracted the changes of apoptosis markers, NOS expression/activation, PI3K and ERK1/2 activation caused by peroxidation. These results were confirmed by cell cycle analysis. CONCLUSION: This study has shown new mechanisms at the basis of protective effects elicited by Aflibercept/Ranibizumab in RPE cells. HUVEC stimulated with Aflibercept/Ranibizumab, could release some paracrine factors that can modulate the RPE cells response in both physiologic and peroxidative conditions.


Assuntos
Comunicação Celular/efeitos dos fármacos , Ranibizumab/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Glutationa/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
2.
Int J Radiat Biol ; 96(1): 155-166, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31216213

RESUMO

Purpose: Evaluation of the pharmacodynamics (PD) and pharmacokinetics (PK) of romiplostim alone and in combination with pegfilgrastim in a non-human primate (NHP) model of acute radiation syndrome (ARS).Materials and methods: Male and female rhesus macaques were subjected to Cobalt-60 γ irradiation, at a dose of 550 cGy 24 h prior to subcutaneous administration of either romiplostim alone as a single (2.5 or 5.0 mg/kg on Day 1) or repeat dose (5.0 mg/kg on Days 1 and 8), pegfilgrastim alone as a repeat dose (0.3 µg/kg on Day 1 and 8), or a combination of both agents (romiplostim 5.0 mg/kg on Day 1; pegfilgrastim 0.3 µg/kg on Days 1 and 8). Clinical outcome, hematological parameters and PK were assessed throughout the 45 d study period post-irradiation.Results: Administration of romiplostim, pegfilgrastim or the combination of both resulted in significant improvements in hematological parameters, notably prevention of severe thrombocytopenia, compared with irradiated, vehicle control-treated NHPs. The largest hematologic benefit was observed when romiplostim and pegfilgrastim were administered as a combination therapy with much greater effects on both platelet and neutrophil recovery following irradiation compared to single agents alone.Conclusions: These results indicate that romiplostim alone or in combination with pegfilgrastim is effective at improving hematological parameters in an NHP model of ARS. This study supports further study of romiplostim as a medical countermeasure to improve primary hemostasis and survival in ARS.


Assuntos
Filgrastim/farmacologia , Neutropenia/tratamento farmacológico , Polietilenoglicóis/farmacologia , Lesões Experimentais por Radiação/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Trombocitopenia/tratamento farmacológico , Trombopoetina/farmacologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/efeitos da radiação , Relação Dose-Resposta a Droga , Interações de Medicamentos , Feminino , Macaca mulatta , Masculino , Neutropenia/sangue , Neutropenia/metabolismo , Lesões Experimentais por Radiação/sangue , Lesões Experimentais por Radiação/metabolismo , Receptores Fc/uso terapêutico , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêutico , Trombocitopenia/sangue , Trombocitopenia/metabolismo , Trombopoetina/farmacocinética , Trombopoetina/uso terapêutico , Fatores de Tempo
3.
Int J Radiat Biol ; 96(1): 145-154, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31021662

RESUMO

Purpose: Rapid depletion of white blood cells, platelets, and reticulocytes are hallmarks of hematopoietic injury of acute radiation syndrome (H-ARS) and, if left untreated, can lead to severe health consequences including death. While the granulocyte colony stimulating factors (G-CSF) filgrastim (Neupogen®), pegfilgrastim (Neulasta®), and sargramostim (Leukine®) are approved to increase survival in patients exposed to a myelosuppressive dose of radiation, no medical countermeasure is currently available for treatment of the thrombocytopenia that also results following radiation exposure. Romiplostim (Nplate®), a thrombopoietin receptor agonist, is the first FDA-approved thrombopoiesis-stimulating protein for the treatment of low platelet (PLT) counts in adults with chronic immune thrombocytopenia. Herein, we present the results of an analysis in mice of romiplostim as a medical countermeasure to improve survival and PLT recovery following acute radiation.Materials and methods: Male and female C57BL/6J mice (11 - 12 weeks of age, n = 21/sex/group) were total body irradiated (TBI) with 6.8 Gy X-rays that reduces 30-day survival to 30% (LD70/30). Vehicle, romiplostim, and/or pegfilgrastim were administered subcutaneously beginning 24 h after TBI for 1-5 days. Evaluation parameters included 30-day survival, pharmacokinetics, and hematology.Results: Full or maximal efficacy with an ∼40% increase in survival was achieved after a single 30 µg/kg dose of romiplostim. No further survival benefit was seen with higher (100 µg/kg) or more frequent dosing (3 or 5 once daily doses at 30 µg/kg) of romiplostim or combined treatment with pegfilgrastim. Pharmacodynamic analysis revealed that the platelet nadir was not as low and recovery was faster in the irradiated mice treated with romiplostim when compared with irradiated control animals (Day 8 versus 10 nadir; Day 22 versus 29 recovery to near baseline). Platelet volume also increased more rapidly after romiplostim injection. Kinetic profiles of other hematology parameters were similar between TBI romiplostim-treated and control mice. Peak serum levels of romiplostim in TBI mice occurred 4 - 24 h (Tmax) after injection with a t1/2 of ∼24 h. Cmax values were at ∼6 ng/ml after 30 µg/kg ± TBI and ∼200 ng/ml after 300 µg/kg. A 10-fold higher romiplostim dose increased the AUClast values by ∼35-fold.Conclusion: A single injection of romiplostim administered 24 h after TBI is a promising radiation medical countermeasure that dramatically increased survival, with or without pegfilgrastim, and hastened PLT recovery in mice.


Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/efeitos da radiação , Contramedidas Médicas , Proteínas Recombinantes de Fusão/farmacologia , Trombopoetina/farmacologia , Animais , Plaquetas/citologia , Relação Dose-Resposta a Droga , Interações de Medicamentos , Feminino , Filgrastim/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contagem de Plaquetas , Polietilenoglicóis/farmacologia , Receptores Fc , Proteínas Recombinantes de Fusão/farmacocinética , Análise de Sobrevida , Trombopoetina/farmacocinética , Raios X/efeitos adversos
4.
Medicine (Baltimore) ; 98(44): e17599, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31689763

RESUMO

This study compares 2 methods of macular function evaluation: the microperimetric examination (mean central retinal sensitivity and fixation stability) and the distance best-corrected visual acuity (BCVA) examination, which is the most frequently used method of assessing macular function in patients with newly diagnosed wet age-related macular degeneration (AMD) who have been treated with anti-vascular endothelial growth factor (VEGF) drug (aflibercept).Prospective analysis was conducted on 44 eyes of 44 patients treated with intravitreal injection of anti-VEGF (aflibercept) because of newly diagnosed neovascular AMD. According to the research protocol, all patients had a 6-month follow-up. The response to treatment was monitored functionallybyMP-1 microperimetry, fixation, and distance BCVA assessment after injection. Improvement of retinal sensitivity and BCVA was found under aflibercept treatment. There was statistically significant improvement in retinal sensitivity in the MP-1 study 3 and 6 months from the beginning of anti-VEGF therapy. Moreover, a significant improvement in retinal sensitivity between 3 and 6 months of observation was demonstrated. At the same time, up to 3 months from the beginning of treatment, BCVA improved significantly compared to the baseline value. In the 6th month of the study BCVA remained stable without further significant improvement.Microperimetric examination with medium sensitivity and fixation stability assessment is a very valuable test determining the retinal function. It is clear that examining the macular morphology itself in modern diagnostics is not enough to assess retinal function. Microperimetry technique is a valuable tool for functional long-term evaluation of retinal function (also for a period of more than 3 months).


Assuntos
Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Retina/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Degeneração Macular Exsudativa/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/farmacologia , Acuidade Visual
5.
Mol Immunol ; 116: 73-79, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31630078

RESUMO

Atherosclerosis is a common comorbidity of type II diabetes and a leading cause of death worldwide. The presence of oxidized low-density lipoprotein (ox-LDL) drives atherogenesis by inducing oxidative stress, mitochondrial dysfunction, expression of proinflammatory cytokines and chemokines including interleukin (IL)-1ß, IL-6, and monocyte chemoattractant protein 1 (MCP-1), adhesion molecules including vascular cellular adhesion molecule 1 (VCAM-1) and E-selectin, and downregulating expression of the Krüppel-like factor 2 (KLF2) transcription factor. Importantly, ox-LDL induced the attachment of THP-1 monocytes to endothelial cells. In the present study, we demonstrate for the first time that the specific glucagon-like peptide 1 receptor (GLP-1R) agonist dulaglutide may prevent these atherosclerotic effects of ox-LDL by preventing suppression of KLF2 by p53 protein in human aortic endothelial cells. KLF2 has been shown to play a major role in protecting vascular endothelial cells from damage induced by ox-LDL and oscillatory shear, and therefore, therapies capable of mediating KLF2 signaling may be an attractive treatment option for preventing the development and progression of atherosclerosis.


Assuntos
Aterosclerose/tratamento farmacológico , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Peptídeos Semelhantes ao Glucagon/análogos & derivados , Fragmentos Fc das Imunoglobulinas/farmacologia , Lipoproteínas LDL/metabolismo , Monócitos/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Aterosclerose/metabolismo , Adesão Celular , Células Cultivadas , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos Semelhantes ao Glucagon/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Monócitos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismo
6.
Int J Mol Sci ; 20(18)2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31547231

RESUMO

NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug's activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.


Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias/irrigação sanguínea , Oligopeptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Inibidores da Angiogênese/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Moleculares , Oligopeptídeos/química , Proteínas Recombinantes de Fusão/química , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/química
7.
Molecules ; 24(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394739

RESUMO

Nanobodies (VHHs) overcome many of the drawbacks of conventional antibodies, and the related technologies represent state-of-the-art and advanced applications in scientific research, pharmaceuticals, and therapies. In terms of productivity and economic cost, the cytoplasmic expression of VHHs in Escherichia coli (E. coli) is a good process for their recombinant production. The cytoplasmic environment of the host is critical to the affinity and stability of the recombinant VHHs in soluble form, yet the effects have not been studied. For this purpose, recombinant anti-ß2 microglobulin VHHs were constructed and expressed in four commercialized E. coli hosts, including BL21 (DE3), Rosetta-gami B (DE3) pLysS, Origami 2 (DE3) and SHuffle T7 Express. The results showed that anti-ß2 microglobulin (ß2MG) VHHs expressed in different hosts exhibited distinctive differences in the affinity and structural characteristics. The VHHs expressed in Rosetta-gami B (DE3) pLysS possessed not only the greatest affinity of (equilibrium dissociation constant) KD = 4.68 × 10-8 M but also the highest yields compared with the VHHs expressed in BL21 (DE3), Origami 2 (DE3) and SHuffle T7 Express. In addition, the VHHs expressed in Rosetta-gami B (DE3) pLysS were more stable than the VHHs expressed in the rest three hosts. Thus far, we have successfully realized the high expression of the active and robust anti-ß2MG VHHs in Rosetta-gami B (DE3) pLysS. The underlying principle of our study is able to guide the expression strategies of nanobodies on the context of industrial large-scale production.


Assuntos
Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia , Microglobulina beta-2/antagonistas & inibidores , Afinidade de Anticorpos , Escherichia coli/genética , Expressão Gênica , Estabilidade Proteica , Proteólise , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/genética , Análise Espectral , Termodinâmica , Tripsina/química , Microglobulina beta-2/química
8.
Molecules ; 24(16)2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31395814

RESUMO

Antibacterial lysins are promising proteins that are active against both antibiotic-susceptible and antibiotic-resistant bacterial strains. However, a major limitation of antibacterial lysins is their fast elimination from systemic circulation. PEGylation increases the plasma half-life of lysins but renders them inactive. Here we report the construction of a fusion protein of lysostaphin, a potent anti-staphylococcal lysin, and an albumin-binding domain from streptococcal protein G. The resulting fusion protein was less active than the parent enzyme lysostaphin, but it still retained significant antibacterial activity even when bound to serum albumin. The terminal half-life of the fusion protein in rats was five-fold greater than that of lysostaphin (7.4 vs. 1.5 h), and the area under the curve increased more than 115 times. Most importantly, this increase in systemic circulation time compensated for the decrease in activity. The plasma from rats that received an injection of the fusion protein retained bactericidal activity for up to 7 h, while plasma from rats that received plain lysostaphin lacked any detectable activity after 4 h. To the best of our knowledge, this is the first report of an antibacterial lysin with both improved pharmacokinetic parameters and prolonged bactericidal activity in the systemic circulation.


Assuntos
Proteínas de Bactérias , Lisostafina , Proteínas Recombinantes de Fusão , Albumina Sérica/química , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacocinética , Proteínas de Bactérias/farmacologia , Feminino , Lisostafina/química , Lisostafina/genética , Lisostafina/farmacocinética , Lisostafina/farmacologia , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia
10.
Invest Ophthalmol Vis Sci ; 60(10): 3547-3555, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415078

RESUMO

Purpose: Current treatments for diabetic retinopathy (DR) have considerable limitations, underpinning the need for new therapeutic options. In this article, the ability of an engineered angiopoietin-1 variant (COMP-Ang1) to ameliorate the injurious effects of hyperglycemia on barrier integrity in a human retinal microvascular endothelial cell (HRMvEC) model is comprehensively investigated. Methods: Confluent HRMvECs were treated (0-72 hours) with d-glucose (5 or 30 mM) in the absence and presence of COMP-Ang1 (10-200 ng/mL). l-glucose (30 mM) was used as osmotic control. Posttreatment, intact cell monolayers were monitored for permeability to FITC-dextran 40 kDa. Cells were also harvested for analysis of interendothelial junction targets by RT-qPCR and Western blotting. The impact of receptor tyrosine kinase Tie2 gene silencing on COMP-Ang1 efficacy was also evaluated. Results: Treatment with 30 mM d-glucose (but not l-glucose) demonstrated a time-dependent elevation in the mean rate of FITC-dextran diffusion across intact HRMvEC monolayers, in parallel with significant reductions in mRNA/protein levels of occludin, claudin-5, ZO-1, and VE-Cadherin. These effects were all attenuated by COMP-Ang1 in a concentration-dependent fashion, with 200 ng/mL recovering barrier function by ∼88%, and recovering reduced interendothelial junction protein levels by more than 50%. Finally, Tie2 knockdown by small interfering RNA silencing blocked the ability of COMP-Ang1 to mitigate against hyperglycemia-induced permeabilization of HRMvECs and depletion of junctional expression levels. Conclusions: In summary, this article presents a reproducible in vitro cell study that quantifies the concentration-dependent efficacy of COMP-Ang1 to mitigate the injurious effects of hyperglycemic challenge on HRMvEC barrier properties via Tie2-mediated signaling.


Assuntos
Barreira Hematorretiniana/fisiologia , Células Endoteliais/efeitos dos fármacos , Hiperglicemia/prevenção & controle , Proteínas Recombinantes de Fusão/farmacologia , Vasos Retinianos/efeitos dos fármacos , Antígenos CD/genética , Western Blotting , Caderinas/genética , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Claudina-5/genética , Dextranos/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Inativação Gênica/fisiologia , Glucose/farmacologia , Humanos , Hiperglicemia/metabolismo , Ocludina/genética , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor TIE-2/genética , Vasos Retinianos/metabolismo
11.
Protein Pept Lett ; 26(12): 930-939, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31441722

RESUMO

BACKGROUND: Supercharged GFP proteins were known as effective carriers for delivery of macromolecules into eukaryotic cells as well as fluorescent fusion tags for in vitro and in vivo detection. OBJECTIVE: Herein, anti-viral effects of +36 GFP and its anti-tumor effects were studied in vitro and in vivo, respectively. METHODS: We evaluated anti-HIV, anti-HSV, and anti-HCV effects of +36 GFP in vitro using ELISA, and real time PCR as common techniques for their detection, respectively. Moreover, we assessed the role of +36 GFP for eliciting HPV-related anti-tumor effects in mice due to the lack of HPV replication in vitro. RESULTS: Our data showed that +36 GFP efficiently enter the cells and augment the transfection rate of HPV16E7 antigen, as well. Furthermore, +36 GFP significantly reduced HCV, HIV and HSV replication up to 75%, 49% and 43% in HCV-infected Huh7.5 cells, HIV-infected Hela cells and HSV-infected Vero cells, respectively. On the other hand, mice immunization with +36 GFP complexed with HPV16 E7 antigen (+36GFP + E7) or fused to HPV16 E7 antigen (+36GFP-E7) elicited a higher Th1 cellular immune response with the predominant IgG2a, IgG2b, IFN-γ and Granzyme B levels than those induced by other groups. These regimens protected mice against TC- 1 tumor challenge (~ 67%) compared to E7 protein alone (~ 33%). These data suggested that +36 GFP can act as an anti-viral agent at certain dose due to its high efficiency in cell penetration in vitro and in vivo. CONCLUSION: Generally, +36 GFP targets viral replication in vitro as well as helps to suppress the growth of HPV-related tumors in vivo.


Assuntos
Antivirais/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas Mutantes/genética , Vacinas contra Papillomavirus/genética , Proteínas Recombinantes de Fusão/genética , Animais , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Linhagem Celular , Feminino , Granzimas/metabolismo , Proteínas de Fluorescência Verde/imunologia , HIV/efeitos dos fármacos , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Hepacivirus/efeitos dos fármacos , Papillomavirus Humano 16/efeitos dos fármacos , Humanos , Imunidade Celular , Interferon gama/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Mutantes/imunologia , Proteínas Mutantes/farmacologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Replicação Viral/efeitos dos fármacos
12.
Int J Oncol ; 55(4): 823-832, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432158

RESUMO

The selective induction of tumor vascular thrombosis using truncated tissue factor (tTF) delivered via a target ligand is a promising novel antitumor strategy. In the present study, an anti­neuropilin­1 (NRP­1) monoclonal antibody (mAb)­streptavidin (SA):tTF­biotin (B) composite system was established. In this system, anti­NRP­1­mAb located tTF to the tumor vascular endothelial cell surface and induced vascular embolization. Due to their high binding affinity, SA and B were used to enhance thrombogenic activity. mAb was conjugated with SA using a coupling method with water­soluble 1­ethyl­3­(3­dimethylaminopropyl) carbodiimide and N­hydroxysulfosuccinimide. Biotinylated tTF (tTF­B) was prepared using a B­labeling kit subsequent to the generation and purification of fusion protein tTF. Confocal microscopy and flow cytometry indicated that the anti­NRP­1­mAb­SA conjugate retained mAb targeting activity. The preservation of B­conjugate binding capacity was confirmed using a competitive ELISA, and factor X­activation analysis revealed that tTF­B retained the procoagulant activity exhibited by tTF. Live imaging was performed to assess mAb­SA distribution and tumor­targeting capability, and this yielded promising results. The results of in vivo studies in mice with subcutaneous xenografts demonstrated that this composite system significantly induced tumor vascular thrombosis and inhibited tumor growth, whereas these histological changes were not observed in normal organs.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Neuropilina-1/imunologia , Tromboplastina/administração & dosagem , Trombose/induzido quimicamente , Animais , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Fator X/metabolismo , Feminino , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/metabolismo , Camundongos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Estreptavidina/química , Tromboplastina/química , Tromboplastina/farmacologia , Trombose/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Exp Clin Cancer Res ; 38(1): 332, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31362764

RESUMO

BACKGROUND: Immunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade. METHODS: We designed a recombinant fusion construct, GrB-Fc-4D5, composed of a humanized anti-HER2 scFv fused to active GrB for recognition of tumor cells and internal delivery of GrB, simulating T and B cell therapy. We assessed the construct's antigen-binding specificity and GrB enzymatic activity, as well as in vitro cytotoxicity and internalization into target and control cells. We also assessed pharmacokinetic and toxicology parameters in vivo. RESULTS: GrB-Fc-4D5 was highly cytotoxic to Her2 positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC50 values of 56, 99 and 27 nM, respectively, and against a panel of HER2+ cell lines regardless of endogenous expression levels of the PI-9 inhibitor. Contemporaneous studies with Kadcyla demonstrated similar levels of in vitro activity against virtually all cells tested. GrB-Fc-4D5 internalized rapidly into target SKOV3 cells within 1 h of exposure rapidly delivering GrB to the cytoplasmic compartment. In keeping with its relatively high molecular weight (160 kDa), the construct demonstrated a terminal-phase serum half-life in mice of 39.2 h. Toxicity studies conducted on BALB/c mice demonstrated no statistically significant changes in SGPT, SGOT or serum LDH. Histopathologic analysis of tissues from treated mice demonstrated no drug-related changes in any tissues examined. CONCLUSION: GrB-Fc-4D5 shows excellent, specific cytotoxicity and demonstrates no significant toxicity in normal, antigen-negative murine models. This construct constitutes a novel approach against HER2-expressing tumors and is an excellent candidate for further development.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Desenvolvimento de Medicamentos , Terapia de Alvo Molecular , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/isolamento & purificação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Expressão Gênica , Vetores Genéticos/genética , Granzimas/administração & dosagem , Granzimas/genética , Humanos , Camundongos , Terapia de Alvo Molecular/métodos , Ligação Proteica/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Expert Rev Clin Pharmacol ; 12(10): 941-946, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31465247

RESUMO

Introduction: CD123 or interleukin 3 receptor alpha is overexpressed in multiple hematologic malignancies. Tagraxofusp is an intravenously administered CD123-directed cytotoxin consisting of the fusion of interleukin-3 with a truncated diphtheria toxin payload and was recently approved by the Food and Drug Administration for the treatment of adults and children aged 2 and older with blastic plasmacytoid dendritic cell neoplasm (BPDCN). Areas Covered: In this review, we discuss the use of tagraxofusp in BPDCN, and active clinical trials involving this agent in several hematologic malignancies are also presented. Tagraxofusp has significant efficacy in patients with BPDCN and manageable safety profile, with the most commonly reported adverse events being asymptomatic elevation of alanine and aspartate aminotransferase levels, hypoalbuminemia, peripheral edema, and thrombocytopenia. The most serious side effect is capillary leak syndrome that can be lethal in some cases but the risk may be mitigated by early recognition and intervention. Expert Opinion: Tagraxofusp has been introduced as a novel treatment of BPDCN, a rare hematologic malignancy, for which no standard therapy previously existed. Many patients treated with this agent were able to be bridged to stem cell transplantation, including older patients. In the future, combinations of tagraxofusp with other targeted agents will be explored.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Hematológicas/tratamento farmacológico , Proteínas Recombinantes de Fusão/administração & dosagem , Adulto , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Criança , Pré-Escolar , Células Dendríticas/patologia , Neoplasias Hematológicas/patologia , Humanos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Terapia de Alvo Molecular , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacologia , Transplante de Células-Tronco/métodos
15.
Int J Mol Sci ; 20(17)2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31454926

RESUMO

Interleukin (IL)-23 is considered an effective therapeutic target for the treatment of psoriasis because of the crucial role of the IL-23/IL-17 axis in the pathogenesis of psoriasis, and it has recently been reported to be involved in ILC3 cell differentiation. In this study, we report that eukaryotically expressed rhIL23R-CHR/Fc, as an endogenous extracellular receptor analogue, could be a natural antagonist in an imiquimod (IMQ)-induced psoriasis-like mouse model, including the antagonizing effect of suppressed inflammation in the skin lesion, decreased production of pro-inflammatory cells, and reduced the expression of pro-inflammatory factors. The rhIL23R-CHR/Fc fusion protein inhibits both innate immune and adaptive immune-mediated inflammatory responses. These findings shed light on rhIL23R-CHR/Fc as a promising candidate therapy for the treatment of psoriasis.


Assuntos
Imunidade Inata/efeitos dos fármacos , Psoríase/etiologia , Psoríase/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Domínios e Motivos de Interação entre Proteínas , Psoríase/tratamento farmacológico , Psoríase/patologia , Receptores de Interleucina/química , Receptores de Interleucina/genética , Proteínas Recombinantes de Fusão/genética , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Pele/patologia , Células Th17/citologia
16.
Int J Med Sci ; 16(7): 1032-1041, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31341417

RESUMO

AP25 is an anti-tumor peptide with a high affinity for integrins. It exerts its anti-tumor activity by inhibiting angiogenesis and by directly inhibiting the growth of tumor cells. Its half-life time in vivo is only about 50 minutes, which limits its clinical application. In order to prolong the half-life time of AP25 while preserving its anti-tumor activity, several fusion proteins of AP25 and IgG4 Fc were designed and expressed; their anti-tumor activity and pharmacokinetics properties were evaluated. Firstly, four AP25-Fc fusion protein sequences were designed, and the corresponding proteins were expressed and purified. Based on the results of HUVEC migration inhibition assay, HUVEC and tumor cell proliferation inhibition assay and yields of expression by HEK293 cells, the fusion protein designated PSG4R was selected for further evaluation. The anti-tumor effect of PSG4R was then evaluated in vivo on HCT-116 nude mice xenograft model. And the pharmacokinetics properties of PSG4R were investigated in rats. The results showed that PSG4R could inhibit the growth of xenografts of human colon cancer cell line HCT-116 in nude mice by intravenous administration of 40 mg/kg once every two days. The half-life time of PSG4R was 56.270 ± 15.398 h. This study showed that the construction of AP25-Fc fusion protein could significantly prolong the half-life of AP25 while retaining its anti-tumor activity, which provides a new direction for new drug development of AP25.


Assuntos
Endostatinas/farmacologia , Imunoconjugados/farmacologia , Imunoglobulina G/farmacologia , Neoplasias/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Administração Intravenosa , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Endostatinas/genética , Endostatinas/uso terapêutico , Feminino , Células HCT116 , Células HEK293 , Meia-Vida , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoconjugados/genética , Imunoconjugados/uso terapêutico , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Masculino , Camundongos , Modelos Animais , Neoplasias/patologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/uso terapêutico , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
17.
PLoS One ; 14(7): e0217205, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31329599

RESUMO

Adjunctive use of antibiotics in periodontal treatment have limitations and disadvantages including bacterial resistance. Antimicrobial peptides (AMPs) are potential new agents that can combat bacterial infection. In this study, antimicrobial activity of different concentrations of conventional antibiotics minocycline (MH), doxycycline (DOX), and antimicrobial peptides LL-37, LL-31, Lactoferrin chimera (LFchimera) and Innate Defense Regulator Peptide 1018 (IDR-1018) against Aggregatibacter actinomycetemcomitans ATCC 43718 were determined using colony culturing assay. Subsequently, in vitro activity of the most effective drug and peptide combination was evaluated by checkerboard technique. Impact of the drug and peptide co-administration on biofilm at different stages, i.e., during adhesion and 1-day old biofilm was compared to each of the agents used alone. Results revealed that the killing effects of all AMPs range from 13-100%. In contrast, MH and DOX at 1 and 5 µM showed no killing activity and instead stimulated growth of bacteria. DOX has better killing activity than MH. LFchimera displayed the strongest killing amongst the peptides. Checkerboard technique revealed that combining DOX and LFchimera yielded synergism. Confocal laser scanning microscopy further showed that the combination of DOX and LFchimera caused significant reduction of bacterial adhesion and reduction of biomass, average biofilm thickness and substratum biofilm coverage of 1-day old biofilm compared to DOX and LFchimera alone. In conclusion, LFchimera alone and in combination with DOX exhibited strong antibacterial and anti-biofilm property against A. actinomycetemcomitans. The findings suggest that LFchimera should be considered for development as a new potential therapeutic agent that may be used as an adjunctive treatment for periodontitis.


Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Lactoferrina/farmacologia , Plâncton/crescimento & desenvolvimento , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/agonistas , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Biofilmes/crescimento & desenvolvimento , Sinergismo Farmacológico , Humanos , Lactoferrina/agonistas , Lactoferrina/química , Lactoferrina/genética , Periodontite/tratamento farmacológico , Periodontite/microbiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia
18.
Toxicon ; 168: 40-48, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31251993

RESUMO

Venoms of spiders and snakes contain toxins extremely active and, thus, provide a natural source for the development of new biotechnological tools. Among the diversity of toxins present in the venom of spiders from genus Loxosceles, the phospholipases D (PLDs) show high hydrolytic activity upon lysophosphatidylcholine (LPC) and sphingomyelin (SM), generating bioactive phospholipids such as cyclic phosphatidic acid (cPA). Since this mediator has been shown to play a major role in complex signaling pathways, including inhibition of tumor cells, the PLDs may hold the key to learn how toxins could be used for therapeutic purposes. However, the strong platelet aggregation of PLDs and their lack of selectivity impose a major limitation. On the other hand, disintegrins present in the venoms of Viperidae snakes are a potent inhibitor of platelet aggregation and possess high affinity and specificity to molecules called integrins that are highly expressed in some tumor cells, such as murine melanoma B16F10. Therefore, disintegrins might be suitable molecules to carry the PLDs to the malignant cells, so both toxins may work synergistically to eliminate these cells. Thus, in this work, a recombinant PLD from Loxosceles gaucho spider was recombinantly fused to a disintegrin from Echis carinatus snake to form a hybrid toxin called Rechistatin. This recombinant toxin was successfully expressed in bacteria, showed binding activity in B16F10 murine melanoma cells and exerted a synergistic cytotoxicity effect on these cells. Therefore, the approach presented in this work may represent a new strategy to explore new potential applications for spider PLDs.


Assuntos
Desintegrinas/genética , Fosfolipase D/genética , Proteínas Recombinantes de Fusão/farmacologia , Animais , Humanos , Melanoma Experimental , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Aranhas , Viperidae
19.
Int Immunopharmacol ; 74: 105649, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31185450

RESUMO

Rheumatoid arthritis is a common autoimmune disease primarily characterized by chronic inflammation, the formation of an invasive pannus, and destruction of the joints. In the present study, we employed real-time PCR and western blot analysis to investigate the role of dulaglutide in human fibroblast-like synoviocytes (FLS). The results of our study show that dulaglutide exerted a powerful protective effect by rescuing mitochondrial membrane potential, inhibiting the production of NOX-4, and abrogating TNF-α-induced downregulation of the antioxidant GSH. Our findings demonstrate that dulaglutide significantly ameliorated the expression of proinflammatory cytokines and chemokines including IL-1ß, IL-6, MCP-1, and HMGB-1. Matrix metalloproteinases mediate cartilage destruction, thereby aiding in pannus formation. Our findings indicate that dulaglutide treatment significantly downregulated the expression of MMP-3 and MMP-13, two crucial degradative enzymes. Importantly, the results of our study demonstrate that the beneficial effects of dulaglutide are mediated through the JNK/NF-κB signaling pathway, which has been suggested as a potential treatment target against RA. Taken together, the results of this study show that dulaglutide may exert significant protective effects against the progression of RA induced by TNF-α.


Assuntos
Anti-Inflamatórios/farmacologia , Peptídeos Semelhantes ao Glucagon/análogos & derivados , Fragmentos Fc das Imunoglobulinas/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Sinoviócitos/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Fibroblastos , Peptídeos Semelhantes ao Glucagon/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sinoviócitos/metabolismo
20.
Mol Cell Biol ; 39(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31208977

RESUMO

Membrane type 1-matrix metalloproteinase (MT1-MMP) and tumor necrosis factor α (TNF-α)-converting enzyme (TACE) are prominent membrane-anchored metalloproteinases that regulate the turnover of extracellular matrix (ECM) components and bioactive molecules required for cancer proliferation. In this study, we describe a novel approach that would allow tissue inhibitor of metalloproteinase 1 (TIMP-1), the endogenous inhibitor of the matrix metalloproteinases (MMPs), to be translocated to the cell membrane for simultaneous MT1-MMP/TACE inhibition. We achieve this by fusing T1TACE, a designer TIMP-1 with superb affinities for MT1-MMP and TACE, to the glycosyl-phosphatidyl inositol anchor of prions to create a membrane-tethered, broad-spectrum inhibitor, named T1Pr αTACE, that colocalizes with MT1-MMP and TACE on the cell surface. Transduction of T1Pr αTACE in human fibrosarcoma cells results not only in a substantial reduction in gelatinolytic and TNF-α/heparin binding epithelial growth factor shedding activities but also in a loss of tubulogenic capability in three-dimensional matrices. In renal carcinoma, T1Pr αTACE triggers cellular senescence and disrupts MMP-mediated proteolysis of ECM components such as fibronectin and collagen I, leading to an impairment in cell motility and survival under both in vitro and in vivo conditions. Taken together, our findings may provide a new perspective in TIMP targeting that could be exploited to halt metastatic renal carcinoma progression.


Assuntos
Carcinoma de Células Renais/terapia , Glicosilfosfatidilinositóis/metabolismo , Neoplasias Renais/terapia , Príons/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Inibidor Tecidual de Metaloproteinase-1/genética , Células A549 , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Renais/metabolismo , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Transporte Proteico , Proteínas Recombinantes de Fusão/farmacologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Transdução Genética , Ensaios Antitumorais Modelo de Xenoenxerto
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