RESUMO
The neutralization of tumor necrosis factor alpha (TNFα) with biopharmaceuticals is a successful therapy for inflammatory diseases. Currently, one of the main TNFα-antagonists is Etanercept, a dimeric TNF-R2 ectodomain. Considering that TNFα and its receptors are homotrimers, we proposed that a trimeric TNF-R2 ectodomain could be an innovative TNFα-antagonist. Here, the 3cTNFR2 protein was designed by the fusion of the TNF-R2 ectodomain with the collagen XV trimerization domain. 3cTNFR2 was produced in HEK293 cells and purified by immobilized metal affinity chromatography. Monomers, dimers, and trimers of 3cTNFR2 were detected. The interaction 3cTNFR2-TNFα was assessed. By microscale thermophoresis, the KD value for the interaction was 4.17 ± 0.88 nM, and complexes with different molecular weights were detected by size exclusion chromatography-high performance liquid chromatography. Moreover, 3cTNFR2 neutralized the TNFα-induced cytotoxicity totally in vitro. Although more studies are required to evaluate the anti-inflammatory effect, the results suggest that 3cTNFR2 could be a TNFα-antagonist agent.
Assuntos
Anti-Inflamatórios/farmacologia , Colágeno/genética , Endotoxinas/antagonistas & inibidores , Etanercepte/farmacologia , Receptores Tipo II do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Colágeno/metabolismo , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Etanercepte/química , Etanercepte/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas/métodos , Multimerização Proteica , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9 nM) and MTV-LRP1 (Kd 399 ± 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.
Assuntos
Anti-Inflamatórios/farmacologia , Endotoxinas/antagonistas & inibidores , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Peptídeos/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Barreira Hematoencefálica/metabolismo , Células CHO , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Expressão Gênica , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Modelos Biológicos , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas/métodos , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
TZP4 is a triazine-based amphipathic polymer designed to mimic the amphipathic structure found in antimicrobial peptides. TZP4 showed potent antimicrobial activity comparable to melittin against antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. TZP4 showed high resistance to proteolytic degradation and low tendency to develop drug resistance. The results from membrane depolarization, SYTOX Green uptake, flow cytometry, and gel retardation revealed that the mechanism of antimicrobial action of TZP4 involved an intracellular target rather than the bacterial cell membrane. Furthermore, TZP4 suppressed the messenger RNA levels of inducible nitric oxide synthase and tumor necrosis factor-α (TNF-α) and inhibited the release of nitric oxide and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. BODIPY-TR-cadaverine displacement and dissociation of fluorescein isothiocyanate (FITC)-labeled LPS assays revealed that TZP4 strongly bound to LPS and disaggregated the LPS oligomers. Flow cytometric analysis demonstrated that TZP4 inhibits the binding of FITC-conjugated LPS to RAW264.7 cells. These observations indicate that TZP4 may exert its antiendotoxic activity by directly binding with LPS and inhibiting the interaction between LPS and CD14+ cells. Collectively, TZP4 is a promising drug candidate for the treatment of endotoxic shock and sepsis caused by Gram-negative bacterial infections.
Assuntos
Anti-Infecciosos , Peptídeos Antimicrobianos , Polímeros , Triazinas , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Anti-Infecciosos/toxicidade , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Farmacorresistência Bacteriana , Endotoxinas/antagonistas & inibidores , Lipopolissacarídeos/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Polímeros/química , Polímeros/farmacologia , Polímeros/toxicidade , Células RAW 264.7 , Triazinas/química , Triazinas/farmacologia , Triazinas/toxicidadeRESUMO
The most potent cell wall-derived inflammatory toxins ("pathogenicity factors") of Gram-negative and -positive bacteria are lipopolysaccharides (LPS) (endotoxins) and lipoproteins (LP), respectively. Despite the fact that the former signals via toll-like receptor 4 (TLR4) and the latter via TLR2, the physico-chemistry of these compounds exhibits considerable similarity, an amphiphilic molecule with a polar and charged backbone and a lipid moiety. While the exterior portion of the LPS (i.e., the O-chain) represents the serologically relevant structure, the inner part, the lipid A, is responsible for one of the strongest inflammatory activities known. In the last years, we have demonstrated that antimicrobial peptides from the Pep19-2.5 family, which were designed to bind to LPS and LP, act as anti-inflammatory agents against sepsis and endotoxic shock caused by severe bacterial infections. We also showed that this anti-inflammatory activity requires specific interactions of the peptides with LPS and LP leading to exothermic reactions with saturation characteristics in calorimetry assays. Parallel to this, peptide-mediated neutralization of LPS and LP involves changes in various physical parameters, including both the gel to liquid crystalline phase transition of the acyl chains and the three-dimensional aggregate structures of the toxins. Furthermore, the effectivity of neutralization of pathogenicity factors by peptides was demonstrated in several in vivo models together with the finding that a peptide-based therapy sensitizes bacteria (also antimicrobial resistant) to antibiotics. Finally, a significant step in the understanding of the broad anti-inflammatory function of Pep19-2.5 was the demonstration that this compound is able to block the intracellular endotoxin signaling cascade.
Assuntos
Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/etiologia , Lipopolissacarídeos/efeitos adversos , Lipoproteínas/efeitos adversos , Peptídeos/uso terapêutico , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Inflamatórios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Endotoxinas/efeitos adversos , Endotoxinas/antagonistas & inibidores , Endotoxinas/química , Humanos , Inflamação/metabolismo , Peptídeos/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Wound healing is a complex process that is essential to provide skin homeostasis. Infection with pathogenic bacteria such as Staphylococcus aureus can lead to chronic wounds, which are challenging to heal. Previously, we demonstrated that the antimicrobial endotoxin-neutralizing peptide Pep19-2.5 promotes artificial wound closure in keratinocytes. Here, we investigated the mechanism of peptide-induced cell migration and if Pep19-2.5 accelerates wound closure in vivo. EXPERIMENTAL APPROACH: Cell migration was examined in HaCaT keratinocytes and P2X7 receptor-overexpressing HEK293 cells using the wound healing scratch assay. The protein expression of phosphorylated ERK1/2, ATP release, calcium influx and mitochondrial ROS were analysed to characterize Pep19-2.5-mediated signalling. For in vivo studies, female BALB/c mice were wounded and infected with methicillin-resistant S. aureus (MRSA) or left non-infected and treated topically with Pep19-2.5 twice daily for 6 days. KEY RESULTS: Specific P2X7 receptor antagonists inhibited Pep19-2.5-induced cell migration and ERK1/2 phosphorylation in keratinocytes and P2X7 receptor-transfected HEK293 cells. ATP release was not increased by Pep19-2.5; however, ATP was required for cell migration. Pep19-2.5 increased cytosolic calcium and mitochondrial ROS, which were involved in peptide-induced migration and ERK1/2 phosphorylation. In both non-infected and MRSA-infected wounds, the wound diameter was reduced already at day 2 post-wounding in the Pep19-2.5-treated groups compared to vehicle, and remained decreased until day 6. CONCLUSIONS AND IMPLICATIONS: Our data suggest the potential application of Pep19-2.5 in the treatment of non-infected and S. aureus-infected wounds and provide insights into the mechanism involved in Pep19-2.5-induced wound healing.
Assuntos
Antibacterianos/farmacologia , Endotoxinas/antagonistas & inibidores , Queratinócitos/efeitos dos fármacos , Peptídeos/farmacologia , Agonistas Purinérgicos/farmacologia , Receptores Purinérgicos P2X7/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Antibacterianos/química , Linhagem Celular Transformada , Feminino , Células HEK293 , Humanos , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Chorioamnionitis (CA) is associated with a high risk for the development of bronchopulmonary dysplasia (BPD) after preterm birth, but mechanisms that increase susceptibility for BPD and strategies to prevent BPD are uncertain. As a model of CA, antenatal intra-amniotic (IA) endotoxin (ETX) exposure alters placental structure, causes fetal growth restriction, increases perinatal mortality, and causes sustained cardiorespiratory abnormalities throughout infancy. Vitamin D (Vit D) has been shown to have both anti-inflammatory and proangiogenic properties. Antenatal IA treatment with Vit D (1,25-(OH)2D3) during IA ETX exposure improves survival and increases vascular and alveolar growth in infant rats. Whether IA ETX causes decreased placental vascular development and if the protective effects of prenatal Vit D treatment are due to direct effects on the fetus or to improved placental vascular development remain unknown. OBJECTIVE: The objective of this study was to determine if IA ETX impairs placental vascular development and Vit D metabolism, and whether 1,25-(OH)2D3 treatment improves placental vascularity after IA ETX exposure during late gestation in pregnant rats. DESIGN/METHODS: Fetal rats were exposed to ETX (10 mg), ETX + 1,25-(OH)2D3 (1 ng/mL), 1,25-(OH)2D3 (1 ng/mL), or saline (control) via IA injection at E20 and delivered 2 days later. To assess placental vascular development, histologic sections from the placenta were stained for CD31 and vessel density per high power field (HPF) was determined and analyzed using Matlab software. To determine the effects of ETX on placental Vit D metabolism, Vit D receptor (VDR) and activity of the Vit D conversion enzyme, CYP27B1, were assayed from placental homogenates. Angiogenic mediators were measured by reverse transcription polymerase chain reaction by RNA extracted from placental tissue. RESULTS: IA ETX reduced placenta and newborn birth weights by 22 and 20%, respectively, when compared with controls (placental weight: 0.60 vs. 0.47 g; p < 0.0001; birth weight: 4.68 vs. 5.88 g; p < 0.0001). IA 1,25-(OH)2D3 treatment increased birth weight by 12% in ETX-exposed pups (5.25 vs. 4.68 g; p < 0.001). IA ETX decreased placental vessel density by 24% in comparison with controls (1,114 vs. 848 vessels per HPF; p < 0.05). Treatment with IA 1,25-(OH)2D3 increased placenta vessel density twofold after ETX exposure (1,739 vs. 848); p < 0.0001), and increased vessel density compared with saline controls by 56% (1,739 vs. 1,114; p < 0.0001). IA ETX decreased both VDR and CYP27B1 expression by 83 and 35%, respectively (p < 0.01). CONCLUSION: IA ETX decreases placental growth and vessel density and decreases placental VDR and CYP27B1 protein expression, and that antenatal 1,25-(OH)2D3 restores placental weight and vessel density, as well as birth weight. We speculate that 1,25-(OH)2D3 treatment preserves placental function in experimental CA and that these effects may be mediated by increased vascular growth.
Assuntos
Indutores da Angiogênese/farmacologia , Displasia Broncopulmonar/prevenção & controle , Corioamnionite/prevenção & controle , Desenvolvimento Fetal/efeitos dos fármacos , Placenta , Vitamina D , Animais , Endotoxinas/antagonistas & inibidores , Feminino , Retardo do Crescimento Fetal/prevenção & controle , Placenta/irrigação sanguínea , Placenta/efeitos dos fármacos , Placenta/patologia , Gravidez , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Vitamina D/farmacologia , Vitaminas/farmacologiaRESUMO
Infectious diseases caused by Gram-negative bacteria and sepsis induced by lipopolysaccharide (LPS) pose a major threat to humans and animals and cause millions of deaths each year. Marine organisms are a valuable resource library of bioactive products with huge medicinal potential. Among them, antibacterial and antiendotoxic peptides or proteins, which are composed of metabolically tolerable residues, are present in many marine species, including marine vertebrates, invertebrates and microorganisms. A lot of studies have reported that these marine peptides and proteins or their derivatives exhibit potent antibacterial activity and antiendotoxic activity in vitro and in vivo. However, their categories, heterologous expression in microorganisms, physicochemical factors affecting peptide or protein interactions with bacterial LPS and LPS-neutralizing mechanism are not well known. In this review, we highlight the characteristics and anti-infective activity of bifunctional peptides or proteins from marine resources as well as the challenges and strategies for further study.
Assuntos
Antibacterianos/farmacologia , Endotoxinas/antagonistas & inibidores , Toxinas Marinhas/farmacologia , Peptídeos/farmacologia , Animais , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Biologia Marinha , Toxinas Marinhas/química , Peptídeos/isolamento & purificaçãoRESUMO
Toll-like receptor (TLR) 4-independent recognition of lipopolysaccharide (LPS) in the cytosol by inflammatory caspases leads to non-canonical inflammasome activation and induction of IL-1 secretion and pyroptosis. The discovery of this novel mechanism has potential implications for the development of effective drugs to treat sepsis since LPS-mediated hyperactivation of caspases is critically involved in endotoxic shock. Previously, we demonstrated that Pep19-2.5, a synthetic anti-endotoxin peptide, efficiently neutralises pathogenicity factors of Gram-negative and Gram-positive bacteria and protects against sepsis in vivo. Here, we report that Pep19-2.5 inhibits the effects of cytoplasmic LPS in human myeloid cells and keratinocytes. In THP-1 monocytes and macrophages, the peptide strongly reduced secretion of IL-1ß and LDH induced by intracellular LPS. In contrast, the TLR4 signaling inhibitor TAK-242 abrogates LPS-induced TNF and IL-1ß secretion, but not pyroptotic cell death. Furthermore, Pep19-2.5 suppressed LPS-induced HMGB-1 production and caspase-1 activation in THP-1 monocytes. Consistent with this observation, we found impaired IL-1ß and IL-1α release in LPS-stimulated primary monocytes in the presence of Pep19-2.5 and reduced LDH release and IL-1B and IL-1A expression in LPS-transfected HaCaT keratinocytes. Additionally, Pep19-2.5 completely abolished IL-1ß release induced by LPS/ATP in macrophages via canonical inflammasome activation. In conclusion, we provide evidence that anti-endotoxin peptides inhibit the inflammasome/IL-1 axis induced by cytoplasmic LPS sensing in myeloid cells and keratinocytes and activation of the classical inflammasome by LPS/ATP which may contribute to the protection against bacterial sepsis and skin infections with intracellular Gram-negative bacteria.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Inflamassomos/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Peptídeos/farmacologia , Piroptose/efeitos dos fármacos , Receptor 4 Toll-Like/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/química , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Endotoxinas/antagonistas & inibidores , Endotoxinas/toxicidade , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Queratinócitos/imunologia , Queratinócitos/metabolismo , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Peptídeos/química , Sulfonamidas/farmacologia , Receptor 4 Toll-Like/metabolismoRESUMO
In this study, we report an interaction study of a 13-residue analogue peptide VG13P (VARGWGRKCPLFG), derived from a designed VG16KRKP peptide (VARGWKRKCPLFGKGG), with a Lys6Gly mutation and removal of the last three residues Lys14-Gly15-Gly16, in lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria and responsible for sepsis or septic shock. VG13P displays an enhanced anti-endotoxin property as evident from significant reduction in LPS-induced TNF-α gene expression levels in a monocytic cell line, while it retains almost unchanged antimicrobial activity as its parent VG16KRKP against Gram-negative bacterial as well as fungal pathogens. In addition, in vitro LPS binding properties of VG13P in comparison to its parent VG16KRKP also remained unhindered, suggesting that the flexible C-terminal end of VG16KRKP may not play a major role in its observed antibacterial and LPS binding properties. An NMR-resolved solution structure of VG13P in LPS reveals two consecutive ß-turns: one at the N-terminus, followed by another at the central region, closely resembling a rocking chair. The crucial Lys6Gly mutation along with C-terminal truncation from VG16KRKP reorients the hydrophobic hub in VG13P in a unique way so as to fold the N-terminal end back on itself, forming a turn and allowing Val1 and Ala2 to interact with Leu11 and Phe12 to bring the hydrophobic residues closer together to form a more compact hub compared to its parent. The hub is further strengthened via CH-π interaction between Gly4 and Phe12. This accounts for its improved anti-endotoxin activity as well as to its uninterrupted antimicrobial activity.
Assuntos
Desenho de Fármacos , Endotoxinas/antagonistas & inibidores , Glicina/metabolismo , Lipopolissacarídeos/química , Micelas , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Bactérias/citologia , Bactérias/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Acute lung injury (ALI) is a serious inflammatory disorder which remains the primary cause of incidence and mortality in patients with acute pulmonary inflammation. However, there is still no effective medical strategy available clinically for the improvement of ALI. Wogonin, isolated from roots of Scutellaria baicalensis Georgi, is a common medicinal herb which presents biological and pharmacological effects, including antioxidation, anti-inflammation, and anticancer. Preadministration of wogonin inhibited not only lung edema but also protein leakage into the alveolar space in murine model of lipopolysaccharide (LPS)-induced ALI. Moreover, wogonin not only reduced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 but also inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) induced by LPS. We further found wogonin inhibited the phosphorylation of p38 MAPK and JNK at a concentration lower than ERK. In addition, inhibition of lung edema, protein leakage, expression of iNOS and COX-2, and phosphorylation of p38 MAPK and JNK were all observed in a parallel concentration-dependent manner. These results suggest that wogonin possesses potential protective effect against LPS-induced ALI via downregulation of iNOS and COX-2 expression by blocking phosphorylation of p38 MAPK and JNK. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 397-403, 2017.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Antioxidantes/farmacologia , Endotoxinas/antagonistas & inibidores , Endotoxinas/toxicidade , Flavanonas/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Animais , Líquido da Lavagem Broncoalveolar , Inibidores de Ciclo-Oxigenase 2/farmacologia , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/prevenção & controleRESUMO
Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.
Assuntos
Bioensaio/métodos , Endotoxinas/análise , Grafite/química , Fator de Necrose Tumoral alfa/análise , Células Cultivadas , Endotoxinas/antagonistas & inibidores , Endotoxinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteínas Filagrinas , Guias como Assunto , Humanos , Teste do Limulus , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxidos/química , Polimixina B/química , Polimixina B/metabolismoRESUMO
Dextrin-colistin conjugates have been developed with the aim of achieving reduced clinical toxicity associated with colistin, also known as polymyxin E, and improved targeting to sites of bacterial infection. This study investigated the in vitro ability of such dextrin-colistin conjugates to bind and modulate bacterial lipopolysaccharide (LPS), and how this binding affects its biological activity. These results showed that colistin and amylase-activated dextrin-colistin conjugate to a lesser extent induced aggregation of LPS to form a stacked bilayer structure with characteristic dimensions, although this did not cause any substantial change in its secondary structure. In biological studies, both colistin and dextrin-colistin conjugate effectively inhibited LPS-induced hemolysis and tumor necrosis factor α (TNFα) secretion in a concentration-dependent manner, but only dextrin-colistin conjugate showed no additive toxicity at higher concentrations. This study provides the first direct structural experimental evidence for the binding of dextrin-colistin conjugates and LPS and gives insight into the mode of action of dextrin-colistin conjugates.
Assuntos
Antibacterianos/química , Bactérias/química , Colistina/química , Colistina/farmacologia , Dextrinas/química , Dextrinas/farmacologia , Lipopolissacarídeos/química , Amilases/metabolismo , Animais , Antibacterianos/farmacologia , Linhagem Celular , Endotoxinas/antagonistas & inibidores , Endotoxinas/química , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Teste do Limulus , Lipopolissacarídeos/antagonistas & inibidores , Ratos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
CONTEXT: Hygrophila auriculata (K. Schum) Heine (Acanthaceae) has been traditionally used for the treatment of various ailments such as inflammation, rheumatism, jaundice and malaria. OBJECTIVE: The present study aims to separate terpenoid fraction (TF) from alcohol (70%) extract of the whole plant of Hygrophila auriculata and assess its anti-inflammatory activity. MATERIALS AND METHODS: HPTLC analysis of TF was performed for the estimation of lupeol. Edema was induced in Wistar albino rats by subplanter injection of 0.1 ml of 1% (w/v) carrageenan into the right hind paw after 1 h of TF administration (100 and 200 mg/kg oral). Septic shock was induced by intraperitoneal administration of LPS (100 µg/kg) in rats and interleukins (IL-1ß and IL-6), tumor necrosis factor (TNF-α), superoxide dismutase (SOD), lipid peroxidation (LPO), and nitric oxide (NO) were measured in serum. AutoDock 4.2 was used for molecular docking. RESULTS: Administration of TF significantly (p < 0.005) restored the serum levels of cytokines, LPO (7.77 ± 0.034 versus 4.59 ± 0.059 nmole of TBARS), NO (9.72 ± 0.18 versus 4.15 ± 0.23 µmol nitrite/mg of wet tissue), and SOD (4.89 ± 0.036 versus 7.83 ± 0.033 Unit/mg protein) compared with the LPS-challenged rats. Analysis of in silico results revealed that TNF-α is the most appropriate target in eliciting anti-inflammatory activity. CONCLUSION: The present findings suggest that TF of Hygrophila auriculata possesses great promise as an anti-inflammatory agent which may be due to its antioxidant effect. Molecular docking results could be exploited for lead optimization and development of suitable treatment of inflammatory disorders.
Assuntos
Acanthaceae , Endotoxinas/antagonistas & inibidores , Endotoxinas/toxicidade , Extratos Vegetais/uso terapêutico , Choque Séptico/tratamento farmacológico , Terpenos/uso terapêutico , Animais , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Endotoxinas/metabolismo , Feminino , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Simulação de Acoplamento Molecular/métodos , Componentes Aéreos da Planta , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Ratos , Ratos Wistar , Choque Séptico/induzido quimicamente , Choque Séptico/metabolismo , Terpenos/isolamento & purificação , Terpenos/farmacologiaRESUMO
Resumo: Objetivo: Analisar o perfil de dez doenças crônicas não transmissíveis investigadas na Pesquisa Nacional de Saúde realizada no Brasil em 2013 e sua associação com a autoavaliação da saúde. Métodos: Estudo transversal de base populacional e abrangência nacional com 60.202 indivíduos com 18 anos ou mais. Foi utilizado processo amostral por conglomerado com três estágios de seleção: setor censitário, domicílio e indivíduo. Calculou-se a prevalência das doenças crônicas e os intervalos de confiança de 95% por idade, sexo e escolaridade, a idade média do primeiro diagnóstico e a proporção de limitação das atividades habituais. Para testar a associação com a autoavaliação de saúde, utilizou-se o procedimento de regressão logística ajustada por sexo e idade. Resultados: As doenças mais prevalentes foram hipertensão arterial (21,4%), depressão (7,6%), artrite (6,4%) e diabetes mellitus (6,2%). Indivíduos com diagnóstico de acidente vascular cerebral (AVC) referiram maior limitação das atividades habituais (38,6%). Observou-se um gradiente na prevalência segundo idade e escolaridade, e todas as doenças foram mais frequentes entre as mulheres. Pior autoavaliação de saúde foi encontrada entre aqueles com diagnóstico de AVC (OR = 3,60; valor de p < 0,001) e nos que referiram duas doenças (OR = 5,53; valor de p < 0,001) ou três ou mais doenças (OR = 10,86; valor de p < 0,001). Conclusões: Por se tratar de doenças associadas a fatores de risco modificáveis, a prevenção com foco populacional é a melhor estratégia para redução da carga dessas doenças.
ABSTRACT: Objective: To analyze the profile of 10 chronic noncommunicable diseases investigated in the National Health Survey carried out in Brazil in 2013 and their association with the self-rated health. Methods: A cross-sectional, population-based nationwide study with 60,202 individuals aged 18 years old or more. Sampling process by conglomerate was carried out in three stages of selection: census tract, household, and individual. The prevalence of chronic diseases by age, gender and educational status and the confidence intervals of 95% , the mean age at the first diagnosis and the proportion of limitation of the usual activities were calculated. To test the association with self-rated health, the logistic regression procedure adjusted for gender and age was used. Results: The more prevalent diseases were hypertension (21.4%), depression (7.6%), arthritis (6.4%), and diabetes mellitus (6.2%). Individuals diagnosed with stroke reported greater limitations in the daily activities (38.6%). There was a gradient in the prevalence by age and educational level, and all the diseases were more frequent among women. A worse self-rated health was observed among those with a diagnosis of stroke (OR = 3.60; p < 0.001) and those who referred two diseases (OR = 5.53; p < 0.001) or three or more diseases (OR = 10.86; p < 0.001). Conclusions: Because these diseases are associated with modifiable risk factors, the prevention with population focus is the best strategy to reduce the burden of these diseases.
Assuntos
Humanos , Antibacterianos/farmacologia , Endotoxinas/antagonistas & inibidores , Peptídeos/farmacologia , Pele Artificial , Sequência de Aminoácidos , Linhagem Celular , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Peptídeos/química , Pseudomonas aeruginosa/efeitos dos fármacos , Trombina/químicaRESUMO
Sepsis, a life-threatening syndrome with increasing incidence worldwide, is triggered by an overwhelming inflammation induced by microbial toxins released into the bloodstream during infection. A well-known sepsis-inducing factor is the membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS), signalling via Toll-like receptor-4. Although sepsis is caused in more than 50% cases by Gram-positive and mycoplasma cells, the causative compounds are still poorly described. In contradicting investigations lipoproteins/-peptides (LP), lipoteichoic acids (LTA), and peptidoglycans (PGN), were made responsible for eliciting this pathology. Here, we used human mononuclear cells from healthy donors to determine the cytokine-inducing activity of various LPs from different bacterial origin, synthetic and natural, and compared their activity with that of natural LTA and PGN. We demonstrate that LP are the most potent non-LPS pro-inflammatory toxins of the bacterial cell walls, signalling via Toll-like receptor-2, not only in vitro, but also when inoculated into mice: A synthetic LP caused sepsis-related pathological symptoms in a dose-response manner. Additionally, these mice produced pro-inflammatory cytokines characteristic of a septic reaction. Importantly, the recently designed polypeptide Aspidasept(®) which has been proven to efficiently neutralize LPS in vivo, inhibited cytokines induced by the various non-LPS compounds protecting animals from the pro-inflammatory activity of synthetic LP.
Assuntos
Antibacterianos/farmacologia , Endotoxinas/efeitos adversos , Endotoxinas/antagonistas & inibidores , Lipoproteínas/efeitos adversos , Lipoproteínas/antagonistas & inibidores , Peptídeos/farmacologia , Sepse/etiologia , Animais , Antibacterianos/síntese química , Citocinas/biossíntese , Modelos Animais de Doenças , Endotoxemia/tratamento farmacológico , Endotoxemia/etiologia , Endotoxemia/metabolismo , Endotoxemia/mortalidade , Feminino , Bactérias Gram-Negativas/imunologia , Células HEK293 , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/química , Lipoproteínas/química , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Peptídeos/síntese química , Peptidoglicano/efeitos adversos , Sepse/tratamento farmacológico , Sepse/metabolismo , Sepse/mortalidade , Staphylococcus aureus/imunologia , Ácidos Teicoicos/efeitos adversosRESUMO
Biomaterials used during surgery and wound treatment are of increasing importance in modern medical care. In the present study we set out to evaluate the addition of thrombin-derived host defense peptides to human acellular dermis (hAD, i.e. epiflex(®)). Antimicrobial activity of the functionalized hAD was demonstrated using radial diffusion and viable count assays against Gram-negative Escherichia coli, Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria. Electron microscopy analyses showed that peptide-mediated bacterial killing led to reduced hAD degradation. Furthermore, peptide-functionalized hAD displayed endotoxin-binding activity in vitro, as evidenced by inhibition of NF-κB activation in human monocytic cells (THP-1 cells) and a reduction of pro-inflammatory cytokine production in whole blood in response to lipopolysaccharide stimulation. The dermal substitute retained its anti-endotoxic activity after washing, compatible with results showing that the hAD bound a significant amount of peptide. Furthermore, bacteria-induced contact activation was inhibited by peptide addition to the hAD. E. coli infected hAD, alone, or after treatment with the antiseptic substance polyhexamethylenebiguanide (PHMB), yielded NF-κB activation in THP-1 cells. The activation was abrogated by peptide addition. Thus, thrombin-derived HDPs should be of interest in the further development of new biomaterials with combined antimicrobial and anti-endotoxic functions for use in surgery and wound treatment.
Assuntos
Antibacterianos/farmacologia , Endotoxinas/antagonistas & inibidores , Peptídeos/farmacologia , Pele Artificial , Sequência de Aminoácidos , Linhagem Celular , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Peptídeos/química , Pseudomonas aeruginosa/efeitos dos fármacos , Trombina/químicaRESUMO
Previous studies have demonstrated that soyasaponin (SoSa) possesses anti-inflammatory properties in lipopolysaccharide (LPS)-stimulated immune cells by influencing the immune sensing of toll-like receptor (TLR) 4. The aim of this study was to investigate the immune modulatory effect of SoSa I on TLR2- and TLR4-induced inflammation within the monocytic MUTZ-3-cell model. MUTZ-3 cells were stimulated with gram-negative (Escherichia coli) or gram-positive (Staphylococcus aureus) bacteria or bacterial pathogen-associated molecular patterns (PAMPs) such as LPS or peptidoglycans (PGN) alone or in combination with SoSa I. Cell morphology was characterized by raster scanning and light microscopy. Cytokine production (IL-1ß, IL-6, TNF-α, IP-10, RANTES and IL-8) was measured by cytometric bead array and the expression of surface markers was assessed by flow cytometry. MUTZ-3 cells revealed a cell maturation-like alteration in morphology and increased expression of CD80, CD86, TLR2 and TLR4 after stimulation with either gram-negative and gram-positive bacteria or bacterial PAMPs. The addition of SoSa I suppressed pro-inflammatory cytokine and chemokine secretions in a dose-dependent manner regardless of TLR2 or TLR4 stimulation. Interestingly, E. coli- and S. aureus-induced inflammation was always inhibited better by SoSa I than that induced by LPS and PGN. Additionally, SoSa I reduced the expression of CD86 in PGN- or LPS-stimulated cells. This study demonstrated that the anti-inflammatory capacity of SoSa I is based on influencing both monocytic TLR2 and TLR4 and that SoSa I inhibits more effectively whole bacteria compared to solely LPS or PGN what points to a broader role of SoSa I in the down-regulation of inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Antígeno B7-2/antagonistas & inibidores , Antígeno B7-2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Quimiocinas/antagonistas & inibidores , Quimiocinas/metabolismo , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Endotoxinas/antagonistas & inibidores , Endotoxinas/toxicidade , Humanos , Fatores Imunológicos/farmacologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Ácido Oleanólico/farmacologia , Concentração Osmolar , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
INTRODUCTION: The current root canal disinfection protocols fail to markedly inactivate bacterial endotoxins from infected root dentin. This study aimed to evaluate the ability of antibacterial photodynamic therapy with chitosan-conjugated rose bengal nanoparticles (CSRBnps) to selectively inactivate endotoxins/lipopolysaccharides (LPSs). METHODS: Antimicrobial agents such as calcium hydroxide (Ca[OH]2), chitosan nanoparticles (CSnps), CSRBnps, and methylene blue (MB) were assessed for their ability to neutralize LPSs obtained from Pseudomonas aeruginosa in a time-dependent interaction with/without photoactivation (20 and 40 J/cm(2)). The inflammatory potential of the treated/untreated LPSs was assessed on macrophage cells (RAW 267.4) using nitric oxide- and enzyme-linked immunosorbent assay (tumor necrosis factor α and interleukin-6 expression)-based analysis. These antimicrobials were tested directly on macrophage cells for cytotoxicity using the mitochondrial activity assay and light microscopy. The data were analyzed using 1-way analysis of variance and the Tukey test. RESULTS: CSnps were least effective in LPS inactivation. Interluekin-6 expression was reduced only with CSRBnp treatment. CSnps and CSRBnps were completely nontoxic, and MB showed slight toxicity to macrophage cells. Ca(OH)2 was highly cytotoxic (P < .005) even at 30 minutes of exposure. CSRBnps and MB with/without photoactivation significantly inactivated LPSs with reduced nitric oxide and tumor necrosis factor α expression (P < .05). Cell death and detachment after Ca(OH)2 treatment resulted in complete absence of all 3 inflammatory markers. CONCLUSIONS: Photodynamically activated CSRBnps caused significant inactivation of endotoxins and the subsequent reduction of all tested inflammatory markers from activated macrophages. Antimicrobial CSRBnps in combination with photodynamic therapy showed the potential to effectively inactivate bacterial endotoxins.
Assuntos
Antibacterianos/farmacologia , Quitosana/farmacologia , Endotoxinas/antagonistas & inibidores , Lipopolissacarídeos/antagonistas & inibidores , Nanopartículas , Fotoquimioterapia/métodos , Hidróxido de Cálcio/farmacologia , Cátions , Células Cultivadas , Corantes Fluorescentes , Humanos , Macrófagos/efeitos dos fármacos , Azul de Metileno/farmacologia , Poliaminas , Polieletrólitos , Pseudomonas aeruginosa , Rosa BengalaRESUMO
Interfering with LPS binding by the co-receptor protein myeloid differentiation factor 2 (MD-2) represents a useful approach for down-regulation of MD-2·TLR4-mediated innate immune signaling, which is implicated in the pathogenesis of a variety of human diseases, including sepsis syndrome. The antagonistic activity of a series of novel synthetic tetraacylated bis-phosphorylated glycolipids based on the ßGlcN(1â1)αGlcN scaffold was assessed in human monocytic macrophage-like cell line THP-1, dendritic cells and human epithelial cells. Two compounds were shown to inhibit efficiently the LPS-induced inflammatory signaling by down-regulation of the expression of TNF-α, IL-6, IL-8, IL-10 and IL-12 to background levels. The binding of the tetraacylated by (R)-3-hydroxy-fatty acids (2 × C12, 2 × C14), 4,4'-bisphosphorylated ßGlcN(1â1)αGlcN-based lipid A mimetic DA193 to human MD-2 was calculated to be 20-fold stronger than that of Escherichia coli lipid A. Potent antagonistic activity was related to a specific molecular shape induced by the ß,α(1â1)-diglucosamine backbone. 'Co-planar' relative arrangement of the GlcN rings was inflicted by the double exo-anomeric conformation around both glycosidic torsions in the rigid ß,α(1â1) linkage, which was ascertained using NOESY NMR experiments and confirmed by molecular dynamics simulation. In contrast to the native lipid A ligands, the binding affinity of ßGlcN(1â1)αGlcN-based lipid A mimetics to human MD-2 was independent on the orientation of the diglucosamine backbone of the synthetic antagonist within the binding pocket of hMD-2 (rotation by 180°) allowing for two equally efficient binding modes as shown by molecular dynamics simulation.
Assuntos
Endotoxinas/antagonistas & inibidores , Lipídeo A/análogos & derivados , Lipídeo A/farmacologia , Antígeno 96 de Linfócito/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Epiteliais/efeitos dos fármacos , Escherichia coli/química , Glucosamina/química , Glucosamina/farmacologia , Humanos , Imunidade Inata/imunologia , Lipídeo A/química , Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/metabolismo , Conformação Molecular , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/metabolismoRESUMO
An increasing number of pathologies have been linked to Toll-like receptor 4 (TLR4) activation and signaling, therefore new hit and lead compounds targeting this receptor activation process are urgently needed. We report on the synthesis and biological properties of glycolipids based on glucose and trehalose scaffolds which potently inhibit TLR4 activation and signaling in vitro and in vivo. Structure-activity relationship studies on these compounds indicate that the presence of fatty ester chains in the molecule is a primary prerequisite for biological activity and point to facial amphiphilicity as a preferred architecture for TLR4 antagonism. The cationic glycolipids here presented can be considered as new lead compounds for the development of drugs targeting TLR4 activation and signaling in infectious, inflammatory, and autoimmune diseases. Interestingly, the biological activity of the best drug candidate was retained after adsorption at the surface of colloidal gold nanoparticles, broadening the options for clinical development.