RESUMO
Aspidasept (Pep19-2.5) and its derivative Pep19-4LF ("Aspidasept II") are anti-infective and anti-inflammatory synthetic polypeptides currently in development for application against a variety of moderate to severe bacterial infections that could lead to systemic inflammation, as in the case of severe sepsis and septic shock, as well as application to non-systemic diseases in the case of skin and soft tissue infections (SSTI). In the present study, Aspidasept and Aspidasept II and their part structures were analysed with respect to their toxic behavior in different established models against a variety of relevant cells, and in electrophysiological experiments targeting the hERG channel according to ICH S7B. Furthermore, the effects in mouse models of neurobiological behavior and the local lymph node according to OECD test guideline 429 were investigated, as well as a rat model of repeated dose toxicology according to ICH M3. The data provide conclusive information about potential toxic effects, thus specifying a therapeutic window for the application of the peptides. Therefore, these data allow us to define Aspidasept concentrations for their use in clinical studies as parenteral application.
RESUMO
The polypeptide Pep19-2.5 (Aspidasept®) has been described to act efficiently against infection-inducing bacteria by binding and neutralizing their most potent toxins, i.e., lipopolysaccharides (LPS) and lipoproteins/peptides (LP), independent of the resistance status of the bacteria. The mode of action was described to consist of a primary Coulomb/polar interaction of the N-terminal region of Pep19-2.5 with the polar region of the toxins followed by a hydrophobic interaction of the C-terminal region of the peptide with the apolar moiety of the toxins. However, clinical development of Aspidasept as an anti-sepsis drug requires an in-depth characterization of the interaction of the peptide with the constituents of the human immune system and with other therapeutically relevant compounds such as antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs). In this contribution, relevant details of primary and secondary pharmacodynamics, off-site targets, and immunogenicity are presented, proving that Pep19-2.5 may be readily applied therapeutically against the deleterious effects of a severe bacterial infection.
Assuntos
Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Endotoxemia/tratamento farmacológico , Inflamação , Peptídeos/farmacologia , Animais , Anti-Infecciosos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Modelos Animais de Doenças , Endotoxemia/imunologia , Humanos , Lipopolissacarídeos , Camundongos , Peptídeos/uso terapêuticoRESUMO
INTRODUCTION: Gram-negative bacterial infections represent still a severe problem of human health care, regarding the increase in multi-resistance against classical antibiotics and the lack of newly developed antimicrobials. For the fight against these germs, anti-infective agents must overcome and/or bind to the Gram-negative outer membrane consisting of a lipopolysaccharide (LPS, endotoxin) outer leaflet and an inner leaflet from phospholipids, with additional peripheral or integral membrane proteins (OMP's). AREAS COVERED: The current article reviews data of existing therapeutic options and summarizes newer approaches for targeting and neutralizing endotoxins, ranging from in vitro over in vivo animal data to clinical applications by using databases such as Medline. EXPERT OPINION: Conventional antibiotic treatment of the bacteria leads to their killing, but not necessary LPS neutralization, which may be a severe problem in particular for the systemic pathway. This is the reason why there is an increasing number of therapeutic approaches, which - besides combating whole bacteria - at the same time try to neutralize endotoxin within or outside the bacterial cells mainly responsible for the high inflammation induction in Gram-negative species.
Assuntos
Antibacterianos/administração & dosagem , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Animais , Antibacterianos/farmacologia , Desenvolvimento de Medicamentos , Endotoxinas/antagonistas & inibidores , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/microbiologia , Lipopolissacarídeos/antagonistas & inibidoresRESUMO
Due to the increase in bacterial resistance to common antibiotics and the lack of newly approved drugs, antimicrobial peptides (AMP) have been shown to be an alternative to combat infections caused by drug-resistant organisms. In particular, synthetic anti-lipopolysaccharide peptides (SALP) with the lead structure Aspidasept (Pep19-2.5) display a high anti-inflammatory activity in vitro and in vivo systems of endotoxemia and bacteremia. This was found not only when SALP were applied systemically (i.e. against sepsis), but also in topical therapies aimed at treating wound infections. A further important application involves combating common pathologies of the gastrointestinal tract, such as chronic infections of the small intestine and the colon (e.g., Crohn's disease). For the necessary oral application, the active pharmaceutical ingredient (API), Aspidasept®, must be encapsulated to ensure its protection against the low pH and the hydrolytic enzymes of the gastrointestinal tract. Here, the encapsulation of Aspidasept in polysaccharide matrices, essentially alginate and pectin, was systematically investigated with a variety of physico-chemical techniques. Specifically, we characterized key features of the nanoparticles such as their sizes and size distributions, as well as their stability in different environments mimicking digestive fluids. Finally, we studied the release of the drug from the polysaccharide matrices and the ability of nanoparticles to neutralize endotoxemia in vitro. We showed that our lead formulations exert an optimum inhibitory activity on immune cells stimulated by lipopolysaccharide.
Assuntos
Peptídeos , Sepse , Antibacterianos , Anti-Inflamatórios , Humanos , LipopolissacarídeosRESUMO
The activity of antimicrobial peptides (AMPs) has been investigated extensively using model membranes composed of phospholipids or lipopolysaccharides in aqueous environments. However, from a biophysical perspective, there is a large scientific interest regarding the direct interaction of membrane-active peptides with whole bacteria. Working with living bacteria limits the usability of experimental setups and the interpretation of the resulting data because of safety risks and the overlap of active and passive effects induced by AMPs. We killed or inactivated metabolic-active bacteria using γ-irradiation or sodium azide, respectively. Microscopy, flow cytometry, and SYTOX green assays showed that the cell envelope remained intact to a high degree at the minimal bactericidal dose. Furthermore, the tumor-necrosis-factor-α-inducing activity of the lipopolysaccharides and the chemical lipid composition was unchanged. Determining the binding capacity of AMPs to the bacterial cell envelope by calorimetry is difficult because of an overlapping of the binding heat and metabolic activities of the bacteria-induced by the AMPs. The inactivation of all active processes helps to decipher the complex thermodynamic information. From the isothermal titration calorimetry (ITC) results, we propose that the bacterial membrane potential (Δψ) is possibly an underestimated modulator of the AMP activity. The negative surface charge of the outer leaflet of the outer membrane of Gram-negative bacteria is already neutralized by peptide concentrations below the minimal inhibitory concentration. This proves that peptide aggregation on the bacterial membrane surface plays a decisive role in the degree of antimicrobial activity. This will not only enable many biophysical approaches for the investigation between bacteria and membrane-active peptides in the future but will also make it possible to compare biophysical parameters of active and inactive bacteria. This opens up new possibilities to better understand the active and passive interaction processes between AMPs and bacteria.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/efeitos da radiação , Raios gama , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Adsorção , Bactérias/ultraestrutura , Fenômenos Biofísicos , Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos da radiação , Membrana Celular/ultraestrutura , Potenciais da Membrana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Fosfolipídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , TermodinâmicaRESUMO
Outer membrane vesicles (OMVs) are secreted by Gram-negative bacteria and induce a stronger inflammatory response than pure LPS. After endocytosis of OMVs by macrophages, lipopolysaccharide (LPS) is released from early endosomes to activate its intracellular receptors followed by non-canonical inflammasome activation and pyroptosis, which are critically involved in sepsis development. Previously, we could show that the synthetic anti-endotoxin peptide Pep19-2.5 neutralizes inflammatory responses induced by intracellular LPS. Here, we aimed to investigate whether Pep19-2.5 is able to suppress cytoplasmic LPS-induced inflammation under more physiological conditions by using OMVs which naturally transfer LPS to the cytosol. Isothermal titration calorimetry revealed an exothermic reaction between Pep19-2.5 and Escherichia coli OMVs and the Limulus Amebocyte Lysate assay indicated a strong endotoxin blocking activity. In THP-1 macrophages and primary human macrophages Pep19-2.5 and polymyxin B reduced interleukin (IL)-1ß and tumor necrosis factor (TNF) release as well as pyroptosis induced by OMVs, while the Toll-like receptor 4 signaling inhibitor TAK-242 suppressed OMV-induced TNF and IL-1ß secretion, but not pyroptosis. Internalization of Pep19-2.5 was at least partially mediated by the P2X7 receptor in macrophages but not in monocytes. Additionally, a cell-dependent difference in the neutralization efficiency of Pep19-2.5 became evident in macrophages and monocytes, indicating a critical role for peptide-mediated IL-1ß secretion via the P2X7 receptor. In conclusion, we provide evidence that LPS-neutralizing peptides inhibit OMV-induced activation of the inflammasome/IL-1 axis and give new insights into the mechanism of peptide-mediated neutralization of cytoplasmic LPS suggesting an essential and cell-type specific role for the P2X7 receptor.
Assuntos
Anti-Inflamatórios/farmacologia , Membrana Externa Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Peptídeos/farmacologia , Membrana Externa Bacteriana/imunologia , Linhagem Celular , Escherichia coli/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/microbiologia , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Piroptose/efeitos dos fármacosRESUMO
Antimicrobial peptides (AMPs) are in the focus of scientific research since the 1990s. In most cases, the main aim was laid on the design of AMP to kill bacteria effectively, with particular emphasis on broadband action and independency on antibiotic resistance. However, so far no approved drug on the basis of AMP has entered the market.Our approach of constructing AMP, called synthetic anti-lipopolysaccharide peptides (SALPs), on the basis of inhibiting the inflammatory action of lipopolysaccharide (LPS, endotoxin) from Gram-negative bacteria was focused on the neutralization of the decisive toxins. These are, beside LPS from Gram-negative bacteria, the lipoproteins (LP) from Gram-positive origin. Although some of the SALPs have an antibacterial action, the most important property is the high-affinity binding to LPS and LP, whether as constituent of the bacteria or in free form which prevents the damaging inflammation, that could otherwise lead to life-threatening septic shock. Most importantly, the SALP may inhibit inflammation independently of the resistance status of the bacteria, and so far the repeated use of the peptides apparently does not cause resistance of the attacking pathogens.In this chapter, an overview is given over the variety of possible applications in the field of fighting against severe bacterial infections, from the use in systemic infection/inflammation up to various topical applications such as anti-biofilm action and severe skin and soft tissue infections.
Assuntos
Antibacterianos/química , Moléculas com Motivos Associados a Patógenos/antagonistas & inibidores , Peptídeos/química , Infecções Bacterianas/tratamento farmacológico , Endotoxinas , Bactérias Gram-Negativas , Humanos , LipopolissacarídeosRESUMO
Increasing failure of conventional antibiotics to combat bacterial infections requires the urgent development of new antibacterial drugs; a promising class of new drugs based on antimicrobial peptides. Here, we studied the molecular interaction of polycationic synthetic antilipopolysaccharide peptides (SALPs) with various gram-negative and gram-positive bacteria, including resistant strains. The analysis of antimicrobial activity by conventional techniques and atomic force microscopy showed a strict dependence on amino acid (aa) sequences, with the type of amino acid, its position within the primary structure, and the sequence length being critical parameters. By monitoring lipopolysaccharide (LPS)- or bacteria-induced cytokine production in human mononuclear cells and whole blood, we found a direct link between the binding of the lead compound Pep19-2.5 to Salmonella enterica and the anti-inflammatory activity of the peptide. Thermodynamic analysis of Pep19-2.5 binding to the bacterial cell envelope showed an exothermic reaction with saturation characteristics, whereas small-angle X-ray scattering data indicated a direct attachment of Pep19-2.5 to the bacterial cell envelope. This binding preferentially takes place to the LPS outer monolayer, as evidenced by the change in the LPS acyl chain and phosphate vibrational bands seen by Fourier-transform infrared spectroscopy. We report here that the anti-inflammatory activity of Pep19-2.5 is not only connected with neutralization of cell-free bacterial toxins but also with a direct binding of the peptide to the outer leaflet of the bacterial outer membrane.
Assuntos
Antibacterianos/farmacologia , Toxinas Bacterianas/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Calorimetria , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/microbiologia , Radioisótopos de Césio/toxicidade , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/microbiologia , Lipopolissacarídeos/farmacologia , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Peptídeos/síntese química , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/metabolismo , Salmonella enterica/efeitos da radiação , Espalhamento a Baixo Ângulo , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Introduction: As part of a study aimed at illuminating at least some of the complex molecular events taking place in COPD, we screened tissues by means of transcriptome analyses. Materials and methods: Tissues were subjected to transcriptome analysis. Candidate genes were identified and validated by immunohistochemistry. Primary human lung cells were subjected to stimulation with cigarette smoke extract for further validation by real time PCR. Results: Six candidate genes were selected for further investigations: Aquaporin 3 (AQP3), extracellular matrix protein 1 (ECM1), four and a half LIM domain 1 (FHL1), milk fat globule epidermal growth factor 8 (MFGE8, lactadherin), phosphodiesterase 4D-interacting protein (PDE4DIP), and creatine transporter SLC6A8. All six proteins were allocated to distinct cell types by immunohistochemistry. Upon stimulation with cigarette smoke extract, human type II pneumocytes showed a dose-dependent down-regulation of MFGE8, while ECM1 and FHL1 also tended to be down-regulated. Although present, none of the candidates was regulated by cigarette smoke extract in primary human macrophages. Discussion: MFGE8 turned out to be an interesting new candidate gene in COPD deserving further studies.
Assuntos
Antígenos de Superfície/genética , Aquaporina 3/genética , Proteínas da Matriz Extracelular/genética , Perfilação da Expressão Gênica/métodos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Proteínas do Leite/genética , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Doença Pulmonar Obstrutiva Crônica/genética , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Proteínas do Citoesqueleto , Regulação para Baixo , Feminino , Alemanha , Humanos , Pulmão , Masculino , Reação em Cadeia da Polimerase em Tempo Real , FumaçaRESUMO
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
Aging affects the core processes of almost every organism, and the functional decline at the cellular and tissue levels influences disease development. Recently, it was shown that the methylation of certain CpG dinucleotides correlates with chronological age and that this epigenetic clock can be applied to study aging-related effects. We investigated these molecular age loci in non-small cell lung cancer (NSCLC) tissues from patients with adenocarcinomas (AC) and squamous cell carcinomas (SQC) as well as in matched tumor-free lung tissue. In both NSCLC subtypes, the calculated epigenetic age did not correlate with the chronological age. In particular, SQC exhibited rejuvenation compared to the corresponding normal lung tissue as well as with the chronological age of the donor. Moreover, the younger epigenetic pattern was associated with a trend toward stem cell-like gene expression patterns. These findings show deep phenotypic differences between the tumor entities AC and SQC, which might be useful for novel therapeutic and diagnostic approaches.
Assuntos
Envelhecimento/genética , Carcinoma de Células Escamosas/genética , Epigênese Genética , Neoplasias Pulmonares/genética , Pulmão/metabolismo , Adenocarcinoma de Pulmão/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Ilhas de CpG , Metilação de DNA , Humanos , Pessoa de Meia-Idade , Transcrição GênicaRESUMO
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
In previous years, we developed anti-infective drugs based on antimicrobial peptides (AMPs), which have been shown to effectively block severe infections and inflammation in vitro as well as in vivo. Besides systemic application, the occurrence of severe local infections necessitates a topical application for example in the case of severe skin and soft tissue infections (SSTI). Recent investigations show that the synthetic anti-lipopolysaccharide peptide (SALP) Pep19-2.5 (Aspidasept® I) and a variant called Pep19-4LF (Aspidasept® II) are able to supress inflammation reactions also in keratinocytes, Langerhans cells, and dendritic cells from the skin. For topical application, a possible formulation represents the drug dispersed into a pharmaceutical cream (DAC base cream). Here, we present investigations on the stability of the peptides using this formulation in dependence on time, which includes the evaluation of the extraction procedure, the quantitative analysis of the peptides after extraction, its sensitivity to protease degradation and its ability to maintain activity against LPS-induced inflammation in vitro. We have developed an extraction procedure for the peptides with an optimum yield and showed that Pep19-2.5 is present as a dimer after extraction from the cream, whereas Pep19-4LF retains its monomeric form. Both peptides show no degradation by chymotrypsin after extraction for at least 1â¯h, which is indicative for an attachment of constituents of the base cream, inhibiting the cutting into peptidic part structures. The extracted peptides and in particular the dimeric Pep19-2.5 are still able to inhibit the LPS-induced inflammation reaction in human mononuclear cells.
Assuntos
Anti-Inflamatórios/química , Pomadas/química , Peptídeos/química , Antibacterianos/química , Anti-Inflamatórios/farmacologia , Células Cultivadas , Química Farmacêutica/métodos , Estabilidade de Medicamentos , Humanos , Inflamação/tratamento farmacológico , QueratinócitosRESUMO
OBJECTIVE: To evaluate (1) levels of the host-defense/antimicrobial peptide LL-37 in patients with trauma and hemorrhagic shock (HS) and (2) the effects of a synthetic host-defense peptide; Pep19-4LF on multiple organ failure (MOF) associated with HS. BACKGROUND: HS is a common cause of death in severely injured patients. There is no specific therapy that reduces HS-associated MOF. METHODS: (1) LL-37 was measured in 47 trauma/HS patients admitted to an urban major trauma center. (2) Male Wistar rats were submitted to HS (90âmin, target mean arterial pressure: 27-32âmm Hg) or sham operation. Rats were treated with Pep19-4LF [66 (n = 8) or 333âµg/kgâ·âh (n = 8)] or vehicle (n = 12) for 4 hours following resuscitation. RESULTS: Plasma LL-37 was 12-fold higher in patients with trauma/HS compared to healthy volunteers. HS rats treated with Pep19-4LF (high dose) had a higher mean arterial pressure at the end of the 4-hour resuscitation period (79â±â4 vs 54â±â5âmm Hg) and less renal dysfunction, liver injury, and lung inflammation than HS rats treated with vehicle. Pep19-4LF enhanced (kidney/liver) the phosphorylation of (1) protein kinase B and (2) endothelial nitric oxide synthase. Pep19-4LF attenuated the HS-induced (1) translocation of p65 from cytosol to nucleus, (2) phosphorylation of IκB kinase on Ser, and (3) phosphorylation of IκBα on Ser resulting in inhibition of nuclear factor kappa B and formation of proinflammatory cytokines. Pep19-4LF prevented the release of tumor necrosis factor alpha caused by heparan sulfate in human mononuclear cells by binding to this damage-associated molecular pattern. CONCLUSIONS: Trauma-associated HS results in release of LL-37. The synthetic host-defense/antimicrobial peptide Pep19-4LF attenuates the organ injury/dysfunction associated with HS.
Assuntos
Anti-Infecciosos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/sangue , Insuficiência de Múltiplos Órgãos/prevenção & controle , Peptídeos/uso terapêutico , Substâncias Protetoras/uso terapêutico , Choque Hemorrágico/tratamento farmacológico , Ferimentos e Lesões/complicações , Animais , Biomarcadores/sangue , Estudos de Casos e Controles , Terapia Combinada , Humanos , Masculino , Insuficiência de Múltiplos Órgãos/etiologia , Ratos , Ratos Wistar , Ressuscitação , Choque Hemorrágico/sangue , Choque Hemorrágico/complicações , Choque Hemorrágico/diagnóstico , Resultado do Tratamento , CatelicidinasRESUMO
Hwang et al. recently showed that VGF substantially contributes to the resistance of human lung cancer cells towards epidermal growth factor receptor kinase inhibitors. This was further linked to enhanced epithelial-mesenchymal transition. Here, we demonstrate that VGF is epigenetically modified in non-small cell lung cancer tissues compared to corresponding tumor-free lung tissues from the same donors by using methylome bead chip analyses. These epigenetic modifications trigger an increased transcription of the VGF gene within the tumors, which then leads to an increased expression of the protein, facilitating epithelial-mesenchymal transition, and the resistance to kinase inhibitors. These results should be taken into account in the design of novel therapeutic and diagnostic approaches.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Neoplasias Pulmonares/genética , Fatores de Crescimento Neural/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Fatores de Crescimento Neural/metabolismo , Regulação para CimaRESUMO
Sepsis, which is induced by severe bacterial infections, is a major cause of death worldwide, and therapies combating the disease are urgently needed. Because many drugs have failed in clinical trials despite their efficacy in mouse models, the development of reliable animal models of sepsis is in great demand. Several studies have suggested that rabbits reflect sepsis-related symptoms more accurately than mice. In this study, we evaluated a rabbit model of acute sepsis caused by the intravenous inoculation of Salmonella enterica. The model reproduces numerous symptoms characteristic of human sepsis including hyperlactatemia, hyperglycemia, leukopenia, hypothermia and the hyperproduction of several pro-inflammatory cytokines. Hence, it was chosen to investigate the proposed ability of Pep19-2.5-an anti-endotoxic peptide with high affinity to lipopolysaccharide and lipoprotein-to attenuate sepsis-associated pathologies in combination with an antibiotic (ceftriaxone). We demonstrate that a combination of Pep19-2.5 and ceftriaxone administered intravenously to the rabbits (1) kills bacteria and eliminates bacteremia 30 min post challenge; (2) inhibits Toll-like receptor 4 agonists in serum 90 min post challenge; (3) reduces serum levels of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor α); and (4) reverts to hypothermia and gives rise to temperature values indistinguishable from basal levels 330 min post challenge. The two components of the combination displayed synergism in some of these activities, and Pep19-2.5 notably counteracted the endotoxin-inducing potential of ceftriaxone. Thus, the combination therapy of Pep19-2.5 and ceftriaxone holds promise as a candidate for human sepsis therapy.
Assuntos
Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Ceftriaxona/uso terapêutico , Peptídeos/uso terapêutico , Salmonella enterica/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Ceftriaxona/administração & dosagem , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Células HEK293 , Humanos , Hiperlactatemia , Hipotermia , Interleucina-6/sangue , Leucopenia , Lipopolissacarídeos/sangue , Masculino , Peptídeos/administração & dosagem , Coelhos , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/sangue , Fator de Necrose Tumoral alfa/sangueRESUMO
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
Septic cardiomyopathy affects up to 70% of patients with septic shock and the derangement of cardiac mitochondrial function contributes to the likelihood of death. However, at present, there is no specific therapeutic drug available. The peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α) and coactivator-1ß (PGC-1ß) modulate members of the PPARs, which regulate mitochondrial energy metabolism and the production of mitochondrial reactive oxygen species in the heart. This study investigated the potential of the newly developed synthetic antimicrobial peptide 19-2.5 (Pep2.5) to attenuate mitochondrial dysfunction in murine cardiomyocytes stimulated with serum from septic shock patients. Pep2.5 treatment attenuated the suppression of PPAR-α, PPAR-γ ( P = 0.0004 and P = 0.0001, respectively) and PGC-1α/ß ( P = 0.0008 and P = 0.0147, respectively) in cardiomyocytes stimulated with serum from septic shock patients compared with untreated cells. Pep2.5 treatment enhanced the mitochondrial maximum respiration ( P < 0.0001), increased cellular ATP levels ( P < 0.0001) and reduced the production of mitochondrial reactive oxygen species. Thus, the administration of Pep2.5 may have the potential as a promising therapeutic approach in septic cardiomyopathy by attenuating mitochondrial dysfunction in the septic heart.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Sepse/terapia , Idoso , Idoso de 80 Anos ou mais , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Linhagem Celular , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/fisiologia , Miócitos Cardíacos/fisiologia , Oxirredução/efeitos dos fármacos , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sepse/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The pattern recognition receptor TLR4 is well known as a crucial receptor during infection and inflammation. Several TLR4 antagonists have been reported to inhibit the function of TLR4. Both natural occurring antagonists, lipopolysaccharide (LPS) from Gram-negative bacteria as well as synthetic compounds based on the lipid A structure of LPS have been described as potent inhibitors of TLR4. Here, we have examined the characteristics of a natural TLR4 antagonist, isolated from Bartonella quintana bacterium by elucidating its chemical primary structure. We have found that this TLR4 antagonist is actually a lipooligosaccharide (LOS) instead of a LPS, and that it acts very effective, with a high inhibitory activity against triggering by the LPS-TLR4 system in the presence of a potent TLR4 agonist (E. coli LPS). Furthermore, we demonstrate that B. quintana LPS is not inactivated by polymyxin B, a classical cyclic cationic polypeptide antibiotic that bind the lipid A part of LPS, such as E. coli LPS. Using a murine LPS/D-galactosamine endotoxaemia model we showed that treatment with B. quintana LPS could improve the survival rate significantly. Since endogenous TLR4 ligands have been associated with several inflammatory- and immune-diseases, B. quintana LPS might be a novel therapeutic strategy for TLR4-driven pathologies.
RESUMO
The stagnation in the development of new antibiotics and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. Antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that Pep19-2.5, a synthetic anti-lipopolysaccharide (LPS) peptide (SALP), efficiently neutralises pathogenicity factors of Gram-negative (LPS) and Gram-positive (lipoprotein/-peptide, LP) bacteria and protects against sepsis. Here, we investigated the potential of Pep19-2.5 and the structurally related compound Pep19-4LF for their therapeutic application in bacterial skin infections. SALPs inhibited LP-induced phosphorylation of NF-κB p65 and p38 MAPK and reduced cytokine release and gene expression in primary human keratinocytes and dermal fibroblasts. In LPS-stimulated human monocyte-derived dendritic cells and Langerhans-like cells, the peptides blocked IL-6 secretion, downregulated expression of maturation markers and inhibited dendritic cell migration. Both SALPs showed a low cytotoxicity in all investigated cell types. Furthermore, SALPs markedly promoted cell migration via EGFR transactivation and ERK1/2 phosphorylation and accelerated artificial wound closure in keratinocytes. Peptide-induced keratinocyte migration was mediated by purinergic receptors and metalloproteases. In contrast, SALPs did not affect proliferation of keratinocytes. Conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections.