In vitro and in vivo evaluation of BMAP-derived peptides for the treatment of cystic fibrosis-related pulmonary infections

Patients with cystic fibrosis require pharmacological treatment against chronic lung infections. The alpha-helical antimicrobial peptides BMAP-27 and BMAP-28 have shown to be highly active in vitro against planktonic and sessile forms of multidrug-resistant Pseudomonas aeruginosa, Staphylococcus aureus, and Stenotrophomonas maltophilia cystic fibrosis strains. To develop small antibacterial peptides for therapeutic use, we tested shortened/modified BMAP fragments, and selected the one with the highest in vitro antibacterial activity and lowest in vivo acute pulmonary toxicity. All the new peptides have shown to roughly maintain their antibacterial activity in vitro. The 1-18 N-terminal fragment of BMAP-27, showing MIC90 of 16 A mu g/ml against P. aeruginosa isolates and strain-dependent anti-biofilm effects, showed the lowest pulmonary toxicity in mice. However, when tested in a murine model of acute lung infection by P. aeruginosa, BMAP-27(1-18) did not show any curative effect. If exposed to murine broncho-alveolar lavage fluid BMAP-27(1-18) was degraded within 10 min, suggesting it is not stable in pulmonary environment, probably due to murine proteases. Our results indicate that shortened BMAP peptides could represent a starting point for antibacterial drugs, but they also indicate that they need a further optimization for effective in vivo use

Mitochondrial Metabolism and EV Cargo of Endothelial Cells Is Affected in Presence of EVs Derived from MSCs on Which HIF Is Activated

Small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) have attracted growing interest as a possible novel therapeutic agent for the management of different cardiovascular diseases (CVDs). Hypoxia significantly enhances the secretion of angiogenic mediators from MSCs as well as sEVs. The iron-chelating deferoxamine mesylate (DFO) is a stabilizer of hypoxia-inducible factor 1 and consequently used as a substitute for environmental hypoxia. The improved regenerative potential of DFO-treated MSCs has been attributed to the increased release of angiogenic factors, but whether this effect is also mediated by the secreted sEVs has not yet been investigated. In this study, we treated adipose-derived stem cells (ASCs) with a nontoxic dose of DFO to harvest sEVs (DFO-sEVs). Human umbilical vein endothelial cells (HUVECs) treated with DFO-sEVs underwent mRNA sequencing and miRNA profiling of sEV cargo (HUVEC-sEVs). The transcriptomes revealed the upregulation of mitochondrial genes linked to oxidative phosphorylation. Functional enrichment analysis on miRNAs of HUVEC-sEVs showed a connection with the signaling pathways of cell proliferation and angiogenesis. In conclusion, mesenchymal cells treated with DFO release sEVs that induce in the recipient endothelial cells molecular pathways and biological processes strongly linked to proliferation and angiogenesis

D-BMAP18 antimicrobial peptide is active In Vitro, resists to pulmonary proteases but loses its activity in a murine model of Pseudomonas aeruginosa lung infection

The spread of antibiotic resistant-pathogens is driving the search for new antimicrobial compounds. Pulmonary infections experienced by cystic fibrosis (CF) patients are a dramatic example of this health-care emergency. Antimicrobial peptides could answer the need for new antibiotics but translating them from basic research to the clinic is a challenge. We have previously evaluated the potential of the small membranolytic peptide BMAP-18 to treat CF-related infections, discovering that while this molecule had a good activity in vitro it was not active in vivo because of its rapid degradation by pulmonary proteases. In this study, we synthesized and tested the proteases-resistant all-d enantiomer. In spite of a good antimicrobial activity against Pseudomonas aeruginosa and Stenotrophomonas maltophilia clinical isolates and of a tolerable cytotoxicity in vitro, D-BMAP18 was ineffective to treat P. aeruginosa pulmonary infection in mice, in comparison to tobramycin. We observed that different factors other than peptide degradation hampered its efficacy for pulmonary application. These results indicate that D-BMAP18 needs further optimization before being suitable for clinical application and this approach may represent a guide for optimization of other anti-infective peptides eligible for the treatment of pulmonary infections

Economic consequences of investing in anti-HCV antiviral treatment from the Italian NHS perspective : a real-world-based analysis of PITER data

OBJECTIVE: We estimated the cost consequence of Italian National Health System (NHS) investment in direct-acting antiviral (DAA) therapy according to hepatitis C virus (HCV) treatment access policies in Italy. METHODS: A multistate, 20-year time horizon Markov model of HCV liver disease progression was developed. Fibrosis stage, age and genotype distributions were derived from the Italian Platform for the Study of Viral Hepatitis Therapies (PITER) cohort. The treatment efficacy, disease progression probabilities and direct costs in each health state were obtained from the literature. The break-even point in time (BPT) was defined as the period of time required for the cumulative costs saved to recover the Italian NHS investment in DAA treatment. Three different PITER enrolment periods, which covered the full DAA access evolution in Italy, were considered. RESULTS: The disease stages of 2657 patients who consecutively underwent DAA therapy from January 2015 to December 2017 at 30 PITER clinical centres were standardized for 1000 patients. The investment in DAAs was considered to equal €25 million, €15 million, and €9 million in 2015, 2016, and 2017, respectively. For patients treated in 2015, the BPT was not achieved, because of the disease severity of the treated patients and high DAA prices. For 2016 and 2017, the estimated BPTs were 6.6 and 6.2 years, respectively. The total cost savings after 20 years were €50.13 and €55.50 million for 1000 patients treated in 2016 and 2017, respectively. CONCLUSIONS: This study may be a useful tool for public decision makers to understand how HCV clinical and epidemiological profiles influence the economic burden of HCV

Optimization of BMAP-18 an anti-infective peptide for the treatment of pulmonary infection

I pazienti con infezioni polmonari richiedono un trattamento farmacologico contro agenti patogeni multiresistenti. Il peptide antimicrobico BMAP27 (1-18), una forma troncata di BMAP-27, ha dimostrato di essere attivo in vitro contro forme planctoniche e sessili di ceppi isolati da pazineti con fibrosi cistica di Pseudomonas aeruginosa multiresistenti e Stenotrophomonas maltophilia. Nonostante la buona attivit\ue0 antimicrobica in vitro, non mostra un'attivit\ue0 protettiva in vivo in un modello murino di infezione polmonare acuta da parte di P. aeruginosa e mostra una tossicit\ue0 residua in vivo. L'inefficienza \ue8 causata dalla sua scarsa stabilit\ue0 nell'ambiente polmonare e ci\uf2 \ue8 confermato dall'osservazione della sua degradazione in presenza di fluido di lavaggio broncoalveolare murino. Per superare questi inconvenienti abbiamo operato in due direzioni. Inizialmente, abbiamo sintetizzato l'enantiomero (D-BMAP-18) per evitare la sua degradazione. Ha una buona attivit\ue0 antimicrobica in vitro contro la forma sessile e planctonica dei ceppi di P. aeruginosa. \uc8 stabile nell'ambiente polmonare ed \ue8 attivo nei media imitando l'ambiente polmonare CF, in questa condizione la sua attivit\ue0 antimicrobica \ue8 aumentata in combinazione con composti mucolitici gi\ue0 approvati nella CF-terapia. Tuttavia, ha mostrato una tossicit\ue0 non trascurabile sia in vitro che in vivo. In secondo luogo, per risolvere parzialmente questo inconveniente il suo pro-form (Pro-D-BMAP18) \ue8 stato sintetizzato al fine di ottenere un peptide inattivo e che viene convertito nella sua forma attiva solo nel sito di infezione / infiammazione grazie all'elastasi, consentendo un lento rilascio del peptide farmacologicamente attivo. Pro-D-BMAP18 sembra risolvere gli effetti collaterali legati al D-BMAP18, mostrando una citotossicit\ue0 ridotta e un'attivit\ue0 antimicrobica conservata. \uc8 correttamente processi in D-BMAP18 in ambiente polmonare e, in questa condizione, mostra una buona attivit\ue0 antimicrobica. Pro-D-BMAP18 sembra essere un composto promettente per il trattamento in vivo di infezione polmonare, ma solo il test in vivo ci consentir\ue0 di comprendere il reale potenziale di Pro-D-BMAP18 nella terapia.Patients with lung infections require pharmacological treatment against multi drug resistant pathogens. The antimicrobial peptide BMAP27(1-18), a truncated form of BMAP-27, have shown to be active in vitro against planktonic and sessile forms of multidrug-resistant Pseudomonas aeruginosa and Stenotrophomonas maltophilia cystic fibrosis strains. Despite the good antimicrobial activity in vitro, it does not show a protective activity in vivo in a murine model of acute pulmonary infection by P. aeruginosa and shows a residual in vivo toxicity. The inefficiency is caused by its scarce stability in the pulmonary environment and this is confirmed by the observation of its degradation in the presence of murine bronchoalveolar lavage fluid. To overcome these drawbacks we operated in two directions. At first, we synthesized the enantiomer (D-BMAP-18) to avoid its degradation. It has a good antimicrobial activity in vitro against sessile and planktonic form of P. aeruginosa strains. It is stable in pulmonary environment and it is active in media mimic the CF lung environment, under this condition its antimicrobial activity is enhanced in combination with mucolytic compounds already approved in CF-therapy. However, it showed a non negligible toxicity both in vitro and in vivo. Secondly, to partially solve this drawback its pro-form (Pro-D-BMAP18) was synthetized in order to obtain a peptide inactive and which is converted to its active form only in the site of infection/inflammation thanks to elastase, enabling a slowly release of the pharmacologically active peptide. Pro-D-BMAP18 seems to solve the side effects linked to the D-BMAP18, showing a reduce cytotoxicity and a conserved antimicrobial activity. It is correctly processes into D-BMAP18 in pulmonary environment and, under this condition, shows a good antimicrobial activity. Pro-D-BMAP18 seems to be a promising compound for the in vivo treatment of pulmonary infection but only the in vivo assay will enable us to understand the real potential of Pro-D-BMAP18 in therapy

Search for Shorter Portions of the Proline-Rich Antimicrobial Peptide Fragment Bac5(1\u201325) That Retain Antimicrobial Activity by Blocking Protein Synthesis

The spread of antibiotic-resistant pathogens has boosted the search for new antimicrobial drugs. Proline-rich antimicrobial peptides are promising lead compounds for the development of next-generation antibiotics, given their very low cytotoxicity and their good antimicrobial activity targeting the bacterial ribosome. Bac5(1\u201325) is an N-terminal fragment of the bovine proline-rich antimicrobial peptide Bac5, whose mode of action has been recently described. In this work we tested a number of Bac5(1\u201325) fragments, and we characterized their antimicrobial activity against Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa. We evaluated their cytotoxicity toward human cells and their efficacy in inhibiting bacterial protein synthesis. This allowed us to identify some shorter fragments of Bac5(1\u201325) with a good balance between antibacterial efficacy, protein synthesis inhibition, and ease/cost-effectiveness of synthesis, suitable as lead compounds to develop new antibacterials

Elastase-Activated Antimicrobial Peptide for a Safer Pulmonary Treatment of Cystic Fibrosis Infections

As bioactive small proteins with antimicrobial and immunomodulatory activities that are naturally produced by all living organisms, antimicrobial peptides (AMPs) have a marked potential as next-generation antibiotics. However, their development as antibacterial agents is limited by low stability and cytotoxicity. D-BMAP18, a membrane-permeabilizing antimicrobial peptide composed of D-amino acids, has shown good antibacterial and anti-inflammatory activities but also a non-negligible cytotoxicity against eukaryotic cell lines. In this study, a prodrug has been developed that extends the peptide with a negatively charged, inactivating sequence containing the cleavage site for neutrophil elastase (NE). The ultimate goal was to allow the activation of D-BMAP18 by endogenous elastase only at the site of infection/inflammation, enabling a slow and targeted release of the pharmacologically active peptide. In vitro activation of Pro-D-BMAP18 was confirmed using purified NE. Its antimicrobial and cytotoxic activities were tested in the presence and absence of elastase and compared to those of the parental form. The prodrug had minimal activity in the absence of elastase, while its proteolysis product retained an appreciable antimicrobial activity but lower cytotoxicity. Moreover, Pro-D-BMAP18 was found to be correctly converted to D-BMAP18 in the presence of CF sputum as a model of the lung environment and showed good antimicrobial activity under these conditions

The Anti-Pseudomonal Peptide D-BMAP18 Is Active in Cystic Fibrosis Sputum and Displays Anti-Inflammatory In Vitro Activity

Most Cystic Fibrosis (CF) patients succumb to airway inflammation and pulmonary infections due to Pseudomonas aeruginosa. D-BMAP18, a membrane-permeabilizing antimicrobial peptide composed of D-amino acids, was evaluated as a possible antibacterial aimed to address this issue. The antipseudomonal activity of D-BMAP18 was tested in a pathophysiological context. The peptide displayed activity against CF isolates of Pseudomonas aeruginosa in the presence of CF sputum when combined with sodium chloride and DNase I. In combination with DNase I, D-BMAP18 discouraged the deposition of new biofilm and eradicated preformed biofilms of some P. aeruginosa strains. In addition, D-BMAP18 down regulated the production of TNF-α, IL1-β, and TGF-β in LPS-stimulated or IFN-γ macrophages derived from THP-1 cells indicating an anti-inflammatory activity. The biocompatibility of D-BMAP18 was assessed using four different cell lines, showing that residual cell-specific cytotoxicity at bactericidal concentrations could be abolished by the presence of CF sputum. Overall, this study suggests that D-BMAP18 may be an interesting molecule as a starting point to develop a novel therapeutic agent to simultaneously contrast lung infections and inflammation in CF patients

An Apple a Day Keeps the Doctor Away: Potential Role of miRNA 146 on Macrophages Treated with Exosomes Derived from Apples

The constant dialogue between the plant world and the animal world (including man among them) has been known since the time of Adam and Eve, where an apple was the origin of the evils of the world. Apart from Snow White—who might have something to object to when it comes to the use of apples—fruits, plants, and natural extracts have been known for millennia as remedies for human health-related ailments. In the light of such evidence, the aim of the present work was to investigate from a biological point of view the potential role of apple exosomes in inflammatory processes on human cells. To this end we isolated and characterized apple exosomes and treated human cells such as macrophages and NCTC L929 as cancer cells in order to evaluate the tumorigenic and anti-inflammatory effect of apple exomes. Microscopic and molecular biology analyses were conducted to characterize exosomes and to assess cell proliferation, death, and miRNA line, as well as gene expression and the uptake of exosomes by cells. The results confirm the absolute biological safety of exosomes and their anti-inflammatory effect, mediated mainly by miRNA146 production by M2 macrophages

Serum from COVID-19 patients promotes endothelial cell dysfunction through protease-activated receptor 2

Background: Endothelial dysfunction plays a central role in the pathophysiology of COVID-19 and is closely linked to the severity and mortality of the disease. The inflammatory response to SARS-CoV-2 infection can alter the capacity of the endothelium to regulate vascular tone, immune responses, and the balance between anti-thrombotic and pro-thrombotic properties. However, the specific endothelial pathways altered during COVID-19 still need to be fully understood. Objective: In this study, we sought to identify molecular changes in endothelial cells induced by circulating factors characteristic of COVID-19. Methods and results: To this aim, we cultured endothelial cells with sera from patients with COVID-19 or non-COVID-19 pneumonia. Through transcriptomic analysis, we were able to identify a distinctive endothelial phenotype that is induced by sera from COVID-19 patients. We confirmed and expanded this observation in vitro by showing that COVID-19 serum alters functional properties of endothelial cells leading to increased apoptosis, loss of barrier integrity, and hypercoagulability. Furthermore, we demonstrated that these endothelial dysfunctions are mediated by protease-activated receptor 2 (PAR-2), as predicted by transcriptome network analysis validated by in vitro functional assays. Conclusion: Our findings provide the rationale for further studies to evaluate whether targeting PAR-2 may be a clinically effective strategy to counteract endothelial dysfunction in COVID-19