Therapeutic Potential of Antimicrobial Peptide PN5 against Multidrug-Resistant E. coli and Anti-Inflammatory Activity in a Septic Mouse Model.

Antibiotic-resistant bacteria have become a public health problem. Thus, antimicrobial peptides (AMPs) have been evaluated as substitutes for antibiotics. Herein, we investigated PN5 derived from Pinus densiflora (pine needle). PN5 exhibited antimicrobial activity without causing cytotoxic effects. Based on these results, we examined the mode of action of PN5 against Gram-negative and -positive bacteria. PN5 exhibited membrane permeabilization ability, had antimicrobial stability in the presence of elastase, a proteolytic enzyme, and did not induce resistance in bacteria. Bacterial lipopolysaccharide (LPS) induces an inflammatory response in RAW 264.7 macrophages. PN5 suppressed proinflammatory cytokines mediated by NF-κB and mitogen-activated protein kinase signaling. In C57BL/6J mice treated with LPS and d-galactosamine, PN5 exhibited anti-inflammatory activity in inflamed mouse livers. Our results indicate that PN5 has antimicrobial and anti-inflammatory activities and thus may be useful as an antimicrobial agent to treat septic shock caused by multidrug-resistant (MDR) Escherichia coli without causing further resistance. IMPORTANCE Antibiotic-resistant bacteria are a global health concern. There is no effective treatment for antibiotic-resistant bacteria, and new alternatives are being suggested. The present study found antibacterial and anti-inflammatory activities of PN5 derived from Pinus densiflora (pine needle), and further investigated the therapeutic effect in a mouse septic model. As a mechanism of antibacterial activity, PN5 exhibited the membrane permeabilization ability of the toroidal model, and treated strains did not develop drug resistance during serial passages. PN5 showed immunomodulatory properties of neutralizing LPS in a mouse septic model. These results indicate that PN5 could be a new and promising therapeutic agent for bacterial infectious disease caused by antibiotic-resistant strains.

ATRA reduces inflammation and improves alveolar epithelium regeneration in emphysematous rat lung.

INTRODUCTION: Pulmonary emphysema characterized by alveolar wall destruction is resultant of persistent chronic inflammation. All-trans retinoic acid (ATRA) has been reported to reverse elastase-induced emphysema in rats. However, the underlying molecular mechanisms are so far unknown. OBJECTIVE: To investigate the therapeutic potential effect of ATRA via the amelioration of the ERK/JAK-STAT pathways in the lungs of emphysematous rats. METHODS: In silico analysis was done to find the binding efficiency of ATRA with receptor and ligands of ERK & JAK-STAT pathway. Emphysema was induced by porcine pancreatic elastase in Sprague-Dawley rats and ATRA was supplemented as therapy. Lungs were harvested for histopathological, genomics and proteomics analysis. RESULTS AND DISCUSSION: In silico docking, analysis confirms that ATRA interferes with the normal binding of ligands (TNF-α, IL6ST) and receptors (TNFR1, IL6) of ERK/JAK-STAT pathways respectively. ATRA restored the histology, proteases/antiproteases balance, levels of inflammatory markers, antioxidants, expression of candidate genes of ERK and JAK-STAT pathways in the therapy group. CONCLUSION: ATRA ameliorates ERK/JAK-STAT pathway in emphysema condition, resulting in alveolar epithelium regeneration. Hence, ATRA may prove to be a potential drug in the treatment of emphysema.

Pseudomonas aeruginosa and epithelial permeability: role of virulence factors elastase and exotoxin A.

Lung injury in bacterial infection is a multifactorial phenomenon that involves bacterial metabolites and host factors. Primary isolates of type II pneumocytes and established cultures of Madin-Darby canine kidney (MDCK) cells were used to study effects of Pseudomonas aeruginosa exoproducts on epithelial paracellular permeability. The results indicate that elastase (PE) and exotoxin A (Exo A) have different, but complementary, actions that diminish epithelial barrier function. We measured transepithelial electrical resistance (TER) and permeability coefficient for mannitol (Pm) across cell monolayers plated on tissue culture membranes. Application of 100 ng/ml of Exo A to the basal side decreased TER from 1,405 +/- 106 to 462 +/- 50 ohm (omega) and increased Pm for mannitol 6-fold in 16 h (P < 0.05). Application of Exo A to the apical side did not affect either TER or Pm. In contrast, PE (6.5 U/ml) applied either apically or basolaterally reduced TER to 353 +/- 66 omega and increased Pm by 10-fold within 90 min (P < 0.05). The increase in permeability correlated with the number of bacteria that traversed the epithelial monolayers. Fluorescent staining and western immunoblot analysis of toxin-treated cells showed that two tight junctional proteins, ZO-1 and ZO-2, were depleted in monolayers treated with enzymatically active PE. The junctional proteins decreased in cells treated overnight with Exo A but were not depleted. Neither agent diminished cell viability as measured by trypan blue staining or release of radioactivity from 51 Cr-labeled cells. Elastase from P. aeruginosa thus seems to increase alveolar epithelial permeability by damaging tight junction-associated proteins. Exo A, through its effect on protein synthesis, may render the cells unable to restore the junctional proteins and thus the functional junctions.

Reduction in virus-neutralizing activity of a bovine colostrum immunoglobulin concentrate by gastric acid and digestive enzymes.

Bovine milk immunoglobulin concentrates have been proposed for inducing passive immunity against various enteric pathogens. In vitro digestion studies were conducted to evaluate the effect of gastrointestinal secretions on the virus-neutralizing activity of a concentrate prepared from the colostrum of cows that were immunized with rotavirus. The proteolytic activity of human gastric and duodenal fluid specimens was used to design a two-stage in vitro digestion model with commercial enzymes for estimating the individual impact of pepsin, gastric acid, and select pancreatic enzymes on antirotavirus activity in bovine milk immunoglobulin concentrates. The rotavirus-neutralizing titer of concentrate was decreased by incubation with pepsin at pH 2, a pool of pancreatic enzymes at pH 7.5, or sequential digestion with pepsin (pH 2) and pancreatic enzymes (from initial titer of 55,210 to 2,030, 19,500, and 320, respectively). Reduction in rotavirus-neutralizing titer after gastric-phase digestion was primarily due to acidic conditions and not to proteolytic cleavage by pepsin. Although both trypsin and carboxypeptidase caused significant proteolysis of concentrate during duodenal-phase digestion, only trypsin caused a significant reduction in rotavirus-neutralizing titer. The extent of digestion was the same for concentrate suspended in water or skim milk. The results demonstrate that the biological activity of bovine milk antibodies is reduced by exposure to acid and trypsin in vitro and suggest that neutralization of both gastric acid and pancreatic trypsin may enhance the effectiveness and economic feasibility of passive oral immunoprophylaxis with bovine milk immunoglobulins.

Pseudomonas and neutrophil products modify transferrin and lactoferrin to create conditions that favor hydroxyl radical formation.

In vivo most extracellular iron is bound to transferrin or lactoferrin in such a way as to be unable to catalyze the formation of hydroxyl radical from superoxide (.O2-) and hydrogen peroxide (H2O2). At sites of Pseudomonas aeruginosa infection bacterial and neutrophil products could possibly modify transferrin and/or lactoferrin forming catalytic iron complexes. To examine this possibility, diferrictransferrin and diferriclactoferrin which had been incubated with pseudomonas elastase, pseudomonas alkaline protease, human neutrophil elastase, trypsin, or the myeloperoxidase product HOCl were added to a hypoxanthine/xanthine oxidase .O2-/H2O2 generating system. Hydroxyl radical formation was only detected with pseudomonas elastase treated diferrictransferrin and, to a much lesser extent, diferriclactoferrin. This effect was enhanced by the combination of pseudomonas elastase with other proteases, most prominently neutrophil elastase. Addition of pseudomonas elastase-treated diferrictransferrin to stimulated neutrophils also resulted in hydroxyl radical generation. Incubation of pseudomonas elastase with transferrin which had been selectively iron loaded at either the NH2- or COOH-terminal binding site yielded iron chelates with similar efficacy for hydroxyl radical catalysis. Pseudomonas elastase and HOCl treatment also decreased the ability of apotransferrin to inhibit hydroxyl radical formation by a Fe-NTA supplemented hypoxanthine/xanthine oxidase system. However, apotransferrin could be protected from the effects of HOCl if bicarbonate anion was present during the incubation. Apolactoferrin inhibition of hydroxyl radical generation was unaffected by any of the four proteases or HOCl. Alteration of transferrin by enzymes and oxidants present at sites of pseudomonas and other bacterial infections may increase the potential for local hydroxyl radical generation thereby contributing to tissue injury.

Evidence by in vivo and in vitro studies that binding of pycnogenols to elastin affects its rate of degradation by elastases.

Procyanidol oligomers and (+) catechin bound to insoluble elastin markedly affect its rate of degradation by elastases. Insoluble elastin pretreated with procyanidol oligomers (PCO) was resistant to the hydrolysis induced by both porcine pancreatic and human leukocyte elastases. The quantitative adsorption of pancreatic elastase was similar on either untreated or PCO-treated elastin suggesting that the binding of this compound to elastin increases the non-productive catalytic sites of elastase molecules. (+) Catechin-insoluble elastin complexes were partially resistant to the degradation induced by human leukocyte elastase but were hydrolysed at the same rate as untreated samples by a constant amount of pancreatic elastase. In addition, the coacervation profile of kappa-elastin peptides as a function of temperature is greatly modified in presence of these flavonoids. We conclusively evidenced that PCOs bind to skin elastic fibres when injected intradermally into young rabbits. As a result, these elastic fibres were found more resistant to the hydrolytic action of porcine pancreatic elastase when injected to the same site. These in vivo studies further emphasized the potential effect of these compounds in preventing elastin degradation by elastase(s) as occurred in inflammatory processes.

Effects of protease treatment on growth, morphology, adhesion, and cell surface proteins of secondary chick embryo fibroblasts.

Several proteolytic enzymes have been studied with regard to their ability to induce DNA synthesis and cell proliferation in resting chick embryo fibroblasts. Of the enzymes examined, thrombin, bromelin, and trypsin exhibit potent mitogenic activity, elastase has significant but less marked activity, whereas thermolysin, papain, and alpha-protease are inactive. The enzymes were also tested for their ability to induce morphological change or to remove two iodinatable proteins of 250,000 and 205,000 daltons. Although the larger protein is removed by some but not all of the proteases examined, every protease tested removed the smaller cell surface proteins; however, loss of the smaller protein does correlate with the reduction of both cytoplasmic spreading and cell-cell interactions observed after protease treatment. A secondary, later event of migration of cells into clumps is observed in those instances when protease treatment did not result in a loss of the 250k protein. Arole for each of these proteins in the processes of cellular adhesion is discussed.