A novel nitric oxide producing probiotic patch and its antimicrobial efficacy: preparation and in vitro analysis

Microbial and fungal infections are a significant consideration in the etiology of all wounds. Numerous antimicrobial and antifungal formulations have been developed with varying degrees of efficacy and stability. Here, we report a nitric oxide producing probiotic adhesive patch device and investigate its antimicrobial and antifungal efficacy in vitro. This probiotic patch utilizes the metabolic activity of immobilized lactic acid bacteria, glucose, and nitrite salts for the production of gaseous nitric oxide (gNO), which is used as an antimicrobial agent against bacterial and fungal pathogens. Results show that application of gNO-producing probiotic patches to cultures of E. coli, S. aureus, P. aeruginosa, MRSA, T. mentagrophytes, and T. rubrum resulted in complete cell death at between 4 and 8 h, and application to cultures of A. baumannii, resulted in fewer than ten colonies detected per milliliter at 6 h. These results demonstrate that a gNO-producing probiotic patch device containing bacteria, glucose, and nitrite salts can produce sufficient levels of gNO over a therapeutically relevant duration to kill common bacterial and fungal wound pathogens in humans.     

Participation of nitric oxide reductase in survival of Pseudomonas aeruginosa in LPS-activated macrophages

Nitric oxide (NO) plays a crucial role in the antimicrobial activity of host defense systems. We investigated the function of Pseudomonas aeruginosa NO reductase as a detoxifying enzyme in phagocytes. We found that the growth of the NO reductase-deficient mutant of P. aeruginosa under a microaerobic condition was inhibited by the exogenous NO. Furthermore, the intracellular survival assay within the NO-producing RAW 264.7 macrophages revealed that the wild-type strain survived longer than the NO reductase-deficient mutant. These results suggest that the P. aeruginosa NO reductase may contribute to the intracellular survival by acting as a counter component against the host's defense systems.     

Astrocyte-derived nitric oxide causes both reversible and irreversible damage to the neuronal mitochondrial respiratory chain

Cytokine-stimulated astrocytes produce nitric oxide (NO), which, along with its metabolite peroxynitrite (ONOO(-)), can inhibit components of the mitochondrial respiratory chain. We used astrocytes as a source of NO/ONOO(-) and monitored the effects on neurons in coculture. We previously demonstrated that astrocytic NO/ONOO(-) causes significant damage to the activities of complexes II/III and IV of neighbouring neurons after a 24-h coculture. Under these conditions, no neuronal death was observed. Using polytetrafluoroethane filters, which are permeable to gases such as NO but impermeable to NO derivatives, we have now demonstrated that astrocyte-derived NO is responsible for the damage observed in our coculture system. Expanding on these observations, we have now shown that 24 h after removal of NO-producing astrocytes, neurons exhibit complete recovery of complex II/III and IV activities. Furthermore, extending the period of exposure of neurons to NO-producing astrocytes does not cause further damage to the neuronal mitochondrial respiratory chain. However, whereas the activity of complex II/III recovers with time, the damage to complex IV caused by a 48-h coculture with NO-producing astrocytes is irreversible. Therefore, it appears that neurons can recover from short-term damage to mitochondrial complex II/III and IV, whereas exposure to astrocytic-derived NO for longer periods causes permanent damage to neuronal complex IV.     

Antimicrobial and anti-inflammatory activities of a Leu/Lys-rich antimicrobial peptide with Phe-peptoid residues

To develop a novel cell-selective antimicrobial peptide with potent anti-inflammatory activity as well as high bacterial cell selectivity, we synthesized a Leu/Lys-rich model peptide, KLW-f (KWKKLLKKfLKLfKKLLK-NH(2)) containing two Phe-peptoid residues in its middle position. KLW-f exhibited high antimicrobial activity (the MIC range: 0.5 approximately 2.0microM) against the tested six bacterial cells. In contrast, KLW-f was no cytotoxic to human red blood cells and HeLa and NIH-3T3 cells. KLW-f caused no or little dye leakage from EYPE/EYPG (7:3, w/w) vesicles (bacterial membrane-mimicking environments), indicating its bacterial-killing action is probably not due to permeabilization/disruption of bacterial cytoplasmic membranes. Furthermore, KLW-f induced a significant inhibition in LPS-stimulated NO production from mouse macrophage RAW264.7 cells at 10microg/ml. Taken together, our results suggest that KLW-f appear to have promising therapeutic potential for future development as a novel antisepsis agent as well as antimicrobial agent.     

Methyl-Hydroxylamine as an Efficacious Antibacterial Agent That Targets the Ribonucleotide Reductase Enzyme

The emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. Ribonucleotide reductase (RNR) is a key enzyme in DNA replication that acts by converting ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks of DNA replication and repair. RNR has been extensively studied as an ideal target for DNA inhibition, and several drugs that are already available on the market are used for anticancer and antiviral activity. However, the high toxicity of these current drugs to eukaryotic cells does not permit their use as antibacterial agents. Here, we present a radical scavenger compound that inhibited bacterial RNR, and the compound''s activity as an antibacterial agent together with its toxicity in eukaryotic cells were evaluated. First, the efficacy of N-methyl-hydroxylamine (M-HA) in inhibiting the growth of different Gram-positive and Gram-negative bacteria was demonstrated, and no effect on eukaryotic cells was observed. M-HA showed remarkable efficacy against Mycobacterium bovis BCG and Pseudomonas aeruginosa. Thus, given the M-HA activity against these two bacteria, our results showed that M-HA has intracellular antimycobacterial activity against BCG-infected macrophages, and it is efficacious in partially disassembling and inhibiting the further formation of P. aeruginosa biofilms. Furthermore, M-HA and ciprofloxacin showed a synergistic effect that caused a massive reduction in a P. aeruginosa biofilm. Overall, our results suggest the vast potential of M-HA as an antibacterial agent, which acts by specifically targeting a bacterial RNR enzyme.     

Improved antimicrobial efficacy with nitric oxide releasing nanoparticle generated S-nitrosoglutathione

Nitric oxide (NO) plays a vital role in mammalian host defense through a variety of mechanisms. In particular, NO can oxidize to form reactive nitrogen species or interact with protein thiols and metal centers, blocking essential microbial processes. S-nitrosoglutathione (GSNO), a potent NO donor formed by the interaction of NO with intracellular glutathione (GSH), is a major factor in this pathway and is considered one of the strongest naturally occurring nitrosating agent. We previously described the broad-spectrum antimicrobial activity of a nanoparticulate platform capable of controlled and sustained release of NO (NO-np). Interestingly, in vivo efficacy of the NO-np surpassed in vitro data generated. We hypothesized that the enhanced activity was in part achieved via the interaction between the generated NO and available GSH, forming GSNO. In the current study, we investigated the efficiency of NO-np to form GSNO in the presence of GSH was evaluated, and assessed the antimicrobial activity of the formed GSNO against methicillin resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. When GSH was combined with NO-np, GSNO was rapidly produced and significant concentrations of GSNO were maintained for >24h. The GSNO generated was more effective compared to NO-np alone against all bacterial strains examined, with P. aeruginosa being the most sensitive and K. pneumoniae the most resistant. We conclude that the combination of NO-np with GSH is an effective means of generating GSNO, and presents a novel approach to potent antimicrobial therapy.     

泊沙康唑药动学-药效学及其治疗药物监测研究进展

泊沙康唑为新一代三唑类广谱抗真菌药,临床主要用于侵袭性曲霉菌病、念珠菌病的预防和难治性口咽念珠菌病的治疗,具有抗菌 活性高、耐受性好、不良反应少等特点,但其口服后生物利用度具有较大的个体差异。综述泊沙康唑混悬液的药动学影响因素、不同患者 人群的药动学特征以及群体药动学特征、药动学 / 药效学特性、治疗药物监测对临床疗效和不良反应的重要影响,以指导临床个体化用药, 提高用药的有效性和安全性。     

Antimicrobial and anti-inflammatory activities of designed antimicrobial peptide P18 analogues

To develop antimicrobial peptides having higher bacterial selectivity than a novel antimicrobial peptide P18, we synthesized several analogues. The P18 analogues are designed by movement of the N-terminal Trp2 residue in P18 (P18-W6, P18-W8 and P18-W15) and the substitution of the central Pro9 residue with D-Pro or Nala (P18-Nala9 and P18-D-Pro9). These analogues retained potent antibacterial activity but displayed less hemolytic activity than P18. From the viewpoint of their therapeutic index, P18 analogues had approximate 3- to 7-fold higher bacterial selectivity compared to P18. The analogues preferentially bind to bacterial membrane-mimicking negatively charged liposomes as well as does P18. Their high specificity to negatively charged phospholipids corresponds well with their high bacterial selectivity. Furthermore, P18-W6, P18-W8 and P18-Nala9 induced a significant inhibition in NO production from LPS-stimulated macrophage RAW264.7 cells, as well as P18. This result suggests that these peptides appear to have promising therapeutic potential for future development as a novel anti-inflammatory agent as well as antimicrobial agent.     

A new method for monitoring nitric oxide production using Teflon membrane microdialysis

The measurement of nitric oxide (NO) by electron spin resonance (ESR) is complicated by potentially toxic spin-trapping agents, which may affect the NO-producing cells per se and/or cause artifacts and systemic side effects. These problems can be addressed by preventing direct interaction between the agent and the biological system. In the present study, we utilized Teflon as a barrier between the spin trap and the living cell, since the material is permeable to gas only. Our aim was to investigate if NO could diffuse across the membrane in sufficient amounts to be trapped and quantified by ESR. We used standard microdialysis equipment and specially designed dialysis probes, or tubing, with Teflon membranes. Sodium nitroprusside was used as a NO donor and Fe-N-dithiocarboxysarcosine (Fe(DTCS)2) as a spin trap. NO readily diffuses through Teflon and could be quantified in concentrations considerably below 50 nM in a reproducible and accurate manner. In cell cultures of activated murine macrophages, NO synthesis from iNOS could be monitored and we noted a huge increase in NO concentration by superoxide dismutase. We conclude that spin trapping of NO by Fe(DTCS)2 across Teflon membranes is an attractive approach for quantifying and monitoring nitric oxide production without interfering with cell viability.     

Regulation of multimeric structure of NO-synthase as a new factor of organogenesis

The effect of NO on organogenesis in Drosophila is discussed. A new model of regulation of the activity of NO-producing enzyme, NO synthase is described, which takes into account endogenous synthesis of its reduced isoform. The reduced isoform of NO synthase is capable of suppressing the enzymatic activity of full-sized NO synthase during formation of a heterodimer in vivo and in vitro. The reduced form of this enzyme inhibits the antiproliferative effect of the full-sized NO synthase isoform during formation of eye structure in Drosophila by affecting the pathways of cell cycle regulation. The reduced form of NO synthase is an endogenous dominant-negative factor of regulation of the NO synthase activity in development of Drosophila.     

Genotoxicity associated with NO production in macrophages and co-cultured target cells

We previously used mouse macrophage-like RAW264.7 cells as an experimental system for the study of nitric oxide (NO)-associated genotoxicity under physiologically relevant conditions, and characterized genotoxic effects in the NO-producing cells. Here we report experiments utilizing a co-culture system enabling parallel studies of cytotoxic and genotoxic responses in co-cultured target cells as well as in macrophages stimulated to produce NO. We found that co-cultivation with macrophages stimulated to produce NO for 38-42 h resulted in significant increases in mutant fraction in the endogenous genes of target human TK6 and hamster CHO-AA8 cells and in the macrophages themselves, accompanied by a substantial decrease in cell viability. Addition of N-methyl-L-arginine, an NO synthase inhibitor, abrogated much of the cytotoxicity and genotoxicity in both target and macrophages cells, verifying the role of NO in the induction of these responses. We also showed that NO-associated genotoxic response in macrophages could be influenced by culture medium. Collectively, these results support the hypothesis that NO production by activated macrophages may contribute to genotoxic risks associated with chronic inflammation.     

Complementary distribution of NADPH-diaphorase and l-arginine in the snail nervous system

Since the interneuronal messenger nitric oxide (NO) can not be stored in neurones, the regulation of the NO-producing enzyme nitric oxide synthase (NOS) is crucial. Neuronal NOS metabolises L-arginine to nitric oxide (NO) and L-citrulline in a Ca(2+)-dependent manner. Thus, availability of L-arginine to NOS may modulate NO production. In this study, we examined the cellular distribution of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, L-arginine and L-citrulline. Using NADPH-diaphorase histochemistry to visualise putative NO-producing cells and immunocytochemistry to localise L-arginine, we showed that the distribution of L-arginine-immunoreactive neurones correlates well with those of NADPH-diaphorase-positive neurones in cerebral ganglia of the pulmonate Helix pomatia. However, substrate and enzyme were visualised in separate but adjacent neurones. We further examined whether NADPH-diaphorase-labelled cells contain the L-citrulline. Following elevation of intracellular Ca(2+) by the Ca(2+) ionophore, ionomycin, or by a high-K(+) solution, the number of L-citrulline-immunoreactive neurones in mesocerebrum and pedal lobe increased up to tenfold. Preincubation of ganglia with the NOS inhibitor N(G)-nitro-L-arginine prevented ionomycin or high-K(+) solution-induced L-citrulline synthesis. Most L-citrulline-immunoreactive neurones contain NADPH-diaphorase activity. In conclusion, these experiments indicate a complementary distribution of NOS and L-arginine and suggest an unknown signalling pathway between neurones to maintain L-arginine and NO homeostasis.     

Mitochondrial nitric oxide synthase, oxidative stress and apoptosis

Nitric oxide (NO) exerts a wide range of its biological properties via its interaction with mitochondria. By competing with O(2), physiologically relevant concentrations of NO reversibly inhibit cytochrome oxidase and decrease O(2) consumption, in a manner resembling a pharmacological competitive antagonism. The inhibition regulates many cellular functions, by e.g., regulating the synthesis of ATP and the formation of mitochondrial transmembrane potential (Delta Psi). NO regulates the oxygen consumption of both the NO-producing and the neighboring cells; thus, it can serve as autoregulator and paracrine modulator of the respiration. On the other hand, NO reacts avidly with superoxide anion (O(2)(-)) to produce the powerful oxidizing agent, peroxynitrite (ONOO(-)) which affects mitochondrial functions mostly in an irreversible manner. How mitochondria and cells harmonize the reversible effects of NO versus the irreversible effects of ONOO(-) will be discussed in this review article. The exciting recent finding of mitochondrial NO synthase will also be discussed.     

Impact of endogenous nitric oxide on microglial cell energy metabolism and labile iron pool

Microglial activation is common in several neurodegenerative disorders. In the present study, we used the murine BV-2 microglial cell line stimulated with gamma-interferon and lipopolysaccharide to gain new insights into the effects of endogenously produced NO on mitochondrial respiratory capacity, iron regulatory protein activity, and redox-active iron level. Using polarographic measurement of respiration of both intact and digitonin-permeabilized cells, and spectrophotometric determination of individual respiratory chain complex activity, we showed that in addition to the reversible inhibition of cytochrome-c oxidase, long-term endogenous NO production reduced complex-I and complex-II activities in an irreversible manner. As a consequence, the cellular ATP level was decreased in NO-producing cells, whereas ATPase activity was unaffected. We show that NO up-regulates RNA-binding of iron regulatory protein 1 in microglial cells, and strongly reduces the labile iron pool. Together these results point to a contribution of NO derived from inflammatory microglia to the misregulation of energy-producing reactions and iron metabolism, often associated with the pathogenesis of neurodegenerative disorders.     

Nitrite modulates bacterial antibiotic susceptibility and biofilm formation in association with airway epithelial cells

Pseudomonas aeruginosa is the major pathogenic bacteria in cystic fibrosis and other forms of bronchiectasis. Growth in antibiotic-resistant biofilms contributes to the virulence of this organism. Sodium nitrite has antimicrobial properties and has been tolerated as a nebulized compound at high concentrations in human subjects with pulmonary hypertension; however, its effects have not been evaluated on biotic biofilms or in combination with other clinically useful antibiotics. We grew P. aeruginosa on the apical surface of primary human airway epithelial cells to test the efficacy of sodium nitrite against biotic biofilms. Nitrite alone prevented 99% of biofilm growth. We then identified significant cooperative interactions between nitrite and polymyxins. For P. aeruginosa growing on primary CF airway cells, combining nitrite and colistimethate resulted in an additional log of bacterial inhibition compared to treating with either agent alone. Nitrite and colistimethate additively inhibited oxygen consumption by P. aeruginosa. Surprisingly, whereas the antimicrobial effects of nitrite in planktonic, aerated cultures are nitric oxide (NO) dependent, antimicrobial effects under other growth conditions are not. The inhibitory effect of nitrite on bacterial oxygen consumption and biofilm growth did not require NO as an intermediate as chemically scavenging NO did not block growth inhibition. These data suggest an NO-radical independent nitrosative or oxidative inhibition of respiration. The combination of nebulized sodium nitrite and colistimethate may provide a novel therapy for chronic P. aeruginosa airway infections, because sodium nitrite, unlike other antibiotic respiratory chain “poisons,” can be safely nebulized at high concentration in humans.     

Nitric oxide (NO) and cartilage metabolism: NO effects are modulated by superoxide in response to IL-1

Nitric oxide (NO) is thought to mediate most effects of interleukin-1 (IL-1) on cartilage. In vitro evidence includes the decreased synthesis of extracellular matrix components, the abnormal cell renewal, the decreased production of IL-1 receptor antagonist, the induction of apoptosis and the enhanced sensitivity of chondrocytes to oxidative stress. Studies in NOS2(-/-) mice or administration of NO synthase inhibitors in animal models of joint disorders have confirmed its potent pathophysiological role in cartilage. Using L-NMMA (1 mM), as a NO synthase inhibitor, and CuDips (10 microM), as a SOD mimetic, we provide evidence that the inhibitory potency of IL-1beta on proteoglycan synthesis and its stimulating effect on COX-2 activity depend both on NO and O2-* production. Peroxynitrite formation is further demonstrated by the occurrence of 3-nitrotyrosines in chondrocytes stimulated in vitro with 2.5 ng/ml IL-1 and in femoral condyles of rats injected locally with 1 microg IL-1. Preliminary data suggest that such contribution of reactive oxygen species is not shared in common by IL-17, another NO-producing cytokine. We conclude that superoxide is a key modulator of NO-mediated effects in chondrocyte stimulated with IL-1 and that a combined therapy with NO synthase inhibitors and antioxidants may be promising for a full cartilage protection.     

Taurine bromamine (TauBr) - its role in immunity and new perspectives for clinical use

This review is an attempt to summarize our knowledge about taurine bromamine (TauBr) properties, its role in innate immunity and its therapeutic potential.TauBr and taurine chloramine (TauCl) are major haloamines generated by eosinophils and neutrophils at a site of inflammation. Both haloamines share anti-inflammatory and anti-oxidant properties. TauBr, similarly to TauCl, decreases the production of proinflammatory mediators. Their anti-inflammatory and anti-oxidant activities are enhanced by their ability to induce the expression of heme oxygenase-1 (HO-1). TauCl is more stable than TauBr. On the other hand, only TauBr was found to be highly membrane-permeable showing stronger microbicidal activity than TauCl.In the light of the anti-inflammatory and antimicrobial properties of TauBr we discuss its therapeutic potential in local treatment of inflammation, especially acne vulgaris, the most common inflammatory skin disorder. TauBr, at non-cytotoxic concentrations, is able to kill Propionibacterium acnes, the skin bacteria involved in pathogenesis of acne vulgaris.As topical antibiotics used in the therapy of acne are associated with the emergence of resistant bacteria, topical TauBr seems to be a good candidate for an alternative therapy.Recently, in a double blind trial, the efficacy of TauBr was compared with the efficacy of clindamycin, one of the most common topical antibiotics used in acne therapy. Comparable reduction of acne lesions was observed in the TauBr and clindamycin groups of patients with mild and moderate inflammatory facial acne vulgaris. We conclude that this pilot study supports our concept that TauBr can be used as a topical agent in the treatment of acne vulgaris, especially in patients who have already developed antibiotic resistance. Further studies are necessary to substantiate the more extended use of TauBr as an anti-inflammatory and anti-oxidant agent in human medicine.     

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.     

The Entamoeba histolytica Dnmt2 Homolog (Ehmeth) Confers Resistance to Nitrosative Stress

Nitric oxide (NO) has antimicrobial properties against many pathogens due to its reactivity as an S-nitrosylating agent. It inhibits many of the key enzymes that are involved in the metabolism and virulence of the parasite Entamoeba histolytica through S-nitrosylation of essential cysteine residues. Very little information is available on the mechanism of resistance to NO by pathogens in general and by this parasite in particular. Here, we report that exposure of the parasites to S-nitrosoglutathione (GSNO), an NO donor molecule, strongly reduces their viability and protein synthesis. However, the deleterious effects of NO were significantly reduced in trophozoites overexpressing Ehmeth, the cytosine-5 methyltransferase of the Dnmt2 family. Since these trophozoites also exhibited high levels of tRNA^Asp methylation, the high levels suggested that Ehmeth-mediated tRNA^Asp methylation is part of the resistance mechanism to NO. We previously reported that enolase, another glycolytic enzyme, binds to Ehmeth and inhibits its activity. We observed that the amount of Ehmeth-enolase complex was significantly reduced in GSNO-treated E. histolytica, which explains the aforementioned increase of tRNA methylation. Specifically, we demonstrated via site-directed mutagenesis that cysteine residues 228 and 229 of Ehmeth are susceptible to S-nitrosylation and are crucial for Ehmeth binding to enolase and for Ehmeth-mediated resistance to NO. These results indicate that Ehmeth has a central role in the response of the parasite to NO, and they contribute to the growing evidence that NO is a regulator of epigenetic mechanisms.