N-acetylcysteine for the prevention of non-contrast media agent-induced kidney injury: from preclinical data to clinical evidence.

PURPOSE: To review available evidence on the effectiveness of N-acetylcysteine (NAC) as a prophylactic agent in the prevention of non-contrast media agent-induced kidney injury. METHOD: Data were collected by searching Scopus, PubMed, Medline, Science direct and Cochrane database systematic reviews. A total of 26 relevant experimental studies up to the date of publication were included in the review. RESULTS: Available evidence shows that NAC has the potential to exert significant protective or ameliorative effects against drug-induced kidney injury in experimental models. The possible suggested renoprotective mechanisms of NAC in different experimental settings were acting as an antioxidant by restoring the pool of intracellular reduced glutathione, scavenging of free radicals, and/or interacting with reactive oxygen species. CONCLUSION: Whether the administration of NAC could be an effective protective clinical strategy to prevent drug-induced kidney injury or not is a question that remains to be answered in future clinical trials.

A hepcidin lowering agent mobilizes iron for incorporation into red blood cells in an adenine-induced kidney disease model of anemia in rats.

BACKGROUND: Anemia is a common complication of chronic kidney disease (CKD) that negatively impacts the quality of life and is associated with numerous adverse outcomes. Excess levels of the iron regulatory hormone hepcidin are thought to contribute to anemia in CKD patients by decreasing iron availability from the diet and from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in human patients. METHODS: We developed a modified adenine-induced kidney disease model with a higher survival rate than previously reported models, while maintaining persistent kidney disease and anemia. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which was previously shown to lower hepcidin levels in rodents, mobilized iron into the plasma and improved iron-restricted erythropoiesis in this model. RESULTS: Adenine-treated rats exhibited increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin (Hb) and inappropriately low erythropoietin (EPO) levels relative to the degree of anemia. LDN-193189 administration to adenine-treated rats lowered hepatic hepcidin mRNA, mobilized stored iron into plasma and increased Hb content of reticulocytes. CONCLUSIONS: Our data suggest that hepcidin lowering agents may provide a new therapeutic strategy to improve iron availability for erythropoiesis in CKD.

Curcumin reduces the antimicrobial activity of ciprofloxacin against Salmonella typhimurium and Salmonella typhi.

OBJECTIVES: Typhoidal and non-typhoidal infection by Salmonella is a serious threat to human health. Ciprofloxacin is the last drug of choice to clear the infection. Ciprofloxacin, a gyrase inhibitor, kills bacteria by inducing chromosome fragmentation, SOS response and reactive oxygen species (ROS) in the bacterial cell. Curcumin, an active ingredient from turmeric, is a major dietary molecule among Asians and possesses medicinal properties. Our research aimed at investigating whether curcumin modulates the action of ciprofloxacin. METHOD: We investigated the role of curcumin in interfering with the antibacterial action of ciprofloxacin in vitro and in vivo. RT-PCR, DNA fragmentation and confocal microscopy were used to investigate the modulation of ciprofloxacin-induced SOS response, DNA damage and subsequent filamentation by curcumin. Chemiluminescence and nitroblue tetrazolium reduction assays were performed to assess the interference of curcumin with ciprofloxacin-induced ROS. DNA binding and cleavage assays were done to understand the rescue of ciprofloxacin-mediated gyrase inhibition by curcumin. RESULTS: Curcumin interferes with the action of ciprofloxacin thereby increasing the proliferation of Salmonella Typhi and Salmonella Typhimurium in macrophages. In a murine model of typhoid fever, mice fed with curcumin had an increased bacterial burden in the reticuloendothelial system and succumbed to death faster. This was brought about by the inhibition of ciprofloxacin-mediated downstream signalling by curcumin. CONCLUSIONS: The antioxidant property of curcumin is crucial in protecting Salmonella against the oxidative burst induced by ciprofloxacin or interferon γ (IFNγ), a pro-inflammatory cytokine. However, curcumin is unable to rescue ciprofloxacin-induced gyrase inhibition. Curcumin's ability to hinder the bactericidal action of ciprofloxacin and IFNγ might significantly augment Salmonella pathogenesis.

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4.

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40 degrees C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu2, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

Effects of ionic and surfactant agents on the antimicrobial activity of polyhexamethylene biguanide.

OBJECTIVES: Polyhexamethylene biguanide (PHMB) exhibits broad-spectrum antimicrobial activity and is included in multipurpose solutions for contact lenses as a disinfectant. Both cationic and hydrophobic features of PHMB are believed to support its association with microbial cell membranes through electrostatic and hydrophobic interactions. We now evaluated the effects of ionic and surfactant agents on the antimicrobial activity of PHMB. METHODS: The antimicrobial activity of PHMB (1 ppm) against various bacteria and fungi was measured with the stand-alone procedure (ISO 14729, 2001). The effect of NaCl as an ionic isotonic agent on such an activity was determined in comparison with that of propylene glycol as a nonionic isotonic agent. The effect of the nonionic surfactant Poloxamer 407 (Px407) was similarly examined in the absence or presence of NaCl. RESULTS: The antimicrobial activity of PHMB increased with time, being especially pronounced after 60 min. This activity was inhibited by NaCl in a concentration-dependent manner but was not affected by propylene glycol. Poloxamer 407 (4%) alone slightly increased the activity of PHMB toward Staphylococcus aureus and fungi. Although Px407 prevented the inhibitory effect of NaCl on PHMB activity against bacteria, it enhanced that observed with Candida albicans. CONCLUSIONS: The antimicrobial activity of PHMB was inhibited by adjustment of osmolality with the ionic agent NaCl but not by that with the nonionic agent propylene glycol. The surfactant Px407 exhibited complex effects on PHMB activity in the presence of NaCl. These findings indicate that the electrostatic interaction with the cell membrane is a dominant factor in the antimicrobial activity of PHMB.

Effects of tonicity-adjusting and surfactant agents on the antimicrobial activity of alexidine.

OBJECTIVES: Alexidine is a bis-biguanide disinfectant with two cationic active sites and hydrophobic ethylhexyl end groups, both of which are believed to support its association with microbial cell membranes through electrostatic and hydrophobic interactions. We evaluated the effects of tonicity-adjusting and surfactant agents on the antimicrobial activity of alexidine to assess its suitability as a disinfectant in multipurpose solutions for contact lenses. METHODS: The antimicrobial activity of alexidine (4.5 ppm) against various bacteria and fungi was measured with the stand-alone procedure (ISO 14729, 2001). The effect of NaCl as an ionic tonicity-adjusting agent on such activity was determined in comparison with that of propylene glycol as a nonionic tonicity-adjusting agent. The effect of the nonionic surfactant Poloxamer 407 (Px407) was similarly examined in the absence or presence of NaCl. RESULTS: Alexidine showed robust antimicrobial activity, with no organisms surviving after 1 hr. Antifungal activity was inhibited by NaCl in a concentration-dependent manner, whereas neither antibacterial nor antifungal activity was affected by propylene glycol. The activity of alexidine was not affected by Px407 (4%) alone but was attenuated by the combination of NaCl and Px407 with all microorganisms tested. CONCLUSIONS: The antifungal activity of alexidine was inhibited by adjustment of osmolality with the ionic agent NaCl but not by that with the nonionic agent propylene glycol. The surfactant Px407 reduced antimicrobial activity only in the presence of NaCl. These findings indicate that electrostatic and hydrophobic interactions with the microbial cell membrane are a key factor in the antimicrobial activity of alexidine.

Mucosal human defensins 5 and 6 antagonize the anti-HIV activity of candidate polyanion microbicides.

Defensins are highly abundant antimicrobial peptides in the female genital mucosa. We have previously shown that human defensins 5 and 6 (HD5 and HD6), produced by cervicovaginal epithelial cells, significantly enhance HIV infectivity in vitro. Candidate polyanion microbicides, including PRO 2000, cellulose sulfate and carrageenan, failed to protect women against HIV infection in large-scale clinical trials, but the molecular basis of ineffectiveness was not clear. We hypothesized that mucosal host factors such as HD5 an HD6 may alter the activity of polyanion microbicides against HIV. Our results demonstrated that HD5 and HD6 but not their linear analogs antagonized the anti-HIV activity of PRO 2000, cellulose sulfate and carrageenan in vitro. Polyanion microbicides also reduced the HIV-enhancing effect of these defensins. We conclude that mucosal host factors could negatively impact the efficacy of topical microbicides against HIV, and their impact on the activity of candidate microbicides needs to be considered during the preclinical evaluation.

Comparison of TNFα to lipopolysaccharide as an inflammagen to characterize the idiosyncratic hepatotoxicity potential of drugs: Trovafloxacin as an example.

Idiosyncratic drug reactions (IDRs) are poorly understood, unpredictable, and not detected in preclinical studies. Although the cause of these reactions is likely multi-factorial, one hypothesis is that an underlying inflammatory state lowers the tolerance to a xenobiotic. Previously used in an inflammation IDR model, bacterial lipopolysaccharide (LPS) is heterogeneous in nature, making development of standardized testing protocols difficult. Here, the use of rat tumor necrosis factor-α (TNFα) to replace LPS as an inflammatory stimulus was investigated. Sprague-Dawley rats were treated with separate preparations of LPS or TNFα, and hepatic transcriptomic effects were compared. TNFα showed enhanced consistency at the transcriptomic level compared to LPS. TNFα and LPS regulated similar biochemical pathways, although LPS was associated with more robust inflammatory signaling than TNFα. Rats were then codosed with TNFα and trovafloxacin (TVX), an IDR-associated drug, and evaluated by liver histopathology, clinical chemistry, and gene expression analysis. TNFα/TVX induced unique gene expression changes that clustered separately from TNFα/levofloxacin, a drug not associated with IDRs. TNFα/TVX cotreatment led to autoinduction of TNFα resulting in potentiation of underlying gene expression stress signals. Comparison of TNFα/TVX and LPS/TVX gene expression profiles revealed similarities in the regulation of biochemical pathways. In conclusion, TNFα could be used in lieu of LPS as an inflammatory stimulus in this model of IDRs.

Cervicovaginal fluid and semen block the microbicidal activity of hydrogen peroxide produced by vaginal lactobacilli.

BACKGROUND: H2O2 produced by vaginal lactobacilli is believed to protect against infection, and H2O2-producing lactobacilli inactivate pathogens in vitro in protein-free salt solution. However, cervicovaginal fluid (CVF) and semen have significant H2O2-blocking activity. METHODS: We measured the H2O2 concentration of CVF and the H2O2-blocking activity of CVF and semen using fluorescence and in vitro bacterial-exposure experiments. RESULTS: The mean H2O2 measured in fully aerobic CVF was 23 +/- 5 microM; however, 50 microM H2O2 in salt solution showed no in vitro inactivation of HSV-2, Neisseria gonorrhoeae, Hemophilus ducreyii, or any of six BV-associated bacteria. CVF reduced 1 mM added H2O2 to an undetectable level, while semen reduced 10 mM added H2O2 to undetectable. Moreover, the addition of just 1% CVF supernatant abolished in vitro pathogen-inactivation by H2O2-producing lactobacilli. CONCLUSIONS: Given the H2O2-blocking activity of CVF and semen, it is implausible that H2O2-production by vaginal lactobacilli is a significant mechanism of protection in vivo.

Beta-lactamase inhibitors: the story so far.

Antimicrobial resistance constitutes one of the major threats regarding pathogenic microorganisms. Gram-negative pathogens, such as Enterobacteriaceae (specially those producing extended-spectrum beta-lactamases), Pseudomonas aeruginosa, and Acinetobacter baumannii have acquired an important role in hospital infections, which is of particular concern because of the associated broad spectrum of antibiotic resistance. beta-Lactam antibiotics are considered the most successful antimicrobial agents since the beginning of the antibiotic era. Soon after the introduction of penicillin, microorganisms able to destroy this beta-lactam antibiotic were reported, thus emphasizing the facility of pathogenic microorganisms to develop beta-lactam resistance. In Gram-negative pathogens, beta-lactamase production is the main mechanism involved in acquired beta-lactam resistance. Four classes of beta-lactamases have been described: A, B, C, and D. Classes A, C, and D are enzymes with a serine moiety in the active centre that catalyzes hydrolysis of the beta -lactam ring through an acyl-intermediate of serine, whereas the class B enzymes require a metal cofactor (e.g. zinc in the natural form) to function, and for this reason, they are also referred to as metallo- beta-lactamases (MBLs). To overcome beta-lactamase-mediated resistance, a combination of beta-lactam and a beta-lactamase inhibitor, which protects the beta-lactam antibiotic from the activity of the beta-lactamase, has been widely used in the treatment of human infections. Although there are some very successful combinations of beta-lactams and beta-lactamase inhibitors, most of the inhibitors act against class A beta-lactamases and remain ineffective against class B, C, and D beta-lactamases. This review constitutes an update of the current status and knowledge regarding class A to D beta-lactamase inhibitors, as well as a summary of the drug discovery strategy currently used to identify new beta-lactamase inhibitors, mainly based on the knowledge of crystal structure of beta-lactamase enzymes.

Impact of protein and lipid on neutralization times of hydrogen peroxide care regimens.

PURPOSE: To investigate the effect of protein, lipid, and lens material on the neutralization kinetics of one-step hydrogen peroxide disinfection systems. METHODS: A UV-based assay was used to determine the rate of neutralization of three one-step hydrogen peroxide systems (CIBA Vision Clear Care; CIBA Vision AOSEPT; Abbott Medical Optics UltraCare). Protein (bovine serum albumin and lysozyme) and various lipids were added to the lens cases during the neutralization phase to determine whether they influenced the rate of neutralization. Finally, rates were determined when the cases contained a silicone hydrogel lens material (lotrafilcon A) or Food and Drug Administration group IV (etafilcon A) lenses. RESULTS: Neutralization for all three systems was complete within 90 minutes. The rate of neutralization for Clear Care and AOSEPT were not significantly different from each other (P=NS). UltraCare exhibited statistically higher levels of peroxide up to the 20-minute time point (P<0.001) Protein, lipid, or lens material did not significantly affect the rate of neutralization for any regimen (P=NS). CONCLUSIONS: Tablet-based one-step disinfection systems neutralize at a slower rate than disc-based peroxide systems, but this difference is only significant during the first 20 minutes after the onset of neutralization. Neither lens deposition nor lens material plays a role in the speed of neutralization of peroxide-based systems.

Anionic C-terminal proregion of nematode antimicrobial peptide cecropin P4 precursor inhibits antimicrobial activity of the mature peptide.

Recently, an anionic proregion was found to be conserved at the C terminus of the antimicrobial peptide, nematode cecropin. Our results suggest that the antimicrobial activity of mature peptide is suppressed by the proregion in its precursor and is released from inhibition after processing. Inhibition is not likely to be due to direct suppression of membrane disruption.

A new family of lysozyme inhibitors contributing to lysozyme tolerance in gram-negative bacteria.

Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme). A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme) of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria interacting with an animal host.

AFM studies of inhibition effect in binding of antimicrobial peptide and immune proteins.

By using atomic force microscopy (AFM), we clearly show that the antimicrobial peptide affects the molecular interaction between lipopolysaccharide (LPS) and immune proteins (lipopolysaccharide binding protein [LBP] and CD14). To reconstruct an in vivo interaction, LBP and LPS (the Ra, Rc, and Re forms from Salmonella minnesota, with varying lengths of the saccharide region) were immobilized onto the AFM tip using a chemical spacer linker. We examined the interaction between the proteins on the tip and model lipid bilayer biomembranes including CD14, in both the presence and absence of the antimicrobial peptide, polymyxin B (PMB). When LPS was present, the binding force between the LBP-LPS complex and CD14 increased dramatically, compared to that seen between LBP and CD14 alone. Longer LPS saccharide regions resulted in higher binding forces. The data suggest that LPS may have an important influence on the binding of LBP to CD14 and that the saccharide region of LPS is influential in this regard. It was also found that the antimicrobial peptide PMB, at or above a particular concentration, specifically inhibited the binding between LBP-LPS and CD14.

Is there a difference in metal ion-based inhibition between members of thionin family: Molecular dynamics simulation study.

Thionins have a considerable potential as antimicrobial compounds although their application may be restricted by metal ion-based inhibition of membrane permeabilizing activity. We previously reported the properties associated with the proposed mechanism of metal ion-based inhibition of beta-purothionin. In this study, we investigated the effects of metal ions on alpha-hordothionin which differs from beta-purothionin by eight out of 45 residues. Three of the differing residues are thought to be involved in the mechanism of metal ion-based inhibition in beta-purothionin. The structure and dynamics of alpha-hordothionin were explored using unconstrained molecular dynamics (MD) simulations in explicit water as a function of metal ions. Although the global fold is almost identical to that of beta-purothionin, alpha-hordothionin displays reduced fluctuating motions. Moreover, alpha-hordothionin is more resistant to the presence of metal ions than beta-purothionin. Mg(+2) ions do not affect alpha-hordothionin, whereas K(+) ions induce perturbations in the alpha2 helix, modify dynamics and electrostatic properties. Nevertheless, these changes are considerably smaller than those in beta-purothionin. The proposed mechanism of metal ion-based inhibition involves the hydrogen bonding network of Arg5-Arg30-Gly27, which regulates dynamic unfolding of the alpha2 C-end which is similar to beta-purothionin response. The key residues responsible for the increased resistance for alpha-hordothionin are Gly27 and Gly42 which replace Asn27 and Asp42 involved into the mechanism of metal ion-based inhibition in beta-purothionin. Comparison of MD simulations of alpha-hordothionin with beta-purothionin reveals dynamic properties which we believe are intrinsic properties of thionins with four disulphide bonds.

Effect of geranylgeranylacetone on gentamycin ototoxicity in rat cochlea culture.

OBJECTIVE: Geranylgeranylacetone (GGA), an anti-ulcer drug, has been reported to induce heat shock proteins (HSPs) in several animal organs. Purpose of this study was to investigate whether GGA has protective effect on gentamycin (GM) ototoxicity and whether it induces HSP70 in rat cochlea. METHODS: We used cochlea explant culture from postnatal 5-day rat. Explants were pre-incubated with GGA, then incubated with GM and GGA. The number of surviving outer hair cells (OHCs) labeled by phalloidin was counted to evaluate the protective effect of GGA. The expression of HSP70 in whole cochlea tissue was investigated by immunoblot analysis. RESULTS: The number of surviving OHCs was significantly high in GGA 10(-5)M group compared to GM only group and GGA 10(-6)M group. In the immunoblot analysis, HSP70 levels in GGA added groups were slightly high compared to simple culture group, but much lower than those in heat shock group. CONCLUSION: It was suggested that GGA-induced HSP70, and had partial protective effects on GM ototoxicity in the cochlea. GGA has possibility to be safe and useful treatment drug for cochlea disorder.

Mathematical formulation of additivity for antimicrobial agents.

The ability to describe and quantify pharmacodynamic interaction objectively is of paramount importance in the study of combination chemotherapy. Current methods based on Loewe additivity and Bliss independence are associated with implicit assumptions of the interacting system. We reviewed the hyperplane theorem that used a generalized equation for defining the isobols (surfaces of equal effect) for 2 noninteracting drugs. We showed that under certain conditions, the original equation might lead to nonintuitive results, which were inconsistent with the definition of additivity. To circumvent these limitations, an alternative model for defining additivity based on effect summation is proposed. This alternative definition for isobols deviates from the Loewe additivity, the null interaction criterion used as the foundation for Berenbaum's isobol equation. However, its results are simple, easily interpreted, and may be more applicable to a wide variety of clinical and experimental circumstances.

Effect of preservative-free artificial tears on the antimicrobial activity of human beta-defensin-2 and cathelicidin LL-37 in vitro.

PURPOSE: Human beta-defensin-2 (hBD-2) and cathelicidin LL-37 are salt-sensitive cationic antimicrobial peptides expressed by ocular surface epithelia. The goal of this study was to investigate the effect of preservative-free artificial tears on hBD-2 and LL-37 antimicrobial activity against Pseudomonas aeruginosa. METHODS: P. aeruginosa was incubated with hBD-2 or LL-37 in the absence or presence (70% vol/vol) of different preservative-free artificial tears--Visine Tears (300 mOsm/kg), Tears Naturale Free (261 mOsm/kg), TheraTears (185 mOsm/kg), and Refresh Plus (325 mOsm/kg)--for 2 hours at 37 degrees C. In some experiments, P. aeruginosa was incubated with hBD-2 or LL-37 and Visine Tears or Tears Naturale Free with or without carboxymethylcellulose (0.5% vol/vol final concentration). Plates were inoculated with samples of each reaction mixture and then incubated for 24 hours at 37 degrees C. RESULTS: Visine Tears and Tears Naturale Free had little or no effect on the antimicrobial activity of 100 microg/mL hBD-2 or LL-37. In the presence of Refresh Plus and TheraTears, the activity of 100 microg/mL hBD-2 or LL-37 was reduced by 90% to 100%. Carboxymethylcellulose, at a concentration comparable to that present in Refresh Plus, reduced the effectiveness of hBD-2 or LL-37 by 40% to 90% in the presence of Tears Naturale Free and Visine Tears. CONCLUSION: Human beta-defensin-2 and cathelicidin LL-37 inhibit the growth of P. aeruginosa in vitro, but this activity is markedly reduced in the presence of Refresh Plus and TheraTears. These results suggest that carboxymethylcellulose-containing artificial tears may reduce the activity of the endogenously produced antimicrobial peptides.