Effects of the microbicide ceragenin CSA-13 on and properties of Bacillus subtilis spores prepared on two very different media.

AIMS: To determine how the microbicide ceragenin-13 (CSA-13) kills Bacillus subtilis spores prepared on growth or sporulation media, and these spores' properties. METHODS AND RESULTS: Spores made on Luria broth (LB) growth or double-strength Schaeffer's-glucose (2xSG) sporulation plates found that spores made on LB plates have coat defects as evidenced by their lower hypochlorite resistance, faster germination with dodecylamine and slower germination with Ca2+ -dipicolinic acid (CaDPA) than 2xSG plate spores. CSA-13 triggered CaDPA release from spores, an early step in germination, but only well at 70°C and better with spores made on LB than on 2xSG plates. Approximately 90% of spores with elevated levels of SpoVA proteins that form a CaDPA release channel, released CaDPA with CSA-13 at 70°C, and faster with spores made on LB than 2xSG plates. Levels of CSA-13 killing of spores made on LB and 2xSG plates were similar to levels of CaDPA release triggered by this agent. CONCLUSIONS: CSA-13 kills bacterial spores, but only at high concentrations and temperatures, and is preceded by CaDPA release. SIGNIFICANCE AND IMPACT OF THE STUDY: CSA-13 is not a direct sporicide as reported previously, but most likely germinates spores via activation of spores' CaDPA channel, albeit inefficiently, and then killing the germinated spores.

Psychosine variants as antigens for natural killer T cells.

Natural killer T (NKT) cells play a central role in the interface between innate and adaptive immunity, and alpha-galactosylceramide was recently shown to be an endogenous antigen for these cells. The source of alpha-galactosylceramide has not yet been determined; however, in vivo degradation of alpha-galactosylceramide involves generation of alpha-psychosine (alpha-galactosylsphingosine). Alpha-psychosine stimulates cytokine release from NKT cells and constitutes an endogenous antigen for these cells. Alpha-psychosine contains a single lipid chain, while most antigens for NKT cells have two lipid chains, and we have investigated if other glycolipids with one lipid chain, derived from know antigens for NKT cells, stimulate cytokine release from NKT cells. Only psychosine variants derived from the most potent NKT cell antigens cause stimulation, and this stimulation occurs in vitro as well as in vivo. Truncated forms of weak antigens for NKT cells are not stimulatory.

A peptide-free, liposome-based oligosaccharide vaccine, adjuvanted with a natural killer T cell antigen, generates robust antibody responses in vivo.

Due to the prevalence of oligo- and polysaccharides on the surfaces of pathogenic organisms, carbohydrates are primary targets for recognition by antibodies generated by the immune systems of higher organisms. Consequently, substantial effort has been expended in efforts to develop vaccines based on carbohydrate epitopes. Typical approaches involve multivalent presentation of carbohydrate targets on antigenic peptides or proteins, which often involve substantial synthetic commitments and/or vaccines that are heterogeneous and difficult to characterize. We have developed a simple, liposome-based approach to generate multivalent carbohydrate vaccines, and in place of an antigenic peptide or protein, we have used a potent antigen for natural killer T cells. This vaccine, based on the Streptococcus pneumoniae serotype 14 polysaccharide, gave a response superior to that from a clinically used vaccine (Prevnar). The dependence of this response on liposome formation was demonstrated by comparison to a simple mixture of the oligosaccharide and the natural killer T cell adjuvant. The importance of the strength of the adjuvant was observed by use of a potent synthetic adjuvant and a weaker, bacterial derived glycolipid adjuvant. These results demonstrate the effectiveness of this novel and relatively simple means of generating carbohydrate-based vaccines.

Effect of pluronic acid F-127 on the toxicity towards eukaryotic cells of CSA-13, a cationic steroid analogue of antimicrobial peptides.

AIMS: CSA-13 is an antimicrobial cationic steroid with some toxicity against eukaryotic cells. The purpose of this work was to test whether pluronic acid F-127 could interfere with the toxicity of CSA-13 on human umbilical vein endothelial (HUVEC) without modifying its bactericidal activity against Pseudomonas aeruginosa. METHODS AND RESULTS: The addition of pluronic acid F-127 slightly decreased the number of dead cells after exposure to CSA-13. Pluronic acid F-127 blocked the permeabilizing effect of CSA-13 on the plasma membrane of HUVEC (uptake of ethidium bromide, release of lactate dehydrogenase) without modifying its toxic effect on their mitochondrial function (MTT test, uptake of tetramethyl rhodamine ethyl ester). CONCLUSION: Pluronic acid F-127 decreased the toxicity of CSA-13 against eukaryotic cells without completely protecting them from mitochondrial damage at high concentrations of the drug. SIGNIFICANCE AND IMPACT OF THE STUDY: This work establishes that studies on the toxic effects of synthetic antimicrobials on eukaryotic cells should not only focus on the permeability of the plasma membrane but also on the integrity of the mitochondria.

Structure-guided design of an invariant natural killer T cell agonist for optimum protection from type 1 diabetes in non-obese diabetic mice.

Because invariant natural killer T (iNK T) cells link innate and adaptive immunity, the structure-dependent design of iNK T cell agonists may have therapeutic value as vaccines for many indications, including autoimmune disease. Previously, we showed that treatment of non-obese diabetic (NOD) mice with the iNK T cell activating prototypic glycolipid α-galactosylceramide (α-GalCer) protects them from type 1 diabetes (T1D). However, α-GalCer is a strong agonist that can hyperactivate iNK T cells, elicit several side effects and has shown only limited success in clinical trials. Here, we used a structure-guided design approach to identify an iNK T cell agonist that optimally protects from T1D with minimal side effects. Analyses of the kinetics and function of a panel of synthetic α-GalCer fatty acyl chain derivatives (C8:0-C16:0) were performed in NOD mice. C16:0 elicited the highest protection from insulitis and T1D, which was associated with a higher frequency and survival of iNK T cells and enhanced activity of tolerogenic dendritic cells (DCs) in draining pancreatic lymph nodes (PLN), inability to transactivate NK cells and a more rapid kinetics of induction and recovery of iNK T cells from anergy. We conclude that the length and structure of the acyl chain of α-GalCer regulates the level of protection against T1D in mice, and propose that the extent of this protection depends on the relative capacity of the acyl chain to accommodate an endogenous spacer lipid of appropriate length and structure. Thus, our findings with the α-GalCer C16:0 derivative suggest strongly that it be considered as a lead glycolipid candidate in clinical trials of T1D.

Potential of ceragenin CSA-13 and its mixture with pluronic F-127 as treatment of topical bacterial infections.

AIMS: Ceragenin CSA-13 is a synthetic mimic of cationic antibacterial peptides, with facial amphiphilic morphology reproduced using a cholic acid scaffold. Previous data have shown that this molecule displays broad-spectrum antibacterial activity, which decreases in the presence of blood plasma. However, at higher concentrations, CSA-13 can cause lysis of erythrocytes. This study was designed to assess in vitro antibacterial and haemolytic activity of CSA-13 in the presence of pluronic F-127. METHODS AND RESULTS: CSA-13 bactericidal activity against clinical strains of bacteria associated with topical infections and in an experimental setting relevant to their pathophysiological environment, such as various epithelial tissue fluids and the airway sputum of patients suffering from cystic fibrosis (CF), was evaluated using minimum inhibitory and minimum bactericidal concentration (MIC/MBC) measurements and bacterial killing assays. We found that in the presence of pluronic F-127, CSA-13 antibacterial activity was only slightly decreased, but CSA-13 haemolytic activity was significantly inhibited. CSA-13 exhibits bacterial killing activity against clinical isolates of Staphylococcus aureus, including methicillin-resistant strains, Pseudomonas aeruginosa present in CF sputa, and biofilms formed by different Gram (+) and Gram (-) bacteria. CSA-13 bactericidal action is partially compromised in the presence of plasma, but is maintained in ascites, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid. The synergistic action of CSA-13, determined by the use of a standard checkerboard assay, reveals an increase in CSA-13 antibacterial activity in the presence of host defence molecules such as the cathelicidin LL-37 peptide, lysozyme, lactoferrin and secretory phospholipase A (sPLA). CONCLUSION: These results suggest that CSA-13 may be useful to prevent and treat topical infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined application of CSA-13 with pluronic F-127 may be beneficial by reducing CSA-13 toxicity.

Ceragenin CSA-13 exhibits antimicrobial activity against cariogenic and periodontopathic bacteria.

INTRODUCTION: Ceragenin CSA-13 is a bile-acid-based mimic of endogenous antimicrobial peptides and shares a mechanism of action with many of these antimicrobial agents. Because CSA-13 is not peptide based, it is not a substrate for the proteases that are found in the oral cavity, which are capable of degrading antimicrobial peptides. Furthermore, the simplicity of the ceragenins makes them easier to prepare and purify than antimicrobial peptides. In this study, we examined the antimicrobial activities of CSA-13 against oral pathogens and found that this compound was bactericidal against all of the strains tested. METHODS: The strains used were isolates of Streptococcus mutans and Porphyromonas species. Minimum inhibitory concentrations (MIC) were determined using agar dilution methods. In susceptibility testing, viable counts were determined after incubation with CSA-13. RESULTS: CSA-13 was potent against all 23 strains tested with MICs of 1-8 microg/ml for S. mutans and 1-16 microg/ml for 24 strains of the genus Porphyromonas. The MIC(50) was 2 and the MIC(90) was 8 mug/ml for S. mutans. MIC ranges for protease-positive P. gingivalis and P. cangingivalis were 2-16 microg/ml, and 1-2 microg/ml for protease-negative P. circumdentaria. CSA-13 interacted with lipopolysaccharide-sensitized erythrocytes at a concentration of 5.0-20.0 microg/ml. CONCLUSION: CSA-13 displays broad-spectrum activity against cariogenic and periodontopathic bacteria. CSA-13 was effective against protease-positive Porphyromonas. It was shown to bind to erythrocytes coated with lipopolysaccharide and lipoteichoic acid from diverse bacterial strains. These results suggest that CSA-13 may be useful for the prevention and treatment of oral microbial diseases.

Enhancement of the efficacy of erythromycin in multiple antibiotic-resistant gram-negative bacterial pathogens.

AIMS: To improve the efficacy of erythromycin, a hydrophobic antibiotic, against multiple antibiotic-resistant gram-negative bacterial pathogens by enhancing their outer membrane permeability. METHODS AND RESULTS: Fifty-one nonrepeat gram-negative bacterial pathogens of various genera, resistant to multiple antibiotics, including erythromycin, were selected by disc agar diffusion tests. The amphiphilic cationic steroid antibiotic, Ceragenin CSA-13, a potent permeabilizer of bacterial outer membranes, reduced the minimum inhibitory concentration of erythromycin in 92% of the bacterial pathogens selected for the test, when supplemented with erythromycin. A synergistic effect of Ceragenin CSA-13 and erythromycin in combination was also observed. Spectrofluorimetric study confirmed that Ceragenin CSA-13 acts by depolarizing the bacterial outer membrane. The toxicity of Ceragenin CSA-13 was evaluated to be insignificant by measuring 'median lethal dose' (LD(50)) on mouse model. CONCLUSIONS: Ceragenin CSA-13 may be useful as an agent to make erythromycin effective against infections caused by multiple antibiotic resistant gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcome of the study suggests erythromycin-Ceragenin combination as a new approach to overcome the problem associated with the rapid emergence of multi-drug-resistant pathogens. The insignificant toxicity of Ceragenin CSA-13, as found, supports the possibility of the application of this compound for human therapeutics.

Prolactin acts as a potent survival factor for human breast cancer cell lines.

Human breast cancer is the leading cause of cancer death in women from Western societies, and a large study of the epidemiology demonstrated strong associations between human prolactin and risk of breast cancer. Using established models of apoptosis of human breast cancer cell lines, we assessed the role of prolactin in breast cancer cell growth and survival. We showed that prolactin had no effect on the metabolic activity or total cell number of any cell lines. We confirmed endogenous prolactin production by these cells and that the levels varied. In the presence of a prolactin-neutralising antibody, each of the cell lines responded with the induction of apoptosis as opposed to growth inhibition. The sensitivity of the cell lines to the physiological inducer of apoptosis, C2-ceramide, appeared relative to the levels of endogenous prolactin that they contained. We then showed that exogenously added prolactin acted as a potent survival factor against apoptosis in all the cell lines examined. In addition, we demonstrated that a prolactin-neutralising antibody in combination with C2-ceramide caused an anticipated, additive increase in cell death. This study demonstrated that prolactin protects human breast cancer cell lines against apoptosis and this may have important implications for cancer treatment.

Adaptations of the IGF system during malignancy: human skeletal muscle versus the systemic environment.

Presented in this study are data derived from a unique cohort of patients both with and without cancer, for whom we not only have serum samples, allowing us to investigate systemic factors impacting on skeletal muscle maintenance, but also primary skeletal muscle cultures giving us a model to mimic the in vivo muscle milieu. Possible local effects of autocrine/paracrine and endocrine IGF system components impacting on myoblast growth and differentiation could therefore be assessed. We report for the first time that the decrease in myoblast stem cell numbers seen with normal aging is lost in cancer patients. We further report that serum IGF-I, IGF-II and IGFBP-3 all show positive correlations with myoblast retrieval in control patients, but that with the exception of IGFBP-3 these correlations are lost in malignancy. Indeed IGF-II switches to a negative correlation with myotube formation in malignancy. Furthermore we provide initial evidence to suggest that there is an apparent altered regulation of local IGFBP-3 production during malignancy which may enable satellite cell proliferation, stem cell infiltration or both. Finally we show the importance of investigations not only monitoring the systemic impact of serum factors on skeletal muscle responses but also critically assessing the role that locally produced muscle IGFBP-3 may have on the systemic environment.

Differential interactions between IGFBP-3 and transforming growth factor-beta (TGF-beta) in normal vs cancerous breast epithelial cells.

In addition to modulating insulin-like growth factors action, it is now clear that insulin-like growth factor-binding protein-3 also has intrinsic effects on cell growth and survival. We have compared the effects of insulin-like growth factor-binding protein-3 and transforming growth factor-beta on cell proliferation and death of Hs578T cells and the normal breast epithelial cell line, MCF-10A. The growth of MCF-10A cells was inhibited at low concentrations of insulin-like growth factor-binding protein-3 but stimulated at high concentrations. These differential effects were unaffected in the presence of an insulin-like growth factor-I receptor antagonist. A synthetic peptide corresponding to the serine phosphorylation domain of insulin-like growth factor-binding protein-3 (that does not bind to insulin-like growth factors) also mimicked these differential actions. The growth of both cell lines was significantly inhibited by transforming growth factor-beta, this was associated with a 14-fold increase of insulin-like growth factor-binding protein-3 secreted by the Hs578T cells but a five-fold decrease of insulin-like growth factor-binding protein-3 secreted by MCF-10A cells. Replacement doses of exogenous insulin-like growth factor-binding protein-3 overcame the transforming growth factor-beta-induced growth inhibition in the MCF-10A cells. Cell death induced by ceramide was significantly reduced by insulin-like growth factor-binding protein-3 in the MCF-10A cells and depleting insulin-like growth factor-binding protein-3 with transforming growth factor-beta in these cells consequently increased their susceptibility to ceramide. In contrast, insulin-like growth factor-binding protein-3 enhanced apoptosis induced by ceramide in the Hs578T cells but transforming growth factor-beta treated Hs578T cells were resistant to apoptosis. The addition of anti-sense mRNA to insulin-like growth factor-binding protein-3 significantly abrogated this effect of transforming growth factor-beta. These data indicate that insulin-like growth factor-binding protein-3 has intrinsic activity capable of inhibiting or enhancing the growth and survival of breast epithelial cells depending on the cell line and exposure to other cytokines.

Bis-8-hydroxyquinoline-armed diazatrithia-15-crown-5 and diazatrithia-16-crown-5 ligands: possible fluorophoric metal ion sensors.

The synthesis and preliminary photophysical properties of a series of diazatrithia-15-crown-5 and diazatrithia-16-crown-5 ligands containing two 8-hydroxyquinoline sidearms are reported. The ligands were prepared by a two-step process. First, diazatrithiacrown ethers 11 and 12 were prepared by treating bis(alpha-chloroamide) 5 with various dimercaptans followed by reduction using a boron-THF complex. Hydroxymethyl-substituted macrocycle 12 was rearranged to hydroxy-substituted diazatrithia-16-crown-5 in refluxing aqueous HCl. Macrocyclic diamines 11-13 were converted to either 5-chloro-8-hydroxyquinolin-7-ylmethyl-substituted diazatrithiacrown ethers 14-16 by a Mannich aminomethylation reaction or to 8-hydroxyquinolin-2-ylmethyl-substituted diazatrithiacrown ethers 17-19 by reductive amination using 8-hydroxyquinoline-2-carboxaldehyde. Preliminary photophysical studies show that ligands 16 and 19 exhibit increased fluorescence in the presence of Zn(2+), indicating that these ligands could be chemical sensors for Zn(2+).

Activities of cholic acid-derived antimicrobial agents against multidrug-resistant bacteria.

Cationic cholic acid derivatives displayed potent and broad-spectrum activity against multidrug-resistant Gram-negative and -positive bacteria. Specific examples were effective permeabilizers of the outer membranes of many strains of multidrug-resistant Gram-negative bacteria and sensitized these to hydrophobic antibiotics. We also prepared a new cholic acid derivative with improved apparent selectivity for prokaryote membranes.

Multidrug-resistant bacteria: overcoming antibiotic permeability barriers of gram-negative bacteria.

Because of the permeability barrier provided by the outer membrane (OM), gram-negative bacteria are inherently resistant to many hydrophobic antibiotics. This resistance limits the arsenal of antibiotics that are effective in treating gram-negative bacterial infections. Compounding this problem, strains of gram-negative bacteria have emerged that display specific resistance mechanisms for effective antibiotics. As a means of expanding the arsenal of effective antibiotics for gram-negative bacteria, compounds that permeabilize the OM to hydrophobic substances have been developed. These compounds are typically cationic, amphiphilic molecules that can be prepared from peptides or steroids. Effective OM permeabilizers sensitize gram-negative bacteria to hydrophobic antibiotics, including erythromycin, fusidic acid, novobiocin and rifampin. These antibiotics are generally not useful in treating gram-negative bacterial infections because they traverse the OM ineffectively. The use of OM permeabilizers, in combination with hydrophobic antibiotics, may provide additional means of controlling growth of gram-negative bacteria. This review describes classes of permeabilizers, including those derived from peptides, and recently reported examples based on steroids.

Cholic acid derivatives: novel antimicrobials.

Mimics of squalamine and polymyxin B (PMB) have been prepared from cholic acid in hope of finding new antimicrobial agents. The squalamine mimics include the polyamine and sulphate functionalities found in the parent antibiotic, however, the positions relative to the steroid nucleus have been exchanged. The PMB mimics include the conservation of functionality among the polymyxin family of antibiotics, the primary amine groups and a hydrophobic chain. Although the squalamine and PMB mimics are morphologically dissimilar, they display similar activities. Both are simple to prepare and demonstrate broad spectrum antimicrobial activity against Gram-negative and Gram-positive organisms. Specific examples may be inactive alone, yet effectively permeabilise the outer membranes of Gram-negative bacteria rendering them sensitive to hydrophobic antibiotics. Problems associated with some of the squalamine and PMB mimics stem from their haemolytic activity and interactions with serum proteins, however, examples exist without these side effects which can sensitise Gram-negative bacteria to hydrophobic antibiotics.

Insulin-like growth factor binding protein-3 (IGFBP-3) potentiates paclitaxel-induced apoptosis in human breast cancer cells.

Variability in response to chemotherapy is poorly understood. Paclitaxel-induced apoptosis was assessed in human Hs578T breast cancer cells, using the MTT assay, cell counting, morphological features and flow cytometry. Pre-dosing cells with non-glycosylated insulin-like growth factor binding protein-3 (ngIGFBP-3) had no effect on the cells per se but accentuated paclitaxel-induced apoptosis. The apoptotic pathway was further examined by measuring caspase-3 activity in cell lysates at time points over 48 hr after dosing with paclitaxel. Activity increased significantly, and Western immunoblots for caspase-3 in conditioned media showed that the inactive precursor decreased after incubation with paclitaxel. Endogenous production of IGFBP-3 by the cells after incubation with paclitaxel was evaluated using Western ligand blotting, specific IGFBP-3 immunoblotting and radioimmunoassay. Paclitaxel increased endogenous IGFBP-3, which was further increased if the cells had been pre-dosed with ngIGFBP-3. These findings suggest that IGFBP-3 may be an important modulator of paclitaxel-induced apoptosis.

Preparation of a protected triamino analogue of cholic acid and sequential incorporation of amino acids in solution and on a solid support.

We have prepared a triamine derivative of cholic acid with protecting groups on the amines that allow sequential amide formation. The triamine was formed from 3 alpha,7 alpha, 12 alpha-trihydroxycholan-24-ol with good stereoselectivity. Sequential removal of the amine protecting groups and amide formation was achieved in high-yielding steps and was performed in solution and on a solid support.

Preparation and characterization of cholic acid-derived antimicrobial agents with controlled stabilities.

[reaction: see text] Novel cholic acid-derived antimicrobial agents that decompose under mildly basic conditions have been prepared. These compounds range in biological properties from potent antibacterial activity to effective permeabilization of the outer membranes of Gram-negative bacteria.

Anionic facial amphiphiles from cholic acid.

[structure:see text] Anionic facial amphiphiles have been prepared from cholic acid. These compounds offer antipodes of recently reported cationic amphiphiles derived from cholic acid. The synthesis of the anionic amphiphiles was accomplished in few steps from a common intermediate. In contrast to many other anionic facial amphiphiles, the cholic acid derived amphiphiles appeared to aggregate at relatively low concentration.

Conjugation of 7-oxodehydroabietic acid to lysine, a haptenation mechanism for an oxidized resin acid with dermal sensitizing properties.

This article explores protein conjugation of 7-oxodehydroabietic acid, a resin acid found in both aerosol from soldering with rosin flux and in rosin solids. In a murine model, conjugation (haptenation) of resin acids to proteins is required to generate antibodies against rosin. Hydroperoxy resin acids are dermal sensitizers, with haptenation thought to occur via radical mechanisms. Dermal sensitization to 7-oxodehydroabietic acid has been observed, although no radical haptenation mechanism has been proposed to explain the sensitizing properties of this compound. Conjugation of L-lysine to 7-oxodehydroabietic acid was predicted, with a Schiff base (or imine) linkage formed between C-7 of the resin acid and a free amino group of lysine. Fast atom bombardment mass spectrometry provided evidence of the conjugate; a small peak was seen for the conjugate (M+H)+ ion in aqueous ethanol with 20 mM concentrations of the free resin and amino acids. A larger conjugate peak was observed with addition of tertiary amine as a mild basic catalyst, and the intensity of the conjugate peak exceeded that of the precursors upon replacement of the ethanol with benzene. Resin acids accumulate in the plasma membrane, a non-aqueous environment apparently conducive to conjugation of 7-oxodehydroabietic acid with lysine side chains of membrane proteins. The result would be dehydroabietic acid covalently bound to protein, which could lead to interaction with immune cells having resin acid specificity. The haptenation mechanism presented may be involved in allergic contact dermatitis and occupational asthma observed from exposure to resin acid solids and aerosols. As sampling and analytical methods have been previously demonstrated for 7-oxodehydroabietic acid, this compound may be a useful exposure marker with relevance to negative health effects such as occupational asthma.