M. Dal Sasso

Inhibitory activity of thymol against the formation and viability of Candida albicans hyphae.

As the capacity of Candida albicans to produce hyphae is considered an important virulence factor in the pathogenesis of candiasis, the aim of this study was to investigate whether thymol, the major component of thyme oil, can interfere with the filamentous forms of Candida albicans and their viability. The morphological transition from yeasts to filamentous forms was investigated by analysing the morphological index (MI), which classifies the differentiated forms and blastoconidia; viability was investigated by means of fluorescence microscopy using a new SYTO‐9 and propidium iodide method previously used to stain only blastoconidia. Without thymol, there was an average of 94.00 ± 3.06% hyphal forms. After 6 h of incubation with 1x MIC (125 μg ml−1), 1/2x MIC and 1/4x MIC of thymol, filamentation was, respectively, 14.33 ± 8.25%, 28.33 ± 7.17% and 45.67 ± 8.09% in comparison with control (all statistically significant). In the absence of thymol, viable cells accounted for an average of 93.00 ± 4.00% whereas, after 6 h of incubation with 1x MIC, 1/2x MIC and 1/4x MIC of thymol, the presence of 54.33 ± 1.86%, 29.00 ± 3.61% and 23.00 ± 2.52% of yellow–orange coloured forms indicated damaged membranes and reduced viability. Our findings show that thymol interferes with the formation and viability of hyphae. This can be attributed to the characteristics of thymol disturbing Candida cell membranes and metabolism, probably by affecting fungal cell‐wall synthesising enzymes.

Influence of Age on Oxidative Bursts (Chemiluminescence) of Polymorphonuclear Neutrophil Leukocytes

The release of reactive oxygen species (ROS) during neutrophil oxidative bursts is the last of a sequence of different steps leading to the neutralization of pathogen microorganisms. Using luminol-amplified chemiluminescence (LACL), the oxidative burst activity of neutrophils in elderly people (≥75 years) was compared with that in younger controls (39 years on average) after activation with both particulate (Candida albicans) and soluble (formyl-methionyl-leucyl-phenylalanine; fMLP) stimulants. After Candida stimulation, a reduction in LACL was observed in the elderly subjects in comparison with the controls, but the difference did not reach statistical significance. After fMLP stimulation, the reduction in LACL was significant, thus suggesting that the Candida pathway of chemiluminescence production seems to be less affected than the fMLP pathway. This finding raises questions concerning the complex differences in the pathways of cell killing and ROS generation, and their efficacy in the elderly. Various possible explanations are discussed, all of which need further investigation.

Antioxidant activity of bisabolol: inhibitory effects on chemiluminescence of human neutrophil bursts and cell-free systems.

Human polymorphonuclear neutrophils (PMN), reactive oxygen species (ROS) and inflammatory reactions are closely interrelated, and increasing attention is being given to the search for new synthetic or natural antioxidant agents, capable of reducing ROS and consequent inflammation. It has been claimed that bisabolol (a monocyclic sesquiterpene alcohol) has an antioxidant/anti-inflammatory activity, but this has almost exclusively been investigated using chemical or biochemical tests. We studied the ability of bisabolol to interfere with ROS production (luminol-amplified chemiluminescence, LACL) during human PMN respiratory bursts induced by both corpusculate(Candida albicans)and soluble stimulants (N-formyl-methionyl-leucyl-phenylalanine, fMLP). LACL was also used to test cell-free systems (SIN-1 and H2O2/HOCl– systems) in order to investigate the presence of scavenging activity. After C. albicans stimulation, significant concentration-dependent LACL inhibition was observed at bisabolol concentrations ranging from 7.7 to 31 μg/ml; after the fMLP stimulus, significant LACL inhibition was observed at bisabolol concentrations ranging from 3.8 to 31 μg/ml. A similar effect was observed in the SIN-1 and H2O2/HOCl– systems. These findings draw the attention to the possible medical use of bisabolol as a means of improving the antioxidant network and restoring the redox balance by antagonising oxidative stress.

Experimental evidences for a role of subinhibitory concentrations of rilopirox, nystatin and fluconazole on adherence of Candida spp. to vaginal epithelial cells.

Candidiasis is frequently localized in the mucosal epithelium which covers the vaginal and oral cavity. The pathogenicity of Candida is correlated with its ability to adhere to epithelial cells and this is the resultant of both fungal and host cell properties and their physicochemical interactions. This study was performed to investigate the ability of subinhibitory concentrations (sub-MICs) of rilopirox, a new antimycotic drug, to interfere with the adhesion of Candida albicans, Candida tropicalis and Candida glabrata to human vaginal cells, in comparison with sub-MICs of nystatin and fluconazole. The three drugs are more active on C. albicans, followed by C. tropicalis and, last, C. glabrata, on which fluconazole was inactive (MIC > 24 micrograms/ml). Rilopirox, nystatin and fluconazole have different mechanisms of action, and different molecular weights, so a comparative analysis of data was performed by means of their sub-MICs. On this basis the order of activity was nystatin [symbol: see text] rilopirox > fluconazole. These findings can be of use for optimizing also the posologic design by regarding sub-MICs which are still active in reducing the adhesiveness of Candida to cells of the vaginal mucosa.

The effects of calcitonin nasal preparations and their excipients on mucociliary clearance in an ex‐vivo frog palate test

Abstract— The topical tolerability of the commercial preparation of 1–7 Asu‐eel and salmon calcitonin with 2% ammonium glycyrrhyzinate and 0·01% benzalkonium chloride, respectively, and of their excipients mixture in solution with increasing concentrations of ammonium glycyrrhyzinate and benzalkonium chloride, respectively, were assessed by investigating their effects on the mucociliary transport velocity in the ex‐vivo frog palate preparation. This preparation provides an integrated biological model readily usable in the laboratory which closely resembles human nasal mucociliary clearance mechanism and can be used for rapid testing and toxicity of agents proposed for topical administration in the upper and lower airways. Frog‐Ringer control, 1–7 Asu‐eel and salmon calcitonin commercial spray preparations and the excipients plus 2% ammonium glycyrrhyzinate and plus 0·01% benzalkonium chloride did not modify significantly the mucociliary transport velocity, confirming their very good tolerability on ciliated epithelium. Higher concentrations of ammonium glycyrrhyzinate (10 and 20%) caused significant slowing, on average —32 and —55%, respectively. Higher concentrations of benzalkonium chloride (0·05 and 0·1%) also caused significant slowing, on average, —43·5 and —87%, respectively.

Influence of subinhibitory concentrations of brodimoprim and trimethoprim on the adhesiveness, hydrophobicity, hemagglutination and motility of Escherichia coli

In the present study the ability of subinhibitory concentrations (sub-MICs) of brodimoprim (a new 2,4-dimethoxybenzylpyrimidine) to interfere with some important aspects of bacterial cell function, such as surface hydrophobicity, fimbriation, motility and adhesiveness to mucosal cells, was investigated in comparison with those of trimethoprim. The inhibitory behavior of both diaminopyrimidines concerning hydrophobicity and hemagglutination (fimbriation) were essentially the same, while for adhesiveness and motility brodimoprim was more effective than trimethoprim. Diaminopyrimidines have high affinity for the bacterial enzyme dihydrofolate reductase, and this reduces the synthesis of essential purines and as a consequence of DNA and proteins. Our findings indicate that the synthesis and/or the expression of surface adhesins, which are proteins, was also affected by both brodimoprim and trimethoprim, the former being more active.

Antinociceptive activity of salmon calcitonin: Electrophysiological correlates in a rat chronic pain model

Experimental and clinical evidence testifies to an antinociceptive action of salmon calcitonin (sCT), administered in different ways, on the central nervous system. These studies were performed almost exclusively in acute pain models. The purpose of the present study was to investigate the effects of sCT, injected directly into the lateral cerebral ventriculi, on the firing of single nociceptive thalamic neurons, detected by electrophysiological techniques in an experimental model of prolonged or chronic pain, such as rats rendered arthritic by injection of Freunds adjuvant into the left hindfoot. The noxious test stimuli used were either extension or flexion of the ankle or mild lateral pressure on the heel. With increasing doses of sCT (5, 10, 20, 40 micrograms, 5 microliters/i.c.v.) it was possible to observe correspondingly increasing inhibitory and long-lasting effects on the evoked firing, with a significant dose-effect relationship. In agreement with electrophysiological findings, preliminary data, obtained with a patch clamp technique, on depression of calcium fluxes through neuronal membrane, induced by sCT, oriented the attention to a direct action of sCT on CNS.

Antioxidant Activity of Calendula officinalis Extract: Inhibitory Effects on Chemiluminescence of Human Neutrophil Bursts and Electron Paramagnetic Resonance Spectroscopy

There is growing interest in natural chemical compounds from aromatic, spicy, medicinal and other plants with antioxidant properties in order to find new sources of compounds inactivating free radicals generated by metabolic pathways within body tissue and cells, mainly polymorphonuclear leukocytes (PMNs) whose overregulated recruitment and activation generate a large amount of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to an imbalance of redox homeostasis and oxidative stress. The aim of this study was to examine whether a propylene glycol extract of Calendula officinalis interferes with ROS and RNS during the PMN respiratory bursts, and to establish the lowest concentration at which it still exerts antioxidant activity by means of luminol-amplified chemiluminescence. Electron paramagnetic resonance (EPR) spectroscopy was also used in order to confirm the activity of the C. officinalis extract. The C. officinalis extract exerted its anti-ROS and anti-RNS activity in a concentration-dependent manner, with significant effects being observed at even very low concentrations: 0.20 μg/ml without L-arginine, 0.10 μg/ml when L-arginine was added to the test with phorbol 12-myristate 13-acetate and 0.05 μg/ml when it was added to the test with N-formyl-methionyl-leucyl-phenylalanine. The EPR study confirmed these findings, 0.20 μg/ml being the lowest concentration of C. officinalis extract that significantly reduced 2,2-diphenyl-1-picrylhydrazyl. These findings are interesting for improving the antioxidant network and restoring the redox balance in human cells with plant-derived molecules as well as extending the possibility of antagonizing the oxidative stress generated in living organisms when the balance is in favor of free radicals as a result of the depletion of cell antioxidants.

Gemifloxacin: effects of sub-inhibitory concentrations on various factors affecting bacterial virulence.

This study investigated the ability of sub-MICs of gemifloxacin to interfere with the bacterial virulence parameters of adhesiveness, haemagglutination, hydrophobicity and motility, as well as their interactions with host neutrophilic defences such as phagocytosis, killing and respiratory bursts. The adhesiveness of both Escherichia coli and Staphylococcus aureus was significantly reduced to a subinhibitory concentration of 1/32 MIC. Indirect fimbriation parameters, such as hydrophobicity and haemagglutination were significantly reduced at a concentration of 1/8 MIC, as was migration (swarming). Phagocytosis and the respiratory burst measured by means of chemiluminescence were not affected, but killing was significantly increased from 1/2 to 1/8 MIC. The interpolation of these pharmacodynamic findings with pharmacokinetic curves indicates that sub-MIC concentrations of gemifloxacin can prolong antimicrobial effects on virulence determinants up to 27 h after the antimicrobial concentration has fallen below the MIC value.

Daptomycin morphostructural damage in Bacillus cereus visualized by atomic force microscopy

Abstract Daptomycin is a novel, rapidly bactericidal in vitro antibiotic that is under investigation for the treatment of serious Gram-positive infections. Although daptomycin appears to disrupt membrane function, the precise mechanism of action has not been fully elucidated. Atomic force microscopy (AFM) is an innovative technique that allows high-resolution visualization and digital image manipulation of cell surface structures in 3 dimensions without the use of photons and electrons. The aim of this study was to use AFM to investigate the morphostructural changes in Bacillus cereus that occur upon daptomycin administration. The effects of daptomycin at 4x and 8x the minimal inhibitory concentration were visualized during an 8-hour incubation period. Atomic force microscopy images showed aberrant bacterial surface formations, including flattening and shrinking of cells and leakage of cytoplasm through the membrane. In addition to structural changes, the destabilization of flagella was also observed. These results support previous data suggesting that daptomycin disrupts membrane function.