Mechanism of activity and toxicity of Nystatin-Intralipid.

A novel lipid formulation of Nystatin (NYT), Nystatin-Intralipid (NYT-IL), which was found to be more active and less toxic in vitro and in vivo, was developed in our laboratory. The aim of the present study was to explore the possible mechanisms underlying its biological activity. To assess mechanisms affecting fungal cells we conducted the following experiments: killing kinetics, scanning and transmission electron microscopy (EM), measurements of potassium ion leakage and susceptibility in the presence of ergosterol. To study mechanisms affecting mammalian cells, we evaluated the effect of NYT-IL on a kidney cell line, with respect to viability, metabolic activity, potassium leakage and internalization of FITC-labeled human transferrin. NYT-IL exhibited killing kinetics patterns against Candida albicans similar to those of NYT and caused disruption of fungal cells and potassium ion leakage. Susceptibility tests showed that NYT-IL had lower antifungal activity in the presence of ergosterol. Thus, NYT-IL acts apparently by damaging fungal membrane, possibly through interaction with ergosterol, and maybe by additional modes of action. NYT-IL did not cause potassium leakage from mammalian kidney cells at any tested concentration and was not cytotoxic, whereas NYT, at high concentrations, led to K(+) leakage and was cytotoxic. Furthermore, the high NYT concentration interfered in the internalization process of human transferrin receptor (hTfnR) while NYT-IL did not. In summary, the Intralipid formulation of NYT diminishes the mechanisms responsible for toxicity to mammalian cells but preserves mechanisms of action against fungi, thereby suggesting superiority of NYT-IL as compared to NYT as an antifungal agent.

Metronidazole-loaded bioabsorbable films as local antibacterial treatment of infected periodontal pockets.

BACKGROUND: Periodontal disease is infectious in nature and leads to an inflammatory response. It arises from the accumulation of subgingival bacterial plaque and leads to the loss of attachment, increased probing depth, and bone loss. It is one of the world's most prevalent chronic diseases. In this study we developed and studied metronidazole-loaded 50/50 poly(DL-lactide-co-glycolide) (PDLGA), 75/25 PDLGA, and poly(DL-lactic acid) (PDLLA) films. These films are designed to be inserted into the periodontal pocket and treat infections with controlled-release metronidazole for >or=1 month. METHODS: The structured films were prepared using the solution-casting technique. Concentrated solutions and high solvent-evaporation rates were used to get most of the drug located in the bulk, i.e., in whole film's volume. The effects of copolymer composition and drug content on the release profile, cell growth, and bacterial inhibition were investigated. RESULTS: The PDLLA and 75/25 PDLGA films generally exhibited a low- or medium-burst release followed by a moderate release at an approximately constant rate, whereas the 50/50 PDLGA films exhibited a biphasic release profile. The drug released from films loaded with 10% weight/weight metronidazole resulted in a significant decrease in bacterial viability within several days. When exposed to human gingival fibroblasts in cell culture conditions, these films maintained their normal fibroblastic features. CONCLUSIONS: This study enabled the understanding of metronidazole-release kinetics from bioabsorbable polymeric films. The developed systems demonstrated good biocompatibility and the ability to inhibit Bacteroides fragilis growth; therefore, they may be useful in the treatment of periodontal diseases.

Preliminary study of activity of the thioredoxin inhibitor pleurotin against Trichophyton mentagrophytes: a novel anti-dermatophyte possibility.

Dermatophyte infections, while not life-threatening, are very common, and there is great interest in developing new antifungal agents. Transcriptional profiling of Trichophyton on keratin has identified some antioxidant genes as induced on this host substrate, including a thioredoxin gene TmTRX1. If thioredoxin is a virulence factor, or necessary for the growth on keratin, thioredoxin inhibitors should act as antifungals. As a first evaluation of this hypothesis, we have tested the activity of a thioredoxin-inhibitory natural product, pleurotin, against a clinical isolate of each of two fungal pathogens: the dermatophyte T. mentagrophytes and Candida albicans. Pleurotin inhibited the growth of the dermatophyte in vitro and in an ex vivo skin model, but had no effect on Candida. It may be possible to develop and optimise thioredoxin inhibitors, some of which are already under study in cancer chemotherapy, as antifungals.

Gentamicin-loaded bioresorbable films for prevention of bacterial infections associated with orthopedic implants.

Adhesion of bacteria to biomaterials and the ability of many microorganisms to form biofilms on foreign bodies are well-established as major contributors to the pathogenesis of implant-associated infections. Treatment of bone infection remains problematic, due to the difficulty of systemically administered antibiotics to locally penetrate bone. The current research addresses this issue by focusing on the development and study of novel gentamicin-loaded bioresorbable films designed to serve as "coatings" for fracture fixation devices and prevent implant-associated infections. Poly(L-lactic acid) and poly (D,L-lactic-co-glycolic acid) films containing gentamicin were developed through solution processing. The effects of polymer type, drug content, and processing conditions on the drug release profile were studied with respect to film morphology. The examined films generally exhibited a burst effect followed by a moderate approximately constant rate of release. The drug contents in the surrounding medium exceeded the required minimal effective concentration. Various gentamicin concentrations that were released from the films with time exhibited efficacy against bacterial species known to be involved in orthopedic infections. The developed systems can be applied on the surface of any metallic or polymeric fracture fixation device, and may therefore comprise a significant contribution to the field of orthopedic implants.

Antifungal effect and possible mode of activity of a compound from the marine sponge Dysidea herbacea.

OBJECTIVES: Assessment of antifungal activity of a compound isolated from the marine sponge Dysidea herbacea against the fungal pathogens Candida (primarily C. albicans) and Aspergillus (primarily A. fumigatus) species, and investigations of the possible mode of activity of the compound. METHODS: Freeze dried sponges were extracted with EtOAc-MeOH. Bioassay guided separation was used to identify the active compound. Antifungal activity was assessed in vitro by a modified NCCLS technique. For determination of the possible mode of activity of the compound we tested the effect on fungal cellular morphology (light, scanning and transmission electron microscopy) and possible site of activity in the fungal cells, such as cell membrane (ion leakage kinetics) as well as toxicity (cytotoxicity tests). RESULTS AND CONCLUSIONS: The active compound was determined to be 3,5-dibromo-2-(3,5-dibromo-2-methoxyphenoxy) phenol. This compound exhibited in vitro activity against the tested fungal pathogens. The experiments on the mode of activity revealed that there are significant changes in fungal cell morphology, as demonstrated by scanning and transmission electron microscopy. The compound, apparently, affects the fungal cell membrane, expressed primarily in leakage of potassium ions from the fungal cells. Two other bromo diphenyl ethers were also found to be active. Further experiments in in vivo models are planned.

Biodegradable soy wound dressings with controlled release of antibiotics: Results from a guinea pig burn model.

There is growing interest in the development of biodegradable materials from renewable biopolymers, such as soy protein, for biomedical applications. Soy protein is a major fraction of natural soybean and has the advantages of being economically competitive, biodegradable and biocompatible. It presents good water resistance as well as storage stability. In the current study, homogenous antibiotic-loaded soy protein films were cast from aqueous solutions. The antibiotic drug gentamicin was incorporated into the films in order to inhibit bacterial growth, and thus prevent or combat infection, upon its controlled release to the surrounding tissue. The current in vivo study of the dressing material in contaminated deep second-degree burn wounds in guinea pigs (n=20) demonstrated its ability to accelerate epithelialization with 71% epithelial coverage compared to an unloaded format of the soy material (62%) and a significant improved epithelial coverage as compared to the conventional dressing material (55%). Our new platform of antibiotic-eluting wound dressings is advantageous over currently used popular dressing materials that provide controlled release of silver ions, due to its gentamicin release profile, which is safer. Another advantage of our novel concept is that it is based on a biodegradable natural polymer and therefore does not require bandage changes and offers a potentially valuable and economic approach for treating burn-related infections.

Hybrid wound dressings with controlled release of antibiotics: Structure-release profile effects and in vivo study in a guinea pig burn model.

Over the last decades, wound dressings have evolved from a crude traditional gauze dressing to tissue-engineered scaffolds. Many types of wound dressing formats are commercially available or have been investigated. We developed and studied hybrid bilayer wound dressings which combine a drug-loaded porous poly(dl-lactic-co-glycolic acid) top layer with a spongy collagen sublayer. Such a structure is very promising because it combines the advantageous properties of both layers. The antibiotic drug gentamicin was incorporated into the top layer for preventing and/or defeating infections. In this study, we examined the effect of the top layer's structure on the gentamicin release profile and on the resulting in vivo wound healing. The latter was tested on a guinea pig burn model, compared to the neutral non-adherent dressing material Melolin® (Smith & Nephew) and Aquacel® Ag (ConvaTec). The release kinetics of gentamicin from the various studied formulations exhibited burst release values between 8% and 38%, followed by a drug elution rate that decreased with time and lasted for at least 7 weeks. The hybrid dressing, with relatively slow gentamicin release, enabled the highest degree of wound healing (28%), which is at least double that obtained by the other dressing formats (8-12%). It resulted in the lowest degree of wound contraction and a relatively low amount of inflammatory cells compared to the controls. This dressing was found to be superior to hybrid wound dressings with fast gentamicin release and to the neat hybrid dressing without drug release. Since this dressing exhibited promising results and does not require frequent bandage changes, it offers a potentially valuable concept for treating large infected burns.

Soy protein films for wound-healing applications: antibiotic release, bacterial inhibition and cellular response.

Use of naturally derived materials is becoming widespread in the biomedical field. Soy protein has advantages over the various types of natural proteins employed for biomedical applications, due to its low price, non-animal origin and relatively long storage time and stability. In the current study, soy protein isolate (SPI) was investigated as a matrix for wound-dressing applications. The antibiotic drug gentamicin was incorporated into the matrix for local controlled release and thus continuous bactericidal effect. Homogeneous high-quality films were cast from aqueous solutions and tested for the effects of gentamicin release on bacterial inhibition. The cytotoxicity and in vitro biocompatibility of these films were also examined. The gentamicin release profiles exhibited a moderate burst effect followed by a decreasing release rate, which was maintained for at least 4 weeks, thus enabling a suitable bacterial inhibition effect. The materials released from the films during an indirect cytotoxicity test were found to be safe, except for a slight inhibitory effect in the presence of high concentrations of glycerol. The biocompatibility test showed confluent cell cultures in close proximity to the SPI films. It is clear that these new antibiotic-eluting SPI films exhibit a high potential for use as wound dressings.

Novel biodegradable composite wound dressings with controlled release of antibiotics: results in a guinea pig burn model.

Approximately 70% of all people with severe burns die from related infections despite advances in treatment regimens and the best efforts of nurses and doctors. Silver ion-eluting wound dressings are available for overcoming this problem. However, there are reports of deleterious effects of such dressings due to cellular toxicity that delays the healing process, and the dressing changes needed 1-2 times a day are uncomfortable for the patient and time consuming for the stuff. An alternative concept in wound dressing design that combines the advantages of occlusive dressings with biodegradability and intrinsic topical antibiotic treatment is described herewith. The new composite structure presented in this article is based on a polyglyconate mesh and a porous poly-(dl-lactic-co-glycolic acid) matrix loaded with gentamicin developed to provide controlled release of antibiotics for three weeks. In vivo evaluation of the dressing material in contaminated deep second degree burn wounds in guinea pigs (n=20) demonstrated its ability to accelerate epithelialization by 40% compared to an unloaded format of the material and a conventional dressing material. Wound contraction was reduced significantly, and a better quality scar tissue was formed. The current dressing material exhibits promising results, does not require frequent bandage changes, and offers a potentially valuable and economic approach to treating the life-threatening complication of burn-related infections.

CD4+ and CD8+ T cells mediated direct cytotoxic effect against Trichophyton rubrum and Trichophyton mentagrophytes.

BACKGROUND: The cellular immune system is the most dominant factor in curing acute dermatophytosis. However, the exact immune mechanisms involved in generating this defense are complex and still obscure. The aim of this study was to investigate the fungicidal mechanism of T cells in the normal population versus patients with chronic fungal infections. METHODS: Thirty patients were included in the study: 15 patients with chronic dermatophytosis and 15 normal healthy patients with a history of acute dermatophytosis. The procedures were performed as follows. 1) Proliferation and cytotoxic activity of lymphocytes cultured with various dermatophytes homogenate such as, Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum gypseum. 2) CD4(+) and CD8(+) T cells were separated by magnetic beads before culture with fresh spores of either T. mentagrophytes or T. rubrum. 3) Routine histology and ultrastructural study were performed to illustrate the mode of activity of the T cells against the dermatophytes. RESULTS: The study showed that both CD4 and CD8 possess cytotoxic activity against dermatophytes. However, the results demonstrated a suppression of lymphocyte proliferation response and a significant lower cytotoxic effect in chronic patients. Ultra structure and histological evaluation of the culture of hyphae with CD4(+) or CD8(+) T cells showed more prominently destructive effects in the culture of cells that had been obtained from normal population than those of patients with long-lasting fungal infections. CONCLUSION: The study suggests a selective impairment of lymphocyte function against dermatophytes, in patients with chronic dermatophytoses.

Gentamicin-eluting bioresorbable composite fibers for wound healing applications.

New gentamicin-eluting bioresorbable core/shell fiber structures were developed and studied. These structures were composed of a polyglyconate core and a porous poly(DL-lactic-co-glycolic acid) (PDLGA) shell loaded with the antibiotic agent gentamicin, prepared using freeze drying of inverted emulsions. These unique fibers are designed to be used as basic elements of bioresorbable burn and ulcer dressings. The investigation focused on the effects of the emulsion's composition (formulation) on the shell's microstructure, on the drug release profile from the fibers, and on bacterial inhibition. The release profiles generally exhibited an initial burst effect accompanied by a decrease in release rates with time. Albumin was found to be the most effective surfactant for stabilizing the inverted emulsions. All three formulation parameters had a significant effect on gentamicin's release profile. An increase in the polymer and organic:aqueous phase ratio or a decrease in the drug content resulted in a lower burst release and a more moderate release profile. The released gentamicin also resulted in a significant decrease in bacterial viability and practically no bacteria survived after 2 days when using bacterial concentrations of 1 x 10(7) CFU/mL. Thus, our new fiber structures are effective against the relevant bacterial strains and can be used as basic elements of bioresorbable drug-eluting wound dressings.

Infection stages of the dermatophyte pathogen Trichophyton: microscopic characterization and proteolytic enzymes.

Dermatophytes are pathogenic fungi that infect human skin, nails and hair and cause dermatophytosis. Trichophyton mentagrophytes is one of the most widespread species that belong to this group. Infection of the skin tissues include several stages, i.e., adhesion to the surface of the skin, invasion into the sublayers by the penetration of fungal elements and secretion of enzymes that degrade the skin components. In this study we have followed the morphology of the fungal elements, such as arthroconidia and hyphae, during the adhesion and invasion stages. Skin explants were inoculated with the dermatophyte and observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Skin explants were also inoculated with a transgenic isolate of T. mentagrophytes expressing the green fluorescent protein (GFP). The infected sublayers were investigated by confocal scanning laser microscopy (CSLM). As an adaptation to the tissue environment, the dermatophyte produced long fibrils when it is on the open surface of the stratum corneum, while short and thin fibrils are produced inside the dense sublayers. The short and long projections might have a role in adhesion. Invasion may be produced by mechanical and biochemical means. Invasion of the tissue showed hyphal branching and growth in multiple directions. The proteolytic profile was assayed by substrate gel and proteolytic activity. Two serine proteases of similar molecular weight were secreted during growth on the epidermal matrix components keratin and elastin. The dermatophyte may use the proteolytic enzymes to invade the surface and also the deep layer of the skin in immunocompromised patients. Dermatophytes, which are well adapted infectious agents, seem to use their mechanical and biochemical capabilities to invade the skin tissue effectively.

Targeting the calcineurin pathway enhances ergosterol biosynthesis inhibitors against Trichophyton mentagrophytes in vitro and in a human skin infection model.

Fluconazole-FK506 or fluconazole-cyclosporine drug combinations were tested in an ex vivo Trichophyton mentagrophytes human skin infection model. Conidia colonization was monitored by scanning electron microscopy over a 7-day treatment period. The fluconazole-FK506 combination demonstrated the most obvious advantage over single-drug therapy by clearing conidia and protecting skin from damage at low drug concentrations.

Markers for host-induced gene expression in Trichophyton dermatophytosis.

Dermatophytes are adapted to infect keratinized tissues by their ability to utilize keratin as a nutrient source. Although there have been numerous reports that dermatophytes like Trichophyton sp. secrete proteolytic enzymes, virtually nothing is known about the patterns of gene expression in the host or even when the organisms are cultured on protein substrates in the absence of a host. We characterized the expression of an aminopeptidase gene, the Trichophyton mentagrophytes homolog of the Trichophyton rubrum Tri r 4 gene. The T. rubrum gene was originally isolated based on the ability of the protein encoded by it to induce immediate and delayed-type hypersensitivity in skin tests. T. mentagrophytes Tri m 4 is closely related to Tri r 4 (almost 94% identity at the protein level). Tri m 4 resembles other protease-encoding genes thought to be virulence factors (for example, DPP V of Aspergillus fumigatus). The Tri m 4 protein was detected immunochemically both in fungal extracts and in the culture medium. Expression of the Tri m 4 gene was induced severalfold when T. mentagrophytes was grown on keratin and elastin. Ex vivo, strong induction was observed after culture on blood plasma, but the use of homogenized skin did not result in a significant increase in Tri m 4 transcript levels. In order to identify additional genes encoding putative virulence factors, differential cDNA screening was performed. By this method, a fungal thioredoxin and a cellulase homolog were identified, and both genes were found to be strongly induced by skin extracellular matrix proteins. Induction by superficial (keratin) and deep (elastin) skin elements suggests that the products of these genes may be important in both superficial and deep dermatophytosis, and models for their function are proposed. Upregulation of several newly identified T. mentagrophytes genes on protein substrates suggests that these genes encode proteins which are relevant to the dermatophyte-skin interaction.

Green fluorescent protein (GFP) as a vital marker for pathogenic development of the dermatophyte Trichophyton mentagrophytes.

Skin infections by dermatophytes of the genus Trichophyton are widespread, but methods to investigate the molecular basis of pathogenicity are only starting to be developed. The initial stages of growth on the host can only be studied by electron microscopy, which requires fixing the tissue. This paper shows that restriction-enzyme-mediated integration (REMI) provides stable expression of the green fluorescent protein (GFP) in a clinical isolate of Trichophyton mentagrophytes. Under control of a constitutively active fungal promoter, GFP renders the hyphae fluorescent both in culture and in a recently developed model using human skin explants. Stages of infection and penetration into the skin layers were visualized by confocal microscopy. The stages of infection can thus be followed using GFP as a vital marker, and this method will also provide, for the first time, a means to follow gene expression during infection of skin by dermatophyte fungi.