M. D. Richardson

ANTIFUNGAL THERAPY IN ‘BONE MARROW FAILURE’

The incidence of severe opportunistic fungal infections in patients with haematological malignancies has increased dramatically over the past 20 years ( Jantunen et al, 1997; Ribaud, 1997; Toren et al, 1997; Tumbarello et al, 1997; Walsh et al, 1996). In neutropenic patients these infections are a major cause of morbidity and mortality. Between 20% and 40% of mycoses in patients with haematological malignancies are disseminated, and more than 70% of these are fatal. Candidosis and aspergillosis are the most common fungal infections in patients receiving immunosuppressive therapy and organ transplantation. Late-onset Pneumocystis carinii pneumonia is also seen (Lyytikainen et al, 1996). These infections are a particular problem in bone marrow transplant (BMT) recipients. Patients receiving conventional-dose cancer chemotherapy alone for a malignancy will often have a more rapid immune recovery and therefore will be less likely to develop opportunistic fungal infections. In contrast, bone marrow transplant patients may be quite immune suppressed for more than a year after transplant, particularly due to delays of lymphocyte recovery and the prolonged use of medications to prevent graft-versus-host disease. Infection remains the leading cause of death in this population. Most clinical centres have adopted an aggressive approach to antimicrobial therapy in these patients (Pizzo, 1993). At the first sign of infection, broad-spectrum agents, covering both gram-negative and gram-positive organisms, are given. An unresponsive fever or clinical deterioration necessitate alternative antibacterial treatment and additional therapy with antifungal or antiviral agents, or a combination of the two. The conventional interval between lack of response to antibacterial agents and the start of antifungal treatment ranges from 72 to 96 h.

Germination of Trichophyton mentagrophytes on human stratum corneum in vitro

The emergence of germ tubes from arthroconidia of Trichophyton mentagrophytes on stripped sheets of stratum corneum from different body areas was measured. Arthroconidia increased in size and started germination by 4 h at 37 degrees C. Germ tubes originated from a point on the arthroconidium surface mid-way between the points of attachment to adjacent conidia. With further incubation arthroconidial germination increased and germ tubes extended across the stratum corneum. Histological staining of transverse sections of infected stratum corneum showed hyphae penetrating longitudinally and perpendicularly through the thickness of the stratum corneum. By 7 days incubation hyphae started to form arthroconidia thereby completing the vegetative growth cycle of the fungus. Scanning electron microscopy revealed penetration of corneocytes by germ tubes resulting in damage to the corneocyte surface.

Adherence of arthroconidia and germlings of anthropophilic and zoophilic varieties of Trichophyton mentagrophytes to human corneocytes as an early event in the pathogenesis of dermatophytosis.

The association (adherence) between human corneocytes and arthroconidia of Trichophyton mentagrophytes strains 121 and 126, and T. interdigitale strain 4 was studied in vitro. Adherence of arthroconidia to corneocytes from either the palm and sole occurred and increased with time up to 6 h, by which time germination of arthroconidia had started. Significant differences were seen between the T. mentagrophytes strains and T. interdigitale in their adherence to corneocytes from the palm. When adherence values for plantar corneocytes were compared, significant differences were found between T. mentagrophytes had T. interdigitale. Not all corneocytes from either site had adherent arthroconidia, although there was a time‐dependent increase in the numbers of corneocytes with adherent fungal cells. By scanning and transmission electron microscopy it was seen that there was a loose association between arthroconidia and corneocytes with no apparent damage to the corneocytes membrane. Adherence of germlings of T. interdigitale to corneocytes from the palm appeared to be mediated by germling outer cell wall fibrils. Hyphal branches and secondary germlings were observed to enhance the attachment of the parent hypha to adjacent corneocytes.

Early events in the invasion of the human nail plate by Trichophyton mentagrophytes.

A new in vitro model for the study of nail invasion by dermatophyte fungi was developed. The dermatophyte Trichophyton mentagrophytes, and fragments of finger‐nails and toe‐nails were used. Arthroconidia were inoculated on the ventral surface of the nails. After 6h. adherence and germination of arthroconidia was observed. By 16th, small germ tubes with side branches were evident. At about 24 h. micro‐colonies had become established. At 48 h, a mycelium had formed, and at about 72 h most of the nail fragment was covered with fungal growth. Nail penetration occurred from the ventral surface through the intercellular spaces, and with longer incubation all three layers were invaded by arthroconidia growing through channels. Nail invasion occurred in the absence of added nutrients. Dermatophyte fungi appeared to invade the nail by a combination of mechanical and chemical factors. The model provides a substrate to study the pharmacokinetics and bioavailability of new antifungal agents in situ.

Opsonizing activity of C-reactive protein in phagocytosis of Aspergillus fumigatus conidia by human neutrophils

Summary. In complement‐free neutrophil mono‐layer assays pathological levels of C‐reactive protein (CRP) were found to promote greater phagocytosis of metabolically‐active conidia of Aspergillus fumigatus than dormant spores. This suggests that metabolically‐active conidia bind CRP which facilitates in vitro phagocytosis by human neutrophils.

Stimulation of neutrophil phagocytosis of Aspergillus fumigatus conidia by interleukin-8 and N-formylmethionyl-leucylphenylalanine.

The effect of the chemoattractants, interleukin-8 (IL-8) and N-formylmethionyl-leucylphenylalanine (fmlp) on neutrophil phagocytosis of serum-opsonised Aspergillus fumigatus conidia were studied. Pre-exposure of neutrophil monolayers to IL-8 (10-100 ng ml-1) or fmlp (10(-6)M) for 20 min increased phagocytosis from 15% to 35%. Furthermore, there was a significant increase in the numbers of conidia ingested by active neutrophils. The chemoattractants did not enhance phagocytosis of conidia when added contemporaneously to neutrophil monolayers. The ability of each chemoattractant to act as a primer for the other was investigated. It was found that fmlp could prime for IL-8 and fmlp enhancement of phagocytosis, but IL-8 could not. An increase from 15% to 55% in phagocytosis was seen at a pre-exposure concentration of 10(-5) M fmlp followed by an incubation period of 30 min in the presence of 20 ng ml-1 IL-8. There was no significant increase in the average number of internalized conidia under these conditions. The response of human neutrophils to priming stimuli may have important implications in the pathogenesis of aspergillosis.

Activity of terbinafine on Trichophyton mentagrophytes in a human living skin equivalent model

Germination of arthroconidia of Trichophyton mentagrophytes in the presence of the allylamine antifungal terbinafine was assessed utilizing a human living skin equivalent model. Arthroconidia were inoculated onto the skin-equivalent previously exposed to low concentrations of terbinafine (0.01-1.0 mg l-1) and then incubated at 28 degrees C for 7 days. An assessment of fungal growth inhibition was made by light and scanning electron microscopy. In the absence of terbinafine, adherence, germination and hyphal extension were observed. Penetration of the model was seen, with hyphae present in the dermal component. In the presence of terbinafine, inhibition of fungal growth was apparent and the drug was seen to act as a barrier against fungal invasion of the dermis. The data indicate that the living skin equivalent model is a promising in vitro system for evaluating new antifungal agents.

Enhanced phagocytosis and intracellular killing of Pityrosporum ovale by human neutrophils after exposure to ketoconazole is correlated to changes of the yeast cell surface

Summary. In seborrhoeic dermatitis an inflammatory response occurs secondary to large numbers of Pityrosporum yeasts appearing within and beneath the epidermis. To study the interaction between human neutrophils and P. ovale and any im‐munomodulating effect of antifungal agents, the yeast was exposed to ketoconazole and then incorporated into neutrophil monolayer assays. Phagocytosis was complement dependent and reached a maximum after 40 min. Ketoconazole at 25, 50 and 100 mg 1‐1 had no significant effect on phagocytosis of P. ovale. However, when yeast cells were pre‐treated with ketoconazole for 2 h before exposure to the phagocyte monolayer there was a significant enhancement of phagocytosis with increasing drug concentration. Intracellular killing of P. ovale was assessed by methylene blue dye exclusion. In the absence of ketoconazole, 5% of intracellular yeast cells were killed following internalization for 2 h. Pretreatment of yeast cells with ketoconazole at 10 and 100 mg 1‐1 for 2 h prior to ingestion significantly increased intracellular killing to a maximum of 23%. This study demonstrates that yeast cells of P. ovale are readily ingested by human neutrophils by a complement dependent process. Phagocytosis is enhanced if the organism is exposed to ketoconazole before opsonisation and ingestion. The inability of neutrophils to kill P. ovale is modulated in the presence of therapeutic concentrations of ketoconazole.

An in vitro model of dermatophyte invasion of the human hair follicle.

A novel in vitro model for the study of hair invasion by Trichophyton mentagrophytes was developed. Hair was obtained by microdissection and plucking. Following inoculation of the hair follicle with arthroconidia growth of the fungus was seen on the hair and within the follicle. Growth was observed to begin at the shaft end and to extend along the hair shaft towards the bulb area. In follicles maintained in organ culture the inner root sheath in particular was invaded by T. mentagrophytes and provided a good substrate for fungal growth. Initially, the cuticle formed a barrier to fungal penetration of the hair. After incubation for 4 days, germlings of T. mentagrophytes were seen to penetrate under the cuticle and in between the layers of cuticular cells to invade the cortex. There was no evidence of intracellular growth; fungal elements were seen intercellularly. There were similarities between the findings in this study of the process of hair invasion by dermatophyte fungi and that in the natural disease.

Inhibitory effect of terbinafine on the invasion of nails by Trichophyton mentagrophytes.

BACKGROUNDnTerbinafine has a broad spectrum of action in vitro and primary fungicidal action against many pathogenic fungi. Its mode of action against dermatophyte fungi in nail keratin is little understood.nnnOBJECTIVEnOur purpose was to determine the bioavailability of terbinafine in a nail fragment model.nnnMETHODSnThe effect of terbinafine on adherence and germination of arthroconidia of Trichophyton mentagrophytes on nail fragments was assessed by gross examination and light and electron microscopy.nnnRESULTSnPreexposure of nail fragments to terbinafine concentrations (0.001 to 10 mg/L) inhibited fungal growth and acted as a barrier to dermatophyte invasion. Damaged arthroconidia and distorted hyphae on the surface of nail fragments were observed.nnnCONCLUSIONnThis in vitro model provides an alternative system for studying the activity of antifungal agents in nail and demonstrates the morphologic changes in dermatophyte fungi after exposure to terbinafine.