ACCEPTED: SEPTEMBER 26, 1992
Activity of topically applied leukotriene B, in experimental dermatophytosis Die Leukotrien B,-Wirkung auf die experimentelle Dermatophytose bei topischer Anwendung G. S. Shankland and M. D. Richardson Key words. Trichophyton mentagrophytes, dermatophytosis, leukotriene B,, guinea pig. Schliisselworter. Trichophyton mentagrophytes, Dermatophytose, Leukotrien B,, Meerschweinchen.
Summary. In a pilot study to assess the antimycotic potential of the chemoattractant leukotriene B, (LTB,), guinea pigs were infected with Trichophyton mentagrophytes var. mentagrophytes and divided into groups of untreated control animals or treated daily with LTB,, isoconazole nitrate or vehicle alone. The animals were assessed clinically, mycologically and histopathologically. Treatment with isoconazole nitrate was the most effective. The LTB,-treated group were clinically worse than the non-treated group and the mycological results remained positive throughout the assessment period. Zusammenfassung. I n einer Pilotstudie zur Aufklarung der antimykotischen Wirksamkeit des Chemoattraktans Leukotrien B, (LTB,) wurden Meerschweinchen mit Trichophyton mentagrophytes var. mentagrophytes infiziert und in folgende Versuchsgruppen eingeteilt: 1. Unbehandelte Kontrolltiere; 2. Tiere, behandelt mit LTB, plus Vehikel; 3. Tiere, behandelt mit Isoconazolnitrat plus Vehikel; 4.Tiere, behandelt rnit dem Vehikel allein (10% Isopropylmyristat in Ethanol). Die Tiere wurden klinisch, mykologisch und histopathologisch bewertet. Die Behandlung mit Isoconazolnitrat war die wirksamste. Die LTB,behandelte Gruppe war klinisch in einem schlechterm Zustand als die nichtbehandelte Gruppe, Regional Mycology Reference Laboratory, Department of Dermatology, University of Glasgow, UK. Correspondence: Dr Gillian S. Shankland, Regional Mycology Reference Laboratory, Department of Dermatology, Robertson Building, University of Glasgow, 56 Dumbarton Road, Glasgow GI 1 6NU, UK.
und die Pilzkulturen blieben die ganze Beobachtungszeit iiber positiv.
Introduction The resolution of a dermatophyte infection normally depends on the inflammatory changes involved. Although specific resistance to dermatophytic infection relies on cell-mediated immunity, clearance of infection is related to the inflammatory response which involves neutrophils and mononuclear cells [ 11. Therefore, the prospect that a chemoattractant for neutrophils may enhance the ability of the host to clear a dermatophyte infection is a concept worthy of investigation. Leukotriene B, (LTB,) is a potent chemoattractant and strongly attracts neutrophils. It aggregates, attracts and stimulates chemokinesis of polymorphonuclear leucocytes and induces exudation of plasma. LTB, is a lipid mediator derived from arachidonic acid, and produced by mast cells, basophils and macrophages. A stimulus, such as antigen or IgE, induces an influx of calcium ions into mast cells. This results in the release of preformed mediators such as histamine and the synthesis of new mediators like LTB,. The alteration of the mast cell membrane after the influx of calcium allows phospholipase A, to release arachidonic acid. This is metabolized by the lipo-oxygenase pathway giving rise to slow-reacting substances of anaphylaxis (LTC, and LTD,) and to LTB,. Katayama  used LTB, as a therapeutic agent for the treatment of dermatophytosis and the results appeared to be impressive especially
G. S. SHANKLAND & M. D. RICHARDSON
as treatment was applied only once a week. Diagnosis of dermatophytosis was based on clinical characteristics and demonstration of fungi on microscopical examination. As no attempt was made to culture clinical specimens it is not known which species of dermatophyte were involved and whether the differences in response noted could be attributed to the causative organisms. The present study was a preliminary investigation to assess if there was indeed any antidermatophytic activity exhibited by topically applied LTB, in a well-tested guinea pig model of experimental dermatophytosis [3-51. The results were compared with those achieved with topically applied isoconazole nitrate.
and/or some scabs; 4 =scabbed, scratched; 5 = scratched, bleeding, scabbed, weeping. Treatment and control animals were photographed and killed at intervals throughout the study. Material from infected sites was scraped for direct microscopical examination and culture on 4% (wtlvol) malt chloramphenicol agar. Further material was removed and fixed for histological examination by the periodic acid Schiff (PAS) and haematoxylin and eosin (H&E) staining methods. The guinea pigs remaining at the end of the study had skin scraped for microscopy and culture after killing.
Results Materials and methods Sixty Duncan Hartley guinea pigs were caged singly and maintained under standard conditions. The hair on their shoulder was shaved. A 4 cm2 site was moistened and inoculated with 0.5 ml of a concentrated microconidial suspension and 1 cm2 of the surface colony of a 10-day-old glucose peptone agar culture of Trichophyton mentagrophytes var. mentagrophytes (strain Glasgow TMMG) . Following inoculation, the animals were left for 3 days to allow the infection to become established. The animals were divided randomly into four groups of 15. These were: LTB, 0.1yo in vehicle; vehicle alone ( 10% isopropylmyristate in ethanol); isoconazole nitrate 2% in vehicle; no treatment control. The concentration of LTB, tested in this pilot study was based on the level shown to induce maximal neutrophil infiltrate in guinea pigs (unpublished data). From 3 days after inoculation the treatments were applied once a day for 12 successive days. Each animal in the treatment groups received 250 p1 of treatment solutions. The animals were examined clinically every day for 3 weeks (7 days after treatment stopped) by which time the normal inflammatory response usually clears the primary infection site [3-51. One animal from the no treatment control group was killed before commencement of treatment, to confirm the infection had become established. Clinical examinations were made of all the animals daily before treatment, observations were recorded and the severity of infection scored on a scale of 0-5. The scoring system was: O = normal skin colour, no scaling; 1 =slightly pink and/or slightly scaly; 2 = pinkish, scaly and/or some thickening; 3 = pink/red scaly, thickened
Clinical observations Before start of treatment all the animals exhibited slight scaling and most showed some degree of erythema.
LTB,. The clinical appearance of these guinea pigs deteriorated to their maximum scores of 4-5 after eight or nine treatments and these high scores were maintained until treatment was completed. The scores fell slightly towards the end of the study period. The finishing scores were always lower than those received on the last treatment day.
Vehicle alone. The animals showed a less acute reaction and tended to have slightly lower scores than the LTB,-treated animals. There was an increase in severity of clinical appearance after ten treatments with the formation of crusts and thickening but the peak scores were not as high as the LTB,-treated animals. There was a slight decrease in clinical scores at the end of treatment. Isoconazole nitrate. Clinically, animals in this group exhibited the most marked improvement. The animals had the lowest clinical scores with the maximum being 3.5 after five treatments and none of the animals developed a severe clinical appearance. All guinea pigs remaining at the end of the study had a normal clinical appearance and a score of 0.
No treatment control. Guinea pigs from this group showed an increase in scaling and erythema followed by the classical reaction of thickening and scabbing. The clinical picture improved slightly when the animals scratched and lost their scabs. mycoses 35, 329-334 (1992)
LEUKOTRIENE B, IN
Mycological investigations The results of microscopical examination and culture of the skin scrapings are given in Table 1.
LTB,. Scrapings from guinea pigs of this group were positive on direct microscopical examination and fungal culture throughout the study.
Vehicle alone. Animals from this group were positive on direct microscopical examination and culture throughout the treatment period. However, the percentage of positive inocula had dropped considerably by the end of the study. Isoconazole nitrate. This group was culture negative after only three treatments, although fungus was still visible after 7 days of treatment.
No treatment control. Scrapings from guinea pigs of this group were positive on direct microscopical examination and fungal culture throughout the study.
LTB,. On the fourth treatment day, infection was present in the stratum corneum and majority of follicles and there were also inflammatory scabs (Fig. 1). As the study progressed there was infiltration of the dermis and areas of spongiosis in the epidermis. By the eleventh treatment day there was marked disruption of the dermal epidermal junction, spongiosis and areas of inflammatory serous crusts (Fig. 2). After the end of treatment there were areas of ulceration (Fig. 3), acanthosis, spongiotic vesicles and parakeratosis. At the end of the assessment period the histological appearance was that of a chronic dermatitis.
Vehicle alone. Even after only three treatments spongiosis, hyperkeratosis and areas of parakeratosis were seen. Inflammatory crusts developed by the eighth treatment day when there was extensive spongiosis and many dilated capillaries. Extensive areas of spongiosis and ulceration, some associated with scratch marks, continued to be seen after the end of treatment. By the sixth day after the end of treatment there was follicular destruction, a general infiltrate, acanthosis hyperkeratosis and parakeratosis. There was some mild spongiosis and some fungus could still be seen in the stratum corneum at the end of the study. Isoconazole nitrate. After three treatments there were many infected follicles and some hyperkeratosis. However, by the eighth treatment day there was no fungus visible, although there was a mild general infiltrate in the upper dermis. O n the tenth treatment day there was no fungus visible but there were some areas of scabbing, a little moderate local spongiosis and some inflammation round follicles. By the end of treatment the stratum corneum appeared to be in good condition and there was only mild acanthosis (Fig. 4).
No treatment control. As this group were untreated they were not subjected to the same disturbance of their infection sites as the other groups. At the start of treatment there was fungus present in the stratum corneum and follicles were infected. By the fourth treatment day there was heavy infection with fungus to the base of the follicles and some inflammatory infiltrate was present. O n the eighth day of treatment there were serous scabs and inflammatory crusts. Focal areas of spongiosis and hyperkeratosis were also seen. Large areas of
Table 1. Mycological findings in guinea pigs infected with Trichophy&onrnenlagrophytes var.
treatment with LTB,, isoconazole or vehicle Treatments
Treatment group LTB,
Start ( 3 days PIS) 3 (6 days PI) 7 (10 days PI) 10 (13 days PI) 12 (17 days PI) 12 (20 days PI) 12 (21 days PI)
+ + + + + +
+ + +
t Per cent of inocula culture positive. $Post infection. mycoses 35, 329-334 (1992)
100 100 100
100 90 60
+ + + +
+ + +
0 0 0 0
85 95 25
+ + + + + + +
+ + +
+ + + +
+vet 95 100 100 100 100 100 100
G . S. SHANKLAND & M. D. RICHARDSON
Figure 1. Section of skin from experimentally induced Trichophyfon rncnfagrophyfesvar. mcnfagrophyks infection in a guinea pig treated with LTB,. Six days post infection, fourth treatment day; there is fungus in the stratum corneurn and the majority of hair follicles are infected. PAS, x 125.
Figure 2. Section of skin from experimentally induced Trichophyfon mmfagrophyfcs var. rnmfagrophyfes infection in a guinea pig treated with LTB,. There are spongiotic vesicles and areas of inflammatory serous crusts. Fungi are visible in the stratum corneurn. PAS, x 240.
ulceration covered with inflammatory slough were seen on the eleventh treatment day. There was a marked infiltration and dilated capillaries in the dermis. After the end of the treatment period there were still infected follicles and some infection of
the stratum corneum. Areas of hyperkeratosis, parakeratosis and acanthosis with dilated capillaries were seen up to the end of the study period. Also present were mild focal spongiosis, inflammatory serous crusts and scratch marks. mycoses 35, 329-334 (1992)
LEUKOTRIENE B, IN
Figure 3. Section of skin from experimentally induced Trichophyfon rnentugrophytcs var. rnenfagrophyfes infection in a guinea pig treated with LTB,. Thirteen days post infection, 11th treatment day; infected follicles are still present and there are areas of hyperkeratosis and parakeratosis. Ulceration and disruption of the dermis can be seen. PAS, x 125.
Figure 4. Section of skin from experimentally induced Trichophyton rnenfugrophytcs var. rnenfagrophytes infection in a guinea pig treated with LTB,. Thirteen days post infection, 11th treatment day; no areas of infection, stratum corneum normal, with only some thickening of epidermis. PAS, x 100.
The present study has established that treatment of experimental dermatophytosis with 2% isoconmycoses 35, 329-334 (1992)
azole was far superior to LTB,. The animals had the lowest clinical scores and none developed a severe infection; after only three treatments mycological investigations were negative. This was the
G. S. SHANKLAND & M. D. RICHARDSON
only group to demonstrate a normal appearance on histological examination by the end of the study. Results from this group are comparable to other groups of guinea pigs which received topical antifungal treatment in other studies [3-51, The group which received LTB, treatment developed a severe infection and the clinical picture remained unpleasant throughout the study. This impression was supported by the mycology results which were always positive by direct microscopy and culture. The vehicle-treated group had severe clinical reactions which took slightly longer to reach a maximum clinical score compared with the LTB,-treated group; again the mycology was positive throughout. The no treatment control group followed the classic course of infection with scabs of fungal debris and inflammatory cells falling off to leave hairless skin with satellite lesions around the primary site of infection. The accumulation of neutrophils in the infected skin is an early event of dermatophytosis [l, 61. Therefore a chemoattractant which promotes the migration of neutrophils to the infection site may suggest that clearing of the infection would occur more rapidly. However, the results of this study in guinea pigs do not agree with the results obtained by Katayama  in humans. Some azoles have been shown to inhibit LTB, generation and metabolism in human polymorphonuclear granulocytes in vitro . Bifonazole was the most active, while other azoles were up to ten times less active. There was a slightly elevated LTB, level in the presence of 0.5 and 1.O pg ml- I bifonazole. Above 2 pg ml- I there was a dose-dependent inhibition of LTB, release and total inhibition of LTB, production by human neutrophils with 64 pg ml-' bifonazole . This may be one reason for the antiinflammatory activity of bifonazole.
The role neutrophils are thought to play in dermatophytosis would indicate that a chemoattractant should enhance the normal inflammatory response of the host and lead to an earlier cure of the infection. However, the results shown here demonstrate an apparent increase in inflammation but no subsequent antifungal activity.
Acknowledgements The authors wish to thank the technical staff of the Medical Mycology Unit and the Department of Dermatology for their assistance, and Schering AG for supporting the study.
References Dahl, M. V. & Carpenter, R. (1986) Polymorphonuclear leukocytes, complement and Trichophyton rubrum. 3. Invest. Derm. 86, 138-141. Katayama, H. (1987) The treatment of tinea with topically applied leukotriene B4. Prostaglandins 34, 797-804. Shankland, G. S. & Richardson, M. D. (1986) Comparative activity of miconazole cream and ointment in the treatment of experimental dermatophytosis in guinea pigs. MpkoStn 29, 579-583. Shankland, G . S. & Richardson, M. D. (1989) Treatment of experimental derrnatophytosis in guinea pigs: a comparative study of bifonazole and a bifonazole/hydrocortisone combination. Mycoses 32, 245-249. Shankland, G. S. & Richardson, M. D. (1990) Comparative activity of clotrirnazole and a clotrimazole/hydrocortisone combination in the treatment of experimental derrnatophytosis in guinea pigs. 3. Antimicrob. Chernother. 25,825-830. Bremm, K. D. & Plempel, M. (1991) Modulation of leukotriene metabolism from human polymorphonuclear granulocytes by bifonazole. Mycoses 34, 41-45.
mycoses 35, 329-334 (1992)