a Nutritional Science Laboratory, Morinaga Milk Industry Co. Ltd, Zama, Kanagawa 228-8583, Japan; b Teikyo University Institute of Medical Mycology, Hachioji, Tokyo 192-0352, Japan
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Abstract |
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Introduction |
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Lactoferrin is an iron-binding glycoprotein of the transferrin family. It is present in milk and other exocrine secretions as well as in neutrophil granules. Bovine lactoferrin can be consumed in a non-denatured form in cheeses or in dietary supplementents. Lactoferrin is thought to play an important role in host defence because it has a variety of biological activities, including antimicrobial activity and immunomodulatory effects.5 We have shown that bovine lactoferrin, a fragment of it (lactoferricin B) and lactoferricin B derivatives have antifungal activity against Candida albicans, another important fungal pathogen, in vitro.6,7 Recently, it has been reported that orally administered bovine lactoferrin or lactoferricin B improves the survival rate of the host or reduces the number of pathogenic organisms in the bodies of hosts infected with bacteria, protozoa or viruses.811
Guinea pigs experimentally infected with T. mentagrophytes are commonly used as models of dermatophytosis to assess the in vivo therapeutic effects of drugs and to study the host defence mechanisms involved.1214 In this study, we attempted to evaluate the in vitro susceptibility of Trichophyton spp. to lactoferrin and its related compounds, and the therapeutic effects of orally administered bovine lactoferrin in guinea pig dermatophytosis models.
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Materials and methods |
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Bovine lactoferrin was produced by Morinaga Milk Industry Co. (Tokyo, Japan). Human lactoferrin was purified from the milk of healthy volunteers by a previously reported method.15 The antimicrobial peptide lactoferricin B was produced from bovine lactoferrin as described previously.16 Human transferrin and griseofulvin were purchased from Sigma Chemical Co. (St Louis, MO, USA). Guinea pig transferrin was obtained from Inter-cell Technologies Inc. (Hopewell, NJ, USA).
Organisms
The following organisms were maintained on Sabouraud glucose agar (1% peptone, 2% glucose and 1.5% agar) slants. Two strains of T. mentagrophytes, TIMM1189 and TIMM2789, isolated from hamster skin, were obtained from the culture collection of Teikyo University Institute of Medical Mycology (Tokyo, Japan). Two strains of T. rubrum, IFO6203 and IFO32409, were obtained from the Institute for Fermentation (Osaka, Japan). A conidial suspension of each strain for in vitro assays was prepared in physiological saline containing 0.05% Tween 80, and the cell concentration was adjusted to 5 x 104 conidia/mL with the test medium. A conidial suspension for inoculation of animals was prepared in physiological saline containing 0.05% Tween 80 from cultures of T. mentagrophytes TIMM2789 grown on modified Sabouraudglucose agar (0.2% peptone, 0.1% glucose, 0.1% KH2PO4, 0.1% MgSO4 and 2% agar) slants at 27°C for 2 weeks. The suspension was filtered through sterilized gauze to remove hyphal fragments and agar debris, and adjusted to 2 x 107 conidia/mL.
In vitro assay of anti-Trichophyton activity
The anti-Trichophyton activities of lactoferrin and reference agents were assayed by a microbroth dilution method, as described below. Aliquots of 170 µL of Sabouraud glucose broth (1% peptone and 2% glucose) or RPMI 1640 supplemented with 0.165 M MOPS, a medium recommended by the NCCLS for susceptibility testing of filamentous fungi,17 10 µL of test agent solution and 20 µL of conidial suspension were added to each well of a 96-well flat-bottomed microtitre plate. The inoculated microtitre plates were incubated at 27°C for 5 days. The MIC was defined as the lowest concentration of a given agent that resulted in no visible growth.
Animals and administration of test agents
Seven- to nine-week-old female Hartley SPF guinea pigs (Japan SLC Inc., Shizuoka, Japan) were used for all animal experiments. One group of guinea pigs was given oral bovine lactoferrin solution (250 mg/mL) twice a day at a daily dosage of 2.5 g lactoferrin per kg body weight by gavage (high-dose lactoferrin group). Another group was given oral lactoferrin (100 mg/mL) once a day at a daily dosage of 0.25 g lactoferrin per kg body weight (low-dose lactoferrin group). A positive control group was given griseofulvin 10 mg/mL in 2% methylcellulose and 0.5% Tween 80, once a day at a daily dosage of 0.025 g griseofulvin per kg body weight. The untreated control group did not receive any test solution.
Guinea pig dermatophytosis models
Three experiments were performed to assess the therapeutic effects of orally administered lactoferrin using infected guinea pigs as models of dermatophytosis. The effect of lactoferrin on animals with dermatophytosis of the back (tinea corporis) was examined in experiments 1 and 2. The effect of lactoferrin on animals with dermatophytosis of the feet (tinea pedis) was examined in experiment 3. These experiments were conducted according to previously reported methods.18,19
In the case of infection of the back, the skin of guinea pigs was shaved with electric clippers; adhesive tape was then applied and removed five times to a circle of shaved skin 2 cm in diameter. One site in each animal was inoculated with 50 µL of T. mentagrophytes conidial suspension, resulting in one lesion per animal. The infected site on each animal was visually examined daily throughout the experimental period to determine the severity of skin lesions. Skin lesions were scored as follows: 0, absence of lesions; 1+, a small number of erythematous papules in the infected site or new hair growth on the bald exposed area; 2+, moderate erythema spreading over the entire infected site accompanied by abrasions; 3+, moderately intense erythema with signs of swelling and scaling; 4+, severely erythematous lesions with extensive and intense crusting spreading over the exposed area. The average lesion score for a group was determined by dividing the sum of the lesion scores by the number of animals.
The guinea pig model of tinea pedis was prepared as follows. The gauze part of an adhesive bandage was wetted with 50 µL of the conidial suspension as inoculum and then fixed on to the soles of the guinea pig's hind feet with adhesive tape. The adhesive bandage was removed 3 days after infection.
On the last day of the experiments, all animals were killed and the skin at infected sites was excised completely. The skin block from each animal's back was divided into 10 pieces. The skin block from each foot was divided into 10 pieces, five from the toe portion and five from the heel portion. Each piece of skin block was placed on a plate of Sabouraudglucose agar containing cycloheximide 500 mg/L, chloramphenicol 50 mg/L and sisomicin 50 mg/L, and plates were then incubated at 27°C for 14 days. Skin pieces that yielded fungal growth were considered culturepositive. The fungal burden was assessed with scores ranging from 0 to 10 or from 0 to 5, based on the number of culture-positive skin pieces among the 10 skin pieces from the back or the five skin pieces from the toe or the heel portion of the foot, respectively. The average score of fungal burden for a group was determined by dividing the sum of the burden scores by the number of animals in the case of tinea corporis or the number of hind feet in the case of tinea pedis.
Statistical analysis
Data are presented as mean ± S.E. Statistical analysis was performed using the MannWhitney U test for comparison of two groups.
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Results |
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Bovine and human lactoferrins and the transferrins tested showed substantial antifungal activity in vitro against test strains of T. mentagrophytes and T. rubrum in RPMI 1640 medium (Table I). The inhibitory activity of lactoferricin B in Sabouraudglucose broth was similar to that reported previously.20,21 The MICs of lactoferrin, transferrin or lactoferricin B for Trichophyton spp. varied markedly depending on the strain and the medium used, while the MIC of the antifungal drug griseofulvin was constant. Although lactoferrin and transferrin were more active in RPMI 1640 medium, lactoferricin B showed higher activity in Sabouraudglucose broth.
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In experiment 1, using the guinea pig model of tinea corporis, the efficacy of two concentrations of lactoferrin was estimated on the basis of the fungal burden of the infected locus of skin tissues. Each group of infected animals was given oral lactoferrin at a low (0.25 g/kg/day) or high (2.5 g/ kg/day) dose or griseofulvin (0.025 g/kg/day), or not given any test agent (untreated control). Daily treatment was started 7 days before infection and was continued for 26 days (until 18 days after infection). Twenty-one days after infection, the skin at the sites of lesions was excised and the level of fungal infection was quantified as described in Materials and methods. Griseofulvin significantly reduced the fungal burden (Table II). High-dose lactoferrin was more effective than low dose lactoferrin at reducing the fungal burden, but was not significantly different from the untreated control (P = 0.060). Based on the results of experiment 1, the high dose of lactoferrin was employed in subsequent experiments.
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Efficacy of orally administered lactoferrin in tinea pedis model
Experiment 3 was conducted to examine the efficacy of orally administered lactoferrin using the guinea pig model of tinea pedis. Because of the severity and the lack of spontaneous healing of infection in the tinea pedis model, prolonged lactoferrin treatment was used. Lactoferrin in a daily oral dose of 2.5 g/kg was administered for 49 days starting 14 days before infection (long treatment) or for 28 days starting 7 days after infection (short treatment). In this experiment, the fungal burden in the untreated control was greater in the heel portion, where the stratum corneum is thick, than in the toe portion (Table III). In the heel portion, the fungal burden decreased significantly in the long lactoferrin treatment group (P = 0.010).
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Discussion |
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Our in vitro susceptibility testing with two assay media, RPMI 1640 and Sabouraudglucose broth, showed that lactoferrin and transferrin are both more active in the former medium, while the lactoferrin-derived peptide lactoferricin B is more active in the latter medium. This result may reflect differences in the mechanisms of antimicrobial action. Lactoferrin and transferrin act on C. albicans mainly in a fungistatic manner by causing depletion of iron, which is essential for the growth of the organism.22,23 This effect may be stronger in medium not supplemented with nutrient sugar (e.g. RPMI 1640), in which fungi grow slowly. It has been reported that supplementation of medium with sucrose increases resistance of Candida spp. to lactoferrin.24 In contrast, lactoferricin B is known to kill fungi by membrane perturbation.21 This effect would operate in glucose-supplemented media such as Sabouraudglucose broth, in which fungi grow rapidly and may become sensitive to fungicidal agents.
The anti-dermatophyte activity of serum is thought to be caused by transferrin,25,26 and dense infiltration of neutrophils is observed in infected areas of the skin in humans and animals with dermatophytosis.27,28 Like transferrin in serum, lactoferrin released from neutrophils accumulating in the epidermis may play an important role in the eradication of dermatophyte fungi growing in the stratum corneum.
Orally administered lactoferrin facilitated cure of dermatophytosis in guinea pigs. This was indicated by accelerated improvement of skin lesions in the tinea corporis model and decreased fungal burden remaining in the lesions in the tinea corporis and tinea pedis models. It will be of interest to elucidate how orally administered lactoferrin exerts these therapeutic effects at sites of infection distant from the gastrointestinal tract, through which lactoferrin passes and is digested. One possibility is that ingested lactoferrin or lactoferricin B-like peptides generated upon digestion of lactoferrin29 are absorbed from the intestinal tract, arriving at sites of infection via the blood circulation, and exerting their antifungal activity there. It has been reported that in germ-free, colostrum-deprived, immunologically naive piglets, substantial amounts of ingested lactoferrin are absorbed, entering the blood circulation as an intact molecule and conferring protection against endotoxin shock.30,31 However, this animal model is a very special case. In normal animals and humans, the extent of absorption of ingested proteins or partially digested peptides from the intestine is very low. In preliminary experiments, we were unable to detect lactoferrin- or lactoferricin B-containing peptide fragments in blood plasma by enzyme-linked immunosorbent assay (ELISA) after feeding lactoferrin to guinea pigs (data not shown).
It seems likely that orally administered lactoferrin affected dermatophytosis by a mechanism other than its direct antifungal effect. Oral administration of bovine lactoferrin or lactoferricin B has a protective effect in animals or humans infected with pathogenic bacteria, protozoa or virus.811 Very little is known about the mechanism responsible for this protective effect of lactoferrin-related compounds. However, some evidence indicates immuno-modulation by po lactoferrin administration in several animal models; this evidence includes enhanced phagocytotic activity of blood neutrophils,32 enhanced natural killer activity of splenic cells,33 an increased number of neutrophil progenitor cells in blood34 and enhanced interferon- production by splenic cells in response to concanavalin A stimulation.35
The development of cell-mediated immunity, correlated with delayed hypersensitivity and an inflammatory response, is associated with clinical cure of dermatophytosis, whereas absent or defective cell-mediated immunity predisposes the host to chronic or recurrent dermatophyte infection.1,36 In the observations reported here, orally administered lactoferrin did not prevent occurrence of symptoms, but promoted the clinical cure of skin lesions after development of symptoms. From this finding, we assume that orally administered lactoferrin does not have direct antifungal activity in vivo, but enhances the inflammatory response involving cell-mediated immunity required for the cure of dermatophytosis. Our hypothesis is that ingested lactoferrin acts on gut-associated lymphoid tissue and thereby modulates systemic immune responses, resulting in an enhancement of the antifungal host defence. Based on this concept, we are now assessing immune responses induced by oral administration of lactoferrin in guinea pigs and mice.
In conclusion, lactoferrin was active against a dermatophyte in vitro. Oral administration of lactoferrin facilitated cure of dermatophytosis by a presently unknown mechanism. Lactoferrin may be useful as a safe food component improving dermatophytosis in human patients.
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Acknowledgments |
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Notes |
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References |
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Received 13 December 1999; returned 23 March 2000; revised 3 May 2000; accepted 20 June 2000