Microbes and 14 (2012) 1144e1151 www.elsevier.com/locate/micinf Original article Dermatophyteehost relationship of a murine model of experimental invasive

James Venturini a,b, Anuska Marcelino A´ lvares c, Marcela Rodrigues de Camargo a,b, Camila Martins Marchetti a, Thais Fernanda de Campos Fraga-Silva a, Ana Carolina Luchini d, Maria Sueli Parreira de Arruda a,*

a Faculdade de Cieˆncias, UNESP e Univ Estadual Paulista, Departamento de Cieˆncias Biolo´gicas, Laborato´rio de Imunopatologia Experimental, Av. Eng. Luiz Edmundo C. Coube 14-01, 17033-360 Bauru, SP, Brazil b Faculdade de Medicina de Botucatu, UNESP e Univ Estadual Paulista, Distrito de Rubia˜o Junior s/n, 18618-970 Botucatu, SP, Brazil c Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP e Universidade Federal de Sa˜o Paulo, R. Botucatu 862, 04023-900 Sa˜o Paulo, SP, Brazil d Instituto de Cieˆncias Biolo´gicas e Naturais, UFTM, Av. Frei Paulino 30, 38025-180 Uberaba, MG, Brazil

Received 15 March 2012; accepted 16 July 2012 Available online 27 July 2012

Abstract

Recognizing the invasive potential of the and understanding the mechanisms involved in this process will help with disease diagnosis and with developing an appropriate treatment plan. In this report, we present the histopathological, microbiological and immunological features of a model of invasive dermatophytosis that is induced by subcutaneous infection of mentagrophytes in healthy adult Swiss mice. Using this model, we observed that the rapidly spreads to the popliteal lymph nodes, spleen, liver and kidneys. Similar to the human disease, the lymph nodes were the most severely affected sites. The fungal infection evoked acute inflammation followed by a granu- lomatous reaction in the mice, which is similar to what is observed in patients. The mice were able to mount a Th1-polarized immune response and displayed IL-10-mediated immune regulation. We believe that the model described here will provide valuable information regarding the dermatophyteehost relationship and will yield new perspective for a better understanding of the immunological and pathological aspects of invasive dermatophytosis. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Dermatophytes; Invasive dermatophytosis; Trichophyton mentagrophytes; Cellular immune response; Experimental dermatophytosis

1. Introduction common skin disorder among children younger than 12 and the second most common skin disorder in adults [2,3]. Dermatophytes are a group of fungi that are able to utilize Dermatophytosis is not an opportunistic infection and keratin as a food source. These fungi are universally distrib- frequently infects the skin, and nails of healthy humans uted and may be categorized as geophilic, zoophilic or and animals, causing superficial [4,5]. The clinical anthropophilic based on their natural habitats [1]. Dermato- manifestations of dermatophytosis range from mild to severe phytes are among the most common zoonotic agents. Der- based on the host reaction to the fungal metabolic products, matophytosis affects approximately 20e25% of the world’s the species of the fungus, the virulence of the fungus and the population and are responsible for 30% of all skin fungal anatomic site of infection [6]. Rarely, in immunocompromised infections; in addition, it is considered to be the third most hosts, the infection can disseminate into deeper skin layers and other organs, resulting in invasive dermatophytosis [7e10]. Experimental models of dermatophytosis have played * Corresponding author. Tel.: þ55 14 31036078; fax: þ55 14 31036092. a crucial role in assessing the efficacy of antimicrobial agents E-mail address: [email protected] (M.S.P. de Arruda). [11,12] and in obtaining a better understanding of disease

1286-4579/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.micinf.2012.07.014 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151 1145 pathogenesis [13e15]. Guinea pigs and rabbits have been the 2.3. Experimental design most frequently employed model animals in studies of derma- tophytosis [16]. However, several points remain to be elicited. In the present study we employed the Swiss mice because One of them is that these experimental models have not been this strain is more susceptible to dermatophytes [18], partic- used to explore the mechanisms involved in immune response ularly to T. mentagrophytes [19]. Thus, the mice were injected against dermatophytes, mainly due to the limited specific tolls in the footpad, subcutaneously, with 0.04 ml of T. menta- for these species such as species-specific recombinant protein grophytes inoculum (TM group) or sterile phosphate-buffered and monoclonal antibodies targeting to these species. The other saline (PBS), pH 7.4, alone (CTL group). The mice were one refers the question of fungal dissemination to internal sacrificed at 6, 24 and 48 h and 7, 14 and 30 days after the organs. According to Hay & Baran [17], ‘the mechanisms inoculation. limiting spread of fungi, under normal circum- stances, to the epidermis remain elusive. The hypothesis linking 2.4. Collection of the biological material a specific clinical form of dermatophyte nail infection with lymphatic spread, although intriguing, needs verification’. Mice were sacrificed via CO2 asphyxiation. Fragments of Cutaneuos dermatophytosis in guinea pigs and rabbits are self- the footpad, popliteal lymph nodes, liver, spleen and kidneys limiting, which restricts their usefulness in the investigation of were dissected and submitted to microbiological analyses. The the aspects of the disseminated infection. In order to overcome footpads were submitted to histological analyses by routine these claims, this study presents a model of invasive dermato- procedures for embedding in paraffin. phytic infection using Swiss mice inoculated subcutaneously with Trichophyton mentagrophytes. The model mimics the 2.5. Colony-forming unit (CFU) determination and human condition by presenting the following aspects: 1) spread frequency of fungal dissemination of the fungus from prior subcutaneous lesion, 2) involvement of various internal organs, 3) resolution of infection associated Fragments of the footpad, popliteal lymph nodes, liver, with development of cellular immune response, 4) in the deep spleen and kidneys were weighed and homogenized in 1 ml of layer of skin, the acute inflammatory lesion progresses to PBS, and 0.1 ml of the homogenate was cultured on granulomatous formations. Once it is well characterized, this Ò 15 90 mm Mycosel agar plates at 25 C for 14 days. Each model will allow studies of underling conditions that exhibit sample was assessed in duplicate. Total colonies were counted, increased susceptibility to dermatophytosis such as . and the results were expressed as the number (log10)ofT. mentagrophytes per gram of tissue. 2. Material and methods The frequencies of fungal dissemination were also reported as the frequencies of T. mentagrophytes-positive mice which 2.1. Mice had one or more internal organ affected and exclude footpad/ total number of mice analyzed. Two-month-old male Swiss mice from the UNESP Animal House at the Laboratorio de Imunopatologia Experimental (LIPE) of UNESP e Univ Estadual Paulista, Bauru, SP, Brazil 2.6. Histological procedures were housed in groups of three to five mice and were provided food and water ad libitum. All protocols used were in accor- Histological sections (4 mm) of mouse footpads were dance with the ethical principles for animal research adopted stained with hematoxylin-eosin (HE) and periodic acid-Schiff by the Brazilian College of Animal Experimentation staining (PAS). (COBEA). This study was approved by the Ethical Committee of Faculdade de Cieˆncias, UNESP e Univ Estadual Paulista. 2.7. Spleen cell culture

2.2. T. mentagrophytes inoculum Fragments of the spleen were collected and homogenized in ice-cold sterile PBS. The red cells were lysed with The T. mentagrophytes strain (2118/99-ILSL) was origi- 0.15 M ammonium nitrate. After washing, the cellular nally obtained from the fungal collection of the Lauro de suspension was centrifuged, and the cells were resuspended in Souza Lima Institute, Bauru, SP, Brazil. The fungi were 1 ml RPMI-1640 (Nutricell, Campinas, SP, Brazil) containing Ò maintained on Mycosel agar slants (Difco Laboratories, 10% heat-inactivated fetal calf serum (Gibco BRL, Grand Detroit, Michigan, USA) and cultured in the same media for Island, NY, USA). The cell concentrations were adjusted to 10 days at 25 C. The fungi were washed carefully by sterile 2.0 106 cells/ml, as determined by 0.1% trypan blue stain- saline solution. Afterward, the suspension were mixed twice ing. A total of 2.0 105 spleen cells were placed into each for 10 s on a vortex-mixer and decanted off for 5 min. The well of 96-well flat-bottom microtiter plates (Costar, Cam- supernatants were collected and washed twice. Fungal bridge, MA, USA) and incubated at 37 C in a 5% CO2 viability was determined by cotton blue staining, and the humidified chamber. The cells were cultured with or without concentrations were adjusted to 5 108 viable conidia of 10 mg/ml Concanavalin A (Sigma, St. Louis, MO, USA) as an T. mentagrophytes/ml. internal control (data not shown). After 48 h, the cell-free 1146 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151 supernatants were harvested and stored at 80 C for cytokine analysis.

2.8. Cytokine analyses

The levels of TNF-a, IFN-g and IL-10 were measured in the cell-free supernatants of the spleen cell cultures using a cytokine Duo-Set Kit (R&D Systems, Minneapolis, MI, USA), according to the manufacturer’s instruction. Each sample was analyzed in duplicate.

2.9. Footpad test

The development of a specific cellular immune response was evaluated using the footpad test (FPT) as described by Arruda et al. [20]. We used the exoantigens produced from the same strain of T. mentagrophytes inoculated in the mice according Venturini et al. [21]. On the 6th, 13th and 29th days after inoculation, the mice were injected with the specific antigen (50 mg of protein /ml, previously standardized e data not showed) in the opposite footpad. After 24 h, the footpads were collected and evaluated histologically. Mice were considered FPT-positive if they showed mononuclear cell infiltration.

2.10. Ouchterlony double immunodiffusion

The humoral immune response was evaluated based on the protocol described by Camargo et al. [22] using the T. men- tagrophytes exoantigens [21].

2.11. Statistical analyses

The microbiological analyses and cytokine production data were analyzed using the non-parametric KruskaleWallis test and Dunn’s post-test. All statistical tests were conducted using GraphPad InStat version 3.0 for Windows (GraphPad Software, San Diego, California, USA), and a two-tailed P- value of less than 0.05 was considered to be statistically significant.

Fig. 1. Microbiological evaluation of the footpads (A), popliteal lymph nodes 3. Results (B) and spleen (C). Swiss mice were inoculated with T. mentagrophytes and evaluated from 6 h to 30 days after fungal inoculation. The various letters 3.1. Microbiological evaluation indicate the statistical differences between the experimental time points. KruskaleWallis test; P < 0.05, n ¼ 6. All TM-mice contained fungi at their sites of inoculation until day 7. The fungal load began to decrease on day 7, 3.2. Histopathological evaluation culminating in fungal clearance by day 30 (Fig. 1AeC). The data regarding the frequency of mice with fungal Changes in the footpad inoculation sites were observed as dissemination are summarized in Table 1. Six hours after early as the first few hours after fungal inoculation. These fungal inoculation, we observed dissemination of the fungus to lesions were characterized by acute inflammatory reactions the popliteal lymph nodes, spleen, liver and kidneys. The leading to suppurative abscess formation. edema and lym- fungal dissemination was more frequent in the popliteal lymph phohistiocytic infiltrates, with polymorphonuclear leukocytes nodes and spleen. The number of mice with affected organs being observed at the limit between the abscess and the upper decreased with time until complete clearance was observed on layer of the skin (Fig. 2B). In the abscesses, hyphae and day 14 after fungal inoculation. conidia were detected by PAS staining (Fig. 3A). J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151 1147

Table 1 incidence of invasive dermatophytic infection is increased in Fungal dissemination frequencies in mice inoculated with T. mentagrophytes. immunocompromised patients; however, the infection is often Microbiological analyses of the popliteal lymph nodes, spleen, liver and undiagnosed, and thus its occurrence may be underestimated. kidneys from 6 h to 30 days after fungal inoculation. The values are reported as the frequencies of T. mentagrophytes-positive mice which had one or more Consequently, little is known about the pathophysiology of internal organ affected and exclude footpad/total number of mice analyzed. invasive dermatophytosis and its associated immune events. (n ¼ 6). Because there are considerable difficulties associated with Organ Infection progress conducting studies in humans, the use of experimental animal Hours Days models has facilitated researchers’ understanding of the phenomena involved in infectious diseases. The experimental 6 244871430 fungal spread in dermatophytosis was documented by Cutsen Popliteal lymph nodes 6/6 4/6 5/6 2/6 0/6 0/6 and Jensen [15], and these authors reported that guinea pigs Spleen 4/6 3/6 3/6 0/6 0/6 0/6 Liver 2/6 1/6 0/6 0/6 0/6 0/6 and rabbits inoculated intravenously with T. mentagrophytes Kidneys 1/6 1/6 1/6 0/6 0/6 0/6 contained fungi in their lungs, kidneys and liver. In contrast with these findings, the spread seen in humans stems from a pre-existing superficial dermatophyte infection, and there are After 24 h, the interstitial infiltration was more intense, and no published data indicating unexpected fungemia due to the epidermis exhibited hyperkeratosis (Fig. 2B). During this dermatophytes [8,17]. In order to better mimic the human time period, the hyphae and conidia decreased (Fig. 3B). condition, we studied the behavior of the fungus in Swiss mice At 48 h, the reaction had spread, and the perilesional after introduction into the subcutaneous footpad tissue. We infiltrates were composed of fibroblasts and mononuclear cells observed that the fungus reached the popliteal lymph nodes, (Fig. 2C). The fungal pattern at this time point was the same as spleen, liver and kidneys soon after T. mentagrophytes footpad that observed at 24 h. inoculation. In a review of 41 human cases with invasive On day 7 and 14, acanthosis was observed in the epidermis, dermatophytosis, the lymph nodes were the most infected sites and the infiltrated area were enlarged with central necrosis and (17%); however, several other organs and tissues were also a peripheral granulomatous reaction (Fig. 2D), which was affected by invasive dermatophyte infection, including the composed of lymphocytes, plasma cells and a small number of bones (7.3%), brain (7.3%), liver (4.9%) and other sites, such modified macrophages (Fig. 2D). Fungal fragments were seen as spleen, muscle, testis and vertebral joints (2.4% each) [8]. inside the modified macrophages on day 14 (Fig. 3C). Thus, our model more closely mimics what occurs in patients On day 30, the lesions showed well-formed granulomas with invasive dermatophytosis. composed of modified macrophage and rare epithelioid cells Histologically, the introduction of subcutaneous T. menta- surrounded by mononuclear cell infiltrates (Fig. 2E and F). No grophytes in Swiss mice initiates an acute inflammatory fungal elements were detected by PAS at this time point process followed by a granulomatous reaction. In the human (Fig. 3D). disease, approximately 44% of the histopathological findings included granuloma formation [8]. In our experimental model, 3.3. Specific cellular and humoral immune responses the number of mice with affected tissues decreased with time, culminating in complete clearance by day 14 after fungal TM-mice showed positive FPTs to specific antigens at 7, 14 inoculation. While spontaneous clearance of fungi from the and 30 days after infection. Sera from these mice were non- organs has not been observed in the human disease, this reactive for the IDD test. Both specific cellular and humoral clearance cannot be ruled out because little is known regarding responses were negative in the CTL-mice. the biology of the dermatophytes in the deeper tissues. Inter- estingly, several authors have suggested that internal organs 3.4. Cytokine determination by spleen cells could act as reservoirs of fungi in recurrent dermatophytic infections [15]. Our results do not confirm this premise, as the In the TM-mice, the TNF-a and IFN-g levels were only internal organs were the first ones to exhibit fungal clearance lower at 48 h after fungal inoculation (Fig. 4A and Fig. 4B). in our model. Also, it is important to highlight that the normal Interestingly, the levels of IFN-g were higher after fungal body temperature is one of the factors involved in the death of clearance than during earlier periods. Conversely, we observed dermatophytes in deep tissue. high levels of IL-10 soon after introduction of the fungus, and The immunological mechanisms involved in the initiation over time, the levels of this cytokine decreased to baseline and clearance of systemic dermatophytosis remain poorly levels (Fig. 4C). understood. Several studies have suggested that the cellular immune response participates in modulating the disease, as 4. Discussion this response could cause increased epidermal proliferation and facilitate dermatophyte elimination [review in [24]]. The Several recent studies have reported that superficial der- data from our study support this premise once that we matophytic infection may result in complications such as observed epidermal hyperplasia in the early stages of infection bacterial superinfection in diabetic patients [23] and in inva- and became more expressive with the development of the sive dermatophytosis [review in 8]. Although rare, the infection and delayed-type hypersensitivity (DTH). 1148 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151

Fig. 2. Histopathological evaluation of the footpad. (A) Normal footpads of Swiss mice inoculated with sterile saline solution and evaluated after 6 h (HE). (B) 6/ 24 h. Hyperkeratosis, suppurative abscess formation, edema and lymphohistiocytic infiltrates with polymorphonuclear leukocytes (HE). (C) 48 h. The reaction spread, and the perilesional infiltrates were composed of fibroblasts and mononuclear cells (HE). (D) Day 7. Acanthosis and enlarged infiltrated areas were observed with central necrosis and peripheral granulomatous reaction (HE). (E) Day 30. Well-formed granulomas composed of modified macrophages and rare epithelioid cells (HE). This observation is depicted in detail in Fig. 2F (HE). (F) Details of the a well-formed granuluma (HE).

Despite our findings, clinical and experimental data have mechanisms underlying invasive dermatophytosis are suggested that the immune response to dermatophytes is not different. Furthermore, patients with chronic dermatophytosis exclusively DTH-dependent. Among patients with AIDS, for may present DTH against trichophytin [25]. These findings example, the characteristic decrease in CD4þ T lymphocytes have also been confirmed experimentally. While athymic nude was expected to lead to higher incidences of invasive derma- BALB/C mice were resistant to T. mentagrophytes infection tophytosis, but in reality, the frequency is not increased in [26], in others strains of mice, the ablation of T-cell mediated these patients. Conversely, invasive dermatophytosis is more pathways leads to chronic and extensive but not internally frequently observed in non-AIDS immunocompromised disseminated dermatophytic infection [27]. patients, such as those suffering from atopic disease, leukemia, In order to expand the discussion about this question, in our diabetes, systemic lupus erythematous, psoriasis and myas- experimental model we carried out other immunological thenia gravis [8,17], suggesting that the immunological approaches. Thus, we investigated the production of the Th1 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151 1149

Fig. 3. Microscopic examination of fungi in the footpad. (A) 6 h. Hyphae and conidia were detected in the abscess (PAS). (B) 24 h. Decreased hyphae and conidia elements (PAS). (C) Day 14. Fungal fragments were seen inside modified macrophages (PAS). (D) Day 30. No detection of fungal elements (PAS). cytokine IFN-g, the Th2/regulatory IL-10, and the pro- method used, the presence of IgG and IgM antibodies specific inflammatory cytokine TNF-a in the cell-free supernatants for the fungus had been previously demonstrated [30e33]. of cultured splenocytes. We did not observe altered IFN-g Similar to other pathologies, the consensus among most levels during the early phase of the infection, but it is possible researchers is that specific humoral immunity does not play that these results may be associated with the high levels of IL- a large role in the resistance to fungal nfections. According to 10, which is a downregulator of the Th1 response. Over time, the authors, the humoral response is weak and directed the response changed, and IL-10 production decreased, while predominantly against polysaccharide antigens. The fact that the levels of IFN-g increased. In the human disease, IFN-g- antibody titers were higher in chronic infections suggests that positive cells were observed in the upper dermis in lesions in these antibodies are not effective at controlling these infec- situ [28]. Interestingly, in our study, the levels of IL-10 were tions [33e35]. notably high during the early phase of the infection when the Taken together, the subcutaneous inoculation of Swiss fungus was present in the viscera, including the spleen. These mice with T. mentagrophytes triggers fungal dissemination to results are similar to the findings of Campos et al. [29], who the internal organs, where the fungus remains until approxi- reported that the interaction of with mately the seventh day. During this period, the mice assem- phagocytic peritoneal cells induced production of IL-10. This bled a Th1-type immune response and IL-10-mediated finding was also supported by our microbiological analyses of immune regulation. Our lab has initiated studies using this the infections in our mice, which showed that when the fungal experimental model to evaluate dermatophytic infection load decreased, the levels of IL-10 returned to basal levels. during a hypoinsulinemia-hyperglycemic (HH) state, a model The role of IL-10 in this process remains unclear. In addition for diabetes, as this metabolic disease is associated with to inducing a Th2-type response, this cytokine plays an a high incidence of dermatophytic infection [36]. We report important role in both innate immunity and the regulation of that HH mice exhibited disseminated dermatophytosis the immune response. By preventing excessive production of following a delay in the infection outcome and that this der- TNF-a and cytotoxic metabolites, IL-10 prevents the devel- matophytosis was associated with a lower percentage of þ opment of a destructive inflammatory response and facilitates peripheral blood CD4 T cells [36]. The data obtained from the proper development of a specific immune response [30]. our experimental model may provide more information With respect to the humoral immune response, we were regarding immunological mechanisms to help researchers unable to determine the presence of specific antibodies in the better understand the immunological and pathological aspects sera of our mice. Even considering the low sensitivity of the of invasive dermatophytosis. 1150 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151

M.A. Pfaller (Eds.), Microbiology Medical, third ed., Mosby Co, St. Louis, MI, 1997, pp. 566e576. [3] B. Havlickova, V.A. Czaika, M. Friedrich, Epidemiological trends in skin mycoses worldwide, Mycoses 51 (2008) 2e15. [4] H. Degreef, Clinical forms of dermatophytosis (ringworm infection), Mycopathologia 166 (2008) 257e265. [5] L.J. Mendez-Tovar, Pathogenesis of dermatophytosis and tinea versi- color, Clin. Dermatol. 28 (2010) 185e189. [6] S. Vermout, J. Tabart, A. Baldo, A. Mathy, B. Losson, B. Mignon, Pathogenesis of dermatophytosis, Mycopathologia 166 (2008) 267e275. [7] M. Ravaghi, Superficial and deep granulomatous lesions caused by Tri- chophyton violaceum, Cutis 17 (1976) 976e977. [8] V.C. Marconi, R. Kradin, F.M. Marty, D.R. Hospenthal, C.N. Kotton, Disseminated dermatophytosis in a patient with hereditary hemochro- matosis and hepatic cirrhosis: case report and review of the literature, Med. Mycol. 48 (2010) 518e527. [9] P. Dan, R. Rawi, S. Hanna, B. Reuven, Invasive cutaneous Trichophyton shoenleinii infection in an immunosuppressed patient, Int. J. Dermatol. 50 (2011) 1266e1269. [10] M.A. Warycha, M. Leger, J. Tzu, H. Kamino, J. Stein, Deep dermatophy- tosis caused by Trichophyton rubrum, Dermatol. Online J. 17 (2011) 21. [11] M.A. Ghannoum, M.A. Hossain, L. Long, S. Mohamed, G. Reyes, P.K. Mukherjee, Evaluation of efficacy in an optimized animal model of Trichophyton mentagrophytes-dermatophytosis, J. Chemother. 16 (2004) 139e144. [12] T. Majima, S. Masui, K. Uchida, H. Yamaguchi, A novel mycological analysis valuable for evaluating therapeutic efficacy of antimycotics against experimental dermatophytosis in guinea pigs, Mycoses 48 (2005) 108e113. [13] S. Fujita, T. Matsuyama, Experimental tinea pedis induced by non- abrasive inoculation of Trichophyton mentagrophytes arthrospores on the plantar part of a guinea pig foot, J. Med. Vet. Mycol. 25 (1987) 203e213. [14] R.J. Hay, R.A. Calderon, C.D. Mackenzie, Experimental dermatophy- tosis in mice: correlation between light and electron microscopic changes in primary, secondary and chronic infections, Br. J. Exp. Pathol. 69 (1988) 703e716. [15] J. Van Cutsen, P.A.J. Janssen, Experimental systemic dermatophytosis, J. Invest. Dermatol. 83 (1984) 26e31. [16] D.M. Saunte, J.P. Hasselby, A. Brillowska-Dabrowska, N. Frimodt- Møller, E.L. Svejgaard, D. Linnemann, S.S. Nielsen, M. Haedersdal, M.C. Arendrup, Experimental guinea pig model of dermatophytosis: a simple and useful tool for the evaluation of new diagnostics and anti- fungals, Med. Mycol. 46 (2008) 303e313. [17] R.J. Hay, R. Baran, Deep dermatophytosis: rare infections or common, but unrecognised, complications of lymphatic spread? Curr. Opin. Infect. Dis. 17 (2004) 77e79. [18] J.A. Schmitt, J.M. Mann, D. Stilwill, Variation in susceptibility to experimental in genetic strains of mice. II. Preliminary Fig. 4. Evaluation of TNF-a (A), IFN-g (B) and IL-10 (C) levels in the results with b alb c and white swiss strains, Mycopathol. Mycol. Appl. 21 e supernatants of spleen cells maintained in culture for five days. Swiss mice (1963) 114 118. Trichophyton mentagrophytes inoculated with T. mentagrophytes were evaluated from 6 h to 30 days after [19] O. Fischman, Z. de Camargo, M. Grinblat, e fungal infection. The CTL group was composed of animals inoculated with infection in laboratory white mice, Mycopathologia 59 (1976) 113 115. sterile saline. The various letters indicate the statistical differences between the [20] M.S.P. Arruda, K.I. Coelho, M.R. Montenegro, Experimental para- experimental time points. KruskaleWallis test; P < 0.05, n ¼ 6. coccidioidomycosis in Sirian hamster inoculated in the cheek pouch, Mycopathologia 128 (1994) 67e73. [21] J. Venturini, R. Rossi-Ferreira, M.S. Arruda, Production of Trichophyton Acknowledgments mentagrophytes antigens and their characterization in mice, J. Venom. Anim. Toxins Incl. Trop. Dis. 14 (2008) 409e422. Supported by FAPESP and FUNDUNESP grants. [22] Z. Camargo, C. Unterkircher, S.P. Campoy, L.R. Travassos, Production of Paracoccidioides brasiliensis exoantigens for immunodiffusion tests, J. Clin. Microbiol. 26 (1988) 2147e2151. References [23] O. Vanhooteghem, G. Szepetiuk, D. Paurobally, F. Heureux, Chronic interdigital dermatophytic infection: a common lesion associated with [1] B.L. Hainer, Dermatophyte infections, Am. Fam. Physician 67 (2003) potentially severe consequences, Diabetes Res. Clin. Pract. 91 (2011) e 101e108. 23 25. [2] P.R. Murray, W.L. Drew, J.G.S. Korbay, Superficial, cutaneous, an [24] S.R. Almeida, Immunology of dermatophytosis, Mycopathologia 166 e subcutaneous , in: P.R. Murray, K.S. Rosenthal, G. Kobayashi, (2008) 277 283. J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151 1151

[25] T. Koga, Immune surveillance against dermatophyte infection, in: [31] G. San-Blas, The cell wall of fungal human : its possible P.L. Fidel, G.B. Huffnagle (Eds.), Fungal Immunology: From an Organ role is host- parasite relationships, Mycopathologia 79 (1982) Perspective, Springer, New York, USA, 2005, pp. 443e452. 159e184. [26] F. Green, K.W. Lee, E. Balish, Chronic T. mentagrophytes dermatophy- [32] T. Hamouda, C.D. Jeffries, E.M. Ekladios, A.M. el-Mishad, M. el- tosis of guinea pig skin grafts on nude mice, J. Invest. Dermatol. 79 Koomy, N. Saleh, Class-specific antibody in human dermatophytosis (1982) 125e129. reactive with Trichophyton rubrum derived antigen, Mycopathologia 127 [27] R.A. Calderon, R.J. Hay, Cell-mediated immunity in experimental (1994) 83e88. murine dermatophytosis. II. Adoptive transfer of immunity to dermato- [33] P. Zrimsek, J. Kos, L. Pinter, M. Drobnic-Kosorok, Serum-specific phyte infection by lymphoid cells from donors with acute or chronic antibodies in rabbits naturally infected with Trichophyton menta- infections, Immunology 53 (1984) 465e472. grophytes, Med. Mycol. 41 (2003) 321e329. [28] T. Koga, H. Duan, K. Urabe, M. Furue, Immunohistochemical detection [34] J.P. Callen, Immunologic testing in the dermatologic patient, Int. J. of interferon-gamma-producing cells in dermatophytosis, Eur. J. Der- Dermatol. 26 (1987) 224e225. matol. 11 (2001) 105e107. [35] M.V. Dahl, Immunological resistance to dermatophyte infections, Adv. [29] M.R.M. Campos, M. Russo, E. Gomes, S.R. Almeida, Stimulation, Dermatol. 2(187) 305e320. inhibition and death of macrophage infected with Trichophyton rubrum, [36] J. Venturini, M.A. Golim, A.M. Alvares, G.A. Locachevic, O.S. Arruda, Microbes Infect. 8 (2005) 372e379. M.S. Arruda, Morphofunctional evaluation of thymus in hyperglycemic- [30] M. Saraiva, A. O’Garra, The regulation of IL-10 production by immune hypoinsulinemic mice during dermatophytic infection, FEMS Immunol. cells, Nat. Rev. Immunol. 10 (2010) 170e181. Med. Microbiol. 62 (2011) 32e40.