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The reconstructed human epidermis infected with Trichophyton rubrum conidia of 4000. (a and b) Stained by H and E and periodic acid-Schiff after 4 days of infection (original magnification, ×400). (c and d) Stained by H and E and periodic acid-Schiff after 10 days of infection (original magnification, ×400). After infection for 4 days, abundance of conidia and hyphae was presented in the stratum corneum. At the 10 th day of infection, the infection extended almost the full epidermis and the epidermis displayed enormous destruction.
Source publication
Background:
Trichophyton rubrum represents the most common infectious fungus responsible for dermatophytosis in human, but the mechanism involved is still not completely understood. An appropriate model constructed to simulate host infection is the prerequisite to study the pathogenesis of dermatophytosis caused by T. rubrum. In this study, we int...
Contexts in source publication
Context 1
... better mimic host dermatophyte infection, we optimized the infection dose by inoculating low-dose (400 conidia) and high-dose (4000 conidia) on the RHE. After 2 days of infection, the results of H and E and PAS staining did not show any conidia or hyphae in the horny layer in either group (data not shown). After T. rubrum was inoculated 4 days, conidia and hyphae fragments were found in the stratum corneum [ Figures 2a, 2b and 3a, 3b]; moreover, infection with 4000 conidia showed more conidia and hyphae fragments compared with infection with 400 conidia. On the 10 th day of co-culture, the histopathology feature exhibited the great difference between the two groups. The group infected by 400 conidia showed that the invasion limited to the cornified layer without penetrating through the stratum corneum to keratinocytes layer [ Figures 2c and 2d], which is in accordance with the pathological characteristics of superficial dermatophytosis caused by T. rubrum in vivo. However, the group infected with 4000 conidia displayed enormous destruction-encroached almost the full epidermis and presented obvious necrosis of keratinocytes [ Figure 3c and 3d]. Hence, the inoculum of 400 conidia was used to imitate host dermatophyte ...
Context 2
... better mimic host dermatophyte infection, we optimized the infection dose by inoculating low-dose (400 conidia) and high-dose (4000 conidia) on the RHE. After 2 days of infection, the results of H and E and PAS staining did not show any conidia or hyphae in the horny layer in either group (data not shown). After T. rubrum was inoculated 4 days, conidia and hyphae fragments were found in the stratum corneum [ Figures 2a, 2b and 3a, 3b]; moreover, infection with 4000 conidia showed more conidia and hyphae fragments compared with infection with 400 conidia. On the 10 th day of co-culture, the histopathology feature exhibited the great difference between the two groups. The group infected by 400 conidia showed that the invasion limited to the cornified layer without penetrating through the stratum corneum to keratinocytes layer [ Figures 2c and 2d], which is in accordance with the pathological characteristics of superficial dermatophytosis caused by T. rubrum in vivo. However, the group infected with 4000 conidia displayed enormous destruction-encroached almost the full epidermis and presented obvious necrosis of keratinocytes [ Figure 3c and 3d]. Hence, the inoculum of 400 conidia was used to imitate host dermatophyte ...
Citations
... For allergic dermatitis, potential contact allergens are evaluated (Peiser et al. 2012). For burn victims, graft generation, and skin infections, different skin layered in-vitro models are being employed for pathogen identification and the development of newer treatment approaches (Boyce et al. 1999;Liang et al. 2016;Faway et al. 2017;Kim et al. 2019). ...
... While the presence of hyphae is a physical marker of disease progression, the release of cytoplasmic lactate dehydrogenase from the KCs is an important biomarker for dermatophytosis. EpiDerm™ revealed that cytoplasmic lactate dehydrogenase didn't indicate location of the fungal infection, utilisation of H and E staining and Periodic acid-Schiff (PAS) obviated this limitation (Liang et al. 2016). ...
Unlabelled:
The success of in vitro 3D models in either recapitulating the normal tissue physiology or altered physiology or disease condition depends upon the identification and/or quantification of relevant biomarkers that confirm the functionality of these models. Various skin disorders, such as psoriasis, photoaging, vitiligo, etc., and cancers like squamous cell carcinoma and melanoma, etc. have been replicated via organotypic models. The disease biomarkers expressed by such cell cultures are quantified and compared with the biomarkers expressed in cultures depicting the normal tissue physiology, to identify the most prominent variations in their expression. This may also indicate the stage or reversal of these conditions upon treatment with relevant therapeutics. This review article presents an overview of the important biomarkers that have been identified in in-vitro 3D models of skin diseases as endpoints for validating the functionality of these models.
Supplementary information:
The online version contains supplementary material available at 10.1007/s10616-023-00574-2.
... Experimental infection models described in the literature use different compositions of inoculum, consisting in suspensions enriched in unicellular infective spores, namely microconidia [17][18][19][20][21][22][23] or arthroconidia [24][25][26][27][28][29], or in a mix of microconidia and hyphae [30][31][32][33][34][35]. Microconidia, as well as macroconidia, are a type of thallic spores resulting from lateral or terminal budding of hyphae. ...
... Nevertheless, after incubation on PDA at 30 • C under 12% CO 2 , identified as the optimal culture condition for viable fungal concentration, dermatophytes produce arthroconidia with percentages ranging from 1% to 68% for T. rubrum IHEM 13894 or 82% for M. canis IHEM 21239. The spore suspensions produced in these conditions are therefore "enriched in arthroconidia" compared to suspensions exclusively composed of microconidia and previously used as inoculum in many models [17][18][19][20][21][22][23]. ...
Dermatophytoses are superficial infections of human and animal keratinized tissues caused by filamentous fungi named dermatophytes. Because of a high and increasing incidence, as well as the emergence of antifungal resistance, a better understanding of mechanisms involved in adhesion and invasion by dermatophytes is required for the further development of new therapeutic strategies. In the last years, several in vitro and in vivo models have emerged to study dermatophytosis pathogenesis. However, the procedures used for the growth of fungi are quite different, leading to a highly variable composition of inoculum for these models (microconidia, arthroconidia, hyphae), thus rendering difficult the global interpretation of observations. We hereby optimized growth conditions, including medium, temperature, atmosphere, and duration of culture, to improve the sporulation and viability and to favour the production of arthroconidia of several dermatophyte species, including Trichophyton rubrum and Trichophyton benhamiae. The resulting suspensions were then used as inoculum to infect reconstructed human epidermis in order to validate their ability to adhere to and to invade host tissues. By this way, this paper provides recommendations for dermatophytes culture and paves the way towards a standardized procedure for the production of infective spores usable in in vitro and in vivo experimental models.
... Macrophages enhance their activities and recruit proinflammatory leukocytes by producing various kinds of cytokines. Macrophages are also able to promote Ag presentation and CD4 1 T cell responses through surface MHC-II molecules and costimulating molecules (18,19). Because TARM-1 is highly expressed in monocytes/macrophages, we assessed the direct role of TARM-1 in macrophage immunity. ...
T cell-interacting activating receptor on myeloid cells 1 (TARM-1) is a novel leukocyte receptor expressed in neutrophils and macrophages. It plays an important role in proinflammatory response in acute bacterial infection, but its immunomodulatory effects on chronic Mycobacterium tuberculosis infections remain unclear. TARM-1 expression was significantly upregulated on CD14high monocytes from patients with active pulmonary tuberculosis (TB) as compared that on cells from patients with latent TB or from healthy control subjects. Small interfering RNA knockdown of TARM-1 reduced expression levels of proinflammatory cytokines IL-12, IL-18, IL-1β, and IL-8 in M. tuberculosis-infected macrophages, as well as that of HLA-DR and costimulatory molecules CD83, CD86, and CD40. Moreover, TARM-1 enhanced phagocytosis and intracellular killing of M. tuberculosis through upregulating reactive oxygen species. In an in vitro monocyte and T cell coculture system, blockade of TARM-1 activity by TARM-1 blocking peptide suppressed CD4+ T cell activation and proliferation. Finally, administration of TARM-1 blocking peptide in a mouse model of M. tuberculosis infection increased bacterial load and lung pathology, which was associated with decreased macrophage activation and IFN-γ production by T cell. Taken together, these results, to our knowledge, demonstrate a novel immune protective role of TARM-1 in M. tuberculosis infection and provide a potential therapeutic target for TB disease.
... Later on, a model of infection by M. canis arthroconidia on reconstructed feline epidermis (RFE) was created to allow investigation of the adhesion mechanisms used by this dermatophyte on its natural preferred host [22]. More recently, two models of dermatophytosis based on commercially reconstructed skin tissue EpiDerm (MatTek) [26] or EpiSkin ® [56] were reported. The first model was explored to evaluate the release of cytokines by keratinocytes during the infection process by several dermatophyte species, including the anthropophilic T. rubrum. ...
... Characterization of the signaling pathways simultaneously involved was also undertaken [26]. By mean of morphological analysis, the second model of dermatophytosis on EpiSkin ® illustrated the different steps of the infection process by T. rubrum [56]. Although both models brought interesting insight and information about dermatophytosis and its pathogenesis, they used aleurioconidia as infective elements. ...
... Simultaneously though, aleurioconidia of T. interdigitale adherent to suspended corneocytes do not exhibit germination yet [19]. In accordance with a delayed germination of aleurioconidia, T. mentagrophytes microconidia inoculated on human skin explant seemed to start germination after 24 h [38], while germination of T. rubrum conidia was observed later, 2 days after infection on human skin [56]. By scanning electron microscopy performed on infected RHE (Fig. 2b), one can observe T. rubrum arthroconidia adhering to the tissue surface as early as after 1 h of contact. ...
Dermatophytosis is a superficial fungal infection of the keratinized structures of the host. Since the last decade, this mycosis became an important health concern due to an increasing prevalence and to the limited number and efficacy of available treatments. Several experimental models have then been developed in order to improve knowledge about this infection and to design new therapeutic strategies. This chapter presents the variety of dermatophytosis experimental models and their contribution in the understanding of mechanisms used by dermatophytes to adhere and to invade the host tissue. Their support to study the establishment of effective antifungal defenses by the host is also summarized. The usefulness of these models for testing the efficacy of antifungal compounds is finally discussed.
... Regarding skin fungal infections with dermatophytes or yeasts of the Malassezia genus, several models of interaction between in vitro reconstructed skin and pathological species have become available for investigation. At least three models of dermatophytosis describe how human tissues prepared in vitro by cell culture can be exposed to several species of anthropophilic and zoophilic dermatophytes (Achterman et al. 2015;Faway et al. 2017;Liang et al. 2016). Studies performed this way have shown morphological analysis of the progressive steps of infection; they were characterized for kinetics and analyzed to monitor multiple parameters that identified the kind of biological responses provided by the infected human tissue when dermatophytes penetrate into the epidermis. ...
... In the context of fungal infection of the skin, in vitro models are available to study the interaction between dermatophytes and the human epidermis (Achterman et al. 2015;Liang et al. 2016). Miconazole was also able to prevent infection by its use as a pre-treatment before the RhE was exposed to dermatophytes (Faway et al. 2017). ...
Investigative skin biology, analysis of human skin diseases, and numerous clinical and pharmaceutical applications rely on skin models characterized by reproducibility and predictability. Traditionally, such models include animal models, mainly rodents, and cellular models. While animal models are highly useful in many studies, they are being replaced by human cellular models in more and more approaches amid recent technological development due to ethical considerations. The culture of keratinocytes and fibroblasts has been used in cell biology for many years. However, only the development of co-culture and three-dimensional epidermis and full-skin models have fundamentally contributed to our understanding of cell–cell interaction and cell signalling in the skin, keratinocyte adhesion and differentiation, and mechanisms of skin barrier function. The modelling of skin diseases has highlighted properties of the skin important for its integrity and cutaneous development. Examples of monogenic as well as complex diseases including atopic dermatitis and psoriasis have demonstrated the role of skin models to identify pathomechanisms and drug targets. Recent investigations have indicated that 3D skin models are well suitable for drug testing and preclinical studies of topical therapies. The analysis of skin diseases has recognized the importance of inflammatory mechanisms and immune responses and thus other cell types such as dendritic cells and T cells in the skin. Current developments include the production of more complete skin models comprising a range of different cell types. Organ models and even multi-organ systems are being developed for the analysis of higher levels of cellular interaction and drug responses and are among the most recent innovations in skin modelling. They promise improved robustness and flexibility and aim at a body-on-a-chip solution for comprehensive pharmaceutical in vitro studies.
... For these reasons, in vitro models have been described as advantageous alternatives to study cells' responses in the presence of dermatophytes, such as systems comprising keratinocytes cultured in monolayers (Firat et al., 2014). In addition, RHE models of dermatophytosis have been reported: Two alternatives have been described and designed by modifying the commercially available systems EpiDerm (Achterman et al., 2015) and Episkin (Liang et al., 2016). Using arthroconidia and reconstructed feline epidermis, another in vitro model of dermatophytosis was developed to investigate the efficacy of a set of antifungal molecules (Tabart, Baldo, Vermout, Losson, & Mignon, 2008). ...
Skin drug delivery is an emerging route in drug development, leading to an urgent need to understand the behaviour of active pharmaceutical ingredients within the skin. Given, As one of the body's first natural defences, the barrier properties of skin provide an obstacle to the successful outcome of any skin drug therapy. To elucidate the mechanisms underlying this barrier, reductionist strategies have designed several models with different levels of complexity, using non‐biological and biological components. Besides the detail of information and resemblance to human skin in vivo, offered by each in vitro model, the technical and economic efforts involved must also be considered when selecting the most suitable model. This review provides an outline of the commonly used skin models, including healthy and diseased conditions, in‐house developed and commercialized models, their advantages and limitations, and an overview of the new trends in skin‐engineered models.
... 12,16,28,30 The experimental GPSE infection was compared with skin biopsies obtained from GP naturally infected with T. benhamiae and is described simultaneously intra-and intercellularly most likely through concerted mechanical and enzymatic forces. [35][36][37][38] Proteases are considered the most important dermatophyte virulence factors especially during the establishment of the infection. 39 Consequently, we confirmed Sub3, Sub6 and MCPA immunohistochemically in skin biopsies of naturally infected GP. ...
Background
Dermatophytoses rank among the most frequent communicable diseases in humans and the zoonotic transmission is increasing. The zoophilic dermatophyte Trichophyton (T.) benhamiae is nowadays one of the main causes of tinea faciei et corporis in children. However, scientific data on molecular pathomechanisms and specific virulence factors enabling this ubiquitous occurrence are scarce.
Objectives
To study tissue invasion and the expression of important virulence factors of T. benhamiae‐isolates that were recovered from two groups of hosts (human vs. guinea pigs (GP)) using an ex vivo skin model.
Methods
After confirmation of species identity by ITS sequencing, CFU suspensions of dermatophyte isolates (n= 20) were applied to the skin infection model and cultured. Employing specific immunofluorescence staining techniques, the expression of subtilisin 3 and 6 and metallocarboxypeptidase A was analysed. The general mode of invasion was explored. Results were compared to biopsies of naturally infected GP.
Results
All isolates were successfully recovered and proliferated well after application to the infection model. Progressive invasion of hyphae through all skin structures and destruction of explants was observed with early events being comparable to natural infection. An increasing expression of the examined virulence factors towards the end of culture was noticed but no difference between the two groups of isolates.
Conclusions
For the first time, important in vivo markers of dermatophytosis were visualised immunohistochemically in an ex vivo skin infection model and in skin biopsies of GP naturally infected with T. benhamiae . More research on the underlying pathomechanisms of dermatophyte infection is urgently needed.
... It is unfortunate that its pathogenic mechanism of invasion into the stratum corneum and dermis is not still totally understood. Different models, such as the animal model, stripped sheets of the stratum corneum, nail plates, monolayer cell culture model, or reconstructed human epidermis, were utilized for the examination of the mechanism of dermatophyte infection [4][5][6][7][8] . Even though they had a few limitations to imitate dermatophyte infections in humans, they can partly exhibit the pathogenic mechanism of dermatophyte infection. ...
... The right model to simulate dermatophyte infection on hosts is a requirement in studying the dermatophytosis infection mechanism. [25] In their in vivo research stated that dermatophyte infection is only limited to stratum corneum. The number of conidia during infection showed the further process of adhesion and stratum corneum invasion to be intuitionistic after infecting 400 conidia. ...
Dermatophytosis is a dermatophyte fungi infection most commonly found in animals and humans. The first step of infection is started from the attachment of arthroconidium into stratum corneum. The attachment of fungi to the host cell is mediated by fungi adhesin and its interaction with the host receptor. The objective of this research was to develop the infection model of Microsporum gypseum and Microsporum canis by inoculating the macroconidia intradermally on the rabbit model. The macroconidia collection method from culture media was re-visualized as fungi ultrastructure using scanning electron microscopy (SEM). Skin lesion analysis was measured from clinical changes of the skin based on primary dermatophytosis signs such as circular alopecia with erythema and squamosal. Clinical confirmation test was done via skin sampling followed by histopathological examination using Methenamine Silver—Grocott’s (GMS) staining. As a result of this research, the in vivo infection model through direct infection of macroconidia applied intradermally was very effective in improving the direct infection to the invasion phase on the skin. This model confirmed the epidermal differentiation process and skin permeability showed primary lesion within 2 hours and aggravated up to 6 hours after inoculation. In conclusion, macroconidium is a potential source of infection to induce the dermatophytosis model and the severity of primary injection correlated with duration and the scale of clinical symptoms exhibited. This is a promising model for further research on the mechanism involved in dermatophyte infection.
... Several models have previously been used to study SFI, such as keratin-soy medium (Zaugg et al., 2009), stratum corneum sheets (Peres et al., 2016), keratinocyte monolayer cell culture (Huang et al., 2015), and reconstructed human epidermis (Liang et al., 2016;Faway et al., 2017). Limitations of these mentioned models include the absence of immune cells or keratinocytes. ...
Human skin fungal infections (SFIs) affect 25% of the world's population. Most of these infections are superficial. The main limitation of current animal models of human superficial SFIs is that clinical presentation is different between the different species and animal models do not accurately reflect the human skin environment. An ex vivo human skin model was therefore developed and standardised to accurately model SFIs. In this manuscript, we report our protocol for setting up ex vivo human skin infections and report results from a primary superficial skin infection with Trichophyton rubrum, an anthropophilic fungus. The protocol includes a detailed description of the methodology to prepare the skin explants, establish infection, avoid contamination, and obtain high quality samples for further downstream analyses. Scanning electronic microscopy (SEM), histology and fluorescent microscopy were applied to evaluate skin cell viability and fungal morphology. Furthermore, we describe a broad range of assays, such as RNA extraction and qRT-PCR for human gene expression, and protein extraction from tissue and supernatants for proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS). Non-infected skin was viable after 14 days of incubation, expressed genes and contained proteins associated with proliferative, immune and differentiation functions. The macroscopic damage caused by T. rubrum had a similar appearance to the one expected in clinical settings. Finally, using this model, the host response to T. rubrum infection can be evaluated at different levels.
