Host defence against Candida albicans and the role of pattern-recognition receptors.

Recognition of Candida albicans is mediated by several classes of pattern-recognition receptors, including Toll-like receptors and C-type lectin receptors. Cell wall components of C. albicans, interact with the pattern-recognition receptors, which are expressed by different cells, primarily antigen-presenting cells. This review aims to discuss the different pattern-recognition receptors responsible for recognition of special structures of C. albicans, which are known to activate intracellular signals that finally lead to directed and efficient host defence.

Hypertrophic scarring and keloids: pathomechanisms and current and emerging treatment strategies.

Excessive scars form as a result of aberrations of physiologic wound healing and may arise following any insult to the deep dermis. By causing pain, pruritus and contractures, excessive scarring significantly affects the patient's quality of life, both physically and psychologically. Multiple studies on hypertrophic scar and keloid formation have been conducted for decades and have led to a plethora of therapeutic strategies to prevent or attenuate excessive scar formation. However, most therapeutic approaches remain clinically unsatisfactory, most likely owing to poor understanding of the complex mechanisms underlying the processes of scarring and wound contraction. In this review we summarize the current understanding of the pathophysiology underlying keloid and hypertrophic scar formation and discuss established treatments and novel therapeutic strategies.

Nail penetration of sertaconazole with a sertaconazole-containing nail patch formulation.

BACKGROUND AND OBJECTIVE: Sertaconazole, an imidazole antifungal drug, has been proven to have broad and potent antifungal activity. In the present study, the pharmacokinetics of sertaconazole nail patches, developed for treatment of onychodystrophy and onychomycosis, were investigated in healthy volunteers. The objective of the study was to investigate the penetration of sertaconazole into the nail and plasma and the residual sertaconazole concentration in patches after 1 week of application onto the nails. METHODS: In a double-blind study, 16 healthy adults were treated with a 2.2 cm2 nail patch containing sertaconazole 3.63 mg and another patch containing no antifungal agent, which were placed on the left and right thumbnail of each subject, respectively (or vice versa), in a randomized order. The treatment period was 6 weeks and the patches were replaced weekly. Nail clippings, used nail patches, and blood samples were investigated to determine sertaconazole concentrations. RESULTS: Sertaconazole was detected in all sertaconazole-treated nail samples with mean concentrations of >100 microg/g, which exceeds the minimum inhibitory concentrations (MICs) for all relevant fungi in this context. Measurements of the residual dose in the patches suggested that 16-71% of the active ingredient had penetrated into the nail. No plasma sertaconazole concentrations could be detected. CONCLUSION: By virtue of their positive influence (occlusion) on water and lipid metabolism in dystrophic nails, nail patches should have beneficial therapeutic effects in onychodystrophic conditions. Addition of the antifungal agent sertaconazole adds broad-spectrum antimicrobial activity. In this study, the concentrations of sertaconazole in the nails were shown to be well above the MIC values for pathogenic fungi relevant to onychomycosis. No systemic absorption of the active ingredient was detectable, which should exclude unwanted systemic effects of the drug.

Infection of human oral epithelia with Candida species induces cytokine expression correlated to the degree of virulence.

A defined and balanced immunomodulatory response is crucial for the protection of mucosal surfaces being in contact with pathogenic microorganisms. This study examined the local host response mechanisms of epithelial cells in experimental Candida albicans, C. tropicalis, and C. glabrata infections by measuring the expression of cytokines at the mRNA and protein level. During the course of infection with active but not with heat-killed C. albicans stimulation of the gene expression levels for interleukin-1alpha, interleukin-1beta, tumor necrosis factor, Exodus-2, P-selectin ligand, granulocyte-monocyte colony-stimulating factor, and interleukin-8 was observed by standard and quantitative reverse transcription-polymerase chain reaction. This cytokine pattern may favor a chemotactic and a T helper 1 response. Initial moderate or weak upregulation of these cytokine genes by reverse transcription-polymerase chain reaction was also observed in epithelial infection with the less virulent species C. tropicalis and C. glabrata. Heat-killed C. albicans failed to induce an epithelial immune response. At the protein level, expression of interleukin-8 protein was strongly enhanced during the course of C. albicans infection, whereas lower levels were seen with C. tropicalis and C. glabrata. The different expression patterns of cytokines were associated with differences in virulence of the Candida strains. This study's data, therefore, show a correlation between the virulence potential of pathogenic fungi, possibly mediated by specific virulence factors (such as proteinases), and the secretion of epithelial cytokines and chemokines, which may initiate in vivo a protective T helper 1 immunologic response and contribute to the recruitment of activated leukocytes and lymphocytes to the site of mucosal infection.

Cytokine expression induced by Candida albicans in a model of cutaneous candidosis based on reconstituted human epidermis.

Skin equivalents based on reconstituted human epidermis have been used recently to establish models for allergic/irritant contact dermatitis and cutaneous candidosis. In the present study the cytokine expression pattern and the morphological alterations in experimental cutaneous candidosis were investigated by RT-PCR and histological analysis. In experimental cutaneous C albicans infection the mRNA expression levels of interleukin (IL)-1a, IL-1beta, IL-8, GM-CSF, Exodus-2, tumour necrosis factor-alpha and PSL (P-selectin ligand) were upregulated. Cytokine profile and histological features of infected skin (separation of keratinocytes, oedema, vacuolisation) were comparable to that seen in experimental contact dermatitis. These immunomodulatory and morphological similarities might reflect a common pathogenesis factor in both diseases.

Different isoforms of secreted aspartyl proteinases (Sap) are expressed by Candida albicans during oral and cutaneous candidosis in vivo.

Distinct isoforms of secreted aspartyl proteinases (Sap) of Candida albicans are important virulence factors for different types of candidosis. Predominant expression of Sap1-3 has been shown to be crucial for superficial infections in experimental mucosal and cutaneous candidosis, whereas Sap4-6 might be important for systemic disease. This in-vivo study investigated Sap expression in two samples from patients with oral candidosis and from cutaneous infection. Two different polyclonal antibodies directed against Sap1-3 and Sap4-6 were used for ultrastructural characterisation of protein localisation and expression. Post-embedding immuno-electron microscopy revealed Sap1-3 and Sap4-6 immunoreactivity in all samples. All C. albicans cells expressed predominantly the proteinases Sap1-3 which were evenly distributed within the cell wall and cytoplasmic membrane. In contrast, Sap4-6 labelling was only evident in a few fungal cells. In particular it was localised at the tips of hyphal cells during invasion. These data suggest a different pathogenetic role for Sap1-3 and Sap4-6 during host-fungal interaction.

Effect of antimycotic agents on the activity of aspartyl proteinases secreted by Candida albicans.

The inhibitory effect of human immunodeficiency virus (HIV) proteinase inhibitors amprenavir and saquinavir and antifungal agents terbinafine, ketoconazole, amphotericin B and ciclopiroxolamine on aspartyl proteinases (Saps) secreted by Candida albicans was tested in an in vitro spectophotometric assay. As expected, both HIV proteinase inhibitors showed a significant inhibitory effect on Sap activity, which was comparable to that of the classical aspartyl proteinase inhibitor pepstatin A (P < 0.001). Antifungal drugs such as ketoconazole, terbinafine and amphotericin B had no, or only minor, inhibitory effects on proteolytic activity. In contrast, a significant reduction in Sap activity could be demonstrated during treatment with the antifungal agent ciclopiroxolamine (P < 0.001). These results point to a multiple effect of this antimycotic agent and might explain the reduced adherence of C. albicans to human epithelial cells at subinhibitory doses.

Reduced expression of the hyphal-independent Candida albicans proteinase genes SAP1 and SAP3 in the efg1 mutant is associated with attenuated virulence during infection of oral epithelium.

The transition of Candida albicans from a yeast to a hyphal form is controlled by several transcriptional factors, including the key regulators Cph1 and Efg1, and is considered an important virulence attribute. These factors, especially Efg1, regulate the expression of hyphal-associated genes e.g. SAP4-SAP6. In order to investigate the relevance of these transcriptional regulators for hyphal-independent SAP genes, recently constructed cph1 and efg1 single mutants and a cph1/efg1 double mutant lacking these factors were tested during interaction with oral epithelium and polymorphonuclear neutrophils. In contrast to the parental wild-type strain and the cph1 mutant, the efg1 and the cph1/efg1 mutants did not produce hyphal forms in all experiments and were less capable of damaging epithelial cells and neutrophil granulocytes. The attenuated epithelial lesions of these mutants were correlated not only with reduced expression of the hyphal-associated gene SAP4, but also with the lack of SAP1 and SAP3 expression previously shown to be important for oral infections. An efg1 mutant strain carrying a plasmid-borne copy of the EFG1 gene regained hyphal growth, damage of keratinocytes, granulocytes and the expression of SAP1 and SAP3. Although efg1 and cph1/efg1 mutants did not produce germ tubes during infection, expression of the hyphal-associated genes SAP5 and SAP6 was not completely abolished. A reduced capacity to stimulate an epithelial immune response manifested by a delayed onset of IL-1beta, IL-8 and TNF expression was only observed in the cph1/efg1-infected tissue. These results provide further evidence for a combined regulation of different virulence factors, such as dimorphism and expression of SAP genes. Furthermore, it could be demonstrated that the lack of Efg1 also caused reduced expression of hyphal-independent SAP genes. Both the EFG1 and the CPH1 gene products are necessary for adequate induction of an immune response.

Dermatomycoses of the glabrous skin : a double-blind, randomised, comparative trial of sertaconazole 2% cream once daily versus vehicle.

OBJECTIVE: The aim of this multicentre, double-blind, vehicle-controlled study was to investigate the safety and efficacy of once-daily topical application of sertaconazole 2% cream compared with corresponding vehicle cream in the treatment of patients with tinea of glabrous skin. PATIENTS AND METHODS: A total of 400 patients were recruited at seven investigational sites; 144 patients in the intent-to-treat (ITT) population and 127 in the per-protocol (PP) population were treated for 3 weeks with either sertaconazole 2% once daily or corresponding vehicle cream. To evaluate therapeutic efficacy and safety, microscopic examination of native preparations, mycological cultures, and clinical assessment of the state of the lesion (objective and subjective monitoring of symptoms) were analysed after 3 weeks of treatment. RESULTS: Based on these parameters, in both the PP and ITT populations, sertaconazole cream 2% once daily induced a higher cure rate than the vehicle cream: 82% of the PP population (vehicle cream control 61%; p = 0.014) and 83% of the ITT population (vehicle cream control 59%; p = 0.003) randomised to sertaconazole with a previously positive mycological culture showed negative culture results after sertaconazole treatment. No statistically significant differences were observed regarding frequency and severity of adverse effects. CONCLUSION: These data indicate that treatment of superficial dermatomycoses with sertaconazole 2% cream for 3 weeks is efficient and safe. The once-daily application regimen may improve patient compliance, which has been shown to be of exceptional importance for successful treatment of dermatomycoses of the glabrous skin.

Design, synthesis, inhibition studies, and molecular modeling of pepstatin analogues addressing different secreted aspartic proteinases of Candida albicans.

The family of secreted aspartic proteinases is known as an important virulence factor of yeast infections by Candida albicans in particular, which is the most common fungal pathogen for humans with respect to systemic disease. Due to the continuing increase of drug resistant strains, these proteinases are currently considered as promising drug target candidates. Based on the known Sap2-substrate specificity data and X-ray analyses of Sap/inhibitor complexes, three libraries of inhibitors were designed and synthesized by modifying the structure of pepstatin A, a common non-selective aspartic proteinase inhibitor, at the P3, P2, or P2' position. These novel inhibitors showed high inhibitory potencies for the isoenzymes Sap1, Sap3, Sap5 and Sap6. Then, the affinity and selectivity of the peptide ligands were investigated by molecular modeling, highlighting new key structural information for the design of potent and selective anti-virulence agents targeting Candida albicans.

In vitro models for human skin disease.

Modern tissue culture technology has made it possible to generate human skin equivalents that represent either epidermis or epidermis plus dermis (full-thickness skin) in vitro. Commercially available skin equivalents and in-house models are used for safety analysis of cosmetics and toxicity screening of various pharmaceutical compounds. Recently, tissue culture technology has also been used to develop in vitro models of skin disease, in particular to promote cutaneous drug research while sparing experimental animals. The spectrum of model diseases available covers a range from inflammatory disease to cancer. It has, thus, been possible to gain more insight into the role of active pharmaceutical ingredients of various dermatologically relevant drug classes as well as conventional and innovative formulations.

Targeting virulence: a new paradigm for antifungals.

In the recent past, we have found ourselves in need of truly novel antifungal drugs as drug resistance in fungi has been evolving. Moreover, effective therapy has become particularly important as the number of immuno-compromised patients with life-threatening fungal infections increases. Fortunately, during the last few years, virulence factors of fungi and their inhibitors have, at least to some extent, been discovered and characterized. This should provide new options for the development of potential antifungal therapeutics. Inhibitors of the secreted aspartic proteinase of Candida albicans might turn out to be particularly rewarding.

Investigation of the synthetic route to pepstatin analogues by SPPS using O-protected and O-unprotected statine as building blocks.

The synthetic route to pepstatin derivatives by a solid phase peptide synthesis using either O-protected or O-unprotected statine as a building block has been investigated. Statine was prepared according to a modified literature procedure, whereas protection of its 3-hydroxyl moiety using tert-butyldimethylsilylchloride (TBSCl) provided the novel O-TBS-protected statine building block. The O-tert-butyldimethylsilyl (TBS)-protected statine approach provides an improved synthetic strategy for the preparation of statine-containing peptides as demonstrated by the synthesis of the pepstatin analogue iva-Val-Leu-Sta-Ala-Sta.

Hydrolytic enzymes as virulence factors of Candida albicans.

Candida albicans is a facultative pathogenic micro-organism that has developed several virulence traits enabling invasion of host tissues and avoidance of host defence mechanisms. Virulence factors that contribute to this process are the hydrolytic enzymes. Most of them are extracellularly secreted by the fungus. The most discussed hydrolytic enzymes produced by C. albicans are secreted aspartic proteinases (Saps). The role of these Saps for C. albicans infections was carefully evaluated in numerous studies, whereas only little is known about the physiological role of the secreted phospholipases (PL) and almost nothing about the involvement of lipases (Lip) in virulence. They may play an important role in the pathogenicity of candidosis and their hydrolytic activity probably has a number of possible functions in addition to the simple role of digesting molecules for nutrition. Saps as the best-studied member of this group of hydrolytic enzymes contribute to host tissue invasion by digesting or destroying cell membranes and by degrading host surface molecules. There is also some evidence that hydrolytic enzymes are able to attack cells and molecules of the host immune system to avoid or resist antimicrobial activity. High hydrolytic activity with broad substrate specificity has been found in several Candida species, most notably in C. albicans. This activity is attributed to multigene families with at least 10 members for Saps and Lips and several members for PL B. Distinct members of these gene families are differentially regulated in various Candida infections. In future, prevention and control of Candida infections might be achieved by pharmacological or immunological tools specifically modulated to inhibit virulence factors, e.g. the family of Saps.

Rate and extent of percutaneous absorption of sertaconazole nitrate after topical administration.

The purpose of this open study was to evaluate the rate and extent of the penetration of sertaconazole nitrate (CAS 99592-32-2, Zalaïn) penetration into the stratum corneum/lucidum of the human skin. Selected areas of 9 cm2 each of the back skin of 12 healthy volunteers were exposed over 8 different time intervals (between 0 and 48 h) to 100 mg of a 2% cream preparation of the compound or to placebo. Using a HPLC-assay the relative amounts of the applied dose of sertaconazole nitrate were determined in the residual cream of the skin surface as well as in 3 layers of the epidermis obtained by the stripping technique. Sertaconazole nitrate was shown to penetrate into the stratum corneum shortly after application, disappearing from the application areas with a mean apparent half-life of approximately 60 h. Immediately after topical application the residual amount of the applied mean dose of 2103 +/- 146.3 microg on the skin's surface was 88.9 +/- 2.3%, decreasing steadily to 52.4 +/- 8.5% after 48 h. A relevant amount of the applied dose (5.3 +/- 3.0%) was recovered from the stratum corneum already 30 min after application, and 3 h after administration a plateau was reached (6.9 +/- 3.2) which could be maintained until 48 h. A gradient from the site of application to the epidermis was apparent since the amounts recovered in The estimated average level of sertaconazole nitrate for a volume of 1 mL of stratum corneum after application of 100 mg cream was 1409 microg immediately after application and reached a plateau at 3 h with 9029 microg. Although not directly measured, the results also gave information about the mean amount of sertaconazole nitrate that penetrated through the stratum corneum and deeper layers allowing an estimate of the total mean amount of compound penetrating into the skin. The relative portion of this amount steadily increased from 1.1% of the applied dose at 0 h to 24.1% at 12 h, 34.2% at 24 h and finally to 37.6% of dose after 48 h of exposure. In view of the high target organ levels of the compound maintained over days, its rapid appearance in the stratum corneum after application and the earlier finding that Sertaconazole nitrate is not distributed into blood in substantial quantities the pharmacokinetic properties of this antifungal preparation therapy can be regarded as favourable.

Candida albicans-secreted aspartic proteinases modify the epithelial cytokine response in an in vitro model of vaginal candidiasis.

Secreted aspartyl proteinases (Saps) are important virulence factors of Candida albicans during mucosal and disseminated infections and may also contribute to the induction of an inflammatory host immune response. We used a model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) to study the epithelial cytokine response induced by C. albicans. In order to study the impact of the overall proteolytic activity and of distinct Sap isoenzymes, we studied the effect of the proteinase inhibitor pepstatin A on the immune response and compared the cytokine expression pattern induced by the wild-type strain SC5314 with the pattern induced by Sap-deficient mutants. Infection of RHVE with the C. albicans wild-type strain induced strong interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha responses in comparison with cytokine expression in noninfected tissue. Addition of the aspartyl proteinase inhibitor pepstatin A strongly reduced the cytokine response of RHVE. Furthermore, SAP-null mutants lacking either SAP1 or SAP2 caused reduced tissue damage and had a significantly reduced potential to stimulate cytokine expression. In contrast, the vaginopathic and cytokine-inducing potential of mutants lacking SAP4 to SAP6 was similar to that of the wild-type strain. These data show that the potential of specific Saps to cause tissue damage correlates with an epithelium-induced proinflammatory cytokine response, which may be crucial in controlling and managing C. albicans infections at the vaginal mucosa in vivo.

Polymorphonuclear leukocytes (PMNs) induce protective Th1-type cytokine epithelial responses in an in vitro model of oral candidosis.

The immune response and the anticandidal activity of keratinocytes and polymorphonuclear leukocytes (PMNs) play a key role in host defence against localized Candida albicans infection. An established model of oral candidosis based on reconstituted human oral epithelium (RHE) was supplemented with PMNs to study the effect of these immune cells during experimental oral candidosis. Infection of RHE with C. albicans induced a strong expression of the chemokine interleukin-8 (IL-8) and the cytokine granulocyte-macrophages colony-stimulating factor (GM-CSF), and a moderate stimulation of interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) by keratinocytes. This immune response was associated with chemoattraction of PMNs to the site of infection, whereas uninfected RHE failed to induce cytokine expression or to attract PMNs. Growth of the pathogen and tissue damage of C. albicans-infected RHE were significantly reduced when PMNs were applied to the apical epithelial surface or when PMNs migrated through a perforated basal polycarbonate filter of the model. Notably, protection against epithelial tissue damage was also observed when PMNs were placed on the basal side of non-perforated filters, which prevented PMN migration into the RHE. Addition of PMNs enhanced a Th1-type immune response (IFN-gamma, TNF-alpha), down-regulated the expression of the Th2-type cytokine interleukin-10 (IL-10), and was associated with protection against Candida-induced tissue damage. This PMN-supplemented model of oral candidosis mimics the in vivo situation, and provides a promising tool for studying the immunological interactions between keratinocytes and C. albicans, as well as the influence of PMNs on C. albicans pathogenesis.

The secreted aspartyl proteinases Sap1 and Sap2 cause tissue damage in an in vitro model of vaginal candidiasis based on reconstituted human vaginal epithelium.

Secreted aspartyl proteinases (Saps) contribute to the ability of Candida albicans to cause mucosal and disseminated infections. A model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) was used to study the expression and role of these C. albicans proteinases during infection and tissue damage of vaginal epithelium. Colonization of the RHVE by C. albicans SC5314 did not cause any visible epithelial damage 6 h after inoculation, although expression of SAP2, SAP9, and SAP10 was detected by reverse transcriptase PCR. However, significant epithelial damage was observed after 12 h, concomitant with the additional expression of SAP1, SAP4, and SAP5. Additional transcripts of SAP6 and SAP7 were detected at a later stage of the artificial infection (24 h). Similar SAP expression profiles were observed in three samples isolated from human patients with vaginal candidiasis. In experimental infection, secretion of antigens Sap1 to Sap6 by C. albicans was confirmed at the ultrastructural level by using polyclonal antisera raised against Sap1 to Sap6. Addition of the aspartyl proteinase inhibitors pepstatin A and the human immunodeficiency virus proteinase inhibitors ritonavir and amprenavir strongly reduced the tissue damage of the vaginal epithelia by C. albicans cells. Furthermore, SAP null mutants lacking either SAP1 or SAP2 had a drastically reduced potential to cause tissue damage even though SAP3, SAP4, and SAP7 were up-regulated in these mutants. In contrast the vaginopathic potential of mutants lacking SAP3 or SAP4 to SAP6 was not reduced compared to wild-type cells. These data provide further evidence for a crucial role of Sap1 and Sap2 in C. albicans vaginal infections.

Candida albicans hyphal formation and the expression of the Efg1-regulated proteinases Sap4 to Sap6 are required for the invasion of parenchymal organs.

The ability to change between yeast and hyphal cells (dimorphism) is known to be a virulence property of the human pathogen Candida albicans. The pathogenesis of disseminated candidosis involves adhesion and penetration of hyphal cells from a colonized mucosal site to internal organs. Parenchymal organs, such as the liver and pancreas, are invaded by C. albicans wild-type hyphal cells between 4 and 24 h after intraperitoneal (i.p.) infection of mice. In contrast, a hypha-deficient mutant lacking the transcription factor Efg1 was not able to invade or damage these organs. To investigate whether this was due to the inability to undergo the dimorphic transition or due to the lack of hypha-associated factors, we investigated the role of secreted aspartic proteinases during tissue invasion and their association with the different morphologies of C. albicans. Wild-type cells expressed a distinct pattern of SAP genes during i.p. infections. Within the first 72 h after infection, SAP1, SAP2, SAP4, SAP5, SAP6, and SAP9 were the most commonly expressed proteinase genes. Sap1 to Sap3 antigens were found on yeast and hyphal cells, while Sap4 to Sap6 antigens were predominantly found on hyphal cells in close contact with host cells, in particular, eosinophilic leukocytes. Mutants lacking EFG1 had either noticeably reduced or higher expressed levels of SAP4 to SAP6 transcripts in vitro depending on the culture conditions. During infection, efg1 mutants had a strongly reduced ability to produce hyphae, which was associated with reduced levels of SAP4 to SAP6 transcripts. Mutants lacking SAP1 to SAP3 had invasive properties indistinguishable from those of wild-type cells. In contrast, a triple mutant lacking SAP4 to SAP6 showed strongly reduced invasiveness but still produced hyphal cells. When the tissue damage of liver and pancreas caused by single sap4, sap5, and sap6 and double sap4 and -6, sap5 and -6, and sap4 and -5 double mutants was compared to the damage caused by wild-type cells, all mutants which lacked functional SAP6 showed significantly reduced tissue damage. These data demonstrate that strains which produce hyphal cells but lack hypha-associated proteinases, particularly that encoded by SAP6, are less invasive. In addition, it can be concluded that the reduced virulence of hypha-deficient mutants is not only due to the inability to form hyphae but also due to modified expression of the SAP genes normally associated with the hyphal morphology.