Ascorbic acid as a modulator of inflammatory response against Candida albicans.

Aim: To evaluate the behavior of oral keratinocytes in the presence of Vitamin C (Vit C) and its anti-inflammatory potential. Materials & methods: Oral keratinocytes were initially exposed to 0.1-2.5 mM of Vit C and the metabolic activity and cell migration were evaluated using MTS assay and Ibidi culture inserts, respectively. After, the cells were challenged with Candida albicans and inflammatory markers were analyzed by qPCR. Results: The treatment was not cytotoxic, and the highest concentrations increased the metabolic activity at 24 h. Vit C delayed the cell migration at 48 and 72 h. Interestingly, it downregulated the genes IL-8 and IL-1ß. Conclusion: Vit C could be an interesting adjuvant to anti-fungal treatment due to its anti-inflammatory potential.

Vitamin C, also known as ascorbic acid, is a vitamin commonly found in fruits and vegetables. It is popular for supporting our immune system, so is commonly taken as a supplement. We looked at the action of vitamin C on cells from the mouth and its potential to reduce inflammation in a fungal disease of the mouth ­ oral candidiasis. We showed that vitamin C is not toxic to cells of the mouth and may reduce inflammation in cells infected by the fungus. This suggests that vitamin C could be used as a complementary therapy for oral candidiasis.

Designed Antimicrobial Peptides for Recurrent Vulvovaginal Candidiasis Treatment.

Recurrent vulvovaginal candidiasis (RVVC) is a widespread chronic infection that has a substantial negative impact on work and quality of life. The development of antimicrobial resistance and biofilm formation are speculated to contribute to Candida pathogenicity and treatment ineffectiveness. Designed antimicrobial peptides (dAMPs) are chemically modified from endogenous antimicrobial peptides that provide the first line of defense against pathogens. The goal here is to identify a dAMP for the topical treatment of RVVC. The dAMP MICs were determined for 46 fluconazole-susceptible and fluconazole-resistant Candida spp. clinical isolates. The possibility of inducing dAMP drug resistance and comparison of dAMP and fluconazole activity against preformed Candida biofilm and biofilm formation were evaluated. Assessment of mammalian cell viability was determined using bioluminescent human keratinocytes. The dAMP effect on fungus was probed via scanning electron microscopy, and topically applied dAMP activity was evaluated in a rodent vulvovaginal candidiasis (VVC) infection model. dAMPs demonstrated broad-spectrum antimicrobial activity against common causative clinical Candida isolates, reduced preformed biofilm, and inhibited biofilm formation. An evaluated dAMP did not induce resistance after repeated exposure of Candida tropicalis The dAMPs were selective for Candida cells with limited mammalian cytotoxicity with substantial activity in a rodent VVC model. dAMPs are described as having potent antifungal and antibiofilm activity, likely direct membrane action with selectivity for Candida cells, with limited resistance development. Combined with activity in a rodent VVC model, the data support clinical evaluation of dAMPs for topical treatment of VCC and recurrent VVC infections.

Novel polymeric dressing to the treatment of infected chronic wound.

Natural rubber latex (NRL) is a natural polymer which has arisen large interest in the biomedical field, mostly, due to its ability to facilitate angiogenesis and therefore, tissue repair. Moxifloxacin (MXF) is a broad-spectrum antibiotic orally administrated. Considering the biological properties of the NRL and its ability to deliver a wide range of compounds, the present study aimed to develop a novel device for infected chronic wound treatment. MXF-loaded NRL was obtained by a casting method. The results demonstrated that the incorporation of MXF in NRL did not promote any molecular interaction, preserving the integrity of the compounds. The mechanical properties of the biomaterial did not show any significant change, indicating enough elasticity for dermal application. The microbiological assays confirmed the ability of the polymer to deliver the drug without influencing its pharmacological properties. Moreover, it has expressed activity against major bacterial strains presented in wound infections. Finally, the biomaterial shown biocompatibility from the in vitro study. Thus, the present work has shown that MXF-loaded NRL membrane is a promising biomaterial to infected wound treatment.

Hop Extract Acts as an Antioxidant with Antimicrobial Effects against Propionibacterium Acnes and Staphylococcus Aureus.

Acne is associated with hyperkeratosis, elevated levels of skin sebum and growth of Propionibacterium acnes (P. acnes) and Staphylococcus aureus (S. aureus). Furthermore, P. acnes promotes inflammation by inducing IL-6 production and oxidative stress. The aim of this study was to assess the antioxidant, anti-inflammatory and antibacterial potential of a hop-CO2-extract with 50% humulone and lupulone. The susceptibility of P. acnes and S. aureus to the hop extract was tested by using the broth microdilution technique. The minimal inhibitory concentrations (MIC) for P. acnes and S. aureus were 3.1 and 9.4 µg/mL, respectively. In addition, the hop extract showed an antioxidative effect with a half maximal inhibitory concentration (IC50) of 29.43 µg/mL as well as additional anti-inflammatory effects by reducing the IL-6 expression (IC50: 0.8 µg/mL). In addition, a gel formulation with 0.3% hop extract (w/w) had antibacterial activity against P. acnes and S. aureus (inhibition zone value: 5.5 mm and 3 mm, respectively) which was significantly superior to the placebo gel. The positive control (a gel with the antibiotic clindamycin) showed an inhibition zone of 9 mm. Due to its antioxidant, anti-inflammatory and antibacterial effects hop extract might be a treatment option for acne-prone skin.

Green tea polyphenols enhance gingival keratinocyte integrity and protect against invasion by Porphyromonas gingivalis.

The gingival epithelium, a stratified squamous tissue that acts as an interface between the external environment and the underlying connective tissue, plays an active role in maintaining periodontal health. The aim of the present study was to investigate the ability of green tea catechins to enhance gingival epithelial barrier function and protect against the disruption of epithelial integrity induced by Porphyromonas gingivalis. Both the green tea extract and epigallocatechin-3-gallate (EGCG) dose- and time-dependently increased the transepithelial electrical resistance (TER) of a gingival keratinocyte model and decreased the permeability of the cell monolayer to fluorescein isothyocyanate-conjugated 4.4-kDa dextran. This was associated with the increased expression of zonula occludens-1 (ZO-1) and occludin, two tight junction proteins. Treating the gingival keratinocyte monolayer with P. gingivalis caused a reduction in TER and affected the distribution of ZO-1 and occludin, allowing P. gingivalis to translocate through the cell monolayer. These deleterious effects mediated by P. gingivalis were abolished by the green tea extract and EGCG. This protection may be in part related to the ability of tea catechins to inhibit the protease activities of P. gingivalis. Given the above properties, green tea catechins may represent promising preventive and therapeutic molecules against periodontal disease.

Fractionated Coptis chinensis Extract and Its Bioactive Component Suppress Propionibacterium acnes-Stimulated Inflammation in Human Keratinocytes.

Coptis chinensis (CC) is widely used in Asian countries to treat inflammatory diseases. We investigated the anti-inflammatory activity of the aqueous fraction separated from CC extract and of berberine, its key bioactive component, in human keratinocytes and the possible molecular mechanisms underlying this. Treating HaCaT keratinocytic cells with heat-killed Propionibacterium acnes induced nitric oxide and proinflammatory cytokine (e.g., tumor necrosis factor-α, interleukin (IL)-1ß, and IL-8) production and their mRNA expression; these effects were suppressed by pretreatment with the aqueous fraction or berberine, which also suppressed the phosphorylation of ERK, JNK, and p38 kinases and the nuclear expression of nuclear factor (NF)-κB p65 in P. acnes-stimulated cells. Thus, the aqueous fraction and berberine effectively exerted anti-inflammatory activities by suppressing mitogen-activated protein kinase and NF-κB signaling pathways in human keratinocytes and may be used for treating P. acnes-induced inflammatory skin diseases.

Intravenous IgG Reduces Pathogenic Autoantibodies, Serum IL-6 Levels, and Disease Severity in Experimental Bullous Pemphigoid Models.

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoantibodies to COL17. Currently, systemic corticosteroids are used as first-line treatments for BP; alternatively, intravenous administration of high-dose IgG (IVIG) has been shown to be effective for patients with steroid-resistant BP in clinical practice. However, the effect of IVIG on BP has not fully been investigated. To examine the effects and mechanisms of action of IVIG against BP, we performed IVIG experiments using two experimental BP mouse models. One is a passive-transfer BP model that reproduces subepidermal separation in neonatal mice by the passive transfer of IgGs against COL17, such as polyclonal or monoclonal mouse IgG or IgG from BP patients. The other is an active BP model that continuously develops a disease phenotype in adult mice. IVIG decreased pathogenic IgG and the disease scores in both models. Injected IVIG distributed throughout the dermis and the intercellular space of the lower epidermis. Notably, IVIG inhibited the increase of IL-6 in both models, possibly by suppressing the production of IL-6 by keratinocytes. These results suggest that the inhibitory effects of IVIG on BP are associated with the reduction of pathogenic IgG and the modulation of cytokine production.

Rhodomyrtus tomentosa Leaf Extract Inhibits Methicillin-Resistant Staphylococcus aureus Adhesion, Invasion, and Intracellular Survival in Human HaCaT Keratinocytes.

Methicillin-resistant Staphylococcus aureus (MRSA) has an ability to invade nonprofessional phagocytic cells, resulting in persistent infections and most likely host cell death. Series of our studies have claimed pronounced antibacterial efficacy of Rhodomyrtus tomentosa leaf extract. This study was to further investigate potency of the extract in intracellular killing of human HaCaT keratinocytes. Pretreatment of MRSA with the extract resulted in a remarkable reduction in the bacterial adhesion to HaCaT keratinocytes, compared with untreated control (p < 0.001). In addition, at least 60% inhibition of the bacterial invasion into HaCaT cells was observed. Intracellular killing assay demonstrated that the extract exhibited strong antibacterial activity against intracellular MRSA at nontoxic concentrations (128 mg/L), which may have resulted from the increase in bactericidal activity under phagolysosomal pH. Transmission electron microscopy displayed the effects of the extract on alterations in the bacterial cell morphology with cell lysis. Fluorescence microscopy revealed that the extract decreased MRSA-induced apoptosis in HaCaT cells. In addition, cytotoxicity of HaCaT cells caused by MRSA supernatant was reduced at least 50% by the extract. The potential activities of R. tomentosa extract may be useful in an alternative treatment of MRSA infections in slight acidic compartments, particularly skin infections.

Anti-inflammatory activity of Crateva adansonii DC on keratinocytes infected by Staphylococcus aureus: From traditional practice to scientific approach using HPTLC-densitometry.

ETHNOPHARMACOLIGICAL RELEVANCE: Leaves of Crateva adansonii DC (Capparidaceae), a small bush found in Togo, are widely used in traditional medicine to cure infectious abscesses. Traditional healers of Lomé harvest only budding leaves early in the morning, in specific area in order to prepare their drugs. AIM OF THE STUDY: The main goal was to validate the ancestral picking practices, and to assess the activity of C. adansonii medicine towards infectious abscesses. MATERIALS AND METHODS: A phytochemical screening of various C. adansonii leaf samples was performed using an original HPTLC-densitometry protocol and major flavonoids were identified and quantified. C. adansonii samples were collected in different neighborhoods of Lomé, at different harvesting-times and at different ages. Radical scavenging capacity, using DPPH assay, was used to quickly screen all extracts. Extracts were tested for anti-Staphylococcus aureus activity and anti-inflammatory effect on human primary keratinocytes infected by S. aureus. IL6, IL8 and TNFα expression and production were assessed by RT-PCR and ELISA assays. RESULTS: Using antioxidant activity as selection criteria, optimal extracts were obtained with budding leaves, collected at 5:00am in Djidjolé neighborhood. This extract showed the strongest anti-inflammatory effect on S. aureus-infected keratinocytes by reducing IL6, IL8 and TNFα expression and production. None of the extracts inhibited the growth of S. aureus. CONCLUSIONS: Those results validate the traditional practices and the potential of C. adansonii as anti-inflammatory drug. Our findings suggest that traditional healers should add to C. adansonii leaves an antibacterial plant of Togo Pharmacopeia, in order to improve abscess healing.

Sacha Inchi Oil (Plukenetia volubilis L.), effect on adherence of Staphylococus aureus to human skin explant and keratinocytes in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Plukenetia volubilis L. (Euphorbiaceae) is a domesticated vine distributed from the high-altitude Andean rain forest to the lowlands of the Peruvian Amazon. Oil from the cold-pressed seeds, sold under the commercial name of Sacha Inchi Oil (SIO) is actually much in favour because it contains a high percentage of omega 3 and omega 6, and is hence used as a dietary supplement. SIO is also used traditionally for skin care, in order to maintain skin softness, and for the treatment of wounds, insect bites and skin infections, in a tropical context where the skin is frequently damaged. AIMS OF THE STUDY: This study was designed in order to verify whether the traditional use of SIO for skin care would have any impact on Staphylococcus aureus growth and skin adherence, as S. aureus is involved in many skin pathologies (impetigo, folliculitis, furuncles and subcutaneous abscesses) being one if the main pathogens that can be found on the skin. Therefore, our objective was to assess SIO bactericidal activity and interference with adherence to human skin explants and the keratinocyte cell line. Cytotoxicity on that cells was also determined. The activity of SIO was compared to coconut oil (CocO), which is widely used for skin care but has different unsaturated fatty acids contents. MATERIALS AND METHODS: Laboratory testing with certified oil, determined antibacterial activity against radio labelled S. aureus. Cytotoxic effects were measured with XTT on keratinocyte cells and with neutral red on human skin explants; phenol was used as cytotoxic control. Adherence assays were carried out by mixing H3-labelled S. aureus bacteria with keratinocyte cells and human skin explants, incubated with oils 2h before (to determine the inhibition of adherence, assimilated to a preventive effect) or 2h after the contact of the biological material with S. aureus (to assess the detachment of the bacteria, assimilated to a curative effect). Residual radioactivity measured after washings made it possible to determine the adherence intensity. Bactericidal effect was determined by colony counting on trypticase soy agar. RESULTS: Laboratory assays showed that SIO and CocO, tested undiluted, were not cytotoxic on keratinocytes nor human explants and were not bactericidal neither. SIO was more active as antiadherent (preventive) than CocO on keratinocytes. There was no significant difference between detachment effects (curative) of both oils on keratinocytes but SIO was almost 5 times more active on the detachment of S. aureus from human skin explants. CONCLUSION: From that study it can be concluded that the use of SIO on dermal cells is safe and efficient in the inhibition of S. aureus adherence. Our results tend to support the traditional use of undiluted SIO in skin care.

Dietary pectin-derived acidic oligosaccharides improve the pulmonary bacterial clearance of Pseudomonas aeruginosa lung infection in mice by modulating intestinal microbiota and immunity.

BACKGROUND: A predominantly T-helper type 2 (Th2) immune response is critical in the prognosis of pulmonary Pseudomonas aeruginosa infection. But the mucosal and systemic immune responses can be influenced by the intestinal microbiota. METHODS: We assessed the effect of microbiota compositional changes induced by a diet enriched in 5% acidic oligosaccharides derived from pectin (pAOS) on the immune response and outcome of chronic pulmonary P. aeruginosa infection in mice. RESULTS: pAOS promoted Th1 polarization by increasing interferon γ release, upregulating t-bet gene expression, decreasing interleukin 4 secretion, and downregulating gata3 gene expression. pAOS also sustained the release of keratinocyte chemoattractant, recruited polynuclear leukocytes and macrophages, stimulated M1 macrophage activation and interleukin 10 release, and decreased tumor necrosis factor α release in the lung. These effects led to increased bacterial clearance after the first and second P. aeruginosa infections. pAOS modified the intestinal microbiota by stimulating the growth of species involved in immunity development, such as Bifidobacterium species, Sutturella wadsworthia, and Clostridium cluster XIVa organisms, and at the same time increased the production of butyrate and propionate. CONCLUSION: These results suggest that pAOS may have beneficial effects by limiting the number and severity of pulmonary exacerbations in patients chronically infected with P. aeruginosa, such as individuals with cystic fibrosis.

Antibacterial and antigelatinolytic effects of Satureja hortensis L. essential oil on epithelial cells exposed to Fusobacterium nucleatum.

The present report examined the effects of essential oils (EOs) from Satureja hortensis L. and Salvia fruticosa M. on the viability and outer membrane permeability of the periodontopathogen Fusobacterium nucleatum, a key bacteria in oral biofilms, as well as the inhibition of matrix metalloproteinase (MMP-2 and MMP-9) activities in epithelial cells exposed to such bacteria. Membrane permeability was tested by measuring the N-phenyl-1-naphthylamine uptake and bacterial viability by using the commercially available Live/Dead BacLight kit. In addition, gelatin zymography was performed to analyze the inhibition of F. nucleatum-induced MMP-2 and MMP-9 activities in HaCaT cells. We showed that 5, 10, and 25 µL/mL of Sat. hortensis L. EO decreased the ratio of live/dead bacteria and increased the outer membrane permeability in a range of time from 0 to 5 min. Treatments with 10 and 25 µL/mL of Sal. fruticosa M. also increased the membrane permeability and 5, 10, and 25 µL/mL of both EOs inhibited MMP-2 and MMP-9 activities in keratinocytes induced after exposure of 24 h to F. nucleatum. We conclude that antibacterial and antigelatinolytic activities of Sat. hortensis L. EO have potential for the treatment of periodontal inflammation.

Micronutrient modulation of NF-κB in oral keratinocytes exposed to periodontal bacteria.

Chronic periodontal diseases are characterised by a dysregulated and exaggerated inflammatory/immune response to plaque bacteria. We have demonstrated previously that oral keratinocytes up-regulate key molecular markers of inflammation, including NF-κB and cytokine signalling, when exposed to the periodontal bacteria Porphyromonas gingivalis and Fusobacterium nucleatum in vitro. The purpose of the current study was to investigate whether α-lipoic acid was able to abrogate bacterially-induced pro-inflammatory changes in the H400 oral epithelial cell line. Initial studies indicated that α-lipoic acid supplementation (1-4 mM) significantly reduced cell attachment; lower concentrations (<0.5 mM) enabled >85% cell adhesion at 24 h. While a pro-inflammatory response, demonstrable by NF-κB translocation, gene expression and protein production was evident in H400 cells following exposure to P. gingivalis and F. nucleatum, pre-incubation of cells with 0.5 mM α-lipoic acid modulated this response. α-Lipoic acid pre-treatment significantly decreased levels of bacterially-induced NF-κB activation and IL-8 protein production, and differentially modulated transcript levels for IL-8, IL-1ß, TNF-α and GM-CSF, TLR2, 4, 9, S100A8, S100A9, lysyl oxidase, NF-κB1, HMOX, and SOD2. Overall, the data indicate that α-lipoic acid exerts an anti-inflammatory effect on oral epithelial cells exposed to periodontal bacteria and thus may provide a novel adjunctive treatment for periodontal diseases.

Study of streptococcal hemoprotein receptor (Shr) in iron acquisition and virulence of M1T1 group A streptococcus.

Streptococcus pyogenes (group A streptococcus, GAS) is a human bacterial pathogen of global significance, causing severe invasive diseases associated with serious morbidity and mortality. To survive within the host and establish an infection, GAS requires essential nutrients, including iron. The streptococcal hemoprotein receptor (Shr) is a surface-localized GAS protein that binds heme-containing proteins and extracellular matrix components. In this study, we employ targeted allelic exchange mutagenesis to investigate the role of Shr in the pathogenesis of the globally disseminated serotype M1T1 GAS. The shr mutant exhibited a growth defect in iron-restricted medium supplemented with ferric chloride, but no significant differences were observed in neutrophil survival, antimicrobial peptide resistance, cell surface charge, fibronectin-binding or adherence to human epithelial cells and keratinocytes, compared with wild-type. However, the shr mutant displayed a reduction in human blood proliferation, laminin-binding capacity and was attenuated for virulence in in vivo models of skin and systemic infection. We conclude that Shr augments GAS adherence to laminin, an important extracellular matrix attachment component. Furthermore, Shr-mediated iron uptake contributes to GAS growth in human blood, and is required for full virulence of serotype M1T1 GAS in mouse models of invasive disease.

Application of orange essential oil as an antistaphylococcal agent in a dressing model.

BACKGROUND: Staphylococcus aureus is the pathogen most often and prevalently involved in skin and soft tissue infections. In recent decades outbreaks of methicillin-resistant S. aureus (MRSA) have created major problems for skin therapy, and burn and wound care units. Topical antimicrobials are most important component of wound infection therapy. Alternative therapies are being sought for treatment of MRSA and one area of interest is the use of essential oils. With the increasing interest in the use and application of natural products, we screened the potential application of terpeneless cold pressed Valencia orange oil (CPV) for topical therapy against MRSA using an in vitro dressing model and skin keratinocyte cell culture model. METHODS: The inhibitory effect of CPV was determined by disc diffusion vapor assay for MRSA and vancomycin intermediate-resistant S. aureus (VISA) strains. Antistaphylococcal effect of CPV in an in vitro dressing model was tested on S. aureus inoculated tryptic soya agar plate. Bactericidal effect of CPV on MRSA and VISA infected keratinocyte cells was examined by enumeration of extra- and intra-cellular bacterial cells at different treatment time points. Cytotoxic effects on human skin cells was tested by adding CPV to the keratinocyte (HEK001) cells grown in serum free KSFM media, and observed by phase-contrast microscope. RESULTS: CPV vapour effectively inhibited the MRSA and VISA strains in both disc diffusion vapour assay and in vitro dressing model. Compared to untreated control addition of 0.1% CPV to MRSA infected keratinocyte decreased the viable MRSA cells by 2 log CFU/mL in 1 h and in VISA strain 3 log CFU/mL reduction was observed in 1 h. After 3 h viable S. aureus cells were not detected in the 0.2% CPV treatment. Bactericidal concentration of CPV did not show any cytotoxic effect on the human skin keratinocyte cells in vitro. CONCLUSIONS: At lower concentration addition of CPV to keratinocytes infected with MRSA and VISA rapidly killed the bacterial cells without causing any toxic effect to the keratinocytes. Therefore, the results of this study warrant further in vivo study to evaluate the potential of CPV as a topical antistaphylococcal agent.

Anti-inflammatory effects of active constituents extracted from Chinese medicinal herbs against Propionibacterium acnes.

Interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) from keratinocyte play important roles in initiating the inflammatory process of acne. They are used as major elements to evaluate the anti-inflammatory activity of drugs. In this study, various active constituents extracted from Chinese medicinal herbs were tested for their anti-inflammatory effects against P. acnes using ELISA. Among the constituents, matrine, baicalin, ursolic acid, sodium danshensu, magnolol, honokiol, hesperidin and andrographolide significantly reduced IL-8 and TNF-α by human HaCaT keratinocyte cells pretreated with heat-killed P. acnes. Excepting hesperidin, these active constituents presented dose-dependent inhibitory effects. Our studies showed that all of them exhibited low cytotoxicity at 5 µg mL⁻¹ in tested cell lines, and even at 50 µg mL⁻¹, in the cases of matrine, baicalin, ursolic acid and sodium danshensu. Based on the obtained results, it can be suggested that these active constituents are potential acne-mitigating candidates for cosmetic applications.

Proanthocyanidin-enriched extract from Myrothamnus flabellifolia Welw. exerts antiviral activity against herpes simplex virus type 1 by inhibition of viral adsorption and penetration.

AIM OF THE STUDY: Extracts from the aerial parts of the South African resurrection plant Myrothamnus flabellifolia Welw. have been used traditionally against infections of the upper respiratory tract and skin diseases. A polyphenol-enriched extract was investigated for potential antiviral effects against herpes simplex virus type 1 (HSV-1) and adenovirus, and the underlying mode of action was to be studied. MATERIALS AND METHODS: Antiviral effects of an acetone-water extract (MF) from Myrothamnus flabellifolia on HSV-1 and adenovirus type 3 were tested in infected Vero cells by plaque reduction assay, MTT test and immunofluorescence. The influence of the extract on the HSV-1 envelope glycoprotein D was shown by Western blot. Organotypic full thickness skin models consisting of multilayer skin equivalents were used for the investigation of MF effects on HSV-1 replication. RESULTS: MF exhibited strong antiviral activity against HSV-1. The HSV-1-specific inhibitory concentration (IC(50)) was determined as 0.4 µg/mL and the cytotoxic concentration (CC(50)) against Vero cells as 50 µg/mL. A selectivity index (SI) (ratio of CC(50) to IC(50)) of approximately 120 was calculated when MF was added to the virus inoculum for 1h at 37°C prior to infection. The replication of adenovirus 3 was not affected by MF. MF abolished virus entry into the host cell by blocking viral attachment to the cell surface. When added after attachment at a concentration of >6 µg/mL, the extract also inhibited penetration of HSV-1 into the host cell. Polyphenolic compounds from MF directly interacted with viral particles, leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein D (gD). Using organotypic full thickness tissue cultures, it was shown that treatment of HSV-1 infected cultures with the MF resulted in reduced viral spread. CONCLUSIONS: A polyphenol-enriched extract from Myrothamnus flabellifolia strongly acts against HSV-1 by blocking viral entry into the cells.

Effects of AV119, a natural sugar from avocado, on Malassezia furfur invasiveness and on the expression of HBD-2 and cytokines in human keratinocytes.

AV119 is a patented blend of two sugars from avocado that can induce human beta-defensin-2 production by normal human keratinocytes. In this study, we analysed the effect of AV119 on growth and invasiveness of Malassezia furfur, a dimorphic, lipid-dependent yeast that is part of the normal human cutaneous commensal flora. The ability to modulate the expression of the proinflammatory and immunomodulatory cytokines in normal human keratinocytes was also investigated. Microbiological assay demonstrated that this sugar induced the aggregation of yeast cells and inhibited the invasiveness of M. furfur, without affecting its growth. Real-time PCR analysis demonstrated that AV119 was able to modulate the HBD-2 response in treated keratinocytes, reaching a maximum after 48-h treatment, and to induce the recovery of a satisfactory proinflammatory response in human keratinocytes. As AV119 can induce aggregation of yeast cells, thus inhibiting their penetration into the keratinocytes, the sugar could be used in the preparation of cosmetics or pharmacological drugs to inhibit colonization of the skin by pathogenic strains of M. furfur.

Antimicrobial photodynamic therapy: assessment of genotoxic effects on keratinocytes in vitro.

BACKGROUND: Work has shown that cutaneous microbial species associated with skin conditions of microbial aetiology are susceptible to killing by antimicrobial photodynamic therapy (APDT) using visible light and methylene blue. OBJECTIVES: To evaluate immediate and delayed genotoxicity of APDT on keratinocytes in vitro. METHODS: A combination of methylene blue (100 microg mL(-1)) and visible light (42 mW cm(-2)), as used in studies of microbe and keratinocyte cytotoxicity, was employed to test a human keratinocyte cell line (H103) for genotoxic damage by comet assay. RESULTS: The comet assay was able to detect genotoxic damage in H2O2-treated keratinocytes (positive control). APDT did not cause either immediate or delayed genotoxic damage in keratinocytes following APDT of up to 180 min. CONCLUSIONS: APDT sufficient to reduce microbes by seven log cycles showed no detectable genotoxic effects on keratinocytes. APDT applied in vivo may represent a useful low-risk alternative to conventional antimicrobial treatment in dermatology.

HPV-18 immortalization of human keratinocytes.

The oncogenic potential of human papillomavirus type 18 which is found in a significant number of cervical and penile cancer biopsies was tested in primary human keratinocytes derived from neonatal foreskin. Viral DNA and a gene for resistance to neomycin were introduced into these cells by calcium phosphate transfection. Selection of cells in G418 led to the isolation of resistant colonies which were propagated in culture. Four cell lines termed FE-A, FEH 18L, FEP18-5, and FEP18-11 have been maintained in culture for 1 1/2-2 years and were selected for further analysis. In all cases the viral DNA was integrated into the cellular genome and the early genes were transcribed, including RNA complementary to the E2, E6, and E7 open reading frames. Radioimmunoprecipitation showed that all cell lines synthesized the E6 and E7 proteins. However, none of the cell lines tested were tumorigenic. The differentiation capacity of these cells was analyzed by assessing their ability to proliferate clonally after exposure to 1.2 mM calcium chloride. All four cell lines were resistant to this stimulus and formed colonies upon return to regular growth medium whereas normal cells differentiated terminally. K6a and K14 keratin RNA expression was down-regulated in the HPV immortalized cell lines compared to primary human epithelial cells.