Examination of the skin barrier repair/wound healing process using a living skin equivalent model and matrix-assisted laser desorption-ionization-mass spectrometry imaging.

OBJECTIVE: Examination of the skin barrier repair/wound healing process using a living skin equivalent (LSE) model and matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to identify lipids directly involved as potential biomarkers. These biomarkers may be used to determine whether an in vivo wound is going to heal for example if infected. METHODS: An in vitro LSE model was wounded with a scalpel blade and assessed at day 4 post-wounding by histology and MALDI-MSI. Samples were sectioned at wound site and were either formalin-fixed paraffin-embedded (FFPE) for histology or snapped frozen (FF) for MSI analysis. RESULTS: The combination of using an in vitro wounded skin model with MSI allowed the identification of lipids involved in the skin barrier repair/wound healing process. The technique was able to highlight lipids directly in the wound site and distinguish differences in lipid distribution between the epidermis and wound site. CONCLUSION: This novel method of coupling an in vitro LSE with MSI allowed in-depth molecular analysis of the skin barrier repair/wound healing process. The technique allowed the identification of lipids directly involved in the skin barrier repair/wound healing process, indicating these biomarkers may be potentially be used within the clinic. These biomarkers will help to determine, which stage of the skin barrier repair/wound healing process the wound is in to provide the best treatment.

Assessment of the antimicrobial activity of acellular vascular grafts.

Several studies have reported biological vascular grafts to be more resistant to microbial infection than synthetic counterparts in vivo. Indeed, small intestinal submucosa (SIS) materials have previously been reported to be antimicrobial. The aim of this study was to assess the antimicrobial activity and the ability to resist biofilm formation of a novel acellular vascular graft and compare it to commercially available alternatives using a range of organisms: MRSA, MSSA, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa and Candida albicans. This was achieved using a modified disk diffusion assay and extraction of the materials into solution followed by minimum inhibitory concentration assays. To assess resistance to biofilm formation a novel biofilm assay was developed which compared the total colony forming units (CFU) recovered from each material and identification of the percentage of CFU which were loosely attached, residing within the biofilm or attached to the biomaterial. The results indicated a lack of antimicrobial activity for all materials tested, including SIS. The biological materials were more resistant to the formation of a biofilm compared to Dacron.

Chloracne in seven organic chemists exposed to novel polycyclic halogenated chemical compounds (triazoloquinoxalines).

Chloracne is an acneiform eruption caused though poisoning by aromatic compounds (usually halogenated) showing a specific molecular configuration. We describe an outbreak of chloracne among seven discovery chemists who synthesized novel polycyclic halogenated chemical compounds which were classified as triazoloquinoxalines, not known to be chloracnegenic. The diagnosis of chloracne, made clinically, elicited a thorough risk assessment and monitoring programme by the occupational health department. The chemists were investigated by serum excretion rates, skin sampling for Propionibacterium acnes, skin biopsy and laboratory blood investigations. Sebum excretion was normal in five cases, raised in one case and severely reduced in another. Skin levels of P. acnes were normal in all patients except for the one subject who had low sebum excretion, in whom they were undetectable. One subject had a slightly raised serum level of alanine aminotransferase. There were no other signs of systemic toxicity. Two subjects were treated with an oral antibiotic, two received topical therapy only and three required no treatment at all. The patients have had thorough health surveillance at 6-monthly and yearly intervals. In each case the chloracne mostly resolved within 18-24 months although on examination about 3 years later, five of the seven still showed minor changes of chloracne. This outbreak emphasizes the need for vigilance in discovery science. The triazoloquinoxalines were not previously recognized as being chloracnegens although their chemical characteristics were subsequently identified as being in keeping with other chemicals that can cause chloracne. Chloracne can be a difficult diagnosis to make when it occurs in a novel setting: occupational physicians and dermatologists need to be vigilant when dealing with unusual eruptions in discovery chemists.

Microbiological effect of photodynamic therapy (PDT) in healthy volunteers: a comparative study using methyl aminolaevulinate and hexyl aminolaevulinate cream.

BACKGROUND: Acne vulgaris is a common skin problem that affects up to 90% of adolescents. Colonization of the duct with Propionibacterium species is one of the factors implicated in the development of acne. Owing to the increasing incidence of antibiotic resistance, there has been an greater interest in the development of new methods to treat acne. Early studies have shown that photodynamic therapy (PDT) with aminolaevulinic acid (ALA) can lead to prolonged improvement in acne. Newer derivatives of ALA such as methyl aminolaevulinate hydrochloride (MAL) and hexyl aminolaevulinate hydrochloride (HAL) have been developed for use in PDT, with the potential benefits of higher lipophilicity and penetration potential. OBJECTIVES: To determine the microbiological effect and tolerability of a single application of HAL-PDT and to compare it with MAL-PDT in healthy volunteers. METHODS: This was a randomised double-blind study to examine the microbiological effects and safety of a single application of MAL-PDT and HAL-PDT on normal skin in 18 healthy volunteers. Bacterial skin samples for Propionibacterium spp. and Micrococceae were obtained at baseline and 2, 4, 7 and 14 days. RESULTS: Following PDT with MAL and HAL, a statistically significant transient reduction in mean density of Propionibacterium spp. 2 days after treatment using each agent (P < 0.05 for both) was found. There were no significant changes in mean number of Micrococceae for the duration of the study period. Treatment with HAL-PDT and MAL-PDT was well tolerated. Overall, HAL-PDT was associated with fewer side-effects compared with MAL-PDT (P < 0.01) over the 14 day study period. CONCLUSION: HAL-PDT and MAL-PDT transiently reduce density of Propionibacterium spp. density to a similar degree in normal healthy individuals. The transient reduction in Propionibacterium spp. suggests that the prolonged antiacne effect of PDT relies on factors independent of bacterial density. HAL-PDT appears to be better tolerated than MAL-PDT.

Topical aminolaevulinic acid-photodynamic therapy for the treatment of acne vulgaris: a study of clinical efficacy and mechanism of action.

BACKGROUND: Acne affects 83-95% of 16-year-olds of both sexes, and many seek help from a clinician. Emerging problems with conventional acne treatments, specifically antibiotic resistance of Propionibacterium acnes and fears over the safety and tolerance of oral isotretinoin, create a demand for novel treatment modalities in acne. OBJECTIVES: To study the efficacy of aminolaevulinic acid-photodynamic therapy (ALA-PDT) in the treatment of acne and to identify the mode of action, looking specifically at the effects on surface numbers of P. acnes and on sebum excretion. METHODS: Ten patients (nine men and one woman, age range 16-40 years) with mild to moderate acne on their backs were recruited. Each patient's back was marked with four 30-cm2 areas of equal acne severity. Each site was then randomly allocated to either ALA-PDT treatment, light alone, ALA alone or an untreated control site. At baseline, numbers of inflammatory and noninflammatory acne lesions were counted, sebum excretion measured by Sebutapes (CuDerm, Dallas, TX, U.S.A.) and surface P. acnes swabs performed. ALA cream (20% in Unguentum Merck) was applied under occlusion to the ALA-PDT and ALA alone sites for 3 h. Red light from a diode laser was then delivered to the ALA-PDT and light alone sites (635 nm, 25 mW cm(-2), 15 J cm(-2)). Each patient was treated weekly for 3 weeks. At each visit acne lesion counts were performed and 3 weeks following the last treatment sebum excretion rates and P. acnes swabs were repeated. RESULTS: There was a statistically significant reduction in inflammatory acne lesion counts from baseline after the second treatment at the ALA-PDT site but not at any of the other sites. No statistically significant reduction in P. acnes numbers or sebum excretion was demonstrated at any sites including the ALA-PDT site. CONCLUSIONS: ALA-PDT is capable of clinically improving acne. An alternative mode of action for ALA-PDT other than direct damage to sebaceous glands or photodynamic killing of P. acnes is suggested from the results of this study.

The effect of lipids on the adherence of axillary aerobic coryneform bacteria.

AIMS: A wide range of lipids are present on the skin surface of human beings and bacterial lipases are known to modify them. The microflora of the underarm (axilla) is often dominated by aerobic coryneforms and whilst many require lipids for growth, they appear not to be utilized as carbon sources. The aim of this study was to investigate the adherence aerobic coryneforms to lipids present on the skin surface of the human axilla to determine whether they contribute to colonization of the skin. METHODS: Aerobic coryneforms were grown in a defined synthetic medium in the presence of (14)C-glucose to produce radio-labelled cells. Adherence to lipids was tested using a thin layer chromatography plate-based assay. SIGNIFICANCE AND IMPACT OF THE STUDY: The mechanism of bacterial adhesion to skin lipids is unknown. The results of this study show that a significant proportion of cutaneous aerobic coryneform isolates from the axilla interact with skin lipids resulting in increased adherence, which may contribute to skin colonization.

The effects of acne treatment with a combination of benzoyl peroxide and erythromycin on skin carriage of erythromycin-resistant propionibacteria.

Concomitant application of 5% w/w benzoyl peroxide and 3% w/w erythromycin has previously been shown to prevent the overgrowth, on the skin of acne patients, of erythromycin-resistant coagulase-negative staphylococci, which occurs when the antibiotic is used alone. Two in vivo studies were carried out to assess the ability of the same therapeutic combination to inhibit the growth of pre-existing erythromycin-resistant propionibacteria and to prevent the selection of resistant strains during treatment. A double-blind clinical trial in 37 patients with mild to moderate acne vulgaris showed that the combination brought about a > 3 log10 c.f.u. reduction in total propionibacterial numbers/cm2 after 6 weeks therapy (P < 0.001, Wilcoxon's matched pairs) and also significantly reduced the number of erythromycin-resistant propionibacteria (P < 0.05). In contrast, erythromycin alone reduced the total propionibacterial count by < 1.5 log10 c.f.u./cm2 after 6 weeks (P < 0.05) and did not affect the number of erythromycin-resistant strains. The combined formulation was significantly more effective at reducing total propionibacterial numbers at 6 (P < 0.01, Mann-Whitney) and 12 weeks (P < 0.05) than erythromycin alone, although, after 12 weeks, the anti-propionibacterial efficacy of both preparations was less marked. Five patients on combination therapy, and five treated with erythromycin alone, acquired erythromycin-resistant strains de novo at week 6 or week 12. In an open study in 21 acne patients, who each carried > 10(3) c.f.u. erythromycin-resistant propionibacteria/cm2 skin pretreatment, the combination of erythromycin and benzoyl peroxide reduced the total propionibacterial count by > 2.5 log10 and the number of erythromycin-resistant strains by a similar amount (P < 0.001, Wilcoxon). This was accompanied by highly significant reductions in acne grade and lesion counts (P < 0.001). These data suggest that the combination of 5% w/w benzoyl peroxide and 3% w/w erythromycin has greater in vivo anti-propionibacterial activity than 3% w/w erythromycin alone, and brings about significant clinical improvement in acne patients with high numbers of erythromycin-resistant propionibacterial strains pretreatment.

The short-term treatment of acne vulgaris with benzoyl peroxide: effects on the surface and follicular cutaneous microflora.

A 28-day treatment regimen was undertaken by 12 volunteers, in which 5% (w/v) benzoyl peroxide (BP) in an aqueous gel was applied daily to the entire face. Clinical efficacy of the treatment was assessed after 2, 4, 9, 14 and 28 days, and the surface and follicular microbial populations were enumerated using established techniques. Viable counts were obtained for propionibacteria and Micrococcaceae. Mean numbers of propionibacteria recovered from the skin surface and follicular casts were significantly reduced after 2 days' treatment (P < 0.01), and the population was maintained at a significantly lower level throughout the study (P < 0.01), with mean values approaching the lower detection limit of the assay. Significant reductions in the surface and follicular Micrococcaceae were observed after 2 days' treatment, and at all subsequent visits (P < 0.05). After 2 days' treatment, only slight reductions in mean acne grade and mean inflamed lesion count were observed. However, at all subsequent visits the mean acne grade was significantly reduced (P < 0.05) compared with T0. The mean non-inflamed lesion count was lower than the pretreatment level at all visits, although the results were variable. The results indicate that significant reductions in surface and follicular microorganisms may be obtained after 48 h treatment with BP. Therefore, the non-specific antibacterial action of BP may be utilized in short intervening courses to reduce the carriage of antibiotic-resistant micro-organisms and thus improve the long-term efficacy of antibiotic acne treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Disruption of the transmembrane pH gradient--a possible mechanism for the antibacterial action of azelaic acid in Propionibacterium acnes and Staphylococcus epidermidis.

The effect of the topical acne treatment azelaic acid on the transmembrane proton gradient (delta pH) of Propionibacterium acnes and Staphylococcus epidermidis was studied in vitro at external pH values found on human skin (pH 4.0-6.0). Bacteria were grown in defined media using continuous culture and delta pH was estimated by measuring the accumulation of [14C] benzoic by the cells using flow dialysis. In both P. acnes and S. epidermidis the addition of 30 mM azelaic acid and the membrane active inhibitors nigericin (150 microM) and CCCP (150 microM) resulted in a rapid release of [14C] label into the dialysate indicating the dissipation of delta pH between external pH values of 4.0-6.0. The addition of 60 mM NaCl as an iso-osmotic control and 150 microM valinomycin did not induce the release of [14C] label. The addition of 30 mM azelaic acid reduced the delta pH of P. acnes by 44% at external pH 4.0 and 28% at external pH 6.0. In S. epidermidis 30 mM azelaic acid reduced delta pH by 88% at external pH 5.0 and 20% at external pH 6.0. Rapid loss of viability occurred in suspensions of P. acnes and S. epidermidis containing 30 mM azelaic acid at pH 4.0 with no viable cells recovered after 60 min incubation. At pH 6.0 little change in viable numbers of P. acnes and S. epidermidis were observed over a 2 h incubation period. The results indicate that the antibacterial activity of azelaic acid is associated with the perturbation of intracellular pH.

Inhibition of erythromycin-resistant propionibacteria on the skin of acne patients by topical erythromycin with and without zinc.

Propionibacteria resistant to high concentrations of erythromycin [minimal inhibitory concentration (MIC) > or = 0.5 mg/ml] are now commonly isolated from the skin of antibiotic-treated acne patients. This double-blind study was carried out to assess the ability of 4% w/v erythromycin with and without 1.2% w/v zinc acetate to reduce the numbers of erythromycin-resistant propionibacteria in vivo, and also to monitor the acquisition of resistant strains de novo during therapy. Under laboratory conditions, erythromycin-resistant propionibacteria were shown to be as sensitive to zinc acetate as fully sensitive strains. In vivo, the erythromycin/zinc complex and erythromycin alone produced highly significant reductions in total propionibacteria (P < 0.001) and in the number of erythromycin-resistant strains (P < 0.001 at 8 weeks). After 12 weeks, resistant propionibacteria were reacquired, or acquired de novo, by three patients treated with erythromycin alone and four patients treated with the erythromycin/zinc complex. In contrast, changes in numbers of Micrococcaceae were slight and, after 12 weeks, erythromycin-resistant strains were predominant in both treatment groups. In vitro MIC determinations suggested that this finding might be explained by the exceptionally high degree of erythromycin resistance displayed by some staphylococcal strains (MIC > 4 mg/ml) and by the relative insensitivity of all staphylococcal strains to zinc acetate. Erythromycin with and without zinc was clinically effective, and both preparations produced significant reductions in acne grade, and inflamed and non-inflamed lesion counts (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Follicular concentrations of azelaic acid after a single topical application.

Follicular concentrations of azelaic acid (AzA) were determined in vivo using a rapid, non-invasive method, after a single topical application of 20% (w/w) AzA cream, in order to establish whether the in vitro antimicrobial effects observed in previous studies are relevant in vivo. Preweighed amounts of 20% (w/w) AzA cream were applied over demarcated areas on the forehead and back of nine young adults, and samples were taken over a period of 5 h. AzA was removed from the skin surface by washing with acetone, and follicular casts were collected using cyanacrylate gel. The samples were centrifuged to remove particulate matter, and the supernatants derivatized for analysis by HPLC. Although the results showed wide-ranging variability, the follicular concentration increased as the amount present on the surface declined. The maximum follicular concentrations of AzA attained ranged from 7.5 to 52.5 ng (micrograms of follicular casts)-1 and 0.5 to 23.4 ng (micrograms of follicular casts)-1 in samples taken from the back and forehead, respectively. Assuming an average density of follicular material of 0.9 g ml-1, the mean maximum follicular concentration attained on the back was between 36 and 251 mmol/l, and on the forehead was between 2 and 112 mmol/l, and indicates that the concentration of AzA attained in follicular casts after a single topical application is comparable with the concentration required to inhibit the growth of Propionibacterium acnes and Staphylococcus epidermidis, in vitro.

The effect of zinc and erythromycin on the growth of erythromycin-resistant and erythromycin-sensitive isolates of Propionibacterium acnes: an in-vitro study.

The effect of zinc and erythromycin on cultures inoculated with mixtures of different ratios of erythromycin sensitive (ES) and resistant (ER) Propionibacterium acnes cells was studied in vitro. Propionibacterium acnes ES outgrew P. acnes ER in the absence of erythromycin and zinc. At low levels of erythromycin ES outgrew ER, whilst the addition of 600 pg/ml zinc further reduced the growth of ER compared to ES. Growth of ER and ES were similar at levels of erythromycin near the minimum inhibitory concentration (MIC) of ES cells. Concentrations above the MIC for ES cells inhibited ES but not ER cells. At the higher concentrations of erythromycin, the addition of 96 ng/ml zinc delayed the growth of ER cells, whilst the addition of 300 micrograms/ml zinc prevented the growth of ER cells. The combination of erythromycin and zinc, at appropriate concentrations, inhibits both ES and ER.

The in-vitro antimicrobial effects of azelaic acid upon Propionibacterium acnes strain P37.

The in-vitro antimicrobial activity of azelaic acid a new topical acne treatment, upon Propionibacterium acnes strain P37 was studied. In phosphate buffer at pH 6.0 500 mM azelaic acid had bactericidal activity whilst the addition of nutrients reduced susceptibility. Bactericidal activity was greatly enhanced by reducing the pH to 5.6. In a simple defined medium growth was inhibited by 100 microM azelaic acid. The accumulation of 14C azelaic acid was pH and temperature dependent with maximum uptake occurring at pH 4.6, 30 degrees C. Valinomycin, nigericin and CCCP (membrane-active inhibitors of energy transduction) inhibited uptake and azelaic acid was not accumulated by non-viable cells. The degradation of azelaic acid was repressed by glucose, and acetic acid was the major end-product of azelaic acid degradation in glucose depleted media. The incorporation of radiolabelled precursors into protein, DNA and RNA were inhibited in a dose dependent manner, and 50% inhibition occurred at 313, 3639 and 9226 microM respectively. The synthesis of proteins was shown to be significantly more sensitive to the action of azelaic acid than both RNA and DNA synthesis.

Azelaic acid: its uptake and mode of action in Staphylococcus epidermidis NCTC 11047.

In vitro tests using Staphylococcus epidermidis as a model have shown that at pH 5.6 the micro-organisms are sensitive to azelaic acid, whilst at pH 6.0 and 7.0 the cells become progressively resistant, especially with nutrients present. In a simple defined medium the growth rate was reduced at 1 mmol/l and growth inhibited at 25 mmol/l. The uptake of azelaic acid was pH dependent, higher transport at lower pH values, and required viable cells. Azelaic acid, 457 mumol/l gave 50% inhibition of protein synthesis and this mechanism could account for the bactericidal and bacteristatic effects. DNA and RNA were affected slightly by 100 mmol/l azelaic acid, and respiration by 500 mmol/l.

The in vitro antimicrobial effect of azelaic acid.

Various strains of cutaneous micro-organisms were tested in vitro for their survival rates in 0.5 mol/l (8.4% w/v) azelaic acid solution. All bacterial strains exhibited large reductions in viability (at least 40-fold) over a 24 h test period, but little response was noted with Pityrosporum ovale. The bactericidal effect of azelaic acid was reduced considerably in the presence of nutrients. Minimum inhibitory concentrations (MICs) and minimum bactericidal (or fungicidal) concentrations (MBCs) were also determined. MICs varied from 0.03 mol/l to 0.25 mol/l; MBCs were all either 0.25 mol/l or greater.