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.

Antibacterial activity and mode of action of tert-butylhydroquinone (TBHQ) and its oxidation product, tert-butylbenzoquinone (TBBQ).

OBJECTIVES: The antioxidant tert-butylhydroquinone (TBHQ) is a food additive reported to have antibacterial activity, and may therefore have application in the healthcare setting. This study sought to characterize the antibacterial activity and mode of action of TBHQ and its oxidation product, tert-butylbenzoquinone (TBBQ). METHODS: The stability of TBHQ/TBBQ was studied in buffer. Susceptibility testing was performed by broth microdilution, and killing and lytic activity were evaluated by viable counting and culture turbidity measurements. Mode of action studies included following the incorporation of radiolabelled precursors into macromolecules. The effect of TBHQ/TBBQ upon bacterial and mammalian membranes was assessed using the BacLight(TM) assay and by monitoring the haemolysis of equine erythrocytes. RESULTS: TBHQ underwent oxidation in solution to form TBBQ. When oxidation was prevented, TBHQ lacked useful antibacterial activity, indicating that TBBQ is responsible for the antibacterial activity attributed to TBHQ. TBBQ demonstrated activity against Staphylococcus aureus SH1000 (MIC 8 mg/L) and against a panel of clinical S. aureus isolates (MIC90 16 mg/L). TBBQ at 4× MIC caused a >4 log10 drop in cell viability within 6 h without lysis, and eradicated staphylococcal biofilms at 8× MIC. TBBQ did not display preferential inhibition of any single macromolecular synthetic pathway, but caused loss of staphylococcal membrane integrity without haemolytic activity. CONCLUSIONS: TBBQ is responsible for the antibacterial activity previously ascribed to TBHQ. TBBQ prompts loss of staphylococcal membrane integrity; it is rapidly and extensively bactericidal, but is non-lytic. In view of the potent and selective bactericidal activity of TBBQ, this compound warrants further investigation as a candidate antistaphylococcal agent.

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.

An observer-blind parallel-group, randomized, multicentre clinical and microbiological study of a topical clindamycin/zinc gel and a topical clindamycin lotion in patients with mild/moderate acne.

INTRODUCTION: A topical acne treatment in which clindamycin phosphate equivalent to 1% clindamycin is presented in a gel formulation has received marketing authorizations in a number of EU and non-EU countries. Clindamycin/zinc gel contains zinc acetate in a formulation that reduces systemic absorption of clindamycin through the skin. OBJECTIVES: To compare the efficacy and safety of a 1% clindamycin/zinc gel when applied to the face once daily or twice daily with a 1% clindamycin lotion applied twice daily for 16 weeks in patients with mild to moderate acne vulgaris. METHODS: This was a randomized, comparative, observer-blind, parallel-group, multicentre study involving 246 acne patients. RESULTS: The study demonstrated therapeutic similarity between clindamycin/zinc gel applied once and twice daily with clindamycin lotion applied twice daily. All three regimens produced a gradual and time-dependent reduction in inflamed lesions, non-inflamed lesions and overall grade. Side effects were similar and minimal, consisting predominantly of mild irritant dermatitis. All regimes produced a time-related significant reduction in skin surface and follicular Propionibacterium spp. and Micrococcaceae. The emergence of resistant strains was less than 5% and was similar with all three regimes. CONCLUSION: The equivalent efficacy and safety of clindamycin/zinc gel either once or twice daily to clindamycin lotion twice daily has been demonstrated. It is suggested that a treatment regime of one application per day may significantly enhance compliance and thus treatment success in acne patients.

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.

Assessment of the biological properties of human split skin allografts disinfected with peracetic acid and preserved in glycerol.

Skin allografts derived from cadaveric human donors are widely used in the treatment of serious burn injuries and other conditions, such as ulcers. In order to render these allografts safe for clinical use, and to enable them to be preserved and banked for long periods, effective methods of decontamination and preservation are required. These methods must not adversely affect graft properties essential for clinical performance. We have investigated the application of a peracetic acid (PAA) disinfection protocol, coupled with preservation in either glycerol or propylene glycol to achieve these goals. An effective decontamination procedure, comprising of a 3h exposure to 0.1% (v/v) PAA in phosphate buffered saline (PBS) at pH 7.0, was developed and had no significant detrimental effects on the structure of skin. Cadaveric skin allografts were then treated with this disinfection protocol and subsequently preserved in either 85% (v/v) glycerol or propylene glycol in PBS, and the biological properties of the allografts thought to be essential to successful clinical performance were assessed. The cytotoxicity of the grafts was assessed using both extract and contact assays; damage to the skin collagen was assessed using a collagenase susceptibility assay and the capacity of the grafts to elicit an inflammatory response in vitro was assessed by quantifying the production of the pro-inflammatory cytokine TNF-alpha by human peripheral blood mononuclear phagocytes. Neither the disinfection protocol nor either of the preservation techniques rendered the grafts cytotoxic or pro-inflammatory. The PAA disinfection and glycerol preservation protocol had no effects on collagenase susceptibility, whereas the disinfection protocol in combination with propylene glycol rendered some of the test samples significantly more susceptible to collagenase digestion. Therefore, this study has demonstrated that PAA disinfection combined with glycerol preservation is suitable for skin allografts. The use of propylene glycol as a preservation agent for skin requires further development.

Clinical benefits of endoscopic vein harvesting in patients with risk factors for saphenectomy wound infections undergoing coronary artery bypass grafting.

OBJECTIVE: The influence of endoscopic harvesting techniques on the prevalence of leg-wound complications after coronary artery bypass grafting remains to be defined for patients at high risk for the development of wound infections. METHODS: Among 1473 patients undergoing coronary artery bypass grafting who had the saphenous vein harvested by either a continuous incision or skip incisions leaving intact skin bridges, we determined the prevalence of wound infections to be 9.6%. The following variables were entered into logistic regression analysis to identify significant risk factors that might be predictive of wound infection: diabetes, peripheral vascular disease, obesity, renal failure, steroid use, age, sex, and type of closure. We then prospectively randomized 132 patients found to be at high risk of wound infection to either endoscopic vein harvesting or a continuous open incision. RESULTS: Univariate analysis showed female sex (P =.04), diabetes (P <.001), and obesity (P <.001) to be predictors of wound infection. In a multivariate model diabetes (P =.02) and obesity (P =.001) were independent predictors. In patients at high risk, the prevalence of wound infection was 4.5% for the endoscopic group versus 20% for the open group (P =.01). Vein procurement time was greater in the endoscopic group (65 minutes vs 32 minutes, P <.001), as was the number of vein repairs required (2.5 vs 0.6, P <.001). CONCLUSION: The use of endoscopic vein harvesting decreases the prevalence of postoperative leg-wound infections in high-risk patients with diabetes and obesity. Whether this translates into an economic benefit that justifies the additional cost of that technology requires further analysis.

Homograft repair of a tuberculous pseudoaneurysm of the ascending aorta.

A 34-year-old HIV-positive black man with pulmonary tuberculosis developed progressive widening of the mediastinum. Evaluation by CT and two-dimensional echocardiography demonstrated a large saccular aneurysm of the ascending aorta. This was repaired with a 26-mm aortic homograft using deep hypothermic circulatory arrest. The microbiology of the aneurysm contents showed this to be a tuberculous pseudoaneurysm.

Ruptured sinus of Valsalva aneurysm simulating endocarditis.

A previously healthy, 31-year-old man was evaluated in the emergency department after being violently assaulted. A harsh, continuous murmur was noted on physical examination. Transthoracic and transesophageal echocardiograms were interpreted as showing a ruptured sinus of Valsalva aneurysm with a shunt into the right atrium and a tricuspid valve vegetation. The patient was treated with antibiotics for presumed endocarditis. Subsequent echocardiographic and surgical evaluation showed no evidence of past or present endocarditis. Rather, the sinus of Valsalva aneurysm and rupture gave the appearance of a valvular mass. This report shows some of the potential pitfalls in the delineation of abnormalities related to sinus of Valsalva aneurysms and rupture.

Treatment of multiple cerebral emboli from an ulcerated, thrombogenic ascending aorta with aortectomy and graft replacement.

Multiple studies link aortic arch atheroma as a donor site for cerebral emboli. However, treatment of this condition remains controversial. We report a patient with recurrent brain emboli from an ulcerated, thrombogenic aorta treated with aortectomy and graft replacement.

Investigation of the mechanism of action of 2% fusidic acid lotion in the treatment of acne vulgaris.

We describe the results of a single-centre, double-blind, vehicle-controlled, parallel group study on the quantitative effects of 2% fusidic acid lotion (Fucidin lotion) in facial acne vulgaris. The trial was completed by 52 patients aged 15-25 years with mild to moderate acne who had been randomized to either Fucidin Lotion (n = 25) or its base (n = 27). Primary outcome measures included colony counts of Propionibacterium acnes and micrococcaceae and measurements of skin surface lipid free fatty acids and sebum excretion rate. Clinical assessment was based on the acne grade, count of inflamed and non-inflamed lesions and evidence of a primary irritant dermatitis. There was a variable but gradual reduction in lesion counts with the maximum improvement at 12 weeks for inflamed lesions, where the reduction was 19.9% for fusidic acid and 24.7% for the placebo. The non-inflamed lesions decreased by 10.8% in the fusidic acid group and increased by 15.9% in the placebo group; this difference was not statistically significant. Although the fusidic acid reduced the micrococcaceae count by 1 log cycle, inferring adequate compliance, there was no reduction in the counts of P. acnes, surface free fatty acids or sebum excretion rate. This study has failed to explain the mechanism of action of topical fusidic acid.

Simultaneous carotid endarterectomy and coronary bypass: perioperative risk and long-term survival.

PURPOSE: The purpose of this article is to examine the outcome of simultaneous coronary bypass-carotid endarterectomy (CABG-CEA) and to compare it with the outcome of endarterectomy alone (CEA alone) in patients at high cardiac risk. METHODS: A retrospective review of the records and follow-up data for 100 consecutive patients who had undergone CABG-CEA and were at high risk and 114 patients who had undergone CEA, had overt coronary artery disease (angina, previous infarct, or ischemic electrocardiographic abnormalities), but had not undergone CABG was carried out. RESULTS: Our CABG-CEA group had a high incidence of symptomatic carotid disease (57%) and contralateral occlusion (28%) when compared with patients in other reports. Patients in the CABG-CEA group were older (67.9 +/- 8.3 years vs 63.6 +/- 15.7 years, p = 0.01) and more often smokers (81% vs 52.6%, p = 0.01) than patients in the CEA alone group. Perioperative mortality was 8% for the CEA-CABG group and for 1.8% for the CEA alone group (p = 0.035). Perioperative stroke morbidity was 9% for the CEA-CABG group and 2.6% for the CEA alone group (p = 0.05). Life table survival at 1,3, and 5 years was 90%, 82%, and 73% versus 96%, 84%, and 76% for the CABG-CEA and CEA alone groups, respectively (p = 0.30). CONCLUSIONS: Selection criteria for CABG-CEA greatly influence perioperative risk. Despite the greater age and more advanced coronary artery disease in the CABG-CEA group, long-term outcome differences are accounted for entirely by differences in perioperative morbidity and mortality. Prospective trials of strategies such as staged CEA and CABG to reduce perioperative risk are needed.

Surgical treatment of systemic atheroembolism from the thoracic aorta.

BACKGROUND: Surgical procedures performed exclusively for atheroembolic events arising from the thoracic aorta rarely have been reported. Presented here are 2 patients who underwent successful operation for these problems. METHODS: The clinical presentation, diagnostic evaluation and surgical approach to 2 patients with different embolic sources in the thoracic aorta are presented. One patient had experienced three strokes and was noted by multiplane transesophageal echocardiography to have protruding atheromas with ulcerations in the transverse arch and origin of the brachiocephalic vessels. The transverse arch was replaced using hypothermic circulatory arrest with individual reimplantation of the brachiocephalic vessels. The second patient presented with "blue toe" syndrome from mobile atheromas in the mid-descending thoracic aorta defined by transesophageal echocardiography. A localized debridement was performed using simple aortic cross-clamping. RESULTS: Both patients had uneventful postoperative courses and had no further atheroembolic events. CONCLUSIONS: When standard diagnostic modalities do not delineate an embolic source for either stroke or peripheral embolization, transesophageal echocardiography is recommended as an excellent means of identifying atheromas in the thoracic aorta that could be the source for emboli. Once these lesions are identified, a surgical procedure should be performed to prevent further embolization.