Use of tissue-engineered skin to study in vitro biofilm development.

BACKGROUND: Biofilms are aggregations of microorganisms that have been identified as potential pathogens in the chronicity of nonhealing wounds. OBJECTIVE To develop an in vitro wound model to study biofilms using Graftskin, a tissue-engineered skin equivalent. MATERIALS AND METHODS: Graftskin constructs were divided into sections, and wounds were created on each section. Bacterial suspensions with a concentration of 10(6) CFU/mL were prepared from cultures of pathogenic isolates of Pseudomonas aeruginosa and Staphylococcus aureus. A 25-microL aliquot of each suspension was deposited in the center of wounds created on the Graftskin. Sections were incubated at various time points, and a biopsy was then taken from the wounded and inoculated area. Sections were visualized with light (hematoxylin and eosin) and epifluorescent microscopy (calcofluor white and ethidium bromide). RESULTS Biofilm was observed on the wound model. Biofilm formation was dependent on time of Graftskin exposure to the bacteria. Biofilm was visualized in the S. aureus group at an earlier time point than in the P. aeruginosa group. CONCLUSIONS: We demonstrated biofilm formation in vitro using a wound model. This model may provide a basis on which future studies may explore therapeutic modalities to prevent and eradicate pathogenic bacterial biofilm. The authors have indicated no significant interest with commercial supporters.

Tumor necrosis factor-alfa in nonhealing venous leg ulcers.

BACKGROUND: Venous leg ulcers are responsible for more than half of all lower extremity ulcerations, affecting more than one million Americans annually. Studies have demonstrated alterations in levels of proinflammatory cytokines in patients with chronic wounds, including tumor necrosis factor-alfa (TNFalpha), which may be implicated in wound chronicity. OBJECTIVE: To test the hypothesis that recalcitrant venous leg ulcers have increased local tissue TNFalpha as compared to normal skin. METHODS: Five patients with nonhealing healing chronic venous leg ulcers were recruited. Two 4-mm punch biopsy specimens were obtained: one from the wound margin and one from noninvolved, non-sun exposed normal skin on the flexor aspect of the forearm. Tissue samples were processed using fixed with formalin stained by immunohistochemistry for TNFalpha. Qualitative and quantitative comparisons were made for the presence of TNFalpha receptor in all tissue samples, specifically comparing the presence of TNFalpha in nonhealing venous leg ulcer samples versus normal skin. RESULTS: The overall staining score for nonhealing venous leg ulcers was significantly higher compared to respective normal skin samples (P = .01). In addition, immunostaining for TNFalpha was significantly less in the two nonhealing venous leg ulcers that were present for the shortest duration compared to the other ulcers of longer duration (P = .048). LIMITATIONS: The small sample size may mitigate the clinical implications of findings. CONCLUSIONS: Increased levels of TNFalpha in nonhealing venous leg ulcers, especially those of longer duration, implies that excessive inflammation may be causal in wound chronicity and suggests potential therapeutic alternatives.

A gene signature of nonhealing venous ulcers: potential diagnostic markers.

BACKGROUND: Venous leg ulcers are responsible for more than half of all lower extremity ulcerations. Significant interest has been focused on understanding the physiologic basis on which patients fail to heal with standard therapy. OBJECTIVE: This study uses complementary DNA microarray analysis of tissue samples from healing and nonhealing venous leg ulcers to identify the genetic expression profiles from these dichotomous populations. METHODS: Ulcer size and chronicity, factors that have been identified as prognostic indicators for healing, were used to distribute venous leg ulcers as healing versus nonhealing. Punch biopsy samples were obtained from the wound edge and wound bed of all venous leg ulcers. The top 15 genes with differential expression greater than 2-fold between the two populations of wounds (P < .05) were reported. RESULTS: Significant differences were demonstrated in the expression of a diverse collection of genes, with particular differences demonstrated by genes coding for structural epidermal proteins, genes associated with hyperproliferation and tissue injury, and transcription factors. LIMITATIONS: Small sample size may mitigate potential clinical implications of findings. CONCLUSIONS: The genetic expression profiles displayed here may have implications for the development of novel therapies for chronic venous leg ulcers, and may also serve as prognostic indicators for wound healing.

A rare presentation of squamous cell carcinoma in a patient with PIBIDS-type trichothiodystrophy.

The clinical presentation of trichothiodystrophy type F includes photosensitivity, ichthyosis, brittle hair, intellectual impairment, decreased fertility, and short stature, often referred to as the PIBIDS syndrome. While many of these patients demonstrate features also found in xeroderma pigmentosum patients, including similar nucleotide excision repair gene defects and photosensitivity, PIBIDS patients rarely demonstrate cutaneous malignancies. This case report demonstrates the rare presentation of squamous cell carcinoma developing in a PIBIDS patient.

Etanercept for the treatment of refractory pyoderma gangrenosum: a brief series.

BACKGROUND: Pyoderma gangrenosum (PG) is a rare ulcerative inflammatory condition of unknown etiology. Therapy for PG involves local wound care along with topical and systemic anti-inflammatory and other immunodulatory agents. Etanercept is one such immunomodulator with activity against the inflammatory cytokine, tumor necrosis factor. OBJECTIVE: To evaluate the efficacy and safety of etanercept in the treatment of PG ulcers. METHODS: A retrospective analysis was performed on seven patients with 11 refractory PG ulcers treated with subcutaneous injections of etanercept (25-50 mg twice weekly). RESULTS: All seven patients with PG responded well to etanercept. Eight of the 11 ulcers (73%) completely healed with the mean time of (12.5 weeks), while the other three ulcers had marked reduction in size (within 8-18 weeks). Etanercept was well tolerated. No serious side-effects were reported. Only one patient discontinued the drug secondary to side-effects. CONCLUSION: Etanercept is an alternative treatment option for patients with refractory ulcers due to PG.

In vivo confocal scanning laser microscopy of a series of congenital melanocytic nevi suggestive of having developed malignant melanoma.

OBJECTIVE: To determine the utility of confocal scanning laser microscopy (CSLM) in the in vivo evaluation of congenital melanocytic nevi (CMNs) that are suggestive of having developed melanoma. DESIGN: The CMNs suggestive of melanoma by clinical and dermoscopic examination were imaged by CSLM, and the findings correlated with the features seen on dermoscopic and histologic examination. SETTING: Dermatology clinic specializing in pigmented lesions. PATIENTS: Seven patients with clinically irregular small to medium CMNs. INTERVENTIONS: The areas imaged by CSLM were sampled with 3-mm punch biopsy specimens. The entire lesion was subsequently excised. The punch biopsy specimens were step sectioned horizontally to correlate with the CSLM images. Excised samples were step sectioned and processed routinely. Histologic features observed on CSLM were correlated with the features seen on dermoscopic and light microscopic examination. MAIN OUTCOME MEASURE: Correlation of the structures seen using CSLM with the dermoscopic and histologic features of CMNs and melanoma. RESULTS: The CSLM illustrated histologic characteristics of CMNs, including the presence of hyperpigmented keratinocytes, nevus cells, melanophages, and a normal "honeycomb" epidermal architecture. Features suggestive of melanoma were not evident by CSLM in 6 histologically proven benign CMNs. Histologic features associated with melanoma, such as an increased number of intraepidermal atypical melanocytes (pagetoid) and loss of normal epidermal cellular architecture, were identified by CSLM in 1 lesion, which on histologic analysis revealed melanoma in association with a CMN. CONCLUSION: Our results illustrate that CSLM may be useful for clinicopathologic correlations and for the preliminary noninvasive diagnosis of pigmented neoplasms in vivo.

Variation in the diagnosis, treatment, and management of melanoma in situ: a survey of US dermatologists.

OBJECTIVE: To assess current practices of US dermatologists regarding the diagnosis, treatment, and management of melanoma in situ (MIS). DESIGN: Survey. PARTICIPANTS: A total of 1200 dermatologists randomly selected from the American Board of Medical Specialists Directory of Board Certified Medical Specialists. MAIN OUTCOME MEASURES: Results based on 597 questionnaires returned. RESULTS: The overall response rate was 63% (597 of 945 eligible participants). To aid in clinical assessment, respondents reported using a magnifying lens (57.4%) and dermoscopy (17.4%). Most dermatologists preferred excisional and saucerization biopsies as the method of choice for sampling. A large percentage of physicians (78.9%) preferentially used dermatopathologists for the evaluation of the majority of pigmented lesions. Although most respondents would not unquestioningly accept a benign pathology diagnosis when there was a clinical suspicion of MIS, 16.1% would accept a pathologist's diagnosis without further action. There was no consensus on the appropriate surgical margins or depth of excision for MIS. Of the respondents who characterized MIS as premalignant and malignant, 63.2% and 46.4%, respectively, did not know what percentage of MISs would progress to metastatic disease if left untreated. CONCLUSIONS: Considerable variability exists in the clinical concept and management of MIS. Dermoscopy is underutilized. The true nature of the evolution of MIS is unknown. Surgical margins and depth of excision need to be standardized to help dermatologists manage disease. Further research in the specific area of MIS is warranted to develop clear guidelines in the management and prevention of further disease.

Use of a living dermal equivalent for a refractory abdominal defect after pediatric multivisceral transplantation.

BACKGROUND: Primary closure is not always possible after pediatric multivisceral transplantation. Reepithelialization may require extended periods of postoperative time, which can be associated with significant morbidity. OBJECTIVE: The objective was to accelerate secondary wound closure thereby minimizing infection or further complications in a pediatric multivisceral transplant patient. METHODS: Five applications of human fibroblast-derived dermis (Dermagraft, Smith and Nephew) were applied to the postsurgical defect of a pediatric multivisceral transplant patient over the course of 8 months. Routine wound care and observation was performed between human fibroblast-derived dermis applications. RESULTS: Human fibroblast-derived dermis stimulated healing and accelerated reepithelialization. Signs of clinical rejection or infection were not observed. CONCLUSION: Reepithelialization can be aided in the postoperative period in pediatric multivisceral transplant patients with human fibroblast-derived dermis, thereby helping to deter complications associated with secondary wound closure. We have illustrated the successful use of a human fibroblast-derived dermis as an adjunct for wound healing in a complicated surgical defect.

Poor prognosis of arthritis-associated pyoderma gangrenosum.

BACKGROUND: The association between pyoderma gangrenosum (PG) and arthritis is well established. We have observed a refractory population of patients with arthritis-associated PG (PGA). We, therefore, tested the hypothesis that differences exist in response to treatment in patients with PGA compared with patients with PG without arthritis. OBSERVATIONS: We performed a review of patients with PG during a 2-year period. Patients had noninfectious chronic ulcerations clinically typical for PG, exclusion of relevant differential diagnoses, and consistent histopathological features. Outcomes compared between patients with arthritis (PGA) and without arthritis (PG) included complete healing, percentage change in wound size, and duration of therapy. Of 10 PG ulcers, 7 healed, compared with 2 of 8 PGA ulcers. There was a greater mean percentage decrease in wound size in the PG vs the PGA ulcers (78.9% vs 23.4%; P =.10) and a shorter mean duration of treatment (8.7 vs 14.8 months; P =.18). CONCLUSIONS: The ulcers of patients with PGA seem more refractory to treatment than the ulcers of patients with PG alone. Those with PGA ulcers represent a refractory subset of patients, and the ulcers are possibly secondary to unique pathophysiological features.

Diagnostic accuracy of patients in performing skin self-examination and the impact of photography.

OBJECTIVE: To determine the sensitivity and specificity of skin self-examination (SSE) to detect new and changing moles with and without the aid of baseline digital photographs in patients with dysplastic nevi. DESIGN AND INTERVENTION: Patients had baseline digital photography and mole counts of their back, chest, and abdomen and were instructed to perform a baseline SSE. Print copies of the images were provided to the patient. Following the baseline examination, the appearance of existing moles was altered and new moles were created using cosmetic eyeliner. The number of moles altered and/or created totaled approximately 10% of each patients' absolute mole count. SETTING AND PATIENTS: Fifty patients with 5 or more dysplastic nevi from the outpatient clinic at Memorial Sloan-Kettering Cancer Center, New York, NY. MAIN OUTCOME MEASURE: Skin self-examinations with and without access to the baseline photographs to identify the number of new and altered moles. RESULTS: The sensitivity and specificity of SSE for detection of both altered and new moles without photography were 60.2% and 96.2%, respectively. Skin self-examination with photography yielded a sensitivity and specificity of 72.4% and 98.4%, respectively. The findings were similar when stratified by site (back vs chest or abdomen). The sensitivity and specificity for new moles were higher compared with altered moles. CONCLUSIONS: Access to baseline photography improved the diagnostic accuracy of SSE on the back and chest or abdomen and improved detection of changing and new moles. Our results suggest that baseline digital photography in tandem with SSE may be effective in improving the diagnostic accuracy of patients performing SSE.

Melanoma or pigmented basal cell carcinoma: a clinical-pathologic correlation with dermoscopy, in vivo confocal scanning laser microscopy, and routine histology.

BACKGROUND/PURPOSE: New techniques are being explored for improving diagnostic accuracy of pigmented skin lesions. Confocal scanning laser microscopy (CSLM) may represent such a novel technique. The purpose of this report was to demonstrate the potential application of CSLM as an aid in the diagnosis of a pigmented skin lesion that is clinically suspicious for melanoma. METHODS: An irregular pigmented lesion was examined clinically and dermoscopically. The lesion was imaged by CSLM and subsequently excised for histologic examination. Findings from CSLM were correlated with features observed on the dermoscopic and histologic examination. RESULTS: Confocal scanning laser microscopy (CSLM) allowed for the non-invasive visualization of the histologic features of superficial pigmented BCC, including buds and "islands" of tumor cells at the dermoepidermal junction and melanin-laden macrophages. Conventional histology confirmed the diagnosis of pigmented BCC. CONCLUSION: Confocal scanning laser microscopy (CSLM) may serve as an aid in the non-invasive diagnosis of pigmented skin lesions clinically suspicious for melanoma.