Carlos A. Charles

Morphologic Features of Melanocytes, Pigmented Keratinocytes, and Melanophages by In Vivo Confocal Scanning Laser Microscopy

Confocal scanning laser microscopy (CSLM) represents a novel imaging technique for in vivo microscopic analysis of skin lesions at a level of resolution that allows morphologic analysis of microanatomic structures. We investigated the feasibility of recognizing the cellular constituents of pigmented skin lesions, such as pigmented keratinocytes, melanocytes, and melanophages, by CSLM. Fifteen pigmented lesions (five pigmented seborrheic keratoses, and 10 compound melanocytic nevi) from 15 patients were studied, as well as normal skin. After the clinical lesions were imaged by CSLM, they were biopsied or excised for examination by conventional histology for comparison of the morphologic features. In images obtained by CSLM, pigmented keratinocytes were seen as polygonal cohesive cells with variably bright granular cytoplasm. Melanocytes appeared as bright round, oval, fusiform, or dendritic cells. The architectural growth pattern of melanocytes could be analyzed. Melanocytes were identified by their nested growth pattern as aggregates of bright round to oval structures at the dermoepidermal junction or in the superficial dermis. Melanocytes were also recognizable as single cells along the dermoepidermal junction, usually separated from each other by a variable number of keratinocytes. Melanophages appeared as large bright plump cells with ill-defined cytoplasmic borders, usually located around or near vessels of the superficial dermis. Our results demonstrate that the cellular constituents of pigmented lesions can be recognized by CSLM. This technique sets a new paradigm for noninvasive quasihistologic examination of pigmented lesions in vivo and merits further evaluation for diagnostic use.

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.

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.

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.

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.

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 106 CFU/mL were prepared from cultures of pathogenic isolates of Pseudomonas aeruginosa and Staphylococcus aureus. A 25‐μL 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.

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

Abstract:  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.