Collision of desmoplastic-neurotropic melanoma and squamous cell carcinoma on the lip.

We report on a case of the collision of a desmoplastic-neurotropic melanoma and a squamous cell carcinoma on the lip. A 46-year-old male developed a multifocal infiltrative squamous cell carcinoma of the lower lip, which also showed sparse melanocyte atypia within the epidermis and an extensive spindle cell proliferation within the dermis, subcutaneous tissues and nerves. An immunohistochemical panel showed that the spindle cells were melanocytes, not derived from the squamous cell carcinoma. Double labeling with AE1/AE3 and S100 showed striking localized proximity of the spindle-cell melanocytic and keratinocyte components in some areas of this tumor. To our knowledge, this is the first report of the collision of a squamous cell carcinoma and desmoplastic-neurotropic melanoma.

Fibroblasts from non-healing human chronic wounds show decreased expression of beta ig-h3, a TGF-beta inducible protein.

BACKGROUND: Increasing evidence shows persistent phenotypic alterations in fibroblasts from non-healing human chronic wounds, which may result in faulty extracellular matrix deposition and keratinocyte migration. We have previously shown that these cells are characterized by morphological changes, low proliferative potential and unresponsiveness to TGF-beta1, and down regulated phosphorylation of Smad 2/3 and p42/44 MAPK from decreased expression of the TGF-beta type II receptor. OBJECTIVE: To identify genes and proteins that may be differentially expressed in chronic wounds and their cultured fibroblasts. METHODS: Differential display analysis with 120 random primer sets was used in fibroblasts from human venous ulcers and acute wounds created on the ipsilateral thighs of the same patients. Positive differential results were confirmed by RT-PCR. Immunohistochemistry of cultured fibroblasts and tissues was used to determine the expression of differentially expressed proteins. RESULTS: A total of 16 differentially expressed genes were identified and cloned. The only candidate gene that was differentially expressed in all patients and confirmed by repeated differential display testing and RT-PCR was beta ig-h3, a TGF-beta-induced gene involved in cell adhesion, migration, and proliferation. Decreased expression of beta ig-h3 in chronic wounds and their fibroblasts was further confirmed by Western blot and immunostaining. CONCLUSION: These findings point to beta ig-h3 as an important gene characterizing the abnormal phenotype of chronic wound fibroblasts. Corrective measures to increase the expression of this protein might have therapeutic potential.

Migration of the epidermal over the dermal component (epiboly) in a bilayered bioengineered skin construct.

A bilayered bioengineered living skin construct (LSC) consisting of viable human neonatal keratinocytes over a collagenous dermis seeded with dermal fibroblasts has been used extensively in difficult-to heal human wounds. Its biological properties include production of several mediators, cytokines, and growth factors and the ability to heal itself upon injury. In this study, we investigated the process of keratinocyte migration in LSC. At baseline, 6-mm punch biopsies of the construct were placed in serum-free medium (AIM-V) or Dulbecco's modified Eagle medium. At varying time points, the LSC samples were processed and analyzed using histology and immunohistochemistry. By 72 h, in a time-dependent manner, the overlying epidermis had migrated over and enveloped the entire underlying dermis, a process known as epiboly. Increasing concentrations of neutralizing antibodies to epidermal growth factor or interleukin-1 alpha down-regulated the extent of epiboly, as measured using computerized planimetry, but antibodies to transforming growth factor-beta 1 did not affect it. The consistent expression of laminin V, alpha3beta1 integrin, and vitronectin (epibolin) and its integrin receptor (alphavbeta5) characterized the tongue of migrating epidermis. Increasing concentrations of antibodies to vitronectin blocked the process of epiboly, as did antibodies to the alphavbeta5 integrin receptor, which mediates vitronectin-driven keratinocyte locomotion. This process of epiboly provides novel mechanisms of action for bioengineered skin constructs.

Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds.

The nonhematopoietic component of bone marrow includes multipotent mesenchymal stem cells (MSC) capable of differentiating into fat, bone, muscle, cartilage, and endothelium. In this report, we describe the cell culture and characterization, delivery system, and successful use of topically applied autologous MSC to accelerate the healing of human and experimental murine wounds. A single bone marrow aspirate of 35-50 mL was obtained from patients with acute wounds (n = 5) from skin cancer surgery and from patients with chronic, long-standing, nonhealing lower extremity wounds (n = 8). Cells were grown in vitro under conditions favoring the propagation of MSC, and flow cytometry and immunostaining showed a profile (CD29+, CD44+, CD105+, CD166+, CD34-, CD45-) highly consistent with published reports of human MSC. Functional induction studies confirmed that the MSC could differentiate into bone, cartilage, and adipose tissue. The cultured autologous MSC were applied up to four times to the wounds using a fibrin polymer spray system with a double-barreled syringe. Both fibrinogen (containing the MSC) and thrombin were diluted to optimally deliver a polymerized gel that immediately adhered to the wound, without run-off, and yet allowing the MSC to remain viable and migrate from the gel. Sequential adjacent sections from biopsy specimens of the wound bed after MSC application showed elongated spindle cells, similar to their in vitro counterparts, which immunostained for MSC markers. Generation of new elastic fibers was evident by both special stains and antibodies to human elastin. The application of cultured cells was safe, without treatment-related adverse events. A strong direct correlation was found between the number of cells applied (greater than 1 x 10(6) cells per cm2 of wound area) and the subsequent decrease in chronic wound size (p = 0.0058). Topical application of autologous MSC also stimulated closure of full-thickness wounds in diabetic mice (db/db). Tracking of green fluorescent protein (GFP)+ MSC in mouse wounds showed GFP+ blood vessels, suggesting that the applied cells may persist as well as act to stimulate the wound repair process. These findings indicate that autologous bone marrow-derived MSC can be safely and effectively delivered to wounds using a fibrin spray system.

Full-thickness wounding of the mouse tail as a model for delayed wound healing: accelerated wound closure in Smad3 knock-out mice.

Experimentally induced wounds in animal models are useful in gaining a better understanding of the cellular and molecular processes of wound healing, and in the initial evaluation of the safety and effectiveness of potential therapeutic agents. However, studying delayed healing has proved difficult in animals, whose wounds heal within a few days. In this report, we describe a novel method for establishing mouse wounds that require up to 3 weeks or more for complete closure, and we show the validity of this model in Smad3 null mice, which are known to display accelerated healing. Full-thickness wounds, measuring 0.3 by 1.0 cm, were made down to fascia on the dorsal aspect of the mouse tail in Smad3 knock-out mice and control littermates, approximately 1 cm distal to the body of the animal. The wounds were left to heal by secondary intention and were assessed histologically by computerized planimetry for wound closure at various times after wounding. The wounds in wild-type mice displayed delayed healing, with full closure occurring between 14 and 25 days after wounding. Complete closure of similar wounds in Smad3 null mice healed 30 percent faster (p < 0.01). By immunostaining for ki67, a marker for proliferation, Smad3 null animals also showed increased proliferation of dermal wound cells by day 4 after wounding. Cultured dermal fibroblasts from Smad3 null mice had increased baseline DNA synthesis and, interestingly, an enhanced response to transforming growth factor-beta1. By Western blot analysis, Smad3 null mice fibroblasts showed a compensatory increase in mitogen-activated protein kinase phosphorylation in response to transforming growth factor-beta1, suggesting that mitogen-activated protein kinase overcompensation together with loss of Smad3 may be involved in the modulation of faster healing. We conclude that this novel tail-wounding model may be useful for studying delayed wound closure.

Human beta-defensin-2 expression is increased in chronic wounds.

First identified in psoriatic epidermis and subsequently in other inflammatory cutaneous lesions, human beta-defensin-2 (hbetaD-2) is one of two endogenous antimicrobial peptides related to defensins in plants and animals. Our objective was to determine the expression of hbetaD-2 after injury and in chronic wounds. Biopsies of normal ipsilateral thigh skin and wound edges were taken from nine consecutive patients with venous leg ulcers (day 1) and from the same biopsy sites 2 days later (day 3). Sequential samples were also obtained from intact or meshed bilayered bioengineered skin consisting of neonatal human keratinocytes and dermal fibroblasts in a collagen matrix. Specimens were processed and immunostained for hbetaD-2 using a polyclonal rabbit antibody. In both human tissues and bioengineered skin, staining for hbetaD-2 was confined to the upper epidermal layers, sparing the basal cells. Analysis of 26 tissue samples from patients showed that normal skin had no hbetaD-2 expression but that marked up-regulation occurred after wounding by day 3. Conversely, chronic ulcers showed moderate-to-strong immunostaining for hbetaD-2 at baseline on day 1, with little or no change in intensity after wounding by day 3. In vitro, bioengineered skin showed increased distribution of cytoplasmic hbetaD-2 immunostaining after meshing. We conclude that the expression of hbetaD-2 is up-regulated after injury. Chronic wounds uniformly show a constitutively high baseline expression of hbetaD-2, possibly due to ongoing tissue injury and bacterial colonization.

Significance of N-methyl-D-aspartate (NMDA) receptor-mediated signaling in human keratinocytes.

Increasing data suggest that glutamate might act as a cell-signaling molecule in non-neuronal tissues such as the skin. Here we demonstrate the presence of functional N-methyl-D-aspartate (NMDA)-type glutamate receptors in human keratinocytes. NMDA receptor expression strongly reflects the degree of cell-to-cell contact. Wounding polarizes the expression of NMDA receptors in keratinocytes involved in re-epithelialization, and the process of re-epithelialization is inhibited by NMDA receptor activation. We also demonstrate that squamous cell carcinomas lack NMDA receptors. Our data suggest that Ca2+ entry through NMDA receptors influences the cycle of keratinocyte proliferation, differentiation, and migration during epithelialization. Moreover, NMDA receptor activation might play a role in contact-mediated inhibition of growth, a process that is absent during neoplastic pathology. This receptor may serve as a pharmacological target for modulating keratinocyte behavior and treating cutaneous disorders.

Histologic progression of B16 F10 metastatic melanoma in C57BL/6 mice over a six week time period: distant metastases before local growth.

B16F10 murine metastatic melanoma in the tails of C57BL/6 mice after subcutaneous injection is a well-established model. However, the histologic progression from injected cells to established local growth of melanoma has not been studied systematically. We therefore have investigated the histologic changes and growth of B16F10 melanoma at the injection site over a six-week time period. One million B16F10 melanoma cells were injected subcutaneously into the dorsal aspect of tails of C57/BL6 mice. Mice were sacrificed at zero, 12, 24, 48, 72 and 96 hours, and at one, two, three, four, five and six weeks. Sections were stained with Hematoxylin and Eosin and immunostained with antibodies to S100. Beginning at time zero, melanoma cells were detected between the dermis and the myofascial bundle of the tail. At week four, distant metastases were clinically evident in the inguinal region, though injection site tumors did not become evident until week six. Histological analysis showed melanoma cells at the injection site at all time periods and no injection site tumor until week six. Indeed, the injection site tumors arose two weeks after distant metastases were clinically apparent. A progression of S100 positivity was also observed. S100 immunostaining was negative in all injection site of B16F10 cells until the cells underwent a morphologic change from small and monomorphic at the injection site, to large, pleomorphic cells at week six in the clinically evident injection site tumors. Inguinal metastases were also S100 positive at week four, though injection site cells were still S100 negative. We conclude that in this particular established model for melanoma, local growth at the injection site may occur after the development of regional metastases. This may prove to be a good model for investigation of local growth of tumor cells and their interaction with metastatic lesions.

Participation of bone marrow derived cells in cutaneous wound healing.

Bone marrow has long been known to be a source of stem cells capable of regeneration of the hematopoeitic system. Recent reports, however, have indicated that bone marrow might also contain early stem cells that can differentiate into other organ tissues such as skin. While these studies have illustrated that bone marrow stem cells could find their way to the skin, they have not addressed the dynamics of how bone marrow stem cells might participate in the homeostatis and regeneration of skin. In this report we followed green fluorescent protein (GFP) labeled bone marrow transplanted into non-GFP mice in order to determine the participation of bone marrow stem cells in cutaneous wounds. Our results indicate that there are a significant number of bone marrow cells that traffic through both wounded and non-wounded skin. Wounding stimulated the engraftment of bone marrow cells to the skin and induced bone marrow derived cells to incorporate into and differentiate into non-hematopoietic skin structures. This report thus illustrates that bone marrow might be a valuable source of stem cells for the skin and possibly other organs. Wounding could be a stimulus for bone marrow derived stem cells to travel to organs and aid in the regeneration of damaged tissue.

Wounding of bioengineered skin: cellular and molecular aspects after injury.

Skin substitutes are increasingly being used in the treatment of difficult to heal wounds but their mechanisms of action are largely unknown. In this study, using histology, immunostaining, flow cytometry, enzyme-linked immunosorbent assay, and reverse transcription polymerase chain reaction, we determined the response to injury of a human bilayered skin substitute. Meshing or scalpel fenestration of the construct was found to stimulate keratinocyte migration and to decrease proliferation. By 24 h, flow cytometry of the keratinocyte component showed that meshing was associated with a 33% decrease in the number of cells in S phase (p < 0.01). An approximately 2-fold decrease in staining for Ki67, a proliferation marker, was observed with meshing of human bilayered skin substitute. The process of reepithelialization was apparent by 12 h, however, the wounded human bilayered skin substitute was healed by day 3, and a stratum corneum and fully stratified epithelium were re-established by day 4. Reverse transcription polymerase chain reaction analysis and enzyme-linked immunosorbent assays showed that the expression of acute proinflammatory cytokines (interleukins 1alpha, 6, and 8, tumor necrosis factor alpha) peaked by 12-24 h postinjury. The levels of mRNA of certain growth factors (transforming growth factor beta1, vascular endothelial growth factor, insulin-like growth factor 2) but not others (platelet-derived growth factors A and B, keratinocyte growth factor, fibroblast growth factors 1 and 7, transforming growth factor beta3) increased by 12 h and peaked by 1-3 d after injury, returning to normal by day 6. Immunostaining for tumor necrosis factor alpha and transforming growth factor beta1 paralleled these findings by reverse transcription polymerase chain reaction. We conclude that human bilayered skin substitute, as a prototypic bilayered skin substitute, is a truly dynamic living tissue, capable of responding to physical injury in a staged and specific pattern of cell migration, reepithelialization, and cytokine expression.

Paraganglioma-Like Adenomas of the Thyroid (PLAT): Incidental Lesions with Unusual Features in a Patient with Nodular Goiter.

We discovered two well-demarcated nodules incidentally in a thyroid removed because of a nodular goiter. Histologically, the nodules showed a pattern of paraganglioma or so-called paraganglioma-like adenoma of the thyroid (PLAT), with lobules of polygonal and oval cells in a vascular stroma, but the immunohistochemical markers typical of paraganglioma, including chromogranin, synaptophysin, Leu 7 and 5-100, and thyroglobulin, characteristic of PLAT, were negative in the tumor cells. C-cell markers calcitonin and somatostatin were also negative. Stain for neuron-specific enolase (NSE), however, showed a distinctive pattern of reactivity within cells at the periphery of the lobules, whereas all tumor cells stained positively for keratins. Stain for carcinoembryonic antigen showed a focal interstitial pattern that corresponded to small intercellular spaces filled by microvilli identified ultrastructurally. This pattern of immunohistochemical staining has not been previously described in paraganglioma or in PLAT, and may have implications about the origin and nature of these controversial entities.