Lillian B. Nanney

Enhancement of wound healing by topical treatment with epidermal growth factor.

Experimental studies in animals have demonstrated that the topical application of epidermal growth factor accelerates the rate of epidermal regeneration of partial-thickness wounds and second-degree burns. We conducted a prospective, randomized, double-blind clinical trial using skin-graft-donor sites to determine whether epidermal growth factor would accelerate the rate of epidermal regeneration in humans. Paired donor sites were created in 12 patients who required skin grafting for either burns or reconstructive surgery. One donor site from each patient was treated topically with silver sulfadiazine cream, and one was treated with silver sulfadiazine cream containing epidermal growth factor (10 micrograms per milliliter). The donor sites were photographed daily, and healing was measured with the use of planimetric analysis. The donor sites treated with silver sulfadiazine containing epidermal growth factor had an accelerated rate of epidermal regeneration in all 12 patients as compared with that in the paired donor sites treated with silver sulfadiazine alone. Treatment with epidermal growth factor significantly decreased the average length of time to 25 percent and 50 percent healing by approximately one day and that to 75 percent and 100 percent healing by approximately 1.5 days (P less than 0.02). Histologic evaluation of punch-biopsy specimens taken from the centers of donor sites three days after the onset of healing supported these results. We conclude that epidermal growth factor accelerates the rate of healing of partial-thickness skin wounds. Further studies are required to determine the clinical importance of this finding.

Elevated cyclooxygenase-2 levels in Min mouse adenomas

BACKGROUND & AIMS Mutations in the APC gene result in an increased propensity to develop intestinal neoplasia; however, a complete understanding of the mechanisms resulting in tumor formation has remained elusive. Min mice possess a mutation in the APC gene and display a neoplastic phenotype similar to that observed in familial adenomatous polyposis coli in humans. Cyclooxygenase (COX) inhibitors decrease tumor multiplicity in the Min mouse intestine. The present study was designed to determine if there was an increase in COX-2 in adenomas harvested from Min mouse intestine. METHODS COX-2 messenger RNA levels were determined by Northern blots and reverse-transcription polymerase chain reactions of B6Min x 129 mouse-derived tumors. Protein levels and localization were determined by Western blots and immunohistochemical staining. RESULTS The Northern blots revealed an approximately threefold increase in the level of COX-2 messenger RNA in Min mouse adenoma compared with normal mucosa. COX-2 protein levels in adenomatous tissues were also approximately threefold higher compared with normal mucosa from the same mouse. Immunohistochemical staining with a monospecific COX-2 antibody confirmed that increases in COX-2 immunoreactivity were restricted to dysplastic and neoplastic foci within intestinal mucosa. CONCLUSIONS These data show that COX-2 levels may be increased at an early stage in colorectal neoplasia during polyp formation and before invasion.

Melanoma, growth factors, acanthosis nigricans, the sign of Leser-Trélat, and multiple acrochordons. A possible role for alpha-transforming growth factor in cutaneous paraneoplastic syndromes

CARCINOMAS have been associated with such paraneoplastic syndromes as skin-tumor growth, ectopic hormone production, arthropathies, myopathies, neuropathies, multiple thromboses, nephrosis, cachexi...

Adhesion formation is reduced after laparoscopic surgery

AbstractBackground: Adhesion formation after abdominal operations causes significant morbidity. Methods: Adhesion formation in pigs was compared after placement of prosthetic mesh during celiotomy (group 1), laparoscopy with large incision (group 2), and laparoscopy (group 3). After peritoneum was excised, polypropylene mesh was fixed to the abdominal wall, then to the opposite abdominal wall in the preperitoneal space followed by peritoneal closure. Adhesion area, grade, and vascularity were measured. Results: More adhesions (p < 0.02) covered intraperitoneal mesh (7.57 ± 1.89 cm2) than covered reperitonealized mesh (2.16 ± 1.13 cm2), and adhesion grade was significantly greater (p < 0.02). Adhesion areas were significantly greater in groups 1 and 2 than in group 3 (p= 0.001 and 0.03, respectively). Adhesion grade was significantly greater in groups 1 and 2 than in group 3 (p= 0.02 and p= 0.04, respectively). Groups 1 and 2 had more vascular adhesions than group 3 (p < 0.01 and p= 0.02, respectively) Conclusions: A foreign body within the peritoneum stimulates more numerous and denser adhesions. Tissue trauma distant from the site of adhesions increases their formation. A major advantage of laparoscopic surgery is decreased adhesion formation.

The tumorigenic and angiogenic effects of MGSA/GRO proteins in melanoma.

Continuous expression of the MGSA/GROα, β, or γ chemokine bestows tumor‐forming capacity to the immortalized murine melanocyte cell line, melan‐a. The mechanism for this transformation is unclear, although both autocrine and paracrine processes are possible because melan‐a cells as well as endothelial cells express a low level of the receptor for this ligand. To further define the role of MGSA/GRO proteins in melanocyte transformation, two types of experiments were designed to neutralize the biological effects of MGSA/GRO in the transfected melan‐a clones: (1) the effect of neutralizing antiserum to MGSA/GRO proteins on melan‐a tumor growth was assessed; (2) the tumor‐forming capacity of melan‐a clones expressing ELR motif‐mutated forms of MGSA/GRO with compromised receptor affinity was compared to the tumor‐forming capacity of clones expressing wild‐type MGSA/GRO. These experiments revealed that SCID mice inoculated with MGSA/GROα‐ or γ‐expressing melan‐a cells and subsequently treated with antiserum to the respective chemokine exhibited decreased tumor growth. This reduction in tumor growth was accompanied by declining angiogenic activity in MGSA/GROγ‐expressing tumors. Moreover, athymic nude mice injected with melan‐a cells expressing ELR‐mutant forms of MGSA/GROα exhibited markedly impaired tumor‐forming capacity compared with those mice injected with melan‐a clones expressing wild‐type MGSA/GRO. These data suggest that continuous expression of MGSA/GRO proteins may facilitate tumor growth by stimulating the growth of microvessels into the tumor (paracrine) and by affecting melanocyte growth (autocrine). J. Leukoc. Biol. 67: 53–62; 2000.

Enhanced tumor-forming capacity for immortalized melanocytes expressing melanoma growth stimulatory activity/growth-regulated cytokine β and γ proteins

Three human MGSA/GRO genes encode 3 highly related chemokines, MGSA/GROα, ‐β and ‐γ. All 3 MGSA/GRO proteins bind to the same receptors, but with differing affinities, and stimulate a number of biological responses including chemotaxis, angiogenesis, and growth regulation. We have previously demonstrated that MGSA/GROα can be isolated from culture medium conditioned by malignant melanoma cells and that continuous secretion of MGSA/GROα contributes to the transformation of immortalized murine melanocytes. The present study was designed to determine whether MGSA/GROβ or ‐γ have similar effects on melanocyte tumorigenicity. Stable Melan‐a clones expressing either human MGSA/GROβ or ‐γ exhibited enhanced ability to form large colonies in soft agar and tumors in nude mice. The clones expressing the MGSA/GROβ or ‐γ transgene formed tumors within 2 months after injection; the tumors were highly pigmented and expressed immunoreactive MGSA/GROβ or ‐γ protein. Furthermore, when conditioned medium from Melan‐a clones expressing MGSA/GROα, ‐β or ‐γ transgenes were examined for the ability to induce angiogenesis in the rat cornea, strong angiogenic responses were observed. This angiogenic response was blocked by antibodies to the respective MGSA/GRO protein, but not by normal rabbit serum. By contrast, angiogenic responses were observed in only 2 of 12 corneal implants (17%) containing medium conditioned by Melan‐a clones expressing the neomycin resistance marker alone. Int. J. Cancer 73:94–103, 1997.

Cell Biology of Wound Healing

Publisher Summary Cutaneous wound repair is a seamless series of events that are initiated whenever tissue integrity is breached. Although extreme diversity exists in the various types of wounds and in their depths of injury, wound repair consists of a predictable set of responses involving migration, proliferation, differentiation, and apoptosis of cell types within the wound environment. This chapter focuses on the roles of these individual cell types throughout the reparative process. Following injury, a series of dynamic interactive processes take place that usually culminates in a mature, healed wound. To study wound repair, wound healing is subdivided into four phases: acute inflammation, epithelialization, granulation tissue formation, and tissue remodeling. These processes follow a specific temporal sequence unless encumbered by various pathologic states such as diabetes, poor arterial perfusion, malnutrition, or sepsis. Following cutaneous injury, the body first acts to stop hemorrhage from blood vessel injury by the action of platelets and by initiation of the clotting cascade. Shortly thereafter, inflammatory cells responding to platelet-released growth and chemotactic factors arrive and predominate in the wound bed. In ideal wounds, monocytes and fixed tissue macrophages reach maximal populations in the next 48 hr. The later phases of inflammation overlap with a period of proliferation that is characterized by migration of fibroblasts and endothelial cells. Epithelialization is the major mechanism of repair in partial loss of skin thickness, whether from accidental abrasions, thermal or chemical injuries, or surgically planned skin graft harvest. The process of resurfacing is of minor consequence in wounds coapted with sutures and is short lived.

Transforming growth factor alpha protection against drug-induced injury to the rat gastric mucosa in vivo.

This study was designed to determine whether transforming growth factor alpha (TGF alpha) protects rat gastric mucosa against ethanol- and aspirin-induced injury. Systemic administration of TGF alpha dose-dependently decreased 100% ethanol-induced gastric mucosal injury; a dose of 50 micrograms/kg delivered intraperitoneally 15 min before ethanol decreased macroscopic mucosal injury by > 90%. At the microscopic level, TGF alpha prevented deep gastric necrotic lesions and reduced disruption of surface epithelium. Pretreatment with orogastric TGF alpha (200 micrograms/kg) only partially (40%) decreased macroscopic ethanol damage. Intraperitoneal administration of TGF alpha at a dose of 10 micrograms/kg, which does not significantly inhibit gastric acid secretion, decreased aspirin-induced macroscopic damage by > 80%. TGF alpha protection does not seem to be mediated by prostaglandin, glutathione, or ornithine decarboxylase-related events, as evidenced by lack of influence of the inhibition of their production. Pretreatment with the sulfhydryl blocking agent N-ethylmaleimide partially abolished (40%) the protective effect of TGF alpha. In addition, systemic administration of TGF alpha resulted in a two-fold increase in tyrosine phosphorylation of phospholipase C-gamma 1 and in a time- and dose-dependent increase in levels of immunoreactive insoluble gastric mucin; these events occurred in a time frame consistent with their participation in the protective effect of TGF alpha.

Particle-mediated gene transfer with transforming growth factor-beta1 cDNAs enhances wound repair in rat skin.

Based on preliminary but variable results with direct DNA transfer into wounds, we evaluated in vivo gene transfer by particle-mediated DNA delivery to rat skin to determine whether overexpression of TGF-beta1 at the site of skin incisions would result in a significant improvement in repair. Optimization of the method with viral promoter-luciferase reporter constructs indicated that expression of luciferase activity persisted up to 5 d and was promoter, pressure, and site dependent (ventral > dorsal). Using cytomegalovirus (CMV)-driven human alpha1-antitrypsin, transgene expression was immunolocalized within keratinocytes of the stratum granulosum at 24 h. We measured tensile strength of skin incisions at 11-21 d in both normal and diabetic rats transfected with TGF-beta1 expression vectors at surgery. Native murine TGF-beta1 under an SV40 promoter produced positive effects, while wound strengthening was more pronounced in diabetic animals using a CMV-driven construct. Transfection of rat skin with constitutively active, mutant porcine TGF-beta1 under the control of the CMV and Moloney murine leukemia virus promoters significantly increased tensile strength up to 80% for 14-21 d after surgery. Transfection 24 h before surgery was more effective. Particle-mediated gene delivery can be used to deliver viral promoter-cytokine expression constructs into rat skin in a safe, efficient, and reproducible fashion. The extent of wound repair, as evidenced by enhanced tensile strength, can be markedly improved in tissues transfected with TGF-beta1 expression constructs.

Molecular Cloning and Functional Expression of a Phorbol Ester-inducible 8S-Lipoxygenase from Mouse Skin

One of the effects of topical application of phorbol ester to mouse skin is the induction of an 8S-lipoxygenase in association with the inflammatory response. Here we report the molecular cloning and characterization of this enzyme. The cDNA was isolated by polymerase chain reaction from mouse epidermis and subsequently from a mouse epidermal cDNA library. The cDNA encodes a protein of 677 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has 78% identity to a 15S-lipoxygenase cloned recently from human skin and approximately 40% identity to other mammalian lipoxygenases. When expressed in vaccinia virus-infected Hela cells, the mouse enzyme converts arachidonic acid exclusively to 8S-hydroperoxyeicosatetraenoic acid while linoleic acid is converted to 9S-hydroperoxy-linoleic acid in lower efficiency. Phorbol ester treatment of mouse skin is associated with strong induction of 8S-lipoxygenase mRNA and protein. By Northern analysis, expression of 8S-lipoxygenase mRNA was also detected in brain. Immunohistochemical analysis of phorbol ester-treated mouse skin showed the strongest reaction to 8S-lipoxygenase in the differentiated epidermal layer, the stratum granulosum. The inducibility may be a characteristic feature of the mouse 8S-lipoxygenase and its human 15S-lipoxygenase homologue.