A microarray analysis of temporal gene expression profiles in thermally injured human skin.

Partial-thickness burns incite a multitude of responses which eventually culminate in cutaneous wound repair. We hypothesized that these events would evoke extensive alterations in gene expression thereby orchestrating the complexity of spatial and temporal events that characterize "normal" human wound healing. In the present study, gene expression from partial-thickness areas at defined temporal periods (1-3 days, 4-6 days, and 7-18 days) after injury were compared to normal non-wounded skin. Gene alterations proved extensive (2286 genes). Statistically significant alterations were noted among increased and decreased genes expressed in the three different temporal groupings. Our foundational data (based on samples from 45 individuals) provide a comprehensive molecular gene expression portrait of the cutaneous reparative responses that are initiated during the first 17 days after injury. Our efforts also represent an initial endeavor to move beyond the historically defined "morphological phases" of wound repair toward reporting molecular clues that define the temporal sequence of healing in human subjects. Further analysis of genes that are either affected or remain not affected following injury to normal skin is expected to identify potential targets for therapeutic augmentation or silencing.

Mechanical abrasion improves early incorporation of small intestinal submucosa.

It has been shown that gross incorporation of porcine-derived small intestinal submucosa (SiS) is limited at 2 weeks. This study evaluates a technique for improving the early incorporation of implanted eight-ply SiS. Six pigs underwent implantation of SiS on the peritoneal surface using three techniques: suture fixation of stock-perforated SiS, suture fixation of manually perforated SiS, and suture fixation of stock-perforated SiS to mechanically abraded peritoneum. Gross incorporation was evaluated and random samples harvested for tensiometric analysis 2 weeks after implantation. SiS placed onto mechanically abraded peritoneum demonstrated significantly greater gross incorporation than both stock-perforated SiS (100% versus 42%, P = 0.015) and manually perforated SiS (100% versus 50%, P = 0.042). There was no difference in gross incorporation between stock and manually perforated SiS. Using tensiometric analysis, the force required to separate the peritoneum from the SiS implant was significantly greater for the SiS placed onto mechanically abraded peritoneum (4.4 +/- 1.7 kg . f/cm2) than for both the stock-perforated SiS samples (1.0 +/- 0.5 kg x f/cm2) and the needle-perforated SiS samples (1.4 +/- 0.9 kg x f/cm2; P < 0.001). There was no difference between stock and manually perforated SiS at 2 weeks. Mechanical abrasion of the peritoneum before SiS onlay leads to improved gross incorporation 2 weeks after implantation in a porcine model of herniorrhaphy. Long-term studies and histologic analysis are needed to validate this method as a means for improving early incorporation of SiS.

The central ACL defect as a model for failure of intra-articular healing.

Intra-articular soft tissues, such as the anterior cruciate ligament (ACL), fail to heal in contrast to the extra-articular medial collateral ligament (MCL), which undergoes classic healing. The goal of this study was to validate a model for failure of intra-articular healing that could be used in the future to test new repair strategies. We conducted a two-part experiment, the first part ex vivo, and the second in vivo. Our initial ex vivo experiments were used to determine the optimal width of the central defect in the canine ACL that would produce reproducible structural properties at time zero. The second experimental series used this optimal scalpel blade width to create a central defect in the canine ACL followed by measurement of structural properties in the ACL after either a 3- or 6-week in vivo healing period. A 3.5-mm beaver blade resulted in a maximum tolerated load of 56.8 +/- 4.7% (mean +/- SEM) of control at time zero. After the 3- and 6-week in vivo healing periods, the maximum load was 74.6 +/- 5.3 at 3 weeks and 64.9 +/- 3.8% at 6 weeks compared to control. Thus, biomechanical parameters tested at 6 weeks after creation of a defect showed no significant gains from defects tested immediately after the creation of injury. The centrally placed ACL defect in this canine model demonstrates failure to mechanically heal, which should prove suitable for future in vivo evaluation of the biomechanical and histological response to tissue engineering repair strategies for intra-articular soft tissues.

Collagen expression and biomechanical response to human recombinant transforming growth factor beta (rhTGF-beta2) in the healing rabbit MCL.

We investigated biomechanical and collagen expression in a healing bilateral rabbit medial collateral ligament (MCL) model to human recombinant transforming growth factor beta (rhTGF-beta2) at three and six weeks. Each rabbit had rhTGF-beta2 in a bioabsorbable pellet administered in one side, with the contralateral side serving as control (no rhTGF-beta2). All MCL healed with rhTGF-beta2 producing a profoundly increased scar mass at three weeks which decreased in size toward control at six weeks. In-situ hybridization demonstrated collagen expression (type I and III) no different than control at three weeks, but by six weeks elevated expression of type I was seen. Biomechanical analysis at three weeks showed no effect of rhTGF-beta2 on structural properties. However, at six weeks rhTGF-beta2 significantly inhibited both the maximum load (p < 0.05) and energy absorbed (p < 0.05) with no change in stiffness. Despite increased type I collagen expression and profound increase in early scar mass, rhTGF-beta2 did not improve the structural properties. Whether the dose or mode of delivery is responsible for decline in structural properties cannot be determined in this design. We hypothesize investigations of healing ligaments to cytokines should have biologic and biomechanical properties correlated in the same study at a minimum of two time points.

An in vivo comparison of topical agents on wound repair.

Selection of the ideal antiseptic or antimicrobial treatment for contaminated wounds remains a controversial decision. Clinical decisions are often made on the basis of in vitro studies and personal preference. Although topical solutions are widely used, their comparative in vivo effects on wound healing are largely unreported.A porcine wound model was used to compare five commonly used topical agents-5% mafenide acetate (Sulfamylon solution), 10% povidone with 1% free iodine (Betadine), 0.25% sodium hypochlorite ("half-strength" Dakin), 3% hydrogen peroxide, and 0.25% acetic acid-with a control group. Reepithelialization, angiogenesis, neodermal regeneration, fibroblast proliferation, collagen production, and bacterial colony counts were analyzed at 4 and 7 days after wounding (n = 4). Reepithelialization was not significantly influenced among the various treatment modalities tested. Sulfamylon and Dakin solutions significantly increased neodermal thickness (p < 0.05), whereas hydrogen peroxide and acetic acid significantly inhibited neodermal formation (p < 0.001). All treatments except hydrogen peroxide significantly increased fibroblast proliferation. Sulfamylon and Betadine significantly enhanced angiogenesis (p < 0.05). Sulfamylon proved most effective in maintaining an aseptic environment while concomitantly increasing angiogenesis, fibroblast proliferation, and dermal thickness compared with control. These data show that selection of a particular topical treatment can affect various aspects of wound repair in an animal model. These results suggest that the selection of topical treatments in the clinical setting should be carefully tailored to match unique wound situations and therapeutic endpoints.

Hepatocellular carcinoma results from chronic cyclin D1 overexpression in transgenic mice.

Cyclin D1 is a known oncogene and a key regulator of cell cycle progression. Amplification of the cyclin D1 gene and its overexpression have been associated with aggressive forms of human hepatocellular carcinoma (HCC). In this study, two independent lines of transgenic mice have been generated that express cyclin D1 under the control of the rat liver fatty acid binding protein promoter. This transgene specifically directs expression in the liver and the intestines. RNA and protein analysis demonstrated increased expression of the cyclin D1 gene product in the liver and bowel when compared with wild-type siblings. Both transgenic lines developed progressive liver disease. Examination of H&E stained sections of the liver and bowel revealed hyperplastic changes in the liver by 3 months of age. By 6 months of age, transgenic mice had obvious hepatomegaly and histological evidence of dysplasia in the liver. These early changes were significantly more dramatic in male animals when compared with female animals. By 9 months of age adenomas of the liver appeared, progressing to HCC over the ensuing 6-month period. By 15-17 months of age, 87% of male and 69% of female animals had either adenomatous nodules or HCCs. By 17 months of age, 31% of male and female animals had disease that had progressed to HCC. These animals represent a unique and significant new model for the study of human HCC. This study demonstrates that overexpression of cyclin D1 is sufficient to initiate hepatocellular carcinogenesis.

Analysis of the effects of deep mechanical massage in the porcine model.

Deep mechanical massage has been advocated as an alternative or adjunctive therapy for the contouring of subcutaneous fat and as a treatment for cellulite. We evaluated the effects of deep mechanical massage using two pig models. Yucatan pigs were divided into three groups (n = 4). One side of each body received 4, 10, or 20 treatments and the other side served as a control. Full-thickness tissue sections, including the underlying muscle, were harvested from identical treated and untreated regions. Examination of these regionally matched samples revealed an accumulation of dense, longitudinal collagen bands in the middle dermal and deep subdermal regions, which progressively increased with the number of treatments. Distortion and disruption of adipocytes was noted. In Yorkshire pigs, force-transducing balloon catheters were surgically placed between the deep subcutaneous tissue and muscle fascia. Catheters were inserted into two regions with different skin and subcutaneous tissue characteristics, the midflank and the hip. Standardized maneuvers were performed at suction settings 3, 5, 7, and 9 to record baseline tissue forces. Each maneuver carried a unique force signature. The measurement of tissue forces was repeated on the opposite side after 10 standardized treatment sessions. Analysis showed a significant reduction of measured forces at the midflank after the treatments. The actual force measured with each particular maneuver varied between different operators but not with different suction settings, suggesting that the technique of administering the treatments is the primary factor in creating the force within the tissue. This leads to the conclusion that deep mechanical massage is highly dependent on the individual operator of the device.

CM101 stimulates cutaneous wound healing through an anti-angiogenic mechanism.

CM101, an anti-pathoangiogenic polysaccharide derived from group B streptococcus, has been shown to inhibit inflammatory angiogenesis and accelerate wound healing in a mouse model and minimize scarring/gliosis following spinal cord injury. To evaluate the in vivo effects of CM101 on cutaneous wound healing in the pig, intravenously delivered CM101 or placebo vehicle was given 1 h after cutaneous wounding and again at 72 h after injury. Tissues from partial-thickness and full-thickness excisions were collected at days 4 and 7 after wounding and evaluated for a variety of standard healing parameters. Both types of CM101-treated wounds showed significantly less evidence of inflammatory angiogenesis when assessed by macroscopic photography of the wound surface, qualitative histological observations, laser doppler perfusion imaging, and quantitative morphometric analysis of microvessel area from endothelium selectively immunostained for factor VIII. Resurfacing was accelerated in partial-thickness and full-thickness excisions that received two doses of CM101 as compared to the placebo-treated excisional wounds. Neodermal thickness was increased in CM101-treated wounds at day 4 and was slightly reduced in comparison with placebo by day 7. New collagen accumulation appeared to be unaffected by the CM101 treatment. Immunohistochemical staining using a polyclonal antisera directed against the anti-pathoangiogenic CM101 target protein HP59 on day 7 indicated a strong immunoreactivity on the microvessels present in the control wounds but not in wounds of the CM101-treated animals. In summary, the immunolocalization HP59 in the microvessels of the cutaneous wound bed in control but not in CM101 treated wounds suggests that CM101 inhibits the pathologic inflammatory angiogenesis accompanying the normal granulation processes. The net biological effect of inhibited inflammatory pathoangiogenesis is a diminished, suggested and purely physiologic, microvascular bed which translates into an enhanced rate of epithelial resurfacing and therefore an overall accelerated rate of wound repair.

Chemokine and chemokine receptor expression in keloid and normal fibroblasts.

Keloids are benign collagenous tumors that occur during dermal wound healing in genetically predisposed individuals. The lesions are characterized by over-proliferation of fibroblasts, some leukocyte infiltration, and prolonged high rates of collagen synthesis. To determine whether leukocyte chemoattractants or chemokines are participating in this disease process, immunohistochemical staining for the CXC chemokine, MGSA/GROalpha, and its receptor, CXCR2, was performed on tissue from keloids, hypertrophic scars and normal skin. Immunoreactive MGSA/GROalpha was not observed in hypertrophic scars or normal dermis, but was present in some myofibroblasts and lymphocytes in nodular areas of the keloid samples. This staining positively correlated with the degree of inflammatory infiltrate in the lesions. Keloids, but not hypertrophic scars or normal dermis, also exhibited intensive immunoreactivity for the CXCR2 receptor in endothelial cells and inflammatory infiltrates with occasional staining of myofibroblasts. In contrast, cultured fibroblasts from either keloids or normal skin did not express detectable amounts of mRNA for MGSA/GRO or CXCR2, although interleukin-1 strongly induced MGSA/GRO mRNA in both cell types. Interleukin-1 induction of MGSA/GRO was inhibited by glucocorticoid in normal and keloid fibroblasts, and the effect was more pronounced in keloid fibroblasts. This event was not correlated with inhibition of nuclear activation of NF-kappaB, AP-1 or Sp1, and might therefore be mediated by another mechanism such as decreased mRNA stability or transcriptional repression through the glucocorticoid response element in the MGSA/GRO promoter. Data from in vitro wounding experiments with cultured normal and keloid fibroblasts indicate that there were no significant differences in MGSA/GRO or CXCR2 receptor levels between normal and keloid fibroblasts. We also show that cultured keloid fibroblasts exhibit a delayed wound healing response. We postulate that the inflammatory component is important in development of keloid lesions and chemotactic cytokines may participate in this process.

RAG2-/-, I kappa B-alpha-/- chimeras display a psoriasiform skin disease.

Nuclear factor-kappa B, a ubiquitous transcription factor involved in inflammatory and immune responses, is inappropriately activated in several immuno-related diseases, such as allograft rejection, or bronchial asthma. As nuclear factor-kappa B activity is regulated by inhibitor of kappa B (I kappa B), the gene encoding I kappa B-alpha was disrupted in mice to observe the in vivo effects of hyperactivation of nuclear factor-kappa B. I kappa B-alpha-/- mice have constitutive nuclear factor-kappa B activity, severe skin disease, and neonatal lethality. To determine the role of I kappa B-alpha deficient immunocytes in the pathogenesis of the skin disease in adult mice, we utilized the RAG2-deficient blastocyst complementation system to generate RAG2-/-, I kappa B-alpha-/- chimeras. These animals display a psoriasiform dermatitis characterized by hyperplastic epidermal keratinocytes and dermal infiltration of immunocytes, including lymphocytes. Skin grafts transferred from diseased chimeras to recipient nude mice produce hyperproliferative psoriasiform epidermal keratinocytes in response to stimulation. Furthermore, adoptive transfer of lymph node cells from diseased chimeras to RAG2-/- recipient mice recapitulates the disease. Taken together, these characterizations provide evidence to suggest that constitutive activation of nuclear factor-kappa B, due to deficiency in I kappa B-alpha, can invoke severe psoriasiform dermatitis in adult mice. J Invest Dermatol 115:1124-1133 2000

Delayed wound healing in CXCR2 knockout mice.

Previous studies demonstrated that the CXC chemokine, MGSA/GRO-alpha and its receptor, CXCR2, are expressed during wound healing by keratinocytes and endothelial cells at areas where epithelialization and neovascularization occur. The process of wound healing is dependent on leukocyte recruitment, keratinocyte proliferation and migration, and angiogenesis. These processes may be mediated in part by CXC chemokines, such as interleukin-8 and MGSA/GRO-alpha. To examine further the significance of CXC chemokines in wound healing, full excisional wounds were created on CXCR2 wild-type (+/+), heterozygous (+/-), or knockout (-/-) mice. Wounds were histologically analyzed for neutrophil and monocyte infiltration, neovascularization and epithelialization at days 3, 5, 7, and 10 postwounding. The CXCR2 -/- mice exhibited defective neutrophil recruitment, an altered temporal pattern of monocyte recruitment, and altered secretion of interleukin-1beta. Significant delays in wound healing parameters, including epithelialization and decreased neovascularization, were also observed in CXCR2 -/- mice. In vitro wounding experiments with cultures of keratinocytes established from -/- and +/+ mice revealed a retardation in wound closure in CXCR2 -/- keratinocytes, suggesting a role for this receptor on keratinocytes in epithelial resurfacing that is independent of neutrophil recruitment. These in vitro and in vivo studies further establish a pathophysiologic role for CXCR2 during cutaneous wound repair.

Boosting epidermal growth factor receptor expression by gene gun transfection stimulates epidermal growth in vivo.

Expression constructs encoding a full-length cDNA encoding the human epidermal growth factor receptor, or reporter gene for green fluorescent protein or luciferase were coated onto gold particles and driven into porcine skin using a gene gun delivery system. Strategies for epidermal growth factor receptor boosting were tested in two types of wounds. For grafted wounds, intact porcine skin was pretreated by the introduction of the epidermal growth factor receptor expression construct 24 hours before its harvesting as a split-thickness skin graft. Partial-thickness excisional wound beds (donor sites) were transfected at the time of their creation. Wound healing parameters were subsequently tested in the presence or absence of excess epidermal growth factor ligand. Initial distributions of gene gun delivered gold particles as well as luciferase expression levels suggested that optimal skin penetrations and expression levels were achieved at 500 psi for intact epidermis and 300 psi for exposed wound beds. At 2 days after gene delivery, visualization of green fluorescent protein by fluorescence microscopy showed focal expression of green fluorescent protein at the advancing epithelial outgrowths found at wound edges or surviving epithelial remnants. Green fluorescent protein expression appeared transient since no green fluorescent protein was noted in specimens removed at 4 days after injury. Northern blot analysis on mRNA isolated from wounds 2 days after introduction of epidermal growth factor receptor coated gold particles by gene gun confirmed the expression of the human epidermal growth factor receptor transgene in both skin grafts and excisional wounds. Skin grafts showed subsequent biological responses to the introduction of excessive epidermal growth factor receptor as well as expression of the human epidermal growth factor receptor construct within healing epidermis. While control autografts (reporter gene treated, epidermal growth factor alone, placebo formula, no treatment) showed few 5'-bromodeoxyuridine-labeled cells, epidermal growth factor receptor autografts showed 5'-bromodeoxyuridine labeling of nearly every basal cell. Favorable wound healing outcomes were also shown within excisional wounds following in vivo boosting of epidermal growth factor receptor. Four days after receiving epidermal growth factor receptor particle growth factor receptor transgene. Application of topical epidermal growth factor ligand resulted in the highest percentage of resurfacing. Maximal re-epithelialization was noted in wound beds receiving both receptor boosting and excessive daily epidermal growth factor ligand. A modest increase in the thickness of the granulation tissue followed gene therapy with epidermal growth factor receptor. In summary these in vivo data suggest that it is possible to boost in vivo expression of a tyrosine kinase receptor during wound repair. Increased epidermal growth factor receptor expression has an integral impact on cell proliferation, rates of resurfacing and dermal components and merits consideration as a possible therapeutic agent.

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

Continuous expression of the MGSA/GROalpha, beta, or gamma 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/GROalpha- or gamma-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/GROgamma-expressing tumors. Moreover, athymic nude mice injected with melan-a cells expressing ELR-mutant forms of MGSA/GROalpha 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).

Basal transepidermal water loss is increased in platelet-type 12-lipoxygenase deficient mice.

The roles of fatty acids in the skin have been under investigation since early reports of the phenotypic abnormalities of mice fed a diet deficient in essential fatty acids. Little is known about the functional significance of fatty acid metabolism by lipoxygenases in epidermis. Here, we have examined the role of platelet-type 12-lipoxygenase which converts arachidonic acid to the oxygenated metabolite 12-hydroperoxyeicosatetraenoic acid, in the skin using platelet-type 12-lipoxygenase-deficient mice generated by gene targeting. Platelet-type 12-lipoxygenase in wild-type mice was localized to the stratum granulosum by immunohistochemical analysis. Platelet-type 12-lipoxygenase-deficient mice lacked immunodetectable platelet-type 12-lipoxygenase in platelets and epidermis, appeared grossly normal, and exhibited an increase in basal transepidermal water loss without alteration in basal mitotic activity. Water loss and mitotic activity in mice with an acetone-disrupted membrane barrier were normal. No defect in ultrastructural properties or content of major fatty acids in dorsal skin or ear inflammation response was apparent in platelet-type 12-lipoxygenase-deficient mice. These results indicate that the platelet-type 12-lipoxygenase pathway in mice is partly responsible for normal permeability barrier function but the mechanism awaits further elucidation.

Differentiating keratinocytes express a novel cytochrome P450 enzyme, CYP2B19, having arachidonate monooxygenase activity.

The novel cytochrome P450, CYP2B19, is a specific cellular marker of late differentiation in skin keratinocytes. CYP2B19 was discovered in fetal mouse skin where its onset of expression coincides spatially (upper cell layer) and temporally (day 15.5) with the appearance of loricrin-expressing keratinocytes during the stratification stage of fetal epidermis. CYP2B19 is also present postnatally in the differentiated keratinocytes of the epidermis, sebaceous glands, and hair follicles. CYP2B19 mRNA is tightly coupled to the differentiated (granular cell) keratinocyte phenotype in vivo and in vitro. In primary mouse epidermal keratinocytes, it is specifically up-regulated and correlated temporally with calcium-induced differentiation and expression of the late differentiation genes loricrin and profilaggrin. Recombinant CYP2B19 metabolizes arachidonic acid and generates 14,15- and 11, 12-epoxyeicosatrienoic (EET) acids, and 11-, 12-, and 15-hydroxyeicosatetraenoic (HETE) acids (20, 35, 18, 7, and 7% of total metabolites, respectively). Arachidonic acid metabolism was stereoselective for 11S,12R- and 14S,15R-EET, and 11S-, 12R-, and 15R-HETE. The CYP2B19 metabolites 11,12- and 14,15-EET are endogenous constituents of murine epidermis and are present in similar proportions to that generated by the enzyme in vitro, suggesting that CYP2B19 might be the primary enzymatic source of these EETs in murine epidermis.

Titanium tetrachloride: an unusual agent with the potential to create severe burns.

Titanium tetrachloride (TiCl4), an intermediate compound in the production of white pigment, can cause severe burns. Two cases are reported in which TiCl4 created 18% to 20% total body surface area burns. These full-thickness injuries were the combined consequence of hydrochloric acid and the heat that was generated in areas where this otherwise stable compound was mixed with perspiration. TiCl4 combined with water is extremely dangerous, and its immediate treatment--towel drying before irrigation--makes it unique among chemicals. Our experience suggests that in most cases grafting will be required. These chemical burns were self-limited and had no notable systemic sequelae. Wound biopsy specimens taken on postburn days 3 and 6 were subjected to immunostaining that showed that TiCl4 did not retard wound healing. Exposure time to TiCl4 vapor will determine the pulmonary and ophthalmologic involvement in each case. Clinical awareness of the propensity of TiCl4 to react with water--even when that water is in the form of perspiration--is vital because prompt management can limit the extent of injury.

Proteolytic cleavage and activation of pro-macrophage-stimulating protein and upregulation of its receptor in tissue injury.

Macrophage stimulating protein (MSP) exists in blood as inactive pro-MSP. Cleavage yields active MSP, the ligand for a membrane receptor (RON) that is expressed on keratinocytes as well as macrophages. Because both cells have roles in tissue injury, we looked for active MSP and expressed RON in wounds. Concentration of pro-MSP + MSP in wound exudates was in the range for optimal activity. Western blot showed that MSP comprised about half the total, in contrast to less than 10% of the total in blood plasma. The presence of MSP was attributed to an exudate pro-MSP convertase that had an inhibitor profile consistent with a trypsin-like serine protease. Exudate evoked morphologic changes in macrophages in vitro like that of MSP. Removal of this activity by an anti-MSP column shows that exudate stimulation of macrophages is due to MSP. RON was infrequently detected in normal skin. RON protein was markedly upregulated in burn wound epidermis and accessory structures, in proliferating cells or differentiated cells, or both. RON was also detected on macrophages and capillaries. Tissue injury leads to cleavage of pro-MSP to MSP, which has potential to act on keratinocytes, macrophages, and capillaries, all components of the wound healing response.

Expression of inducible nitric oxide synthase in human burn wounds.

Nitric oxide is produced by various cell types and can initiate either beneficial or deleterious effects. Because cultured human keratinocytes express an inducible isoform of nitric oxide synthase, it was postulated that keratinocytes within a burn wound would express increased levels of inducible nitric oxide synthase following the injury. Immunohistochemical staining identified the sites of cellular expression and temporal sequence of inducible nitric oxide synthase protein within partial- and full-thickness burns excised from 29 patients. While migrating keratinocytes at the immediate edge of the wounds showed decreased or undetectable levels of inducible nitric oxide synthase, the immediately adjacent proliferative population and upwardly growing keratinocytes from surviving hair follicles showed increasingly greater cytoplasmic staining for inducible nitric oxide synthase at 4-21 days after injury. Noninjured skin showed minimal inducible nitric oxide synthase staining. Within the wound, detectable inducible nitric oxide synthase protein appeared to decrease as keratinocytes assumed a differentiated phenotype in the outer newly resurfaced epidermis, in inner root sheath layers of hair follicles, or in epithelium of eccrine sweat ducts. Within granulation tissue, immunoreactive inducible nitric oxide synthase was detected in capillary endothelium and in arterial smooth muscle layer. Focal increases in inducible nitric oxide synthase expression were noted in association with inflammatory infiltrates. In conclusion, the cellular and temporal distributions of immunoreactive inducible nitric oxide synthase suggest that nitric oxide may play a role in the regulation of wound repair processes beyond the acute burn injury.

Immunoperoxidase evaluation of lichen planus biopsies for hepatitis C virus.

BACKGROUND: Lichen planus is a papulosquamous dermatosis which has recently been linked to infection with hepatitis C virus. It is unclear whether or not viral antigens may be present in the cutaneous lesions of lichen planus. MATERIALS AND METHODS: Twenty-five paraffin-embedded samples of glabrous lichen planus were evaluated using immunoperoxidase staining for the presence of hepatitis C virions. Control tissues consisted of hepatitis C-infected hepatic tissue (n = 2), normal hepatic tissue (n = 2), normal human skin (n = 1), and two cutaneous biopsies of lichen planus from persons known to be infected with hepatitis C. RESULTS: The sections of hepatitis C-infected liver tissue stained positive for hepatitis C virions. The 25 biopsies of glabrous lichen planus, the two biopsies of lichen planus from hepatitis C patients, the two sections of normal liver, and the one normal skin sample all failed to take up the stain. CONCLUSIONS: Cutaneous lesions of lichen planus are more probably reactive to the underlying infection than a manifestation of skin involvement by this disease. This theory is supported by the histologic findings in a lichenoid drug eruption, which are virtually identical to those of idiopathic lichen planus. Insufficient sensitivity by the immunoperoxidase procedure used is a possible explanation for our results; however, it appears more probable that no virus exists at the sites of cutaneous involvement.