Treatment of giant congenital melanocytic nevi with cultured epithelial autografts: Clinical and histopathological analysis.

INTRODUCTION: Curettage and dermabrasion are effective in the treatment of giant congenital melanocytic nevi (GCMN); however, local infection and hypertrophic scar formation are major issues. Thus, we applied cultured epithelial autografts (CEA) on skin defects after curettage or abrasion of GCMN and assessed the postoperative outcomes. METHODS: Seven nevi lesions of five patients (aged 3 months to 24 years) were treated with CEA after curettage or abrasion with a dermatome or a surgical bar, respectively. We assessed the postoperative outcomes, including CEA take ratio, erosion and/or ulcer formation in the acute phase, hospitalization days, Vancouver scar scale, and color improvement one year after the operation. In addition, a histological evaluation of a skin biopsy was performed over one year after the operation. RESULTS: The CEAs took well on the wound, and the wound surface was mostly epithelized by postoperative day 7 in all cases. While hypertrophic scar formation and slight pigmentation were observed in some lesions, the color was improved in all of the treated lesions. Histopathological examination revealed that the regenerated epidermis had stratified keratinocytes with rete ridges, and the dermal layer without nevus cells regenerated above the remaining dermis layer. CONCLUSIONS: In this study, we found that early epithelialization and regeneration of the dermal layer was achieved after the application of CEA, suggesting that CEA could be an effective option after curettage or abrasion of GCMN.

Molecular defect in siblings with prolidase deficiency and absence or presence of clinical symptoms. A 0.8-kb deletion with breakpoints at the short, direct repeat in the PEPD gene and synthesis of abnormal messenger RNA and inactive polypeptide.

Prolidase deficiency is an autosomal recessive disorder with highly variable symptoms, including mental retardation, skin lesions, and abnormalities of collagenous tissues. In Japanese female siblings with polypeptide negative prolidase deficiency, and with different degrees of severity of skin lesions, we noted an abnormal mRNA with skipping of 192 bp sequence corresponding to exon 14 in lymphoblastoid cells taken from these patients. Transfection and expression analyses using the mutant prolidase cDNA revealed that a mutant protein translated from the abnormal mRNA had an Mr of 49,000 and was enzymatically inactive. A 774-bp deletion, including exon 14 was noted in the prolidase gene. The deletion had termini within short, direct repeats ranging in size of 7 bp (CCACCCT). The "slipped mispairing" mechanism may predominate in the generation of the deletion at this locus. This mutation caused a 192-bp in-frame deletion of prolidase mRNA and was inherited from the consanguineous parents. The same mutation caused a different degree of clinical phenotype of prolidase deficiency in this family, therefore factor(s) not related to the PEPD gene product also contribute to development of the clinical symptoms. Identification of mutations in the PEPD gene from subjects with prolidase deficiency provides further insight into the physiological role and structure-function relationship of this biologically important enzyme.

Biochemical basis of prolidase deficiency. Polypeptide and RNA phenotypes and the relation to clinical phenotypes.

Cultured skin fibroblasts or lymphoblastoid cells from eight patients with clinical symptoms of prolidase deficiency were analyzed in terms of enzyme activity, presence of material crossreacting with specific antibodies, biosynthesis of the polypeptide, and mRNA corresponding to the enzyme. There are at least two enzymes that hydrolyze imidodipeptides in these cells and these two enzymes could be separated by an immunochemical procedure. The specific assay for prolidase showed that the enzyme activity was virtually absent in six cell strains and was markedly reduced in two (less than 3% of controls). The activities of the labile enzyme that did not immunoprecipitate with the anti-prolidase antibody were decreased in the cells (30-60% of controls). Cell strains with residual activities of prolidase had immunological polypeptides crossreacting with a Mr 56,000, similar to findings in the normal enzyme. The polypeptide biosynthesis in these cells and the controls was similar. Northern blot analyses revealed the presence of mRNA in the polypeptide-positive cells, yet it was absent in the polypeptide-negative cells. The substrate specificities analyzed in the partially purified enzymes from the polypeptide-positive cell strains differed, presumably due to different mutations. Thus, there seems to be a molecular heterogeneity in prolidase deficiency. There was no apparent relation between the clinical symptoms and the biochemical phenotypes, except that mental retardation was present in the polypeptide-negative patients. The activities of the labile enzyme may not be a major factor in modifying the clinical symptoms.

Differential expression of human cornifin alpha and beta in squamous differentiating epithelial tissues and several skin lesions.

Cornifins/small proline-rich proteins (SPRRs) belong to a family of proline-rich proteins that function as cornified envelope precursors. We report here an immunohistochemical analysis of human cornifin-alpha and -beta expression in several stratified squamous epithelia. In normal human skin, cornifin-alpha was expressed in the granular layer of the epidermis of palmoplantar skin, in the inner lining cells of the follicular infundibulum, and in the inner root sheath of the hair follicle. It was also expressed in the upper squamous layers of the oral, esophageal, and vaginal epithelia. Cornifin-beta was detected in oral, esophageal, and vaginal epithelia, but not in normal skin. Immunoblot analysis revealed quantitative differences in cornifin-alpha expression in skin from different regions. Studies of specimens from various skin diseases showed that (i) cornifin-alpha was upregulated in inflammatory skin diseases, hyperplastic lesions, and in well-differentiated squamous cell carcinomas (SCCs), (ii) the expression of cornifin-beta was absent in inflammatory skin but was detected in highly differentiated keratinocytes in well-differentiated SCCs of the skin and some other hyperproliferative skin lesions, and in SCCs of the oral mucosa and esophagus. Northern blot analysis revealed that cornifin-alpha mRNA was present in all the squamous epithelial tissues studied, whereas cornifin-beta mRNA was expressed in oral mucosal epithelia and verrucous carcinoma of the skin but neither in normal nor in psoriatic skin. These results indicate that (i) the amount of cornifin alpha/SPRR1 expression in normal human skin depends on the body region, (ii) cornifin-alpha/SPRR1, but not cornifin-beta, contributes to the integrity of the hair follicle, and (iii) the expression of cornifin-beta is induced in some hyperplastic skin diseases only when the keratinocytes undergo extensive squamous differentiation.

Increased type 2 cytokine expression by both CD4+ CD45RO+ T cells and CD8+ CD45RO+ T cells in blood circulation is associated with high serum IgE but not with atopic dermatitis.

Type 2 cytokines, such as interleukin-4 (IL-4) and IL-13, are associated with immunoglobulin E (IgE) production. This association has also been observed in CD8+ T cells from patients infected with leprosy and human immunodeficiency virus (HIV). Using intracellular cytokine staining and flow cytometry, the cytokine profile [IL-2, IL-4, IL-10, IL-13, and interferon (IFN)-gamma] of both CD4+ and CD8+ memory/effector T cells circulating in atopic dermatitis (AD) patients was investigated at the single cell level. The levels of type 2 cytokines in CD4+ T cells or CD8+ T cells in AD patients with high levels of serum IgE (AD-H), low levels of serum IgE (AD-L), and healthy controls were compared. Increased production of IL-4 and IL-13 in both CD4+ CD45RO+ T cells and CD8+ CD45RO+ T cells after 4 h in vitro stimulation with phorbol 12-myristate 13-acetate and ionomycin, was more prominent in AD-H patients than in AD-L patients or healthy controls, whereas IFN-gamma-producing CD4+ CD45RO+ T cells and CD8+ CD45RO+ T cells were relatively diminished in AD-H patients. CD4+ T cells and CD8 + T cells from AD-H patients, cultured for 48 h with phorbol 12-myristate 13-acetate and ionomycin, released larger amounts of IL-4 and IL-13 but smaller amounts of IFN-gamma than both types of cells from AD-L patients or healthy controls. In addition, when stimulated with immobilized anti-CD3 monoclonal antibody (MoAb) and anti-CD28 MoAb, CD4+ CD45RO+ T cells and CD8+ CD45RO+ T cells from AD-H patients contained more IL-4-producing cells but fewer IFN-gamma-producing cells compared with healthy controls. Finally, spontaneous mRNA expression of IL-4 in blood CD8+ CD45RO+ T cells isolated from AD-H patients was increased, as determined by reverse transcriptase-polymerase chain reaction. Therefore, in AD patients with high IgE levels, type 2 cytokine (IL-4 and IL-13) expression is associated with IgE production, in both CD4+ CD45RO+ T cell and CD8+ CD45RO+ T cell subsets.

Expression of retinoic acid receptor genes in keratinizing front of skin.

We found, by an in situ hybridization method with riboprobes synthesized from human cDNA of the retinoic acid receptor (RAR), that the RAR genes (predominantly gamma-subtype) are intensively expressed in the epidermis of normal and psoriasic human skins, and also in keratinizing fronts of 4-day-old mouse skins, nail matrices and hair follicles. Thus, target cells of retinoic acid in the skins are concluded to be keratinocytes, which is quite consistent with the fact that retinoic acid regulates keratinization of epidermis in vivo and also modulates expression of the keratin gene in vitro.

Correlation between expression of peroxisome proliferator-activated receptor beta and squamous differentiation in epidermal and tracheobronchial epithelial cells.

Previously, several members of the nuclear receptor superfamily have been implicated in the regulation of epidermal differentiation. In this study, we analyze the expression of members of the PPAR nuclear receptor subfamily in relation to the process of squamous differentiation in normal human epidermal keratinocytes (NHEK), human tracheobronchial epithelial (HBE) cells and the epidermis in vivo. Our results demonstrate that induction of differentiation in NHEK by either treatment with the phorbol ester phorbol 12-myristate-13-acetate (PMA), suspension culture or confluence greatly enhances the expression of PPARbeta mRNA. Likewise, topical treatment of mouse skin with PMA results in increased PPARbeta mRNA expression in the epidermis. In addition, the induction of squamous differentiation in HBE cells was also associated with an upregulation of PPARbeta mRNA expression. Finally, in situ hybridization analysis localized PPARbeta mRNA to the suprabasal layers of normal human skin. Our results demonstrate that the expression of PPARbeta is associated with squamous differentiation suggesting a regulatory role for this receptor in the control of specific genes during this differentiation process.

Role of fibronectin in the adherence of Staphylococcus aureus to dermal tissues.

Staphylococcus aureus has a fibronectin receptor on its surface. Fibronectin seems to play a role in the initiation and modification of infection with S. aureus. We studied the role of fibronectin in the binding of S. aureus (clinical isolates) to dermal tissues in mice, and the relationship between the fibronectin binding ability of S. aureus and clinical features of S. aureus skin infections. Mice were inoculated with S. aureus incubated with gold-particles bound fibronectin and skin specimens were taken for electron microscopic examination. The number of gold particles surrounding the S. aureus cells decreased with time, with none detected 4 h after inoculation. At both 5 min and 1 h after inoculation, gold particles were only found on the free surface of S. aureus cells and not in the interface between S. aureus cells and fibroblasts. Fibronectin-bound gold particles were bound more extensively to S. aureus strains isolated from furunculosis or furuncle than to those from bullous impetigo. These results suggest that the matrix fibronectin on the surface of the fibroblasts of mice contributes to the adherence of S. aureus to the fibroblasts, and that the number of fibronectin binding sites on S. aureus cells is related to the degree of local invasiveness.

Production of staphylococcal impetigo-like lesion on human skin explants in culture.

We produced a highly reproducible experimental impetigo-like lesion in normal human skin explants in culture. The three Staphylococcus aureus strains we used were an isolate from a human impetigo (E strain), an isolate from a human furunculosis (N strain) and ATCC 29213 strain. E strain was a protein A positive, coagulase type V, producer of exfoliative toxin (ET) and beta-toxin. N strain was a coagulase type IV, ET non-producer and alpha-toxin positive. ATCC 29213 was a coagulase type II, ET non-producer, and alpha-, beta-, and delta-toxin positive. Normal human skin samples were obtained from 8 adult skin surgery patients. One specimen was obtained from human oral mucosa. Small pieces of the samples were slightly abraded on the epidermal surface and cultured on lens paper rafts floating in Eagle's Minimum Essential Medium in an atmosphere of 5% CO2 and 95% air. Fifty microliters of the respective bacterial suspensions were applied to the epidermal surfaces of the explants. The inoculated surfaces were then occluded under sterile plastic plaster. Histologically, the formation of intraepidermal blisters at the granular layer level with acantholytic cells was observed in all 8 of the skin specimens at 10 h after inoculation with E strain. The specimen from an oral mucous membrane did not produce similar changes with any of the three S. aureus strains. Neither N or ATCC strains developed bullae in the epidermis at 6, 10 or 18 h after inoculation. Immunofluorescent examination revealed that the inner surfaces of blisters in the epidermis were lined with anti-ETA antibody. Under the electron microscope, the blisters of the specimens which had been inoculated with strain E contained only a few S. aureus cells. These results suggest that blister formation at the granular layer level with acantholytic cells is mediated by ET action at the granular layer level and occurs without invasion of lymphocytes or neutrophils, or the involvement of any serum components. Therefore, under appropriate conditions, impetigo could develop even in adults.

Interaction of Staphylococcus aureus cells and silk threads in vitro and in mouse skin.

Staphylococcus aureus cell suspension was epicutaneously inoculated on the back skin of cyclophosphamide-treated mice with silk stitches and these sites were occluded. Biopsy specimens were taken from three mice at 1, 3, 6, 12, 24, 48, and 72 h after inoculation and were examined by electron microscopy. Fibril-like structures (glycocalyx) were seen around the S. aureus cells at 1 h. At 3 h, they had extended towards the silk threads. There were microcolonies on the surfaces of the silk threads and at 12 h the S. aureus cells were enclosed in membrane-like structures. The electron density of the membrane-like structures increased over time. After ruthenium red staining, the membrane-like structures and the fibril-like structures were stained positive, suggesting that these structures contain polysaccharide components. With a combination chemotherapy using clarithromycin and ofloxacin, S. aureus cells in the membrane-like structures were degenerated, whereas the use of clarithromycin or ofloxacin alone had little effect. Chlorhexidin gluconate and povidone iodine were effective if they were able to reach the biofilm. The fibril-like structures appeared in vitro only in the presence of silk threads, and were enhanced by the presence of mouse plasma. These structures did not form with formaldehyde-killed S. aureus cells. Thus, S. aureus cells may interact with foreign bodies to form biofilms, thereby evading the effect of antibacterial agents.

Localization of prolidase gene expression in scar tissue using in situ hybridization.

We investigated prolidase gene expression in human skin by means of Northern blot analysis and in situ hybridization. Northern blot analysis revealed that an mRNA species that was specific for prolidase was present in cultured human skin fibroblasts and keratinocytes. In situ hybridization using non-isotopic riboprobes labeled with digoxigenin and an isotopic riboprobe labeled with [35S]UTP localized prolidase gene expression to fibroblasts and endothelial cells of small vessels in scar tissue. Prolidase mRNA was also prominently expressed in keratinocytes near the basal layer overlying scar tissue. These findings indicate that prolidase may have an important role in wound healing.

Production of experimental staphylococcal impetigo in mice.

We produced a staphylococcal impetigo model by epicutaneous inoculation in mature mice. A strain isolated from a human impetigo was used. Five-week-old female mice (ddy-strain) were used with and without pre-treatment by cyclophosphamide (Cy) (2 mg/mouse) for 5 days. The back skin of mice was shaved by a razor blade and slightly abraded by sand paper. Bacterial suspension (1.4 x 10(7) CFU/0.05 ml) was applied on the abraded areas which were then occluded under sterile plastic plaster. Although intraepidermal blisters developed in non-Cy-treated mice, massive neutrophil infiltration obscured the changes there. Development of subcorneal bullae in Cy-treated mice inoculated with Staphylococcus aureus was first observed at 3h and enlargement of bullae was apparent at 12 h after inoculation. The bullae produced in Cy-treated mice contained numerous S. aureus bacilli. Electronmicroscopically, S. aureus cells invaded the horny layer at 1/4 h. A clear halo was seen between S. aureus cells and horny cells. S. aureus cells attached to surrounding horny cells by fibril-like structures. The halo-like spaces became larger, coalesced and then developed into an intraepidermal blister. Our new method to produce human impetigo-like blister in Cy-treated adult mice may contribute to disclosing the mechanisms of blister formation in epidermis by S. aureus. Due to the thin structure of mouse epidermis, only specimens taken earlier than 24 h after inoculation were considered appropriate.

Expression pattern of the bcl-2 homologous protein bad in normal skin, psoriasis vulgaris and keratinocytic tumors.

Previous investigations focused on the mechanisms and regulation of apoptotic process have found that bcl-2 and its homologous proteins are central regulators of the mitochondrial phase of apoptosis. Expression of several members of the bcl-2 family has been studied in various tissues including skin under normal as well as disease conditions. In this report, we investigated the expression of bad, the pro-apoptotic member of the BH3 subfamily, in normal and psoriatic epidermis, keratoacanthoma, and basal and squamous cell carcinomas. Normal and psoriatic epidermis showed accentuation of the staining in the lower suprabasal compartment. A weak, predominantly diffuse staining pattern was observed in the upper epidermis of psoriatic plaques. Keratoacanthoma showed strong but diffuse immunostaining for pro-apoptotic bad, however we found only weak bad expression in squamous cell carcinoma. Seven out of 15 basal cell carcinomas failed to express bad protein. There was no correlation between bad positivity and depth of tumour infiltration. Our observation suggests that the pro-apoptotic bad may function as one of regulators involved in the maintenance of epidermal homeostasis and this function could be altered depending on the disease state.

Adherence characteristics and susceptibility to antimicrobial agents of Staphylococcus aureus strains isolated from skin infections and atopic dermatitis.

We examined the adherence characteristics and susceptibility to various antimicrobial agents of 130 strains of Staphylococcus aureus isolated from infective skin lesions and 135 strains of S. aureus isolated from non-infective eczematous lesions of atopic dermatitis (AD) patients. The isolation rate of methicillin-resistant S. aureus (MRSA) was 27.7% in strains from clinical sources excluding AD and 31.1% in those from AD. Coagulase type II strains were most frequently observed in MRSA strains isolated from all sources excluding AD, and coagulase type III strains were most frequently observed in those isolated from AD. We proposed that antimicrobial treatment for AD patients should be carefully designed to prevent MRSA infection. Plasma coagulation ability was lowest in S. aureus strains isolated from abscesses, suggesting that the lower production of fibrin observed in abscesses may assist the infiltration of neutrophils into skin tissues and that a decrease in plasma coagulation ability may enable abscess formation. Adherence to polypropylene tubes with slime production was most evident in S. aureus strains isolated from felon and least evident in those isolated from cellulitis and lymphangitis. Tube adherence was characteristic of the S. aureus strains attached to superficial skin tissues, but not necessarily for strains that had infiltrated the deep skin tissues. Fusidic acid demonstrated significant antimicrobial activity against the MRSA strains, but rifampicin was the strongest antimicrobial agent.

The production of superantigenic exotoxins by coagulase-negative staphylococci isolated from human skin lesions.

We examined the production of superantigenic exotoxins in 136 coagulase-negative staphylococci isolated from various skin lesions in humans using a reversed passive latex agglutination test (Denka Seiken). As a control we examined the same in 50 Staphylococcus aureus strains isolated from non-infective skin ulcers in humans. Of the 136 strains of coagulase negative-staphylococci, 9 (6.6%) produced one or more identifiable exotoxins. In contrast, 21 (42%) out of the 50 S. aureus strains produced one or more identifiable exotoxins (P<0.01).

Adhesion of Staphylococcus aureus to horny layer: role of fibrinogen.

Staphylococcus aureus cells attach to and invade the epidermis more easily under conditions of abrasion or occlusion or in the presence of irritant dermatitis than when the epidermis is intact. This fact strongly suggests that exuded plasma components may play an important role in the adherence of S. aureus cells to the horny layer. S. aureus cells (Cowan 1 strain, Wood 46 strain, and the protein A-deficient mutant, C7 strain, which was isolated from the Cowan 1 strain) were epicutaneously inoculated on the backs of mice. Biopsy specimens were taken from the mice at 1 h, 3 h, and 6 h after inoculation and examined using immunoelectron microscopy. Gold particles for fibrinogen gathered in a time-dependent manner at the interfaces of S. aureus cells and horny material in the lesions inoculated with the Cowan 1 and C7 strains but not in the lesions inoculated with the Wood 46 strain. These results suggest that fibrinogen plays a role in the binding of S. aureus cells to the horny layer.

Staphylococcus aureus infection on cut wounds in the mouse skin: experimental staphylococcal botryomycosis.

Staphylococcus aureus cells were inoculated on the cut wounds in the skin of cyclophosphamide-treated mice. Biopsy specimens were taken from three mice at 1, 3, 6, 12, 24, 36, 48 and 60 h after the inoculation and were examined by light and electron microscopies. One hour after the inoculation Staphylococcus aureus cells were seen around the cut wound and deeper into the subcutaneous tissue. By 6 h after the inoculation, Staphylococcus aureus cells formed clusters of bacterial colonies. By 36 h after the inoculation inflammatory cells, mainly polymorphonuclear leukocytes and macrophages, were seen around the clusters. Electron microscopic examination revealed fibril-like structures around the Staphylococcus aureus cells at 1 h. The Staphylococcus aureus cells were enclosed in membrane-like structures at 3 h. The membrane-like structures and the fibril-like structures were positive for Ruthenium red. By 12 h after the inoculation, the membrane-like structures increased in thickness and in electron density. Inflammatory cells were seen around but outside of the membrane-like structures at 24, 36 and 48 h. At 60 h the tissues around the membrane-like structures were degenerated and almost necrotic. These results suggest that Staphylococcus aureus cells may form biofilm in dermal or subcutaneous tissues in a neutropenic condition.

Characteristics in adherence of streptococci and Staphylococcus aureus isolated from various infective skin lesions: serum IgA decreases adherence of Streptococcus pyogenes but not Staphylococcus aureus.

We characterized adherence of streptococci and Staphylococcus aureus strains isolated from various infective skin lesions in terms of hydrophobicity, negative charge, tube adherence, slime production, and influence on adherence to coverslips by plasma and serum immunoglobulins. High hydrophobicity was more frequently observed in Streptococcus pyogenes strains than in Streptococcus agalactiae strains (P < 0.01) and S. aureus strains (P < 0.001) and slime production was more frequently observed in S. agalactiae strains than in S. pyogenes strains (P < 0.05). Serum IgA decreased adherence to coverslips of S. pyogenes strains but not that of S. aureus strains.

Calcium oxide and magnesium oxide inhibit plasma coagulation by Staphylococcus aureus cells at the lower concentration than zinc oxide.

We examined the effect of ceramic powder slurries on the coagulation of plasma by Staphylococcus aureus cells. Plasma coagulation by S. aureus strains or their cultured supernatant was inhibited in the plasma with 0.12% calcium oxide or 0.25% magnesium oxide after incubation for 24 h at 37 degrees C. Inhibition of plasma coagulation by calcium oxide and magnesium oxide was observed at the lower concentration than zinc oxide.

Staphylococcus aureus infection on experimental croton oil-inflamed skin in mice.

Staphylococcus aureus cells were inoculated on the surface of skin inflamed by application of croton oil in cyclophosphamide-treated mice. Skin specimens were taken at 1, 3, 6, 12, and 24 h inoculation and each specimen was examined by microscopy. The S. aureus cells which attached to the surface of the skin immediately after inoculation had invaded the horny layer within 1 h. The cells gradually penetrated deeper into the epidermis. Electron microscopy revealed fibril-like structures around the S. aureus cells and the cells which adhered to the horny layer and fibrin by means of Ruthenium red-positive, fibril-like structures. A combined application of 0.1% gentamicin ointment, 2% fusidic acid ointment, and clobetasol propionate ointment was more effective in decreasing the number of S. aureus cells in the lesions than was an application of clobetasol propionate ointment alone. However, a combined application of 0.1% gentamicin ointment and 2% fusidic acid ointment without clobetasol propionate ointment showed almost the same efficacy as that with clobetasol propionate ointment. Although povidone iodine killed S. aureus in vitro at a concentration of 0.01% (100 micrograms/ml) in 40 s, its in vivo efficacy was limited.