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Cryofixed, freeze-dried and paraffin-embedded skin enables successful immunohistochemical staining of skin basement membrane antigens.

Conventional chemical fixation and paraffin-embedding procedures give good preservation of morphology, although the antigenicity of many proteins in the tissue sample is destroyed. On the other hand, fresh frozen sections can preserve the antigenicity, but provide poor morphological preservation. To overcome this dilemma, cryofixation and freeze drying were used on human skin tissue, applying methodology which has only been used to study lymphoid tissue. First, fresh human skin was cryofixed in liquid isopentane (-160 degrees C) cooled by liquid nitrogen. The skin was then freeze-dried at -40 degrees C and 10(-2) atmospheric pressure for 72 h, followed by embedding in paraffin. Sections 4 microns thick taken from this cryofixed, freeze-dried, and paraffin-embedded skin were stained with hematoxylin-eosin or used for immunolabeling with antibodies against basement membrane antigen, including type IV and type VII collagen, bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and GB3 antigen. The morphological preservation of these sections was as good as that of routine formalin-fixed and paraffin-embedded skin sections. The basement membrane was clearly immunostained with all antibodies used, and the intensity of the reaction was as strong as that seen in frozen sections. Evaluation of antigen distribution in conjunction with the detailed skin structure was therefore possible in the same sections.

Antibodies, Monoclonal↗

[Topically applied arginine hydrochloride. Effect on urea content of stratum corneum and skin hydration in atopic eczema and skin aging].

BACKGROUND AND OBJECTIVE: Currently, there are no data on how the topical application of amino acids influences the complex moisture retaining system of the skin in vivo. PATIENTS/METHODS: An open study was performed to investigate the effects of topical application of arginine hydrochloride on epidermal stratum corneum urea content, transepidermal water loss, skin hydration, and clinical status of patients with atopic dermatitis and dry elderly skin. RESULTS: Treatment of patients with atopic dermatitis with 2.5% arginine hydrochloride ointment over 4 weeks showed a significant increase in urea in the stratum corneum as well as a continuous increase in skin moisture. CONCLUSIONS: The urea deficit in the stratum corneum in atopic dermatitis and elderly skin was corrected not by applying the moisturizer urea itself but instead by using arginine - its precursor in the Krebs-Henseleit urea cycle. This topical treatment also improved the clinical symptoms of dry skin.

Administration, Topical↗

Myeloperoxidase activity in skin lesions. I. Influence of the loss of blood, depth of excoriations and thickness of the skin.

The measurement of the myeloperoxidase activity was used to quantify the acute granulocyte reaction. A 35% loss of blood resulted in a clear decrease of the myeloperoxidase activity at the edges of experimental incision wounds in rat skin in the first day after inflicting the wounds. After 12 h only about one-third and after 24 h about half of the activity observed in the wounds without the loss of blood remained. After 3 days however the activity had returned to the same level as in the wounds without any loss of blood. In very deep excoriations of the rat skin where only a narrow zone of the dermis was left about double the activity increase was observed after 12 and 24 h when compared to values observed in very superficial or moderate excoriations. When the same type of excoriations were made in both thin inguinal skin and thick dorsal skin then a much higher peroxidase activity increase was observed over 4, 12 and 24 h in the thin inguinal skin than in the excoriations made in the thick dorsal skin.

Animals↗

Organotypical engineering of differentiated composite-skin equivalents of human keratinocytes in a collagen-GAG matrix (INTEGRA Artificial Skin) in a perfusion culture system.

BACKGROUND: The production of autologous composite skin equivalents for the treatment of full-thickness skin defects in burns is time consuming and costly because of laboratory procedures which have to be performed manually. In the present study keratinocytes were seeded into INTEGRA Artificial Skin and placed in a perfusion culture system in order to evaluate the possibility of producing composite grafts in an automated system with the aim of establishing a cost-effective method of industrial production. METHODS: Composite grafts of INTEGRA and human keratinocytes were raised in perfusion culture and grafted onto athymic mice to evaluate their potential to reconstitute a full-thickness skin substitute in vivo compared to grafts from standard stagnant cultures. RESULTS: Cultured composites from perfusion cultures showed no significant histological differences compared to those from stagnant cultures; however, a tendency of improved cell growth and a more surface-oriented localization was observed. Cell proliferation and surface-bound differentiation were not impaired by the use of carbonate-independent buffering (HEPES), which is necessary for perfusion culture. The composite grafts from perfusion culture exhibited identical wound adherence and complete healing and histologically represented a multi-layered, keratinizing human epidermis. CONCLUSION: Engineering of differentiated composite skin equivalents is possible in a perfusion culture system, which offers technical and procedural and possibly even biological advantages compared to standard stagnant culture methods. The development of automated perfusion culture systems for the production of composite grafts in sizes required clinically (scale-up) will be the next step in the cost-effective engineering of large-scale composite skin equivalents.

Animals↗

Systemic antimicrobial therapy in skin and skin structure infections: comparison of temafloxacin and ciprofloxacin.

Temafloxacin is an investigational fluoroquinolone with in vitro activity against common skin pathogens and a favorable pharmacokinetic profile. A randomized, double-blind, multicenter study was conducted in 492 patients to compare the safety and efficacy of temafloxacin 600 mg with ciprofloxacin 750 mg for the treatment of bacterial infections of the skin or skin structure. Both drugs were given twice daily for 7-28 days. The most common diagnoses were abscess and superficial skin infection, which were usually caused by staphylococci. In evaluable patients, clinical success (cure plus improvement) rates were 96% in the temafloxacin group and 99% in the ciprofloxacin group. Bacteriologic eradication rates were 95% and 93% in the temafloxacin and ciprofloxacin groups, respectively. Both regimens were well tolerated. Temafloxacin appears to be safe and effective for the management of bacterial infections of the skin and skin structure.

Adolescent↗

Rabbit F(ab')2 antihuman IgE is a universal skin test reagent in the evaluation of skin mast cell degranulation in vivo.

Antihuman IgE is often used to study basophil- and mast cell-mediator release in vitro but is infrequently used in vivo. To evaluate in vivo skin reactivity to anti-IgE, an affinity-purified rabbit F(ab')2 fragment of antihuman IgE was injected intradermally in 22 nonallergic and 27 allergic subjects. All 49 subjects (including a subject with less than 1 ng/ml of total serum IgE) had positive immediate cutaneous reactions to anti-IgE. Although total serum IgE level was weakly correlated (r = -0.51; p less than 0.005) with in vivo skin reactivity to anti-IgE for the entire population, allergic subjects did not have significantly increased skin reactivity compared to nonallergic subjects (p = 0.18), despite having higher total serum IgE levels (p less than 0.002). A late-phase cutaneous response (LPR) to anti-IgE occurred in 60% of the allergic and in 50% of the nonallergic subjects. Subjects with an LPR required approximately tenfold higher concentrations of anti-IgE to produce an immediate wheal of 10 mm compared to subjects who did not develop an LPR (p = 0.02), suggesting that the concentration of the stimulus injected is more important for the development of a LPR than the size of the immediate cutaneous response. Skin reactivity to codeine phosphate (a non-IgE-dependent secretagogue) was correlated with skin reactivity to anti-IgE (r = 0.47; p less than 0.05), suggesting that in vivo skin mast cell degranulation is partially a function of mast cell releasability.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

A controlled trial of skin-to-skin contact in extremely preterm infants.

BACKGROUND: Extremely preterm birth, even in the absence of significant neurological impairment, is associated with altered pain responses and impaired memory and behaviour. Preterm birth increases the risk of maternal depression and may impede the development of the mother-infant relationship, factors that in turn are also associated with impaired infant outcome. Mother-infant skin-to-skin contact has been recommended as a simple means of ameliorating these effects. METHODS: We conducted a pragmatic, prospective, controlled, intention-to-treat trial in two neonatal intensive care units. Infants born below 32 weeks gestation were recruited within the first week after birth and assigned to a control group receiving standard care, or an intervention group in which mothers were encouraged to provide a session of skin-to-skin contact once daily for 4 weeks. We assessed infant behaviour at time of discharge from hospital, responses to immunisation at 4 and 12 months of age, and memory, behaviour and development at 1 year corrected (postmenstrual) age. Indices of maternal depression, stress, anxiety, lactation performance and infant interaction were assessed at time of infant discharge, 4 months and 1 year. RESULTS: No significant difference was identified in any infant or maternal measure at any time point. CONCLUSIONS: Mother-infant skin-to-skin contact after extremely preterm birth results in neither benefit nor adverse consequences. Although there is no reason to dissuade mothers who wish to provide STS contact, we are unable to recommend resource allocation for the implementation of STS programmes for extremely preterm infants in a neonatal intensive care unit setting.

Adult↗

Endogenous DNA damage clusters in human skin, 3-D model, and cultured skin cells.

Clustered damages-two or more oxidized bases, abasic sites, or strand breaks on opposing DNA strands within a few helical turns-are formed in DNA by ionizing radiation. Clusters are difficult for cells to repair and thus pose significant challenges to genomic integrity. Although endogenous clusters were found in some permanent human cell lines, it was not known if clusters accumulated in human tissues or primary cells. Using high-sensitivity gel electrophoresis, electronic imaging, and number average length analysis, we determined endogenous cluster levels in DNA from human skin, a 3-D skin model, and primary cultured skin cells. DNA from dermis and epidermis of human skin contained extremely low levels of endogenous clusters (a few per gigabase). However, cultured skin fibroblasts and keratinocytes-whether in monolayer cultures or in 3-D model skin cultures-accumulated oxidized pyrimidine, oxidized purine, and abasic clusters. The levels of endogenous clusters were decreased by growing cells in the presence of selenium or by increasing cellular levels of Fpg protein, presumably by increasing processing of clustered damages. These results imply that the levels of endogenous clusters can be affected by the cells' external environment and their ability to deal with DNA damage.

Animals↗

Novel antibacterial agents for skin and skin structure infections.

UNLABELLED: With the continuing development of clinical drug resistance among bacteria and the advent of resistance to the recently released agents quinupristin-dalfopristin and linezolid, the need for new, effective agents to treat multidrug-resistant gram-positive infections remains important. With treatment options limited, it has become critical to identify antibiotics with novel mechanisms of activity. Several new drugs have emerged as possible therapeutic alternatives. This review focuses on agents newly introduced and those presently in clinical development for the treatment of skin and skin structure infections. Linezolid, quinupristin-dalfopristin, and daptomycin have been approved by the Food and Drug Administration for the treatment of skin and skin structure infections. Two newer compounds, oritavancin and dalbavancin, are in clinical development for this indication. In addition, the quinolones moxifloxacin and gatifloxacin recently were approved for cutaneous infections. LEARNING OBJECTIVE: At the conclusion of this learning activity, participants should be familiar with the modes of action, clinical indications, dosage regimens, and contraindications and cautions for several novel antibacterial agents for skin and skin structure infections.

Acetamides↗

Influence of preoperative showers on staphylococcal skin colonization: a comparative trial of antiseptic skin cleansers.

We undertook a prospective randomized observer-blinded study comparing the ability of preoperative showers with chlorhexidine gluconate (Hibiclens), povidone-iodine (Betadine), and a lotion soap (Safe 'N Sure) to diminish the staphylococcal skin flora of patients. By block randomization, patients scheduled for an elective cardiac operation or coronary artery angioplasty were assigned to shower with one of the study skin cleansers either once (evening only) or twice (both evening and morning) before the procedure. Semiquantitative samples for culture were obtained from the subclavian and inguinal sites on the evening before the procedure (baseline culture) and again the next morning before the operation. The chlorhexidine skin cleanser consistently reduced staphylococcal colony counts at both the subclavian and inguinal sites before the procedure. This reduction was significant for patients showering both evening and morning (p less than 0.05). The use of the povidone-iodine skin cleanser inconsistently affected skin flora. Patients using lotion soap either experienced no change or had an increase in colony counts. Chlorhexidine is more effective than povidone-iodine in diminishing skin colonization with staphylococci in patients before operation. Repeated applications of chlorhexidine are superior to a single shower with this agent.

Anti-Infective Agents, Local↗

Inhibition of delayed hypersensitivity reaction in skin (DNCB test) by 8-methoxypsoralen photochemotherapy. Possible basis for pseudo-promoting action in skin carcinogenesis?

Fifty-five of a hundred and two subjects undergoing photochemotherapy with 8-methoxypsoralen and near ultraviolet showed an abnormally low or undetectable delayed cellular hypersensitivity reaction in the skin as judged by the dinitrochlorobenzene test. It is suggested that photochemotherapy may act as a pseudo-promotor by blocking an immunologically based control process in the skin so allowing the relatively rapid appearance of squamous skin tumours, documented elsewhere, in individuals whose skin already contains a population of potentially tumorous cells. Immune surveillance of a kind may thus operate in human skin. Impairment of delayed cellular hypersensitivity to dinitrochlorobenzene was more likely to occur with more intensive treatments and in patients with less skin pigmentation.

Adolescent↗

Surfactant-induced skin irritation and skin repair: evaluation of a cumulative human irritation model by noninvasive techniques.

BACKGROUND: Although surfactant-induced acute irritant dermatitis has been extensively studied, our understanding about the induction and repair of the clinically more relevant chronic form is limited. OBJECTIVE: Our purpose was to investigate qualitative and quantitative differences in surfactant-induced irritant skin reactions from cumulative exposure to structurally unrelated surfactants and to compare the maximal irritant responses from this model with corresponding reactions noted in a previously reported acute irritation model. METHODS: Sodium lauryl sulfate (SLS), dodecyl trimethyl ammonium bromide (DTAB), and potassium soap were the model irritants. Surfactant solutions (7.5%) were applied for 20 minutes daily (for 8 consecutive days excluding the weekend) to the volar aspect of the forearm of 11 volunteers. Irritant reactions were repeatedly assessed until complete healing was indicated by visual assessment and by measurements of transepidermal water loss (TEWL), erythema (skin color reflectance), and stratum corneum hydration (electrical capacitance). Maximum irritant responses were compared with corresponding reactions from an acute irritation model. RESULTS: TEWL was increased by SLS and DTAB to the same extent, but erythema was significantly higher in DTAB-treated skin. Skin dryness, as demonstrated by decreased capacitance values and increased scores for scaling and fissuring, was significantly more pronounced than in an acute irritation model for SLS and DTAB, although no difference was detected between the two surfactants. Potassium soap led to a slight increase in TEWL, whereas the remaining features were not significantly changed. CONCLUSION: This chronic irritation model appears to represent the clinical situation of irritant contact dermatitis with pronounced skin dryness more closely than the acute irritation model. The present study confirms that an extended time is needed for complete healing of irritant skin reactions. We also demonstrated that the evaluation of the irritation potential of diverse surfactants depended significantly on the feature evaluated (erythema vs electrical capacitance and TEWL), on the mode of application (acute vs cumulative), and, in the cumulative model, on the point of observation.

Adult↗

Cytokine mRNA profiles in cultured human skin component cells exposed to various chemicals: a simulation model of epicutaneous stimuli induced by skin barrier perturbation in comparison with that due to exposure to haptens or irritant.

The skin protects our body by producing an efficient barrier membrane, the stratum corneum, from desiccation as well as from various damaging effects of environmental chemicals. Although the skin expresses various cytokines after barrier perturbation, exact cell types producing each cytokine have not been determined. Using a cell culture system, we analyzed the initial responses of various cutaneous cells to treatments simulating epicutaneous stimuli induced by a barrier perturbation of the skin in comparison with those caused by irritant or hapten exposure. We used cultured normal human epidermal keratinocytes (NHEK), human microvascular endothelial cells (HMVEC) and normal human dermal fibroblasts (NHDF). We treated them with the following chemicals and examined their cytokine mRNA levels 6 h later: high osmotic (0.5 molar) NaCl and hydrogen peroxide (H2O2), which simulate desiccation and exposure to high oxygen pressure, respectively, that may take place in vivo after perturbation of the barrier. In addition, we also studied their response to two representive haptens, nickel chloride (NiCl2) and dinitrochlorobenzene (DNCB), and an irritant, sodium dodecyl sulfate (SDS). We found that 0.5 M NaCl treatment increased mRNA levels of proinflammatory cytokines such as IL-1alpha, IL-6 and IL-8 as well as ICAM-1 in NHEK and IL-1alpha, IL-1beta and IL-6 mRNA levels in NHDF. In contrast, H2O2 treatment remarkably increased IL-10, GMCSF and ICAM-1 mRNA levels in NHEK, and IL-6 mRNA levels in HMVEC and NHDF. The exposure to haptens did not induce any remarkable increase in mRNA levels of the proinflammatory cytokines in NHEK. But NiCl2 increased IL-1alpha, IL-6 and IL-8 mRNA levels in HMVEC, while DNCB increased only their IL-6 mRNA levels. By contrast, SDS stimulated all the cell types to increase at least some of these proinflammatory cytokine mRNA levels. Our present data suggest that each skin component cell participates in inflammatory processes of the skin through its distinctive cytokine production profile when the skin barrier is compromized physically or chemically.

Cells, Cultured↗

Iontophoresis of a model peptide across human skin in vitro: effects of iontophoresis protocol, pH, and ionic strength on peptide flux and skin impedance.

This study deals with effects of electrical (current density, frequency and duty cycle) and chemical (buffer pH and ionic strength) conditions on the flux of the octapeptide, 9-desglycinamide, 8-arginine-vasopressin (DGAVP), through dermatomed human skin. A pulsed constant current was applied during iontophoresis. The anode faced the anatomical surface of the skin samples inside the diffusion cells. The resistive and capacitative components of the equivalent electrical circuit of human skin could be calculated by fitting the voltage response to a bi-exponential equation. The skin resistance prior to iontophoresis varied between 20 and 60 k omega.cm2. During iontophoresis a decrease of skin resistance and an increase of the series capacitances was observed, which were most pronounced during the first hour of iontophoresis; thereafter both quantities gradually levelled off to an apparent steady state value. The reduction of the resistance during iontophoresis increased non-linearly with increasing current density between 0.013-0.64 mA.cm-2. The steady state resistance and capacitances did not vary significantly with frequency and duty cycle of the current pulse. There was no pH dependence of skin resistance at steady state. Between pH 4 and 10, the steady state peptide flux had a bell-shaped pH-dependence with a maximum of 0.17 nmol.cm-2.h-1 at pH 7.4, which is close to the I.E.P. of the peptide. Lowering the ionic strength from 0.15 to 0.015 M NaCl increased the steady state flux at pH 5 and pH 8 by a factor 5 to 0.28 +/- 0.21 and 0.48 +/- 0.37 nmol.cm-2.h-1, respectively. Together these observations suggested that DGAVP is transported predominantly by volume flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine Vasopressin↗

Prevalence of whole-body skin self-examination in a population at high risk for skin cancer (Australia).

OBJECTIVE: Whole-body skin self-examination (SSE) with presentation of suspicious lesions to a physician may improve early detection of melanoma. The aim of this study was to establish the prevalence and determinants of SSE in a high-risk population in preparation for a community-based randomised controlled trial of screening for melanoma. METHODS: A telephone survey reached 3110 residents older than 30 years (overall response rate of 66.9%) randomly selected from 18 regional communities in Queensland, Australia. RESULTS: Overall, 804 (25.9%) participants reported whole-body SSE within the past 12 months and 1055 (33.9%) within the past three years. Whole-body SSE was associated in multivariate logistic regression analysis with younger age (< 50 years); higher education; having received either a whole-body skin examination, recommendation or instruction on SSE by a primary care physicial; giving skin checks a high priority; concern about skin cancer and a personal history of skin cancer. CONCLUSION: Overall, the prevalence of SSE in the present study is among the highest yet observed in Australia, with about one-third of the adult population reporting whole-body SSE in the past three years. People over 50 years, who are at relatively higher risk for skin cancer, currently perform SSE less frequently than younger people.

Adult↗

Mice with skin-specific DNA repair gene (Ercc1) inactivation are hypersensitive to ultraviolet irradiation-induced skin cancer and show more rapid actinic progression.

Ercc1 has an essential role in the nucleotide excision repair (NER) pathway that protects against ultraviolet (UV)-induced DNA damage and is also involved in additional repair pathways. The premature death of simple Ercc1 mouse knockouts meant that we were unable to study the role of Ercc1 in the skin. To do this, we have used the Cre-lox system to generate a skin-specific Ercc1 knockout. With a Cre transgene under control of the bovine keratin 5 promoter we achieved 100% recombination of the Ercc1 gene in the epidermis. Hairless mice with Ercc1-deficient skin were hypersensitive to the short-term effects of UV irradiation, showing a very low minimal erythemal dose and a dramatic hyperproliferative response. Ultraviolet-irradiated mice with Ercc1-deficient skin developed epidermal skin tumours much more rapidly than controls. These tumours appeared to arise earlier in actinic progression and grew more rapidly than tumours on control mice. These responses are more pronounced than have been reported for other NER-deficient mice, demonstrating that Ercc1 has a key role in protecting against UV-induced skin cancer.

Animals↗

Expression of sarco/endo-plasmic reticulum Ca2+-ATPase type 2 isoforms (SERCA2) in normal human skin and mucosa, and Darier's disease skin.

BACKGROUND: The recent report that mutations in ATP2A2, which encodes the Ca2+ transporting sarco/endo-plasmic reticulum pump type 2 isoforms (SERCA2), cause Darier's disease (DD) suggests that SERCA2 plays an important role in epidermal cell adhesion and differentiation. However, no data exist regarding SERCA2 expression in normal human skin, mucosa and DD. OBJECTIVES: We have therefore investigated SERCA2 expression in normal human skin (40 samples), oral and vaginal mucosa (13 samples) and DD lesional skin (six samples). MATERIALS AND METHODS: These investigations were performed with a mouse monoclonal antibody specific for human SERCA2, using a standard ABC immunoperoxidase technique. RESULTS: SERCA2 was expressed in all specimens. SERCA2 expression was pronounced in the subnuclear aspect of basal epidermal keratinocytes, with variable suprabasal expression. SERCA2 expression was also observed in the infundibulum and outer root sheath of hair follicles; germinative and mature cells of sebaceous glands; secretory coil and duct of eccrine glands; apocrine gland cells, and arrector pili muscle. Fibroblasts and blood vessels (endothelium and muscle) expressed SERCA2, whereas nerves did not. SERCA2 expression was observed throughout oral and vaginal mucosa. In DD skin, strong SERCA2 positivity was detected in the basal, suprabasal and acantholytic lesional cells. Perilesional DD skin was comparable to normal skin. CONCLUSIONS: These findings support the hypothesis that SERCA2 is an important player in cutaneous biology, and provide baseline data that will facilitate the design and interpretation of functional studies of cutaneous SERCA2.

Calcium-Transporting ATPases↗

Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in mouse skin: does S. aureus generally produce a biofilm on damaged skin?

BACKGROUND: Bacteria that adhere to damaged tissues encase themselves in a hydrated matrix of polysaccharides, forming a slimy layer known as a biofilm. This is the first report of detection of glycocalyx production by Staphylococcus aureus using confocal laser scanning microscopy (CLSM) on damaged skin tissues. OBJECTIVES: To analyse glycocalyx production by S. aureus cells on damaged skin tissues and the influence of polymorphonuclear leucocytes (PMNs) and various antimicrobial agents on its production using CLSM in cyclophosphamide (Cy)-treated (neutropenic) or non-Cy-treated (normal) mice. METHODS: S. aureus cells were inoculated on damaged skin tissues in neutropenic or normal mice with or without topical application of antimicrobial agents. S. aureus cells were stained with safranine, and positive staining with fluorescein isothiocyanate-conjugated concanavalin A was considered to indicate the presence of glycocalyx. RESULTS: All S. aureus cells tested on damaged skin tissues formed microcolonies encircled by glycocalyx. The colony counts of S. aureus cells on croton oil dermatitis in normal mice treated with 2% fusidic acid ointment were about 100 times lower than those in neutropenic mice (control). CONCLUSIONS: As S. aureus cells can generally produce a biofilm on damaged skin tissues, antimicrobial agents may not eradicate S. aureus cells without the help of PMNs. S. aureus glycocalyx may play a crucial role in colonization and adherence to damaged skin tissues.

Animals↗