Skin Of Normal Individuals Research Articles

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

Cutaneous HPV and skin cancer

Papillomaviruses (HPVs) are small non-enveloped icosahedral viruses that infect the keratinocytes of skin and mucosa. The cutaneous HPV types are represented mainly by the beta and gamma genera, which are widely present in the skin of normal individuals. More than 40 beta-HPV types and 50 gamma-HPV types have been isolated, and these numbers are continuously growing. The main cause of non-melanoma skin cancer is exposure to ultraviolet radiation (UVR). However, cutaneous HPVs that belong to the beta genus may act as a co-carcinogen with UVR. The association between beta-HPVs and skin cancer was first reported in patients with epidermodysplasia verruciformis (EV), who frequently develop cutaneous squamous cell carcinoma (SCC) on sun-exposed areas. Isolation of HPVs from the lesions suggested that HPVs might act as a co-carcinogen with UVR in EV patients. Beta-HPVs may also play a role in cutaneous SCC in immunocompromised non-EV and in immunocompetent individuals. Several studies have reported an association of viral DNA and/or antibodies to beta HPV types with SCC. Interestingly, HPV prevalence and viral load decrease during skin carcinogenesis, being significantly higher in actinic keratosis than in SCC, suggesting that the virus may play a role in the early stages of tumour development (the "hit-and-run" hypothesis). Concordantly, in vivo and in vitro studies have shown that E6 and E7 from certain cutaneous HPV types display transforming activities, further confirming their potential role in carcinogenesis.

UV emissions from low energy artificial light sources

Energy efficient light sources have been introduced across Europe and many other countries world wide. The most common of these is the Compact Fluorescent Lamp (CFL), which has been shown to emit ultraviolet (UV) radiation. Light Emitting Diodes (LEDs) are an alternative technology that has minimal UV emissions. This brief review summarises the different energy efficient light sources available on the market and compares the UV levels and the subsequent effects on the skin of normal individuals and those who suffer from photodermatoses.

Collagens, collagen-binding heat shock protein 47 and transforming growth factor-beta 1 are induced in cicatricial pemphigoid: possible role(s) in dermal fibrosis.

Cicatricial pemphigoid (CP) is an autoimmune mucocutaneous blistering disease associated with scarring. Heat shock protein 47 (HSP47) is thought to play an important role in fibrogenesis, but its role in skin lesions of cicatricial pemphigoid is not yet known. In the present study, we examined the role of HSP47 in dermal fibrosis in cutaneous lesions of a CP patient. Skin biopsies from a patient with CP, and from normal subjects were studied for the expression of HSP47, and interstitial collagens (type I and type III collagens) by immunohistochemistry. Dermal fibroblasts isolated from skin of normal individuals and from fibrotic skin of a CP patient were used to study the expression of HSP47, transforming growth factor beta 1 (TGF-beta 1), type I and type III collagens. Compared to the control skin sections, an increased expression of HSP47 was associated with an increased deposition of interstitial collagens in the fibrotic skin section of the CP patient. Similarly, in contrast to control dermal fibroblasts, the fibroblasts isolated and cultured from fibrotic skin of the CP patient, and grown in vitro, exhibited increased expression of HSP47, type I and type III collagens. Furthermore, compared to the normal control fibroblasts, an increased expression of TGF-beta 1 was detected in the dermal fibroblasts isolated from fibrotic skin of the CP patient. When dermal fibroblasts were treated with various concentrations of TGF-beta 1 (6.25, 12.5, 25, 50 and 100 ng/ml for 24 h), it induced the expression of both type I collagen and HSP47, as determined by quantitative real-time PCR. In conclusion, the expression of TGF-beta 1, HSP47, type I collagen and type III collagen was up-regulated in the fibrotic skin of CP patient, and a complex interaction of these molecules may initiate and propagate the fibrotic cascade in the skin of CP patients.

Bacterial infections and atopic dermatitis.

Bacterial infections and atopic dermatitis.

Mosaic Pattern of Maternal and Paternal Keratinocyte Clones in Normal Human Epidermis Revealed by Analysis of X-Chromosome Inactivation

During early development of the female embryo, one X-chromosome is randomly inactivated in each cell. As a result of growth, migration, and differentiation, the adult female becomes a mosaic of cells with either the paternal or the maternal X-chromosome inactivated. It is not known what structure the X-chromosome inactivation pattern has in skin of normal individuals. We investigated normal skin from four healthy females, heterozygous for the HUMARA microsatellite on the X-chromosome. Following careful microdissection, DNA from adjacent epidermal samples consisting of approximately 35 basal keratinocytes was digested with the methylation-sensitive enzyme HpaII. The inactivated X-chromosome remained intact due to extensive methylation. The enzyme-digested DNA was amplified using polymerase chain reaction and fragments were analyzed for size. Through examination of adjacent samples and consecutive sections, we found normal human skin to be composed of a fine mosaic of tiles with either maternal or paternal X-chromosome inactivated. The sizes of these tiles were between 20 and 350 basal cells. The method described has the potential to resolve the clonal status in normal as well as pathologic conditions.

Genotypic and phenotypic characterization of Staphylococcus aureus strains isolated in subjects with atopic dermatitis. Higher prevalence of exfoliative B toxin production in lesional strains and correlation between the markers of disease intensity and colonization density

Staphylococcus aureus strains generally colonize eczematous lesions of subjects with atopic dermatitis much more frequently than in the skin of normal individuals. The aim of this study was to provide a detailed genotypic and phenotypic analysis of S. aureus strains colonizing four different sites (lesional and non-lesional skin areas, nasal and pharyngeal mucosas) of 49 patients with atopic dermatitis. The 88 isolates were analyzed in duplicate by pulsed field gel electrophoresis and in their exfoliative toxin A or B production by latex test. The patients were characterized by age, sex, severity scoring of atopic dermatitis and serum eosinophil cationic protein. Fourteen (28.6%) of the patients were completely negative for S. aureus while 35 (71.4%) were positive in at least one site. The severity scores and eosinophil cationic protein levels were significantly correlated variables ( P<0.001), linked to the colonization intensity ( P ranging between 0.05 and <0.001 depending on the site) and to the number of colonized sites ( P at least <0.01). The genotypic patterns, widely heterogeneous, showed no restriction to peculiar patterns. Only eight strains produced exfoliative toxin B which was significantly restricted to the lesional isolates ( P=0.012).

Human papillomaviruses: General features

Papillomaviruses (HPVs) are small non-enveloped icosahedral viruses that infect the keratinocytes of skin and mucosa. The cutaneous HPV types are represented mainly by the beta and gamma genera, which are widely present in the skin of normal individuals. More than 40 beta-HPV types and 50 gamma-HPV types have been isolated, and these numbers are continuously growing. The main cause of non-melanoma skin cancer is exposure to ultraviolet radiation (UVR). However, cutaneous HPVs that belong to the beta genus may act as a co-carcinogen with UVR. The association between beta-HPVs and skin cancer was first reported in patients with epidermodysplasia verruciformis (EV), who frequently develop cutaneous squamous cell carcinoma (SCC) on sun-exposed areas. Isolation of HPVs from the lesions suggested that HPVs might act as a co-carcinogen with UVR in EV patients. Beta-HPVs may also play a role in cutaneous SCC in immunocompromised non-EV and in immunocompetent individuals. Several studies have reported an association of viral DNA and/or antibodies to beta HPV types with SCC. Interestingly, HPV prevalence and viral load decrease during skin carcinogenesis, being significantly higher in actinic keratosis than in SCC, suggesting that the virus may play a role in the early stages of tumour development (the “hit-and-run” hypothesis). Concordantly, in vivo and in vitro studies have shown that E6 and E7 from certain cutaneous HPV types display transforming activities, further confirming their potential role in carcinogenesis.

Changes in the secretion and glycosylation of fibronectin by human skin fibroblasts associated with tuberous sclerosis.

Fibroblasts from skin and skin lesions of patients with tuberous sclerosis (TS) and from skin of normal individuals were grown in culture. ELISA showed that the spent medium of those derived from TS skin lesions contained significantly more fibronectin (FN) than spent medium from the other cells. Amino acid compositional analysis of the FN from TS and normal sources revealed no substantial differences. However the FN of fibroblasts from TS-skin lesions was shown by HPAEC to contain a two- to three-fold increased content of carbohydrate. The changed monosaccharide composition was consistent with an increased content of N- and O-linked glycans and with the former containing polylactosamine chains. Fibroblasts from a normal individual were shown to proliferate more slowly and to produce larger cells when grown on FN from a TS skin lesion compared to growth on FN from normal skin.

Alpha 1-adrenoceptors in normal and hyperalgesic human skin.

1. Evidence of an adrenergic component of cutaneous hyperalgesia has recently been obtained in animal models of painful peripheral neuropathy. These findings have prompted speculation that an increased density or sensitivity of peripheral alpha-adrenoceptors contributes to sensory abnormalities and chronic neuropathic pain in conditions such as reflex sympathetic dystrophy. However, it is not known whether alpha-adrenoceptors are present at the site of nociception, either in hyperalgesic or normal skin. 2. We used the selective radioligand 125I-hydroxyphenyl-ethyl-aminomethyl-tetralone (HEAT) to label alpha 1-adrenoceptors, and quantitative autoradiography to assess the relative density of these receptors in skin samples from seven normal individuals and from the hyperalgesic and pain-free limbs of five patients with reflex sympathetic dystrophy. The distribution of autoradiographic grains over the epidermis and dermis was investigated in 10 microns serial transverse sections. 3. alpha 1-Adrenoceptors were identified in the epidermis and dermal papillae of normal individuals, and in the hyperalgesic and pain-free skin of patients with reflex sympathetic dystrophy. The density of alpha 1-adrenoceptors was greater in the epidermis and dermal papillae than further down in the dermis. 4. The mean density of alpha 1-adrenoceptors was significantly greater in the hyperalgesic skin of patients than in the skin of normal individuals (35.4 grains/1000 microns2 compared with 15.5 grains/ 1000 microns2, P < 0.01). The mean density of alpha 1-adrenoceptors in the pain-free skin of patients (26.9 grains/1000 microns2) fell midway between receptor density in hyperalgesic skin and in the skin of normal individuals, and did not differ significantly from either. 5. Our findings indicate that alpha 1-adrenoceptors are present in the epidermis, and suggest that their numbers may be increased in the hyperalgesic skin of patients with reflex sympathetic dystrophy. Further studies need to identify the dermal and epidermal cell types that express high densities of alpha 1-adrenoceptors, and to investigate their normal function and role in neuropathic pain.

Adhesion molecules in atopic dermatitis: VCAM-1 and ICAM-1 expression is increased in healthy-appearing skin

In the skin of normal and atopic individuals, the expression of E-selectin (ELAM-1), L-selectin (LECAM-1), P-selectin (CD62), CD31 (PECAM), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and cutaneous lymphocyte antigen (CLA) were compared by immunostaining of skin biopsies which were taken from normal individuals (n = 17), the healthy-appearing skin of patients with atopic dermatitis (n = 10), and their acute (n = 5) and chronic (n = 6) skin lesions. In contrast to ELAM-1, the expression of VCAM-1 and ICAM-1 was found to be significantly increased in nonlesional atopic skin in comparison to the skin of normal individuals. Moreover, in contrast to normal skin of healthy individuals, nonlesional atopic skin showed a further increase of VCAM-1, ICAM-1, and ELAM-1 when cultured with medium alone. This suggests that certain adhesion molecules are constitutively upregulated in healthy-appearing skin of patients with atopic dermatitis. In addition, atopic skin appears to respond to nonspecific stimuli (such as culture with medium alone) with upregulation of VCAM-1, ICAM-1, and ELAM-1. It is suggested that the observed upregulation of adhesion molecules is mediated by the release of cytokines such as interleukin-4 from cells which reside in atopic skin. The question of whether the inherent upregulation of adhesion molecules in atopic skin contributes to the development of Th2 cells, which have been found to predominate in atopic inflammation, has to be further investigated.

Protein catabolism in fibroblasts cultured from patients with mucolipidosis II and other lysosomal disorders

Protein catabolism in fibroblasts cultured from the skin of normal individuals and of patients with mucolipidosis II (I-cell disease) and several other lysosomal storage diseases was examined by metabolic labelling with [3H]leucine and following the fate of radioactive proteins in pulse-chase experiments. In mucolipidosis II cells, overall protein degradative rates were found to be distinctly lower than in normal control cells. To distinguish lysosomal from non-lysosomal degradation, labelling experiments were carried out in the presence and absence of 10 mM NH4Cl, an inhibitor of lysosomal function. It was found that mucolipidosis II fibroblasts exhibited a markedly reduced rate of lysosomal protein degradation, whereas the rate of nonlysosomal degradation appeared normal. Serum and amino acid starvation led to a marked increase in lysosomal protein degradation in normal cells, but had only a minimal effect on that in mucolipidosis II fibroblasts. The specific activities of cathepsins B, H and L were profoundly diminished in all mucolipidosis II cell lines tested. Lysosomal protein degradation in a mucolipidosis III cell line was impaired to a similar degree as in mucolipidosis II cells, whereas it was decreased to a lesser extent in fibroblasts from patients with mucopolysaccharidoses I and VI, galactosialidosis and GM1-gangliosidosis. We conclude that fibroblasts from patients with mucolipidosis II and III have a severely compromised capacity for endogenous lysosomal protein degradation that appears to result from multiple cathepsin deficiency. This lysosomal defect is likely to have pathophysiological consequences.

In vivo Modulation of the High-Affinity Receptor for IgE (FcεRI) on Human Epidermal Langerhans Cells.

The presence of IgE-bearing epidermal Langerhans cells (LC) has been reported not only in patients with atopic eczema (AE) but also in patients with various inflammatory dermatoses unrelated to atopic diathesis. We have shown very recently that normal LC express the high-affinity receptor for IgE (FcεRI) which accounts for the binding of monomeric IgE on these cells. In the present study, we investigated the IgE-binding capacity of resident LC on cryosections from skin of normal individuals and from uninvolved and involved skin of patients with atopic eczema, psoriasis, lupus erythematosus and mycosis fungoides. When compared to LC from uninvolved or normal skin, an increased binding of IgE was observed in LC from lesional skin of all four diseases studied here. This binding could be blocked by anti-FcεRI mAb. Furthermore, increased IgE binding capacity of LC correlated to an increased expression of FcεRI on these cells. These results clearly demonstrate that the expression of FcεRI on LC is increased in inflammatory skin areas. Since this phenomenon is observed in several unrelated diseases, it suggests the presence in these conditions of a similar microenvironment which up-regulates the FcεRI expression on LC.

The metabolism of fibroblasts from normal and fibrotic skin is inhibited by minoxidil in vitro

The effects of minoxidil in vitro were studied using fibroblasts grown from the lesional skin of patients with lichen sclerosus et atrophicus, morphoea and from the skin of normal individuals. The proliferation of all fibroblast lines over 3 days was inhibited in proportion to the concentration of minoxidil, being 20% or less of controls at 1 mM, where cell viability was only marginally reduced (84 +/- 2% vs. 88 +/- 2% (SEM) in controls). At 5 mM there was usually a net loss of cells and only 72% of those remaining were viable. In contrast, minoxidil at 0.1-1 mM stimulated the proliferation of foreskin keratinocytes by up to 130%. Contraction of collagen lattices containing the three types of fibroblasts was inhibited by 22-26% with 1 mM minoxidil after 5 days and by 50-94% with 5 mM. Secretion of glycosaminoglycans by normal fibroblasts showed concentration-dependent reduction, being 25 +/- 6% of that of untreated cultures with 1 mM minoxidil. These findings show that minoxidil has a range of inhibitory effects on both normal and abnormal skin fibroblasts in vitro, which contrast with its stimulation of skin epithelial cells, and support suggestions that it may provide a useful topical treatment for keloids and other fibroses.

The Effects of Antipsoriatic Treatment on Cutaneous Blood Flow in Psoriasis Measured by 133Xe Washout Method and Laser Doppler Velocimetry

In 8 patients with psoriasis vulgaris, the cutaneous blood flow (CBF) was measured simultaneously in both involved and uninvolved psoriatic skin before (i.e., on the first day of hospitalization) and on the 3rd, 7th, 14th, and 28th days of treatment with tar. The 133Xe washout method was used after epicutaneous labeling and compared to the laser Doppler velocimetry (LDV) technique. Control experiments were performed in 10 normal individuals. Before treatment the mean CBF in involved psoriatic skin was 62.6 +/- 18.7 SD ml X (100 g X min)-1, which is significantly higher than CBF of uninvolved skin in psoriatic patients, 9.5 +/- 4.0 SD ml X (100 g X min)-1, (p less than 0.01) and is 13.6 times higher than CBF in the normal individuals (p less than 0.01). Fifty hours following onset of treatment (i.e., after only 2 applications of tar), mean CBF of the involved psoriatic skin had decreased significantly to 35.0 +/- 13.9 SD ml X (100 g X min)-1, (p less than 0.01), which was not statistically different from the CBF on the 7th day. During the following weeks, the CBF in involved psoriatic skin decreased at a more moderate rate than that observed during the first week and was 15.0 +/- 6.1 SD ml X (100 g X min)-1 on the 28th day. This value is not significantly different from the CBF of uninvolved skin in these patients. At the end of treatment, the CBF of the uninvolved skin had decreased significantly (p less than 0.05) in all the patients to values similar to those observed in the skin of normal individuals. A parallel decline was observed in a clinical psoriatic score index; however, it is not known whether the observed decrease in CBF was preceded or succeeded by the clinical improvement. A comparison of the 133Xe measurements and LDV measurements in the normal individuals by linear regression analysis yielded a correlation coefficient of -0.24 (N = 20, p greater than 0.05). In the skin of psoriatic patients the correlation coefficient was 0.01, (N = 47, p greater than 0.05) for unaffected skin, and 0.61 (N = 47, p less than 0.001) in the involved psoriatic skin sites. The LDV measurements did not reflect changes in the uninvolved skin in psoriatic patients during treatment and resulted in a remarkably high C.V. in the bilateral measurements of skin in normal individuals (43.8%) compared to the C.V. of 133Xe flow measurements (14.8%). It is concluded that while the LDV method gives a rough estimate of blood flow in cutaneous tissue with a high capillary perfusion rate, inaccurate measurements are made in skin areas with normal to twice the normal CBF range.

DNA Repair in Skin of Lupus Erythematosus Following In Vivo Exposure to Ultraviolet Radiation

The in vivo repair of pyrimidine dimers induced in the DNA of skin of 9 patients diagnosed as systemic or discoid lupus erythematosus (LE) was measured. A small area of the buttock was exposed to radiation emitted from a Burdick UV-800 sunlamp. The number of pyrimidine dimers was measured by incubating the epidermal skin DNA with UV-specific endonuclease and sedimenting the DNA through alkaline sucrose gradients. The initial number of dimers induced following sunlamp exposure was 7.6 +/- 1.8 per 10(8) daltons DNA. The level of photorepair was measured by illuminating an area of the skin with greater than 450-nm radiation immediately following sunlamp exposure. We found that 56.5 +/- 9.5% of the dimers are photorepaired with 5 min of illumination. Excision repair was measured in an area of the skin covered for 2 and 24 h postirradiation. Approximately 44 and 81% of the dimers induced immediately following sunlamp exposure were removed at these respective times. These observations in LE are similar to those observed in the skin of normal individuals.

Transvascular transport and distribution of fluid and protein in psoriasis

By combining the suction blister technic with studies of tracer protein kinetics, we have investigated the leakiness of the microvasculature and the distribution of extracellular water and plasma proteins in patients with psoriasis vulgaris. As in patients with extensive, erythrodermic skin diseases, we found a shift of fluid from the intra- to the extravascular spaces and an increased transcapillary escape rate of albumin (TER_alb), which is a measure of the relative efflux of macromolecules from the total microvasculature. The appearance rate of intravenously injected labeled albumin in the extravascular fluid of newly formed suction blisters was significantly elevated in involved psoriatic skin as compared to uninvolved skin and skin of normal individuals. The blister fluid concentration of four endogenous plasma proteins (albumin, transferrin, IgG, and α₂-macroglobulin) was within normal range in involved skin and decreased in uninvolved skin. The calculated intravascular and extravascular masses of albumin were decreased but distributed normally within and outside the vascular system. It is suggested that the reduction in total albumin mass might be induced by an increased extravasation of albumin through the psoriatic skin vessels, causing an increased fractional catabolism of endogenous albumin. It is concluded that the leakiness of the microvasculature of involved psoriatic skin is increased, and this increase is compensated by an increased lymphatic drainage. These findings are discussed in the light of previously described histologic alterations in psoriatic skin.

Comparative Studies of Keratins Isolated from Psoriasis and Atopic Dermatitis

Keratin proteins were extracted from scales of normal skin, clinically active psoriatic lesions, and atopic dermatitis. Filaments prepared by in vitro assembly upon dialysis of the proteins against a low ionic strength buffer were comparatively characterized by electron microscopy, SDS gel electrophoresis, and amino acid analysis. Filaments formed using keratin obtained from the skin of normal individuals were thin and wavy, whereas those formed using keratin isolated from the scales of psoriatic patients were straight and showed a tendency to assemble side by side. Filaments of atopic dermatitis were indistinguishable from those of normal individuals. Filaments of both normal and atopic dermatitis contained the protein band of 67,000 daltons, which was absent in filaments of psoriasis. In contrast, 2 protein bands of 54,000 and 57,000 daltons were only detectable in psoriasis. Amino acid analysis of these filaments further demonstrated that filaments of psoriasis differ from those of normal individuals in that they have a glycine content that is 60% of normal.

Characterization of human fibroblasts from cancer patients: Loss of fibrin clot retractile activity after ‘ in vitro’ spontaneous transformation

Human fibroblast-like cells cultured from skin and stromal tissue of lung carcinomas, are able to induce fibrin clot retraction similarly to fibroblasts from the skin of normal individuals. Following in vitro spontaneous transformation, these cells loose their retractile property; fibrin clot retraction could be a rather specific parameter to identify fibroblastic cells and to monitor their in vitro oncogenic transformation.

Male pseudohermaphroditism presumably due to target organ unresponsiveness to androgens. Deficient 5alpha-dihydrotestosterone binding in cultured skin fibroblasts.

Maximum specific 5alpha-dihydrotestosterone (DHT) binding activity (Bmax) had been measured in intact confluent monolayers representing fibroblast strains derived form nongenital and genital (labium majus) skin of normal individuals and of 11 patients fulfilling the clinicogenetic criteria of complete testicular feminization (TF). Nine labium majus strains from adult females had a mean Bmax value three times greater than that of seven nongenital strains from adult females (33 vs. 11 fmol/mg cell protein). The Bmax results for 13 adult nongenital strains varied from 5.6 to 23.3 fmol/mg protein; the values for males and females had very similar means and ranges. The variation could not be correlated with the chronologic age of adult skin explant donors or with the in vitro age (mean population doubling level) of the cultures assayed. The Bmax activities of three nongenital strains from normal infants (two male, one female) did not exceed 5 fmol/mg protein. Seven of eight nongenital TF strains had Bmax values below 2 fmol/mg protein; the value for the eighth coincided with the lower limit of normal adults. The lower limit of DHT binding in normal labium majus strains was 15 fmol/mg protein. Three of five labial strains from patients with TF had Bmax values close to zero; the other two fell between 10 and 15 fmol/mg protein. It is apparant that labial skin fibroblast strains from clinically homogeneous patients with TF had highly variable degrees of DHT binding deficiency, and that they permit a more reliable diagnosis of severe and intermediate degrees of DHT binding deficiency than do strains of nongenital skin fibroblasts.