Acceleration of de novo cholesterol synthesis in the epidermis influences desquamation of the stratum corneum in aged mice.

Cholesterol, a component of intercellular lipids, is important for stratum corneum (SC) homeostasis, including its barrier function and desquamation. However, cholesterologenesis in the epidermis decreases under basal conditions with aging. We found that the number of horny layers in murine SC increased with the decrease of desquamation in the outermost corneocytes associated with aging. The cholesterol content decreased and the cholesterol sulfate content increased in the horny layer with aging, which resulted in an increase in the ratio of cholesterol sulfate to cholesterol. Moreover, we investigated the effects of accelerated cholesterologenesis on desquamation in aged murine skin following topical application of mevalonic acid. The ratio of cholesterol sulfate to cholesterol in aged murine SC significantly decreased following topical treatment with mevalonic acid, which resulted from an increase in cholesterol content via the acceleration of cholesterologenesis. Treatment with mevalonic acid also significantly reduced the number of cell layers in the SC along with the acceleration of desquamation, as measured by desmoglein I content, corneocyte surface area and proteinase activity. These results indicate that an improvement in the ratio of cholesterol sulfate to cholesterol content by de novo cholesterologenesis may be important for desquamation of the SC in aged epidermis.

Topical mevalonic acid stimulates de novo cholesterol synthesis and epidermal permeability barrier homeostasis in aged mice.

Extracellular lipids of the stratum corneum, which are composed of cholesterol, fatty acid, and ceramides, are essential for the epidermal permeability barrier function. With damage to the barrier, a decreased capacity for epidermal lipid biosynthesis in aged epidermis results in an impaired repair response. Mevalonic acid is an intermediate after the rate-limiting step in cholesterol biosynthesis, which is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase. In the present study, we investigated the effect of topical mevalonic acid on the murine epidermal permeability barrier function, comparing it with that of cholesterol. Topical treatment with acetone caused linear increases in transepidermal water loss, in proportion to the number of treatments more rapidly in aged mice than in young mice. Administration of mevalonic acid on aged murine epidermis enhanced its resistance against damage and the recovery rate of barrier function from acute barrier disruption. In contrast, although cholesterol also had the same effect, it required a much higher amount than mevalonic acid. In young mice, neither mevalonic acid nor cholesterol had any effect on resistance against acetone damage nor the recovery rate from acetone damage. In the skin of mice topically administered with mevalonic acid, stimulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were both observed, whereas none was seen with stimulation by equimolar cholesterol. These data indicate that a topical application of mevalonic acid enhances barrier recovery in aged mice, which is accompanied by not only acceleration of cholesterol synthesis from mevalonic acid but also stimulation of the whole cholesterol biosynthesis.

Growth factors in infant germinal matrix: relationship to extracellular matrix and cell adhesion molecules.

We examined the expression of selected growth factors, growth factor receptors, elements of extracellular matrix and cell adhesion molecules in the germinal matrix layer (GML) utilizing immunohistochemistry and reverse transcriptase polymerase chain reaction. At autopsy brain samples from 10 neonatal infants were used. Epidermal growth factor receptor (EGFR) was significantly expressed in the matrix cells. While transforming growth factor alpha and heparin-binding epidermal growth factor-like growth factor were found in the matrix cells or vascular wall as ligands, epidermal growth factor was not expressed. EGFR and its ligands are thought to be important factors for the maintenance of the matrix cells and cell-to-cell interactions. Insulin like growth factor I, its receptor Ibeta and tenascin were found in the stroma of the GML and periventricular region. Vascular endothelial growth factor and receptor Flk-1, laminin A and B2, fibronectin, collagen type IV and integrins such as beta3, alpha5beta1 and alphaVbeta3 were found mainly in or around the vascular wall indicating their important roles for vascularization. Transforming growth factor beta2 and its receptor II were expressed in the matrix cells and/or vascular wall suggesting a role in proliferation and/or regression of the vasculature. CD44 and Thy-1 were also expressed in the matrix cells.

Cytologic and genetic study of polyomavirus-infected or polyomavirus-activated cells in human urine.

OBJECTIVE: To carry out a morphologic and genetic study of human polyomavirus infection or activation in child and adult urine specimens. DESIGN AND SETTING: The study was carried out on 16 urine samples from children with human polyomavirus infection and 104 samples from adults with virus activation identified among 18800 consecutive urine samples (0.64%). RESULTS: All specimens from children showed numerous typical intranuclear inclusion-bearing (INIB) cells. All adult specimens with cytological features similar to childhood specimens were defined as type 1 adult cases. We identified 14 adult cases with marked immunologic suppression as type 1 cases. The inclusions were large, homogeneous, and basophilic, and they were mainly attributable to the BK virus, as demonstrated by a polymerase chain reaction. These infected or activated cells revealed features demonstrating their origin in the superficial transitional epithelium of the urinary tract. Adult cases with different cytologic features were designated as type 2 adult cases. In type 2 adult cases, the number of virus-activated cells was lower, and degenerated intranuclear inclusion-bearing cells with a coarse chromatin pattern were observed in most cases. These characteristics were identified in 90 adults without immunologic suppression. A polymerase chain reaction with BamHI digestion demonstrated JC virus DNA in nearly all of these specimens. CONCLUSION: The JC virus-activated cells found in type 2 adult cases and the BK virus-infected cells found in childhood cases were not of clinical importance. However, the BK virus-activated cells associated with immunologic suppression may have prognostic significance.

Galactocerebroside and not glucocerebroside or ceramide stimulate epidermal beta-glucocerebrosidase activity.

Glycosphingolipids including glucocerebroside (GluCer) and galactocerebroside (GalCer) have been recognized as bioreguratory lipids by our group and others. In addition, our recent study demonstrated that GalCer corrects dry skin conditions in humans. The processing of stratum corneum lipids, which occurs when beta-glucocerebrosidase (beta-GluCer'ase) changes GluCer to ceramide (Cer), is required to form the epidermal permeability barrier. We herein investigated the effects of GluCer, GalCer and Cer on the processing of GluCer to Cer by assaying epidermal beta-GluCer'ase in mice (155%, P < 0.01) when compared to vehicle treated controls, while neither GluCer nor Cer had this effect. Studies using inhibitors of beta-GluCer'ase or beta-galactosidase and measuring the optimum pH of the enzyme verified that GalCer specifically activated beta-GluCer'ase. We confirmed that GalCer significantly increased beta-GluCer'ase activity in the outer epidermal fraction (172%, P < 0.01) and that the activation of beta-GluCer'ase is not due to a direct activating effect of GalCer on the enzyme. Furthermore, the induction of beta-GluCer'ase activity by GalCer was also observed in cultured normal human deratinocytes (123%, P < 0.01). Finally, acylceramide content in stratum corneum was increased in mice treated with GalCer (194%, P < 0.0005). These results indicate that GalCer appears to affect the Cer construct in the stratum corneum by the activation of beta-GluCer'ase, which ultimately contribute to an enhancement of barrier formation.

Structural and biochemical basis for the UVB-induced alterations in epidermal barrier function.

Ultraviolet light (UVR) induces a myriad of cutaneous changes, including delayed disruption of the permeability barrier with higher doses. To investigate the basis for the UVB-induced barrier alteration, we assessed the epidermal lamellar body secretory system at various time points before and after barrier disruption with a single high dose of UVB (7.5 MED) to murine epidermis. Morphological data were correlated with changes in epidermal proliferation and lipid synthesis, indicative of lamellar body generation. Twenty-four hours following UVB, the stratum corneum (SC) is normal, but a layer of abnormal, vacuolated, and lamellar body (LB)-deficient cells is present, immediately beneath the stratum granulosum (SG)/SC interface. Immediately subjacent to this band of damaged cells, normal keratinocytes that contain intact LBs are present. By 72 h, concomitant with the appearance of a barrier abnormality, extensively damaged cells persist at the SC/SG interface, and abnormal lamellar membrane structures appear in the lower SC. Upper stratum spinosum (SS) and lower SG cells appear normal, with increased numbers of LBs. A barrier abnormality is still present at 96 h, in association with membrane abnormalities in the lower SC interstices, but up to four normal appearing, subjacent SG cell layers are present. By 120 h, accelerated LB formation and precocious LB extrusion occur throughout the thickened SG; normal lamellar membranes are present in the lower SC; and barrier recovery is almost complete. Whereas, epidermal synthesis of the major barrier lipid species (i.e., cholesterol, fatty acids, and ceramides, including acylceramides) is reduced or unchanged at 24 and 48 h, it increases significantly 72 h after exposure to UVB. Therefore, the delayed disruption of the permeability barrier following acute UVB exposure results from the arrival of a band of lamellar body-incompetent (i.e., damaged) cells at the SG/SC interface. The subsequent, rapid recovery of the barrier, in turn, results from compensatory hyperplasia of subjacent, undamaged SS/SG cells, generating increased numbers and contents of LB. These results underscore the critical role of the stratum compactum in mediating barrier function, and suggest that beneficial therapeutic effects of UV exposure may be due to enhanced lipid production and barrier regeneration.

UVB-induced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response.

UV irradiation induces a variety of cutaneous responses, including disruption of epidermal permeability barrier function, the basis for which is not known. Herein, we investigated the separate roles of hyperproliferation and inflammation in the pathogenesis of UVB-induced barrier disruption. Adult hairless mice were exposed to increasing doses of UVB (1.5-7.5 MED), and transepidermal water loss (TEWL) was monitored daily for up to 7 d. The extent of TEWL increase was dependent on the UVB dose, but with all doses, the increase began after > or =48 h and peaked at 96 h, decreasing by 120 h. Epidermal [(3)H]thymidine incorporation increased at 24 h and peaked at 48 h (570%), preceding the maximal increase in TEWL. Cyclosporin A, methotrexate, 5-fluorouracil, or arabinosylcytosine significantly diminished the UVB-induced TEWL increase. Athymic nude mice also displayed a markedly diminished response to UVB, and DNA synthesis did not increased at 48 h. Transplantation of athymic mice with T-cell-enriched mixed immune cells significantly restored sensitivity to both the UVB-induced hyperproliferation and the barrier defect. Finally, although UVB exposure increased PGE2 levels in whole skin samples (2- to 3-fold within 1-3 h; p < 0.005), this increase was completely blocked by topical indomethacin, and neither topical indomethacin nor topical glucocorticoids blocked development of the barrier abnormality. These results show that (i) UVB produces delayed alteration in barrier function and (ii) both an epidermal proliferative response and thymocyte-mediated events (but not PGE2 production and nonspecific inflammation) appear to contribute to UVB-induced abrogation of the permeability barrier.

Intrinsically aged epidermis displays diminished UVB-induced alterations in barrier function associated with decreased proliferation.

Ultraviolet (UV) irradiation of the skin induces a variety of responses in the epidermis, including sunburn cell formation, epidermal hyperplasia, and a dose-dependent permeability barrier abnormality, an effect that appears to be dependent upon both UVB-induced hyperplasia and T-cell activation. Since intrinsically aged epidermis displays decreased epidermal turnover, diminished inflammatory response to various stimuli, including UVR, and impaired immune function, we investigated the effects of UVB on both epidermal barrier function and proliferation in hairless mice of increasing chronologic age (27, 61, and 90 wk). After a single UVB exposure (0.15 J/cm2 7.5 MED), a barrier abnormality developed (i.e., increased transepidermal water loss; TEWL), after a delay of > or = 48 h, regardless of age. In young mice (27 wk old), TEWL levels peaked at 72-96 h (9.9-fold over untreated controls), whereas increased epidermal [3H]thymidine incorporation preceded the peak TEWL increase (i.e., approximately 570% increase over controls at 48 h). In contrast, the UVB-induced increased in both TEWL and DNA synthesis were significantly diminished, with decreased epidermal hyperplasia evident, in intrinsically aged versus young mouse epidermis. Baseline epidermal thickness decreased with animal age (i.e., 16.8 +/- 3.1 vs. 27.9 +/- 0.7 microm for 90- vs. 27-wk-old animals, respectively; p < 0.02), suggesting that the diminished barrier response with aging reflects an attenuation of events subsequent to initial UVB exposure, rather than an increase in the UV dose delivered. These results demonstrate that (i) murine epidermis becomes less sensitive to UVB-induced barrier alterations with age and (ii) decreased DNA synthesis after UVB correlates with the age-related decrease in barrier dysfunction.

Effects of ethyl alpha-D-glucoside on skin barrier disruption.

Daily treatments of skin in hairless mice with concentrates of rice wine, Japanese traditional alcohol, lowered transepidermal water loss levels compared to the controls on the 3rd day after ultraviolet B (UVB) irradiation. These findings indicate that the concentrates of rice wine suppress the murine skin barrier disruption caused by UVB. Ethyl alpha-D-glucoside (alpha-ethylglucoside), one of the peculiar components in rice wine, showed the same effect, whereas beta-ethylglucoside had no effect. In order to clarify the functions of alpha-ethylglucoside on murine skin, we examined the effects of this compound on the expression of some phenotypes in human keratinocytes in vitro. As a result, alpha-ethylglucoside as well as beta-ethylglucoside enhanced cell proliferation weakly, and the formation of cornified envelopes and differentiated type keratin (K1) in keratinocytes was accelerated by alpha-ethylglucoside but not by beta-ethylglucoside. From the results, we conclude that alpha-ethylglucoside enhanced the differentiation of keratinocytes, which might be related to reduced barrier disruption by UVB.

Age-related changes in branched-chain fatty acid concentration of the skin surface lipid from hairless mouse.

Age-related changes in branched chain fatty acid (BCFA) concentration were studied with the skin surface lipid from hairless mice. A large proportion of BCFA was present in the cholesterol ester (CE) and wax diester (WDE) fraction of the skin surface lipid from hairless mice. The concentration of iso-series BCFA was highest at infancy and decreased with advancing age in both CE and WDE fraction. The concentration of anteiso-series BCFA appeared to be constant throughout the experiment.

1-O-alkyl-2,3-diacylglycerols in the skin surface lipids of the hairless mouse.

A neutral lipid class was isolated by thin-layer chromatography from the skin surface lipids of the hairless mouse. The fraction migrated faster than triglycerides and had a migration rate similar to that of diacyl alkanediols (diester wax). Upon deacylation, however, the long-chain diols were identified as 1-alkylglycerol ethers based on their chromatographic properties and on the mass spectra of their nicotinylidene derivatives. Thus, the skin lipid fraction was identified as 1-O-alkyl-diacylglycerol. The alkyl moieties were all saturated and even-numbered and ranged in chainlength from C16 to C22 with 1-O-hexadecylglycerol amounting to 34% of the total glycerol ether moieties. The fatty acids derived from this lipid fraction were mostly monoenoic with chainlengths ranging from C16 to C24. The major acyl component was eicosenoic acid (20:1) representing 61% of the total fatty acids.

Elastofibroma dorsi. Cytologic, histologic, immunohistochemical and ultrastructural studies.

Cytologic, light and electron microscopic, and immunohistochemical studies were conducted on a case of elastofibroma. Aspiration cytology showed a characteristic "braidlike" or "fern leaf-like" structure. Immunohistochemically the accumulate was shown to be elastin. Transmission electron microscopy indicated electron-dense, granular aggregates surrounded by microfilaments and collagen, while scanning electron microscopy revealed balls with a ball-of-yarn-like structure consisting of small fibrils, probably of elastin. These structures are unique to this disease and useful for diagnosis.

S-100 protein in glands within decidua and cervical glands during early pregnancy.

Strong immunoreactivity with polyclonal S-100 protein antisera and monoclonal S-100 alpha subunit antiserum was found in glandular cells of the decidua basalis and cervical polyps during early pregnancy. Immunoreactive S-100 protein was negative in glandular cells of the endometrium and cervix of nonpregnant women. It was also negative in endometrial hyperplasia and endometrial carcinoma. While the function of S-100 protein is not known, a relationship between humoral factors related to pregnancy and expression of S-100 protein gene is suggested by the results of this study.