In vivo percutaneous absorption: a key role for stratum corneum/vehicle partitioning

Summary Percutaneous absorption of five compounds was studied in the hairless rat in vivo: benzoic acid, caffeine, hydrocortisone, inulin and thiourea. The results clearly demonstrate that, as with in vitro experiments, a steady-state flux can be achieved in vivo. This steady-state flux is strongly molecule dependent. Thus, the values for inulin and benzoic acid differ by a factor of about 40. In contrast, although the physicochemical properties of the studied compounds vary widely, their lag times were not significantly different. The mean lag time was 11±2 min. Different compounds could be considered to have approximately the same apparent diffusion coefficient with regard to their percutaneous absorption in vivo. Thus, for a given thickness of stratum corneum and a given anatomical site, the penetration flux value of a substance depends only on its stratum corneum/vehicle partition coefficient. Using a classical model, we have demonstrated that the amount of substance present in the stratum corneum (Q _sc) at equilibrium (30 min) is related to this partition coefficient. There is also a linear relationship between steady-state flux and Q _sc. In practice, the in vivo steady-state flux of penetration of a compound can be predicted from the simple measurement of the amount present in the stratum corneum after a contact time of 30 min.     

In vivo ultrastructural localization of the desmoglein 3 adhesive interface to the desmosome mid-line

Desmoglein (Dsg) is a cadherin cell-cell adhesion molecule located in desmosomes and its precise mechanism for cell-cell adhesion still remains to be elucidated. Opposing cadherin molecules may adhere to the N-terminal EC1 domains, or the entire length of the extracellular (EC) domains may overlap. To solve this controversy, we performed immunoelectron microscopy to map the Dsg3 epitopes in desmosomes. Three different hybridoma cell lines producing anti-Dsg3 monoclonal antibodies (mAb) were intraperitoneally injected into immunodeficient mice and the precise ultrastructural location of bound IgG between the mucosal epithelial cells in vivo was statistically measured and analyzed. The binding site of the AK23 mAb that recognizes the N-terminal EC1 domain was localized to the electron-dense mid-line of desmosomes. The binding sites of AK7 and AK18, which recognize the C-terminal membrane proximal and middle portions of the EC domains, were localized to the desmosomal region proximal to the membrane and the region between the plasma membrane and the dense mid-line, respectively. These results indicate that the N-terminal regions of Dsg3 from opposing cells interact at the dense mid-line of desmosomes where EC1 overlaps.     

pU-VEGF-siRNA对裸鼠恶性黑素瘤生长影响的研究

目的 研究pU-VEGF-siRNA对恶性黑素瘤成瘤和凋亡的影响及其机制.方法 构建针对血管内皮生长因子(VEGF)的发卡样siRNA真核表达载体pU-VEGF-siRNA,通过电穿孔法将构建重组体导入人恶性黑素瘤细胞系A375,并建立pU-VEGF-siRNA转染细胞荷瘤裸鼠模型,应用免疫组织化学方法检测荷瘤裸鼠肿瘤组织VEGF和血管内皮细胞特异性Ⅷ因子相关抗原(FⅧRAg)的表达,依据FⅧRAg的表达评价肿瘤微血管密度,末端脱氧核苷酸转移酶标记法(TUNEL)定量检测荷瘤裸鼠模型的肿瘤组织凋亡.结果 体内实验表明,实验组成瘤率明显低于对照组,且其肿瘤生长速度也明显减慢(P<0.01).实验组VEGF表达和肿瘤微血管密度明显低于对照组(P<0.01).实验组可见大量凋亡细胞,对照组仅见少许凋亡细胞,实验组凋亡指数与对照组相比差异有统计学意义(P<0.01).结论 通过RNA干扰技术阻断VEGF的表达,在裸鼠体内可显著抑制恶性黑素瘤生长.     

Preparation of [3H]collagen for studies of the biologic fate of xenogenic collagen implants in vivo

Reduction of a commercially available, pepsin-solubilized, bovine dermal collagen (Vitrogen 100) with sodium [3H]borohydride provided radiolabeled collagen preparations with specific activities ranging from 7.1-12.0 muCi/mg collagen. These specific activities were 2-3 times greater than those obtained by reduction of intact rat tail tendon collagen under similar conditions. The alpha, beta, and higher aggregate components of type I collagen were radiolabeled as well as the alpha component of a small amount of type III collagen present in the samples. Fractionation of cyanogen bromide peptides showed that alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB3,5 were the predominant peptides labeled by this procedure. Amino acid analysis indicated that the majority of the radioactivity was in reducible cross-links, precursors of these cross-links, and in hexosyllysine residues. Reconstitution experiments comparing this radiolabeled collagen with nonlabeled collagen showed them to be indistinguishable. Bacterial collagenase digestion of this reconstituted fibrillar collagen in both a lightly cross-linked (glutaraldehyde 0.0075%) and noncross-linked form provided evidence that digestion of labeled and nonlabeled collagens proceeded at similar rates. Thus, labeling did not change the properties of the collagen. Cross-linking made the preparation refractory to proteolytic degradation. Injection of fibrillar collagen preparations, spiked with radiolabeled collagen, into the guinea pig dermis followed by quantitation of the amount of radioactivity recovered from implant sites as a function of time, indicated that the lightly cross-linked samples also were more resistant to degradation in vivo than the noncross-linked preparation. The half-life of noncross-linked collagen was about 4 days while that of the cross-linked collagen was about 25 days. These degradation rates were much faster than observed for similar, nonlabeled samples injected into the dermis of humans, presumably due to a higher metabolic activity in the guinea pig dermis.     

A role for the androgen receptor in collagen content of the skin

Collagen, the major macromolecular component of skin, is responsible for maintaining the structural integrity of the tissue as well as for providing important functional characteristics, such as pliability and thickness. We have been studying the structure and regulation of collagen in mouse mutations affecting the skin. In the course of these studies, we found that there are significant differences in collagen content between the skin of wild-type male and female mice, which become evident at puberty. Furthermore, male mice with an X-linked mutation in the androgen receptor gene (formerly called testicular feminization and abbreviated as Ar(Tfm)) showed decreased levels of collagen, indicating that the androgen receptor pathway contributes to the observed differences. These findings demonstrate that there are striking differences in the collagen content of skin between male and female mice, and provide a biochemical explanation for these differences.     

In vivo reflectance confocal microscopy detects pigmentary changes in melasma at a cellular level resolution

Please cite this paper as: In vivo reflectance confocal microscopy detects pigmentary changes in melasma at a cellular level resolution. Experimental Dermatology 2010; 19 : e228–e233. Abstract: Melasma is a frequent pigmentary disorder caused by abnormal melanin deposits in the skin. In vivo reflectance confocal microscopy (RCM) is a repetitive imaging tool that provides real‐time images of the skin at nearly histological resolution. As melanin is the strongest endogenous contrast in human skin, pigmentary disorders are the most suitable candidates for RCM examination but RCM features of melasma have never been reported. This study investigates the pilot use of RCM in melasma to provide a set of well‐described morphological criteria with histological correlations. RCM images were acquired from melasma skin and compared to adjacent control skin in 26 patients. Skin biopsies were obtained from eight patients. In the epidermis, RCM showed in all patients a significant increase in hyperrefractile cobblestoning cells. These cells corresponded to hyperpigmented basal keratinocytes in histology. In six patients, dendritic cells corresponding to activated melanocytes were also found in the epidermis. In the dermis, RCM identified in nine patients plump bright cells corresponding to melanophages. Interestingly, for a given patient, the topographic distribution of melanophages in melasma lesions was very heterogeneous. RCM also showed a significant increase in solar elastosis and blood vessels in the dermis. RCM is a non‐invasive technique that detects pigmentary changes in melasma at a cellular level resolution. Therefore, RCM provides an innovative way to classify melasma by pigment changes.     

Localization of the alpha 3 (V) chain of type V collagen in human skin

Serum from goats immunized with human type V collagen chains that were cut out of polyacrylamide gels contained an antibody that recognized only type V collagen in an enzyme-linked immunoabsorbent assay and did not label laminin, fibronectin, or types I and IV collagen. Western blot analysis of the antibody showed that its determinant was the alpha 3 (V) chain of type V collagen. Indirect immunofluorescent staining of intact human skin with the antibody produced staining of the dermal blood vessels but not of the dermal-epidermal junction (DEJ). In contrast, both the dermal blood vessels and the DEJ were labeled by the antibody if the skin substrate was first split through the lamina lucida region of the DEJ by incubation in 1 M NaCl solution. Indirect immunoelectron microscopy confirmed the staining pattern found by immunofluorescence and defined the ultrastructural localization of type V collagen in skin. Type V collagen is localized within the DEJ to the lamina lucida region and polar aspects of the basal cell keratinocyte plasma membrane.     

Filamentous aggregates of collagen. Ultrastructural evidence for collagen-fibril degradation in situ

Summary Filamentous aggregates of collagen are distinct structures in the pathological dermis. These aggregates are distinguishable from fibrous long-spacing collagen (in vitro and at biopsy) and the Luse body. The aggregates are produced from dermal collagen fibrils by clostridial collagenase and culture-medium extract, which supposedly contains cellular collagenase at a neutral pH, as well as by organ cultures. In vitro experiments showed that carrageenan granuloma contains fibrous long-spacing collagen and segment long-spacing collagen. The granuloma also contains the aggregates. The aggregates were found in skin biopsies from syphilitic chancres, acrosclerotic scleroderma, morphea, mycosis fungoides, myeloid leukemia, mastocytosis and malignant melanoma. These findings indicate that the aggregates are products of the in situ degradation of collagen fibrils by some collagenolytic factor. This factor may originate in fibroblast-like cells, reticulum cells, leukemia cells, mast cells and melanoma cells.     

In vivo reflectance confocal microscopy image interpretation for the dermatopathologist

Reflectance confocal microscopy (RCM) is a technology utilized for bedside diagnosis of cutaneous pathology by non‐invasive, in vivo, cellular‐level imaging. With the recent establishment of reimbursement codes by the US Centers for Medicaid and Medicare Services, RCM is now likely to be employed by clinical dermatologists and impact decision making on skin cancer management. Dermatopathologists, therefore, would benefit from learning how to interpret RCM images and how RCM findings correlate with histopathological criteria of diagnosis. This review briefly explains the principles behind RCM image acquisition, describes the key RCM features of normal skin, and delineates the RCM characteristics of frequently observed benign and malignant neoplasms.     

In vivo persistent pigment darkening method: a demonstration of the reproducibility of the UVA protection factors results at several testing laboratories

BACKGROUND/PURPOSE: The aims of the present studies were to check that persistent pigment darkening (PPD) method can produce accurate and reproducible results for high UVA protection factors (UVAPF) and to provide data on the variability between laboratories and on the influence of skin types. METHODS: The Japanese Cosmetic Industry Association (JCIA) PPD method was used to determine the UVAPF in different laboratories of different sunscreen formulations with increasing UVAPF. Two formulations were tested at seven independent laboratories and five products within two laboratories. The influence of skin types on the UVAPF of two products was tested within one laboratory on two panels of volunteers. All laboratories complied with the JCIA method specifications. RESULTS: Reproducible results have been obtained between the different labs and a low and satisfactory variability was achieved with a panel size of 10 subjects. Furthermore, skin type was demonstrated to have no influence within the defined selection criteria. CONCLUSIONS: From this multiple center testing, the PPD method has been shown to be appropriate for testing sunscreen formulations with UVAPFs above 20. It is reasonable to expect that test results will be consistent if an identical protocol is used between laboratories.     

In vivo and in vitro evidence for epidermal H2O2‐mediated oxidative stress in piebaldism

Please cite this paper as: In vivo and in vitro evidence for epidermal H₂O₂‐mediated oxidative stress in piebaldism. Experimental Dermatology 2010; 19 : 883–887. Abstract: Piebaldism is characterised by the absence of pigment in patches on the skin, usually present at birth. Mutations in the kit gene are documented. Clinically this disorder can mimic vitiligo. Here, we show for the first time the presence of oxidised pteridine‐induced fluorescence in association with H₂O₂‐mediated stress in piebald patches employing Wood’s light and in vivo FT‐Raman spectroscopy. In situ immunofluorescence data revealed low catalase and methionine sulphoxide reductase A (MSRA) levels whereas thioredoxin reductase and methionine sulphoxide reductase B (MSRB) are not affected. We also show low superoxide dismutase levels in these patients. The presence of thioredoxin reductase provides capacity to reduce H₂O₂, a mechanism which is absent in vitiligo. Importantly, this enzyme reduces biopterin back to the functioning cofactor 6‐tetrahydrobiopterin. The absence of MSRA indicates deficient methionine sulphoxide repair in the cytosol, meanwhile the presence of MSRB is helpful to protect the nucleus. Taken together, we have identified H₂O₂‐mediated stress in piebald skin with distinct differences to vitiligo.     

Pretibial epidermolysis bullosa: is this case a new subtype with loss of types IV and VII collagen?

Pretibial epidermolysis bullosa (PEB) is an extremely rare subtype of dominant dystrophic epidermolysis bullosa (DDEB), in which recurrent blistering with scarring predominantly involves the pretibial skin. Nail dystrophy, albopapuloid lesions, and hypertrophic scars may also occur. In PEB, immunohistochemical and electron microscopic studies demonstrate the complete or partial loss of the anchoring fibril (AF) in the basement membrane zone, suggesting disturbed synthesis or excessive degradation of collagen VII, the main component of AF. Interestingly, we report a case of PEB with unusual results of joint loss of types IV and VII collagen.     

In vivo stimulation of de novo collagen production caused by cross-linked hyaluronic acid dermal filler injections in photodamaged human skin

OBJECTIVE: To determine whether endogenous synthesis of new extracellular matrix may contribute to the degree and duration of clinical benefits derived from cross-linked hyaluronic acid dermal filler injections. DESIGN: In vivo biochemical analyses after filler injections. SETTING: Academic referral center. PARTICIPANTS: Eleven healthy volunteers (mean age, 74 years) with photodamaged forearm skin. Interventions Filler and vehicle (isotonic sodium chloride) injected into forearm skin and skin biopsy specimens taken 4 and 13 weeks later. MAIN OUTCOME MEASURES: De novo synthesis of collagen, the major structural protein of dermal extracellular matrix, was assessed using immunohistochemical analysis, quantitative polymerase chain reaction, and electron microscopy. RESULTS: Compared with controls, immunostaining in skin receiving cross-linked hyaluronic acid injections revealed increased collagen deposition around the filler. Staining for prolyl-4-hydroxylase and the C-terminal and N-terminal epitopes of type I procollagen was enhanced at 4 and 13 weeks after treatment (P<.05). Gene expression for types I and III procollagen as well as several profibrotic growth factors was also up-regulated at 4 and 13 weeks compared with controls (P<.05). Fibroblasts in filler-injected skin demonstrated a mechanically stretched appearance and a biosynthetic phenotype. In vitro, fibroblasts did not bind the filler, suggesting that cross-linked hyaluronic acid is not directly stimulatory. CONCLUSIONS: Injection of cross-linked hyaluronic acid stimulates collagen synthesis, partially restoring dermal matrix components that are lost in photodamaged skin. We hypothesize that this stimulatory effect may be induced by mechanical stretching of the dermis, which in turn leads to stretching and activation of dermal fibroblasts. These findings imply that cross-linked hyaluronic acid may be useful for stimulating collagen production therapeutically, particularly in the setting of atrophic skin conditions.