Time course effect and sequential morphological changes of subepidermal nerve fibers in guinea pig skin after application of histamine ointment: a laser scanning confocal fluorescence microscopic and electron microscopic study

This study was undertaken to elucidate the morphological effects of histamine on subepidermal nerve fibers. A 10% histamine ointment was topically applied to the back skin of 17 adult male Hartley guinea pigs. Biopsy specimens were obtained at times from 5 min to 24 h, and were examined by conventional immunofluorescence (IF), laser scanning confocal fluorescence microscopy (LSCM) and transmission electron microscopy. On IF and LSCM, marked diminutions in the immunoreactivity of protein gene product 9.5-immunoreactive (PGP 9.5-IR) fibers as well as of substance P-immunoreactive (SP-IR) and calcitonin gene-related peptide-immunoreactive (CGRP-IR) substances were observed 5 min after histamine application. By 30 min, immunoreactivity of PGP 9.5, SP and CGRP was completely lost. By 2 h, however, immunoreactivity of PGP 9.5-IR fibers and CGRP-IR substances started to show recovery. By 4 h, immunoreactivity of PGP 9.5, SP and CGRP had almost recovered, but the recovery time for each substance was slightly different (PGP 9.5 first, CGRP next, and SP last). By 6 h after histamine application, immunoreactivity of all these substances had fully recovered. Ultrastructurally, 5 min after histamine application, axonal and mitochondrial swelling and glycogen deposition were seen in the axons of subepidermal nerve fibers. By 30 min, severe axonal degeneration had occurred in some of the axons. It was only by 4 to 6 h that almost normal ultrastructural features were observed. Schwann cells and perineurial cells did not show any pathological changes. These findings demonstrate that 10% histamine ointment produced organic changes in the axons in the subepidermal nerve fibers of guinea pig skin, but these morphological changes were short-lived, reversible and transitory.     

The subbasement membrane distribution of type IV collagen in normal human skin

Samples of normal human skin were obtained from 48 sites in 26 subjects ranging in age from 2 to 85 years. The samples were examined by indirect immunofluorescence and immunoelectron microscopy using anti-human type IV collagen antibodies produced by immunizing rabbits with type IV collagen extracted from human placenta. Fluorescence was observed as granular or fine fibrous patterns, not only in the basement membrane at the dermo-epidermal junction, around the vessels, and the accessory organs of the skin, but also in the dermal regions in the vicinity of the basement membranes. This suggests the presence of type IV collagen in the dermis deep to the basement membrane. Ultrastructurally, the extrabasal lamina distribution of type IV collagen was noted as a partial distribution around the fibroblasts that existed close to the basal lamina. These findings are considered to be important in examining the function of this collagen in the dermis and the dynamics and metabolism of the basement membrane under normal and abnormal conditions.     

Effect of UVB 311 nm irradiation on normal human skin

Ultraviolet radiation B (UVB) on the skin induces erythema, inflammation and modifications of the immune system. These changes have been reported after excessive short-term or long-term exposure to broad spectrum UVB. In this study, we examined the effects of local repetitive UVB irradiation of 311 nm wavelength on the skin of seven young volunteers. Skin biopsies were taken before and after UVB irradiation, and we immunohistochemically analyzed the expression of CD1a and HLA-DR antigens of Langerhans cells (LC), the possible infiltration of dermis/epidermis by CD11b macrophages, the modifications or the induction of intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1) involved in the binding of leukocytes to the endothelial surface and the development of perivascular infiltrates of LFA-1⁺ mononuclear cells. We also determined the expression of substance P receptors (SPR) using biotinylated substance P (SPB). Exposure of UVB 311 nm induced a drastic reduction of CD1a⁺ cells and a moderate increase of HLA-DR⁺ dendritic cells in the epidermis without infiltration by CD11b macrophages. An increase of the binding of SPB to upper layer epidermal cells was noted in five of seven biopsies. In the dermis, vessel-associated ICAM-1 expression increased and an induction of E-selectin occurred on nearly 20 to 40% of endothelial cells, but VCAM-1 expression remained undetectable. The percentage of LFA-1⁺ cells did not change significantly after irradiation. These observations may be compatible with a selective role of UVB 311 nm on the skin immune response.     

细胞角蛋白在正常皮肤组织中的表达

目的 观察7种细胞角蛋白（cytokeratins，CK）在正常皮肤组织的表达，并探讨其意义。方法 应用免疫组织化学S-P法对20例不同部位的正常皮肤组织进行CK7（K72．2）、CK8（C-51）、CK10（DE-K10）、CK14（LL002）、CK17（E3）、CK18（DC10）、CK19（KS19-1）标记，观察不同细胞角蛋白的表达情况。结果 在正常皮肤上皮组织中，7种细胞角蛋白的表达和分布有所不同。结论 有选择地检测一组CK的联合表达，有助于各种皮肤上皮组织的识别。     

Immunolocalization of aquaporin-5 in normal human skin and hypohidrotic skin diseases

Aquaporin (AQP)‐5 has been shown to be expressed in the secretory parts of mouse, rat and horse sweat glands. However, the precise localization of AQP‐5 in normal and diseased human skin has not been fully determined. The aim of the present study was to further clarify the immunolocalization of AQP‐5 in normal human skin and hypohidrotic skin diseases. Normal human scalp skin and biopsies from skin affected by hypohidrotic diseases were analyzed for AQP‐5 and/or dermcidin expression by immunohistochemistry, immunofluorescence and/or immunoelectronmicroscopy. AQP‐5 was expressed on the apical and basolateral plasma membranes of the clear cells in eccrine sweat coils, but not in ductal components or apocrine glands. Numbers of AQP‐5‐positive coils in the secretory part of eccrine sweat glands were decreased in Sjögren’s syndrome, but not in skin affected by idiopathic segmental anhidrosis or idiopathic pure sudomotor failure. AQP‐5 was mostly localized to the plasma membranes of clear cells in the secretory coils of eccrine sweat glands, suggesting that it plays a role in producing the primary sweat fluid.     

Staphylococci of the normal human skin flora

Summary 352 strains of Staphylococci of the normal human skin flora were sampled from one volunteer by single scrabbing in a ca. 3 cm² measuring area. They were biotyped by the scheme of Pelzer et al. (1973)—a modified Baird-Parker-Scheme (1963)— and the resistance to antibiotics was investigated by the method of Bauer et al. (1966).All the nine biotypes of Staphylococci were found in variable quantities. It seems problematic to call one biotype as the main type. Morphologically identical colonies of Staphylococci from the indigenous flora of the human skin were not identical in their biotypes as previously described by Pelzer (1976).Only the investigation of all Staphylococci colonies from the culture plate can evaluate all biotypes of Staphylococci of the normal human skin flora, and can give the right quantitative correlation. Staphylococci were found to be sensitive and resistant up to four antibiotics, and one biotype did not show one type of antibiogram.     

Immunohistochemical localization of cathepsin L and cystatin A in normal skin and skin tumors

Cathepsin L, a cysteine proteinase, and cystatin A, an inhibitor of cysteine proteinases, are thought to regulate the invasion and metastasis of malignant cells. In this study, the expression of cathepsin L and cystatin A in skin tumors was investigated immunohistochemically in order to examine the relationship between these two enzymes in the pathophysiology of malignant cells. Formalin-fixed and paraffin embedded specimens from normal skin, seborrheic keratoses, and squamous cell carcinomas were reacted with polyclonal antibodies against rat cathepsin L or cystatin a which cross-react to human cathepsin L and cystatin A, respectively. The consequent immunostaining of these enzymes was observed to be strong in normal skin (4 cases) and seborrheic keratosis (6 cases). In well-differentiated squamous cell carcinoma (SCC) (9 cases), staining for cathepsin L and cystatin A was moderately positive in differentiated tumor cells and negative in undifferentiated SCC (5 cases). The degree of staining of these enzymes was inversely correlated with the differentiation of the malignant cells. These results suggest that the immunohistochemical analysis of cathepsin L and cystatin A is a useful indicator for an aspect of malignancy in human epidermal keratinocytes.     

Immunohistochemical localization of type V collagen in normal human skin

Summary Tissue distribution of type V collagen in normal human skin was studied using an indirect immunofluorescent technique to determine whether type V collagen is present in the interstitium or in the basement membrane. Type V collagen was isolated from the human placenta by pepsin digestion and was purified with fractioning salt precipitations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that type V collagen contained 1(V) and 2(V) chains, but not the 3(V) chain. Specificity of the rabbit antibodies to type V collagen was assessed using enzyme-linked immunosorbent assay (ELISA) and an immunoblotting method. Antibodies showed no cross-reactivity to other collagens, laminin, and fibronectin. With an indirect immunofluorescent technique, type V collagen was found to be widely distributed throughout the dermis. Intense fluorescent staining was noted in the papillary dermis and adnexal dermis surrounding hair follicles and eccrine glands. The basement membrane of the dermoepidermal junction, skin appendages, and capillaries was not stained. By indirect immunoperoxidase double staining, type V collagen was not found to be deposited on type IV collagen present in the basement membrane. Immunoelectron microscopic studies showed that type V collagen was not located in the basal lamina. These results suggest that type V collagen is distributed in the interstitium, but not in the basement membrane of normal human skin.     

Topical application of emollients prevents dry skin-inducible intraepidermal nerve growth in acetone-treated mice

In common dermatoses, such as atopic dermatitis (AD), a decline in skin barrier function often accompanies an increased severity of clinical symptomatology, including pruritus [1]. Skin barrier disruption alters epidermal innervation and increases nerve density in the skin [2]. These findings are indicative of increases in sensory receptors responsive to exogenous trigger factors, suggesting that hyperinnervation is partly responsible for intense itch sensations [2]. Therefore, the abnormal innervation associated with skin barrier dysfunction such as dry skin has been considered as a target of antipruritic therapy. Moreover, recent studies have demonstrated that epidermal innervation is probably regulated by a fine balance of nerve elongation factors (e.g., nerve growth factor (NGF), amphiregulin, gelatinase) [2] and nerve repulsion factors (e.g., semaphorin 3A (Sema3A), anosmin-1) [2] and [3]. Although many people use emollients daily to alleviate symptoms of clinically and subjectively dry skin [4], the effects of emollients on nerve fiber density and nerve growth activity in dry skin remain unclear. We therefore examined the anti-nerve growth effects of petrolatum and heparinoid cream in the epidermis of acetone-treated mice, an animal model of acute dry skin [5].     

Presence of immunoreactive beta-endorphin in human skin

The production and its induction by ultraviolet radiation (UVR) of proopiomelanocortin (POMC)-derived peptides by keratinocytes has been reported, albeit not consistently. Recently we demonstrated that only under specific culturing conditions human keratinocytes are capable of producing a beta-endorphin (betaE)-like peptide with the characteristics of beta-lipotropin (betaLPH). Here the presence and UV-induction of betaE-immunoreactivity (betaE-IR) in keratinocytes in human skin in vivo was investigated. betaE-IR was detectable by immunohistochemistry in keratinocytes of the follicular matrix and to some extent in cells of sweat ducts, but was absent from epidermal keratinocytes. Absence of betaE-IR was confirmed by radioimmunoassay of HPLC-fractionated extracts of normal epidermis. Repeated exposure to solar-simulated UVR had no effect. This investigation is the first to demonstrate the presence of betaE-immunoreactive material in the follicular matrix of corporal hairs and in duct cells of sweat glands. The possible meaning of these results is discussed.     

Distribution of carbohydrate residues in normal skin

Summary Sections of biopsies of normal skin obtained from 11 individuals were incubated with 8 lectins using an avidin-biotin complex (ABC). All sections when incubated with the appropriate lectin showed the presence of the following carbohydrate residues: l-fucose, -(1–4)-d-GlcNAc)₂ (N-acetylglucosamine), acetylneuraminic acid, Gal--(1–3)-GalNAc (N-acetyl-galactosamine), -d-galactose, -d-glucose, and -d-mannose. In addition, sections of individuals with blood group A showed -d-GalNAc and sections of individuals with blood group B showed -d-galactose. In the stratum (str.) basale, carbohydrates were present in small quantities, but as the cells matured and moved upward, the incorporation of carbohydrates into the cell membranes increased considerably. In the str. granulosum, lectin reactivity was absent in many sections, probably due to masking by phospholipids. The dark cells in the eccrine glands showed reactivity with all lectins except in the one nonsecretor with blood group A₁, whose dark cells showed no l-fucose and -d-GalNAc. The endothelial cells of the blood vessels showed lectin reactivity except when incubated with concanavalin A. The sebaceous glands showed both cytoplasmic and membrane staining when incubated with various lectins.     

Clinico-histopathological correlation of skin and nerve in leprosy

The histopathological features of skin tissue sections in patients clinically diagnosed as leprosy were correlated with the histopathological features of nerve specimens obtained from the same patients. Fifty untreated leprosy patients attending the Outpatient Department of the Department of Dermatology and Sexually Transmitted Diseases of Smt. Sucheta Kriplani and Kalawati Saran Children's Hospitals, New Delhi, India were included in the study. On correlating the histological features of skin and nerve tissue sections, concordant findings were found in 24 out of the 50 patients (48%) but discordance between the histopathological features of skin and nerve tissue sections were found in 26 out of 50 cases (52%). Of these 26 cases, the nerve tissue histology when compared with the skin histology showed features lower down the disease spectrum in 17 (34%) cases. Seven of the 50 patients (14%) showed histological features of leprosy higher in the disease spectrum in the nerve tissue sections than in the skin biopsy sections. One patient clinically LL leprosy demonstrated histopathological features of Histoid leprosy in the skin sections and LL in the nerve sections. The remaining one patient had features of TT leprosy in the skin tissue sections while the nerve tissue histopathology showed non-specific changes. Histological features of the skin tissue sections were consistent with the clinical diagnosis in 33 out of 50 cases (66%). When the clinical groups were correlated with the histological features of the nerve tissue sections, concordance was found in 30 of the 50 cases (60%). On comparison of the histological features of skin and nerve tissue sections with the clinical diagnosis, concordance was still lower i.e., 19 out of 50 cases (38%). Thus the histological features of the skin tissue sections correlated more frequently with the clinical diagnosis than did those of the nerve sections. The importance of neural histology lies in the fact that it shows a higher BI and a lower histological grading in some cases and if not performed the lapse can result in inadequate treatment, drug resistance and even relapse.     

Decreased p53 expression in chronically sun-exposed human skin after topical photoprotection

UV-induced DNA damage appears to play an essential role in skin car-cinogenesis. Following acute UV irradiation, there is an overexpression of normal p53 protein in epidermal keratinocytes, representing a physiological response to DNA damage. Sun protection through topical sunscreens or clothing is believed to reduce the hazardous effects of UV irradiation and subsequently the risk of skin cancer. We have examined the effect of an SPF 15 topical sunscreen and blue denim fabric (SPF 1700) in chronically sun-exposed human skin after sun exposure during a normal summer. Skin biopsies from sun-protected and sun-exposed skin were compared with respect to immunohistochemically detectable p53. This method provides a model for assessing the significance of different degrees of UV protection under physiological conditions. Our results show a significant reduction of p53-positive cells in sun-protected skin as compared with sun-exposed skin. The reduction of p53-positive keratinocytes differed between topical sunscreen (33% reduction) and blue denim fabric (66% reduction). Interindividual variations were large, possibly because of variations in sun exposure. These variations also suggest that mechanisms determining UV damage at the cellular level are complex. The role of residual p53-positive keratinocytes after 2 months of total sun-protection (i.e., SPF 1700) is discussed.     

Distribution of monohydroxy fatty acids in specific human epidermal phospholipids

Abstract Monohydroxy derivatives of polyunsaturated fatty acids such a s arachidonic acid (AA) and linoleic acid (LA) can inodulate inflamation and epidermal proliferation. The purpose o f this study was to determine the in vito distribution orthc AA derivatives; 12- and 15-hydroxy- eicosatetraenoic acid (1 2-HETE and 15-HETE) and the LA derivatives; 9- and 13-liydroxyotadecadienoic acid (9-IHODE and 13-HODE) in specific phospholipids of normal humoii skin. Lipids were extracted from 6 normal keratoine skin biopsies and phospholipids were separated into the major classes by two-dimensional thin layer chroiiiatograpliy. Monohydroxy fatty acids (MHFAs) released from specific phospholipids after treatment with phospholipase A₂ were identified by reversed phase and straight phase high-performance liquid chromatography and UV-absorption spec- tra. Unesterified MIHFAs were deteriiiined in a similar way. 9-MODE, 13-HODE and 15-IHETE were detectable in pliospliatidylcholamine (PC), pliospliatidylinositol (PI) and pliosphatidylethanolamine (PE). Interestingly. 12-NETE was not detectable in these phospholipids, although the Linesteriried 12-HETE was detectable in amounts similar to uncstcrilied 15-HETE. Eskrilied I 5-HETE was equally distributed between PI arid PC, in which IS-HETE was predominant, accounting for 60% and 69% of the total MHFAs, respectively (p < 0.05). These results demonstrate that the LA dcrivntives 9-MODE and 13-HODE. as well as the AA derivative 15-1HETE, are esterified to PC, PI and PE of normal human epidermis in vito. The possibility remains that 9-HODE. 13-HODE anrl 15-HETE, may incdiatc their biological cfrccts by being incorporated into specific I pli osplioli picis.     

金属硫蛋白I、II在正常人皮肤中的表达及其意义

目的:探讨金属硫蛋白II、I(Metallothionein II、I,MT-I、II)在正常人皮肤的表达及其意义。方法:采用组织芯片、免疫组化灰度分析方法,研究正常皮肤MT-II、I表达情况。结果:(1)正常皮肤MT-II、I主要表达于表皮基底层和基底层上1~2层角质形成细胞。真皮极少数成纤维细胞、外毛根鞘、小汗腺导管上皮细胞以及皮脂腺外单层嗜碱性细胞也有MT表达。(2)同性别同部位正常皮肤MT-II、I表达强度与年龄呈显著负相关(面r=-0.73,臀部r=-0.98,P<0.01),40岁之前面部皮肤表达强度随年龄增长而快速下降。(3)同年龄同部位正常皮肤MT-II、I表达强度男性显著高于女性(t=6.95,P<0.01)。(4)同年龄同性别正常皮肤MT-II、I表达强度遮光部位显著高于曝光部位(t=4.14,P<0.01)。结论:正常皮肤MT-II、I的表达可能存在人种、性别和年龄差异。老化皮肤合成MT的能力降低,MT-II、I在对抗皮肤老化方面可能有重要作用。皮肤光老化与MT-II、I表达降低有关。     

金属硫蛋白Ⅰ、Ⅱ在正常人皮肤中的表达及其意义

目的：探讨金属硫蛋白Ⅰ、Ⅱ（Metallothionein Ⅰ、Ⅱ，MT—Ⅰ、Ⅱ）在正常人皮肤的表达及其意义。方法：采用组织芯片、免疫组化灰度分析方法，研究正常皮肤MT—Ⅰ、Ⅱ表达情况。结果：（1）正常皮肤MT—Ⅰ、Ⅱ主要表达于表皮基底层和基底层上1-2层角质形成细胞。真皮极少数成纤维细胞、外毛根鞘、小汗腺导管上皮细胞以及皮脂腺外单层嗜碱性细胞也有MT表达。（2）同性别同部位正常皮肤MT—Ⅰ、Ⅱ表达强度与年龄呈显著负相关（面r=-0．73，臀部r=-0．98，P〈0．01），40岁之前面部皮肤表达强度随年龄增长而快速下降。（3）同年龄同部位正常皮肤MT—Ⅰ、Ⅱ表达强度男性显著高于女性（t=6．95，P〈0．01）。（4）同年龄同性别正常皮肤MT—Ⅰ、Ⅱ表达强度遮光部位显著高于曝光部位（t=4．14，P〈0．01）。结论：正常皮肤MT—Ⅰ、Ⅱ的表达可能存在人种、性别和年龄差异。老化皮肤合成MT的能力降低，MT—Ⅰ、Ⅱ在对抗皮肤老化方面可能有重要作用。皮肤光老化与MT—Ⅰ、Ⅱ表达降低有关。     

Heat-separation of normal human skin for epidermal and dermal prostaglandin analysis

Summary Heat-separation was introduced as a simple, reliable method of obtaining pure epidermis and dermis for prostaglandin (PG) analysis. Heating of normal human skin at 60°C for 1 min resulted in a distinct separation of the epidermis from the dermis. After heat-separation the mean concentration ±SEM of PGE₁ activity in normal epidermal tissue was 2906±281 pg/mg dry weight. The PGE₁ level in the corresponding dermal samples was 30±4 pg/mg dry weight and the mean leakage of PGE₁ from the tissue into the buffer used during heating was 426±54 pg/ml.This work was supported by grant 512-8125 from the Danish State Research Foundation     

Aminolaevulinic acid diffusion characteristics in 'in vitro' normal human skin and actinic keratosis: implications for topical photodynamic therapy

Background: The response rate of aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) in certain subtypes of actinic keratosis (AK), such as hypertrophic and hyperkeratotic lesions, is variable, an effect attributable to a supposed lack of ALA penetration. A detailed and depth-related profile of spatial ALA permeation in AK following drug administration would lead to a greater understanding of concentrations achievable before protoporphyrin IX biosynthesis and subsequent PDT.
Methods: ALA penetration through excised normal human skin (NS) and AK lesions was evaluated using a cryostatic sectioning technique and radio-isotope counting following drug delivery using a novel, bioadhesive patch, loaded with 19, 38 or 50 mg/cm² ALA.
Results: Distinct differences in ALA concentration with respect to depth between AK and NS samples were shown, particularly within the superficial layers of the tissue structure, down to a depth of 1.0 mm. Patch application times were shown to influence ALA concentrations in tissue, but there was no clear correlation between ALA penetration in AK lesions taken from different body locations and from patients of different age. Similarly, the thickness of stratum corneum was not related to the ALA distribution profiles.
Conclusions: Sizable variation in ALA concentration was a prominent feature of profiles through AK lesions, which may explain the variation of observed protoporphyrin IX production seen in the clinical implementation of AK PDT. That said, the results of this study show sufficient ALA penetration to a depth of 1.0 mm, which should be satisfactory for successful treatment of the majority of non-hyperkeratotic, hypertrophic AK using patch-based delivery methods.