Skin penetration of infective hookworm larvae. II. The path of migration of infective larvae of Ancylostoma braziliense in the metacarpal foot pads of dogs

The hairless metacarpal foot pads of six hookworm-free puppies were exposed to infective larvae of Ancylostoma braziliense. Serial sections of the biopts stained with Harris' haematoxylin and eosin showed that the infective larvae are able to penetrate the toughest region of canine skin. Pores of eccrine sweat glands did not seem to constitute sites of entry and no larvae were detected in these glands. Larvae were only observed in the epidermis. The histopathology of the infected skin of the foot pads of the puppies was similar to that in human skin with "creeping eruption" as described by Fülleborn (1927). The biopts appeared to consist of hairy skin as well. In the unexposed adjacent hairy skin of the foot pads, larvae were also observed. They were found in the epidermis, hair follicle systems and dermis, suggesting that the migration from the epidermis into deeper tissue depends on the presence of the hair follicle systems.     

Immunohistochemical localization of activated EGF receptor in human eccrine and apocrine sweat glands.

Epidermal growth factor (EGF) is secreted into sweat from secretory cells of human sweat glands. The function of EGF in sweat is poorly understood. The biological function of EGF is exerted by the binding of EGF to the receptor (EGFR) and its activation. Therefore, we immunohistochemically localized the activated form of EGFR in human eccrine and apocrine sweat glands to assess the functional importance of the EGF-EGFR system in human sweat glands. Frozen sections of human skin were stained with a monoclonal antibody (MAb) specific for tyrosine-phosphorylated (activated) EGFR and with an MAb that stains both activated and non-activated EGFR. In the secretory portion of eccrine sweat glands, nuclei of the secretory cells were stained with the anti-activated EGFR MAb. In coiled and straight portions of eccrine sweat ducts, nuclei of luminal and peripheral cells were stained with the antibody specific for activated EGFR. Luminal cell membranes and luminal cytoplasm of inner ductal cells possessed non-activated EGFR. In the secretory portion of apocrine sweat glands, activated EGFRs were present in cytoplasm and nuclei of secretory cells. These data suggest that EGF, already known to be present in the cytoplasm of secretory cells in eccrine and apocrine sweat glands, activates EGFR in the nuclei of secretory cells themselves in an intracrine manner. Because ductal cells do not express EGF, EGF in the sweat secreted from the secretory cells should activate EGFR in the ductal cells in a paracrine manner. (J Histochem Cytochem 49:597-601, 2001)     

Immunohistologic studies of the distribution of proliferative compartments in the appendages of adult human skin

Both, calmodulin (CaM) as well as the antigen Ki67 show a close relationship to cell proliferation. By means of specific antibodies against them, it has become possible to study the spatial distribution of proliferative compartments in tissues. We performed an indirect immunofluorescence study on unfixed frozen sections of human adult skin to gain more informations about the spatial distribution of immunoreactive CaM and Ki67 in skin appendages, i.e. anagen hair follicle, sebaceous and eccrine sweat gland. Two major patterns of immunoreactivity were seen: Type (1) or epidermis-like, which was present in the interfollicular epidermis and the pilosebaceous unit. Type (2) or sweat gland type, which was seen in eccrine sweat glands. Both types disclosed significant differences in the relative number of proliferative cells in S-phase, which might be a consequence of a quiet different tissue architecture. Furthermore, myoepithelial cells of secretory coils were likely to represent mainly SQ-cells. Their immunoreactivity in human skin was quiet different from other parts of eccrine sweat glands suggesting another ontogenetic pathway.     

Peripheral choline acetyltransferase in rat skin demonstrated by immunohistochemistry

Conventional choline acetyltransferase immunohistochemistry has been used widely for visualizing central cholinergic neurons and fibers but not often for labeling peripheral structures, probably because of their poor staining. The recent identification of the peripheral type of choline acetyltransferase (pChAT) has enabled the clear immunohistochemical detection of many known peripheral cholinergic elements. Here, we report the presence of pChAT-immunoreactive nerve fibers in rat skin. Intensely stained nerve fibers were distributed in association with eccrine sweat glands, blood vessels, hair follicles and portions just beneath the epidermis. These results suggest that pChAT-positive nerves participate in the sympathetic cholinergic innervation of eccrine sweat glands. Moreover, pChAT also appears to play a role in cutaneous sensory nerve endings. These findings are supported by the presence of many pChAT-positive neuronal cells in the sympathetic ganglion and dorsal root ganglion. Thus, pChAT immunohistochemistry should provide a novel and unique tool for studying cholinergic nerves in the skin.     

Localization of sex steroid receptors in human skin

Sex steroid hormones are involved in regulation of skin development and functions as well as in some skin pathological events. To determine the sites of action of estrogens, androgens and progestins, studies have been performed during the recent years to accurately localize receptors for each steroid hormone in human skin. Androgen receptors (AR) have been localized in most keratinocytes in epidermis. In the dermis, AR was detected in about 10% of fibroblasts. In sebaceous glands, AR was observed in both basal cells and sebocytes. In hair follicles, AR expression was restricted to dermal papillar cells. In eccrine sweat glands, only few secretory cells were observed to express AR. Estrogen receptor (ER) alpha was poorly expressing, being restricted to sebocytes. In contrast, ERbeta was found to be highly expressed in the epidermis, sebaceous glands (basal cells and sebocytes) and eccrine sweat glands. In the hair follicle, ERbeta is widely expressed with strong nuclear staining in dermal papilla cells, inner sheath cells, matrix cells and outer sheath cells including the buldge region. Progesterone receptors (PR) staining was found in nuclei of some keratinocytes and in nuclei of basal cells and sebocytes in sebaceous glands. PR nuclear staining was also observed in dermal papilla cells of hair follicles and in eccrine sweat glands. This information on the differential localization of sex steroid receptors in human skin should be of great help for future investigation on the specific role of each steroid on skin and its appendages.     

Three-dimensional reconstructed eccrine sweat glands with vascularization and cholinergic and adrenergic innervation

Functional integrity of the regenerated tissues requires not only structural integrity but also vascularization and innervation. We previously demonstrated that the three-dimensional (3D) reconstructed eccrine sweat glands had similar structures as those of the native ones did, but whether the 3D reconstructed glands possessing vascularization and innervation was still unknown. In the study, Matrigel-embedded eccrine sweat gland cells were implanted under the inguinal skin. Ten weeks post-implantation, the vascularization, and innervation in the 10-week reconstructed eccrine sweat glands and native human eccrine sweat glands were detected by immunofluorescence staining. The results showed that the fluorescent signals of general neuronal marker protein gene product 9.5, adrenergic nerve fiber marker tyrosine hydroxylase, and cholinergic nerve fiber markers acetylcholinesterase and vasoactive intestinal peptide embraced the 3D reconstructed glands in circular patterns, as the signals appeared in native eccrine sweat glands. There were many CD31- and von Willebrand factor-positive vessels growing into the plugs. We demonstrated that the 3D reconstructed eccrine sweat glands were nourished by blood vessels, and we for the first time demonstrated that the engineering sweat glands were innervated by both cholinergic and adrenergic fibers. In conclusion, the 3D reconstructed eccrine sweat glands may have functions as the native ones do.     

Structure and function of human sweat glands studied with histochemistry and cytochemistry

The basic structure and the physiological function of human sweat glands were reviewed. Histochemical and cytochemical techniques greatly contributed the elucidation of the ionic mechanism of sweat secretion. X-ray microanalysis using freeze-dried cryosections clarified the level of Na, K, and Cl in each secretory cell of the human sweat gland. Enzyme cytochemistry, immunohistochemistry and autoradiography elucidated the localization of Na,K-ATPase. These data supported the idea that human eccrine sweat is produced by the model of N-K-2Cl cotransport. Cationic colloidal gold localizes anionic sites on histological sections. Human eccrine and apocrine sweat glands showed completely different localization and enzyme sensitivity of anionic sites studied with cationic gold. Human sweat glands have many immunohistochemical markers. Some of them are specific to apocrine sweat glands, although many of them stain both eccrine and apocrine sweat glands. Histochemical techniques, especially immunohistochemistry using a confocal laser scanning microscope and in situ hybridization, will further clarify the relationship of the structure and function in human sweat glands.     

Multipotent Nestin-Positive Stem Cells Reside in the Stroma of Human Eccrine and Apocrine Sweat Glands and Can Be Propagated Robustly In Vitro

Human skin harbours multiple different stem cell populations. In contrast to the relatively well-characterized niches of epidermal and hair follicle stem cells, the localization and niches of stem cells in other human skin compartments are as yet insufficiently investigated. Previously, we had shown in a pilot study that human sweat gland stroma contains Nestin-positive stem cells. Isolated sweat gland stroma-derived stem cells (SGSCs) proliferated in vitro and expressed Nestin in 80% of the cells. In this study, we were able to determine the precise localization of Nestin-positive cells in both eccrine and apocrine sweat glands of human axillary skin. We established a reproducible isolation procedure and characterized the spontaneous, long-lasting multipotent differentiation capacity of SGSCs. Thereby, a pronounced ectodermal differentiation was observed. Moreover, the secretion of prominent cytokines demonstrated the immunological potential of SGSCs. The comparison to human adult epidermal stem cells (EpiSCs) and bone marrow stem cells (BMSCs) revealed differences in protein expression and differentiation capacity. Furthermore, we found a coexpression of the stem cell markers Nestin and Iα6 within SGSCs and human sweat gland stroma. In conclusion the initial results of the pilot study were confirmed, indicating that human sweat glands are a new source of unique stem cells with multilineage differentiation potential, high proliferation capacity and remarkable self renewal. With regard to the easy accessibility of skin tissue biopsies, an autologous application of SGSCs in clinical therapies appears promising.     

Differential expression of dipeptidyl peptidase IV in human versus cynomolgus monkey skin eccrine sweat glands

Dipeptidyl peptidase IV (DPP4) is a peptidase whose inhibition is beneficial in Type II diabetes treatment. Several evidences suggest potential implication of DPP4 in skin disorders such as psoriasis, keloids and fibrotic skin diseases where its inhibition could also be beneficial. DPP4 expression in human skin was described mainly in dermal fibroblasts and a subset of keratinocytes in the basal layer. Of importance in the perspective of preclinical experimentation, DPP4 distribution in skin of non-human primate species has not been documented. This report evidences unexpected differences between a set of human and cynomolgus monkey skin samples revealing a major expression of DPP4 in eccrine sweat glands of cynomolgus monkeys but not in humans. This represents a unique distinctive feature compared to the conserved expression of dipeptidyl peptidases 8 and 9 and potential relevant DPP4 substrates such as neuropeptide Y (NPY) and receptors (NPY-receptor 1 and Neurokinin receptor). Finally the observation that cathepsin D, an unrelated protease, shows the opposite expression compared to DPP4 (present in human but not in cynomolgus monkey eccrine sweat glands) could indicate that human eccrine sweat glands evolved a divergent protease repertoire compared to non-human primates. These unexpected differences in the eccrine sweat glands protease repertoire will need to be confirmed extending the analysis to a major number of donors but could imply possible biochemical divergences, reflecting the functional evolution of the glands and the control of their activity. Our findings also demonstrate that non-human primates studies aiming at understanding DPP4 function in skin biology are not readily translatable to human.     

Vitiligo-related neuropeptides in nerve fibers of the skin

Skin distribution of substance P (SP)-, somatostatin (SOM)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivity (LI) in vitiligo patients was studied by an indirect immunofluorescence technique. Immunocytochemical characteristics of the epidermis, dermal-epidermal junction, papillary and reticular dermis and skin appendages were analyzed in lesional and marginal vitiligo areas, as well as in healthy skin. In healthy pigmented skin, SP-, SOM-, CGRP-, and NPY-LI nerve fibers were observed with specific distributional patterns. In uninvolved vitiligo skin, thin SP-containing fibers were evident in dermal papillae, extending into the epidermis, and SP-LI fibers were seen around blood vessels and sweat glands. SOM-LI varicose nerve fibers were associated with Meissner corpuscles in the dermal papillae, while CGRP-LI was demonstrated in the free subepidermal nerve terminals and in sensory nerve fibers around blood vessels, hair follicles and sweat glands. Autonomic NPY-nerve fibers innervated the eccrine sweat glands and blood vessels. The distribution of these neuropeptides in both marginal and lesional areas of vitiliginous skin was the same as in the skin of healthy control subjects, except for an increased immunoreactivity against NPY and, to a lesser extent, against CGRP in the skin depigmentation lesions. The elevated NPY levels in skin affected by vitiligo suggest that this peptide may serve as a neurochemical marker in the pathogenesis of the disease, thus supporting the neuronal theory of vitiligo.     

Gross Cystic Disease Fluid Protein 15 in Stratum Corneum Is a Potential Marker of Decreased Eccrine Sweating for Atopic Dermatitis

It is well known that eccrine sweating is attenuated in patients with atopic dermatitis (AD). We have reported by using proteome analysis that gross cystic disease fluid protein 15 (GCDFP15), a substance secreted from eccrine sweat glands, is decreased in tape-stripped stratum corneum (SC) samples from AD patients. The aim of this study was to evaluate GCDFP15 production by eccrine glands with SC samples and to assess sweating in AD. SC samples were obtained from 51 healthy control (HC) and 51 AD individuals. Sweat samples were from 18 HC and 12 AD subjects. GCDFP15 was quantified by ELISA. By immunohistochemistry, the expression of GCDFP15 in eccrine glands was examined in normal and AD skin specimens. To identify GCDFP15-producing cells, double immunofluorescence staining for GCDFP15 and S100 protein was performed in frozen sections. To address the mechanism underlying the decreased eccrine sweating in AD patients, we examined the expression of cholinergic receptor M3 (CHRM3), a receptor for acetylcholine-induced sweating, in eccrine sweat glands. The amounts of GCDFP15 in the SC extracts were significantly lower in AD than HC (P < 0.0001). The sweat samples from AD patients also had lower levels of GCDFP15 concentration (P < 0.05). Immunohistochemistry showed positive GCDFP15 staining in the eccrine gland secretory cells and the ductal and acrosyringial lumen in normal skin, but AD lacked clear staining. Immunofluorescence staining revealed that GCDFP15 was co-expressed with S100 protein, suggesting that the clear cell of eccrine glands produces GCDFP15. Finally, we found that the expression of CHRM3 was depressed in AD, suggesting contribution to the low sweating. The SC of AD patients contains a low amount of GCDFP15 due to both low sweating and low GCDFP15 concentration in the sweat. GCDFP15 in SC is a potential marker for dysregulated sweating in AD.     

VIP-immunoreactivity in the skin of various mammals: immunohistochemical, radioimmunological and experimental evidence for a dual localization in cutaneous nerves and merkel cells

In the present study VIP-immunoreactive (IR) nerve fibers were found in the skin of several mammalian species (cat, dog, pig and man). They supplied predominantly the arteries and arterial portions of arteriovenous anastomoses. Far fewer VIP-IR nerve fibers innervated veins and arterioles. Capillaries were supplied by VIP-IR fibers only in sweat and Meibomian glands. Some non-vascular VIP-IR nerve fibers were seen in contact with dermal smooth muscle strands. In eccrine sweat glands and in Meibomian glands VIP-IR fibers were targeting glandular cells. In addition, VIP-IR nerve fibers innervated the upper parts of facial hair follicles. In non-neuronal localization VIP-IR occurred in Merkel cells in all species and sites, while the intraepidermal axons consistently were not VIP-IR. Radioimmunoassay of different skin regions of cats also suggested both a neuronal and a Merkel cell origin of VIP-IR. Under physiological conditions VIP which is released from its neuronal and non-neuronal cutaneous pools may have an impact on thermoregulation by influencing blood flow and sweat production. It may also modulate axon-endings in Merkel cell-axon complexes and hair follicle receptors. Under pathological conditions an enhanced release of cutaneous VIP may lead to local inflammatory processes partly mediated via release of histamine from cutaneous mast cells.     

Three-dimensional culture and identification of human eccrine sweat glands in matrigel basement membrane matrix

Interactions between the extracellular matrix (ECM) and epithelial cells are necessary for the proper organization and function of the epithelium. In the present study, we show that human eccrine sweat gland epithelial cells cultured in matrigel, a representation of ECM components, constitute a good model for studying three-dimensional reconstruction, wound repair and regeneration and differentiation of the human eccrine sweat gland. In matrigel, epithelial cells from the human eccrine sweat gland form tubular-like structures and then the tubular-like structures coil into sphere-like shapes that structurally resemble human eccrine sweat glands in vivo. One sphere-like shape can be linked to another sphere-like shape or to a cell monolayer via tubular-like structures. Hematoxylin and eosin staining has revealed that the tubular-like structures have a single layer or stratified epithelial cells located peripherally and a lumen at the center, similar to the secretory part or duct part, respectively, of the eccrine sweat gland in sections of skin tissue. Immunohistochemical analysis of the cultures has demonstrated that the cells express CK7, CK19, epithelial membrane antigen and actin. Thus, matrigel promotes the organization and differentiation of epithelial cells from the human eccrine sweat gland into eccrine sweat gland tissues.     

An analysis on the rates and regulation of insensible water loss through the eccrine sweat glands

An analysis is presented on insensible water loss from the human body at rest through exposed skin surfaces into still air. Possible sites of moisture release are identified as the stratum corneum of the skin, free surfaces of dilute sweat liquids perpetually present in the microscopic ducts of a large population of eccrine sweat glands, and moist microvillous processes which line part of the periductal surfaces in the glands, particularly in the helical coils within the stratum spinosum of the epidermis. Water supply to the sites involves transepidermal migration across skin tissue layers, secretion and partial reabsorption of solutes and water within eccrine glands, and transport across periductal lining of eccrine glands from the surrounding connective tissues respectively. Evaporation and gas phase diffusion within eccrine ducts were modelled. Basal loss rates of water (as regulated by the ambient temperature and relative humidity and by aspects of the anatomy of and physiological factors for eccrine glands, the epidermis and the dermis) were calculated at between 1 and 20 g hr-1 at an ambient temperature of 25 degrees C and a relative humidity of 60% as an example. Such rates are significant fractions of experimental values for insensible water loss rates reported at between 4 and 35 g hr-1 in air at 22-30 degrees C and a relative humidity of 30-60%.     

The skin of primates. XXX. The skin of the woolly monkey. (Lagothrix lagotricha)

The anatomical and histochemical features of the skin of the woolly monkey are intermediate between those of the Cercopithecoidea and the Pithecoidea. The animal has a prehensile tail, the glabrous, friction surface of which is similar to that of the fingers. The epidermis is heavily pigmented. The dermal vascularization is relatively well-developed and similar to that of the skin of the Cercopithecoidea. Hair follicles grow in groups of 4 to 15, as in the skin of the Pithecoidea. In the hairy skin, eccrine sweat glands occur only in the tail and genitalia. The woolly monkey, like the green monkey, possesses only acetylcholinesterase-containing nerve fibers around its eccrine sweat glands.     

The skin of primates. XXXIV. The skin of the golden spider monkey (Ateles geoffroyi)

The skin of the golden spider monkey (Ateles geoffroyi) has many histological and histochemical similarities to that of the woolly monkey (Lagothrix lagotricha) and howler monkey (Alouatta caraya); however, this monkey possesses certain peculiar properties such as large sebaceous glands, a combined distributional pattern of eccrine and apocrine sweat glands, and abundant alkaline phosphatase in the sebaceous glands, apocrine and eccrine sweat glands. In brief, the anatomical and histochemical properties of the skin of this animal are more similar to those of the howler monkey than to the woolly monkey. In addition, the skin of these three Ceboids falls phylogenetically between that of the Cercopithecoidea and Pithecoidea.     

Immunoelectron microscopic localization of epidermal growth factor in the eccrine and apocrine sweat glands.

We studied the localization of the epidermal growth factor (EGF) in eccrine and apocrine sweat glands with light microscopic and electron microscopic immunohistochemistry. Anti-human EGF (anti-hEGF) polyclonal antiserum and anti-hEGF monoclonal antibody (MAb) were used for the study. Light microscopic immunohistochemistry with monoclonal and polyclonal antibodies showed that hEGF-like immunoreactivity was strongly positive in the myoepithelial cells and weakly positive in the secretory cells of eccrine sweat glands. In apocrine sweat glands, it was strongly positive in the secretory cells as well as in the myoepithelial cells. Immunoelectron microscopy with polyclonal antibody showed that hEGF-like immunoreactivity was present in secretory granules of apocrine secretory cells. These granules had mitochondrion-like internal structure. No reactivity was observed on the eccrine secretory cells by immunoelectron microscopy. Neither dark cell granules nor mitochondria in eccrine secretory cells were labeled with anti-hEGF antibody. In both eccrine and apocrine sweat glands, hEGF-like immunoreactivity was diffusely present in the cytoplasm of myoepithelial cells. However, nuclei and mitochondria of myoepithelial cells were devoid of immunoreactivity for hEGF. Our observations indicate that apocrine sweat glands may secrete more hEGF in the sweat than eccrine sweat glands.     

Notes on Technic: Differential staining of Fungus and host cells Using a modifciation of Pianeze IIIB

Intercellular secretory capillaries in parotid glands, eccrine sweat glands and intracellular secretory capillaries in parietal cells of gastric glands were demonstrated histo-chemically by the use of the Wachstein-Meisel adenosinetriphosphatase (ATPase) technique in the rabbit, rat and guinea pig. However, with the Wachstein-Meisel 5-nucleotidase technique, secretory capillaries were not stained. For parotid glands, optimal incubation in ATPase substrate mixture was: in rabbit, 15 min; in rat, 2.5 hr; and in guinea pig, 2 hr. For eccrine sweat glands, optimal incubation was 15 min in rabbit, 30 min in rat and 15 min in guinea pig. For parietal cells of gastric glands, optimal incubation was 3 hr for all three species. Secretory capillaries were best demonstrated in the parotid by using rabbit tissue; in eccrine sweat glands, with rat tissue, and in parietal cells, guinea pig tissue. Since ATPase activity in cell membranes of secretory cells may play a part in the mechanism of transport of secretory products from their place of formation in the acini to the excretory ducts, the Wachstein-Meisel ATPase technique can therefore be used successfully for staining secretory capillaries in many of the exocrine glands of laboratory mammals.     

Staining Secretory Capillaries of Exocrine Glands with Techniques for Specific Phosphatases

Intercellular secretory capillaries in parotid glands, eccrine sweat glands and intracellular secretory capillaries in parietal cells of gastric glands were demonstrated histo-chemically by the use of the Wachstein-Meisel adenosinetriphosphatase (ATPase) technique in the rabbit, rat and guinea pig. However, with the Wachstein-Meisel 5-nucleotidase technique, secretory capillaries were not stained. For parotid glands, optimal incubation in ATPase substrate mixture was: in rabbit, 15 min; in rat, 2.5 hr; and in guinea pig, 2 hr. For eccrine sweat glands, optimal incubation was 15 min in rabbit, 30 min in rat and 15 min in guinea pig. For parietal cells of gastric glands, optimal incubation was 3 hr for all three species. Secretory capillaries were best demonstrated in the parotid by using rabbit tissue; in eccrine sweat glands, with rat tissue, and in parietal cells, guinea pig tissue. Since ATPase activity in cell membranes of secretory cells may play a part in the mechanism of transport of secretory products from their place of formation in the acini to the excretory ducts, the Wachstein-Meisel ATPase technique can therefore be used successfully for staining secretory capillaries in many of the exocrine glands of laboratory mammals.