Involvement of aquaporin 5 and Na-K-2Cl cotransporter 1 in the pathogenesis of primary focal hyperhidrosis: evidence from the primary sweat gland cell culture.

People with primary focal hyperhidrosis (PFH) usually have an overactive sympathetic nervous system, which can activate the sweat glands through the chemical messenger of acetylcholine. The role of aquaporin 5 (AQP5) and Na-K-2Cl cotransporter 1 (NKCC1) in PFH is still unknown. The relative mRNA and protein levels of AQP5 and NKCC1 in the sweat gland tissues of three subtypes of patients with PFH (primary palmar hyperhidrosis, PPH; primary axillary hyperhidrosis, PAH; and primary craniofacial hyperhidrosis, PCH) were detected with real-time PCR (qPCR) and Western blot. Primary sweat gland cells from healthy controls (NPFH-SG) were incubated with different concentrations of acetylcholine, and the relative mRNA and protein expression of AQP5 and NKCC1 were also detected. NPFH-SG cells were also transfected with si-AQP5 or shNKCC1, and acetylcholine stimulation-induced calcium transients were assayed with Fluo-3 AM calcium assay. Upregulated AQP5 and NKCC1 expression were observed in sweat gland tissues, and AQP5 demonstrated a positive Pearson correlation with NKCC1 in patients with PPH (r = 0.66, P < 0.001), patients with PAH (r = 0.71, P < 0.001), and patients with PCH (r = 0.62, P < 0.001). Upregulated AQP5 and NKCC1 expression were also detected in primary sweat gland cells derived from three subtypes of patients with PFH when compared with primary sweat gland cells derived from healthy control. Acetylcholine stimulation could induce the upregulated AQP5 and NKCC1 expression in NPFH-SG cells, and AQP5 or NKCC1 inhibitions attenuated the calcium transients induced by acetylcholine stimulation in NPFH-SG cells. The dependence of ACh-stimulated calcium transients on AQP5 and NKCC1 expression may be involved in the development of PFH.NEW & NOTEWORTHY The dependence of ACh-stimulated calcium transients on AQP5 and Na-K-2Cl cotransporter 1 (NKCC1) expression may be involved in the development of primary focal hyperhidrosis (PFH).

Fingerprint ridges allow primates to regulate grip.

Fingerprints are unique to primates and koalas but what advantages do these features of our hands and feet provide us compared with the smooth pads of carnivorans, e.g., feline or ursine species? It has been argued that the epidermal ridges on finger pads decrease friction when in contact with smooth surfaces, promote interlocking with rough surfaces, channel excess water, prevent blistering, and enhance tactile sensitivity. Here, we found that they were at the origin of a moisture-regulating mechanism, which ensures an optimal hydration of the keratin layer of the skin for maximizing the friction and reducing the probability of catastrophic slip due to the hydrodynamic formation of a fluid layer. When in contact with impermeable surfaces, the occlusion of the sweat from the pores in the ridges promotes plasticization of the skin, dramatically increasing friction. Occlusion and external moisture could cause an excess of water that would defeat the natural hydration balance. However, we have demonstrated using femtosecond laser-based polarization-tunable terahertz wave spectroscopic imaging and infrared optical coherence tomography that the moisture regulation may be explained by a combination of a microfluidic capillary evaporation mechanism and a sweat pore blocking mechanism. This results in maintaining an optimal amount of moisture in the furrows that maximizes the friction irrespective of whether a finger pad is initially wet or dry. Thus, abundant low-flow sweat glands and epidermal furrows have provided primates with the evolutionary advantage in dry and wet conditions of manipulative and locomotive abilities not available to other animals.

Syringotropic Melanoma: A Diagnostic Challenge With Prognostic Implications.

ABSTRACT: The presence of neoplastic melanocytes within the eccrine apparatus into the reticular dermis and/or subcutaneous tissue is extremely rare. The staging of syringotropic melanomas and their biological behavior are still controversial. We present 6 new cases of syringotropic melanoma and their main histopathologic features; review the previous literature; and discuss about the origin, staging, and prognosis of this rare variant of melanoma.

Trichilemmal Cysts With Divergent Ductal Differentiation: A Series of 4 Cases.

ABSTRACT: Trichilemmal cysts are common clonal tumors with a predilection for the scalp. They are composed of an outer epithelial wall resembling the outer root sheath in the isthmus of the hair follicle and a central core of compact keratin. Sweat duct differentiation is exceptional with only one convincing case reported to date. Here, we sought to characterize the clinicopathological characteristics of sweat duct differentiation in trichilemmal cysts. We reviewed all cases of trichilemmal cyst diagnosed at our institution between 2008 and 2019. Ductal structures were found in 4 of 411 cases (0.97%). Subjects included 2 male and 2 female patients with a median age of 37.5 years (range 34-55). The ducts were lined by attenuated epithelial cells and immunoreactive for polyclonal carcinoembryonic antigen and cytokeratin 7. Ductal differentiation involved a median of 7.5% (range 1%-50%) of the cyst wall. All 4 cases were from the scalp and treated with local excision. No recurrence was identified with a median follow-up period of 1.5 years (range 1-12 years). In summary, sweat duct differentiation in trichilemmal cysts is rare but likely under recognized. Conceptually, we suggest it represents a type of divergent cellular differentiation within a clonal neoplasm rather than a retention cyst or hybrid cyst.

Chemical Visualization of Sweat Pores in Fingerprints Using GO-Enhanced TOF-SIMS.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used in imaging of small molecules (<500 Da) in fingerprints, such as gunshot residues and illicit drugs. However, identifying and mapping relatively high mass molecules are quite difficult owing to insufficient ion yield of their molecular ions. In this report, graphene oxide (GO)-enhanced TOF-SIMS was used to detect and image relatively high mass molecules such as poison, alkaloids (>600 Da) and controlled drugs, and antibiotics (>700 Da) in fingerprints. Detail features of fingerprints such as the number and distribution of sweat pores in a ridge and even the delicate morphology of one pore were clearly revealed in SIMS images of relatively high mass molecules. The detail features combining with identified chemical composition were sufficient to establish a human identity and link the suspect to a crime scene. The wide detectable mass range and high spatial resolution make GO-enhanced TOF-SIMS a promising tool in accurate and fast analysis of fingerprints, especially in fragmental fingerprint analysis.

Overexpression of AQP5 Was Detected in Axillary Sweat Glands of Primary Focal Hyperhidrosis Patients.

BACKGROUND: The expression of aquaporin 5 (AQP5) in human axillary sweat glands has never been studied so far. OBJECTIVE: To detect the expression of AQP5 in axillary sweat glands of patients with primary focal hyperhidrosis (PFH) relative to control subjects. METHODS: The morphological characteristics and the number of sweat coils in axillary sweat glands were compared between two groups by using transmission electron microscopy. The expression of AQP5 was detected by immunohistochemistry, Western blot analysis, and real-time transcription polymerase chain reaction. RESULTS: There were no significant differences between the two groups in terms of morphological characteristics and the number of sweat coils in axillary sweat glands. The expressions of AQP5 protein and AQP5 mRNA were significantly higher in the patient group than in the control group. CONCLUSION: AQP5 is involved in the secretion of human axillary sweat glands. The overexpression of AQP5 in sweat glands is probably one pathogenetic mechanism underlying PFH.

What determines human body odour?

Human body odour and earwax type are genetically dependent on a single-nucleotide polymorphism (SNP) located in the ABCC11 gene. So far, it still remains to be clear how SNP in the ABCC11 gene is associated with human malodour. In a recent issue of Experimental Dermatology, Baumann et al. propose one of the underlying molecular pathways. Although one of the amino acid conjugated of the odorants, Cys-Gly-3-methyl-3-sulfanylhexanol (3M3SH), was not taken up by the transporter ABCC11, glutathione conjugate of 3MSH (SG-3MSH) was transported by ABCC11. Moreover, SG-3MSH was processed to 3M3SH by γ-glutamyl-transferase 1 (GGT1), which was abundantly expressed in apocrine sweat glands. These findings may pave a way for the pharmacogenetics of human body odour and the development of innovative deodorant products.

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.

Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins.

The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.

Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.

Dependence on p75 for innervation of some sympathetic targets.

The low-affinity neurotrophin receptor p75 binds all neurotrophins with similar affinity. For elucidation of its function, mice bearing a null mutation in the p75 locus were generated. Examination of sympathetic innervation of target tissues revealed that pineal glands lacked innervation and sweat gland innervation was absent or reduced in particular footpads. The absence of adult innervation reflects the failure of axons to reach these targets during development rather than a target deficit. These results indicate that p75 facilitates development of specific populations of sympathetic neurons, for which it may support axon growth.

Refined Quantitation of Sweat Gland Innervation.

Skin biopsies have gained increasing popularity as a tool to evaluate disorders affecting small nerve fibers. While reports on sweat gland nerve fiber density (SGNFD) to quantitate sudomotor innervation have been promising, methodologies vary significantly. Although conventional stereology is commonly used, no standard technique has been established. We sought to develop an accurate and reproducible technique to quantify SGNFD. Skin punch biopsies from healthy individuals were cut and stained. Images of sweat glands (SGs) were acquired using confocal and widefield microscopes, and optimized using deconvolution. Nerve fibers were reconstructed and nerve fiber length (NFL) was quantified using three-dimensional (3D) automated software. SGNFD was obtained by dividing NFL by SG volume. SGNFD was also assessed using stereology for comparison. Ninety-two SGs from 10 healthy subjects were analyzed by independent observers. Using confocal microscopy, the software reliably traced nerve fibers. In contrast, rendering of nerve fibers was inferior using widefield microscopy. Interobserver reliability was suboptimal using widefield images compared to confocal (ICC = 0.82 vs ICC = 0.98). Correlation between 3D-reconstruction and stereology was poor (ICC = 0.38). The newly developed technique of SGNFD quantitation using 3D reconstruction of SG innervation with confocal microscopy reliably traces nerve fibers, shows outstanding reproducibility, is almost completely unbiased, and superior to conventional stereology methods.

The in Vitro Immune-Modulating Properties of a Sweat Gland-Derived Antimicrobial Peptide Dermcidin.

The epidermal barriers of the skin serve as the first layer of defense by limiting the access of many pathogens to the blood circulation. In addition, human skin also contains sweat glands that can secrete a wide array of antimicrobial peptides to restrain the growth of various microbes. In the case of microbial infection, macrophages and monocytes constitute the first line of defense by producing a wide array of proinflammatory cytokines and chemokines. This process is triggered either by pathogen-associated molecular pattern molecules (such as bacterial endotoxin) or damage-associated molecular pattern molecules (such as HMGB1). In light of our findings that a sweat gland-derived antimicrobial peptide, dermcidin, affected both pathogen-associated molecular pattern and damage-associated molecular pattern-induced cytokines/chemokines by macrophages/monocytes, we propose that dermcidin may play an important role in the regulation of the innate immune responses to infection and injury. Future investigations are warranted to further test this understudied hypothesis in both preclinical and clinical settings.

Aberrant gating, but a normal expression pattern, underlies the recessive phenotype of the deafness mutant Connexin26M34T.

Mutations in the gene GJB2, encoding the gap junction protein Connexin26 (Cx26), are the most prevalent cause of inherited hearing loss, and Cx26M34T was one of the first mutations linked to deafness (Kelsell et al., 1997; Nature 387, 80-83). We report the first characterization of the gating properties of M34T, which had previously been reported to be nonfunctional. Although homotypic mutant channels did not produce detectable currents, heterotypic pairings with wtCx26 confirmed that M34T formed intercellular channels, although the gating properties were altered. Cx26M34T displayed an inverted response to transjunctional voltage (Vj), mediating currents that activate in a time- and Vj-dependent manner. These characteristics suggest that the channel population is only partially open at rest, consistent with previous reports that dye transfer in M34T-expressing cells is reduced or abolished (e.g., Thonnissen et al., Human Genet. 111, 190-197). To investigate the controversial recessive/dominant behavior of this mutant, we coexpressed M34T with wtCx26 RNA at equimolar levels, mimicking the situation in heterozygotic individuals. Under these conditions, M34T did not significantly reduce Cx26/Cx26 coupling, or alter the electrophysiological properties of the wt channels, consistent with the recessive nature of the allele. Overexpression of the mutant did have some inhibitory effects on conductance, possibly explaining some of the previous reports in exogenous expression systems and some patients. Consistent with its electrophysiological behavior, we also show that M34T localizes to cell junctions in both transfected HeLa cells and patient-derived tissue.

Changes of expression of intermediate filament proteins during ontogenesis of eccrine sweat glands.

The intermediate filament expression in fetal and adult human eccrine sweat glands was studied by immunoperoxidase microscopy performed on cryostat sections using monoclonal antibodies against various cytokeratins (CK), vimentin, and actin. In palmar skin of 14-week-old fetuses, the early dermal cords showed a primitive CK pattern similar to that of epidermal basal cells. From week 15 on (distal finger skin), inner cells of the proximal (ductal) portion of the glandular anlagen expressed CK 1/10/11 and 19 (markers of adult eccrine ductal luminal cells). In addition, CK 4 was expressed in ductal luminal cells mainly in the fetal period. In the distal portion of the sweat gland anlagen the increased or new expression of the simple-epithelium-type CK 7, 8, 18, and 19 was detected at week 15, indicating the onset of the secretory differentiation pathway. Two subsegments of the prospective secretory portion could be distinguished (elongated part and end bud). Interestingly, in fetuses, most secretory portion cells co-expressed vimentin in addition to CK. From week 22 on, peripheral cells of the secretory portion were stained for CK 17 and smooth-muscle-type actin, suggesting myoepithelial differentiation. In newborn and adult eccrine glands, secretory cells expressed mainly CK 7, 8, 18, and 19, whereas myoepithelial cells were conspicuous by their co-expression of certain CK (including CK 5 and 17), vimentin, and smooth-muscle-type actin and sometimes even glial filament protein (GFP), similar to myoepithelial cells of other glands. These results throw further light onto the complex processes of fetal development of eccrine sweat glands and their cellular diversification. The possible biologic significance of the differential CK expression in the various glandular cell types is discussed.

Interleukin-8 immunoreactivity in the skin of healthy subjects and patients with palmoplantar pustulosis and psoriasis.

Previous studies have shown that neutrophil-activating peptide 1/interleukin-8 (IL-8) is present in psoriatic scales and to a lesser extent in normal human epidermis. A panel of monoclonal antibodies and polyclonal antisera raised against IL-8 was used to localize IL-8 with immunoperoxidase techniques in non-lesional and lesional skin of patients with psoriasis and palmo-plantar pustulosis (PPP), and in corresponding sites from healthy subjects. Intracellular IL-8 immunoreactivity was found in all epidermal cell layers in biopsies of healthy subjects and in non-lesional and lesional skin in both PPP and psoriasis. The most intense immunolabeling was regularly found in the basal cell layer. Intercellular epidermal IL-8 immunolabeling was regularly detected in lesional biopsies in PPP and psoriasis, but not in healthy subjects or non-lesional skin in PPP and psoriasis. No intercellular immunolabeling was detected after successful treatment of lesional skin. The majority of cells along the eccrine sweat glands, dermal mononuclear cell infiltrates, and endothelial cells were IL-8 immunoreactive in all biopsies studied. The present study suggests that IL-8, its precursor form, or, alternatively, a degradation product is present in normal human epidermis.

Cystic fibrosis transport regulator and its mRNA are expressed in human epidermis.

Cystic fibrosis transport regulator is a cAMP-dependent chloride channel protein. Normal (non cystic fibrosis) human epidermis stained positive for cystic fibrosis transport regulator as densely as did the eccrine sweat gland when three monoclonal antibodies for R (regulatory) and C (C-terminus) domains of cystic fibrosis transport regulator were used. All the layers of the epidermis took up staining uniformly. A peptide for C-epitope completely blocked the staining with monoclonal antibodies for C. Nested reverse transcription polymerase chain reaction of freshly isolated human epidermal fragments and the eccrine sweat glands amplified the cystic fibrosis transport regulator mRNA sequence derived from exons 13 and 14 to comparable extents. The 526 base pair antisense, but not sense, RNA probe derived from exons 10-13 stained cystic fibrosis transport regulator mRNA in both the epidermis and the sweat gland to a similar extent. In the epidermis, the cytoplasm of basal cells, stratum spinosum cells, and granular layer cells were all stained uniformly, but not corneocytes in the stratum corneum. In the sweat secretory coils, both clear and dark cells were stained but not the myoepithelium, with the dark cells staining more densely than the clear cells as in a previous study. In the duct, both luminal and basal ductal cells took up cystic fibrosis transport regulator staining uniformly but luminal cytoplasm of luminal ductal cells was devoid of cystic fibrosis transport regulator mRNA. Although the function of cystic fibrosis transport regulator in the epidermis is totally unknown, its recently proposed role as a universal regulator of a variety of cellular and membrane functions necessitates further studies on its regulation and function in health and disease.

Topography of mammalian connexins in human skin.

We have explored the expression of gap junction proteins in normal human skin by immunostaining cryostat sections (indirect immunofluorescence) or lyophilized epidermis (Western blotting) with antibodies against four mammalian connexins Cx26, Cx32, Cx40, Cx43; and by hybridizing total epidermal RNA (Northern blotting) with cRNA probes for Cx26, Cx32, and Cx43. We found that epidermal keratinocytes express Cx43 but not Cx26, Cx32, or Cx40. This expression was minimal in the basal layer, much higher in the spinous layer, reduced in the granular layer, and absent in the stratum corneum. Immunostaining for Cx43 was also observed in sebaceous glands, hairs, and eccrine sweat ducts. The two latter epidermal adnexae were also markedly labeled by antibodies against Cx26, a gap junction protein that was undetectable by immunofluorescence in interfollicular keratinocytes. Immunoblots of polyacrylamide gel electrophoresis-separated epidermal proteins and hybridization of epidermal RNA confirmed the presence of Cx43 in epidermis. These observations indicate that 1) Cx43 and Cx26 are components of human keratinocyte gap junctions; 2) these two proteins are differentially expressed in the interfollicular epidermis and the skin adnexae; 3) in interfollicular epidermis, Cx43 is a predominant gap junction protein, mostly expressed by the differentiating spinous cells; 4) Cx43 distribution is in accordance with the extensive dye coupling previously observed in this epidermal compartment.