Assessment of markers expressed in human hair follicles according to different skin regions.

BACKGROUND: Body region-dependent hair follicle (HF) characteristics are concerned with follicular size and distribution, and have been demonstrated to have characteristics for each region of the body. OBJECTIVES: The aim of the present study was to investigate the expression patterns of the markers called cytokeratin 15 (K15), cytokeratin 6 (K6) and monoclonal antibody Ki-67, and also apoptosis in HFs, which can be observed in different parts of the human body. MATERIAL AND METHODS: In this study, healthy human HFs were taken by biopsy from 5 various donor sites of the human body: the scalp, the leg, the abdomen, the back and waist. HF-containing skin specimens taken using cryosection were stained with hematoxylin & eosin (H&E) and K15, K6, Ki-67 and terminal deoxynucleotidyl transferase-mediated digoxigenin-dNTP nick end-labelling (TUNEL) immunofluorescence staining protocol was performed. RESULTS: Different skin regions from the human body were examined histologically. While the HFs of scalp tissue showed anatomically obvious hair layers, some hair sections from other regions, like the leg, the abdomen, back and waist, were not as distinct as in the scalp region. According to our findings, K15 expression was highest in the scalp. In addition, the immunoreactivity (IR) intensity of K15 was significantly decreased in the HFs on the waist and abdominal regions, compared to the scalp and back regions (p < 0.001). However, the IR intensity of K6 in the scalp region was statistically significantly higher than the IR intensity of K6 in the abdomen region (p < 0.05). Moreover, we showed intraepithelial apoptosis and proliferation of keratinocytes in the bulge of HF. In the study, Ki-67-positive and TUNEL-positive cell numbers were not statistically significant (p > 0.05). CONCLUSIONS: Our findings are important for further investigation of molecular aspects of the human hair follicle stem cells compartments in health and disease, which might be a promising model for comparative studies with different human diseases.

Eradication of damaged keratinocytes in cutaneous lichen planus forms demonstrated by evaluation of epidermal and follicular expression of CK15, indices of apoptosis and regulatory protein S100.

The study of cytoskeleton arrangement and its contribution to survival of cell-to-cell contacts appears to be essential for understanding of numerous cellular and tissue processes. Applying CK15, S100 labeling and TUNEL reaction to cutaneous lichen planus subtypes, we found CK15 expression in the outer and inner root sheath of hair follicles, the basal epidermal layer, and eccrine glands. Its follicular expression was decreased in nearby inflammatory infiltrates. The CK15 immunopositivity was mostly described as weak (92.3%) for lichen planus but equally subdivided into weak, moderate and strong in lichen planopilaris (2 = 32.514; df = 4; p < 0.001). The greatly varying apoptotic index was the highest in the lichen planopilaris involving the scalp: 81.2 ±10.7; 87.8 ±10.7 and 88.0 ±10.5 for the basal, spinous and upper epidermal layers, respectively. S100 positive epidermal and follicular cells did not differ in the lesions demonstrated in the study groups; still immunoreactivity was more pronounced in the scalp region of lichen planopilaris. Damage of cell-to-cell contacts was confirmed by electron microscopy. Apart from immunocyte-mediated keratinocyte death, cytoskeleton-based injury and loss of cell-to-cell and matrix contacts may be of great importance, leading to eradication of degrading cells and thus contributing to the pathogenesis of lichen planus.

N(1)-methylspermidine, a stable spermidine analog, prolongs anagen and regulates epithelial stem cell functions in human hair follicles.

Spermidine (Spd), the prototypic polyamine, has been shown to be essential for hair follicle (HF) growth. However, Spd can be readily converted into other polyamines, and is physiologically unstable. Therefore, to assess its individual functions on HFs, we used the metabolically stable Spd analog N(1)-methylspermidine (N(1)-MeSpd). N(1)-MeSpd was confirmed to be a metabolically stable compound, with a half life of 90 h. 0.5 µM N(1)-MeSpd strongly prolonged anagen and decreased cell apoptosis in HFs in culture after 6 days, accompanied by specific stimulation of the expression of the epithelial stem cell-associated keratin, K15. N(1)-MeSpd also reduced lactate dehydrogenase activity in the culture supernatant, a parameter of cell death and cell lysis. N(1)-MeSpd diminished intracellular reactive oxygen species production in cultured keratinocytes, and reduced tumor necrosis factor-α, interleukin (IL)-1ß and IL-6 gene and protein expression after lipopolysaccharide stimulation. This suggests that some effects of N(1)-MeSpd may be mediated by anti-oxidative and anti-inflammatory effects. These additional properties of N(1)-MeSpd could be clinically important for the treatment of inflammatory alopecias and inflammatory scalp diseases.

Cellular and biological evaluation and diagnostic immunohistochemistry of cytokeratin 15/19 expression in distinguishing cutaneous basal cell carcinoma.

Recent studies have investigated the expression of proliferative markers, but little is known about the expression of cytokeratin 15 and 19 in different histological subtypes of basal cell carcinoma (BCC). We conducted cellular, biological, clinicopathological and immunohistochemical analysis on the manifestations of 8 BCC by hematoxylin and eosin stain (H&E) staining and immunohistochemistry and reviewed relevant literature. Microscopically, the tumor cells were multiple remarkable foci of epidermolytic hyperkeratosis with large pleomorphic nuclei and scant cytoplasm together with peripheral palisading and forming solid nests. Furthermore, the most tumors were composed of highly cellular areas with a homogenous population of round, ovoid and spindle cells, hyperchromatic nuclei, high cellular pleomorphism, high mitotic index and various morphologic patterns. Moreover, the tumors displayed an invasive growth, with positive expression of Cytokeratin 19 (CK19) and negative expression of CK15. Our study revealed that the expression of CK19 was associated with progression and invasion in cases with BCC and immunohistochemistry is indispensable in distinguishing this tumor from other types of cutaneous carcinoma. To our best knowledge, it may be a considerable biomarker to assess invasiveness of cutaneous-surface BCC and to guide clinical management of such tumors.

The changes in the expression levels of follicular markers in keratoacanthoma depend on the stage: keratoacanthoma is a follicular neoplasm exhibiting infundibular/isthmic differentiation without expression of CK15.

BACKGROUND: Although the precise etiology of keratoacanthoma (KA) is unknown, KA is generally assumed to differentiate toward hair follicles based on previous studies of experimental carcinogenesis. METHODS: We performed a comprehensive immunohistochemical study of various follicular markers in all stages of KA. A total of 67 tumors, including 16 early or proliferative stage lesions, 43 well-developed stage lesions, five regressing stage lesions and three regressed stage lesions, were subjected to the analysis. RESULTS: CK15 (clone C8/144B), CK19 and CD34 were not expressed at any stage. CK1, CK10, CK16, CK17, CK15 (clone LHK15) and calretinin showed dynamic changes in their expression in KA depending on the stage. CONCLUSIONS: KA is a follicular neoplasm with infundibular/isthmic (upper segmental region of hair follicles) differentiation. It is considered that early or proliferative stage tumors show keratin-filled invaginations with infundibular differentiation and gradual isthmic differentiation. Well-developed examples of KA generally show isthmic differentiation in the whole lesions. The regressed stage KAs lose the features of this type of follicular differentiation and show epidermal characteristics. No expression of CK15 (clone C8/144B) was observed in KAs, although this finding is insufficient to completely rule out the correlation between the regression of KA and the hair follicle cycle.

The ventral proximal nail fold: stem cell niche of the nail and equivalent to the follicular bulge--a study on developing human skin.

BACKGROUND: Although the bulge is well characterized as a stem cell niche of the hair follicle, comparatively little is known about the location of stem cells in the nail. Herein, we describe the spatiotemporal expression pattern of six stem cell markers in the developing human nail and compared it with the embryonic and fetal human hair follicle. The areas of proliferative activity were additionally examined using labeling with Ki-67. METHODS: We examined immunohistochemically samples from embryonic and fetal human nail, hair and skin for the expression of cytokeratin 15 (CK15, two clones), cytokeratin 19 (CK19), PHLDA1, CD200, nestin and Ki-67 using standard techniques. RESULTS: CK15 (clone LHK15), CK19 and PHLDA1 are negative in the nail and hair matrix but positive in the ventral proximal nail fold and in the follicular bulge. Over the course of embryogenesis they display a highly specific spatiotemporal expression pattern both in the nail and in the hair follicle. CONCLUSIONS: We propose that at least during embryogenesis the proximal ventral nail fold represents the niche for the nail stem cells. In contrast to animal experiments, autoradiographic pulse-chasing studies cannot be performed in human, and immunohistochemical studies are a valid alternative although they have their limitations. Further studies on adult human nail units are suggested.

Expression of basal cell keratin 15 and keratin 19 in oral squamous neoplasms represents diverse pathophysiologies.

Human epithelium contains keratin, which is expressed during differentiation. Depending on the target cell type, different types of keratin are expressed, and their alterations seem to represent changes in cell properties. The basal cells of oral epithelium express keratin 5 (K5), K14, K15 and K19, but their alterations in tumors are unclear. To address this issue and to seek possible diagnostic application, we examined the expression of these keratins in oral squamous cell carcinoma (OSCC) and squamous intraepithelial neoplasm (SIN). cDNA microarray analysis of 43 OSCC revealed slight upregulation of KRT14, downregulation of KRT15 and KRT19, and unaltered KRT5 expression. There were great variations in KRT15 and KRT19 expression across each cancer. Well-differentiated OSCC tended to express more KRT15 and less KRT19 compared to moderately- or poorly-differentiated OSCC. KRT15 was positively correlated with differentiation-related keratin, KRT13. These observations were further investigated by immunohistochemical examination. K5 and K14 were ubiquitously expressed in all 50 OSCC and 50 SIN examined. K15 and K19 were generally downregulated, but were considerably retained in about half of the cases and showed diverse expression patterns. K15-positive cancers tended to show a well-differentiated phenotype, and K19-positive cancers tended to show more invasive tumor fronts. Most K19-positive cancers appeared to develop with little associating SIN. K19 was consistently downregulated in SIN, while K15 was downregulated mainly in high grade SIN. In summary, K15 and K19, unlike K5 or K14, are expressed variably in both SIN and OSCC, which reflects the differences in their pathogenesis and biological behaviors, suggesting their prospective applications as markers for subclassifying OSCC and SIN.

Cytokeratin 15 expression in central, centrifugal, cicatricial alopecia: new observations in normal and diseased hair follicles.

BACKGROUND: Cytokeratin 15 (CK15) is a useful marker for the bulge zone (BZ) and has been used to examine follicles in cicatricial alopecia. We studied the expression of CK15 in hair follicles of patients with central, centrifugal, cicatricial alopecia (CCCA) in an attempt to define BZ integrity. METHODS: A commercially available antibody to CK15 was used on formalin-fixed, paraffin-embedded tissue from clinically and histologically 'normal' scalps, clinically diseased scalps from patients with CCCA and clinically 'normal' scalps from patients with CCCA. RESULTS: In both normal and diseased follicles, CK15 expression was closely linked to anatomical zone cellular morphology. Normal and abnormal inner root sheath (IRS) desquamation occurred in concert with predictable cellular morphological changes and CK15 expression. In most abnormal follicles, once the IRS desquamated, the morphology of BZ epithelium changed and CK15 expression disappeared. CONCLUSIONS: CK15 highlights BZ cells in normal human follicles, but may be unreliable for this purpose in diseased follicles. CK15 should not be the sole marker for studying stem cells in cicatricial alopecia because any disease-induced structural changes could alter CK15 expression. More sophisticated studies of stem cells will be required to reliably define their role in the pathogenesis of cicatricial alopecia.

Endocrine controls of primary adult human stem cell biology: thyroid hormones stimulate keratin 15 expression, apoptosis, and differentiation in human hair follicle epithelial stem cells in situ and in vitro.

Here we demonstrate that physiological concentrations of the thyroid hormones T3 and T4 enhance the KERATIN 15 promoter activity and expression in epithelial stem cells of adult human scalp hair follicles in situ and in vitro. Additionally, T3 and T4 stimulate expression of the immuno-inhibitory surface molecule CD200. Subsequently, T3 and T4 induce apoptosis and differentiation and inhibit clonal growth of these progenitor cells in vitro. These data suggest that human hair follicle bulge-derived epithelial stem cells underlie profound, previously unknown hormonal regulation by thyroid hormones, and show that primary human keratin 15-GFP+ progenitor cells can be exploited to further elucidate fundamental endocrine controls of human epithelial stem cells.

Expression of the hair stem cell-specific marker nestin in epidermal and follicular tumors.

Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells give rise to the outer-root sheath. Nestin-expressing hair follicle stem cells that are negative for the keratinocyte marker keratin 15 (K15) can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Recent studies suggest that the epithelial stem cells are important in tumorigenesis. In this study, we immunohistochemically examined the expression of three hair follicle stem cell and progenitor cell markers, nestin, K15, and CD34, in normal human epidermis and hair follicles and in epidermal and follicular tumors, trichilemmoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). In normal human skin, the cells in the epidermal basal layer were positive for K15 and negative for nestin and CD34. The hair follicle cells below the sebaceous glands were also positive for nestin and K15 and negative for CD34. The outer-root sheath cells under this area could be divided into three parts: an upper part of the outer-root sheath cells that was partially positive for nestin and positive for K15 and negative for CD34; a middle part that was CD34-positive and K15-negative; and a lower part that was positive for K15 and negative for CD34. In the tumor tissues, nestin immunoreactivity was observed in trichilemmoma but not in BCC. Also, immunoreactivity for K15 was strong in BCC and weak in trichilemmoma, and SCC was negative for nestin and partially positive for K15. No CD34 immunoreactivity was observed in any of the cases. These results suggested that trichilemmoma originates in the nestin-positive/K15-positive/CD34-negative outer-root sheath cells below sebaceous glands, BCC tumor cells from the more mature nestin-negative/K15-positive/CD34-negative outer-root sheath cells, and SCC from the nestin-negative/K15-positive/CD34-negative keratinocytes of the basal cell layer in the epidermis.

Changes in the expression of stem cell markers in oral lichen planus and hyperkeratotic lesions.

Despite the pivotal role of stem cells in homeostasis of oral epithelia the location of this cell population within the tissue is uncertain. How disease influences these cells in vivo also remains to be elucidated. In this study we have used six molecular markers to identify stem cells in normal and diseased buccal mucosa. Samples of normal oral mucosa (NOM), hyperkeratosis (OHK) and oral lichen planus (OLP) were immunostained for alpha6 and beta1 integrins, keratin 15 (K15), melanoma-associated chondroitin sulphate proteoglycan (MCSP), NG2 the rat homologue of human MCSP and notch 1. K15, NG2 and beta1 staining was continuous in the basal layer of NOM whilst alpha6 and MCSP were limited to basal cells at the tips of connective tissue papillae. K15 was downregulated in OLP whereas alpha6, beta1 and MCSP were upregulated in both OLP and OHK. NG2 remained unchanged and notch 1 was absent in all samples. Therefore, the stem cell phenotype in OLP and OHK maybe altered in response to pathological signaling. Classification of these changes is essential to understand the role of adult stem cells in the pathogenesis of oral diseases characterised by abnormal keratinocyte proliferation and differentiation.

Expression of COUP-TF-interacting protein 2 (CTIP2) in mouse skin during development and in adulthood.

COUP-TF-interacting protein 2 (CTIP2), also known as Bcl11b, is a transcriptional regulatory protein that is highly expressed in and plays a critical role(s) during development of T lymphocytes and the central nervous system. We demonstrate herein that CTIP2 is also highly expressed in mouse skin during embryogenesis and in adulthood as revealed by immunohistochemical analyses. CTIP2 expression in the ectoderm was first detected at embryonic day 10.5 (E10.5), and became increasingly restricted to proliferating cells of the basal cell layer of the developing epidermis in later stages of fetal development and in adult skin. In addition, CTIP2 expression was also detected in some cells of the suprabasal layer of the developing epidermis, as well as in developing and mature hair follicles. Relatively fewer cells of the developing dermal component of skin were found to express CTIP2, and the adult dermis was devoid of CTIP2 expression. Some, but not all, of the cells present within hair follicle bulge were found to co-express CTIP2, keratin K15, but not CD34, indicating that a subset of K15(+) CD34(-) skin stem cells may express CTIP2. Considered together, these findings suggest that CTIP2 may play a role(s) in skin development and/or homeostasis.