This experiment developed the methodology of double staining for senescence associated-beta-galactosidase (SA-beta-gal) activity and keratin 10 (K10) or involucrin. To prove the usefulness of the double staining, the author investigated the relationship between senescence and differentiation in monolayer and organotypic cultured keratinocytes. The results were as follows: K10 and involcrin together with SA-beta-gal were doubly stained in most of monolayer cultured keratinocyte. This fact indicated that the senescence and differentiation had simultaneously occurred in the same keratinocyte. In spite of the advantages to preserving structures, the paraffin specimen was not suitable for double staining because of the limitation of SA-beta-gal reactivity. Although the cryosectioned specimen did not have the morphology as good as the paraffin specimen, it was suitable for double staining due to the goodness of SA-beta-gal reactivity. Double staining well reflected the disturbances of senescence and differentiation which could be caused by deranged organizations of the organotypic cultured skin. The organotypic cultured skin which showed deranged organizations such as stratified basal layer, no typical cell features in each epdermal layer, and wide intercellular spaces had SA-beta-gal activity in epidermis and K10 or involucrin reaction in basal cell. But the skin which showed well arranged organizations resembling in vivo skin had no SA-beta-gal activity and no K10 or involucrin reaction in basal cells. In conclusion, it might be suggested that the double staining for SA-beta-gal activity and K10 or involucrin could be used for detecting the extent of senescence and differentiation in the same cell.
Aging* ; Epidermis ; Extracellular Space ; Keratin-10* ; Keratinocytes* ; Paraffin ; Skin

In human skin, specific keratin markers reflect on normal differentiation and pathologic conditions. This experiment focused on the expressional pattern of keratin 10 (K10: normal differentiation marker), and keratin 8 & 13 (K8 & K13: pathologic differentiation marker) together with their cellular localization after treating HaCaT cells with 12-Otetradecanoylphorbol 13-acetate (TPA). The cells were treated with TPA at 0, 0.1, 1 microgram/ml for 2 hours or 6 hours. Morphologic studies revealed that TPA treatment changed the shape of cells into the fibroblast-like cells with highly folded nuclear membrane and reduced number of the desmosome. The results of indirect immunofluorescent staining and Northern blotting analysis showed that TPA considerably down-regulated the expression of K10, while markedly up-regulating the expression of K8 and K13 both at protein and mRNA levels. Furthermore, by simultaneous staining for keratins and DNA content in flow cytometry, it was found that TPA increased the expression of K8 and K13 dramatically at the S-G2-M phase of the cells. In conclusion, these changes induced by TPA in HaCaT cells may indicate a close relationship between the morphologic change and the altered expression of keratin subfamilies. It also suggests that TPA known as a tumor promotor may directly induce the potentially malignant cells even without the support of tumor initiator.
Blotting, Northern ; Desmosomes ; DNA ; Flow Cytometry ; Humans ; Keratin-10 ; Keratin-8 ; Nuclear Envelope ; RNA, Messenger ; Skin

PURPOSE: To reconstruct a cultured conjunctival equivalent that closely resembles normal conjunctival epithelium in three-dimensional culture systems. METHODS: Human conjunctival epithelial cells were cultured on dead de-epidermized dermis in the air-exposed state. After 2 weeks of culture, the sections were stained with hematoxylin and eosin. Immunohistochemical and electron microscopic studies were performed. The results were compared with those of normal conjunctiva and cultured eyelid skin equivalent. RESULTS: In the cultured conjunctival equivalent, nonkeratinizing stratified epithelium was formed similarly to normal conjunctival epithelium. Keratin 13 was expressed, but not keratin 10, in the cultured conjunctival equivalent, similarly to normal conjunctival epithelium. However, in the cultured eyelid skin equivalent, keratinizing stratified epithelium was formed. In addition, keratin 10 was expressed, but not keratin 13, contrary to those of the cultured conjunctival equivalent. In the cultured conjunctival equivalent, ultrastructurally, keratin intermediate filaments and desmosomes were found. In addition, microvilli were seen in the uppermost epithelial cells. CONCLUSIONS: These findings demonstrate that the cultured conjunctival equivalent resembles normal conjunctival epithelium morphologically, biochemically and ultrastructurally, thereby suggesting that the cultured conjunctival equivalent may have a great potential in the study of conjunctival epithelium.
Conjunctiva ; Dermis ; Desmosomes ; Eosine Yellowish-(YS) ; Epithelial Cells ; Epithelium* ; Eyelids ; Hematoxylin ; Humans ; Intermediate Filaments ; Keratin-10 ; Keratin-13 ; Microvilli ; Skin

BACKGROUND: The differential diagnosis of psoriasis and seborrheic dermatitis can be difficult when both conditions are localized to the scalp without the involvement of other skin sites. OBJECTIVE: We aimed to evaluate the histopathological differences between psoriasis and seborrheic dermatitis on the scalp and identify favorable criteria for their differential diagnosis. METHODS: We evaluated 15 cases of psoriasis and 20 cases of seborrheic dermatitis of the scalp that had been clinicopathologically diagnosed. Skin biopsy sections stained with H&E were examined. Additional immunohistochemistry was performed, including Ki-67, keratin 10, caspase-5, and GLUT-1. RESULTS: On histopathological examination, mounds of parakeratosis with neutrophils, spongiform micropustules of Kogoj, and clubbed and evenly elongated rete ridges were significantly more frequently observed in psoriasis. Follicular plugging, shoulder parakeratosis and prominent lymphocytic exocytosis were significantly more common in seborrheic dermatitis. Moreover, significantly higher mitotic figures were observed in psoriatic lesions than in seborrheic dermatitis. Immunohistochemistry did not show any difference between psoriasis and seborrheic dermatitis. CONCLUSION: Histopathological features favoring psoriasis include mounds of parakeratosis with neutrophils, spongiform micropustules of Kogoj, clubbed and evenly elongated rete ridges, and increased mitotic figures (≥6/high-powered field). Features indicating seborrheic dermatitis are follicular plugging, shoulder parakeratosis and prominent lymphocytic exocytosis. Immunohistochemistry was not helpful in differentiating psoriasis from seborrheic dermatitis.
Biopsy ; Dermatitis, Seborrheic* ; Diagnosis, Differential* ; Exocytosis ; Immunohistochemistry ; Keratin-10 ; Neutrophils ; Parakeratosis ; Psoriasis* ; Scalp* ; Shoulder ; Skin

OBJECTIVETo investigate the gene mutation in one sporadic case of bullous congenital ichthyosiform erythroderma (BCIE), and to explore the relationship between the genotype and phenotype.

METHODSDNA was extracted from the blood samples of the patient with BCIE, unaffected members of the pedigree, and 50 unrelated healthy controls. PCR was used to amplify the hot spot fragment of keratin 1 (KRT1) and keratin 10 (KRT10) gene. The PCR products were directly sequenced to detect the mutations.

RESULTSA heterozygous 467G>A mutation was found in the patient, resulting in the substitution of arginine (R) by histidine (H) in codon 156 (R156H) in the 1A domain of the KRT10 protein but not in the healthy individuals from the family and the 50 unrelated individuals.

CONCLUSIONThe mutation of 467G>A in exon 1 of KRT10 gene identified may play a major role in the pathogenic mechanism of this case of BCIE.

Adolescent ; Base Sequence ; DNA Mutational Analysis ; Exons ; genetics ; Female ; Humans ; Hyperkeratosis, Epidermolytic ; genetics ; pathology ; physiopathology ; Keratin-10 ; genetics ; Mutation

OBJECTIVETo investigate the different location and the expression characteristics of epidermal stem cells in normal adult skin and scar tissue.

METHODSSkin tissue specimens were harvested from the corresponding sites from 6 healthy volunteers and from scar tissue of 6 patients 1 year after major deep burn. beta1 integrin and keratin 19 (K19) were employed as the biochemical markers for stem cells and transit amplifying cells identification and keratin 14 (K14) and keratin 10 (K10) as markers for post-mitotic cells and terminally differentiated cells respectively. Integrin and keratin were determined by Elivision two-step immunohistochemistry.

RESULTSThe expression of beta1 integrin and the K19 positive cell count in the epithelial basal layers of scar tissue were evidently decreased and weakened than those in normal adult healthy skin. Furthermore, the positive cells expressing K14 in epidermis of scar tissue were only located in 2 - 3 layers of basal epidermis, and their number was much less than that in normal adult skin. Whereas the cells positively expressing K10 were distributed wider in area than that in normal healthy skin. The epidermal stem cells and transit amplifying cells in scar epidermis were much less in number than that in normal skin. The differentiation process of scar epidermal stem cells was different from that of normal skin. And the proportions of post-mitotic cells and terminally differentiated cells were abnormal.

CONCLUSIONThe results indicated that the self-renewal ability of the scar epidermis was decreased, and the differentiation process of it was in disorder, which may be a reason for the abnormality of structure and function of the epidermis in scar, and a reason for the decreased ability of wound healing of scar tissue.

Adult ; Burns ; complications ; metabolism ; pathology ; Cicatrix ; etiology ; metabolism ; pathology ; Epidermis ; chemistry ; cytology ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Keratin-10 ; Keratin-14 ; Keratins ; analysis ; Middle Aged ; Skin ; chemistry ; cytology ; Stem Cells ; chemistry ; cytology

BACKGROUND: Calcium plays a role in the proliferation and differentiation of keratinocytes. In a normal situation, the calcium concentration forms a gradient across the epidermal layers. Calcium is sparse in the basal layer and spinous layer. Skin organ culture is a useful model for conducting research on various aspects of skin biology. Skin organ culture systems are used for defining factors that affect homeostasis when elucidating the modulatory effects of biologic response modifiers, drugs and physical agents on the skin and also when studying complex aspects of cutaneous biology in normal and diseased skin. OBJECTIVE: In this study, we investigated the effects of extracellular calcium on the epidermis in a skin organ culture. METHODS: We compared the skin organ culture patterns under various culture conditions (calcium 0.1, 0.7, 1.4 and 2.0 mM). RESULTS: H&E staining showed different phenotypes according to the calcium concentration and IHC also showed different phenotyes compared to that of keratin 10, involucrin, filaggrin, loricrin and PCNA. CONCLUSION: As a result, we concluded that the calcium gradient is also an important factor in skin organ culture to maintain the vivo-like environment and the appropriate calcium concentration is 1.4 mM.
Biology ; Calcium ; Epidermis ; Homeostasis ; Intermediate Filament Proteins ; Keratin-10 ; Keratinocytes ; Membrane Proteins ; Organ Culture Techniques ; Phenotype ; Proliferating Cell Nuclear Antigen ; Protein Precursors ; Skin

OBJECTIVETo investigate the distribution of epidermal stem cells (ESCs) in different degrees of burn wounds in scalded rats.

METHODSThirty-two Sprague-Dawley (SD) rats were employed in the study. First degree (I), shallow (shallow II) and deep partial thickness (deep II) and full thickness burn wounds (III) were created on the rat skin. Burn wound samples were harvested at 24 postburn hours (PBHs) from all the wounds and were processed to tissue slices. The tissue slices were stained by immunohistochemistry technique. The expression and distribution of ESCs in different degrees of burn wounds were observed with integrins alpha 2 beta 1 and keratin 10 (K10) as first antibodies.

RESULTSK10 positive cells were found to distribute in the strata spinosum, granulosum and lucidum in the first degree burn wound (I) with large amounts of integrins alpha 2 beta 1 positive cells in the residual basal layer and skin appendages (hair follicles) in shallow partial thickness burn wound (shallow II degree), and there were less integrins alpha 2 beta 1 positive cells in the remaining skin appendages in deep dermis in deep partial thickness burn wound (deep II degree). Finally, integrins alpha 2 beta 1 positive cells were sparsely found in the III degree burn wound.

CONCLUSIONThe distribution of ESCs in burn wounds was closely related to the depth of burn wound. The residual ESCs might be the origin of burn wound regeneration and reepithelization.

Animals ; Burns ; metabolism ; pathology ; Female ; Immunohistochemistry ; Integrin alpha2beta1 ; analysis ; Keratin-10 ; Keratins ; analysis ; Male ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; pathology

Adult ; Cell Transformation, Neoplastic ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Keratin-10 ; metabolism ; Male ; Ovotesticular Disorders of Sex Development ; metabolism ; pathology ; surgery ; Teratoma ; metabolism ; pathology ; surgery ; Vimentin ; metabolism

OBJECTIVE: The expression of C/EBPα, CK10 in nasal inverted papilloma (NIP) were detected in the study. Further discussed their significance in genesia, development and recurrence of NIP. METHOD: Three groups including nasal cavity mucosae (NM 10 cases), nasal polyp (NP 20 cases) and NIP (30 cases) were selected in the study. Expretion of C/EBPα, CK10 were detected by immunohistochemisty PV-6000 method. RESULT: (1) The different expression of C/EBPα and CK10 in the group of NM, NP and NIP was statistically significant (P < 0.05). (2) The different expression of C/EBPα, CK10 in the group of benign NIP and NIP with atypical hyperplasia was statistically significant (P < 0.05). (3) The different expression of C/EBPα and CK10 in the group of NIP with recurrence and NIP with no recurrence was statistically significant, P < 0.05, respectively. (4) Our result indicate that the relationship of C/EBPα and CK10 (r = 0.578, P < 0.01) was direct correlation. The difference was statistically significant. CONCLUSION In conclusion, the present results describe C/EBPα, CK10 expression in NIP and their possible implication in the regulation of tumor growth and differentiation. C/EBPα and CK10 production may prove useful in terms of a prognostic marker for the recurrence in nasal inverted papilloma.
CCAAT-Enhancer-Binding Proteins ; genetics ; metabolism ; Humans ; Keratin-10 ; genetics ; metabolism ; Nasal Polyps ; genetics ; metabolism ; Neoplasm Recurrence, Local ; Nose ; Nose Neoplasms ; genetics ; metabolism ; Papilloma, Inverted ; genetics ; metabolism