Notch Signaling May Be Involved in the Abnormal Differentiation of Epidermal Keratinocytes in Psoriasis

Localization of each keratin isoform differs among epidermal layers. Proliferating basal cells synthesize keratin 14 (K14) and suprabasal cells express keratin 10 (K10) in normal skin. Notch signaling is essential for keratinocyte differentiation. Notch1 is expressed in all epidermal layers, Notch2 in the basal cell layer and Notch3 in basal cell and spinous cell layers in normal epidermis. It has been poorly elucidated how localization and expression levels of Notch molecules are related to epidermal molecular markers K10 and K14 in psoriatic skin with abnormal differentiation of epidermal tissue. This study aimed to investigate the relationship between abnormal differentiation of epidermal cells in psoriatic skin and expression of Notch molecules. We investigated keratins (K14 and K10) and Notches (1, 2, 3 and 4) using immunohistochemistry in psoriatic skin (n=30) and normal skin (n=10). In normal skin, K14 and K10 were discretely observed in the basal cell layer and suprabasal layer, respectively. In psoriatic skin, K14 was expressed in the pan epidermal layer while it and K10 were co-expressed in some middle suprabasal layer cells. Notch1, 2, 3, and 4 localized in all epidermal layers in normal skin. In psoriatic skin, Notch1, 2, and 4 mainly localized in suprabasilar layers and Notch3 is lacalized in pan epidermal, suprabasilar, and basilar layers. Protein and mRNA of Notch1, 2, and 3 isoforms decreased in psoriatic epidermis compared with normal epidermis. These data suggest that decrements in these Notch molecules might cause aberrant expression of K10 and K14 leading to anomalous differentiation of the epidermis in psoriatic lesions.     

Monoclonal antibodies (BL7 and BL9) having crossed reactivity with cells of the human epidermis

The relationships between epithelial cells and immunocompetent cells could be approached by studying of the common antigens expressed by these two cell types. This paper reports the study of two monoclonal antibodies (BL7 and BL9) which react with epidermal cells. BL7 was obtained after immunization of mice with human thymic cell suspensions and BL9, after immunization with Raji cells. BL7 stained the epithelial network of the thymus and the basal cell layer of the epidermis. BL7 also reacted with the endothelial cells of the vessels of the dermis. This reactivity against the basal cell layer of the epidermis was observed in man, mice and rabbit. BL9 showed a reactivity against thymic epithelial cells and stained the membrane of the keratinocytes of the human epidermis. The antigenic expression revealed by BL9 decreased during the epidermal cell differentiation and disappeared in the horny layer. BL9 showed no reactivity with the epidermis of mice and rabbit. These two monoclonal antibodies are new tools in cutaneous immunopathology: BL7 is the first monoclonal experimental marker which identifies the basal cells of the epidermis, BL9 identifies an antigen related to the human epidermal cell differentiation.     

Alterations in the expression of two epidermal differentiation antigens in human epidermal disorders

The expression of an epidermal keratin subunit and a specific antigen of the keratinocyte membrane, two differentiation antigens in normal human epidermis, was studied in benign and malignant epidermal lesions by use of monoclonal antibodies KL1 (anti 55-57 Kd keratins) and KL3 (anti keratinocyte membrane antigen). In normal human epidermis, KL1 labelled all keratinocytes from the suprabasal layers, KL3 stained the intercellular spaces in all epidermal layers with a fluorescence intensity increasing from the basal to the more upper layers and recognized a keratinocyte membrane antigen as demonstrated in electron microscopy. Frozen or deparaffinized sections of basal cell carcinomas (BCC), squamous cell carcinomas (SCC) malignant melanomas, warts, and skin biopsies from benign lesions (psoriasis, lichen planus, bullous pemphigoid, lupus erythematodes, pemphigus, vasculitis) were tested with either KL1 or KL3 by indirect immunofluorescence and/or immunoperoxidase. Benign and malignant lesions in which modifications of the keratinization process and cell differentiation are known to occur (BCC, SCC, warts, psoriasis) showed the most severe alterations as compared to normal epidermis. With KL1 we observed an irregular staining of basal cells; a reorganization of keratin filaments and variable staining intensities within tumoral cells which did not express high MW keratins. With KL3 drastic alterations in the epidermal intercellular patterns and loss of reactivity of tumoral cells were noted. Conversely, the positivity of epidermal basal cells with KL1, in some cases, was the only modification noted in other skin lesions.     

Expression of human epithelial characteristics in late passages of interspecific somatic hybrids derived from normal and hand wart keratinocytes

The expression of epithelial features has been investigated from the 9th to the 30th passage in cells derived from the fusion of mouse 3T3.4E cells with normal or hand wart human keratinocytes. These cells stratified and grew either on plastic or collagen substrate without modifications. Such growth characteristics are similar to those observed in the epidermis. The doubling time of hybrid cells is nearer to that of 3T3.4E than to that of keratinocytes. Some epithelial markers were detected by immunofluorescence staining. Bullous pemphigoid antigen characteristic of the basal keratinocyte was detected, and cytoplasmic antigen of basal cells was positive with BL7 monoclonal antibody. Among the antigens of the suprabasal cell layers of the epidermis, the pemphigus antigen was present in all hybrids; epidermal keratins recognized by the monoclonal antibodies KL1 and KL2 were detected in wart hybrids up to the 9th passage. When hybrids were cultured in delipidized serum or in methylcellulose, few cells (less than 10%) reacted with KL2. Karyological analysis revealed both murine acrocentric and human submetacentric chromosomes. Thus, hybrid cells obtained from normal and wart keratinocytes were new cell types with a phenotype between keratinocytes and 3T3.4E cells.     

Human melanocytes and melanoma cells constitutively express the Bcl-2 proto-oncogene in situ and in cell culture.

The Bcl-2 proto-oncogene regulates cell survival by antagonizing events that lead to apoptotic cell death and has been reported to be expressed in situ in lymphoid tissues, glandular epithelium, neurons, and basal epidermal cells. When we performed immunostaining on cryostat sections of normal skin, anti-Bcl-2 reactivity was confined to scattered dendritic cells in the basal epidermal layer. Double-staining experiments showed that the Bcl-2+ cells were positive for vimentin but negative for cytokeratins, CD1a, and CD45 antigens, excluding keratinocytes and Langerhans cells as possible candidates for constitutive Bcl-2 expression. Bcl-2+ epidermal cells also reacted with the monoclonal anti-melanocyte antibody NKI/beteb, and were absent from lesional skin in vitiligo, confirming that they represented epidermal melanocytes. Western blot analysis of cultured melanocytes and melanoma cell lines revealed a 26-kd protein specifically reacting with the anti-Bcl-2 monoclonal antibody. Immunostaining of pigmented lesions revealed strong expression of Bcl-2 by five of five nevocellular nevi and seven of seven melanomas. Our observations demonstrate that, within normal human epidermis, melanocytes are the only cells that express Bcl-2 constitutively and that Bcl-2 is expressed in benign and malignant pigmented tumors of the skin in situ.     

The role of epithelial cell differentiation in the expression of herpes simplex virus type 1 in normal human oral mucosa in culture

Summary We have examined by immunofluorescent antibody staining technique the expression of herpes simplex virus type 1 (HSV-1) in organ cultures of the normal human oral mucosa. The expression of HSV-1 antigen was found selectively in the epithelial cell layers in relatively undifferentiated states such as basal layer and lower prickle cell layer in addition to the basement membrane. When the epithelial cells dissociated from the oral mucosa were infected with HSV-1 and association of the HSV-1 expression with the cellular differentiation was examined, the epithelial cells containing laminin in an undifferentiated state were permissive for the expression of HSV-1 antigen whereas terminally differentiated epithelial cells with the cornified envelope did not express HSV-1 antigen. These findings indicate that the expression of HSV-1 antigen is restricted in the mucosal epithelial cells in a differentiated state, although the possibility that the cornified envelope might protect the cells from infection is not excluded.With 4 Figures     

CD5− dendritic epidermal T cells are derived from CD5+ precursor cells

Murine Thy-1⁺, TcR Vγ3/Vδ⁺ dendritic epidermal T cells (DETC) differ from most other T cell subsets by the absence of CD4 and CD8 antigens as well as the lack of CD5 expression. To see whether negativity for those antigens is an intrinsic feature of a given T cell population or if such triple-negative T cells go through a maturational stage where they express these antigens, we determined the phenotype of TcR Vγ3⁺ fetal thymocytes which are the precursor cells of DETC. We found that TcR Vγ3⁺ fetal thymocytes phenotypically differ from mature DETC in that they are CD5⁺, mostly CD8⁺ and partly CD4⁺. The injection of fetal thymic suspensions containing TcR Vγ3⁺/CD5⁺ (but not TcR Vγ3⁺/CD5^?) thymocytes into Thy-1-disparate athymic nude mice resulted in the appearance of donor-type TcR Vγ3⁺/CD5^? dendritic cells in the recipients' epidermis, indicating that TcR Vγ3⁺ thymocytes are indeed the precursors of CD5^? DETC. Tracing CD5 expression on DETC precursors during their intrathymic maturation and their migration to the fetal skin, we found that (i) the earliest DETC precursor cells as defined by TcR Vγ3 expression express high levels of CD5 antigen (day 15 of gestation), (ii) after day 16 of gestation 70% of TcR Vγ3⁺ thymocytes express high and 30% express intermediate levels of CD5, (iii) TcR Vγ3⁺ cells in the fetal blood express low levels of CD5, (iv) the first TcR Vγ3⁺ cells entering the epidermis express very low levels of this antigen and (v) TcR Vγ3⁺ epidermal cells later than day 19 of gestation are CD5^?. A similar down-regulation of CD5 expression on DETC precursors was also noted when TcR Vγ3⁺ cells were cultured in vitro. Even the addition of PMA and ionomycin, which up-regulates CD5 expression on TcR α/β-bearing thymocytes and lymph node T cells, could not prevent down-regulation on DETC precursors. The described cell system may serve as a useful tool in further experiments aimed to clarify the function of the CD5 glycoprotein as well as the mechanism(s) regulating its expression.     

Expression of cyclin kinase inhibitor p27(Kip1) in skin tumours of dogs

Skin tumours (n=148) of epidermal or hair follicle origin were examined immunohistochemically to determine the expression of p27(Kip1)(p27), a cyclin-dependent kinase inhibitor (CDKI), and of Ki-67. In normal skin, a large number of basal cells of the epidermis and hair follicles were positive for Ki-67 and many suprabasal epithelial cells were positive for p27. Most of the hair matrix cells were positive for Ki-67 but negative for p27. Hair papillae were strongly positive for p27. Squamous cell carcinomas had a p27 positive index (PI) significantly lower than that of trichoepitheliomas (P<0.005), basal cell tumours (P<0.05) and intracutaneous cornifying epitheliomas (P<0.001). In contrast, Ki-67 PIs of squamous cell carcinomas and pilomatrixomas were significantly higher than those of trichoepitheliomas, basal cell tumours and intracutaneous cornifying epitheliomas (P<0.01 to P<0.001). No significant difference was observed between the Ki-67 PI values of squamous cell carcinomas and pilomatrixomas. The results suggested that p27 is capable of suppressing cell proliferation in the differentiation of normal canine skin. In spite of being a benign neoplasm, pilomatrixomas had a low p27 expression; this may be a reflection of the proliferative potential of the hair matrix. The expression of p27 may be a useful marker for the analysis of cell kinetics.     

羊膜上皮细胞和羊膜间充质干细胞构建组织工程皮肤

背景：由于人胎盘来源的间充质干细胞具有多方面的优点,近年来已成为干细胞研究的热点。 目的：分析鉴定羊膜间充质干细胞和羊膜上皮细胞的生物学特性,探讨其作为皮肤种子细胞在三维气液培养构建组织工程皮肤中的应用情况。 方法：用胰酶胶原酶多步消化法获取羊膜间充质干细胞和羊膜上皮细胞,通过流式细胞术、反转录-聚合酶链反应和免疫荧光染色技术,鉴定两种细胞的表面分子标记、干细胞特性、与皮肤角质形成细胞的相似性,并利用两种细胞为种子细胞以鼠Ⅰ型胶原为基质进行三维气液培养。 结果与结论：①流式细胞术检测体外培养羊膜间充质干细胞和羊膜上皮细胞均高表达CD90、CD73、CD105,不表达造血干细胞标志CD34以及MHC-Ⅱ类分子HLA-DR。②反转录-聚合酶链反应检测到羊膜间充质干细胞表达干细胞特性基因CMCY和NANOG,羊膜上皮细胞表达干细胞特性基因CMCY和 KLF4,两种细胞均有干细胞特性。③反转录-聚合酶链反应检测羊膜间充质干细胞表达皮肤角质形成细胞特性基因K19、β1-integrin、K8,羊膜上皮细胞表达K19、β1-integrin、K5、K8,免疫荧光染色见羊膜上皮细胞表达与角质形成细胞增殖相关的的特性蛋白K14,说明羊膜上皮细胞与皮肤角质形成细胞更具相似性, 在特定条件下更易于分化为皮肤角质形成细胞。④利用两种细胞成功构建组织工程皮肤,苏木精-伊红染色切片显示其具有一定的皮肤结构,且羊膜上皮细胞发生了初步分化。以上结果说明羊膜间充质干细胞与羊膜上皮细胞通过三维培养构建人皮肤组织是可行的。     

Human embryonal tissues of all three germ layers can express the CD30 antigen. An immunohistochemical study of 30 fetuses coming after therapeutic abortions from week 8th to week 16th of gestation

Originally, expression of the CD30 antigen was shown to be typical of the tumor cells of Hodgkin disease and of anaplastic large cell lymphomas. In reactive lymphoid tissue, CD30 is expressed only in a small population of activated lymphoid blasts. Since then, several reports have been published describing CD30 expression in non lymphoid tissues and neoplasms, such as embryonal carcinomas, seminomas, cultivated macrophages, histiocytic neoplastic cells, deciduals cells, and mesothelioma cells. In order to gain insight into the functions of CD30, given that it can mediate signals for cell proliferation and apoptosis, we studied the distribution of the antigen in different fetal archival paraffin-embedded tissues from week 8th to 16th of gestation. We investigated the immunohistochemical expression of CD30 in 30 paraffin-embedded tissue samples representing all three germ layers, using the monoclonal antibody Ber-H2 CD30 is expressed early in human fetal development (8th-10th week) in a wide variety of tissues, with the exception of the skin and thymus in which it is expressed later on. This is consistent with the observation that these organs are not fully differentiated before 10th and 13th week, respectively. No expression was observed in the cardiovascular and respiratory systems. The finding of CD30 expression in the terminal period of organogenesis, period, which is highly hormone related, implies that the antigen has an important role in cell development, maturation, and pathway to terminal differentiation in almost all fetal tissues and structures.     

Mast cells and vasculature in atopic dermatitis--potential stimulus of neoangiogenesis

BACKGROUND: Atopic dermatitis skin lesions are characterized by inflammatory changes and epithelial hyperplasia requiring angiogenesis. As mast cells may participate in this process via bidirectional secretion of tissue-damaging enzymes and pro-angiogenic factors, the present study aimed to assess the occurrence and possible function of mast cells in the papillary dermis and in epidermal layers of atopic dermatitis lesions. METHODS: Semi-thin and serial sections in combination with immunohistochemistry, histochemistry and proliferating cell nuclear antigen (PCNA)-activity assays were used and related to epidermal thickness and targeted gene expression studies. RESULTS: Mast cells were located in the papillary dermis and migrated through the basal lamina into the epidermis of atopic dermatitis lesions. An increased PCNA-activity in cells of superficial epidermal layers indicated an activation of keratinocytes and stimulation of endothelial growth. Only approximately 30% of the papillary mast cells stained with the tryptase were toluidin-blue-positive, and approximately 80% were chymase positive. A high number of mast cells expressed c-kit. Most papillary and epidermal mast cells were localized close to endothelial cells. Vascular expression of endoglin (CD105) demonstrated neoangiogenic processes. Mast cells stimulation led to the expression of proangiogenic factors. Also, gene expression of tissue-damaging factors such as matrix metalloproteinases was increased. CONCLUSIONS: These data suggest that in atopic dermatitis, mast cells are abundantly localized close to and within the epidermis where they may stimulate neoangiogenesis. Via the new vessels, inflammatory cells, together with complement components and antibodies, can be transported to the epidermis to aid in the defense against environmental antigens and to maintain chronic inflammation.     

The intercellular adhesion molecule, cadherin-10, is a marker for human prostate luminal epithelial cells that is not expressed in prostate cancer.

During the normal turnover of prostate epithelium, stem cells in the basal cell layer produce an intermediate cell population that gives rise to fully differentiated secretory luminal cells. This process is extensively studied in relation to the development of prostate disease, in particular, to elucidate the origin and nature of prostate cancer. We previously showed that the mRNA of a poorly characterised intercellular adhesion molecule, cadherin-10, is strongly expressed in human prostate. Using anticadherin-10 antibodies, immunohistochemistry, and confocal microscopy, we have examined the pattern of cadherin-10 expression in relation to human prostate epithelial differentiation markers (E-cadherin, CD44, and cytokeratins (CK) 14, 18 and 19) in archival paraffin-embedded and fixed-frozen histopathological specimens in individual and serial sections. In non-neoplastic prostate, E-cadherin is expressed by all basal and luminal epithelial cells, while cadherin-10 is variably expressed in luminal cells where it is colocalised with E-cadherin at basolateral plasma membranes. Cadherin-10 is absent in CK14- and/or CD44-positive basal cells, but is expressed in CK18-positive luminal cells (differentiated secretory cells), a subset of CK19-positive intermediate/luminal cells, but not CK19-positive basal cells. Small foci of prostate cancer express E-cadherin, CK19 and CK18, but cadherin-10 expression is low or undetectable. These findings suggest that the expression of cadherin-10 is associated with the later stages of differentiation of luminal secretory cells, indicating a specific role in secretory cell terminal differentiation. While prostate cancer cells express secretory cell markers (eg, CK18, prostate-specific antigen) and the more generally expressed E-cadherin, their failure to express cadherin-10 further emphasises a role for this cadherin in normal prostate organisation and function.     

Immunohistochemical localization of thrombomodulin in normal human skin and skin tumours

Thrombomodulin (TM) expression has been investigated in sections of normal human skin, in cultured normal human keratinocytes, and in a variety of skin tumours. TM was present in squamous epithelial cells in the spinous layer of normal epidermis and in the outer root sheath of hair follicles, but was absent in the cells of the basal layer. It appeared to be predominantly localized to the cell membrane and the intercellular bridges in these areas. Cultured normal human keratinocytes demonstrated functionally active constitutive TM expression on their cell surface. Immunoperoxidase staining of skin tumours using anti-human TM antibodies demonstrated a typical cell membrane positivity in tumours with squamous or hair follicle differentiation. Basal cell carcinomas showed TM expression only in areas where incomplete squamoid metaplasia occurred. Sweat gland tumours and lesions of the melanogenic system failed to express TM. The localization of TM by immunostaining in various benign and malignant skin tumours typically correlated with their normal skin element of origin. The physiological significance of TM expression in the epidermis is currently undefined.     

Transgenic Expression of Cyclin-Dependent Kinase 4 Results in Epidermal Hyperplasia, Hypertrophy, and Severe Dermal Fibrosis

In a previous report we have described the effects of expression of D-type cyclins in epithelial tissues of transgenic mice. To study the involvement of the D-type cyclin partner cyclin-dependent kinase 4 (CDK4) in epithelial growth and differentiation, transgenic mice were generated carrying the CDK4 gene under the control of a keratin 5 promoter. As expected, transgenic mice showed expression of CDK4 in the epidermal basal-cell layer. Epidermal proliferation increased dramatically and basal cell hyperplasia and hypertrophy were observed. The hyperproliferative phenotype of these transgenic mice was independent of D-type cyclin expression because no overexpression of these proteins was detected. CDK4 and CDK2 kinase activities increased in transgenic animals and were associated with elevated binding of p27(Kip1) to CDK4. Expression of CDK4 in the epidermis results in an increased spinous layer compared with normal epidermis, and a mild hyperkeratosis in the cornified layer. In addition to epidermal changes, severe dermal fibrosis was observed and part of the subcutaneous adipose tissue was replaced by connective tissue. Also, abnormal expression of keratin 6 associated with the hyperproliferative phenotype was observed in transgenic epidermis. This model provides in vivo evidence for the role of CDK4 as a mediator of proliferation in epithelial cells independent of D-type cyclin expression.     

Effects of all-trans retinoic acid and Ca++ on human skin in organ culture.

In this study, we have established an organ culture model of human skin and examined the effects of both all-trans retinoic acid (RA) and extracellular Ca++ on the epidermal and dermal components of the organ-cultured skin. Our data show that while organ cultures maintained in serum-free, growth factor-free culture medium containing 0.15 mM Ca++ degenerated rapidly, those treated with concentrations of RA that have been shown previously to stimulate fibroblast and keratinocyte proliferation in monolayer culture (J Invest Dermatol 1989, 93:449; 1990, 94:717; Am J Pathol 1990, 136:1275) demonstrated a healthy appearance for up to 12 days. Degeneration of the control cultures was characterized by separation of the epidermis from the underlying dermis, progressive cell necrosis leading to a complete absence of viable cells from both the dermal and epidermal compartments, disintegration and fibrillation of the dermal connective tissue, and a cessation of protein synthesis. RA-treated organ cultures contained large numbers of healthy-appearing cells in both the epidermal and dermal compartments. One or several layers of viable basal cells in the epidermis could be seen at least through day 12. However, the upper layers of the epidermis frequently separated from the cells in the basal layer. The dermal connective tissue was histologically well-preserved. Furthermore, the level of protein synthesis was higher in the RA-treated cultures than in the control cultures. In addition to treating organ cultures with RA, other cultures were exposed to serum-free, growth factor-free culture medium containing 1.4 mM Ca++. The presence of the elevated Ca++ concentration also preserved cellular and connective tissue structures in the dermal and epidermal compartments. In comparison to RA there was better preservation of the overall epidermal structure. The upper layers of epidermal cells did not separate from the basal cells, and the various stages of epithelial differentiation could be seen. Histologically, the dermis was well-preserved in the presence of elevated extracellular Ca++. Specimens treated with a combination of Ca++ and RA demonstrated features consistent with the features induced by each treatment separately. This included an expanded basal layer of epithelial cells and a prominent keratotic layer with a fairly orderly pattern of differentiation. The tendency of the upper epidermis to separate from the basal cells was partially mitigated. Taken together, these data indicate that both RA and extracellular Ca++ act to prevent the degeneration of human skin in organ culture but probably do so through different mechanisms.     

Immunohistochemical localization of syndecan in mouse skin tumors induced by UV irradiation. Loss of expression associated with malignant transformation.

Immunoreactivity for syndecan, a cell surface proteoglycan, which binds extracellular matrix molecules and growth factors, was studied in hairless (hr/hr) mice exposed to UV-A and UV-B irradiation. Positive staining was observed at the surface of normal epidermal cells as well as in the dermal abortive hair follicle cysts characteristic to this mouse strain. Early reaction to UV-irradiation showing hyperplastic epidermis with slight cellular atypia showed also positive, although reduced, staining of epidermal cell surfaces. Specimens with severe dysplasia showed weak staining in the granular cell layer, whereas the basal cell layer was negative. In papillomas and keratoacanthomas, immunoreactivity for syndecan was observed in the benign hyperplastic epidermal cells as well as in the proliferating epidermal cells of the horn cysts. Malignant transformation of epithelium, expressed as the formation of early invasive and anaplastic squamous cell carcinomas, was uniformly associated with loss of syndecan staining. These results are consistent with the previous findings of reduced expression of syndecan associated with malignant transformation of cultured epithelial cells, but also suggest an important role for syndecan in the maintenance of normal tissue architecture and differentiation pattern of the skin.     

Clear cell papulosis of the skin: a case report from Singapore

Clear cell papulosis of the skin is a rare condition; to our knowledge only 12 cases have been reported. Here, we report for the first time a case of clear cell papulosis with cytokeratin 7 expression and provide a comprehensive literature review. A 16-month-old girl presented with 3 hypopigmented lesions in the pubic region that were 3 to 9 mm in diameter; 1 lesion was papular, and the other 2 were macular. A skin biopsy revealed acanthosis with a proliferation of clear cells along the basal and suprabasal layers of the epidermis occurring in small clusters and singly. The cells had round to oval regular nuclei with abundant to moderate lightly eosinophilic to clear cytoplasm and intracytoplasmic mucin. Immunostaining produced positive results for carcinoembryonic antigen, AE1/3, epithelial membrane antigen, cell adhesion molecule 5.2, and cytokeratin 7 and negative results for gross cystic fluid disease protein, S100, and HMB-45. Clear cells of clear cell papulosis are mucin-positive and S100-negative glandular-secretory epithelial cells with histogenetic features of Toker cells of nipple and Paget cells. Immunohistochemical features support an eccrine secretory cell origin because the clear cells are consistently and strongly positive for carcinoembryonic antigen, positive for cell adhesion molecule 5.2, and negative or rarely positive for gross cystic fluid disease protein.     

Phenotypic expression of human epidermal growth factor in foetal submandibular gland and pleomorphic adenoma of salivary gland

Summary The phenotypic expression of the human epidermal growth factor (EGF) was investigated immunohistochemically in human foetal submandibular glands from the 5th to 10th month of gestation, adult normal submandibular glands and 48 cases of pleomorphic adenomas. In foetal submandibular glands, both the terminal buds and primary ducts at the intermediate stage of gestation were positive for EGF, and in particular, the outer layer cells of primary ducts showed strong EGF-immunoreactivity. EGF-positive cells decreased as the gestational stage advanced and only ductal cells were weakly positive for EGF at the terminal stage of gestation. In the adult normal submandibular gland, weak immunoreactivity for EGF was restricted to ductal cells. However, 41 (86%) of the 48 pleomorphic adenomas had EGF-positive cells which were distributed among the ductal, chondroid and myxoid portion. No EGF-immunoreactivity was detected in the solid portion of pleomorphic adenomas. These results suggest that EGF may play an important role in the growth and differentiation of foetal cells as well as the proliferation of tumour cells in pleomorphic adenomas.     

Demonstration of intermediate cells during human prostate epithelial differentiation in situ and in vitro using triple-staining confocal scanning microscopy

In human prostate epithelium, morphologically basal and luminal cells can be discriminated. The basal cell layer that putatively contains progenitor cells of the secretory epithelium is characterized by the expression of keratins (K) 5 and 14. Luminal cells represent the secretory compartment of the epithelium and express K8 and 18. We developed a technique for the simultaneous analysis of K5, 14, and 18 to identify intermediate cell stages in the prostate epithelium and to study the dynamic aspects of its differentiation in vitro. Nonmalignant prostate tissue and primary epithelial cultures were immunohistochemically characterized using triple staining with antibodies for K5, K14, and K18. Antibodies for K18 and K5 were conjugated directly with fluorochromes Alexa 488 and 546. K14 was visualized indirectly with streptavidin-Cy5. Keratin expression was analyzed by confocal scanning microscopy. The occurrence of exocrine and neuroendocrine differentiation in culture was determined via antibodies to prostate-specific antigen (PSA), chromogranin A, and serotonin. We found that basal cells expressed either K5(++)/14(++)/18+ or K5(++)/18+. The majority of luminal cells expressed K18(++), but colocalization of K5+/18(++) were recognized. Epithelial monolayer cultures predominantly revealed the basal cell phenotype K5(++)/14(++)/18+, whereas intermediate subpopulations expressing K5+/14+/18(++) and K5+/18(++) were also identified. On confluence, differentiation was induced as multicellular gland-like buds, and extensions became evident on top of the monolayer. These structures were composed of K18(++)- and K5+/18(+)-positive cell clusters surrounded by phenotypically basal cells. Few multicellular structures and cells in the monolayer showed exocrine differentiation (PSA+), but expression of chromogranin A and serotonin was absent. We conclude that simultaneous evaluation of keratin expression is useful for analyzing epithelial differentiation in the prostate. During this process, putative stem cells phenotypically resembling K5(++)/14(++)/18+ differentiate toward luminal cells (K18(++)) via intermediate cell stages, as identified by up-regulation of K18 and down-regulation of K5 and 14.