CD44在某些皮肤肿瘤中表达的初步研究

目的：探讨CD44在皮肤肿瘤中的表达情况。方法：免疫组化法。结果：在鳞癌和基癌中,癌巢距表皮越近,CD44的表达越强;反之,CD44的表达越弱。在痣细胞痣和恶性黑素瘤（恶黑）标本中,CD44标准型（CD44S）均表达。在其它皮肤肿瘤中,CD44的表达同正常皮肤。结论：在鳞癌和基癌中,CD44的表达与癌巢距表皮的远近有关。CD44S的表达与痣细胞的良性或恶性无关。     

皮肤鳞状细胞癌CD44v6蛋白表达

采用免疫组织化学及图像分析技术对66例皮肤鳞状细胞癌、12例淋巴结转移灶、11例假性上皮瘤样增生、10例正常皮肤进行检测，探讨CD44v6与皮肤鳞状细胞癌的发生、发展与转移的关系。结果表明鳞状细胞癌CD44v6表达下调，且分化越差下调越明显，与是否转移及发病部位无关。提示CD44v6对维持表皮正常结构起重要作用，并与细胞增殖分化程度 有关，可以作为一个研究皮肤鳞状细胞癌发生发展的分子标志。     

CD44v6和PCNA在皮肤鳞状细胞癌中的表达及其临床意义

目的 :　研究CD4 4v6和PCNA与皮肤鳞状细胞癌 (SCC)临床病理特征的关系。方法 :　免疫组化法研究 4 8例SCC原发灶、11例淋巴结转移灶和 10例正常皮肤组织中CD4 4v6和PCNA的蛋白表达。结果 :　SCC标本中 ,CD4 4v6表达下调 (P <0 .0 1) ,分化越差下调越明显 (P <0 .0 5 ) ;PCNA表达增高 (P <0 .0 5 ) ,分化越差增高越明显 (P <0 .0 1)。二者与临床分期、淋巴结转移及发病部位均无关 (P >0 .0 5 ) ,且CD4 4v6和PCNA的表达无相关性 (r=- 0 .176 ,P >0 .0 5 )。结论 :　CD4 4v6和PCNA均可作为研究SCC发生与发展的独立的生物学指标     

Hyaluronan and CD44 in psoriatic skin. Intense staining for hyaluronan on dermal capillary loops and reduced expression of CD44 and hyaluronan in keratinocyte-leukocyte interfaces

The distributions of hyaluronan (HA) and its presumptive receptor, CD44, were studied in skin samples from 13 psoriasis vulgaris patients, using an HA-specific probe (HABC), and monoclonal antibodies, respectively. The general distribution of HA and CD44 in psoriatic lesional epidermis resembled that in normal epidermis. However, areas of epidermis invaded by leukocytes showed a local depletion of HA and CD44, particularly at the contact areas of keratinocytes to lymphocytes and neutrophils. Removal by cellular uptake or extracellular degradation of CD44 and HA may be required for tight adherence between a keratinocyte and a leukocyte. On the dermal side, the tips of the prolonged dermal papillae in psoriatic lesions were intensively stained with HABC. The dilated capillaries and the space below the tip basal lamina, in particular, were heavily covered with HA. Occasionally, a similar intense staining was seen around an enlarged capillary in uninvolved psoriatic skin. CD44-positive leukocytes were found around the affected capillaries. The accumulation of HA in the dermal papillae may support the growth of psoriatic lesions, since HA stimulates the growth of capillaries as well as attracting inflammatory cells.     

Changes in CD44 isoform expression during inflammatory skin disease

The CD44 family of cell surface glycoproteins is widely-expressed in epithelial, mesothelial and haemopoietic tissues and is thought to function primarily as adhesion molecules. The molecule has an intracellular, a trans-membrane and an extracellular domain. The membrane proximal region of the extracellular domain is of variable structure depending on which of 10 variable exons are involved in coding for this region. Both in vitro stimulated T cells and cytokine stimulated keratinocytes are known to express certain isoforms. In this study we have investigated whether specific isoforms of the CD44 molecule are expressed on epithelial cells and lymphocytes in the course of two inflammatory skin diseases, namely lupus erythematosus and lichen planus. Monoclonal antibodies, specific to the epitopes of the CD44 molecule encoded by v3, v4/5, v6 and v8/9 variable exons and a pan CD44 marker, were used on 10 lupus and 8 lichen planus frozen skin samples and compared with normal skin from 9 different body sites. Results failed to show detectable levels of variant isoforms of CD44 on lymphocytes in either inflammatory skin disease, despite evidence of T cell activation. All CD44 variant isoforms were reduced on the keratinocytes in some sections of lupus and lichen planus. The results are discussed in the context of the current models for the role of CD44 in the immune response.     

骨桥蛋白和CD44v6在乳房外Paget病中的表达及其生物学意义

目的探讨骨桥蛋白(OPN)和CD44v6在乳房外Paget病中的表达特点及其生物学意义。方法应用免疫组化二步法分别检测34例乳房外Paget病患者皮损及20例正常对照皮肤中OPN和CD44v6的表达情况。结果 OPN和CD44v6在乳房外Paget病中阳性表达分别为70.59%和82.35%,显著高于正常皮肤组织的40%和0,差异有统计学意义(P0.05)。乳房外Paget病中OPN和CD44v6阳性表达与淋巴结转移有关(P0.05),OPN和CD44v6的表达呈正相关(P0.05)。结论 OPN和CD44v6高表达在乳房外Paget病的发生发展中起着重要的协同作用,两者的高表达与乳房外Paget病的淋巴结转移有关。     

Hyaluronan,CD44 and versican in epidermal keratinocyte tumours

BACKGROUND: The high molecular weight polysaccharide hyaluronan is a major component of the extracellular matrix between the vital cells of human skin epidermis. The levels of hyaluronan, and those of the hyaluronan receptor CD44 and the hyaluronan binding proteoglycan versican, correlate with the aggressiveness of different human carcinomas of epithelial origin. OBJECTIVES: To study skin keratinocyte tumours for the expression of hyaluronan, the hyaluronan receptor CD44 and the hyaluronan binding proteoglycan versican. METHODS: Paraffin-embedded sections of 114 basal cell carcinomas (BCC), 31 in situ carcinomas (ISC) and 35 squamous cell carcinomas (SCC) were stained with a hyaluronan specific probe, biotinylated hyaluronan binding complex, and with monoclonal antibodies against CD44 and versican. RESULTS: Compared with normal epidermis, ISC and well differentiated SCCs showed an enhanced hyaluronan signal on carcinoma cells while CD44 expression level resembled that of normal skin. Less differentiated SCCs showed reduced and irregular expression of both hyaluronan and CD44 on carcinoma cells. In BCCs, hyaluronan and CD44 signals were absent or very low on the surface of carcinoma cells. However, hyaluronan was frequently present on BCC cell nuclei, a feature completely absent in ISC, SCC and normal epidermis. An accumulation of hyaluronan in the connective tissue stroma around the tumour was more frequent in SCCs than BCCs. Versican staining was positive around hair follicles and dermal blood vessels of normal skin. Peritumoral versican signal was present in a part of the BCCs but not in other tumours. CONCLUSIONS: The completely different hyaluronan and CD44 expression patterns in BCC and SCC probably reflect the different origins of the tumours, with BCC an undifferentiated keratinocyte and SCC a keratinocyte at an early stage in the differentiation pathway. The difference in hyaluronan and CD44 expression between these tumours may also contribute to the difference in their capacity to metastasize.     

Detection of CD44 splice variants in formalin-fixed, paraffin-embedded specimens of human skin cancer

The standard form of CD44 (CD44s) and CD44 isoforms, containing sequences encoded by one or several of 10 different variant CD44 exons (v1-v10), are thought to play a crucial role in the growth and metastasis of certain human tumors. Recently, monoclonal antibodies (mAbs) directed against all CD44 isoforms (panCD44), or against epitopes encoded by specific variant exons (CD44v) have been developed, which unfortunately only stain cryopreserved tissues. We wished to develop a technique to unmask chemically CD44s and CD44v epitopes in paraffin-embedded specimens of human skin cancers, so that they would be accessible for these mAbs. To address this issue, CD44s and CD44v expression was compared in cryopreserved and in formalin-fixed, paraffin-embedded biopsies obtained from the same basal cell carcinomas (BCC), squamous cell carcinomas (SCC), primary malignant melanomas (PMM) and metastatic malignant melanomas (MMM). Formalin-fixed tumors were de-paraffinized and treated briefly with an antigen retrieval fluid (TUF™) at 95°C or left untreated. In untreated paraffin-embedded tissues, no CD44s or CD44v staining was detected. In contrast, in antigen retrieval fluid-treated biopsies CD44s and CD44v expression was identical to that in cryopreserved specimens of the same tumor with the exception of mAbs detecting v7/8 and v10. We conclude that antigen retrieval unmasks certain epitopes in formalin-fixed, paraffin-embedded tissues, thus facilitating future research on the relevance of CD44s and CD44v expression for human skin carcinogenesis.     

Depletion of cell surface CD44 in nonmelanoma skin tumours is associated with increased expression of matrix metalloproteinase 7

Background  Expression of matrix metalloproteinase (MMP)-7 and MMP-9 is low in the normal epidermis and is induced by physiological processes such as wound healing, but also malignant transformation of epidermal cells. The activity of both MMPs has been associated with the hyaluronan (HA) receptor CD44. We previously reported that the levels of CD44 and HA differ between the two types of epidermal tumours, basal (BCC) and squamous cell carcinoma (SCC), as well as between different grades of SCC.
Objectives  To investigate if the immunostaining patterns of MMP-7 and MMP-9 correlate to those of CD44 and HA in BCC and SCC.
Methods  Paraffin sections from 71 BCCs, 21 in situ SCCs and 27 SCCs were immunostained for MMP-7 and -9.
Results  Positive immunostaining for MMP-7 and MMP-9 was found in tumour cells of both BCC and SCC, while the staining intensity tended to be stronger in SCC. The staining intensity of MMP-7 was inversely correlated with that of CD44 in both tumour types. In well-differentiated SCC, the intensity of MMP-7 was generally weak, while CD44 staining was strong and homogeneously distributed. In poorly differentiated SCC, an increase in MMP-7 was seen, and the staining intensity of CD44 became weak and was locally absent. No correlation was seen between MMP-9 and CD44 or either of the two MMPs and HA.
Conclusions  Our results show that in nonmelanoma skin tumours MMP-7 and -9 are present in the tumour cells, and suggest a link between MMP-7 activity and the depletion of cell surface CD44.     

Distribution of CD44 variant isoforms in human skin: differential expression in components of benign and malignant epithelia

Expression of cell adhesion molecules regulates epithelial cell differentiation and organization of complex tissues such as skin. The CD44 family of adhesion molecules is generated by alternative splicing of up to 10 variant exons encoding inserts into the extracellular domain. Expression of CD44 variant exons has been correlated with metastatic potential of some epithelial malignancies. We studied the distribution of total and variant CD44 isoforms containing exons v4, v6, and v9 in normal skin, basal cell carcinoma, and in control tissues using immunohistologic assays. While normal epidermis and other stratified squamous epithelia reacted strongly with antibodies specific for standard CD44 (CD44S) and CD44 isoforms containing exons v4, v6, and v9, the epithelium of eccrine glands was reactive, often in a polarized distribution, only with antibodies specific for CD44S and isoforms containing exon v9. These studies suggest that differential expression of CD44 variant exons may be important in development and organization of epithelial structures within skin. Malignant cells in basal carcinoma tissues were found to have low reactivity with antibodies specific for CD44S or variant CD44 molecules. The low expression of CD44 molecules in basal cell carcinomas may play a role in the relatively low probability of metastasis of these neoplasms.     

The expression of CD44 glycoprotein adhesion molecules in basal cell carcinomas is related to growth pattern and invasiveness

Basal cell carcinomas (BCCs) of the skin exhibit a wide range of histological growth patterns as well as a highly variable rate of invasiveness. A large body of experimental and clinical studies supports a role for the CD44 glycoprotein family in the latter process. In the present study, we explored the distribution and the level of expression of pan-CD44, CD44v3, CD44v5 and CD44v6 in BCCs. The use of paraffin sections, combined with an antigen retrieval procedure, yielded far more detailed data than would have been possible with frozen sections. On average, the level of expression of the four CD44 isoforms studied appeared to differ relatively little. However, tumours or tumour areas consisting of thin tumour cell strands showed a significantly stronger expression of all four isoforms than those consisting of solid tumour cell groups. Furthermore, the highest CD44 expression was frequently observed in the smallest tumour cell strands in the tumour periphery. In these strands, the label seemed to be located not only at the tumour cell-tumour cell interface, as in other tumour areas, but also on the tumour cell surfaces facing the stroma. We are presently assessing the exact localization of CD44 at the cellular level by immunoelectron microscopy. In most cases, different growth patterns with significantly different levels of CD44 expression were found side by side within individual tumours. CD44 expression is therefore not a static tumour cell characteristic but is correlated with tumour architecture and tumour-stroma interaction.     

CD44V6在表皮肿瘤及恶性黑素瘤的表达

目的　研究CD44V6在鳞状细胞癌、基底细胞癌和恶性黑素瘤的表达。方法　免疫组织化学对石蜡包埋组织标本进行检测。结果　 1 0例鳞状细胞癌CD44V6膜表达阳性 ,且随癌细胞分化程度降低CD44V6表达下调。 1 0例基底细胞癌和 8例恶性黑素瘤不表达CD44V6。结论　CD44V6的表达与皮肤肿瘤的类型有关     

Hyaluronan-independent adhesion of CD44H+ and CD44v10+ lymphocytes to dermal microvascular endothelial cells and keratinocytes.

We have recently shown the CD44 variant isoform 10 (CD44v10) to be expressed on reactive as well as malignant cutaneous lymphocytes; however, the functional consequences of CD44v10 expression on lymphocytes are not elucidated. By using appropriately transfected lymphatic cells we analyzed the role of CD44v10 on lymphocytes in cell-matrix adhesion and homotypic and heterotypic cell-cell adhesion assays. Despite a low binding affinity to hyaluronan, CD44v10-expressing lymphocytes exhibited heterotypic cell-cell adhesion to inflamed dermal microvascular endothelium and keratinocytes, as indicated by Stamper-Woodruff assays on tissue sections of delayed type hypersensitivity reactions and adhesion assays with cultured keratinocytes and cytokine-stimulated human dermal microvascular endothelial cells. Antibody-blocking assays excluded interaction of CD44v10 with the principal CD44 ligand hyaluronan as well as involvement of selectins or integrins in these heterotypic cell-cell adhesion assays. In contrast, cellular aggregation assays with fluorescence-labeled CD44v10- and CD44H-expressing lymphocytes revealed homotypic CD44v10/CD44v10 binding as well as binding of CD44v10 with CD44H. Heterotypic cell-cell adhesion assays with ultraviolet-A-irradiated CD44v-negative cytokine-stimulated endothelial cells demonstrated binding kinetics of CD44v10-expressing lymphocytes paralleling those of endothelial CD44H expression. These results imply that a hyaluronan-independent CD44v10/CD44H-mediated pathway is involved in lymphocyte infiltration into the dermis and epidermis of inflamed skin and suggest modulation of CD44H expression on inflamed dermal microvascular endothelium as a mechanism of ultraviolet-A-induced therapeutic effects on the skin.     

CD44 Expression in Normal Human Skin and Skin Tumors

CD44 is thought to be a principal cell surface receptor for hyaluronic acid. Although the distribution of hyalulonic acid has been studied, little is known about the distribution of the CD44 molecule in the human skin and skin tumors. This study was undertaken to investigate the distribution of the CD44 molecule in normal human skin as well as in benign and malignant skin tumors. In normal skin, CD44 was expressed on 1) keratinocyte cell surfaces throughout the epidermis except for the granular and horny layers, 2) hair follicular cells, 3) eccrine sweat gland cells, and 4) cell surfaces of dendritic cells in the dermis. In skin tumors, although CD44 was expressed on the tumor cell surface of seborreic keratosis, Bowen's disease, and squamous cell carcinoma as in normal skin, we could not detect any CD44 expression on the cell surface of the tumor cells of basal cell carcinoma. However, CD44 positive dendritic cells were observed in the tumor islands of basal cell carcinoma. Phenotypic analysis suggested that these CD44 positive cells were melanocytes.     

CD44分子在银屑病患者表皮的表达

采用免疫组化染色研究银屑病患者损害、无损害及治愈后皮肤的ＣＤ４４分子表达。发现ＣＤ４４呈现在表皮的棘细胞和基底细胞表面；银屑病损害处比无损害处及治愈后皮肤要显著得多，呈高表达。用逆转录聚合酶链反应分析ＣＤ４４的类型，结果主要为ＣＤ４４的标准型，即ＣＤ４４Ｓ，也有ＣＤ４４Ｅ型，未见到ＣＤ４４Ｖ型。本研究提示ＣＤ４４的高表达可能与银屑病损害表皮细胞过度增生有关，ＣＤ４４Ｓ型及ＣＤ４４Ｅ型可能提示良性增生。     

Expression of integrin subunits and CD44 isoforms in psoriatic skin and effects of topical calcitriol application

Increasing evidence suggests involvement of integrins and CD44 isoforms in the pathogenesis of psoriasis, contributing to uncontrolled keratinocyte proliferation, neovascularization, and invasion of inflammatory cells. We have analyzed immunohistochemically in situ expression of integrins (CD29, CDw49b, CDw49c, CDw49e, CDw49f) and CD44 isoforms (CD44 standard, CD44 var/v6, CD44 v10) on frozen sections of normal and psoriatic skin (nonlesional skin, lesional skin before and along with topical calcitriol treatment). We did not observe visual changes of immunoreactivity in normal as compared to nonlesional psoriatic skin, while the staining pattern of CDw49c, CDw49f, and CD29 was severely altered in untreated lesional psoriatic skin. Most markedly, CDw49c, CDw49f, and CD29 were focally upregulated in suprapapillar epidermal compartments of lesional psoriatic skin, a staining pattern that is in accordance with the phenomenon that was described by Pinkus as 'squirting papilla'. Additionally, an increased proportion of inflammatory and endothelial cells revealed immunoreactivity for CD44(std.) in untreated lesional psoriatic as compared to nonlesional psoriatic or normal skin. After 8 weeks of topical calcitriol treatment (15 μg/g ointment), the staining pattern for CDw49c, CDw49f and CD29 was markedly changed in epidermis of lesional psoriatic skin, reverting to the staining pattern characteristic for the nonlesional psoriatic or normal human skin, although epidermal expression of CDw49f was still upregulated and CDw49e-, CDw49f-, CD29-, and CD44(std.)- immunoreactive inflammatory and endothelial cells were still to be found in the dermal compartment.     

Lichen sclerosus: evidence that immunological changes occur at all levels of the skin

An immunohistochemical approach was used to characterize the inflammatory infiltrate in vulval lichen sclerosus, using monoclonal antibodies to CD3, CD4, CD8, CD68 and HLA-DR. Significant numbers of CD4 + and CD8 + lymphocytes were observed in the dermal band of inflammatory cells in approximately equal proportions. Less numerous CD4 + and CD8 + lymphocytes also occurred adjacent to the dermoepidermal junction and occasionally in the lower epidermis. Increased numbers of cells staining with the monocyte/macrophage marker CD68 were also present in the band of inflammatory cells as well as being scattered diffusely throughout the sclerotic region. Expression of HLA-DR in the lichen sclerosus specimens was increased within the inflammatory infiltrate and around blood vessels in the dermis. All the vulval lichen sclerosus specimens also demonstrated some HLA-DR expression around the keratinocytes, suggesting that these keratinocytes might be involved in antigen presentation. We also studied the expression of CD44 and its isoforms 3G5 (marker of V3), 8G5 (marker of V6), 3D2 (marker of V4/5) and IE8 (marker of V8/9). CD44 has been proposed to play a part in lymphocyte homing, cell-matrix interaction (particularly with hyaluronic acid), lymphocyte activation and malignant progression of certain tumours. The epidermis of the lichen sclerosus specimens appeared to demonstrate a greater intensity of staining with the pan-CD44 marker F10-44, and reduced staining with 3G5, 3D2 and IE8 compared with normal skin. Like normal skin, the dermis of the lichen sclerosus specimens did not demonstrate staining with 3G5, 3D2, 8G5 or 1E8, but did show staining with F10-44. However, the pattern of the dermal staining with F10-44 reflected the position of the inflammatory infiltrate and was sparse in the five sections where there was a prominent sclerotic zone, but increased in the three sections where there was a prominent band of inflammation cells. Our results demonstrate evidence of immunological changes at all levels of skin involved by lichen sclerosus, including the epidermis.     

CD44 and variants in melanocytic skin neoplasms

Expression of cell surface molecules that mediate cell-matrix and cell-cell interactions largely contributes to the ability of melanoma cells to migrate and spread beyond the primary site of the tumor. CD44, the principal cell-surface receptor for hyaluronate, and its numerous splice variants have been reported to play a crucial role in invasion and the metastatic process of different human neoplasms, including primary malignant melanoma (PMM). The aim of this study was to clarify which isoforms of CD44 (standard CD44 and CD44 variants) are distributed in PMM with a vertical tumor thickness of >1.4 mm. Staining of CD44 standard (CD44s) and splice variants was further examined for diagnostic and prognostic relevance in a panel of melanocytic skin lesions. Ten cases of PMM with Breslow >1.4 mm were analysed by immunohistochemistry using monoclonal antibodies specific for CD44s and the splice variants v3, v5, v6, v7, v7-8, and v10. In addition, using anti-CD44s, v5, and v6 antibodies, 55 meianocytic lesions, including dermal nevi (n=12), Clark nevi (dysplastic nevi) (CN; n=11), melanoma in situ (Mis; n=8), PMM (n=18), and cutaneous metastasis of malignant melanoma (cM-MM; n=6) were assessed. Staining intensities were scored visually and evaluated by means of a staining index. In ten cases of PMM with a Breslow index >1.4 mm positive staining was ascertained for CD44s, v5 and for v6 in three cases. No staining was found for v3, v7, v7-8, and v10. Examination of CD44s, v5, and v6 in 55 melanocytic skin lesions revealed a high index for CD44s in all specimens and a weak staining of v5 in Mis; dermal nevi and CN did not stain for v5. However, in PMM and cMMM we found v5 to be strongly positive. The isoform v6 showed a variable index only in PMM, but without connection to established prognostic criteria. We conclude that CD44s and splice variants can not be regarded as indicators for tumor progression in malignant melanomas. However, v5 may potentially serve as a diagnostic marker for meianocytic skin lesions.     

精神刺激降低裸鼠表皮CD44水平

目的确定精神刺激对表皮CD44的表达是否有影响。方法利用限制裸鼠自由活动的方法建立精神刺激模型,并于造模结束时用胶带破坏其躯干部表皮通透屏障功能。用免疫组化法观察CD44在完整的表皮及屏障功能破坏6h后表皮的表达。结果与对照组相比,在精神刺激后,无论是完整的,还是表皮通透屏障功能破坏的皮肤,其表皮CD44的表达都明显降低;两组之间,透明质酸的表达没有明显差别。结论精神刺激能降低表皮CD44的表达。     

外阴白色病变、鳞状上皮内瘤样变及外阴鳞状细胞癌中CD44v6的表达

目的　探讨CD44v6与外阴鳞状细胞癌发生、发展和侵袭转移的关系。方法　采用免疫组化SP法对外阴正常皮肤、外阴白色病变、鳞状上皮内瘤样变及外阴鳞状细胞癌中CD44v6进行检测。结果　正常皮肤无CD44v6表达 ;在外阴白色病变中其阳性率为 2 6.7%;鳞状上皮内瘤样变中为 5 3 .3 %;外阴鳞状细胞癌中为 73 .3 %。在外阴鳞状细胞癌中CD44v6阳性率与临床分期呈正相关 ;有淋巴结转移者阳性率为 92 .3 %,明显高于无淋巴结转移者 (P <0 .0 5 )。结论　CD44v6与外阴鳞状细胞癌淋巴结转移密切相关。