Immunolocalization of nestin, mesothelin and epithelial membrane antigen (EMA) in developing and adult serous membranes and mesotheliomas

The spatial and temporal distribution of epithelial membrane antigen (EMA), mesothelin and nestin was immunohistochemically analyzed in developing and adult human serous membranes and mesotheliomas in order to detect possible differences in the course of mesenchymal to epithelial transformation, which is associated with differentiation of mesothelial cells during normal development and tumorigenesis. Pleura and pericardium developing from the visceral mesoderm gradually transform into mesothelial cells and connective tissue. EMA appeared in mesothelium of both serous membranes during the early fetal period, whereas during further development, EMA expression was retained only in the pericardial mesothelium. It increased in both pleural mesothelium and connective tissue. Mesothelin appeared first in pericardial submesothelial cells and later in surface mesothelium, while in pleura it was immediately localized in mesothelium. In adult serous membranes, EMA and mesothelin were predominantly expressed in mesothelium. Nestin never appeared in mesothelium, but in connective tissues and myocardial cells and subsequently decreased during development, apart from in the walls of blood vessels. Mesothelial cells in the two serous membranes developed in two separate developmental pathways. We speculate that submesothelial pericardial and mesothelial pleural cells might belong to a population of stem cells. In epithelioid mesotheliomas, 13% of cells expressed nestin, 39% EMA and 7% mesothelin.     

Similarities of antisera to casein and epithelial membrane antigen

Summary Antisera raised against human milk fat globule membranes and against the casein fraction of human milk have been compared. Using an immunohistochemical stain of tissue sections it has been shown that many of the antigenic determinants detected by the different antisera are identical. A radioimmunoassay for epithelial membrane antigen (EMA) showed that casein preparations are associated with small quantities of EMA. Antisera to casein frequently contained appreciable concentrations of antibodies to EMA and this accounts for the immunohistochemical staining of non-mammary tissues.     

Distribution of epithelial membrane antigen in benign and malignant lesions of the salivary glands

Summary An antiserum against epithelial membrane antigen has been used to stain a variety of lesions arising in the salivary glands. In normal major and minor glands staining was localised to the ductal systems. There was no evidence of myoepithelial cell staining. The mucous elements of the submandibular and sublingual glands were negative, but in the mucous elements of the minor glands there was focal cytoplasmic positivity. There was no cytoplasmic staining of serous elements in major or minor salivary glands. In pleomorphic adenomas the luminal membrane of ductal elements was strongly positive, with focal cytoplasmic positivity in some myxoid areas. In mucoepidermoid tumours both adjacent cell membranes and cytoplasm were strongly positive. The ductal structure of adenoid cystic carcinomas were clearly delineated while the pseudoducts produced by enclosed areas of stroma were negative. All mesenchymally derived tumours were negative and a tumour previously considered as a chondroma was strongly positive. The results are discussed in relation to phenotypic heterogeneity and the histogenesis of salivary gland tumours.     

微管相关蛋白2和巢蛋白在人胚胎脊髓的表达

目的 探讨微管相关蛋白2(MAP-2)和巢蛋白在人胚胎脊髓发育阶段的分布规律及其表达意义.方法 应用免疫组织化学法,检测第2、3、4月龄段共16例人胚胎脊髓前角、中央管及后角中MAP-2和巢蛋白的表达、分布状况.结果 第2～4个月龄段,人胚胎脊髓内均可见巢蛋白表达阳性的神经纤维分布,随着胎龄的增大,脊髓前角处巢蛋白阳性...     

成人侧脑室下区Nestin阳性细胞的免疫组化观察

目的　探讨成人侧脑室下区 (SVZ)Nestin阳性细胞的分布及其化学特点。方法　采用 3 3 1B、10C2、Rat4 0 1、GFAP、NSE和viminten等神经细胞标志物对成人SVZ区进行免疫组织化学研究。结果　SVZ区存在较多Nestin反应阳性细胞 ,GFAP免疫阳性细胞及较少的NSE免疫阳性细胞。Nestin免疫反应阳性细胞可分为两类 ,一类为卵圆形 ,胞体较大 ,少有纤维突起 ;一类为星形 ,胞体较小 ,发出多支放射状的纤维突起。Nestin免疫阳性细胞均不与GFAP及NSE发生交叉反应。成人SVZ区细胞未见有viminten的表达。结论　成人SVZ区可能存在神经前体细胞和星形胶质样Nestin免疫阳性细胞。     

Epithelial markers in primary skin cancer: an immunoperoxidase study of the distribution of epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA) in 65 primary skin carcinomas

Sixty-five primary malignant skin tumours have been stained for carcinoembryonic antigen (CEA) and epithelial membrane antigen (EMA) using rabbit polyclonal affinity-purified antibodies and an indirect immunoperoxidase technique. The tumours consisted of 15 invasive squamous carcinomas, 23 basal cell carcinomas, 16 malignant eccrine poromas (porocarcinomas), and 11 sebaceous carcinomas. The basal cell carcinomas were negative for CEA and EMA except where there was keratotic or sebaceous differentiation. All the sebaceous and squamous carcinomas and 15/16 porocarcinomas contained EMA. 12/15 squamous carcinomas were positive for CEA. The malignant poromas were negative for CEA except on the ulcerated surface of two. In tumours classified as sebaceous carcinomas there was positive staining for CEA in some cells, cyst contents and/or keratotic foci. These findings have implications for the use of immunoperoxidase localization of epithelial markers in the differential diagnosis of primary and metastatic skin cancer.     

微管相关蛋白2和巢蛋白在人胚胎小肠中的表达

目的: 探讨微管相关蛋白2(MAP-2)和巢蛋白(nestin)与人胚胎小肠壁组织发育的关系。 方法: 应用免疫组织化学PV法和图像分析(NIS-DR)软件检测第2、3、4个月龄段,人胚胎小肠组织内MAP-2和nestin的表达分布规律,数据的组间比较采用单因素方差分析。 结果: 第2、3、4个月龄段,MAP-2主要表达于人胚胎小肠壁黏膜下和肌间神经丛内神经细胞和纤维,随胎龄的增大,肌间神经丛内神经细胞的阳性强度增强, 两两比较, 差异有统计学意义(P<0.05)。Nestin蛋白在小肠壁各层均有阳性表达,其黏膜下层和肌间神经丛内阳性细胞数量随胎龄增大而呈先增高再降低的趋势,两两比较, 差异有统计学意义(P<0.05)。 结论: MAP-2和nestin参与调节人胚胎小肠壁组织的生长发育过程。     

Epithelial membrane antigen in normal and proteinuric glomeruli and in damaged proximal tubules

Epithelial membrane antigen was detected in normal glomeruli by a polyclonal antiserum to the antigen and by the monoclonal antibodies Ca 1, DAKO-EMA and HMFG 2, but not HMFG 1, using an indirect immunoperoxidase method. The antigen was in the form of a thin ring or collar at the junction of glomerulus and tubule. In a series of 47 renal biopsies from patients with proteinuria, the antigen could still be seen in glomeruli, provided that there were adequate numbers of glomeruli in the sections. The main object of study was the glomerular tip lesion, in which tip adhesions were seen to be just adjacent to the patch of epithelial membrane antigen. This suggested that the antigen may be important in pathogenesis of the lesion. Normal proximal tubules did not express epithelial membrane antigen but it was detected on the luminal border of acutely damaged proximal tubules. Thus the distribution of epithelial membrane antigen in the kidney is more complex than was previously thought.     

Alcian blue and epithelial membrane antigen are useful markers in differentiating benign from malignant papillae in thyroid lesions

Summary Immunohistochemistry for epithelial membrane antigen (EMA) and histochemistry for alcianophilic substances were performed in 17 cases of papillary thyroid carcinoma (PTC) and 11 cases of benign thyroid lesions showing papillary changes (7 diffuse hyperplastic goitres-Graves'disease; 4 colloid cystic goitres). In all PTCs the glycocalix of the cells lining the papillary structures was stronly positive with anti-EMA antiserum. Alcian blue pH 2.5 stain (AB 2.5) was also positive in 15 of these cases. In contrast, no cases of benign thyroid lesions showed AB 2.5 positivity in the cells lining the papillary structures and the positivity with anti-EMA antiserum, present in only 5 out the 11 cases, was focal and very weak. These results indicate that the presence and distribution of EMA and alcianophilic substances may be useful in distinguishing benign from malignant thyroid lesions containing papillae.     

Immunohistochemical observations of keratins,involucrin, and epithelial membrane antigen in urinary bladder carcinomas from patients infected withSchistosoma haematobium

Summary Squamous cell carcinomas of the urinary bladder and the epithelial lesions associated with infection bySchistosoma haematobium were histopathologically and immunohistochemically described for keratin proteins (TK, 41–65 kDa; KL1, 55–57 kDa; PKK1, 40, 45 and 52.5 kDa), involucrin, and epithelial membrane antigen (EMA). Normal urothelial epithelium was positive for all keratins, and showed absent or slight reactions for involucrin and EMA in superficial umbrella cells. The intestinal type of epithelium was composed of columnar cells and small basal cells; TK was positive in the basal cells, KL1 staining was positive in the columnar cells, whereas PKK1 was negative or slight in the columnar cells. Involucrin was confined to columnar cells. Squamous metaplastic epithelium showed a rather regional keratin distribution: TK was distributed in all layers, KL1 decorated upper spinous and granular layers, but PKK1 did not bind, and involucrin staining existed only in upper spinous and granular cells. Keratin expression in squamous cell carcinomas indicated heterogeneity and its stainability was dependent on the degree of keratinization: The G 1 type revealed strong reaction, the G 2 type showed a similar distribution pattern, but the staining intensity was less, and the G3 type showed irregular staining with decreased intensity. Involucrin staining was limited to keratinized cells of carcinoma as was that for EMA.     

微管相关蛋白2、巢蛋白与人胚胎食管肌层发育的关系

目的: 探讨微管相关蛋白2(MAP-2)和巢蛋白(nestin)与人胚胎食管肌层发育关系。方法: 应用免疫组织化学PV法检测第2、3、4月龄段人胚胎食管组织内MAP-2和nestin的表达分布规律。结果: 第2、3、4月龄段,MAP-2在食管肌层的肌细胞呈阴性表达,肌间神经丛内神经细胞处呈阳性表达;nestin在第2-3月龄食管肌层的肌细胞处呈阳性表达,到第4月龄转阴性表达;而在食管肌间神经丛内大部分神经细胞,第2-4月龄段呈弱阳性或阴性表达。结论: MAP-2和nestin可能参与调节人胚胎食管肌层的形成过程。     

神经元核心抗原和神经元特异性烯醇化酶在人胚胎脊髓发育阶段的表达

目的 探讨神经元核心抗原（NeuN）和神经元特异性烯醇化酶(NSE)在人胚胎脊髓发育阶段的分布规律及其表达意义。 方法 应用免疫组织化学法检测第2、3、4个月龄段,共16例人胚胎,脊髓前角、后角中NeuN和NSE的表达、分布状况。 结果 在脊髓后角处：第2个月胚龄段,NeuN呈阴性表达, 部分细胞呈NSE阳性表达;第3个月胎龄段,部分细胞开始呈NeuN阳性表达,NSE则呈广泛阳性表达;第4个月胎龄段,NeuN阳性细胞表达数量和强度均较前增高,NSE阳性表达与第3个月龄相似。在人胚胎脊髓前角处： 第2～4个月龄段,NeuN和NSE呈广泛阳性表达。 结论 人胚胎脊髓发育第2～4个月龄段,NeuN和NSE阳性表达在脊髓的前角处早于后角,NeuN和NSE参与脊髓神经元的发育成熟过程。     

中国人前列腺特异膜抗原基因的克隆及序列测定

目的 克隆人前列腺模特异抗原(prostate-specific membrane antigen,PSMA）的编码区cDNA片段。方法 从前列腺癌组织提取总RNA,利用RT-PCR技术扩增出人PSMA基因编码区序列,将其克隆至pcDNA3.1载体中,并行序列测定。结果 序列测定表明,克隆获得的2253bp片段与文献报道的人PSMA基因编码区cDNA序列一致。结论 本研究为进一步研究 PSMA的生物学性能和用于前列腺癌诊断治疗的可能性打下了基础。     

神经元核心抗原和神经元特异性烯醇化酶在人胚胎小肠发育阶段的表达

 目的：探讨神经元核心抗原（neuronal nuclear antigen,NeuN）和神经元特异性烯醇化酶(neuron-specific enolase, NSE)在人胚胎小肠发育阶段的分布规律及其表达意义。方法：应用免疫组织化学法检测第2~4月龄段共16例人胚胎小肠壁细胞NeuN和NSE的表达、分布状况。结果：第2~4月胎龄段,NSE在人胚胎小肠肌间神经丛内的神经元及神经纤维均呈强阳性表达,在黏膜下层,随着胎龄的增大,NSE阳性表达细胞和纤维数量逐渐增多,在肠腺内均有少量散在分布的NSE阳性细胞;在黏膜层的腺体和上皮组织内均有散在的NeuN阳性细胞分布,随着胎龄的增大,NeuN阳性细胞数量增多;在肌间神经丛,第3月龄段开始,有少量NeuN阳性细胞,随着胎龄的增大, NeuN阳性细胞数量逐渐增多;在黏膜下层,未见NeuN阳性细胞分布。结论: 人胚胎小肠发育阶段,NeuN和NSE在小肠壁的阳性表达和分布不一致,均参与小肠壁神经元及神经内分泌细胞的发育过程。     

Expression of extracellular matrix-degrading proteins in classic, atypical, and anaplastic meningiomas

Although the majority of meningiomas, commonly benign tumors (WHO I), are amenable to surgical resection, a percentage of up to 3% will recur as higher-grade meningiomas with potential brain invasion. Our study aims at the in situ identification of proteolytic, extracellular matrix-degrading enzymes in a broad spectrum of meningiomas. We examined 80 meningiomas (50 classic meningiomas WHO I, 19 meningiomas WHO II, including atypical, chordoid, and clear cell types, as well as 11 anaplastic meningiomas WHO III) for the immunohistochemical expression patterns of cathepsin D and metalloproteinases MMP-2 and MMP-9. Meningiomas of all types and grades revealed a distinct expression of MMP-9 and cathepsin D, while MMP-2 was found predominantly in WHO II and III meningiomas. There was a significant increase in positive tumor cells from WHO grade I to II and III for MMP-2 (p<0.001), but not for cathepsin D (p=0.099). MMP-9 displayed an increased number of positive tumor cells from WHO grade I to II, but a decrease in WHO III meningiomas (p<0.002). Routine screening for the expression of metalloproteinases and cathepsin D will not reveal any new diagnostically or prognostically relevant information. However, these factors may represent a potential target for pharmacological blocking as an anti-invasive therapy.     

An immunohistochemical comparison of cytokeratin 7, cytokeratin 15, cytokeratin 19, CAM 5.2, carcinoembryonic antigen, and nestin in differentiating porocarcinoma from squamous cell carcinoma

The distinction of porocarcinoma from squamous cell carcinoma is clinically relevant but can often be a diagnostic dilemma. Current markers reported to be helpful in diagnosing porocarcinoma include carcinoembryonic antigen and cytokeratin 7; however, their expression has been demonstrated in 30% to 80% and 13% to 22% of squamous cell carcinoma cases, respectively. In this study, we assessed immunohistochemical expression of cytokeratin 7, cytokeratin 15, cytokeratin 19, CAM 5.2, carcinoembryonic antigen, and nestin in 67 cases (39 porocarcinomas and 28 moderately differentiated squamous cell carcinomas) to determine their use as histologic adjuncts. Expression of carcinoembryonic antigen, cytokeratin 19, cytokeratin 7, CAM 5.2, cytokeratin 15, and nestin was seen in 77%, 67%, 64%, 51%, 49%, and 13% of porocarcinomas, respectively; and in 57%, 18%, 26%, 32%, 30%, and 37% of squamous cell carcinomas, respectively. Of these, cytokeratin 19 was the most specific (specificity, 82%) in detecting porocarcinomas, and carcinoembryonic antigen was the most sensitive (sensitivity, 77%). By χ(2) test, statistically significant P values (<.05) were observed for cytokeratin 7, cytokeratin 19, and nestin in the distinction of porocarcinoma from squamous cell carcinoma. However, in a logistic regression and stepwise selection for predicting a porocarcinoma, statistical significance was observed only for cytokeratin 19 (P = .0003). In conclusion, we found cytokeratin 19 to be a helpful marker in the distinction of porocarcinoma from squamous cell carcinoma, although a focal staining pattern can be seen in a third of cases. The diagnostic sensitivity and specificity appear to be significantly improved using a selected panel of immunohistochemical stains that include cytokeratin 7, cytokeratin 19, and nestin.     

构建PSMA shRNA慢病毒载体转染293T细胞后PSMA mRNA和蛋白的表达变化

目的：利用基因工程技术筛选获得阻断前列腺癌细胞株LNCaP中前列腺癌特异性膜抗原 (PSMA) 表达的shRNA序列，并构建慢病毒载体。观察转染前列腺癌细胞后对PSMA mRNA和蛋白表达的影响。方法：〖HTSS〗根据PSMA基因信息，设计siRNA1、siRNA2、siRNA3三条针对PSMA基因cds区的siRNA序列，组建shRNA对应的4对互补的单链DNA，包括siRNA的正义链和反义链；正义链序列按5向3顺序依次为：酶切位点（BamHⅠ）、干扰序列（19 bp）、loop环（TTCAAGAGA）、干扰序列的反向互补序列（19 bp）、终止信号（TTTTT）、酶切位点（EcoRⅠ）。将合成的序列插入空载体pSIH1-H1-copGFP shRNA vector中，转染前列腺癌细胞后，通过real-time PCR检测对PSMA mRNA表达，通过Western blotting检测PSMA蛋白的表达。结果：〖HTSS〗设计的3条针对PSMA的序列中第2条的抑制效果最好，目的序列位于PSMA（NM_004476）的1207到1226，茎环序列为5-GATCC GTCTCAAAGTGCCCTACAA TTCAAGAGA TTGTAGGGCACTTTGAGAC TTTTT G-3。其对前列腺癌细胞株中PSMA mRNA表达的抑制率为60%，对PSMA蛋白表达的抑制率为86%。转染细胞后，细胞可以稳定低表达PSMA。结论：〖HTSS〗成功获得阻断前列腺癌细胞株LNCaP PSMA表达的shRNA序列，并构建慢病毒载体，pSIH-PSMA-siRNA2转染前列腺癌细胞后对PSMA mRNA 表达的抑制率为60%，对PSMA蛋白表达的抑制率达86%。为后期研究PSMA在前列腺癌发病中的作用机制以及免疫导向治疗等提供了实验基础。     

Expression of vimentin and epithelial membrane antigen in human malignant lymphomas

Immunoreactivity with monoclonal antibodies against the intermediate filament protein, vimentin, and epithelial membrane antigen (EMA) was examined in 330 cases of lymphoma (317 non-Hodgkin's and 13 Hodgkin's lymphomas), 12 reactive lymph nodes and mononuclear cells of the peripheral blood using either indirect immunoperoxidase staining or the avidin-biotin immunoperoxidase complex technique. The cell origin of each tumor was established using a panel of monoclonal antibodies against lymphocyte differentiation antigens. There were 41 T-cell, 247 B-cell and 29 undetermined lymphomas, and 13 cases of Hodgkin's disease in the series. Vimentin was expressed in 24 T-cell lymphomas (58.5%) and 60 B-cell lymphomas (24.2%). This difference in frequency was statistically significant. Vimentin expression in follicular lymphomas was less frequent than in diffuse B-cell lymphomas. In diffuse lymphomas, small and medium cell types were more reactive with anti-vimentin than large cell types. Reed-Sternberg cells (R-S cells) in Hodgkin's disease were positive for vimentin in 11 cases (84.6%). The frequency of EMA reactivity in lymphomas was low, particularly in T-cell lymphomas. No positive cases were found among follicular lymphomas. In diffuse non-Hodgkin's lymphomas, EMA was expressed only in mixed and large cell types, but never in smaller ones. In conclusion, monoclonal antibodies against vimentin and EMA appear to be of limited usefulness for the diagnosis of non-Hodgkin's lymphomas, but anti-vimentin antibody may be used as an adjunct to the diagnosis of R-S cells in Hodgkin's disease.     

Detailed investigation of the diagnostic value in tumour histopathology of ICR.2, a new monoclonal antibody to epithelial membrane antigen

The production and detailed immunostaining properties of a new rat monoclonal antibody (ICR.2) to epithelial membrane antigen are reported. The antibody was selected for its ability to compete with the polyclonal antiserum (M7), used in the original immunohistological studies, in order that it might serve as a direct replacement in diagnosing epithelial tumours. Most of the staining reactions on normal tissues were identical to those previously reported with M7 but there were some important differences. They included: positivity of renal and adrenal capsular fibroblasts, perineurium, some myoepithelial and smooth muscle cells, occasional osteoblasts and squamous and thyroid follicular epithelium in the normal state. The intercellular canaliculi of sweat glands and secretory canaliculi of gastric oxyntic cells were clearly demonstrated. These staining reactions could be obtained with M7 when a sensitive detection system was used although the results were usually weak and inconsistent. Nearly all adenosquamous and transitional carcinomas were positive. The remaining tumours fell into three major groups: (1) those which were consistently or nearly consistently negative--melanoma, seminoma, rhabdomyosarcoma, alveolar soft part sarcoma, adrenal cortical carcinoma, granulocytic sarcoma, paraganglioma, non-Hodgkin's lymphoma. Hodgkin's disease and embryonal carcinoma: (2) those which were either negative or positive with distinctive patterns of staining--basal cell carcinoma, embryonal tumours: and (3) non-epithelial tumours that were consistently positive--epithelioid sarcoma, synovial sarcoma, osteosarcoma, chordoma and myeloma--or positive in a significant minority of cases--leiomyosarcoma, malignant fibrous histiocytoma, clear cell sarcoma of tendon sheath, various neuroectodermal tumours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Usefulness of epithelial membrane antigen (EMA) to discriminate between perineural invasion and perineural indentation in prostatic carcinoma

AIMS: Perineural invasion (PNI) is one of the few unequivocal criteria to diagnose adenocarcinoma of the prostate. Distinguishing PNI from perineural indentation (PNIn), however, is sometimes difficult. The aim of this study was to discriminate between PNI and PNIn using EMA immunohistochemistry. METHODS AND RESULTS: We selected representative sections from 87 prostatectomies with prostatic adenocarcinoma. Normal peripheral nerves were continuously encircled with perineurium, which was immunoreactive for EMA. We identified 1319 PNI by carcinomas, 368 PNIn by carcinomas, and 303 PNIn by benign glands. We categorized the EMA immunoreactivity patterns into three classes: samples that displayed discontinuity or complete loss of the perineurium (Type A), samples where there were carcinomas or benign glands in the perineural space or peripheral nerves (Type B) and samples that showed no changes in the perineurium (Type C). For PNI we observed Type A, Type B, and Type C patterns in 55.3%, 24.8% and 19.9% of carcinomas, respectively. The incidence of each of those patterns in PNIn by carcinomas was 32.1%, 14.9% and 53.0%, respectively. Cases of PNIn by benign glands showed Type A or Type C patterns. They did not, however, exhibit Type B patterns. CONCLUSION: EMA immunostaining will aid the diagnosis of prostatic adenocarcinoma.