骨肉瘤中p53、p21WAF1、cyclinA蛋白的表达及其意义

目的探讨骨肉瘤中p53、p21WAF1、cyclinA蛋白的表达及相互关系.方法应用免疫组化方法对骨肉瘤组织及正常软组织中p53、p21WAF1、cyclinA的蛋白表达进行检测.结果正常软组织中p53表达均阴性,28%(14/50)骨肉瘤中可检测到p53蛋白的异常积累;正常软组织中均有不同程度的p21WAF1蛋白的阳性表达,52%(26/50)骨肉瘤p21WAF1阴性,骨肉瘤中p21WAF1的蛋白表达表现为p53蛋白依赖性的方式;正常组织中cyclinA为阴性,75.6%(28/37)骨肉瘤中存在cyclinA蛋白过表达,cyclinA与p21WAF1的表达呈负相关(r=-0.874,P＜0.01);p21WAF1蛋白的表达与骨肉瘤的分化呈正相关(r=0.687,P＜0.01).结论p53蛋白的异常积聚、p21WAA1的失表达及cyclinA的过表达参与了骨肉瘤失控的增生及肿瘤的形成.     

Podocalyxin-like protein 1 expression in primary hepatic tumours and tumour-like lesions

AIMS: The differential diagnosis of benign hepatic lesions and well-differentiated hepatocellular carcinomas can be a challenge, especially in small biopsy specimens. Recently, novel proteins expressed by the neovasculature in hepatocellular carcinoma (HCC) have been identified. The aim of this study was to compare the expression of podocalyxin-like protein 1 (PODXL1), a CD34-related sialomucin, in HCC and benign liver tumours or tumour-like lesions. METHODS AND RESULTS: Vascular marker expression was examined using tissue microarrays as well as standard paraffin sections from formalin fixed paraffin-embedded liver tissue samples. Expression of PODXL1 was compared with anti-CD34, CD31 and von Willebrand factor VIII staining by immunohistochemistry. PODXL1 is expressed in tumour-associated microvasculature endothelial cells in HCC, as well as in capillarized sinusoidal endothelium of focal nodular hyperplasia (FNH) and hepatic adenoma. Expression in cirrhotic nodules correlates with CD34 and highlights endothelium in the inflow area. In dysplastic nodules CD34 and PODXL1 are not or only focally expressed. CONCLUSIONS: Expression patterns of CD34 and PODXL1 are almost identical in primary hepatic tumours and tumour-like lesions. The presence of CD34+ and PODXL1+ sinusoidal endothelial cells aids in the diagnosis of HCC. Sinusoidal expression of PODXL1 is also seen in a less diffuse pattern in FNH and adenoma.     

Diagnostic utility of WT-1 cytoplasmic stain in variety of vascular lesions

Vascular lesions are commonly encountered in routine pathologic practice and often pose diagnostic challenges owing to their morphologic diversity. Although WT-1 expression was reported in some vascular tumors, little is known about its staining patterns in a spectrum of vascular lesions from various locations. We examined WT-1 immunostain in 95 cases of vascular lesions including angiosarcomas (AS, 19 cases), hemangioendotheliomas (HE, 5), Kaposi’s sarcomas (KS, 4), cavernous hemangiomas (CVH, 12), capillary hemangiomas (CPH, 7), pyogenic granulomas (PG, 4), lymphangiomas (LA, 4), hemangiopericytomas (HP, 5), glomus tumors (GT, 8), vascular malformation (VM, 13) and granulation tissue (GRT, 14). Strong WT-1 cytoplasmic stain was invariably observed in all cases of malignant and borderline vascular tumors including AS (19/19), KS (4/4) and HE (5/5). WT-1 was also consistently expressed in CPH (7/7), PG (4/4), and GRT (14/14), while it became weaker in VM (10/13) and often negative in CVH (2/12) and LA (0/4). WT1 stain was not demonstrated in HP (0/5) and rarely in GT (2/8). We conclude that consistent and diffuse WT-1 cytoplasmic stain in AS, HE and KS can be useful in distinguishing these tumors from poorly differentiated tumors with mimicking features. On the other hand, reliable WT-1 stain in CPH, PG and GRT may help in differential diagnosis with non-endothelial vascular tumors such as GT and HP. Recognizing the WT-1 cytoplasmic stain in a broad spectrum of benign and neoplastic tissues is critical in formulating appropriate immunohistochemical panels and avoiding misinterpretation of results.     

流体切应力下血管内皮单核细胞趋化蛋白-1的表达

目的研究流体切应力对人血管内皮细胞的单核细胞趋化蛋白-1(MCP-1)表达的影响,探讨血流动力在动脉粥样硬化(AS)发生早期中的作用.方法利用平行板流动腔对血管内皮细胞施加不同切应力,分别采用夹心酶联免疫吸附测定(EL,ISA)及逆转录-聚合酶链反应(RT-PCR)法检测MCP-1蛋白及mRNA水平.结果以0.72Pa切应力进行不同时间作用,0.5h后MCP-1mRNA即达到静态时(0.160±0.037)的4倍(0.684±0.033),5h时略有升高,达0.707±0.089,12h后下降到低于对照组的水平(0.036±0.006,P＜0.001).灌流液中MCP-1蛋白时间依赖性增加,但5h后增长趋缓;而在不同切应力(0.30、0.72、2.40Pa)相同时间(5h)下,0.72Pa的作用最显著,MCP-1蛋白比静态对照增加了2倍多,mRNA水平则升高达4倍.结论MCP-1的表达对切应力的变化反应强烈,持续稳定的切应力则下调MCP-1的表达,此研究结果表明血流紊乱可促进AS病变的发生发展.     

DLG1 polarity protein expression associates with the disease progress of low-grade cervical intraepithelial lesions

Human Discs large tumour suppressor (DLG1) participates in regulating cell polarity and proliferation, suggesting an important connection between epithelial organization and cellular growth control. However, it was demonstrated that DLG1 could acquire oncogenic attributes in some specific contexts. In this work, we evaluated the expression of DLG1 and its contribution to the progress of cervical lesions in order to investigate a potential role of this polarity protein in human oncogenic processes.We analyzed cervical biopsies from women with low-grade squamous intraepithelial lesion (LSIL) diagnosis (n = 30), for DLG1 expression by immunohistochemistry. These results were correlated with the clinical monitoring of the patients during a 24-month follow-up period. Our data indicate that while all LSIL patients with a DLG1 staining pattern similar to normal tissues are significantly more likely to regress (n = 23, Pattern I), all LSIL biopsy specimens showing a diffuse and intense DLG1 staining likely progress to high-grade lesions (n = 4, Pattern II). Finally, all persistent LSIL analyzed showed an undetermined DLG1 staining, with a diffuse distribution without a strong intensity (n = 3, Pattern III). We found a significant association between the expression pattern of DLG1 and the evolution of the lesion (p < 0.00001).This work contributes to the knowledge of DLG1 biological functions, suggesting that its expression may have an important role in the progression of early dysplastic cervical lesions, giving prognostic information.     

Melan-A/Mart-1 expression in various melanocytic lesions and in non-melanocytic soft tissue tumours

AIMS: The purpose of this study was to test different malignant non-melanocytic tumours with the commercially available antibody Melan-A to examine its diagnostic specificity and to compare the S100, Melan-A and HMB-45 reactivity in various melanocytic lesions. METHODS AND RESULTS: Seventy-three benign and malignant melanocytic lesions and 31 cases of non-melanocytic tumours, sarcomas, carcinomas and carcinoids, were selected. Immunohistochemical staining of paraffin sections, following a high temperature antigen unmasking technique, was performed. Melan-A stains junctional and dermal melanocytes in all benign melanocytic lesions with the exception of neuro-naevoid areas. The epithelioid and the spindle cells in malignant melanomas did not show considerable difference in their Melan-A reactivity. The predominantly spindle cell type mucosal melanomas contained more Melan-A-positive cells than HMB-45-positive cells and similar results were observed in metastatic malignant melanomas. In desmoplastic melanomas the positivity of Melan-A was not consistent. None of the sarcomas, carcinomas and carcinoids expressed Melan-A. Almost all soft tissue tumours, except for two malignant gastrointestinal stromal tumours, were unreactive for HMB-45. These two cases did not react with Melan-A antibody. CONCLUSIONS: Melan-A is a useful additional marker to differentiate non-melanocytic tumours from primary or metastatic melanoma. In melanocytic lesions the Melan-A staining pattern is similar to S100, but seems to be more specific. In desmoplastic melanomas, however, the variable Melan-A staining further necessitated detailed histological examination and the use of the S100 reaction.     

Tumour suppressor gene TP53 mutations in atypical vascular lesions of breast skin following radiotherapy

Santi R, Cetica V, Franchi A, Pepi M, Cesinaro A M, Miracco C, Paglierani M, De Giorgi V, Delfino C, Difonzo E M, Pimpinelli N, Bianchi S, Sardi I, Santucci M & Massi D
(2011) Histopathology 58 , 455–466
Tumour suppressor gene TP53 mutations in atypical vascular lesions of breast skin following radiotherapy Aims: Atypical vascular lesions (AVL) occurring at the site of radiotherapy represent an uncommon but well‐documented complication in the setting of breast‐conserving therapy for breast carcinoma. Although the biological behaviour of AVL has been regarded as benign, it has been suggested that AVL may represent a precursor of angiosarcoma. A better understanding of the biology of AVL is essential in order to assess appropriate patient management. The aim of the present study was to investigate alterations of tumour suppressor gene TP53 in a series of radiation‐induced AVL and angiosarcomas (AS). Methods and results: Direct sequencing analysis of the TP53 gene showed the presence of at least one variation in 10 of 12 (83.3%) AVL and in seven of eight (87.5%) AS. The most common alteration in both categories was the P72R polymorphism in exon 4. One angiosarcoma sample carried a pathogenetically relevant disruptive mutation c.592delG, a frameshift deletion in exon 6, causing a premature stop codon. Conclusions: The presence of TP53 alterations suggests that its mutational inactivation may be implicated in the pathogenesis of radiation‐associated vascular proliferations. The common mutational pathway suggested by our data supports the hypothesis that AVL and AS are biologically related entities, most probably representing the extremes of a morphological continuum.     

p27/kip1 expression in normal epithelium, benign and neoplastic breast lesions.

The development of cancer in the breast and in other sites is a complex process requiring a number of different genetic and epigenetic alterations. The accumulation of the genetic changes is thought to underlie the progression from precancerous lesions to carcinomas. The expression of p27/kip1 protein, a cyclin-dependent kinase inhibitor, was investigated by immunohistochemistry in normal epithelial specimens, benign alterations, and malignant lesions of the breast. The number of p27/kip1-positive cells ranged from none to more than 98% in the overall series. Wide ranges of p27/kip1-positive cells were consistently observed within each histological category, but the median value progressively decreased in typical hyperplasia and fibroadenoma, with an even more marked reduction in malignant lesions, compared with normal epithelium. Moreover, the percentage of cells expressing p27/kip1 in tumours was about three times lower in invasive than in in situ lesions and was inversely related to tumour size, but not to lymph node involvement. In conclusion, the degree to which p27 expression is altered in typical hyperplastic lesions and fibroadenomas indicates that the deregulation of p27 may occur very early on during breast cell transformation, but the usefulness of its determination to categorize subgroups of lesions at different risk of evolution remains somewhat doubtful.     

胃癌中BRG1蛋白的表达及意义

目的 探讨BRG1基因蛋白在胃癌组织中的表达及与pRb、p53基因的相互关系。方法 应用S-P免疫组化法检测30例胃癌组织中BRG1及pRb和p53的表达情况，分析其相关性，并且结合临床病理因素，初步探讨其和胃癌发生、发展及预后的关系。结果 BRG1与pRb的表达有相关性，而和p53无关。BRG1在胃癌中的表达率为76.67％(23/30)，与正常组织相比呈高表达。BRG1和pRb的表达与胃癌的浸润、淋巴结转移、分级和分期有关。结论 BRG1蛋自在胃癌中高表达，对胃癌的发生、发展可能起重要作用。BRG1及pRb的表达可作为判断胃癌恶性程度及预后的重要指标。     

尤文肉瘤EWS-FLI1融合蛋白B淋巴细胞表位的预测及鉴定

目的:预测及鉴定尤文肉瘤EWS-FLI1融合蛋白的B淋巴细胞表位。方法:采用综合法预测EWS-FLI1蛋白的二级结构及B淋巴细胞表位,运用标准Fmoc方案合成预测的表位肽,HPLC和MS进行表位肽的纯度分析及分子量鉴定,ELISA法检测表位肽的抗原性,并测定表位肽特异性免疫血清效价,Western blot鉴定免疫血清与EWS-FLI1蛋白亲合力。结果:通过综合法预测得到3个高分值的B淋巴细胞表位,HPLC分析合成的表位肽纯度>85%,MS鉴定表位肽的分子量无误,ELISA法检测证实3个B淋巴细胞表位肽均可获得强的抗原抗体反应,其中表位肽P2的抗原性最强,在1∶40时A450=2.46,达到最高,1∶10 240稀释后抗原抗体反应仍呈阳性;用这3个B淋巴细胞表位肽免疫新西兰兔也能获得理想的抗体效价,其中表位肽P2获得的抗体效价最高,1∶512 000稀释时A450=1.11;Western blot鉴定表位肽P1、P2免疫血清能够结合EWS-FLI1蛋白。结论:尤文肉瘤EWS-FLI1蛋白的B淋巴细胞表位肽P1、P2具有潜在的抗原性和免疫原性。     

Epidermotropism of T cells correlates with intercellular adhesion molecule (ICAM1) expression in human papillomavirus (HPV)-induced lesions.

Adhesion molecules play an important role in inflammatory reactions. Among them, ICAM1, a ligand for the lymphocyte function-associated antigen (FLA1) of leucocytes, may be expressed by antigen-presenting cells and keratinocytes in various inflammatory disorders. As cell-mediated immune responses play a great role in HPV infections, we investigated the expression of ICAM1 and correlated it with the presence of LFA1-positive cells by immunohistochemistry on serial frozen sections of a series of non-regressing cutaneous and mucosal HPV-induced lesions. ICAM1 expression by keratinocytes was observed only in intensely infiltrated lesions of condylomas and laryngeal papillomas. Its induction was usually correlated with the presence of LFA1-positive cells (mainly CD8-positive cells) which were in close apposition to ICAM1-positive proliferative epithelial cells expressing also, in some cases, HLA-DR antigen. ICAM1 was not correlated with the presence of HPV DNA or viral antigen. In moderately infiltrated lesions, keratinocytes did not express ICAM1, and LFA1-positive cells were not observed in the epidermis. In all lesions, ICAM1 was more intense on endothelial cells than in normal skin; infiltrating cells (lymphocytes and dendritic cells) may also express this antigen but intraepithelial Langerhans cells were devoid of any labelling. These studies provide further evidence that T-lymphocyte mechanisms are important in the host response to HPV-induced lesions. ICAM1 expression correlates with a lesional infiltrate but not with HPV infection and probably results in a more efficient initiation of the immune reaction.     

白细胞介素1,肿瘤坏死因子α和脂多糖对人脐静脉内皮细胞表达 …

目的 观察细胞因子白细胞介素１（ＩＬ－１）β、肿瘤坏死因子（ＴＮＦ）α和脂多糖（ＬＰＳ）是否诱导人脐静脉内皮细胞表达单核细胞趋化蛋白１（ＭＣＰ－１）ｍＲＮＡ及蛋白。方法 选取生长汇合的人脐胸脉内皮细胞,在其培养基中分别加入终浓度为２ｎｇ／ｍｌ的ＩＬ－１β、２０ｎｇ／ｍｌ的ＴＮＦβ和１００ｎｇ／ｍｌ的ＬＰＳ,３７℃共育４ｈ后,按照一步法提取其总ＲＮＡ,用γ－＾２２Ｐ标记的寡核苷酸ｄｏｔ ｂｌｏｔ分析     

Role of ROC1 protein in the control of cyclin D1 protein expression in skin melanomas

A decrease in the level of the ROC1 protein, which is involved in cyclin D1 degradation, might explain an increase in cyclin D1 protein in the absence of gene overexpression. This study aimed to investigate the relationship between ROC1 and cyclin D1 expression in skin melanomas. A total of 62 cases of primary skin melanomas and 58 cases of compound melanocytic nevi were assessed. Immunohistochemistry was performed using cyclin D1 and ROC1 antibodies, and fluorescent in situ hybridization was used to assess the amplification of the CCND1 gene. ROC1 was expressed in >50% of cells in 87.9% of the melanocytic nevus cases and in 45.2% of the melanoma cases (p = 0.0014). There was a significant negative correlation between ROC1 and cyclin D1 expression in all cases (p = 0.0008985). In comparison with cyclin D1, ROC1 expression was increased in 86.2% of the melanocytic nevi and in 45.2% of the melanomas (p < 0.001). Among the non-amplified melanomas, 50% expressed cyclin D1 in >50% of the cells and expressed ROC1 in <25%. ROC1 expression is negatively correlated with cyclin D1 expression, demonstrating its importance in the degradation of cyclin D1 in melanomas.     

Overexpression of vascular adhesion protein‐1 is associated with poor prognosis of astrocytomas

Vascular adhesion protein‐1 (VAP‐1) is one of the endothelial adhesion molecules that is believed to play a role in tumor progression and metastasis, supporting cancer cell extravasation. Very few studies have been performed on analyzing the contribution of VAP‐1 in brain tumor. Astrocytomas are the most common type of brain tumors, which are classified by World Health Organization (WHO) into four grades according to the degree of malignancy. This study was designed to investigate VAP‐1 expression level in different astrocytoma grades and its correlation with clinicopathological features as well as prognosis of astrocytoma patients. Eighty‐seven patients with different grades of astrocytoma (WHO Grade I–Grade IV) were enrolled in this study. The expression of VAP‐1 was assayed by immunohistochemistry. The correlation between VAP‐1 expression and clinicopathological features was evaluated by Chi‐square test, and overall survival was analyzed by Kaplan–Meier method. Cox regression analysis was applied to analyze the independent influence of each parameter on overall survival. The expression level of VAP‐1 was significantly higher in diffuse astrocytoma than those of pilocytic astrocytoma (p < 0.0001). In the subgroup analysis, upregulated VAP‐1 expression was frequently found in older age patients (≥50 years). The VAP‐1 expression was found to be significantly correlated with the overall survival (p = 0.0002). There was a statistical correlation between VAP‐1^high tumors in diffuse astrocytoma and VAP‐1^low tumors in pilocytic astrocytoma (p < 0.0001). Multivariate Cox analysis indicated VAP‐1 was an independent predictive marker for poorer prognosis (p = 0.0036). Therefore, VAP‐1 could be a promising prognostic biomarker in astrocytoma.     

IL-1通过PKC/MAPK通路上调泡沫细胞ACAT-1的表达及活性

 摘要 目的 研究白细胞介素1(Interleukin 1, IL-1)是否通过蛋白激酶C/丝裂原激活蛋白激酶（PKC/MAPK）信号通路上调泡沫细胞中脂酰CoA胆固醇酯酰转移酶-1（ACAT-1）的表达及活性。方法 复苏并培养人单核细胞株THP-1细胞,与200nM乙酸肉豆蔻佛波酯（PMA）共孵育48h,再与氧化低密度脂蛋白（Ox-LDL）共孵育24h,油红O染色观察细胞质内脂质沉积;检测泡沫细胞、泡沫细胞加IL-1及泡沫细胞加IL-1/IL-1单克隆抗体三组细胞中PKC和MAPK的活性;在三组细胞中分别加入PKC和MAPK抑制剂,分别以Western blot及液相闪烁计数法检测ACAT-1的蛋白表达及酶活性。结果 与PMA共孵育48h后,THP-1细胞逐渐伸出突起,由圆形、悬浮式生长转变为多角形、梭形,呈阿米巴样贴壁生长;经Ox-LDL诱导24h后,油红O染色胞质内可见大量吞噬的脂质小滴。与泡沫细胞组相比,泡沫细胞加IL-1组PKC活性（P<0.05）、MAPK活性（P<0.05）、ACAT-1蛋白表达及活性增加（P<0.05）;PKC抑制剂和MAPK抑制剂能显著抑制IL-1上调ACAT-1表达（P<0.05）及活性（P<0.05）的作用。结论 IL-1上调泡沫细胞ACAT-1表达及活性的作用是通过PKC/MAPK信号通路途径实现的。     

Thrombospondin-1 inhibits Kaposi's sarcoma (KS) cell and HIV-1 Tat-induced angiogenesis and is poorly expressed in KS lesions

Kaposi's sarcoma (KS), a neoplasm often associated with iatrogenic and acquired immunosuppression, is characterized by prominent angiogenesis. Angiogenic factors released by both KS and host cells, as well as HHV-8 and HIV viral products, have been implicated in the pathogenesis of this lesion. Angiogenesis is the result of imbalance among angiogenesis promoters and inhibitors, which disrupts homeostasis. The aim of this study was to investigate the expression and mechanism of KS control of thrombospondin-1 (TSP), a physiological inhibitor of angiogenesis. Immunohistochemical analysis of four KS lesions showed only spotty reactivity for TSP in the stroma and in less than 10 per cent of lesional blood vessels. In addition, the typical KS spindle cells were not stained. In agreement with these findings, decreased levels of TSP were measured with an ELISA assay in the supernatants of cultured KS cells, compared with endothelial cells. In vitro, TSP inhibited the endothelial cell proliferation and motility induced by KS cell supernatants. TSP also prevented endothelial cell motility induced by Tat, a product of HIV-1 endowed with angiogenic potential and implicated in the pathogenesis of AIDS-KS. In vivo, TSP inhibited the angiogenic activity exerted by Tat in the Matrigel sponge model. These results suggest that TSP down-regulation might be permissive for the development of KS-associated angiogenesis. Copyright © 1999 John Wiley & Sons, Ltd.     

Vascular and myofibrillar lesions in acute myoglobinuria associated with carnitine-palmityl-transferase deficiency

Summary A case of severe exercise-induced myoglobinuria in a 14-year-old boy suffering from a carnitine-palmityl-transferase (CPT) defect is reported. Biopsies of the forearm muscle were examined using light and electron microscopy in the acute and recovery phases of the illness. The first biopsy showed the presence of scattered foci of necrosis where necrotic fibres with occasional disruptions of the basal lamina were seen around injured capillaries. Various degrees of damage and different stages of evolution were found in these foci, which also contained regenerating muscle fibres. In the second biopsy, performed 2 weeks later, most of the fibres displayed a normal structure. Necrosis was no longer present. However, in some areas perivascular fibrosis was prominent, the fibres were small and irregularly shaped, and their nuclei often centrally located. These data strongly suggest that circulatory disorders and ischaemia, brought about by premature acute metabolic imbalance, could be involved in the development of exerciseinduced myolysis observed in CPT deficiency. The risk of fibrous cardiomyopathy in these patients is pointed out.     

组蛋白脱乙酰化酶GST-HDAC1融合蛋白载体的构建及其在大肠杆菌中的表达

目的 构建GST-HDAC1融合蛋白表达载体,并在大肠埃希菌(E.coli)中诱导表达.方法以质粒pcDNA3.1- HDAC1为模板进行PCR,通过EcoR1单酶切位点将HDAC1插入p GEX-5X-1中,构建原核表达质粒pGEX-5X-1-HDAC1,并转化点coli DH5 α,通过利用载体上的BamH1酶切位...