Detection of Human Polyomavirus Dna In Papanicolaou Stained Smears of Urinary Sediment By In Situ Hybridization

Papanicolaou stained smears of urinary sediment containing inclusion bearing urothelial cells suggestive of human polyomavirus infection were destained and reprocessed for in situ hybridization using a biotinylated probe for human polyomavirus DNA. Seven slides were processed in this way. A hybridization signal for viral DNA was noted in each case, even in smears that had previously been stored for 11 years. This simple and rapid non-radioactive detection system is a valuable supplement to routine urinary cytology for the definitive diagnosis of this virus infection.     

Screening For High- and Low-Risk Human Papillomavirus Types In Single Routine Cervical Smears By Non-Isotopic In Situ Hybridization

Routine cervical smears (n = 262) from a Sexually Transmitted Diseases clinic were screened by non-isotopic in situ hybridization (NISH) stratifying human papillomavirus (HPV) infections into HPV6/11 (low risk) and HPV16/18/33 (high risk) categories. Of 188 patients with cytologically normal smears, HPV sequences were demonstrated in 41%. Of the 128 cases analysed by dual NISH, 16% contained low risk, 20% high risk and 5% both groups. In patients with cytological evidence of wart virus infection (WVI) only, 54% (n = 50) contained high-risk and 22% low-risk HPV types. The comparable incidences in CIN1/2 plus WVI (n = 24) were not significantly different: 54% and 17%, respectively. Cytological criteria underestimate the prevalence of HPV infection in patients with cytologically normal smears. This represents either 'occult' or 'latent' infection. The identical prevalence of HPVB16/18/33 in WVI only, and CIN1/2 plus WVI, suggests that the cytopathic effect induced by these HPVs may represent one end of a spectrum of morphological change which progresses to cervical intraepithelial neoplasia (CIN).     

Human Papillomavirus Detection In Cervical Cells By In Situ Hybridization With Biotinylated Probes

We have investigated the applicability of human papillomavirus (HPV) DNA detection by in situ hybridization with biotinylated probes in epithelial cells obtained from the cervix using a cotton tip swab. We describe a simple procedure for obtaining homogeneous cell samples and good preservation of cellular structure. This is achieved by pretreatment of cells with L-cysteine before hybridization. Separate denaturation of cellular DNA and probe DNA is also necessary for satisfactory results. Both benign HPV DNA 6/11 and potentially oncogenic HPV DNA 16/18 could be identified in our series. In situ hybridization on cervical scrapes is a rapid, simple and very specific method for detecting patients infected with oncogenic HPV types.     

Anorectal Human Papillomavirus: Current Concepts

Increased anorectal human papillomavirus (HPV) infection is related to the recent trends in sexual behavior in both homosexual and heterosexual groups and prevalence of infection with human immunodeficiency virus (HIV). Clinical presentation and natural history depend on the serotype involved. HPV 6 and 11 are found in the benign wart. Local control can be achieved with a wide selection of surgical and topical techniques. HPV 16, 18, and 31 are found in dysplastic lesions and have the potential to progress to invasive anal squamous cell carcinoma. Recognition and early management of dysplastic lesions is crucial to prevent the morbidity and mortality associated with anal cancer. While low-grade lesions can be closely observed, high-grade lesions should be eradicated. Different strategies can be used to eradicate the disease while preserving anorectal function. Studies on the efficacy of vaccination on anorectal HPV showed promising results in select population groups and led to the recent expansion of current vaccination recommendations.     

应用树状DNA杂交(DDH)对生殖道尖锐湿疣中HPV DNA的分型检测

从手术切除的50例生殖道尖锐湿疣新鲜标本中,以及15例正常人血清中,提取基因组DNA,同时用树状DNA杂交(dendrimer DNA hybridizalion,DDH)技术和PCR进行HPV DNA的分型检测.结果50例尖锐湿疣中,以DDH方法检测,感染HPV6型者20例,感染11型者24例,6/11型混合感染者3例,阴性3例,总检测率达94%;以PCR方法检测,HPV6型感染者21例,11型感染者24例,6/11型混合感染者3例,阴性2例,总检测率为96%.15例正常人血清中,以DDH方法检测,HPV感染的假阳性率为0%;以PCR检测,假阳性率为6.67%.还以HPV阳性标本对DDH方法做了敏感度的测定,结果阳性病例DNA检测最低浓度为97.28pg/ml.研究表明,DDH技术具有较高敏感性和高特异性,且成本较低,操作安全简便,可适用于基层中小医院较大样本量筛查.     

人乳头瘤病毒16型假病毒中和实验的建立和初步应用

探讨了应用多质粒磷酸钙共转染方法在293FT细胞中生产HPV16(human papillomavirus type 16)假病毒。蛋白印迹检测显示在转染后细胞的裂解上清中具有很好的L1蛋白活性,通过透射电镜可观察到形态与天然病毒粒子相似的假病毒颗粒。对293FT细胞的感染实验显示,该假病毒可有效将EGFP报告质粒导入靶细胞中进行表达,经测定其滴度约为2×107TU/mL。通过与4株HPV16对照单抗的中和实验证明该假病毒可有效应用于中和实验。应用该方法从18株抗HPV16L1的单克隆抗体中鉴定获得了2株中和单抗3D10、PD1。所建立的HPV16假病毒生产和中和实验方法具有快速高效、低成本和易于检测的优点,适于进行较大规模应用,为快速准确鉴定HPV16中和单抗和候选疫苗的免疫保护效果提供了有效手段。     

The Diagnosis of Pneumocystis Carinii Pneumonia By Cytologic Evaluation of Papanicolaou and Leishman-Stained Bronchoalveolar Specimens In Patients With the Acquired Immunodeficiency Syndrome

The presence of foamy alveolar casts or flocculent material in Papanicolaou and Leishman-stained smears of bronchoalveolar lavage (BAL) fluid is said to be indicative of infection with Pneumocystis carinii. We have investigated the sensitivity and specificity of this method of diagnosing pneumocystis pneumonia in patients with the acquired immunodeficiency syndrome (AIDS). Patients (n= 114) with diffuse lung infiltrates were submitted to fibreoptic broncoscopy and BAL. Seventy of them were patients with AIDS. the other 44 individuals were not infected by the human immunodeficiency virus (HIV). Pneumocystis carinii organisms were identified on Grocott's methenamine silver (GMS)-stained BAL smears in 30 patients with AIDS. Flocculent material was present in the Papanicolaou and Leishman-stained smears from all of these cases. Conversely, P. carinii were not seen on GMS-stained smears in the remaining 84 individuals with or without AIDS. No flocculent material was observed in Papanicolaou or Leishman-stained smears in these 84 patients. We concluded that the presence of flocculent material in Papanicolaou or Leishman-stained smears of BAL fluid is indicative of P. carinii pneumonia in patients with AIDS. La présence de cylindres alvéolaires spumeux ou de matériel floculé dans les étalements de liquide de lavage bronchoalvéolaire (LBA) colorés selon Papanicolaou ou Leishman est considérée comme symptomatique d'une infection par Pneumocystis carinii. Nous avons étudié la sensibilité et la spécificité de cette méthode de diagnostic de l'infection par Pneumocystis carinii chez des patients atteints de syndrome de déficience immunitaire acquise (SIDA). Cent quatorze malades avec des infiltrats pulmonaires diffus ont subi une fibroscopie bronchique et un lavage broncho-alvéolaire. Soixante dix d'entre eux edtaient atteints de SIDA, 44 n'étaient pas infectés par le Virus de l'Immunodéficience Humaine (VIH). Le Pneumocystis carinii a été identifiié par la coloration de Grocott chez 30 patients atteints de SIDA. Chez ces patients, la présence d'un matériel floculé est constante sur les étalements colorés au Papanicolaou et au Leishman. A l'inverse, Pneumocystis carinii n'a pas été retrouvé chez les 84 autres malades, atteints ou non du SIDA et les étalements de LBA ne contenaient pas de matériel floculé. En conclusion, la présence de matériel floculé dans les étalements de LBA colorés selon Papanicolaou ou Leishmanest associée à une pneumpathie àPneumocystis carinii chez les patients atteints de SIDA. Sensitivität und Spezifität des Nachweises schaumiger oder flockiger Alveolarausgüsse bei Pneumocystis carinii wurden in 114 Fällen diffuser Lungeninfiltrate untersucht. 70 Patienten waren an AIDS erkrankt, 44 weitere waren HIV-negative. In 30 der AIDS-Fälle wurde P. carinii mit der Grocott'schen Färbung nachgewiesen. Die typischen Eiweißniederschläge waren in all diesen Fällen nachweisbar. Umgekehrt ergab die Grocottfärbung in 84 Fällen mit oder ohne AIDS ein negatives Ergebnis. In all diesen Fällen war kein Eiweißniederschlag nachweisbar. Daraus ergibt sich, daß die Eiweißniederschläge in Präparaten, die nach Papanicolaou oder Leishman gefärbt wurden, kennziechned sind für die P. carinii Pneumonie.     

Identification of Genetic Variants of Human Papillomavirus in a Group of Mexican HIV/AIDS Patients and Their Possible Association with Cervical Cancer

Infections caused by the human immunodeficiency virus (HIV) and human papillomavirus (HPV) cause thousands of deaths worldwide each year. So far, there has been no consensus on whether there is a direct relationship between the incidence of neoplasms and the immunosuppression caused by HIV that could help understand if coinfection increases the likelihood of cervical cancer. The objective of the study was to identify the presence of genetic variants of HPV in a group of HIV-positive women and their possible association with cervical cancer. Cervical samples were taken from HIV-positive patients for cytological analysis to identify the HPV genotype by polymerase chain reaction (PCR) and sequencing. The most prevalent L1 capsid protein mutations in the HPV genotype were analyzed in silico. Various types of HPV were identified, both high-risk (HR) and low-risk (LR). The most prevalent genotype was HPV51. Analysis of the L1 gene sequences of HPV51 isolates showed nucleotide variations. Of the samples analyzed in Puebla, Mexico, HPV51 had the highest incidence (17.5%, 7/40). Different mutations, which could be used as population markers, were detected in this area, and they have not been reported in the L1 databases for HPV51 in Mexico. Genotypes 6, 14, 86, 87, 89, and 91, not detected or reported in samples from patients with HPV in Mexico, were also identified. Data from the population analyzed suggest no direct relationship between HIV immunosuppression and cervical cancer, regardless of the high- or low-risk HPV genotype. Furthermore, it is possible to develop regional population markers for the detection of HPV based on the mutations that occur in the sequence of nucleotides analyzed.     

ELISPOT检测人乳头瘤病毒特异性的记忆T细胞

应用ELISPOT方法检测人乳头瘤病毒感染后自发清除者外周血中抗原特异性的记忆T细胞。收集人乳头瘤病毒（HPV）感染后自发清除者外周血（病毒清除后74个月）,分离外周血单个核细胞（peripheralblood mononuclear cells,PBMC）。体外应用已鉴定的表位肽刺激PBMC,10 d后计数细胞,去除表位肽,继续培养。第11天,ELISPOT方法检测PBMC中HPV抗原特异性的记忆T细胞。PBMC经表位肽刺激10 d后,细胞数量有明显增加,由最初的4.1×105增加为4.2×106。第11天,细胞数量增加为4.65×106,为抗原刺激前细胞数的11.3倍。ELISPOT结果显示,PBMC中的记忆T细胞活化后,能够识别抗原递呈细胞递呈的抗原肽,并分泌IFN-γ。此HPV自发清除者外周血中抗原特异性的记忆T细胞的频数为0.007%。人乳头瘤病毒（HPV）感染后自发清除者外周血存在抗原特异性记忆T细胞,抗原肽可激活记忆T细胞,使之数量增加,分泌IFN-γ。ELISPOT可用于检测外周血中HPV特异性的记忆T细胞。     

人乳头瘤病毒在宫颈鳞癌宫旁组织中的表达及意义

为研究高危型人乳头瘤病毒(high-risk human papillomavirus,hr-HPV)在宫颈鳞癌宫旁组织中的表达及意义,采用免疫组织化学技术对51例宫颈鳞癌患者术后宫旁组织不同位点的hr-HPV表达情况进行检测,应用重复测量设计的方差分析及单因素分析方法进行统计。hr-HPV在宫旁组织中的表达随着距宫颈距离的增加呈明显的梯度下降趋势(P0.05),在不同临床分期、宫颈浸润深度、淋巴结转移组内表达存在差异(P0.05),当临床分期ⅠB1期、宫颈浸润深度≤1/2以及无淋巴结转移时,hr-HPV在距离宫颈2 cm处主韧带、骶韧带及距离宫颈3 cm处阴道中表达开始与正常对照组无差异(P0.05)。结果表明,部分早期宫颈鳞癌患者可选择Ⅱ型改良式根治性子宫切除术,但阴道切除仍推荐3 cm。     

Borderline Nuclear Abnormality In Cervical Smears: A Cytological Review of 200 Cases With Histological Correlation

The cervical cytology and histology specimens from 200 patients referred to colposcopy with borderline nuclear abnormality were reviewed. Human papillomavirus (HPV)-associated changes were identified in 103 of 200 (53%) referral smears and in 139 of 150 (91%) biopsy specimens. Cervical intraepithelial neoplasia (CIN) was less frequently diagnosed on review compared with the original histopathology reports (30.7%vs 45.4%); the discrepancy was largely attributable to a lower incidence of CINI. There was agreement in the grading of borderline nuclear abnormality in 161 of 200 referral smears following review. Twenty-three smears were upgraded to mild dyskaryosis, whereas 16 were reclassified as negative.     

人乳头瘤病毒6型L1基因的克隆及蛋白表达

目的：在大肠杆菌中表达经密码子优化的人乳头瘤病毒6型（HPV6）L1的融合蛋白。方法：PCR方法扩增HPV6 L1,基因,测序及序列比对后,对基因进行密码子优化并合成优化后的基因HPV6mLI,将其克隆入原核表达载体pGEX4T-1,IPTG诱导融合蛋白在大肠杆菌BL21（DE3）中表达,SDS-PAGE鉴定表达产物。结果：酶切和测序结果证实HPV6 mL1基因的原核表达载体构建正确;以1mmol／L IPTG于37℃诱导4h,蛋白以包涵体形式表达;表达产物的相对分子质量与预期值一致,为80000。结论：获得大肠杆菌表达的HPV6L1蛋白,为其结构功能研究和疫苗研发提供了基础。     

人乳头瘤病毒（HPV）58型中和单克隆抗体的制备及初步应用

人乳头瘤病毒（human papillomavirus,HPV）58型是宫颈癌的主要诱因之一. HPV58在亚洲地区宫颈癌组织中的检出率仅次于HPV16/18. HPV58中和单克隆抗体可用于 HPV病毒样颗粒（virus-like particle,VLP）疫苗的研究,并为病毒感染入侵机制的 研究提供实验材料. 本研究采用HPV58 L1 VLP免疫BALB/c小鼠,取其脾细胞进行杂交瘤 细胞的制备,通过VLP-ELISA和假病毒中和实验筛选杂交瘤细胞株;经rProtein A纯化 阳性杂交瘤细胞培养上清获得单抗;采用ELISA测定型别特异性中和单抗的亲和力,采用相加实验及变性VLP-ELISA分析单抗识别表位的性质;选取高亲和力单抗建立定量分 析HPV58 L1 VLP的ELISA方法. 获得了2株HPV58特异性中和单抗XM-22和XM-23,亲和常数分别为2.7×107 mol-1·L和1.9×106 mol-1·L,二者识别表位可能不同. 同时获得2株具有交叉中和活性的单抗XM-21和XM-24,除可较高水平中和HPV58外,还可分别交叉 中和亲缘关系较远的HPV18和HPV6. 以XM-22建立的ELISA方法定量分析HPV58 L1 VLP的检测范围为0.05 μg/mL~0.40 μg/mL. 本研究建立的ELISA方法可用于HPV58 L1 VLP疫苗生产的质量控制研究,获得的4株具有不同特点的中和单抗可用于HPV58感染入侵机制 的研究.     

Tetramer技术检测人乳头瘤病毒抗原特异性T细胞条件的优化

人乳头瘤病毒(human papillomavirus,HPV)感染是宫颈癌发生的首要因素。针对病毒早期蛋白E6的特异性细胞毒性T淋巴细胞(cytotoxic Tlymphocyte,CTL)在清除HPV感染细胞和病毒转化形成的肿瘤细胞过程中发挥重要作用,因此检测体内抗原特异性CTL的频数和功能有助于了解病毒感染者或宫颈癌患者体内的特异性细胞免疫反应。利用加载HPV16 E6表位抗原肽(E6 133-142;HNIRGRWTGR)的HLA-A6801四聚体(Tetramer),即HPV16 E6 133-142/HLA-A6801-PE四聚体,通过检测混有HPV特异性CTL的PBMC标本,优化Tetramer染色的实验条件,探讨染色的最佳温度及Tetramer浓度。结果显示,染色温度(4℃、室温及37℃)对Tetramer与CTL的结合无明显影响。Tetramer稀释度为1∶1 600时,HPV特异性CTL荧光强度保持高水平,且非特异性染色少,为最佳染色浓度。研究结果为进一步检测HPV感染者或宫颈癌患者体内抗原特异性的CTL打下基础。     

新疆维族妇女感染的HPV 型别分布的研究

目的:探讨人乳头瘤状病毒(Human papillomavirus,HPV)在新疆南部维族妇女人群中的型别分布情况。方法:以年龄在30-59岁的新疆伽师县夏普吐勒乡维吾尔族妇女人群为基础进行筛查,签署知情同意书后,采集受试者宫颈脱落细胞,利用PCR和基因芯片技术检测HPV DNA并分型。结果:共2473名妇女入选。HPV总的感染率为9.1%,高危型中HPV-16的感染率最高为6.9%,其他高危型的感染率从高到低依次为:HPV-59、HPV-56、HPV-18、HPV-33、HPV-58、HPV-51、HPV-31、HPV-45、HPV-52、HPV-68、HPV-35、HPV-39。低危型中HPV11感染率最高,其他低危型的感染率从高到低依次为HPV-42、HPV-43、HPV-6、HPV-53、HPV-66、HPV-73。HPV-44、-83、-MM4没检测到。多重感染率为34.2%。结论:新疆维吾尔族妇女人群中以HPV16感染为主,其次为HPV59、56、18、33等。HPV59可能是新疆维吾尔族妇女较易感染的类型。体现了新疆维吾尔族妇女感染HPV的特殊性。     

Normal Human Fibroblasts Immortalized by Introduction of Human Papillomavirus Type 16 (HPV-16) E6-E7 Genes

This report demonstrates that normal human fibroblasts can be immortalized by the introduction of HPV-16 E6-E7 genes. We designed zinc-inducible expression plasmids with HPV-16 E6, E7 or both. Each plasmid was introduced into normal human fibroblasts (TIG-3 cells) using lipofection methods. Only trans-fectants with the HPV-16 E6-E7 zinc-inducible expression plasmid, which were cultured in medium supplemented with 100 μM ZnSO₄, overcame crisis and could be cultured over 200 population doubling levels (PDLs). These cell lines showed the reactivation of telomerase after crisis, and morphological alterations were also observed.     

空泡细胞在诊断宫颈人乳头瘤病毒感染中的意义

本文对空泡细胞(Koilocyte)的形态进行了光镜和电镜观察,并应用分子杂交技术和PAP染色的方法对宫颈疾患中人乳头瘤病毒(HPV)DNA和抗原进行了检测,分析HPV DNA及抗原检测结果与空泡细胞出现的关系发现,空泡细胞阳性的病例中HPV DNA及抗原的检出率较低,并且部分空泡细胞阳性的病例中也可测出疱疹病毒Ⅱ型(HSV_2)抗原,结果提示:空泡细胞并不是HPV感染所特有的,空泡细胞作为诊断宫颈HPV感染的特征性指标值得怀疑。     

用昆虫-杆状病毒系统表达的HPV6型L1蛋白检测尖锐湿疣患者血清中抗体

用杆状病毒表达系统表达人乳头瘤病毒6型（human papillomavirus type 6,HPV6）主要衣壳蛋白（major capsid protein,L1）作为抗原,对尖锐湿疣（condyloma acuminata,CA）患者抗体进行检测。采用昆虫杆状病毒系统表达HPV6L1蛋白,通过镍柱亲和层析法获得纯化抗原;以酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）检测30例CA、20例献血员和10例儿童血清中的HPV6 LI IgG抗体。感染重组杆状病毒的昆虫细胞经SDS-PAGE和Western blot检测,在大约55kD处有明显的外源蛋白表达条带。ELISA结果显示,CA组的血清阳性率为66．7％（20／30）,献血员组的阳性率为15％（3／20）,两组之间有显著性差异（P〈0．05）。22例HPV6型感染的CA患者有15例血清阳性（68．2％）,6例HPV11型CA患者4例阳性（66．7％）,1例混合感染者为阳性,1例HPV16型患者为阴性。女性CA患者的血清抗体阳性率高于男性（P=0．0052）。本研究建立的ELISA体系具有敏感性和针对低危型HPV感染的特异性。这不仅对于HPV血清流行病学研究是有价值的,而且对于临床诊断HPV感染可能具有一定的应用价值。     

目视化纳米探针检测宫颈癌组织HPV16E6基因的研究

目的：建立目视化纳米探针检测宫颈癌组织HPV16E6基因的方法,为临床降低HPV感染者宫颈癌的发病率提供技术支持。方法：采用固定于硅烷化片基上的捕获探针,特异性结合单链人乳头瘤病毒16型新疆株E6基因(HPV16E6)扩增片断,再与互补的纳米金颗粒标记的探针结合,通过银染加强显色。结果：初步建立了金标银染法快速检测HPV16E6 DNA的技术。结论：金标银染法灵敏,可以快速特异地检测出各类宫颈组织中人乳头瘤病毒的感染,为进一步应用于临床检测降低新疆维吾尔妇女宫颈癌的发病率奠定了基础。     

宫颈癌组织人乳头瘤病毒的荧光偏振基因分型

采用荧光偏振人乳头瘤病毒(human papillomavirus,HPV)分型新方法探讨了8种常见型别HPV在陕西宫颈癌患者中的流行情况。首先,用HPV GP5 ／GP6 通用引物PCR扩增65例早期宫颈癌(Ⅱa期内)和72例慢性宫颈炎病变组织DNA粗提物,继之将模板指导的末端延伸反应与荧光偏振检测技术结合(TDI-FP),用GP5 ／GP6 扩增区内的HPV6、11、16、18、31、33、35和58型特异性探针与PCR产物杂交后,荧光素标记的特异碱基(TAMRA-ddTTP或R110-ddGTP)在GP5 ／GP6 产物中相应的模板指导下,掺入延伸至相应探针末端,致使对应的TAMRA或R110 FP值升高,从而对扩增的HPV阳性产物进行HPV分型。65例宫颈癌患者中检出HPV57例,阳性率87．69％,72例慢性宫颈炎患者中检出HPV28例,阳性率38．89％,两组间HPV阳性率有显著性差异。宫颈癌与慢性宫颈炎患者中4种最常见的HPV型别分别是HPV 16(45．6％)、HPV 18(22．8％)、HPV 58(17．5％)、HPV 31(7．02％)和HPV 16(35．7％)、HPV 11(32．1％)、HPV 6(21．4％)、HPV 18(10．7％)。慢性宫颈炎患者中检出的HPV型别57．14％属高危型。HPV 16在两组中均最为多见。中国陕西宫颈疾病患者中HPV感染有其特点,世界范围内少见的HPV 58在陕西宫颈癌与慢性宫颈炎患者中均较为多见,在进行HPV新诊断方法及疫苗研制时应考虑到这种特点。