山东省女性HPV6/11和HPV16/18感染情况分析

目的:对山东省女性人乳头状瘤病毒(human papillomavirus,HPV)6/11和HPV16/18感染的检测结果进行回顾分析,以了解山东省各年龄段、各地区女性HPV的感染状况。方法:采用实时荧光定量PCR技术,对2012年1月至2016年6月间济南金域医学检验中心收集的3995例和7508例样本,分别进行HPV6/11或HPV16/18检测,并对检测结果进行回顾分析。结果:HPV6/11检测的阳性率为27.2%(1085/3995),明显高于HPV16/18检测的5.1%(384/7508)。两种HPV检测的阳性率呈现不对称双峰分布,均以20岁组阳性率最高,分别为35.5%和21.7%。随后HPV6/11和HPV16/18的阳性率随年龄增长而逐渐下降,后HPV6/11的阳性率又逐渐升高至51~60岁组的34.3%和60岁组的43.8%;HPV16/18检测的阳性率在60岁组也略有上升,为3.4%。各地区中鲁中(31.6%)和鲁西(30.6%)HPV6/11的阳性率要高于鲁东(22.1%)和鲁北(21.7%);HPV16/18的检出率以鲁中(16.3%)最高,而鲁东最低(4.1%)。结论:本研究结果反映了山东省女性HPV6/11、HPV16/18感染的最新分布状况,对掌握山东省女性HPV感染的流行病学特征,为子宫颈癌筛查和HPV疫苗的接种提供科学依据。     

Focal epithelial hyperplasia by human papillomavirus (HPV)‐32 misdiagnosed as HPV‐16 and treated with combination of retinoids,imiquimod and quadrivalent HPV vaccine

Focal epithelial hyperplasia (FEH) or Heck's disease is a rare, benign and asymptomatic mucosal proliferation associated with human papillomavirus (HPV) infection, mainly with genotypes 13 and 32. We report a florid case of FEH in an 11‐year‐old Haitian girl with systemic lupus erythematosus receiving immunosuppressive therapy. Cryotherapy was previously performed on numerous occasions with no results. We decided to prescribe a non‐invasive and more comfortable treatment. A combination of topical retinoid and imiquimod cream was well tolerated and led to an important improvement. The evidence of infection by HPV‐16 detected by polymerase chain reaction (PCR) technique, prompted us to prescribe the quadrivalent HPV vaccine (types 6, 11,16 and 18). Subsequent PCR sequencing with generic primers GP5–GP6 and further BLAST comparative analysis confirmed that genomic viral sequence in our case truly corresponded with HPV‐32. This molecular misdiagnosis can be explained by the similarity between genomic sequences of both HPV‐16 and ‐32 genotypes. At the 1‐year follow up, we observed total clinical improvement and no recurrences of the disease. Complete healing in this case may correspond to a potential action of topical retinoid, imiquimod and the cross‐protection mechanism of the quadrivalent HPV vaccine.     

An overview of human papillomavirus infection for the dermatologist: disease,diagnosis, management,and prevention

Genital human papillomavirus (HPV) is a common, usually transient, dermatologic infection transmitted by genital contact that can cause a variety of anogenital diseases, including warts (condyloma), dysplasia (cervical, vaginal, vulvar, anal), and squamous cell carcinoma. A number of treatment modalities are available to treat anogenital warts, both patient‐ and provider‐applied. Treatment is efficacious, but lesions can recur. Bivalent and quadrivalent vaccines are approved to prevent HPV infection. Both are indicated to prevent cervical cancer, while the quadrivalent vaccine is also approved to prevent vaginal/vulvar cancers as well as genital warts in males and females. Providers should clearly explain the natural history and potential sequelae of HPV disease, counsel patients on prevention strategies, and recommend vaccination as an effective method of prevention to their patients.     

人乳头瘤病毒6b亚型L1重组质粒的构建及免疫原性分析

目的：构建人乳头瘤病毒(HPV)6b亚型L1的重组质粒．并了解其对小鼠的免疫原性。方法：将HPV6b的L1基因插入真核表达载体pcDNA3.1中，构建L1的重组质粒；答定后转染COS-7细胞，并用免疫印迹法进行表达分析；18只BALB/巾雌性小鼠用作重组质粒的免疫原性检测。结果：重组质粒可被内切酶EcoRI和BamHI切开，测序发现插入片段与L1基因一致；转染24h后十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)及免疫印迹出现了阳性条带；经PcDNA3.1-HPV6b L1免疫的小鼠可测到L1抗体(滴度为1：100)，IL-4水平明显升高。结论：HPV6b L1的重组质粒构建成功，能在真核细胞内表达，并能有效激发小鼠产生体液免疫和细胞免疫。     

男性尖锐湿疣患者疣体和手指人乳头瘤病毒6/11型DNA的检测

目的：确定男性尖锐湿疣患者疣体和手指人乳头瘤病毒6/11型的感染。方法：采用PCR荧光法对46例男性尖锐湿疣患者疣体和手指进行HPV6/11型核酸定性、定量检测,同时以20名无尖锐湿疣的男性手指作对照。结果：尖锐湿疣患者39例疣体(39/46＝84.78％)阳性,手指25例(25/46＝54.35％)阳性。无尖锐湿疣患者的手指仅2人阳性,差异有显著性意义( P<0.05)。 HPV6/11型疣体病毒载量为6.60×107±3.77×108,病毒载量与病程长短无相关性;尖锐湿疣患者的手指病毒载量为5.72×103±1.75×104,低于疣体的病毒载量(P<0.05)。结论：男性尖锐湿疣患者的手指携带HPV6/11型,其在尖锐湿疣的传播意义有待研究。     

In situ DNA hybridization analysis of human papillomavirus (HPV) sequences in benign oral mucosal lesions

Summary A series of 144 surgically treated benign oral mucosal lesions were analysed using an in situ DNA hybridization technique with ³⁵S-labeled human papillomavirus (HPV) DNA probes to demonstrate the DNA of HPV types 6, 11, 13, and 16, in routinely processed, paraffin-embedded biopsy specimens. These lesions and an additional 62 benign oral mucosal biopsy specimens (total, 206 specimens) were also assessed by the indirect immunoperoxidase (IP-PAP) technique to detect the expression of HPV structural proteins (viral antigens). A total of 54/206 (26.2%) lesions were observed to express HPV antigens, being found in 45/92 (48.9%) of the squamous cell papillomas/condylomas, in 1/54 fibrous hyperplasias, in 1/8 true fibromas, and in 7/8 (87.5%) of the focal epithelial hyperplasia (FEH) lesions. Of the HPV DNA-positive lesions, 15/45 (33.3%) expressed HPV antigens, the expression not being related to any particular HPV type. HPV DNA sequences were found in 45/144 (31.3%) of the lesions. HPV DNA was present with the highest frequency in FEH (83.3%), papillary hyperplasia (28.6%), fibrous hyperplasia (24.4%), and true fibromas (14.3%). The most frequent HPV type was HPV 11, representing 37.8% of the DNA-positive lesions. HPV 13 DNA, previously regarded as specific to FEH, was disclosed as a single HPV type in seven cases, and as a double infection by HPV 11 and 13 in an additional three cases, including all five morphologically distinct entities. Noteworthy is the discovery, of the high-risk HPV type 16 DNA in 17.8% of the DNA-positive lesions, four papilloma/condyloma lesions, three fibrous hyperplasias, and one FEH. The results confirm the previously reported evidence regarding HPV involvement in oral mucosal lesions. The implications of these findings are discussed in terms of the epidemiology, HPV etiology, and proper classification of the oral mucosal lesions, with special emphasis on the discovery of the high-risk HPV type 16 in the benign lesions as well as in oral cancer. The use of the in situ DNA hybridization as a powerful tool in detecting the specific HPV DNA sequences in routinely processed oral biopsy specimens is strongly recommended.     

Distinctive distribution of human papillomavirus type 16 and type 20 DNA in the tonsillar and the skin carcinomas of a patient with epidermodysplasia verruciformis

BACKGROUND: Epidermodysplasia verruciformis (EV) is a rare skin disease characterized by disseminated pityriasis versicolor-like or flat wart-like lesions and by the development of skin carcinomas. It is well established that specific cutaneous human papillomaviruses (EV-HPVs) are associated with both benign and malignant skin lesions in EV patients. However, little is known of the relationship between HPV and the mucosal lesions of EV patients. OBJECTIVES: To detect and identify HPV types associated with skin and mucosal lesions of an EV patient. PATIENT/METHODS: We investigated the skin carcinoma and the coexisting tonsillar carcinoma of a 41-year-old man with EV. Histopathologically, both lesions were squamous cell carcinomas. We analysed these two lesions by immunohistochemistry, in situ hybridization, and by molecular virology. RESULTS: Neither skin nor tonsillar lesions exhibited positivity for HPV capsid antigen by immunohistochemistry. By Southern blot hybridization, however, the skin carcinoma harboured 'EV-specific' HPV20 DNA, while the tonsillar carcinoma harboured 'genital' HPV16 DNA. In addition, in situ hybridization localized the respective viral DNA in the corresponding lesion. CONCLUSIONS: The results indicate that EV-HPV could be responsible for the development of the skin carcinoma, but not the mucosal carcinoma in this patient.     

Deregulation of E‐cadherin by human papillomavirus is not confined to high‐risk,cancer‐causing types

Background E‐cadherin is a tumour suppressor protein, which is normally expressed on keratinocytes and antigen‐presenting Langerhans cells (LCs) in the epidermis. We have previously shown that E‐cadherin is lost from tissues infected with the high‐risk cancer‐causing human papillomavirus (HPV) type 16. Objectives To test if E‐cadherin dysregulation is associated with the cancer risk of the infecting HPV and to establish if it is conserved among HPVs in the α, β, γ and μ genera. Methods Forty‐seven lesions infected with low‐ or high‐risk HPV types spanning four HPV genera were stained for E‐cadherin, P‐cadherin and CD1a to detect LCs. Results Surface E‐cadherin was reduced in tissues infected with members of the α4, α7 and α9 species and the γ and μ genera but was equivalent to normal epidermis in the β only‐infected lesions tested and patchy in α10‐infected tissues. There was a direct relationship between atypical E‐cadherin expression and a significant reduction in LCs. Expression of P‐cadherin, a protein that is increased in the E‐cadherin constitutive knockout mouse, was increased in lesions with reduced E‐cadherin. Conclusions These data show that E‐cadherin dysregulation by HPV is widely conserved across the majority of HPV genera. E‐cadherin expression was reduced or lost in epidermis irrespective of the cancer risk of the infecting HPV type or the ability of the virus to degrade retinoblastoma protein or p53. A correlation between dysregulated E‐cadherin and reduced numbers of LCs supports viral regulation of surface E‐cadherin contributing to viral evasion of the host immune system.     

p16ink4a expression decreases during imiquimod treatment of anal intraepithelial neoplasia in human immunodeficiency virus-infected men and correlates with the decline of lesional high-risk human papillomavirus DNA load

BACKGROUND: Human papillomavirus (HPV)-associated anogenital cancers and their precursor lesions occur in excess in human immunodeficiency virus (HIV)-infected patients despite the initiation of highly active antiretroviral therapy. In this context, a drastically increased relative risk for anal intraepithelial neoplasia (AIN) exists in HIV-infected men having sex with men (MSM). In a pilot study, imiquimod, a topical immune response modifier, has been reported to be beneficial in the treatment of AIN. OBJECTIVES: To investigate the role of several biomarkers as potential adjuncts in the course of imiquimod treatment for AIN, and to determine whether these markers correlate with the course of high-risk HPV DNA load during imiquimod therapy. METHODS: Immunohistochemical staining was performed for p16(ink4a), minichromosome maintenance protein (MCM), Ki67, proliferating cell nuclear antigen (PCNA) and p21(waf1) expression before and after 16 weeks of imiquimod treatment for AIN. High-risk HPV DNA load determinations were performed by real-time polymerase chain reaction with type-specific primers and probes for HPV types 16, 18, 31 and 33. RESULTS: Histopathological and virological analyses were performed in 21 HIV-infected MSM with histologically confirmed AIN. Eighteen (86%) patients had a complete histological clearance of AIN after imiquimod therapy. As previously shown, lesional high-risk HPV DNA load significantly decreased during imiquimod therapy. Moreover, a significant decline of p16(ink4a), Ki67, MCM and PCNA expression after treatment was observed, while p21(waf1) expression changed nonsignificantly after imiquimod therapy. A significant correlation between the course of high-risk HPV DNA load and p16(ink4a) expression was observed during imiquimod treatment of AIN, whereas the decline of high-risk HPV DNA load did not significantly correlate with MCM, Ki67, PCNA or p21(waf1) expression. CONCLUSIONS: The significant decrease in p16(ink4a) expression in correlation with the drop of lesional high-risk HPV load suggests that p16(ink4a) may be a useful adjunct for the evaluation of treatment response in HPV-associated malignancies and their precursor lesions.     

紫外线和人乳头状瘤病毒16 E6协同诱导、促进裸鼠皮肤鳞状细胞癌的机制研究

【摘要】 目的 构建裸鼠皮肤鳞状细胞癌（CSCC）移植瘤模型,探讨紫外线（UV）损伤及人乳头瘤病毒（HPV）感染诱导、促进CSCC的协同作用机制。方法 将人CSCC细胞A431分成3组,即用 HPV16 E6腺病毒转染的HPV16 E6 过表达组,空白腺病毒转染的空白载体组（简称空载组）,未进行腺病毒转染的空白对照组。使用无血清DMEM培养基将空载组及HPV16 E6过表达组（LV-OE-HPV16 E6组）A431细胞制成单细胞悬液,分别接种于SKH-1裸鼠左侧臀部皮下作为空载组（n = 16）和LV-OE-HPV16 E6组（n = 16）。每3天观察并记录小鼠肿瘤生长情况,当瘤体达到150 mm3时,视为建模成功。建模成功后,每组取8只小鼠进行UV照射,分为4组,即空载组、空载 + UV组、LV-OE-HPV16 E6组、LV-OE-HPV16 E6 + UV组,UV照射剂量为1 440 mJ/（cm2·d）,每次12 min,持续4周后处死裸鼠,测量瘤重及体积,绘制肿瘤生长曲线,免疫组化、Western印迹和qRT-PCR检测验证Wnt1、β联蛋白mRNA及蛋白在裸鼠CSCC中的表达。数据若符合正态分布,多组间比较采用方差分析,组间两两比较采用LSD-t检验;数据若不符合正态分布,采用秩和检验对数据进行统计分析。结果 空载 + UV组瘤重为（2.90 ± 0.36） g,LV-OE-HPV16 E6组（3.19 ± 0.32） g,LV-OE-HPV16 E6 + UV组（4.41 ± 0.18） g,与空载组（2.20 ± 0.24） g比较,差异均有统计学意义（t值分别为4.39、6.77、20.11,均P＜0.001）;空载 + UV组瘤体积为（1 033.12 ± 400.15） mm3,LV-OE-HPV16 E6组（1 119.21 ± 447.57） mm3,LV-OE-HPV16 E6 + UV组（1 464.29 ± 409.98） mm3,与空载组（688.94 ± 319.31） mm3比较差异均有统计学意义（t值分别为1.90、2.21、4.22,均P＜0.001）。免疫组化显示,4组间Wnt1、β联蛋白表达水平差异无统计学意义（F值分别为0.76、0.71,均P > 0.05）;Western印迹显示,4组间Wnt1、β联蛋白水平差异有统计学意义（F值分别为16.74、49.90,均P＜0.05）,且LV-OE-HPV16 E6 + UV组Wnt1、β联蛋白水平高于空载组、空载 + UV组和LV-OE-HPV16 E6组（均P＜0.05）。mRNA水平分析显示,4组间组织中Wnt1、β联蛋白mRNA水平差异均有统计学意义（F值分别为7.77、8.38,均P＜0.05）,且LV-OE-HPV16 E6 + UV组Wnt1 mRNA水平高于空载组、空载 + UV组和LV-OE-HPV16 E6组（均P＜0.05）。结论 UV和HPV感染在诱导、促进CSCC中具有协同作用。     

Detection of human papillomavirus and response to topical 5% imiquimod in a case of stucco keratosis

Stucco keratosis is a skin disorder with papular warty lesions that usually appear on the lower limbs in elderly people. The aetiology, pathogenesis and treatment is still a matter of debate. We report a 75-year-old non-immunosuppressed man with extensive lesions all over his body, which had not responded to curettage or electrodesiccation. To determine the possible role of human papillomavirus (HPV) in stucco keratosis, we used nested polymerase chain reaction (PCR) to identify HPV DNA in the lesions. To include a broad range of both cutaneous and mucosal HPV types, PCR was performed with two sets of degenerate primers. Using this approach we detected HPV types 9, 16, 23b, DL322 and a variant of HPV type 37 in multiple stucco keratoses. Imiquimod (5% cream), a new compound that modifies the immune response by stimulating production of cytokines, applied overnight, three times a week for 5 weeks, resulted in resolution of all treated lesions.     

Use of the polymerase chain reaction to detect human papillomavirus type 16 in the cervical scrapes of Irish women with varying grades of cervical intraepithelial neoplasia

Objective To assess the prevalence of HPV type 16 among Irish women with various degrees of CIN.
Subjects One hundred and five women with varying degrees of CIN, detected on prior cytological screening, and thirty-two women with cytologically normal cervical smears.
Methods HPV 16 DNA was detected by the PCR technique, with controls against contamination.
Setting The colposcopy clinic in the Rotunda Maternity Hospital, Dublin, and the Virus Reference Laboratory, U.C.D., from October 1991 to April 1992.
Results HPV 16 DNA was detected in 66% of subjects with abnormal cervical cytology, and in 31% of those with normal cervical smears. A higher HPV 16 prevalence was found among those women with CIN III (74%), than among those with CIN I/CIN II (60%).
Conclusions The prevalence of HPV 16 among Irish women with normal cytology and CIN was similar to previous published studies from other areas, with a higher prevalence in those with abnormal cervical cytology. The usefulness of detection of HPV 16 as an indicator of a higher risk of carcinoma of the cervix is discussed.     

Immunoglobulin A, G, and M responses to L1 and L2 capsids of human papillomavirus types 6, 11, 16, 18, and 33 L1 after newly acquired infection

OBJECTIVES: We performed a study to establish the pattern of serological reactivity for immunoglobulins (Ig), to capsids of human papilloma virus (HPV) after new HPV infection in two groups of subjects. METHODS: The pattern of serological reactivity after acquisition of infection with HPV was investigated by measuring IgA, IgM, and IgG antibodies to capsids containing L1 and L2 proteins of HPV types 6, 11, 16, 18, and 33 in longitudinal studies of groups with different patterns of sexual activity. Individuals who tested negative for HPV DNA by the polymerase chain reaction at enrolment, but who became HPV DNA positive during follow up, were examined for antibodies to HPV capsids by enzyme linked immunosorbent assay. One group consisted of 15 young girls (with eight controls who remained HPV DNA negative) who were becoming sexually active and the other comprised 12 male (with five controls) and 35 female (with seven controls) heterosexual attenders of a sexually transmitted disease clinic who had had multiple sexual partners. RESULTS: The sexually inexperienced girls showed IgA and IgG responses, but seldom an IgM response to infection with HPV types 6/11, 16, and 18. No consistent pattern of serological reactivity was apparent for the heterosexuals with multiple partners. The lack of association between current HPV DNA positivity and detectable antibodies in these individuals was possibly related to the duration of infection or to prior exposure to HPV. For the latter group serological reactivity to HPV capsids was significantly greater in women than in men (p = 0.001, p = 0.003, and p = 0.024, for IgG to HPV 6, 11, and 16, respectively). CONCLUSION: The sex difference in antibody response detected in previous studies with assays based on peptide antigens was thus corroborated in the present study with capsid based serological assays. This sex difference might reflect a difference in sexual activity and prior exposure to HPV between men and women in this particular group.     

Detection of alpha- and beta-human papillomavirus (HPV) in cutaneous melanoma: a matched and controlled study using specific multiplex PCR combined with DNA microarray primer extension

Abstract:  There are few contradictory studies investigating the involvement of HPV in melanoma. We designed a controlled study to evaluate the HPV DNA prevalence in melanoma. One hundred patients with cutaneous malignant melanoma diagnosed between 2002 and 2006 were included. Complementary wide excision (healthy skin) was performed in 85 patients and was used as internal control. After DNA extraction, 68 different HPV types were studied using a multiplex PCR combined with microarray primer extension. We did not observe any statistical significant difference in terms of HPV DNA prevalence in melanoma (38.8%) and in healthy skin from wide excision (42.4%). Twenty-one different HPV types were detected but only one type was present in the majority of our samples (80/85 melanoma vs 59/66 HS). The distribution of HPV genera and types was similar in melanoma and HS, and beta-HPV was predominant (30.6% and 31.8%). Among alpha-HPV (10.6%), high-risk mucosal HPV16 was predominant. Among beta-HPV, melanoma harboured significantly more type 22 than control normal skin from the same patients and significantly less type 21 than paired control normal skin. No correlation between clinical and pathological melanoma characteristics and HPV DNA prevalence was found. Our data do not support a role of HPV infection in melanocarcinogenesis, but confirm the previous data suggesting that HPV DNA is widely distributed among the population and that occult HPV infections are frequent. Furthermore, specific HPV types, such as a-HPV16 and beta-HPV species 2 may be involved in a sub-group of melanoma.     

Detection of human papillomavirus (HPV) DNA in oral squamous cell carcinomas by in situ hybridization and polymerase chain reaction

Summary A series of 156 formalin-fixed, paraffin-embedded biopsies from 40 patients with surgically-treated oral squamous cell carcinomas was analysed for the presence of human papillomavirus (HPV) infection by histopathological evaluation, in situ DNA hybridization and polymerase chain reaction (PCR). Epithelial changes suggesting a HPV lesion within, or adjacent to, the carcinoma lesions were found in 16 out of 40 patients (40%). Morphological signs of a flat HPV lesion were found in four cases (10%), those of inverted type in three cases (7.5%), and those of papillary type in nine cases (22.5%). HPV DNA was demonstrated in one of the lesions by in situ hybridization with biotin-labelled DNA cocktail probe containing HPV types 6, 11, 16 and 18. With the PCR technique, samples from 11 (27.5%) of the 40 patients proved to contain HPV DNA. Of these, HPV 6 was demonstrated in one case, HPV 16 in ten cases and HPV 18 in one case. HPV DNA was exclusively detected in the biopsies showing carcinoma tissue or its adjacent precancer lesions. No viral DNA was found in the biopsies derived from the tumour-free resection margins. These results provide further evidence to support the concept of HPV involvement in the aetiology of oral squamous cell carcinomas, most probably acting synergistically with other carcinogens.     

High viral load of human wart-associated papillomaviruses (PV) but not β-PV in cutaneous warts independent of immunosuppression

Background  A broad spectrum of human papillomaviruses (HPV) has been detected in warts from immunocompetent patients and a much more diverse range from immunosuppressed organ transplant recipients (OTR).
Objectives  To determine the HPV types in warts from OTR, we assessed present infections of mucosal (α-PV), wart-associated (α-, μ- and ν-PV) and cutaneous HPV types (β-/γ-PV) in immunocompetent patients and OTR.
Patients/methods  Forty-one warts from 29 immunocompetent patients (non-OTR) and 53 warts from 33 OTR were analysed for DNA of human α-, β-, γ-, μ- and ν-PV. For frequent types viral load was determined by quantitative real-time PCR.
Results  Compared with non-OTR prevalence of cutaneous HPV (79% vs. 49%, P  <   0·01) and the number of multiple infections (62% vs. 17%, P  <   0·0001) were significantly increased. The mean viral load of the wart-associated HPV was more than 10⁵-fold higher compared with human β-PV in both cohorts.
Conclusions  The high load of wart-associated HPV suggests an active role of these viruses rather than cutaneous types in warts independent of immunosuppression; however, the substantial fraction of warts with low HPV genome copies remains to be explained.