Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens.

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.

Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein

In the majority of cervical cancers, DNAs of high-risk mucosotpropic human papillomaviruses (HPVs), such as type 16, are maintained so as to express two viral proteins, E6 and E7, suggesting an essential importance to carcinogenesis. The high-risk HPV E6 proteins are known to inactivate p53 tumor suppressor protein but appear to have an additional, molecularly unknown function(s). In this study, we demonstrate that these E6 proteins can bind to the second PDZ domain of the human homologue of the Drosophila discs large tumor suppressor protein (hDLG) through their C-terminal XS/TXV/L (where X represents any amino acid, S/T serine or threonine, and V/L valine or leucine) motif. This finding is similar to the interaction between the adenomatous polyposis coli gene product and hDLG. E6 mutants losing the ability to bind to hDLG are no longer able to induce E6-dependent transformation of rodent cells. These results suggest an intriguing possibility that interaction between the E6 protein and hDLG or other PDZ domain-containing proteins could be an underlying mechanism in the development of HPV-associated cancers.

RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin

Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC50 for HeLa cells treated with cisplatin was 9.4 mu M, but after the addition of a lentivirus-delivered shRNA against E6, the IC50 was reduced almost 4-fold to 2.4 mu M. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with individual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.

God's gift to women: The human papillomavirus vaccine

An Australian newspaper recently bestowed Ian Frazer the title of God's gift to women for his research team's part in developing a vaccine to help control cervical cancer. Here Frazer discusses this work and the science behind the vaccine.

Serologic response to human papillomavirus 16 among Australian women with high-grade cervical intraepithelial neoplasia

This study evaluated the detection of human papillomavirus (HPV) 16 antibody in HPV 16-associated cervical intraepithelial neoplasia (CIN) in Australian women. Seroreactivity to HPV 16 L1 virus-like particles was assessed in patients with CIN 2 (n = 169) and CIN 3 (n = 229) lesions previously tested for the presence of HPV DNA. Seropositivity was significantly commoner in women with HPV 16 DNA-positive lesions (98/184) than in women with no HPV DNA in the lesion (15/47) or with HPV of types other than 16 in the lesion (43/167) (P = 0.0004). In addition, seropositivity was observed in 33% (55/169) of women with CIN 2 and 46% (106/229) of women with CIN 3, in keeping with the lower fraction of CIN 2 (57/169) than CIN 3 (127/229) biopsies positive for HPV 16 DNA. HPV 16 seropositivity is most common in women with HPV 16-associated CIN, but many patients with HPV-associated CIN 3 are seronegative, and HPV 16 seropositivity is common in women with CIN associated with other HPV types. Overall, HPV 16 serology is a poor predictor of presence of HPV 16-associated CIN 3 in patient population studied.

Genital human papillomavirus: Women's knowledge and attitudes of this sexually transmitted disease

The purpose of this study was to investigate women's knowledge and attitudes regarding genital human papillomavirus (n=100). Using a descriptive design, the Health Education Questionnaire was administered to 100 female patients (Mean Age = 33, SD = 7.17) at a physicians office in South Florida. The results indicated a lack of knowledge regarding genital human papillomavirus with 21 patients (21%) reported having knowledge and 79 (79%) having never heard of this disease. In addition, the group familiar with genital human papillomavirus also possessed a low level of knowledge with only 57% acknowledging an association of genital human papillomavirus and cervical cancer, 52% aware that a pap smear can detect the virus, 42% knowing that antibiotics can not treat the disease and 57% aware that it is not associated with a family history. An association was found between attitudes and health seeking behaviors. Subjects stating that they would take all measures to prevent genital human papillomavirus, were more likely to have a pap smear within the last year (Chi-square (1) = 4.33, p < .05). Higher levels of education and income were associated with increased knowledge regarding genital human papillomavirus when subjects were categorized according to sociodemographic characteristic (Chi-square (1) =9.45, p < .05; Chi-square (1) = 6.75, p < .05). There was no significant correlation between knowledge and ethnicity, marital status or age. Findings indicated the need for improved education and promotion of positive attitudes regarding human papillomaviurs in order to improve health seeking behaviors among women.

Molecular epidemiology of sexually transmitted human papillomavirus in a self referred group of women in Ireland

Background: Human papillomavirus (HPV) causes cervical cancer and external genital warts. The purpose of this study is to document the genotype distribution of HPV in females aged between 18 and 34 who self-referred to an STI clinic with visible external genital warts (EGW). Scrapings were taken from visible external genital warts (EGW). These scrapings were analysed by PCR for the presence of HPV DNA. Positive samples were then genotyped by means of a commercially available assay (LiPA). A comparison of genotyping results determined by the LiPA assay and direct amplicon DNA sequencing was also performed. Results: Ninety-two patients out of 105 samples (88%) had detectable levels of HPV DNA. The majority of individuals with EGW (66%) showed the presence of two or more genotypes. The most common HPV genotypes present in the study population were HPV-6, HPV-11, HPV-16, HPV-18, HPV-33 and HPV-53. Potential effects of vaccination on HPV molecular epidemiology indicate that 40% of the patients could have been protected from the high risk genotypes HPV-16 and HPV-18.Conclusion: This is the first report of the molecular epidemiology of external genital warts in women aged between 18 and 34 from Ireland based on results from a LiPA assay. The study shows that most individuals are infected with multiple genotypes including those with high oncogenic potential and that the newly available HPV vaccines could have a significant impact on prevalence of the most common HPV genotypes in this study population.

Risk of Human Papillomavirus-Associated Cancers Among Persons With AIDS

Background Although risk of human papillomavirus (HPV)–associated cancers of the anus, cervix, oropharynx, penis, vagina, and vulva is increased among persons with AIDS, the etiologic role of immunosuppression is unclear and incidence trends for these cancers over time, particularly after the introduction of highly active antiretroviral therapy in 1996, are not well described. Methods Data on 499 230 individuals diagnosed with AIDS from January 1, 1980, through December 31, 2004, were linked with cancer registries in 15 US regions. Risk of in situ and invasive HPV-associated cancers, compared with that in the general population, was measured by use of standardized incidence ratios (SIRs) and 95% confidence intervals (CIs). We evaluated the relationship of immunosuppression with incidence during the period of 4–60 months after AIDS onset by use of CD4 T-cell counts measured at AIDS onset. Incidence during the 4–60 months after AIDS onset was compared across three periods (1980–1989, 1990–1995, and 1996–2004). All statistical tests were two-sided. Results Among persons with AIDS, we observed statistically significantly elevated risk of all HPV-associated in situ (SIRs ranged from 8.9, 95% CI = 8.0 to 9.9, for cervical cancer to 68.6, 95% CI = 59.7 to 78.4, for anal cancer among men) and invasive (SIRs ranged from 1.6, 95% CI = 1.2 to 2.1, for oropharyngeal cancer to 34.6, 95% CI = 30.8 to 38.8, for anal cancer among men) cancers. During 1996–2004, low CD4 T-cell count was associated with statistically significantly increased risk of invasive anal cancer among men (relative risk [RR] per decline of 100 CD4 T cells per cubic millimeter = 1.34, 95% CI = 1.08 to 1.66, P = .006) and non–statistically significantly increased risk of in situ vagina or vulva cancer (RR = 1.52, 95% CI = 0.99 to 2.35, P = .055) and of invasive cervical cancer (RR = 1.32, 95% CI = 0.96 to 1.80, P = .077). Among men, incidence (per 100 000 person-years) of in situ and invasive anal cancer was statistically significantly higher during 1996–2004 than during 1990–1995 (61% increase for in situ cancers, 18.3 cases vs 29.5 cases, respectively; RR = 1.71, 95% CI = 1.24 to 2.35, P < .001; and 104% increase for invasive cancers, 20.7 cases vs 42.3 cases, respectively; RR = 2.03, 95% CI = 1.54 to 2.68, P < .001). Incidence of other cancers was stable over time. Conclusions Risk of HPV-associated cancers was elevated among persons with AIDS and increased with increasing immunosuppression. The increasing incidence for anal cancer during 1996–2004 indicates that prolonged survival may be associated with increased risk of certain HPV-associated cancers.

Human papillomavirus vaccines in plants

Human papillomaviruses are the etiological agents of cervical cancer, one of the two most prevalent cancers in women in developing countries. Currently available prophylactic vaccines are based on the L1 major capsid protein, which forms virus-like particles when expressed in yeast and insect cell lines. Despite their recognized efficacy, there are significant shortcomings: the vaccines are expensive, include only two oncogenic virus types, are delivered via intramuscular injection and require a cold chain. Plant expression systems may provide ways of overcoming some of these problems, in particular the expense. In this article, we report recent promising advances in the production of prophylactic and therapeutic vaccines against human papillomavirus by expression of the relevant antigens in plants, and discuss future prospects for the use of such vaccines. © 2010 Expert Reviews Ltd.

Human papillomavirus prevalence, viral load and pre-cancerous lesions of the cervix in women initiating highly active antiretroviral therapy in South Africa : a cross-sectional study

Background Cervical cancer and infection with human immunodeficiency virus (HIV) are both important public health problems in South Africa (SA). The aim of this study was to determine the prevalence of cervical squamous intraepithelial lesions (SILs), high-risk human papillomavirus (HR-HPV), HPV viral load and HPV genotypes in HIV positive women initiating anti-retroviral (ARV) therapy. Methods A cross-sectional survey was conducted at an anti-retroviral (ARV) treatment clinic in Cape Town, SA in 2007. Cervical specimens were taken for cytological analysis and HPV testing. The Digene Hybrid Capture 2 (HC2) test was used to detect HR-HPV. Relative light units (RLU) were used as a measure of HPV viral load. HPV types were determined using the Roche Linear Array HPV Genotyping test. Crude associations with abnormal cytology were tested and multiple logistic regression was used to determine independent risk factors for abnormal cytology. Results The median age of the 109 participants was 31 years, the median CD4 count was 125/mm3, 66.3% had an abnormal Pap smear, the HR-HPV prevalence was 78.9% (Digene), the median HPV viral load was 181.1 RLU (HC2 positive samples only) and 78.4% had multiple genotypes. Among women with abnormal smears the most prevalent HR-HPV types were HPV types 16, 58 and 51, all with a prevalence of 28.5%. On univariate analysis HR-HPV, multiple HPV types and HPV viral load were significantly associated with the presence of low and high-grade SILs (LSIL/HSIL). The multivariate logistic regression showed that HPV viral load was associated with an increased odds of LSIL/HSIL, odds ratio of 10.7 (95% CI 2.0 – 57.7) for those that were HC2 positive and had a viral load of ≤ 181.1 RLU (the median HPV viral load), and 33.8 (95% CI 6.4 – 178.9) for those that were HC2 positive with a HPV viral load > 181.1 RLU. Conclusion Women initiating ARVs have a high prevalence of abnormal Pap smears and HR-HPV. Our results underscore the need for locally relevant, rigorous screening protocols for the increasing numbers of women accessing ARV therapy so that the benefits of ARVs are not partially offset by an excess risk in cervical cancer.

Re: Human papillomavirus infection and incidence of squamous cell and basal cell carcinomas of the skin

Human papillomaviruses (HPVs) cause cervical cancer and some other types of epithelial cancers. HPV types from the phylogenic beta genus (beta-PVs), formerly known as epidermodysplasia verruciformis–associated HPV types, are frequently detected in nonmelanoma skin cancers, especially in squamous cell carcinomas (SCCs). An etiologic relationship with beta-PV infection is suspected...

Automatic classification of human epithelial type 2 cell indirect immunofluorescence images using cell pyramid matching

This paper describes a novel system for automatic classification of images obtained from Anti-Nuclear Antibody (ANA) pathology tests on Human Epithelial type 2 (HEp-2) cells using the Indirect Immunofluorescence (IIF) protocol. The IIF protocol on HEp-2 cells has been the hallmark method to identify the presence of ANAs, due to its high sensitivity and the large range of antigens that can be detected. However, it suffers from numerous shortcomings, such as being subjective as well as time and labour intensive. Computer Aided Diagnostic (CAD) systems have been developed to address these problems, which automatically classify a HEp-2 cell image into one of its known patterns (eg. speckled, homogeneous). Most of the existing CAD systems use handpicked features to represent a HEp-2 cell image, which may only work in limited scenarios. We propose a novel automatic cell image classification method termed Cell Pyramid Matching (CPM), which is comprised of regional histograms of visual words coupled with the Multiple Kernel Learning framework. We present a study of several variations of generating histograms and show the efficacy of the system on two publicly available datasets: the ICPR HEp-2 cell classification contest dataset and the SNPHEp-2 dataset.

Awareness and knowledge of Human Papillomavirus (HPV) among ethnically diverse women varying in generation status

Although Human papillomavirus (HPV) is a common sexually transmitted infection, there is limited knowledge of HPV with ethnic/racial minorities experiencing the greatest disparities. This cross-sectional study used the most recent available data from the California Health Interview Survey to assess disparities in awareness and knowledge of HPV among ethnically/racially diverse women varying in generation status (N = 19,928). Generation status emerged as a significant predictor of HPV awareness across ethnic/racial groups, with 1st generation Asian-Americans and 1st and 2nd generation Latinas reporting the least awareness when compared to same-generation White counterparts. Also, generation status was a significant predictor of HPV knowledge, but only for Asian-Americans. Regardless of ethnicity/race, 1st generation women reported lowest HPV knowledge when compared to 2nd and 3rd generation women. These findings underscore the importance of looking at differences within and across ethnic/racial groups to identify subgroups at greatest risk for poor health outcomes. In particular, we found generation status to be an important yet often overlooked factor in the identification of health disparities.

Uterine cervix nitric oxide and human papillomavirus infection in women

Most women acquire genital high risk human papillomavirus (HPV) infection during their lifetime, but seldom the infection persists and leads to cervical cancer. However, currently it is not possible to identify the women who will develop HPV mediated cervical cancer and this often results to large scale follow-up and overtreatment of the likely spontaneously regressing infection. Thus, it is important to obtain more information on the course of HPV and find markers that could help to identify HPV infected women in risk for progression of cervical lesions and ultimately cancer. Nitric oxide is a free radical gas that takes part both in immune responses and carcinogenesis. Nitric oxide is produced also by cervical cells and therefore, it is possible that cervical nitric oxide could affect also HPV infection. In the present study, including 801 women from the University of Helsinki between years of 2006 and 2011, association between HPV and cervical nitric oxide was evaluated. The levels of nitric oxide were measured as its metabolites nitrate and nitirite (NOx) by spectrophotometry and the expression of nitric oxide producing enzymes endothelial and inducible synthases (eNOS, iNOS) by Western blotting. Women infected with HPV had two-times higher cervical fluid NOx levels compared with non-infected ones. The expression levels of both eNOS and iNOS were higher in HPV-infected women compared with non-infected. Another sexually transmitted disease Chlamydia trachomatis that is an independent risk factor for cervical cancer was also accompanied with elevated NOx levels, whereas vaginal infections, bacterial vaginosis and candida, did not have any effect on NOx levels. The meaning of the elevated HPV related cervical nitric oxide was evaluated in a 12 months follow-up study. It was revealed that high baseline cervical fluid NOx levels favored HPV persistence with OR 4.1. However, low sensitivity (33%) and high false negative rate (67%) restrict the clinical use of the current NOx test. This study indicated that nitric oxide favors HPV persistence and thus it seems to be one of the cofactor associated with a risk of carcinogenesis.