Objective. To evaluate associations between common vaginal infections and human papillomavirus (HPV). Study Design. Data from up to 15 visits on 756 HIV-infected women and 380 high-risk HIV-uninfected women enrolled in the HIV Epidemiology Research Study (HERS) were evaluated for associations of bacterial vaginosis, trichomoniasis, and vaginal Candida colonization with prevalent HPV, incident HPV, and clearance of HPV in multivariate analysis. Results. Bacterial vaginosis (BV) was associated with increased odds for prevalent  and incident  HPV and with delayed clearance of infection . Whereas BV at the preceding or current visit was associated with incident HPV, in an alternate model for the outcome of incident BV, HPV at the current, but not preceding, visit was associated with incident BV. Conclusion. These findings underscore the importance of prevention and successful treatment of bacterial vaginosis. Candida albicans is the most prevalent species in most cases of asymptomatic colonization and vulvovaginal candidiasis; however certain species of Candida are more pathogenic than others and induce hyphae formation and have enhanced proteolytic activity and antigen modulation, enabling Candida to penetrate the mucosal surface and induce mucosal swelling, erythema, and exfoliation of cells [Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) among young adult women, and it plays a critical role in the development of cervical cancer , 2. Biolof cells –12. The of cells , 13–19. The HIV Epidemiology Research Study (HERS) was a longitudinal multi-site investigation of the biological, psychological, and social effects of HIV infection among US women . Brieflyn = 17) or negative (n = 72) test results for hybridization with the control, B-globin probe were considered of unsatisfactory quality, and the HPV results were set to missing. HPV testing used polymerase chain reaction (PCR) to amplify virus obtained by cervicovaginal lavage . The ampFor the purposes of the current analysis, visit-specific prevalent HPV infection was defined by detection of any HPV DNA by general probe at a given visit. Incident infection was defined as detection of a new type of HPV not present at any prior visit. Thus, HPV type-specific negative women at enrollment were at risk for infection with the respective HPV type. Women who were HPV-negative by all type-specific probes and generic probe prior to testing positive by generic probe only  were classified as having incident HPV at that visit. For the evaluation of time to HPV clearance, women contributed event times for each incident HPV type. Time to clearance was defined as the interval from the type-specific incident visit to the first of two consecutive type-specific negative visits. The flagging negative visit was required to avoid misclassification from fluctuating detection of HPV . HPV infCandida colonization was based on a positive culture for Candida species or detection of yeast or pseudohyphae on gram stain. Presence of trichomoniasis was determined by detection of trichomonas on wet mount  or on culture (visits 4 and above). Each vaginal infection was defined using standard criteria. Presence of bacterial vaginosis was determined by Nugent scoring of a gram-stained slide . A Nugenn = 1256). Of these women, 108 who did not have a cervix at baseline and one woman who had cervical treatment and a hysterectomy within 6 months before and after enrollment, respectively, were excluded. For simplicity, an additional 11 women who seroconverted to HIV-seropositive during follow-up were excluded, leaving 1136 subjects for this analysis. Person-time was censored at the date of cervical treatment  or hysterectomy for 111 subjects. Women were eligible for the current analysis if they had baseline data on HPV DNA and bacterial vaginosis, vulvovaginal candidiasis, or trichomoniasis test results , illicit drug use, cigarette smoking, use of hormonal contraceptives, immunologic predisposing factors , and demographic indicators for HPV prevalence in the USA  [A priori models for time to HPV clearance also included a covariate indicating coinfection with another HPV type within the type-specific duration and its cancer risk classification. All statistical models included the study design variables: study site and risk behavior cohort . In addition, models included a covariate for visit number, year of visit, or year of HPV onset to account for period effects.Potential confounders of the relationship between vaginal infections and prevalent HPV, incident HPV, and clearance of HPV were selected based on the literature and a proposed causal directed acyclic diagram (not shown) used for identification of sufficient sets of confounders . The a p status) , 25. A pa priori, other factors related to cervicovaginal health were evaluated in bivariate and multivariable analysis using SAS versus 9.2 . These factors included potential hormonal influences , sexual behavior , genital tract infections , and other risk behaviors or conditions . The importance of a covariate was evaluated in terms of the causal directed acyclic graph, collinearity with other covariates, and its coefficient magnitude, statistical significance, and impact on the effect estimates for vaginal infections upon addition to and removal from the statistical model. In addition to the variables selected A generalized linear mixed model with a random intercept and a slope based on a toeplitz matrix was used to model associations between current vaginal infections and prevalent HPV while accounting for between-subject variation and repeated measures correlation. To evaluate associations between vaginal infections at the preceding visit and incident HPV, as well as to explore the temporal relationship between HPV and bacterial vaginosis, the case-crossover analytic approach was used. The case-crossover approach is useful for identifying associations between recent, time-varying exposures and an incident outcome while adjusting for all time-independent factors by comparing each woman's case  visits to her own noncase visits. To evaluate temporality, separate case-crossover models were run for incident HPV and incident bacterial vaginosis that were adjusted for a few shared time-dependent predictors: a combined indicator for HIV status and CD4 category  and self-reported crack or cocaine use, new or multiple sex partners, and consistency of condom use since the prior study visit.Analyses of time to clearance of a type-specific HPV infection used the Wei, Lin, and Weissfeld extended Cox model. Hazard ratios less than 1.0 indicate lower clearance rates . Vaginal infections, and most of the covariates, were modeled as time-dependent variables allowing the instantaneous effects to vary with changes in the time-dependent variables. P < 0.15) using the Wald and Likelihood Ratio tests. Due to effect measure modification of the association between certain vaginal infections and HPV outcomes by HIV status, tabular results are presented separately for HIV-infected and HIV-uninfected women. Upon finalizing the main effects models, modification of the effect estimates for vaginal infections across levels of biologically relevant covariates  was evaluated for magnitude and significance (n = 756) were similar to HIV-uninfected women (n = 380) in age, number of live births, cigarette smoking status, and prevalence of bacterial vaginosis, trichomonas vaginalis, Chlamydia trachomatis, and Neisseria gonorrhoeae infections  was over twice that of HIV-uninfected women (29%), and HIV-infected women were more likely to be coinfected with multiple HPV strains  era, so none of the women were on HAART regimens at baseline. However, one-third of the HIV-infected women were taking antiretrovirals.At baseline, HIV-infected women  = 1.53, 95% CI: 1.25, 1.86). There was no modification of the odds ratio for bacterial vaginosis on prevalent HPV by HIV status . In contrast, when trichomoniasis was removed from the model, vaginal Candida colonization was not associated with prevalent HPV .The prevalence of HPV infection at visits 1–15 ranged from 38% to 56% (49–68% for HIV-infected women and 12–32% for HIV-uninfected women). Longitudinal, multivariable analysis for prevalent HPV demonstrated a strong association with HIV status and immunodeficiency. HIV-infected women with CD4 cell counts below 200 cells/V status . In a molent HPV . Howevermoniasis . When vaCandida colonization was not significantly associated with incident HPV, regardless of timing of vaginal infection relevant to HPV infection . There was no modification of the odds ratio for prior bacterial vaginosis on incident HPV by HIV status . In a monfection . μL were half as likely to clear an HPV infection as those with CD4 counts above 500 cells/μL (adjusted hazards ratio (aHR) = 0.51, 95% CI: 0.40, 0.64). HIV status and CD4 category did not significantly modify the hazard ratios for vaginal infections with HPV clearance. However, there was a statistical interaction between bacterial vaginosis and vaginal Candida colonization , and vice versa, among women with bacterial vaginosis, vaginal Candida colonization was associated with delayed HPV clearance . When vaginal Candida colonization was removed from the model, bacterial vaginosis remained significantly associated with delayed clearance of HPV among all HERS women . In contrast, when bacterial vaginosis was removed from the model, vaginal Candida colonization was not associated with HPV clearance . We did not detect a significant association between trichomoniasis and time to clearance of HPV infection, regardless of HIV status (The majority of incident HPV infections cleared over subsequent study visits (63.1% clearance among HIV-infected women and 74.8% among HIV-uninfected women). Among HIV-infected women, those with CD4 counts below 200 cells/nization . Among aV status .Consistent with a recent meta-analysis of primarily cross-sectional studies, bacterial vaginosis was associated with increased odds for prevalent HPV among women in HERS . FurtherWhile trichomoniasis infection was not significantly associated with HPV outcomes among HIV-uninfected women in HERS, HIV-infected women with trichomoniasis had decreased odds for prevalent HPV. A similar finding was reported from a WIHS analysis which combined 1736 HIV-infected and 493 HIV-uninfected women into one analytic group , 15, 18.Candida colonization alone was not significantly associated with HPV outcomes; however when concurrent with bacterial vaginosis or trichomoniasis, it negatively impacted their effects on certain HPV outcomes. The decreased odds for prevalent HPV among women with trichomoniasis was not observed among women with vaginal Candida colonization, and the association between bacterial vaginosis and delayed clearance of an HPV infection was strongest among women with vaginal Candida colonization. Standard antibiotic treatment for bacterial vaginosis or trichomoniasis may precipitate a vaginal yeast infection, and a high incidence of asymptomatic and symptomatic vulvovaginal yeast infection has been reported in patients with recurrent bacterial vaginosis [Candida colonization may indicate more severe or difficult-to-treat bacterial vaginosis or trichomoniasis. The presence of vaginal aginosis . TherefoThe HERS cohort, two-thirds of which are HIV-infected, represents a population at increased risk for vaginal infections and HPV. As such, there are two primary concerns regarding these study's findings: (1) the potential dependency of the results on HIV disease and (2) the generalizability of the results to the general population of women. Several methods were used to reduce potential confounding of the relationships between vaginal infections and HPV by HIV-associated factors: (1) by design, HIV-infected women were frequency matched at enrollment to HIV-uninfected women on sexual and injection drug use behavior, (2) self-reported risk behavior between study visits was included in multivariable analyses, (3) models among all HERS women were evaluated for statistical interaction with a composite variable of HIV status and CD4 count, and (4) separate models were built for HIV-infected and HIV-uninfected women, with the former adjusted for CD4 category and logarithm of viral load. Despite these efforts, disentangling the interrelationships between HIV disease and concurrent vaginal infections was limited by decreased statistical power to detect significant associations among HIV-uninfected women separately. While the HERS cohort is a population at high-risk for genital tract infections, the primary findings involve two infections for which the general female population is also at high risk: bacterial vaginosis and HPV. Moreover, there is mounting evidence for an association between bacterial vaginosis and HPV across diverse study populations . This is the second large, prospective study among HIV-infected and HIV-uninfected women to report consistent findings for bacterial vaginosis with increased prevalent and incident HPV . In the Bacterial vaginosis is associated with increased odds for prevalent and incident HPV as well as delayed clearance among women in the HIV Epidemiology Research Study.


In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R Human papillomaviruses (HPV) are causally involved in the induction of cervical cancer and its precursor lesions. Currently, 12 HPV types are classified as carcinogenic to humans and an additional 8 types as probably or possibly carcinogenic to human The mode of action of both vaccines is considered to be the induction of neutralizing antibodies directed against L1 surface loops of the viral capsid. With more than six years history on papillomavirus prophylactic vaccination, monitoring long term development of protective titers of neutralizing antibodies is of increasing importance. Thus, there is a need for the evaluation of such antibody responses, specifically for functional assays analyzing neutralizing antibodies.Papillomaviruses cannot be replicated in simple cell culture systems. Therefore, in the past a number of functional assays have been developed to measure antibody-mediated neutralization of papillomaviruses. These assays involved the use of authentic viruses The current ‘gold standard’ for measuring neutralizing anti-HPV antibodies is a manually performed pseudovirion-based neutralization assay manPBNA;  using seSeveral arguments make a case for the need of a high-throughput neutralization assay with improved sensitivity: (i) requirement of larger serum sample numbers for follow-up studies on current vaccines, (ii) detection of cross-neutralizing antibodies induced by the commercial vaccines, and (iii) monitoring the effect of simplified vaccination schemes. Also, induction of neutralizing antibodies by second generation vaccines, e.g. based on the L2 protein needs to be assessed. Finally, large scale neutralization assays would allow addressing questions on naturally occurring protective immunity against HPV infections. Especially in respect to antibody responses against natural papillomavirus infections there are high demands for sensitivity and reproducibility in a neutralization assay. To date, high-throughput detection of HPV capsid-specific antibodies has been possible only with the aid of surrogate detection assays such as ELISA and competitive Luminex immunoassay (cLIA) using VLPs as antigen. Here we describe the adaptation of the PBNA to a high-throughput (HT) setting. We developed a purely add-on system in which the serial dilution of serum samples is separated from the cell-based assay, providing a high degree of flexibility. The high-throughput assay demonstrates high robustness with little intra- and inter-assay day variability. Also, the HT-PBNA shows higher sensitivity compared to the manually performed assay using SEAP as reporter. In its current format, the neutralization titer of 110 serum samples for seven HPV types can be determined in a single run. The HT-PBNA will allow the execution of large studies on vaccine- and naturally-induced immunity against HPV infections.Ethics statementhttp://www.ethikkommission.uniklinikum-jena.de/Ethik_KommissionderFSUJena.html). All investigations involving humans have been performed in accordance with the principles embodied in the Declaration of Helsinki. The study was performed in compliance with the guidelines of Good Clinical Practice as described in the International Conference on Harmonization of Good Clinical Practices guidelines step 4 (1996). All patients signed an informed consent form. Study participation took 24 weeks with 2 optional follow-up visits after 36 and 48 weeks. Blood samples were collected at baseline, 2 weeks after each vaccination and after 24 weeks. Peripheral blood mononuclear cells (PBL) and plasma were stored frozen until analysis.Base line sera from 35 women with HPV 16 mono-infected high grade cervical intraepithelial neoplasia (CIN 2/3) participating in a vaccination trial with chimeric HPV 16 L1-E7 virus-like particles A post-vaccination serum collected after obtaining written informed consent from a 21 years old female volunteer 3 years post completion of a 3-dose Gardasil vaccination course was used as positive standard on all assay plates. Approval for obtaining and testing this serum derives from ‘Ethik-Kommission der Bayerischen Landesärztekammer’, Munich, Germany.6 293TT cells using TurboFect (Fermentas) according to the manufacturer’s instructions with minor modifications. Briefly, 10–15 µg DNA were mixed with 2 volumes of TurboFect and 1 ml of unsupplemented DMEM and added to 293TT cells which were subsequently incubated at 37°C, 5%CO2 for 72 hours. Absence of cell culture contaminations and authentication of the cell line were determined by the Multiplex cell Contamination Test Pseudovirions (PSV) were prepared as previously described For pseudovirion extraction, 293TT cells were harvested and the cell pellet was resuspended in an equal volume of lysis buffer. Lysis buffer was prepared by adding 58,3 µl of 10% Brij58 (w/v) (Sigma) and 6,6 µl RNAse A/T (Fermentas) to every ml of DBPS (Invitrogen). Cells were rotated overnight at 37°C to induce pseudovirion maturation. On the next day, salt extraction was performed by adding 0,17 volumes of 5 M NaCl, followed by the addition of 250 units of benzonase for 1 h at 37°C. The pseudovirions were purified by Optiprep gradient and fractions were stored at −80°C.alone−Psvserum)/*100%. Sera with at least 70% neutralizing activity were regarded as neutralizing.The SEAP manPBNA was essentially performed as described Antibodies binding to HPV L1 proteins were measured in a multiplexed glutathione S-transferase capture immunosorbent assay with fluorescent bead-based Luminex technology as previously described in detail The 293TT cells originally described as target cell line for the PBNA were not suited for the add-on assay layout for the HT-PBNA since attachment of the 293TT cells to the assay plates was very variable and resulted in high assay variability. Therefore, we generated a HeLa cell line stably transfected with a linearized expression construct for the SV 40 large T-antigen. HeLa cells (tested for absence of contaminations and for authenticity as described above http://www.slas.org/community/microplate_standards.cfm) to columns 3 and 13 of 384 well intermediate plates  to 1/157,464 and finally 5 µl of the serial dilutions were transferred with the EP3 robot to each of nine assay plates . The plates were sealed immediately with a polypropylene cover foil  and stored at −20°C.2. The plates where then removed from the incubator and equilibrated to RT before 20 µl/well glow substrate solution  for the Gaussia luciferase was added with the FlexDrop dispenser. Luminescence was read directly in the assay plates after 11 min in an Envision plate reader (PerkinElmer) equipped with stacker and internal barcode reader.Assay plates containing the serum dilutions were equilibrated to room temperature (RT), centrifuged for 1 min at 1.000×g and the cover foil was removed before 15 µl PSV diluted in fully supplemented cell culture medium were added with a FlexDrop bulk dispenser (PerkinElmer) and incubated for 1 h at RT . Thereaf50-value (effective dilution giving 50% inhibition) for each serum according to the four parameter logistic curve fit model: y = A+(B−A)/(1+(C/x)∧D) .Three identical assay plates originating from the same serum dilution plate were used for each PSV- type. Inhibition (%) was calculated by normalization of the luminescence in serum-dilution containing wells to the mean of the negative control wells without serum present on each plate. The median of the triplicate values was used for the calculation of the EDPlasmids and cell lines are available on request for non-commercial users following standard material transfer agreement.In order to translate the  PBNA to high-throughput format we first identified, then optimized and standardized critical parameters of the assay including pseudovirion (PSV) concentration, serum-PSV incubation time, cell lines and cell density.In preceding experiments we tested different cell lines in the HT-PBNA. The 293TT cell line 50 is minimal and no correction for position effects is necessary.Typically, for the luminescence signal of the Gaussia luciferase reporter assay in 384 well plates we observed a coefficient of variation (CV) of less than 3% within rows and less than 6% for the whole plate (data not shown). The higher CV for the whole plate is mainly a consequence of the decay of the Gaussia luciferase luminescence during the time that is necessary to read the 384 well plate (75 sec), even when employing a glow substrate with reduced turnover rates. The luminescence reader measures the plate row-wise so that the decrease in signal is negligible within a row. Sera are diluted within a row and therefore the effect of a decreasing luminescence signal on the calculation of the ED50) of a serum derived from a Gardasil-immunized individual for different dilutions of HPV 16 and HPV 18 PSV  PSV activity in the HT-PBNA, irrespective of whether the sera originated from HPV-vaccinated or non-vaccinated persons. In addition, at a 1∶20 dilution, these sera also neutralized PSV of all other tested HPV types. This effect was not due to reduced viability of the reporter cell line as high serum concentrations rather stimulated cell growth (data not shown). Pastrana and colleagues observed a similar “cross-species inhibition” at 1∶20 dilution for some sera in their SEAP PBNA 50-values of 58 repeats with both, the HPV 16 and HPV 18 HT-PBNA. The CV for the intra-run variability with 8–10 repeats per run ranged from 6 to 12%. For a cell-based assay, these CV indicate a low variability. As the underlying assay is newly developed, no data on reproducibility in other laboratories are yet available.The reproducibility of titers obtained with the HT-PBNA for HPV 16 and HPV 18 was determined with our serum standard tested a total of 58 times on individual plates in seven independent runs over a period of two months in the course of a larger serological study . A mean 50>80 . Considering the neutralization of BPV 1 in the PBNA as unspecific we defined an ED50 of 80 as the cut off value to classify sera positive in the neutralization assay for all HPV types.Previously, we analyzed a total of 1271 sera from a different vaccine trial  for neutralization of HPV 16, HPV 18 and BPV 1 PSV in the HT-PBNA and found only 8 sera (0.6%) with a BPV 1-specific ED50 of 463 , 6 (17%) and 8 (23%) were found positive for neutralization of HPV 16, HPV 18 and HPV 31 PSV, respectively (50 = 111). The HPV 31 DNA status of the patients has not been determined, but the well-documented cross-reactivity of HPV 16 antibodies against HPV 31 might explain neutralization of HPV 31. These HPV types are closely related and induction of cross-protection against HPV 31 has been demonstrated by the two commercial HPV-vaccines containing HPV 16 virus-like particles Using an EDectively . The geo50 GMT of 258 and a maximal ED50 of 1703. All 15 manPBNA positive sera (GMT (ED50) = 250) were also positive in the HT-PBNA (GMT (ED50) = 1895). This indicates a higher sensitivity of the HT-PBNA in the detection of sera with low neutralization titers.In general there is a direct, linear correlation between the titers determined in HT-PBNA and manPBNA based on using SEAP, especially for the vaccine-induced high titer sera . However2-value of 0.7 (0.67 for the 29 HT-PBNA positive sera only) was determined for the linear regression analysis between GST-HPV 16 L1 antibody reactivity  and the HT-PBNA ED50 values. Of the 35 sera, 28 (80%) were considered HPV 16 seropositive by the GST-HPV 16 L1 antibody binding assay (reactivity >109 MFI) and 29 (83%) sera neutralization antibody positive by the HT-PBNA (ED50>80). A Cohen’s kappa coefficient of 0.72 indicated good agreement between these two assays.The HT-PBNA also showed a good agreement with a Luminex-based total IgG-assay using GST-HPV 16 L1 as antigen . An R2-v50-values of 135 and 204, respectively.For the 72 analyzed post-vaccination sera of the trial, covering a very wide range of titers, a direct correlation of the manPBNA and the HT-PBNA was observed, but again the HT-PBNA yielded approximately 10 fold higher titers . All exc2 of 0.61 for the linear regression  and competitive immunoassays with VLPs as antigen were used There is good reason to believe that anti-L1 antibodies are the key mediators in providing vaccine-induced protection. First, these antibodies have been detected in serum and genital secretion of individuals vaccinated with Gardasil or Cervarix in vitro pseudovirion-based assay that allowed detection of neutralizing antibodies and this assay is currently considered the gold standard for assaying protective anti-L1 response in vitro detection of neutralizing antibodies as a correlate for clinical protection is still missing. The PBNA described by Pastrana et al. quantitatively detects neutralizing antibodies against a number of different HPV types, but the assay is laborious thereby limiting its use in respect to the number of samples, replicates, titration steps and papillomavirus types. To our knowledge the largest study to date has been reported by Dessy et al. In 2004, Pastrana and colleagues described an In general, there is a good correlation between non-functional and functional detection of anti-L1 responses when analyzing sera from vaccinees. However, this correlation is the result of (a) the specific induction of conformation-specific antibodies using well-structured antigens with (b) a detection system monitoring antibodies against properly folded antigens. Still, non-functional assays tend to measure a higher degree of cross-type reactivity.Previously, we demonstrated that a second generation L2 vaccine induces high titers of L2-binding antibodies but only a fraction of these are neutralizing Gaussia princeps it was possible to quantify reporter expression with high sensitivity directly in the cell culture plate with high absolute signals and excellent signal to background ratios. Adding the substrate for Gaussia princeps luciferase directly to the cell culture plate also avoids the tedious transfer of small volumes of cell culture supernatants to separate luminescence plates, an error-prone step often introducing variation between replicate samples. The compatibility of HT-PBNA with a 384 well format and bulk dispension not only reduces reagent consumption and costs generated by tip dispensing but also accelerates assay assembly and throughput. Furthermore, a high throughput 384 well format is preferable to a manual 96 well format when testing many different pseudovirion types and sera as required, for example, in L2 cross-protection analyses.To fulfill the need for an assay able to detect functional, i.e. neutralizing antibodies, we modified the assay originally described by Pastrana and colleagues. To establish the pseudovirion-based neutralization assayin a high throughput setting, the protocol should be as simple as possible while at the same time sensitivity and reproducibility should be as high as possible. A key parameter for increasing the throughput of our HT-PBNA was the separation of assay plate preparation and performance of the neutralization assay itself. Large batches of 384-well assay plates with serially diluted serum samples can be prepared in advance by automated liquid handling and subsequently stored at −20°C. After thawing, all reagents for the succeeding steps are added consecutively with a bulk dispenser to the assay plates in a pure add-on process, without the requirement for removal of supernatants or pre-incubation on separate plates. With the luciferase from Neutralization titers of sera were determined from full titration curves as effective dilutions giving 50% inhibition (ED50). Our standard layout included 10 titration steps, which can be 2-fold, 3-fold or 4-fold. By this, a large range of serum reactivity can be captured, and even high titer immune sera will be fully titrated, a prerequisite for accurate curve fitting. At the same time activity in low titer sera is still determined. To ensure high data quality the curve fit was performed from the median inhibition of triplicates on identical replicates of assay plates. Barcoding of plates enabled complete sample tracking and automated data evaluation with specialized data calculation and management software such as ActivityBase (IDBS).While we observed a good correlation of HT-PBNA with manPBNA and GST-L1 Luminex assays, the HT-PBNA showed a significant higher sensitivity compared to the manPBNA originally described by Pastrana et al, which uses SEAP as reporter system. Of note, performing the neutralization assay manually but following the protocol for the HT-PBNA we also reached higher sensitivity indicating that the automated processing is not the prime parameter for sensitivity. Still, automation leads to an increase in robustness and decrease in assay variation which itself allows the analysis of low reactive sera. While sensitivity in detecting vaccine-type-specific neutralizing antibodies in vaccinees at early time points after vaccination might not be a critical issue, it becomes very important when analyzing either antibody titers in long-term follow-up studies, natural immunity or vaccine induced cross-protection against non-vaccine HPV types. For example the cross-neutralization induced by Gardasil® and Cervarix® against HPV types 31, 33 and 45 would probably have been missed using the conventional manPBNA. Thus, HT-PBNA delivers more sensitive and therefore comprehensive analyses of natural and vaccine-derived immunity.in vitro neutralization assay (L2-assay) with a much higher sensitivity for L2 but not for L1-specific antibodies in vivo challenge model described by Roberts et al. th International Papillomavirus Conference, Puerto Rico, 2012). These pseudovirions might be very well suited for HT-PBNA, but it remains to be determined whether these modified pseudovirions can be produced for different HPV types.As a proof of concept, we demonstrated that HT-PBNA is suitable for detection of L2-directed neutralizing antibodies. As discussed above, functional assays are critical for L2 immunity due to the discrepancy of non-functional and functional assays for this antigen. Recently, Day et al. presented a modified In conclusion, we present a protocol for a mostly automated high throughput pseudovirion-based assay for the detection of papillomavirus neutralizing antibodies. The increased sensitivity and reproducibility will allow the design of large, comprehensive studies for evaluating natural and vaccine-induced immunity.


Although contributions of the integrin to the microenvironment cannot be excluded, our studies indicate that α2β1 integrin expression by HPV-transformed keratinocytes modulates SCC growth and progression.Expression of the α2β1 integrin, a receptor for collagens and laminin, is altered during tumor progression. Recent studies have linked polymorphisms in the α2 integrin gene with oral, squamous cell carcinoma (SCC). To determine the α2β1 integrin's role in SCC progression, we crossed α2-null mice with K14-HPV16 transgenic animals. Pathological progression to invasive carcinoma was evaluated in HPV-positive, α2-null (HPV/KO) and HPV-positive, wild-type (HPV/WT) animals. α2β1 integrin expression stimulated progression from hyperplasia and papillomatosis to dysplasia with concomitant dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% in HPV/KO, compared to HPV/WT, animals. To evaluate the integrin-specific impact on the malignant epithelium versus the microenvironment, we developed primary tumor cell lines. Although transition from dysplasia to carcinoma was unaltered during spontaneous tumor development, isolated primary HPV/KO SCC cell lines demonstrated decreased migration and invasion, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cells were orthotopically injected into WT or KO hosts, tumor α2β1 integrin expression resulted in decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage  Arbeit, Coussens, and Hanahan developed a transgenic mouse model of epithelial carcinogenesis in which the HPV 16 early region genes were expressed in basal keratinocytes under the control of the keratin 14 promoter Cancers arise from the accumulation of genetic mutations that alter cell proliferation, differentiation, and tissue organization. Infection with Human Papilloma Virus (HPV) causes 100% of cervical cancer, 90% of anal cancer, 40% of vulvar and vaginal cancer, 15%–35% of oropharyngeal cancers, and approximately 3% of oral cancers in vivo during progression of breast, lung, and prostate cancers et al. demonstrated that human SCC cell lines that expressed high levels of the α2β1 integrin were more invasive than cells with low integrin expression Cancer progression and metastasis do not solely rely upon the genetic and epigenetic events within the tumor cell, but also on changes in the microenvironment To determine the role of α2β1 integrin expression in squamous epithelial carcinogenesis, we chose the K14-HPV16 model for several reasons: 1. Expression of the α2β1 integrin on squamous epithelial cells is regulated in a differentiation-dependent manner. 2. The K14-HPV16 model investigates the complex interplay between the malignant cells and the host immune system, including a requirement for mast cells in the progression towards invasive SCC. 3. Prior data from our laboratory demonstrated α2β1 integrin expression on connective tissue mast cells and showed that the integrin participated in mast cell activation in response to specific pathogens. 4. HPV-stimulated carcinoma is highly relevant to human disease and represents a significant public health burden.in vivo and orthotopic primary tumor cell transplantation in vivo models support a role for α2β1 integrin expression by the HPV oncogene-transformed SCCs in malignant progression. The differences between the spontaneous model and the orthotopic injection model suggest that many factors play a role in tumor progression in vivo.We now show that α2β1 integrin expression promotes early preneoplastic dysplasia from hyperplasia and papillomatosis to dysplasia. Decreased dysplasia in the HPV/KO mice was associated with decreased recruitment of mast cells at early time points. Although, loss of α2β1 integrin expression did not affect tumor latency, prevalence, tumor growth, or histologic grade, metastasis to the regional lymph nodes was decreased by 31.3%. Since these studies were conducted in animals in which the α2β1 integrin was globally deleted, primary tumor cell lines were developed. Isolated, primary HPV/WT, but not HPV/KO SCC cell lines, migrated rapidly and invaded through a matrix of type I collagen. Following orthotopic injection of HPV/WT and HPV/KO cells into either WT or KO mice, HPV/WT SCC cells formed tumors with a short latency and rapid growth. In contrast, HPV/KO SCC cells either failed to form tumors or grew significantly slower than the HPV/WT tumor cells. Integrin status of the host animal did not influence tumor development. Therefore, our data from both the spontaneous Mice were housed in pathogen-free conditions at Vanderbilt University Medical Center in strict compliance with national and institutional animal welfare regulations; all animal experiments were approved by Vanderbilt's IACUC protocol M/10/002.2, where V is the volume, A is the largest diameter, and B is the shortest diameter. Once tumors reached a maximum diameter of at least 10 mm, affected animals were sacrificed, and tumors, blood, ears, and superficial lymph nodes were harvested.The α2β1 integrin-deficient mice on the FVB/N background were crossed with congenic K14-HPV16 transgenic mice, a generous gift from Lisa Coussens, to generate K14-HPV16, α2-null (HPV/KO) or wild-type (HPV/WT) mice Tumor and ear morphology was evaluated on formalin-fixed, paraffin-embedded, and hematoxylin and eosin stained sections. Tumors were classified according to the WHO system of differentiation  Immunohistochemical identification of wide-spectrum cytokeratin expressing metastatic tumor cells was performed on lymph node sections using anti-bovine WSCK antibody, EnVision+ polyclonal labeled polymer, and DAB substrate . Immunofluorescence analysis of primary tumor cells was conducted on cells plated on coverslips with anti-LYVE-1 , anti-WSCK (Dako), and DAPI (Invitrogen). Secondary antibodies included Alexa Fluors 568 and 488 (Invitrogen). Imaging was performed using a Nikon Eclipse 80i microscope and quantitated using ImageJ .Flow cytometric analyses of peripheral blood and infiltrates into ear skin and tumors were performed on a 3-laser, BD LSR II Flow Cytometer in the Vanderbilt Medical Center Flow Cytometry Shared Resource, and data analysis was performed using FlowJo . Tumor-bearing animals were sacrificed and tumor, ear, and peripheral blood leukocytes were collected and processed, as described previously 2.Primary tumors were dissected under sterile conditions, finely minced, and cultured in primary tumor media containing DMEM-F12  supplemented with 5% FBS , 0.01 µg/mL human EGF , 1 µg/mL hydrocortisone , 0.1 µg/mL cholera toxin , 0.3 units of insulin , and 100 units of penicillin/streptomycin  at 37°C in 5% CO3) were plated on either collagen type I  (100 µg/mL), fibronectin (BD Biosciences Discovery Labware) (100 µg/mL), or plastic and incubated in primary tumor media. Cell proliferation was evaluated every 24 hours using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay , according to the manufacturer's protocol. Plates were read at 490 nm on a Molecular Devices Emax Precision Microplate Reader, and data was analyzed using Softmax (Molecular Devices).HPV/WT or HPV/KO tumor cells (2×10The mouse full-length α2 (mα2) integrin subunit (NCBI GenBank Z29987), a gift from Jeffrey Bergelson, was inserted into the mammalian pSRα expression vector. Clones were selected and verified by sequencing. HPV/KO-2 cells were transfected with 20 µg of either pSRα-mα2 or empty pSRα vector using Lipofectamine 2000  according to manufacturer's protocol.4 cells/well) to adhere at 37°C for 1 hour to either collagen type I (20 µg/mL) or fibronectin (20 µg/mL) was determined using established protocols in the presence of 2 mM MgCl2 (Sigma-Aldrich) or 2 mM EDTA (Sigma-Aldrich) The ability of primary tumor cells (1.7×104) in primary tumor media containing 1.5% FBS were placed on the upper membrane of 0.8 µm pore transwell filters , which was either uncoated or coated with collagen type I (100 µg/mL). Primary tumor media containing 5% FBS was placed in the lower chamber. The number of cells migrating to the lower chamber at 37°C after 18 hours was determined by counting the number of cells in 3 random high power (10X magnification) fields.Cell migration and invasion assays were performed using a modification of the protocol described previously 6) derived from two separate HPV/WT or HPV/KO lines were injected subcutaneously into the interscapular region of either WT or KO, 4-5-week-old FVB/N hosts. Tumors were measured twice a week; volumes were calculated as previously described.Tumor cells  and GraphPad Prism .FVB/n background with congenic K14-HPV16 mice to generate K14-HPV16/wild-type (HPV/WT) and K14-HPV16/α2-null (HPV/KO) mice. Preneoplastic progression, including hyperplasia, papillomatosis, or dysplasia, was defined in the ear skin of HPV/WT and HPV/KO mice at 3-, 6-, or 9-months-of-age, or at the time of sacrifice due to the development of invasive, squamous carcinoma at another location. By 6-months-of-age, there were significant differences in dysplasia and papillomatosis between the two genotypes: approximately 15% of HPV/WT animals (n = 20), but none of the HPV/KO animals (n = 25), developed dysplasia. In contrast, the incidence of papillomatosis was almost double in the HPV/KO animals (p = 0.0384). Differences in papillomatosis and dysplasia between HPV/KO and HPV/WT ears were also present at later time points   and finally to invasive and metastatic cancer 0.00169) .Inflammation has been shown to be responsible for driving neoplastic progression in K14-HPV16 transgenic animals To determine the impact of α2β1 integrin expression on progression from dysplasia to invasive carcinoma, tumor latency and prevalence in HPV/KO and HPV/WT animals were determined. Tumor latency was similar in HPV/KO and HPV/WT animals (p = 0.11) . No diffPrevious studies demonstrated that α2β1 integrin expression may be associated with normal, regulated, epithelial differentiation and that altered expression of the integrin may be seen in different subtypes of cancer. To determine whether α2β1 integrin expression or lack thereof affected tumor cell differentiation, invasive HPV/WT and HPV/KO tumors were analyzed histologically. SCC differentiation was graded based on the Broder's four-tier system Previous studies have shown that approximately 30% of SCCs in the K14-HPV16 mouse metastasize to regional lymph nodes 2+ dependent and EDTA2+-inhibitable manner, as did a positive control, NMuMG-X2C2 (derived from the NMuMG3 line stably transfected with full length human α2 integrin subunit)  and invaded through a barrier of type I collagen (p<0.0001)  and two HPV/KO (HPV/KO-1 and HPV/KO-2) squamous carcinoma cell lines were developed. The epithelial origin of the tumor cells was confirmed by cytokeratin staining . The HPVsubunit)  [40]. Alsubunit) . Therefo<0.0001) .+) or control vector (HPV/KO-2-VC). As determined by flow cytometric analysis, HPV/KO-2-mα2+ cells expressed high levels of the murine α2β1 integrin   . Restora= 0.015) .To determine the impact of α2β1 integrin expression by the tumor cells on tumor growth and latency, the primary tumor cell lines derived from HPV/WT and HPV/KO animals (HPV/WT-1 and -2 and HPV/KO-1 and -2) were orthotopically injected into nontransgenic, wild-type or α2-null mice. The HPV/WT tumor cells grew rapidly when placed in either wild-type or α2-null mice. In contrast, the HPV/KO tumor cells demonstrated increased latency (p = 0.0003) and markedly decreased tumor growth rates (p = 0.034) when compared to mice injected with HPV/WT SCC cells, regardless of recipient mouse integrin status . The shoin vivo, primary tumor cells derived from HPV/KO animals demonstrated diminished cell migration and invasion in vitro and decreased tumor formation and growth when implanted orthotopically into non-K14-HPV16 transgenic wild-type or α2-null animals. Additionally, the host's integrin status did not impact tumor formation or growth, thereby suggesting that α2β1 integrin expression by the tumor microenvironment is not responsible for tumor progression in this model.Using the K14-HPV16 cancer model, we demonstrate that lack of α2β1 integrin expression results in decreased progression from epithelial papillomatosis to dysplasia, increased formation of sebaceous adenocarcinomas instead of SCCs, and modestly decreased lymph node metastasis. Although global loss of the α2β1 integrin in all HPV/KO mouse cells did not affect tumor latency, growth, or multiplicity in vivo murine studies, demonstrating that dysregulated expression of the α2β1 integrin did not alter malignant conversion in SCC, α2β1 integrin expression in the K14-HPV16 model did not affect later aspects of tumor progression Diminished epithelial dysplasia and enhanced papillomatosis in HPV/KO mice suggest that the α2β1 integrin plays a role in regulating epithelial differentiation and promoting the initial steps of neoplasia. The mast cell reduction in 6-month-old HPV/KO mice may promote papillomatosis. On one hand, the reduction in mast cells may limit the further progression of papillomas to carcinoma. On the other hand,mast cell deficient animals have been shown to be more susceptible to papilloma formation than their wild-type counterparts in other models vivo, when primary squamous carcinoma cells isolated from HPV/WT or HPV/KO mice were reintroduced orthotopically into either non-K14-HPV16 transgenic, wild-type or α2-null animals, the HPV/WT tumor cells, but not the HPV/KO tumor cells engrafted and grew rapidly. The HPV/WT tumor cells were significantly more migratory and invasive in vitro. Integrin loss on SCC cells resulted in reduced migration but even more striking deficiencies in invasion through collagen type I. in vivo, thus preventing the analysis of integrin rescue in a SCC model of in vivo tumor formation and growth.Although no difference in SCC progression was noted in In patients with SCC, the development of lymph node metastasis is a predictor of poor outcome The discordant contributions of the α2β1 integrin to metastasis in the HPV-stimulated model of SCC versus the neu-driven model of breast cancer raise interesting questions. First, the two models deal with distinctly different subtypes of carcinoma, SCC and adenocarcinoma, which arise from two disparate cells types. Perhaps the α2β1 integrin's role in regulating the multistep process of tumorigenesis is different depending on the cell of origin. Second, the metastatic route and mechanisms of dissemination are different between the two disease models. In the K14-HPV16 model, SCCs primarily metastasize via the lymphatics to regional lymph nodes. In the MMTV-neu model, cancer metastasizes primarily via the hematogenous route to the lungs. Third, the two tumor models are driven by different oncogenes that function distinctly. K14-HPV16 oncogenesis is triggered by expression of early region HPV16 oncogenes, E6 and E7, which inactivate two important tumor suppressor genes p53 and retinoblastoma, respectively. In contrast, the MMTV-Neu model of mammary cancer is driven by overexpression of the Neu tyrosine kinase that stimulates activation of the ras/map kinase cascade. Fourth, progression to carcinoma in the K14-HPV16 model requires the protumorigenic activity of inflammatory cells. Neither tumorigenesis nor metastasis in the MMTV-Neu model is dependent on recruitment of inflammatory cells. Fifth, the interaction of cancer cells with the specific collagen content of their microenvironment may be different. SCCs expressing the α2β1 integrin may have increased migration and invasion along collagen type I fibers in the skin, whereas receptor ligation with collagen type I may not be as important in breast cancer cell motility.In summary, the α2β1 integrin plays a complex role in tumor progression through its contributions to both the malignant epithelial cell and within the tumor microenvironment. Our data are the first to suggest that integrin dependent regulation of tumor progression may be specific to the tissue type and to the mechanism of oncogenesis. In c-neu/HER2-positive breast cancer, the α2β1 integrin is a metastasis suppressor. In contrast, the α2β1 integrin promotes tumor metastasis in HPV-induced squamous cancer, likely by increasing the migratory and invasive ability of cells along collagen type I.Figure S1The K14-HPV16 transgene, not the α2β1 integrin, mediates a robust inflammatory response.A–C, Flow cytometric analysis of inflammatory cells was performed on the blood and preneoplastic ears of non-K14HPV16 transgenic wild-type (WT Ctrl) or α2-null (KO Ctrl) mice and HPV/WT and HPV/KO animals, either with (SCC+) or without tumors (SCC−). Similar analysis was also performed on the tumor tissue of HPV/WT and HPV/KO mice. The percentage of inflammatory cell subsets in HPV/WT and HPV/KO animals was compared to non-transgenic controls. Inflammation was highly dependent upon the presence of the K14-HPV16 transgene. Loss of the α2β1 integrin in HPV/KO ears increased the percentage of NK1.1+ cells relative to HPV/WT ears in non-tumor bearing animals (p = 0.014). Additionally, there was a significant increase in CD3ε+ T cells in HPV/KO tumor infiltrates, when compared to HPV/WT SCCs (p = 0.033). (TIF)Click here for additional data file.Figure S2Loss of the α2β1 integrin does not alter SCC growth, multiplicity, or grade.A, Tumor volumes were measured weekly. The rate of tumor growth over time was calculated from tumor volume regression slopes and plotted as a function of time. No significant differences existed in the rates of SCC growth between HPV/WT (n = 22) and HPV/KO (n = 22) mice (p = 0.37). B, Total tumor burden for each HPV/WT (n = 97) and HPV/KO mouse (n = 73) was quantitated at the time of sacrifice. No significant differences were found for the multiplicity of tumor development (p = 0.45). C, Since multiple tumors may form on an animal, the highest grade scored was considered for analysis of differentiation loss. No significant differences were observed when considering the highest grade of SCC that developed in HPV/WT (n = 97) or HPV/KO (n = 73) mice (p = 0.57).(TIF)Click here for additional data file.Figure S3In vitro proliferation of primary SCC cells was unaffected by loss of the α2β1 integrin. Proliferation of the HPV/WT-1 and -2 and HPV/KO-1 and -2 SCC lines when adherent to collagen, fibronectin, or tissue culture plastic was determined in vitro. Proliferation in vitro of HPV/WT and HPV/KO lines was similar irrespective of the matrix .(TIF)Click here for additional data file.Table S1Detailed Analysis of Inflammatory Cell Populations in Blood, Preneoplastic Ears, and Tumors. WT Ctrl and KO Ctrl animals were used to verify and establish baseline inflammatory populations independent of the K14-HPV16 transgene. Chi2 probability with ties analysis was performed on all 6 groups for each specific tissue; those found to be significant or close to p<0.05 were analyzed further through inter-comparison of the 6 groups by Mann-Whitney tests. The groups in which significance was found are denoted as Genotype 1 vs. Genotype 2. Differences in inflammatory cells were found between non-K14-HPV16 transgenic, control animals and those expressing the K14-HPV16 transgene. Integrin-dependent differences were identified in the NK1.1-positive and CD3ε-positive cell populations. Non-neoplastic ear tissue in HPV/KO, SCC− mice had increased NK1.1-positive cells than HPV/WT, SCC− ears (p = 0.014). HPV/KO SCCs contained more CD3ε-positive cells than HPV/WT tumors (p = 0.033). T regulatory cells were defined as CD4, CD25, and Foxp3 triple-positive cells as a percentage of CD4-positive cells. . * represents p<0.05 ** represents p<0.001 *** represents p<0.0001.(DOCX)Click here for additional data file.


Data on the current burden of adenocarcinoma (ADC) and histology-specific human papillomavirus (HPV) type distribution are relevant to predict the future impact of prophylactic HPV vaccines.We estimate the proportion of ADC in invasive cervical cancer, the global number of cases of cervical ADC in 2015, the effect of cervical screening on ADC, the number of ADC cases attributable to high-risk HPV types -16, -18, -45, -31 and -33, and the potential impact of HPV vaccination using a variety of data sources including: GLOBOCAN 2008, Cancer Incidence in Five Continents (CI5) Volume IX, cervical screening data from the World Health Organization/Institut Català d'Oncologia Information Centre on HPV and cervical cancer, and published literature.ADC represents 9.4% of all ICC although its contribution varies greatly by country and region. The global crude incidence rate of cervical ADC in 2015 is estimated at 1.6 cases per 100,000 women, and the projected worldwide incidence of ADC in 2015 is 56,805 new cases. Current detection rates for HPV DNA in cervical ADC tend to range around 80–85%; the lower HPV detection rates in cervical ADC versus squamous cell carcinoma may be due to technical artefacts or to misdiagnosis of endometrial carcinoma as cervical ADC. Published data indicate that the five most common HPV types found in cervical ADC are HPV-16 (41.6%), -18 (38.7%), -45 (7.0%), -31 (2.2%) and -33 (2.1%), together comprising 92% of all HPV positive cases. Future projections using 2015 data, assuming 100% vaccine coverage and a true HPV causal relation of 100%, suggest that vaccines providing protection against HPV-16/18 may theoretically prevent 79% of new HPV-related ADC cases  and vaccines additionally providing cross-protection against HPV-31/33/45 may prevent 89% of new HPV-related ADC cases .It is predicted that the currently available HPV vaccines will be highly effective in preventing HPV-related cervical ADC. Invasive cervical cancer (ICC) is one of the leading causes of cancer in women and according to global estimates from 2008, there are approximately 530,000 new cases and 275,000 deaths annually . Cervicain situ (AIS) [The natural history of cervical ADC is very different from that of SCC and this may explain the lower rates of detection of premalignancies during cytologic screening. The earliest precursor lesions of ADC are more difficult to define than those of SCC; lesions are more diverse, and invasive ADC is thought to develop particularly from a small focus of adenocarcinoma tu (AIS) ,11,12. Atu (AIS) . As a retu (AIS) -19.Cervarix®) has also shown significant type-specific cross-protection for several non-vaccine oncogenic HPV types, including HPV-31, -33, -45 and -51, using both virological and histopathological endpoints [Gardasil®), significant cross-protection has also been reported for HPV-31, but not for other oncogenic HPV types to date [Two prophylactic human papillomavirus (HPV) vaccines for the prevention of cervical cancer are now available and licensed in more than 100 countries. In clinical trials, these vaccines have shown good efficacy against high grade cervical lesions associated with HPV-16 and/or -18 -22. The ndpoints ,23. For  to date .However, no data are currently available regarding the efficacy of these vaccines specifically against ADC. Although AIS lesions were included within the definition of high-grade lesions in clinical trials evaluating the efficacy of these vaccines, the number of such lesions was relatively small ,25. TherIn this article, we estimate the global burden of disease due to ADC, investigate the effect of cervical screening on ADC, summarize data on HPV type-distribution in ADC and estimate the potential impact of HPV vaccination on ADC.The projected number of ADC cases in 2015 was estimated using data from a number of published sources. Firstly, estimates of ICC cases by country were downloaded from GLOBOCAN 2008 . The IARIn IARC CI5, ASC was categorized as “other” specified carcinoma rather than ADC, therefore, it was not possible to estimate the global burden of disease due to ADC including ASC .Registry-specific age-standardized incidence rates (ASIRs) of ADC are now published by the IARC in CI5 volume IX . WeighteData on screening coverage rates from cervical screening programs collated by World Health Organization/Institut Català d'Oncologia (WHO/ICO) Information Centre on HPV and Cervical Cancer were used to investigate whether there was an effect of cervical screening on the proportion of ADC among ICC cases  by countWe summarized the global HPV type distribution in cervical ADC using data from two published sources ,30. The 10 broad-spectrum primers [SPF10-DEIA/LiPA25-polymerase chain reaction (PCR) system . HPV prevalence data (19 high-risk and 8 low-risk types) were reported for those cases which were positive for HPV DNA, and data analyses included algorithms of multiple infections to estimate type-specific relative contributions.The second source was a retrospective cross-sectional study, performed by de Sanjosé and colleagues, of 10,575 cases of ICC (951 cases of ADC or ASC) paraffin-embedded tissue blocks collected between 1949 and 2009 from worldwide historic archives in 38 countries . AlthougCrude data from Li and colleagues were adjusted to facilitate comparison across the two data sources. Firstly, data were adjusted so that each HPV type-specific prevalence was expressed as a proportion of HPV positive cases, i.e., each crude estimate was multiplied by 100/82 . SecondFinally, to consolidate the HPV-type specific estimates from these two sources ,30 into Gardasil® and Cervarix®) were sourced from published data from double-blind, randomized, controlled, Phase III clinical studies [Cervarix® and Gardasil®, respectively, due to adjustments for multiplicity. 95% confidence intervals were used for estimates of vaccine efficacy against other HPV types. Estimates of efficacy from these published sources were as follows: 98% (86 to 100%) associated with HPV-16 and -18 [Vaccine efficacies against HPV types -16, -18, -45, -31 and -33 for the two currently available licensed HPV vaccines . This trend was not strong and was influenced by Finland . When developed and developing countries were considered separately .Information regarding cervical screening coverage was available from 48 countries  of the 61 countries that had data reported for the proportion of ADC in ICC  cases of ADC and 392,688  cases of SCC per year for a vaccine providing protection against HPV-16, -18 -45, -31 and -33  and low viral load [10 and other less sensitive PCR based detection methods [In the two data sources we selected, HPV DNA positivity in cases of ADC (including ASC) were 65.6%  and 82.0ral load . Li and ral load , which i methods -41.10[The low HPV detection rates may also be partly attributed to misdiagnosis of endometrial carcinoma as cervical ADC ,43, but 10,49.If any falsely HPV DNA negative samples were attributable to a particular HPV type, the missing HPV types could theoretically bias the overall HPV type distribution . AlthougAlphapapillomavirus 9  species characterized by HPV-16 and including types -31, -33, -35, -52 and -58 and the alpha-7 species characterized by HPV-18 and including types -39, -45, -59 and -68 [HPV types are traditionally grouped into phylogenetically-related species based on the genetic similarity of their L1 genes . High-ri and -68 . It is w and -68 .The incidence of multiple infections amongst overall ICC was comparable in both data sources   of routiPrevention of cervical ADC through detection of preneoplastic stages has proved to be largely unsatisfactory, as observed by the high proportion of ADC in well-screened populations. We predict that the currently available HPV vaccines will be highly effective in preventing cervical HPV-related ADC. Extended protection against HPV-45, -31 and -33 would also be of benefit.Cervarix is a registered trade mark of the GlaxoSmithKline group of companies.Gardasil is a registered trade mark of Merck & Co Inc.in situ; ASC: Adenosquamous carcinoma; ASIR: Age-standardized incidence rate; CI5: Cancer Incidence in Five Continents; IARC: International Agency for Research on Cancer; ICC: Invasive cervical carcinoma; HPV: Human papillomavirus; R2: Coefficient of determination; SCC: Squamous cell carcinoma; WHO/ICO: World Health Organization/Institut Català d'Oncologia.ADC: Adenocarcinoma; AIS: Adenocarcinoma All costs related to the development of this manuscript were met by GlaxoSmithKline Biologicals SA.JMP is employed by GlaxoSmithKline Research and Development, and holds shares in GlaxoSmithKline; CG and SMT are employed by GlaxoSmithKline Vaccines, and hold shares in GlaxoSmithKline. DJ is a former employee of GlaxoSmithKline Vaccines and has in the past acted as a consultant to GlaxoSmithKline Vaccines. He has not received payment for work on this study.JMP and DJ conceived the investigation. JMP and CG conducted the analyses. JMP, CG and SMT drafted the manuscript. DJ critically reviewed the manuscript and revised it for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/553/prepub


Inverted ductal papilloma of the oral cavity is an infrequent benign neoplasm of papillary appearance that originates in the secretory duct of a salivary gland. The etiology is unknown, though some authors have related it to human papillomavirus (HPV) infection. We present the case of a 40-year-old woman with a tumor of the lower lip mucosa. Histopathological study of the lesion diagnosed inverted ductal papilloma of the oral cavity. Human papillomavirus DNA detection and typing based on tumor lesion DNA amplification and posterior hybridization, revealed no presence of viral DNA. The antecedents of trauma reported by the patient could have played an important role in the development of this tumor. Key words:Inverted ductal papilloma, intraductal papilloma, oral papilloma, papillary epidermoid adenoma. Oral papilloma is a benign tumor located in the oral mucosa. It normally shows exophytic growth with a poly-poid or verrucous appearance. However, there have been reports of cases characterized by endophytic or inverted growth , most ofA 40-year-old caucasian woman was reported to our service of Oral Surgery for evaluation and treatment of a tumor located in the lower left lip mucosa. She had the habit of nibbling her lower lip, and had suffered trauma in this same region, upon impacting with the head of her young child. After three months the patient noticed slight changes in the size of the lesion. She appeared to be in good health, and no maxillary asymmetries or tumors, or neck adenopathies were detected. At clinical exploration, the lesion showed a nodular aspect, of soft consistency, with no adherence to deep layers, and no pain in response to palpation Fig. . There w- Differential diagnosisBased on the location, the history of trauma and the clinical characteristics of the lesion, a lower lip mucocele was initially diagnosed. However, the differential diagnosis also included other conditions such as pleomorphic adenoma, traumatic fibroma and lipoma.- Diagnosis and treatmentComplete removal of the lesion was carried out with the CO2 laser under local anesthesia using 4% articaine with 1:100.000 adrenalin . The specimen sent to the pathology laboratory was of a pinkish color, rounded and measured 1 x 0.5 cm in size. The following postoperative medication was prescribed: ibuprofen 600 mg in tablets, one every 8 hours for 5 days  and chlorhexidine gel, three applications a day for 10 days . The postoperative course was free of complications, and the patient is presently subjected to periodic controls to detect possible relapse.- Histological studyThe surgical specimen was stained with hematoxylin-eosin (HE) and examined under an Olympus CX31RBSF light microscope . The histological study revealed an endophytic lesion with extensive, rounded and regular margins, exhibiting an expansive growth pattern. The lesion was located in the transition zone between the secretory duct of a minor salivary gland and the surface epithelium of the oral cavity Fig. . The tum- Detection of HPVDNA extraction was carried out from the sample fixed in formalin solution and embedded in paraffin, based on the conventional technique. HPV detection was carried out with the HPV LA genotyping test . This process involves amplification of the viral DNA sequence via polymerase chain reaction (PCR) and posterior hybridization using probes corresponding to a total of 37 HPV subtypes – 18 of which are considered to be of high risk , and 19 of low risk . The procedure for typing HPV DNA was carried out based on the same methodology as described above, and using the primers PGMY 09/11 for viral DNA amplification, and the primers PCO3, PCO4 and PCO5 for amplifying the β-globin gene, which was used as internal control. The biotinylated amplicons were denaturalized with 0.4 N NaOH and hybridized in an immobile matrix with probes corresponding to 37 different HPV subtypes, according to the protocol recommended by the manufacturer . Hybridization positivity was detected by streptavidin – horseradish peroxidase precipitation onto the probe membrane. The sample DNA was correctly amplified, though no HPV DNA was detected.Inverted ductal papilloma of the oral cavity (IDPOC) was first described in 1982 by White et al. . Regezi The patient age at appearance of the lesion is usually between the fourth and fifth decade of life and no particular gender predilection has been observed ,5. In ou


KAI1 was initially identified as a metastasis-suppressor gene in prostate cancer. It is a member of the tetraspan transmembrane superfamily (TM4SF) of membrane glycoproteins. As part of a tetraspanin-enriched microdomain (TEM), KAI1 inhibits tumor metastasis by negative regulation of Src. However, the underlying regulatory mechanism has not yet been fully elucidated. CUB-domain-containing protein 1 (CDCP1), which was previously known as tetraspanin-interacting protein in TEM, promoted metastasis via enhancement of Src activity. To better understand how KAI1 is involved in the negative regulation of Src, we here examined the function of KAI1 in CDCP1-mediated Src kinase activation and the consequences of this process, focusing on HIF-1 α and VEGF expression.μg/ml G418. Protein levels were assessed by immunoblotting and VEGF reporter gene activity was measured by assaying luciferase activitiy. We followed tumor growth in vivo and immunohistochemistry was performed for detection of HIF-1, CDCP1, and VHL protein level.We used the human prostate cancer cell line PC3 which was devoid of KAI1 expression. Vector-transfected cells (PC3-GFP clone #8) and KAI1-expressing PC3 clones (PC3-KAI1 clone #5 and #6) were picked after stable transfection with KAI1 cDNA and selection in 800 in vivo xenograft model, KAI1 inhibited the expression of CDCP1 and HIF-1α.We demonstrated that Hypoxia-inducible factor 1α (HIF-1α) and VEGF expression were significantly inhibited by restoration of KAI1 in PC3 cells. In response to KAI1 expression, CDCP1-enhanced Src activation was down-regulated and the level of von Hippel-Lindau (VHL) protein was significantly increased. In an These novel observations may indicate that KAI1 exerts profound metastasis-suppressor activity in the tumor malignancy process via inhibition of CDCP1-mediated Src activation, followed by VHL-induced HIF-1α degradation and, ultimately, decreased VEGF expression. More than 20 metastasis-suppressor genes encoding products with specific metastasis-suppressing functions have been discovered . KAI1 , CD81, and CD9 . CDCP1 aRecent studies have revealed that KAI1 attenuates EGFR, integrin, and c-Met signaling, confirming the involvement of KAI1 in the intracellular signaling pathways associated with these molecules ,5. Moreoin vivo tumor xenograft model. These novel findings may indicate that KAI1 functions as a profoundly effective metastasis suppressor in the process of tumor malignancy and angiogenesis through inhibition of CDCP1-mediated Src activation and a subsequent dramatic reduction in HIF-1α and VEGF expression via functional activation of VHL. These studies may shed light on the mechanism by which KAI1 suppresses prostate cancer metastasis.To better understand how KAI1 is involved in the negative regulation of Src and inhibition of metastasis, we postulated an interrelationship between KAI1 and CDCP1 in regulation of Src activity in TEM. To test this supposition, we here examined the function of KAI1 in CDCP1-mediated Src kinase activation and the consequences of this process, focusing on VEGF expression. Restoration of KAI1 expression in PC3 human prostate carcinoma cells blocked Src activation through negative regulation of CDCP1. Notably, HIF-1α and VEGF expression were significantly inhibited by KAI1-mediated Src inactivation and subsequent VHL up-regulation. Importantly, KAI1 inhibited the expression of CDCP1 and HIF-1α in an ® CRL-1435) were maintained in RPMI medium 1640 supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. Before exposure to hypoxia, culture medium was replaced by a thin layer of fresh medium (0.15 ml/cm2). Cells were transferred to a Bactron Anaerobic/Environmental Chamber , which was flushed with 1% O2, 5% CO2, and 95% N2 at 37°C. In hypoxia-mimic experiments, cells were incubated with 100 μM of CoCl2 under normoxic conditions.PC3 cells . For stable transfection of KAI1, PC3 (KAI1-/-) cells were transfected with GFP-KAI1 expression plasmid. Vector-transfected cells (PC3-GFP clone #8) and KAI1-expressing PC3 clones (PC3-KAI1 clone #5 and #6) were picked after selection in 800 μg/ml G418.Plasmid of GFP tagged KAI1 was gifted by Dr. Kyung Keun Kim . VEGF-luc promoter-reporter construct (2.2 kb) and HA tagged VHL expresson vector were also gifted by Dr. Joohun Ha  and Dr. Hong-Duk Youn  respectively. HIF-1α expression plasmid was provided by Addgene. Predesigned small interfering RNA (si-RNA) for KAI1, VHL, and PKCδ were purchased from Santa Cruz Biotechnology, Inc . Si-RNA for CDCP1 was purchased from Dharmacon . For transient transfection, PC3 cells were plated on 6-well plate 1 day before transfection as a density of 10397 ; anti-CDCP1, anti-phospho-p130CASY410, anti-phospho-SrcY416, anti-Src, and anti-VHL ; anti-GFP, anti-PKC δ, anti-KAI1, anti-HA, anti-FAK, anti-snail, anti-VHL, anti-paxillin, and anti-β actin .For polyacrylamide gel electrophoresis (PAGE), the cells were washed with PBS twice and extracted in RIPA buffer . Cell lysates were centrifuged and supernatants were collected. For immunoprecpiation, cell extracts were incubated with antibody (1:1000 dilutions) with constant agitation at 4°C overnight. After electrophoresis, proteins were transferred to nitrocellulose membrane. Membranes were blocked with 1% bovine serum albumin. Then, protein levels were detected using the following commercial antibodies: anti-HIF-1 α, anti-integrin β1, anti-E-cadherin, anti-N-cadherin, and anti-FAKCell migration assays were performed using Boyden chamber as previously described . Cells wCell invasion assay was performed on a matrigel invasion chamber . This experiment was set up identically to the cell migration assay. After 72 hours, cells remaining upper face of the membrane were scraped using a cotton swab and cells that had invaded to the lower face of the membrane were stained with DiffQuick  Wright-Giemsa Solution.Total RNA was extracted with Trizol reagent . The cDNA fragment was amplified by PCR using following specific primers: CDCP1, sense 5'-CTTCAACCTCTCCAACTGTG-3' and antisense 5'-TGGTCTGTGCAGCTTATGGT-3', HIF-1α, sense 5'-CTTGCTCATCAGTTGCCACTT-3', and antisense 5'-GCCATTTCTGTGTGTAAGCAT-3'; VEGF, sense 5'- CGAAGTGGTGAAGTTCATGGATG-3', and antisense 5'- TTCTGTATCAGTCTTTCCTGGTGAG -3'; GLUT1, sense 5'-CGGGCCAAGAGTGTGAA-3', and antisense 5'- TGACGATACCGGAGCCAATG-3', GAPDH, sense 5'-TGCTGAGTATGTCGTGGAGTCTA-3', and antisense 5'-AGTGGGAGTTGCTGTTGAAGTCG-3'; β-actin, sense 5'-GTGGGGGCGCCCAGGCACCA-3', and antisense 5'-CTCCTTAATGTCACGCACCATTTC-3'. PCR was initiated in a thermal cycle programmed at 95°C for 5 min, 94°C for 45 s, 60°C for 2 min, and 72°C for 2 min and amplified for 30 cycles. The amplified products were visualized on 1% agarose gels.5 cells/well), and the following day cells were co-transfected with a VEGF-luc promoter-reporter construct and KAI1 expression vector. Forty-eight hours after transfection, reporter gene activity was measured by assaying luciferase activitiy , and normalized to β-galactosidase activities .PC3 cells were plated onto 6-well plates . Tumor measurements were done with precision calipers and animals were sacrificed after 35 days of injection. Experiments were conducted according to the guidelines for ethical use of animals of our Institution under an approved protocol . Tumors were harvested and fixed overnight in 10% buffered formalin, embedded in paraffin, and sectioned. Primary tumor volumes were calculated with the formula: V = length × (width)2O2 followed by blocking in goat serum and primary incubation at 4°C overnight. The slides were then washed in Tris-NaCl buffer and incubated at room temperature for 60 minutes with anti-CDCP1 antibody  at a 1:50 dilution, anti-HIF-1 antibody  at a 1:50 dilution, anti-VHL antibody  at a 1:50 dilution. Slides were rinsed in Tris-NaCl buffer and incubated for 60 minutes with a biotinylated secondary antibody, biotin polyclonal anti-rabbit immunoglobulin (for CDCP1 and VHL)  at a 1:200 dilution, or biotin polyclonal anti-mouse immunoglobulin (for HIF-1)   and 3,3'-diaminobenzidine (DAB)-H2O2 substrate . Slides were then counterstained with hematoxylin, dehydrated through graded alcohols and xylene and mounted. Slides were studied and imaged under brightfield microscopy.Deparaffinized sections were rehydrated and antigen retrieval was performed by 15 minutes incubation in warm trypsin followed by microwave in 10 mM citrate buffer for total of 10. Slides were then blocked with 3% Hp- values were < of 0.05. Densitometry of immunoblots was performed by scanning of the exposed film and using The Quantity one 1-D analysis software Data are expressed as means ± standard deviations (SD) of at least three experiments. Statistical significance was determined using Student's t-test for comparisons between two means. The null hypothesis was rejected in cases where 416) was reduced by KAI1 expression. To provide further support for this interpretation, we also monitored the Src substrates, FAK and p130Cas. The level of the tyrosine-phosphorylated, active forms of FAK and p130Cas were decreased by KAI1 expression , which mimics hypoxia, increased HIF-1α levels in PC3 cells, an effect that was dramatically inhibited by transient or stable expression of KAI1. Next, GFP vector clones (PC3-GFP#8) and KAI1 stable transfectants (PC3-KAI1 #5 andPC3-KAI1 #6) were exposed to hypoxia (1% O2) for 24 hours, then the effects of KAI1 on hypoxia-induced HIF-1α up-regulation were determined. Similar to the effects observed with CoCl2, HIF-1α protein levels under hypoxic conditions were significantly decreased by KAI1 expression  induced an increase in VEGF promoter activity that was reduced by KAI1 expression , a metastasis-suppressor gene initially discovered in prostate cancer, inhibits Src and affects the metastasis of prostate carcinoma. Clinical cancer studies have linked a high level of KAI1 expression with a good prognosis. Conversely, the loss of KAI1 expression is correlated with metastasis in tumor progression ,5. HowevCurrent studies have proposed that KAI1 functions to inhibit Src kinase activity, but the identified mechanisms have been limited to associations with integrins, EGFR, and c-Met . It has Together with KAI1, CDCP1 has been reported to be a component of the TEM in colon cancer, but its function in this context has remained poorly characterized . CDCP1 iCDCP1 gene regulation has been investigated in various cancer cell lines. PC3 cells show abundant expression of CDCP1 protein and a low frequency of methylation in transcription-initiation sites . We founin vivo xenograft model, which showed that KAI1-expressing tumor volumes were significantly reduced compared with those of controls (Figure Angiogenesis is an indispensible step in the progression of prostate cancer, and VEGF has emerged as a critical proangiogenic growth factor in prostate carcinogenesis ,42,43. Hs Figure . Moreoves Figure . How KAIs Figure . VHL proWe report here that KAI1 acts in the process of tumor malignancy and angiogenesis by profoundly suppressing metastasis via inhibition of CDCP1-enhanced Src activation and functional activation of VHL. This KAI1-induced VHL activation causes HIF-1α degradation and, ultimately, down-regulation of VEGF expression. These results provide insight into how KAI1 suppresses prostate cancer metastasis. Although we focused our efforts here on CDCP1-Src inhibition by KAI1 and their effects on VEGF expression, KAI1 expression may also influence other key processes in prostate carcinogenesis.CDCP1: CUB-domain-containing protein 1; HIF-1α: Hypoxia inducible factor 1 α; VHL: Von Hippel-Lindau; VEGF: Vascular Endothelial Growth Factor; TEM: Tetraspanin-Enriched Microdomain; GAPDH: Glyceraldehydes-3-phosphate dehydrogenase; GLUT1: Glucose Transporter 1; siRNA: small interfering RNAThe authors declare that they have no competing interests.JJP performed experiments; YBJ did tumor xenograft and immunohistochemistry; YJL helped performing experiments. JSL helped designing the research; YSL helped designing the research; YGK designed, analyzed data, and wrote paper; ML designed, analyzed data, and wrote paper. All authors have read and approved this manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/81/prepub


We conducted a baseline prevalence survey of unvaccinated 11- to 18-year olds to inform effectiveness studies for the new human papillomavirus (HPV) immunisation programme in Scotland.Participants were recruited from schools and colleges and invited to provide demographic data and an anonymous urine sample for type-specific HPV testing.P=0.045). Among males aged 11–14 years, the weighted prevalence was 1.4% overall; 1.0% for high-risk types and 0.7% for types 16 and 18. Among 15- to 18-year old males, the weighted prevalence was 3.9% overall; 2.4% for high-risk types and 0.7% for types 16 and 18.Among females aged 11–14 years, the weighted prevalence was 1.1% overall; 0.9% for high-risk types and no infections were associated with types 16 and 18. Among 15- to 18-year old females, the weighted prevalence was 15.2% overall; 12.6% for high-risk types and 6.5% for types 16 and 18. Among females aged 16–18 years, infection was more frequently associated with attending college and rural schools, and showed a trend towards increasing prevalence with increasing social deprivation (Human Papillomavirus prevalence is low among 11- to 14-year olds, which includes the age group targeted for routine vaccination. The prevalence in males and correlation with deprivation require further investigation. Persistent infection with high-risk types of human papillomavirus (HPV) is recognised as the principal cause of cervical cancer, the second most common cancer among women globally . CurrentIn Scotland, the bivalent HPV vaccine, which protects against the high-risk HPV types 16 and 18, was introduced into the national immunisation schedule in September 2008, targeting females aged 12–13 years. To facilitate the ongoing monitoring of HPV infection in vaccinated cohorts, before the introduction of the immunisation programme, we undertook a comparative baseline cross-sectional survey of unvaccinated Scottish adolescents to measure the type-specific prevalence of HPV infection and its association with age, sex and social deprivation. We report here the findings of this survey, which we believe is the first population-based prevalence survey of HPV infection among adolescent males and females in the United Kingdom and one of only a few surveys  attending publicly funded (state) and private fee-paying (independent) schools. Approximately 35% of young people in Scotland leave school by the age of 16 years , and leaWe sampled education authorities (EAs) using population proportional to size and, within them, we invited all state and independent schools and colleges to participate. The target sample was stratified by age, with oversampling in older age groups. We aimed to recruit a sample size that would provide sufficient power to estimate the overall prevalence of HPV infection, the prevalence by age, sex and social deprivation and the specific genotypes in circulation. For state schools, we collected data on the denominational (religious) affiliation of the school and whether it was in an urban or rural setting. The achieved sample had a precision of ±3% on the estimated prevalence of HPV and a power of 80% to detect differences in prevalence of at least 8% between the two subgroups with 1000 participants.Participants gave written informed consent, as did the parents of students aged <16 years. All the students within a selected year were invited to a presentation given by the study team, in which they were asked to take part in the study by providing an anonymous specimen of urine for HPV testing along with information on their age, sex and postal code using a standardised data-collection form. College students were recruited directly on campus on the designated study days. Samples and data forms were labelled and linked with a unique anonymous study number. Participants were not given their results. A urine sample was chosen, as it is a non-invasive method to access the population of interest.As urine contains relatively few cells, an HPV assay  with a high analytical sensitivity was used . This asData were entered into a Microsoft Access database. Postal codes were automatically assigned a point on the Scottish Index of Multiple Deprivation (SIMD)  at data χ2-tests. Prevalence estimates and 95% CIs were calculated. A crude and weighted stratified analysis of HPV prevalence by sex was conducted using the survey package in R, with schools nested within EAs. Sample weights were calculated to account for the sampling proportional to size of the EA and response bias. We compared the prevalence of HPV in 11- to 14-year olds  with the prevalence in 15- to 18-year olds (which includes those targeted as part of the catch-up immunisation campaign). The percentage of infections and 95% CIs due to infections with the bivalent vaccine types 16 and 18, with high-risk types and with multiple types were calculated as ratio estimates for females aged 15–18 years. Weighted odds ratios (OR) for HPV infection were calculated using logistic regression accounting for the survey design. Two separate regressions were carried out, one restricted to females attending schools and another on all females aged 16–18 years (including those attending college), as there is confounding between age, attending a college and deprivation. No regression analysis was carried out using the data for males and females together, as the data for males were inconsistent and did not exhibit the same trends. All estimates were weighted to account for the age-stratified sampling strategy and to adjust for the lower response rate associated with high deprivation. Weights were calculated on the basis of the mid-year population estimates for Scotland  and R version 2.9.0 . The institutional and student response rates by type of educational establishment were determined and the characteristics of the sample described. A comparison was made between the characteristics of the total sample and the 1866 samples included in the final analysis using Fisher's exact and Scotland . Only thOf the 32 EAs in Scotland, 8 were invited and all agreed to participate in the study; 51% (58/114) of state schools, 75% (3/4) of independent schools and 60% (9/15) of colleges agreed to take part. Within schools, the response rate, calculated on the basis of the number of registered students in a year and not on the number who actually attended the study presentation, was estimated at 15% (2219/14 417) overall. The response rate was 14% (1980/13 713) for state schools  and 34% (239/704) for independent schools . The response rate at colleges was not recorded, as reliable denominator data was not available.P<0.001).Excluding leaking samples and those with insufficient volume, 2447 (95%) of 2575 submitted samples were suitable for processing; 1866 (72%) contained amplifiable DNA and were classified as valid for HPV detection. Submission of an invalid sample was not related to age, deprivation, recruitment from an urban or rural setting or attending a denominational school . Of the In the general Scottish population of 11- to 18-year olds, ∼21% are from areas of high deprivation ; 42.5% (95% CI: 32.7–52.2) of all infections were with types 16 and 18 . The weighted prevalence of HPV-6 and -11 was 2.9% (95% CI: 0.7–5.1). High-risk infections accounted for 82.7% (95% CI: 76.0–89.3) of all infections ; 50.3% (95% CI: 40.9–59.6) had multiple infections . Human papillomavirus 16 was most prevalent , with a much higher prevalence among those aged 17–18 years.Among females attending school , age wasP=0.017) towards higher positivity with deprivation, although this trend was not strictly linear. In comparison to females in the most affluent quintile, those in the medium quintile of deprivation had a greater OR of being HPV positive  than those in the second most deprived quintile . Overall, compared with those in the most affluent quintile, the OR of being HPV positive was greatest among those in the most deprived quintile .There was a trend  towards increased risk of infection with increasing deprivation. Again this trend was not linear, as the risk of infection was highest in those from the second most deprived quintile  rather than in those from the most deprived quintile . A sizeable proportion of 16- to 18-year olds (21% of the sample) attend colleges. We observed an association between HPV infection and attending a college rather than an urban school . All colleges are in urban areas. Adjusting for deprivation, females attending rural schools were more likely to be infected with HPV than females in urban schools . Again, denominational affiliation of the school had no association with HPV status.Among females aged 16–18 years, those aged 17–18 years had increased odds of being positive  compared with those aged 16 years. There was also a statistically significant trend . The weighted prevalence of types 16 and 18 was 0.7% (95% CI: 0.0–1.5). The weighted prevalence of HPV-6 and -11 was 0.9% (95% CI: 0.0–1.9). The weighted prevalence of high-risk infections was 2.4% (95% CI: 0.0–4.7) and of multiple infections was 1.2% (95% CI: 0.0–2.4). Human papillomavirus 70 was most prevalent  and few have included males.In our sample of 11- to 14-year olds, the overall weighted prevalence of HPV was low (1%); no infections were associated with the bivalent vaccine types 16 and 18, although one infection with HPV type 6  was detected. In this age group, most of those who were infected were infected with a high-risk type. Among females aged 15–18 years, the overall weighted prevalence of HPV was substantially higher (15%); over 80% were infected with high-risk types and almost half of all infections were with types 16 and 18.There is some variation in the HPV prevalence estimates reported in surveys of the general population of adolescent women, and as most of these have used cervical swabs or brush samples, which have a higher sensitivity than urine, a direct comparison with our results is difficult. Nonetheless, a survey of 15- to 17-year-old female secondary school students in Sweden . This is important because it is recognised that those from deprived areas are less likely to attend cervical screening  and moreThe HPV prevalence rates detected for males in both age groups was low and is lower than rates reported in most other studies  and specificity (63.2%) of the assay in male samples compared with female samples , as determined from the assay validation study, would have led to the systematic underdetection of HPV infection among males. In addition, samples from males in our study were twice as likely as samples from females to contain no amplifiable DNA, as determined by the absence of the cellular housekeeping gene, and therefore to be invalid for HPV detection. Given this, it is likely that we have underestimated the prevalence of HPV, particularly in our sample of males, although we expect the distribution of HPV types to be accurate, as the assay validation study demonstrated a similar distribution of HPV types in urine compared with gold standard specimens.Those assumed to be most at risk of HPV infection were underrepresented in our sample because of the lower response rate from those from areas of high social deprivation, in which indicators of sexual health are known to be poorer , and alsWe estimated the response rate to the study  to be ∼14%. Although this is likely to be an underestimate, the overall response was still low. This was, in part, due to the low acceptability of donating a urine sample in this population. Similar problems have previously been encountered by other population-based surveys of HPV, particularly those employing genital swabs, with response rates as low as 4% being reported in recent literature (HPV prevalence is low in the 11–14 year age group, which includes the age group targeted for routine vaccination and so supports previous evidence used to recommend the optimum age to offer the vaccine. The relationship between deprivation and HPV prevalence requires further investigation, especially in relation to the need to ensure a high uptake of HPV immunisation among those who are less likely to attend cervical screening. The picture of HPV infection among males is unclear and requires further investigation.