Chronic infection with oncogenic HPV genotype is associated with the development of anal dysplasia. Antiretroviral therapy (ART) has been shown to decrease the incidence of cervical carcinoma in women with HIV. We sought to: 1) describe the prevalence and grade of anal dysplasia and HPV infection in our study subjects; 2) analyze the grade of correlation between anal cytology, PCR of high-risk HPV, and histology; 3) identify the factors associated with the appearance of ≥AIN2 lesions.Cross-sectional, prospective study.A cohort of HIV-positive males  who have sex with males (MSM) had epidemiological, clinical and analytical data collected. Anal mucosa samples were taken for cytology, HPV PCR genotyping, and anoscopy for histological analysis.in situ; 74.2% had high-risk (HR), 59.7% low-risk (LR) HPV genotypes and 46.8% had both. The combination of cytology with PCR identifying HR-HPV better predicts the histology findings than either of these factors alone. Logistic regression highlighted ART as a protective factor against ≥AIN2 lesions . Anal/genital condylomas , and HPV68 genotype  were identified as risk factors.Within the cohort, 77.1% were being treated with ART, 8.5% anoscopy findings were AIN2, and 11.4% carcinoma In our cohort, ART has a protective effect against dysplastic anal lesions. Anal/genital warts and HPV68 genotype are predictors of ≥AIN2 lesions. Introducing PCR HPV genotype evaluation improves screening success over that of cytology alone. Over the last few decades there has been an increase in the incidence of anal cancer due, in large part, to the increase in risk groups such as males who have sex with males (MSM), the immunocompromised and, especially, patients with HIV infection The incidence of anal cancer in HIV-positive patients varies between 40 and 137 per 100,000 person/years, which is much higher than in the general seronegative population, with a predominance of males with AIDS staging Anal cancer in HIV patients is currently considered one of the most-frequent non-AIDS-defining malignancies Hence, we proposed as principal objective: 1) to describe the prevalence and grade of anal dysplasia, and of the infection by HPV, in our cohort of HIV-positive MSM patients; and as secondary objectives: 2) to analyze the degree of correlation between anal cytology and PCR of HR-HPV with the histology findings from biopsy and 3) to study the factors associated with the appearance of ≥AIN2 lesions in our cohort. The factors analyzed included that of ART.Hospital Universitario Virgen de las Nieves  the ethics committee of which approved the study.Cross-sectional study conducted between May 2010 and Sept. 2013 in a cohort of 140 patients with HIV-MSM recruited consecutively into a program of screening, diagnosis, treatment and follow-up of dysplastic lesions of the anal mucosa. The HIV-positive patients were from among those receiving attention in the Infectious Disease Unit of the The inclusion criteria were: adult (≥18 years of age) MSM infected with HIV. The exclusion criteria were: females, heterosexual HIV-positive males, and history of anal canal neoplasia at the time of recruitment into the study.The objectives and conditions of the study were explained to the patients at the first clinical visit, and written informed consent was obtained (as outlined in PLoS consent form). Clinical, epidemiological and analytical data were obtained and codified for anonymity according to the laws on protection of personal information currently in existence in Spain.The variables collected were: age, history of perianal or genital condylomatosis, number of different sexual partners in the previous 12 months, condom use in sexual intercourse, tobacco use, alcohol use , intravenous drug abuse (IDA), HIV acquisition route, months since HIV diagnosis, HIV stage according to the CDC Atlanta; months on ART, virologic treatment failure , use of concomitant treatments, other infections including chronic hepatitis B and C , luetic positive serum (syphilis), other sexually transmitted diseases (STD), latent tuberculosis or tuberculosis under treatment or active infection.The laboratory analytes measured included: lymphocyte number, CD4 nadir, CD4, CD8, and viral load at the time of HIV diagnosis together with CD4, CD8 and viral load at the time of inclusion into the study.At the clinical visit, 2 mucosa samples were taken from the anal canal with cotton swabs soaked in physiologic saline, and stored in liquid medium (thin layer liquid) for the detection and genotyping of the HPV using the polymerase chain reaction (PCR) technique , and for cytology using the ThinPrep Pap Test . Both samples were sent to the anatamo-pathology laboratory where the same senior pathologist of the research team (JE) carried out the cytology evaluation, validation of the PCR, and histology analyses. The genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51–53, 56, 58, 59, 66, 68, 73 and 82 were considered high risk (HR-HPV). Genotypes 6, 11, 34, 40, 42–44, 54, 55, 57, 61, 70–72, 81, 83, 84 and 89 were considered low risk (LR-HPV) Subsequently, anoscopy by the digestive tract specialist of the research team (MLdeH) was performed within an interval of between 4 and 12 weeks from the cytology assessment. A standard endoscope of 9 mm with working channel of 2.8 mm was used, without any image enhancement. A short exploration of 15–20 cm was performed with retrovision maneuver to better visualize the pectinea line. Samples were taken for histology examination using an endoscopic retrograde cholangio-pancreatography (ERCP) catheter not only from the lesions (color change with irrigation with 5% acetic acid), but also from other parts of the quadrant with ostensibly-normal mucosa.The cytology classification was that of Bethesda in situ), and invasive carcinoma in situ. We considered lesions ≥AIN2 those that proceeded beyond AIN2 to carcinoma in situ.The histology classification employed divides the lesions into LSIL (AIN1/condyloma), HSIL  for the quantitative variables, and the absolute frequencies with 95% confidence intervals (95%CI) for the qualitative variables. The prevalence and 95%CI were calculated for HPV, dysplasias obtained from cytology, and dysplasias obtained from the histology evaluation. Diagnostic success, not only from cytology but also from PCR of HR-HPV of dysplasias that included lesions ≥AIN1 and ≥AIN2, was defined by the receiver operating characteristics (ROC) curves. The results were considered poor: 0.5 to 0.6, acceptable: 0.6 to 0.75, good: 0.75 to 0.9, very good: 0.9 to 0.97, and excellent 0.97 to 1. The degree of concordance between cytology, PCR of HR-HPV, and biopsy results was analyzed using the Kappa index. The results of the test were evaluated using the classification of Landis and Koch in which a value of k<0.20 would be considered poor; 0.21 to 0.40 weak; 0.41 to 0.60 moderate; 0.61 to 0.80 good; and 0.81 to 1.00 very good t-test for independent samples was applied for quantitative variables that followed a normal distribution, while the Mann-Whitney test was employed for those variables that did not follow normal distributions. The Kolmogorov-Smirnov test was used to assess whether the different variables fulfilled the criteria of normal distribution. Comparison of differences between variables was with the Pearson χ2 test, or the Fisher exact test if the application criteria were not fulfilled.We used bivariate analyses to assess the relationship between the possible risk factors and the presence of dysplastic lesions ≥AIN2. The Student Logistic multivariate regression was applied based on the classic formula of Freeman [n = 10*(k+1)], A value of p<0.05 was considered statistically significant in all the analyses. The statistical software used was the SPSS (version 15.0).Outcomes data and details of methods are available from the corresponding author, on request.Included were 140 patients, mean age 37 years, CD4 nadir 356 cells/μL, median time of clinical evolution of the HIV infection around 33 months (IQR: 11–84), 78% on treatment with combination anti-retroviral therapy (ARTc) over a median period of 23.5 months at which time there were 652 cells/μL CD4, and only 6% in virologic treatment failure The characteristics are summarized in in situ  of whom had high-risk genotype, 59.7% (95%CI: 51–70) low-risk genotype and 46.8% (95%CI: 39–58) both genotypes. The HPV genotypes isolated most frequently in anal mucosa were: 16 (30.6%), 6 (16.1%), 51 (15.3%), 84 (13.7%), 11 (12.9%), 18 (12.1%), and 61 (12.1%) . Of the in situ .The diagnostic performance curves of the cytology, and those from PCR of HR-HPV genotypes used to predict the presence of dysplasia in the histology, revealed acceptable results. The area under the curve (AUC) values of the receiver operating characteristics (ROC) were not clearly discriminatory; the majority of values being around 6. The combination of HR-HPV PCR with cytology better predicts the grade of dysplasia than the cytology alone i.e. in those with normal cytology and HPV-negative PCR, the AUC of the ROC was 0.74 (p = 0.003) while, in the case of normal cytology alone, it was 0.65 (p = 0.04)  and 2.in situ (3.4%) and AIN2 (5.2%). However, when we analyzed cytology and PCR of the HR-HPV in combination, the diagnostic efficiency was considerably improved. In case of normal cytology with PCR of HR-HPV-positive, 24.4% presented carcinoma in situ/AIN3 and 6.8% AIN2 i.e. 31.1% had lesions ≥AIN2. Of further note is that in case of normal cytology with PCR of HR-HPV-negative, 75% of the patients had normal histology, and none had lesions ≥AIN2  especially genotype 68 (p = 0.01). Longer-term ART was a protective factor (p = 0.02), as was a longer time since HIV diagnosis (p = 0.007) .Finally, in the logistic regression analysis assessing appearance of lesions ≥AIN2, we identified the risk factors as: the presence of perianal condylomas during the study  and the HPV genotype 68  whose prevalence was around 6.5%. The factors protective against the appearance of these lesions were identified as: receiving ART , and having had a previous diagnosis of syphilis .Our cohort of HIV-positive MSM patients had a high prevalence not only of dysplastic lesions (50% LSIL in cytology and 20% biopsy lesion ≥AIN2) but also infection by high-risk HPV genotypes of around 74%. The results were very similar to those published by other authors who had observed the percentage of anal dysplasia with low grade cytology (LSIL) at around 40%, 19% ≥AIN2 lesions and the most-frequently identified HPV16 genotype in situ/AIN3 in our group of patients, was 11.4% (95%CI: 4–19) which translates into 1/9 patients having anal cancer in situ. AIN2 was 8.5%, i.e. 1/5 of patients included had pre-malignant lesions (HSIL). These findings highlight the need to implement routine consultations in HIV clinics which, currently in Spain, are not performed systematically for screening, diagnosis, treatment and follow-up of dysplastic lesions of the anal mucosa. Anal cancer is one of the most frequent non-AIDS defining malignancies in HIV, and has become so probably because of the increase in survival of HIV patients The prevalence of anal carcinoma The HPV genotype most frequently identified in the anal mucosa of our patient cohort was the oncogenic 16, present in about 30.6% of the patients included in this study. It is one of the most frequent, high-risk genotypes isolated in anal mucosa of HIV-positive MSM patients in situ. Conversely, when the patient with normal cytology had high-risk HPV in anal mucosa, only 20% had a normal histology. Of the rest, 55.5% had AIN1, 6.8% AIN2 and 24.4% AIN3/carcinoma in situ. Of further note is that in patients with normal cytology with PCR of high-risk HPV-negative, 75% had normal histology, and none had ≥AIN2 lesions. Our results have some important consequences for recommendations for screening of premalignant lesions and carcinoma of the anal canal in HIV patients. To date, the different AIDS scientific societies recommend only anal cytology The correlation between the results of the cytology and histology that we observed are slightly more encouraging; only 48.3% of the patients with normal cytology had a normal biopsy . Of the rest, 43.1% were AIN1, 5.2% AIN2 and 3.4% carcinoma We observed the presence of anal condylomatosis as well as HPV68 genotype as being risk factors associated with the appearance of ≥AIN2 lesions. Genital-anal condylomatosis is due to the infection by the HPV genotypes and, similar to those that present in the anal canal mucosa, are associated with the appearance of dysplasia not only in women We observed that receiving ART and previous diagnosis of luetic infection were factors predisposing against the appearance of ≥AIN2 lesions. With respect to ART, we support the findings of the Swiss study Treponema pallidum can be a protective factor against the appearance of ≥AIN2 lesions, could be interpreted as syphilis being a surrogate marker for early diagnosis of HIV and, thus, of the infection by HPV which is encountered in an earlier phase in which the appearance of the dysplastic lesions have not, as yet, occurred.That previous infection with Finally, a weak point of our study needs to be addressed. The study was cross-sectional and, as such, no causal relationships between the measured variables could be demonstrated. The strength of the study is the consistency of the data. All the anoscopies, biopsy and cytology of anal mucosa, as well as the PCR of HPV, microbiology laboratory assessments, and clinical interviews were each performed by the relevant specialist, and this minimizes inter-observer bias.In conclusion, in our cohort of HIV-positive MSM patients, 3/4 were infected with HPV genotypes of high-risk; 1/5 had high-grade lesions. There was a low correlation between the cytology findings and the anal histology. Adding PCR of the HPV genotypes to the screening for ≥AIN2 lesions would increase the diagnostic efficiency above that of cytology alone. Antiretroviral treatment could exercise a protective role against the presence of lesions ≥AIN2. The presence of genital or perianal warts and HPV68 genotype in anal mucosa are 2 factors predictive of ≥AIN2 lesions. These findings warrant further investigation.


An epidemic of human papillomavirus (HPV)-related oropharyngeal squamous cell cancer (OPSCC) has been reported worldwide largely due to oral infection with HPV type-16, which is responsible for approximately 90% of HPV-positive cases. The purpose of this study was to determine the rate of HPV-positive oropharyngeal cancer in Southwestern Ontario, Canada.A retrospective search identified ninety-five patients diagnosed with OPSCC. Pre-treatment biopsy specimens were tested for p16 expression using immunohistochemistry and for HPV-16, HPV-18 and other high-risk subtypes, including 31,33,35,39,45,51,52,56,58,59,67,68, by real-time qPCR.Fifty-nine tumours (62%) were positive for p16 expression and fifty (53%) were positive for known high-risk HPV types. Of the latter, 45 tumors (90%) were identified as HPV-16 positive, and five tumors (10%) were positive for other high-risk HPV types (HPV-18 (2), HPV-67 (2), HPV-33 (1)). HPV status by qPCR and p16 expression were extremely tightly correlated . Patients with HPV-positive tumors had improved 3-year overall (OS) and disease-free survival (DFS) compared to patients with HPV-negative tumors . HPV-16 related OPSCC presented with cervical metastases more frequently than other high-risk HPV types (p = 0.005) and poorer disease-free survival was observed, although this was not statistically significant.HPV-16 infection is responsible for a significant proportion of OPSCC in Southwestern Ontario. Other high-risk subtypes are responsible for a smaller subset of OPSCC that present less frequently with cervical metastases and may have a different prognosis. The incidence of head and neck squamous cell carcinoma is generally decreasing ,2, likelThere is a wide discrepancy in the fraction of OPSCC that are HPV-positive world-wide, with incidence rates ranging from 11% to as high as 93% ,8. In NoStudy approval was obtained from the University of Western Ontario Research Ethics Board. A retrospective search of the London Regional Cancer Program (LRCP) database was performed to identify patients diagnosed with OPSCC from 2003 to 2009. Patient eligibility required: 1) a histologically confirmed diagnosis of squamous cell carcinoma, 2) no prior history of head and neck cancer, and 3) the availability of a pre-treatment primary site biopsy specimen for analysis. Patient data was extracted from a retrospective chart review, which included age at diagnosis, use of tobacco and alcohol, AJCC TNM stage, treatment regimen, and post-treatment follow-up information.After the completion of cancer therapy, patients were followed closely at six to twelve week intervals by either a radiation oncologist or head and neck surgeon. Treatment response was evaluated by physical exam as well as computed tomography imaging as needed. Salvage neck dissection was undertaken within three months of the completion of radiation treatment if evidence of residual adenopathy was present radiographically or on physical exam. The presence of neck disease more than three months after diagnosis was considered a regional persistence.The formalin fixed paraffin embedded blocks from each patient’s primary site were sectioned (5 μm thick) and mounted on slides. The slides were then deparaffinized with 3 minute washes in xylene (100%) twice, followed by a 1:1 xylene:ethanol mix, then ethanol (100%) twice, followed by single washes in ethanol at 95%, 70% and 50%. Lastly, the slides were washed in water for 5 minutes.202 prior to primary antibody incubation. Specimens were incubated with a primary mouse monoclonal antibody against human p16  at 1:500 dilution. The Vectastain ABC kit, with a goat anti mouse secondary antibody, was used as per the manufacturer’s instructions. Antigen detection was done using diaminobenzidine chromogen . Tissues were counterstained with Carazzi’s hematoxylin. Immunohistochemistry scoring was conducted by two head and neck pathologists (KK and BW) blinded to HPV status and patient outcome. Scoring was as described by Begum et al., with strong and diffuse staining (>80 percent of tumour cells) regarded as a positive result, and negative if absent or focal [Citrate buffer was applied to the deparaffinized slides for heat induced epitope retrieval for 30 minutes, followed by treatment with 0.1% Triton X-100 in PBS. Endogenous biotin was quenched using an avidin/biotin kit  and the endogenous peroxidase activity was blocked by Hor focal .Deparaffinized tissue was scraped into a 1.5 ml eppendorf tube containing 50–100 μl (depending on the amount of tissue) of TE and proteinase K  and incubated overnight at 65°C. Following proteinase K treatment, the samples were heated at 95°C for 10 minutes and allowed to cool to room temperature. 0.2 μl of each sample was used directly in the qPCR reactions.In an effort to screen clinical samples for the presence of human papillomavirus (HPV), we designed a multiplex quantitative PCR to identify those samples that were HPV positive and to confirm the HPV type in the positive samples. We designed primer/probe sets Table  against Tumors that tested positive for HPV infection by qPCR with the broad spectrum primer/probe set designed against the E2 region of HPV were tested with a second broad spectrum set of primers against the E1 region of HPV by conventional PCR  was defined as the time of completion of treatment to local, regional, or distant recurrence. Survival curves were created using the method of Kaplan and Meier. The Cox proportional hazard model was used to estimate the relative hazard of mortality or recurrence over the follow-up period and to perform univariate and multivariate analysis. Multivariate analysis began with a fit to all patient variables, followed by backward stepwise regression using the Akaike information criterion (stepAIC function) and retention of variables in the final model having p values of 0.05 or less.Statistical analysis was performed using SPSS with the exact tests package . For all analyses, a T < 30) and negative tumors (CT > 35). Overall, fifty tumors were positive for HPV, including 45 positive for HPV-16 (90%) and 2 positive for HPV-18. The broad-spectrum primer/probe set detected three other HPV-positive tumors, which were determined to be type 33 (1 tumor) and type 67 (2 tumors) by sequencing of PCR products. The identification of HPV-67 was surprising because the high-risk HPV-67 sequence was not included in our efforts to design broad-spectrum primers. Regardless, sequencing of the 150 nt product was highly consistent with the published HPV-67 sequence by BLAST analysis [-39, accession D21208). p16-positivity was tightly correlated with HPV status , further supporting its use as a surrogate marker of HPV infection. Analysis of patient characteristics and HPV-positive OPSCC revealed associations with age <60, tonsil or base of tongue primary site, and minimal smoking history  as opposed to primary surgery  Table . Patientanalysis  (p < 10-Median follow-up time for the study was 39 months (range: 1 to 91 months). A total of 26 of 95 patients (27%) recurred, and eleven 42%) of these patients died of disease. Five patients died of other causes. The three-year disease-free (DFS) and overall survival (OS) for all patients was 75 % and 85%, respectively. Univariate analysis identified age, p16 status and the presence of any high-risk HPV type as predictors of disease-free and overall survival  in North America and beyond ,4. UsingHowever, a fraction of these patients still relapse and succumb to disease following conventional therapy. Ang et al  demonstrAlthough limited conclusions can be drawn given the small numbers of HPV types other than HPV-16 in our cohort, our data suggests that HPV-16 cancers may present more frequently with lymph node metastases and may have a poorer outcome compared with non-16 high-risk HPV types. Given the low frequency of HPV-positive OPSCC presentations that are not due to HPV-16, a very large multi-institutional series with HPV genotyping would need to be assembled in order to answer this question definitively.A tremendous step forward in the battle against the multiple cancers caused by HPV is the implementation of HPV vaccination programs worldwide. The quadrivalent HPV vaccine is highly effective in preventing benign and precancerous lesion lesions due to HPV types 6, 11, 16 and 18 including cervical and anal dysplasia . Due to HPV-positive cancers are fundamentally distinct from traditional tobacco and alcohol related cancers . Viral oHPV infection is the etiologic factor for a significant proportion of OPSCC in Southwestern Ontario as has been observed worldwide. The majority of cases are attributable to HPV-16, while other high-risk subtypes are responsible for the remainder. HPV-16-positive OPSCC may present at a more advanced stage than those due to other high-risk HPV types. Larger studies are required to confirm this observation and determine if different HPV types influence prognosis.This study was supported by an investigator initiated grant from Merck Canada, Inc. The authors have no other disclosures.This project has been a coordinated effort designed by the London Health Sciences Centre Head and Neck multidisciplinary team. Specifically, medical oncologists , radiation oncologists , head and neck surgeons  and translational scientists  planned this study together from its initiation. Dr. Marina Salvadori (MI), an HPV and vaccine expert, was also involved with the study design. Pathologists (BW and KK) were responsible for screening all specimens for the presence of tumor. As this study involved a large retrospective chart review, specimen retrieval, DNA extraction, and HPV testing multiple trainees were involved. Specifically, patient data was extracted by SSD, CC, JB, and DNA was extracted by AN and JWB. All HPV testing was done by the senior author JWB. The manuscript was written by AN and JWB and critically revised by all authors. All authors read and approved the final manuscript.This study was supported by a research grant from Merck Canada Inc.This material has never been published and is not currently under evaluation in any other peer-reviewed publication.


Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5–3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes  in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-β1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-β1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded. Idiopathic pulmonary fibrosis is a chronic and devastating disease without a known etiology We and others have applied genome scale transcript profiling techniques of human IPF lungs to better understand the disease, identify novel targets for therapeutic interventions as well as new biomarkers Cartilage oligomeric matrix protein (COMP) is an extracellular matrix protein that is mainly localized to tendon, cartilage, and pericartilage tissues Increases in COMP have been reported in several diseases Based on these observations, we decided to investigate the role of COMP in IPF. We analyzed the expression of COMP in a larger set of lungs, localized its protein over-expression in IPF lungs and determined its regulation and effects on normal human lung fibroblasts and determined the relationship between elevated COMP serum levels and measures of disease severity in IPF.The IPF and control lung tissues were obtained from University of Pittsburgh Health Sciences Tissue Bank . The experimental materials, procedures, samples collection, IRB, and statistical analysis have been previously described by us https://www.lung-genomics.org). 500 ng of total RNA were hybridized to a 3′ biotinylated capture probe and a 5′ reporter probe tagged to a fluorescent barcode. Following overnight hybridization at 65°C, the samples were transferred to the nCounter® Prep Station, excess probes were washed out, and the probe-RNA complexes were bound and immobilized on streptavidin-coated cartridges. The cartridges were scanned in the nCounter® Digital Analyzer using 1155 fields of vision. The data was normalized using GUSB as the housekeeping gene.We extracted total RNA from 154 Control lungs and 115 IPF lung tissues obtained from the Lung Tissue Research Consortium (LTRC) for nCounter® Analysis System  validation of COMP mRNA expression. Details about the samples are available at the LGRC (Lung Genomics Research consortium) website  at 37°C and maintained as per the supplier’s instructions. For hypoxic conditions, cells were placed in hypoxic conditions with 1% of oxygen for 24 hours. All cells were grown until 70–80% confluence. Whenever indicated, cells were stimulated with recombinant TGF-β1  and/or transfected with 50 nM siCOMP and their corresponding negative controls  using Lipofectamine 2000  according to the manufacturer’s instructions.Early passages (1–3) of primary normal human lung fibroblast (NHLF)  were cultured in a humidified atmosphere containing 5% of COTotal RNA were extracted from NHLF. All reagents (including primers for COMP) and analysis software used for qRT-PCR experiment were obtained from ABI,  and performed according to the vendor recommendation as previously described by us http://rsbweb.nih.gov/ij/).Lung tissues and NHLF were lysed, harvested following the manufacturers’ protocol . The concentrations of protein were measured by using Pierce’s Bicinchoninic acid (BCA) . For Western blot analysis, equal amounts of cellular extracts (10 µg) were separated on 10% SDS-PAGE gels and transferred to PVDF-Plus membranes . Western blots were performed with antibodies against COMP , β-actin , P-SMAD3 . After incubation with the respective secondary antibodies, specific bands were visualized by autoradiography using enhanced chemiluminescence according to the manufacturer’s instructions . Densitometry was performed using the shareware, ImageJ . Tissue slides were deparaffinized in serials: 100%, 90%, 80%, 70%, 60% and 50% ethanol and rehydrated three times in PBS each time for 10 minutes. Slides were incubated for 45 minutes with 5% donkey serum in Tris-buffered saline (TBS) pH 7.4 containing 3% bovine serum albumin (Sigma-Aldrich) and incubated for 4 hours with primary antibody. After five washes with 0.5% BSA in TBS for 5 minutes each time, slides were incubated in a biotinylated donkey anti-rat secondary antibody for 30 minutes. After 2 washes with 0.5% BSA in TBS for 10 minutes each, the slides and arrays were then incubated with streptavidin-linked alkaline-phosphatase . Slides were washed again and incubated for 15 minutes in Fast Red substrate  to detect the activity of alkaline-phosphatase. Briefly, tissue sections and arrays were washed for five minutes in water and counterstained using Mayer’s Hematoxylin . The images were visualized with Olympus™ microscope, PROVIS .Frozen IPF lung slides were fixed in 2% Paraformaldehyde  for 20 minutes and permeabilized using 0.1% Triton X in PBS for 15 minutes, followed by rehydration in PBS, washes with 0.5% BSA in PBS and blocking with 5% donkey serum (Sigma) in 3% BSA in PBS for 45 minutes. Slides were incubated with anti-COMP , Vimentin , or pSMAD3  antibodies in a blocking solution overnight at 4°C. Secondary antibodies, nucleus staining (DAPI) and confocal imagining were performed as previously described by us All patients were evaluated at the University of Pittsburgh Medical Center, Pittsburgh, PA and studies were approved by the Institutional Review Board (IRB) at the University of Pittsburgh. The diagnosis of IPF was established on the basis of American Thoracic Society (ATS) and European Respiratory Society (ERS) Criteria The IPF patients were recruited in longitudinal study as described in “Study Population”. Participants were followed up for 2.5 years while their blood was drawn and PFT were examined. We measured COMP level in serum of IPF patients and controls using COMP ELISA kit . The experimental procedures were followed as recommended by the vendor and data were analyzed by utilizing Delta soft 111 version 2.243 .All values were presented as mean ± SD. Group comparisons were made using an unpaired, two-tailed Student’s t-test for normally distributed data. A level of p<0.05 was considered statistically significant. Longitudinal study of COMP and all the PFT for 23 patients within 120 days of any blood draw were used for correlation analysis of COMP level to FVC %. A data set with one record per PFT occasion, associating each PFT with COMP level from the blood draw nearest it, was generated for all PFT of 23 patients within 120 days of any blood draw. A generalized estimating equation was used to fit to the data, based on the reference of Yan et al COMP was one of the most significantly increased genes in our previously published microarray data After co-localizing COMP in fibroblasts of IPF lungs, we wanted to determine whether TGF-β1 regulates COMP expression. Stimulation of NHLF with TGF-β1 (5 ng/ml) induced increase in mRNA and protein levels of COMP in a time dependent manner as determined by qRT-PCR, western blot and ELISA . To deteTo determine the dose response of COMP induction by TGF-β1, we stimulated NHLF with 2, 5, and 10 ng/mL of TGF-β1. We observed increased expression of COMP with 2 ng/mL of TGF-β1, and an additional significant induction when we used a concentration of 5 ng/mL TGF-β1. Increasing the TGF-β1 concentration to 10 ng/mL had no additional effect on COMP expression . In cont2) for 24 hours induced a significant increase COMP gene expression  and controls (n = 20). We used COMP ELISA Assay and determined that COMP concentrations were significantly increased in the serum of IPF patients when compared with controls  and multiple epiphyseal dysplasia (MED) While we could not directly demonstrate COMP induction by TGF-β1 in human IPF lungs, we demonstrated that COMP mRNA and protein levels were increased after stimulation of human lung fibroblasts with TGF-β1. This is consistent with other reports that demonstrated induction of COMP after TGF-β1 stimulation in keloid and dermal fibroblast The fibrotic lung is characterized by the intensive accumulation of extracellular matrix (ECM). The pathologic ECM depositions in fibrotic lungs include collagens , fibronectin, elastin, and cartilage related proteins in-vitro did also induce COMP, but it is unclear whether this mechanism would be relevant in-vivo. Clinically, our findings are also limited, while we offer compelling evidence of the relationship between COMP protein levels in serum and FVC, we are aware that the numbers are limited and we did not capture patients for this study in earlier stages of the disease. Our center is a tertiary medical facility, which gets regional and national referrals, and thus the populations of the study were at later stages of the disease. Despite this shortcoming, we were able to show the continuous increase of serum COMP level in 2.5 years. Considering its role in regulation of matrix rigidity, the observation that elevated COMP concentrations in serum of IPF patients are associated with a decline of FVC provides support to our hypothesis that COMP plays a role in pathogenesis of IPF and should be further evaluated as a biomarker for disease activity in IPF.It is critically important to acknowledge few limitations of our study. First, we provide evidence on COMP regulation through TGF-β1 and its involvement in TGF-β1 signaling cascade in NHLF, but we do not provide similar data in-vivo. We feel that the co-localization of vimentin, pSMAD3 and COMP in fibrotic foci in human IPF lungs suggests that TGF-β1 induces COMP secretion mostly in fibrotic regions of IPF lungs and to some extent more informative than using a limited model of lung fibrosis. To that extent we did observe that in-vitro exposing NHLF to extreme hypoxia in-vitro serves as an enhancer of TGFβ1 signaling as previously proposed. Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded.In this manuscript we demonstrate that the mRNA and protein expression levels of COMP, an extracellular matrix protein that accentuates TGF-β1 signaling and is associated with extracellular matrix polymerization and stiffness, are high in IPF lungs compared to controls. COMP serum protein concentrations are increased in IPF patients and correlate with the decline of FVC over time in individuals with IPF. We also demonstrate data that COMP may be induced by TGF-β1 in the IPF lung and that at least


As the result of basic researches, several intravital fluorophores have been determined so far in human colorectal tissue. Autofluorescence endoscopy (AFE) can detect slight alterations in their distribution and concentration during the colorectal carcinogenesis process and, thus facilitate noninvasive screening colonoscopies without the need for fluorescent substances or staining reagents to be administered. While detecting faint autofluorescence intensity by conventional fiberoptic endoscopy remains challenging, the latest AFE system with high-resolution videoendoscope capabilities enables such detection by using a false-color display algorithm. To this end, the diagnostic benefits of AFE have been reported in several multicenter randomized controlled studies of colorectal cancer (CRC) screening and differential diagnosis. CRC screening using the latest AFE technology could, therefore, lead to future reductions in CRC mortality. Early detection and removal of colorectal adenomatous polyps is essential in reducing the mortality rate of colorectal cancer (CRC). Although there are several modalities for CRC screening, colonoscopy is considered the most effective procedure, allowing direct visualization and on-site treatment of the encountered lesions. However, minute or flat-type polyps are hard to detect even by conventional colonoscopy . Narrow-Autofluorescence endoscopy (AFE) is now attracting attention for its potential in improving diagnostic yields for CRC. This technology was shown to detect slight alterations in autofluorescence intensity in the colorectal wall during the carcinogenesis process , 9.When light is focused onto a molecule, part of the light energy is reflected or scattered, and the rest is absorbed. The energy status of the molecule shifts from a ground state to a high-vibration energy state—this is known as excitation. Excess energy is emitted as thermal energy or consumed as vibrational energy when the molecules revert to their ground state. However, naturally fluorescent molecules in tissue release such excess energy as autofluorescence, which can be detected and measured.Fluorophores determined so far in human colorectal tissue include collagen, which forms the basement membrane and the submucosal layer, NADH and FAD, which exist mainly in gland cell mitochondria and lysosomal granules, and porphyrin in the mitochondria of red blood cells and gland cells.Autofluorescence emission has been reported mainly with respect to collagen distributed throughout the submucosal colorectal layer , with loSeveral studies focusing on fluorescence endoscopy combined with topical application of fluorescent substances such as porphyrin , tetracyAFE detects intravital fluorescent substances without administration of exogenous fluorescent agents. Firstly developed as an autofluorescence bronchoscope  , the tecThe autofluorescence imaging system from Olympus (AFI system) is the latest AFE system and is equipped with high-resolution videoendoscope capabilities .Based on the advantage of AFE that colorectal lesions are displayed in purple, which is like a “red flag” in the surrounding normal colorectal mucosa shown in green, several randomized clinical trials have focused on the diagnostic utility of AFE in screening by colonoscopy. In a randomized controlled study using the AFI system , a modifP = 0.001). In addition, the additionally detected adenomas with AFE were significantly smaller than the adenomas detected by WLE .A back-to-back comparative study by Ramsoekh et al.  analyzedAlthough early detection and removal of colorectal adenomas is considered the most effective way of preventing colorectal cancer progression , 23, then = 22) than in neoplastic lesions , n = 81) (P < 0.001). Under receiver operating characteristic analysis, with a cut-off value of 1.01 for G/R ratio, it was shown that AFE had a sensitivity and specificity of 98.8% and 86.4%, respectively. This result indicated that the color tone in AFE might directly visualize pathological features of colorectal lesions, and its analysis may facilitate the automated optical diagnosis of colorectal neoplastic lesions in the future.The false color range in AFE is determined based on the calculation of balance between autofluorescence intensity and reflected green light intensity , and this balance could be affected by thickness of the lesion, degree of vascularity, and glandular density. We numerically analyzed the color tone of colorectal lesions in AFE using special color analysis software . A totalP = 0.023).Despite more advantages with the latest AFE technology, the system still has some limitations that need to be overcome for its full potential to be realised. The outside diameters of the distal end and insertion tube are relatively thick  compared to those used in conventional colonoscopy. This might limit maneuverability and, thus, hinder polyp detection, especially of those lesions harbored behind folds or flexures. Use of a transparent hood (TH) in AFE was shown to improve detection rates for colorectal neoplasms . In this The AFI system has two other limitations—delayed display and low image resolution. In our system, both video-frame rate and image resolution were reduced to create false-color images employing very faint autofluorescence intensity. In the future these factors should be overcome with system refinements so that CRC screening using this technology becomes more practical.In this paper, we reviewed several papers that focused on the diagnostic value of AFE for CRC screening. We anticipate that AFE may contribute to future reductions in CRC mortality.


Objective. To estimate the prevalence and genotypes of high-risk human papillomavirus (HPV) focusing HPV 16, 18, 52, and 58 in Japan. Methods. Liquid-base cytology specimens were collected from Japanese women (n = 11022), aged 14–98. After classifying cytodiagnosis, specimens were analyzed for HPV DNA by the multiplex polymerase chain reaction method, where 1195 specimens were positive for cervical smear, except adenomatous lesions. Result. HPV genotypes were detected in 9.5% of NILM and 72.2% of ASC-US or more cervical lesions. In positive cervical smears, HPV genotypes were HPV 52 at 26.6%, HPV 16 at 25.2%, HPV 58 at 21.8%, and HPV 18 at 7.1%. Most patients infected with HPV 16 were between 20–29 years old, decreasing with age thereafter. As for HPV 52 and 58, although the detection rate was high in 30- to 39-year-olds, it also was significant in the 50s and 60s age groups. Conclusion. In Japan, as a cause of abnormal cervical cytology, HPV52 and 58 are detected frequently in addition to HPV 16. In older age groups, HPV 52 and 58 detection rates were higher than that observed for HPV 16. After widespread current HPV vaccination, we still must be aware of HPV 52 and 58 infections. Cervical cancer is the major cause of death from gynecological cancer worldwide, even though screening with cervical cytological testing (the Papanicolaou (Pap) test) has been available for over 50 years. In recent years, molecular biology has firmly established a causal relationship between persistent infection with high-risk human papillomavirus (HPV) genotypes and cervical cancer. Hence, geographic variations in HPV type distributions should be an important consideration . A meta- Recently, we have started a new trial for liquid-based cytology (LBC) with ThinPrep, in conjunction with a novel, one-step HPV-typing method using multiplex polymerase chain reaction method (PCR) in Japan (CCLBC study) under the financial support of the National Hospital Organization of Japan. The CCLBC study has been designed to perform a multihospital analysis to further assess the effort of LBC and the prevalence of HPV of the cervix. In this paper, we report the prevalence of the HPV genotypes focusing on HPV 16, 18, 52, and 58, using LBC in Japan.This study was based on a CCLBC study. That is, cervical cytology samples were obtained by a Broom Brush from 8 hospitals and institutes . To compare LBC with the conventional Pap smear, a split-sample method was employed. Cytology was diagnosed by the most severe lesion, as identified by LBC or the conventional Pap smear. Between October 2007 and March 2010, 11022 specimens , 1195 abnormal smear findings, except adenomatous lesions) excluding inadequate specimens were available for analysis. Written informed consent was obtained from all patients. Pap-stained specimens were screened by cytotechnologists and were classified according to the Bethesda system 2001. All cytotechnologists and cytopathologists participating in the study had been authorized by Cytyc Corporation for use of the ThinPrep test. We randomly selected 2068 specimens from the 9760 samples of normal cytology. These 2068 normal cytology specimens and 1195 abnormal cytology specimens were used for detecting HPV genotypes . The resThis analysis included 11022 Japanese women. The mean age of the study subjects was 44.6 years . Of these, 9760 cases did not exhibit abnormal cytological findings (NILM). ASC-US or more were found in 1262 cervical smears. Of the total, 1195 were squamous lesions , 447 low-grade squamous intraepithelial lesion (LSIL), 449 high-grade squamous intraepithelial lesion (HSIL), and 105 squamous cell carcinoma (SCC)) and 67 were adenomatous lesions. The age and distribution were almost the same in the two groups . There wHPV prevalence was 9.4% in 2068 women with NILM, 45.6% (77/169) in ASC-US, 68.0% (17/22) in ASCH, 65.1% (291/447) in LSIL, 83.3% (374/449) in HSIL, and 78.1% (82/105) in SCC . In NILM, the detected HPV genotypes and positive rates were HPV 52 , HPV 58 , HPV 16 , HPV 18 , and HPV 39 , in order of frequency. In abnormal smear findings, HPV genotypes and positive rates were HPV 16 , HPV 52 , HPV 58 , HPV 51 , and HPV 31 , in order of frequency. HPV genotypes and rates of SCC in HPV-positive cases were HPV 16 , HPV 18 , HPV 52 , and HPV 58 , in order of frequency . In NILM, the prevalence of HPV 16, 18, 52, and 58 was high among the 20- to 39-year olds. HPV 16, 18, 52, and 58 detection rates in HSIL or more severe cytological findings were higher than those in women with cytological abnormalities across all age groups. In HSIL or more, the prevalence of HPV 16 was highest among women aged 20 to 29 years old, decreasing with age thereafter. As for HPV 52 and HPV 58, although the detection rate was high in 30- to 39-year olds, it had increased in the 50s and 60s again .Recent studies revealed that at least 13 HPV types, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 are commonly associated with ICC . While H In abnormal smear cases, HPV genotypes and positivity were HPV 16 at 27.5%, 52 at 27.3%, 58 at 26.9%, 51 at 9.6%, and 31 at 8.6%, in order of frequency. These data indicate that these HPV subtypes mainly involve abnormal cervical lesions. Miura et al. performed a meta-analysis of HPV type prevalence and type-specific risks for cervical carcinogenesis in Japan. They reported that 21 kinds of HPV types were detected from invasive cervical cancer, where the HPV genotypes and positive rates were HPV 16, 18, 52, 58, 33, 31, 35, and 51, in order of frequency , 13. The Recently, the interpretation of geographical variations of HPV type distribution has been requiring careful consideration in view of the differences in sensitivity of the HPV detection assays . For exa Clinical studies of  HPV vaccines have demonstrated close to 100% protection against HPV 16- and HPV 18-related infections and diseases –19, impl We also need to focus on HPV prevalence by age. HPV infection was most frequently detected in young women aged 15–25 years, and a second peak was observed in women aged 55 years or older, which is also consistent with results from African, American, and European populations , althoug In the future, HPV typing in conjunction with cervical cytology testing could be used for uterine cancer screening or in a follow-up program after conservative treatment of uterine cervical lesions since around 10% of NILM patients were HPV-positive. The natural history of HPV 52 and 58 is still unknown. Even after current widespread HPV vaccination, cross-protection against HPV 52 and 58 after the age of 30 remains incompletely understood, so attention should be paid to geographical distribution and causal significance of HPV types for uterine cervical lesions. A more sensitive and accurate method for HPV typing with corresponding clinical research will permit better understanding of the effects of the HPV genotype.In Japan, as a cause of abnormal cervical cytology, HPV 52 and HPV 58 cannot be ignored. Even after current widespread HPV vaccination, smear abnormalities focusing on HPV 52 and HPV 58 infection should be carefully followed up.


The rationale behind current worldwide human papilloma virus (HPV) vaccination programs starts from two basic premises, 1) that HPV vaccines will prevent cervical cancers and save lives and, 2) have no risk of serious side effects. Therefore, efforts should be made to get as many pre-adolescent girls vaccinated in order to decrease the burden of cervical cancer. Careful analysis of HPV vaccine pre- and post-licensure data shows however that both of these premises are at odds with factual evidence and are largely derived from significant misinterpretation of available data. The recent Editorial by Silvia de Sanjosé*  is problregardless of the vaccination status [et al. [In spite of much unwarranted and premature optimism, the fact is however that HPV vaccines have not thus far prevented a single case of cervical cancer . Instead, what the clinical trials have shown is that HPV vaccines can prevent some of the pre-cancerous CIN 2/3 lesions associated with HPV-16 and HPV-18 infection, a large fraction of which would spontaneously resolve n status . For exan status . Moreoven status . Accordi [et al.  CIN 2 is [et al. .if proven effective against cervical cancer as claimed), will result in 70% reduction of cervical cancers appears to be largely based on premature, exaggerated and invalid surrogate marker-based extrapolations [Indeed, the optimistic assumption that HPV vaccination  reliability of surrogate-markers ;(2) efficacy against oncogenic HPV strains not covered by the vaccine;(3) possibility of increased frequency of infections with these types;(4) efficacy in women acquiring multiple HPV types;(5) effects in women with pre-existing HPV infectionsFast Track approval by the U.S. Food and Drug Administration (FDA) after a 6-month review process, despite the fact that it failed (and still continues to fail) to meet a single one of the four criteria required by the FDA for Fast Track approval. Gardasil is demonstrably neither safer nor more effective than Pap screening combined with the loop electrosurgical excision procedure (LEEP) in preventing cervical cancers, nor can it improve the diagnosis of serious cervical cancer outcomes [unwarranted confidence in the newHPVvaccines led to the impression that there was no need to actually evaluate their effectiveness” [It is also noteworthy that Merck’s HPV vaccine Gardasil received priority outcomes . In thisiveness” .Similarly, the notion that HPV vaccines have an impressive safety profile can only be supported by highly flawed design of safety trials ,13 and iAlthough a report to a vaccine safety surveillance system does not by itself prove that the vaccine caused an adverse reaction, the unusually high frequency of adverse reactions related to HPV vaccines reported worldwide, as well as their consistent pattern (i.e. nervous system-related disorders rank the highest in frequency), points to a potentially causal relationship . FurtherIn summary, the optimistic claims that HPV vaccines will prevent cervical cancers and save lives, and that they are extremely safe, rest on assumptions which are misinterpreted and presented to the public as factual evidence. We thus conclude that further reduction of cervical cancers might be best achieved by optimizing cervical screening  and targeting other factors of the disease rather than by the reliance on vaccines with questionable efficacy and safety profiles ,25.To those who wish to promote HPV vaccination as a means for reducing cervical cancer burden, perhaps the following should be asked:1. HPV vaccines have not been demonstrated to prevent any cervical cancers so why are they being promoted as cervical cancer vaccines?2. If the majority of HPV infections and a great proportion of pre-cancerous lesions clear spontaneously and without medical treatment and are thus not a reliable indication of cancer later in life, then how can these end-points be used as a reliable indicator of the number of cervical cancer cases that will be prevented by HPV vaccines?3. How can the clinical trials make an accurate estimate of the risk associated with HPV-vaccines if they are methodologically biased to produce type-2 errors ?4. Can a passive monitoring system such as that used by most vaccine surveillance systems world-wide allow the medical regulatory agencies to make accurate estimates on the real frequency of HPV-vaccine related adverse reactions?5. Can an accurate estimate of the real frequency of HPV-vaccine related adverse reactions be made if appropriate follow-up and thorough investigation of suspected vaccine related ADRs is not conducted but instead, these cases are a-priori dismissed as being unrelated to the vaccine?6. Why are women not informed of the fact that in some circumstances , HPV vaccination may accelerate the progression of cervical abnormalities ,26-28?7. How can women make a fully informed decision about whether or not to consent to vaccination if crucial information regarding HPV vaccine efficacy and safety is not being disclosed to them?8. Should the medical health regulators and authorities rely solely on data provided by the vaccine manufacturers to make vaccine-policy decisions and recommendations ,29?The authors declare that they have no conflict of interests.LT was involved in choosing the topic and drafting the initial manuscript. CAS, JW, EV and TB were involved in critically revising the manuscript and additional content. The authors have read and approved the manuscript. This work was supported by the Dwoskin and Katlyn Fox Family Foundations.


High-risk human Papillomavirus infection is a necessary factor for cervical squamous intraepithelial lesions and invasive cervical cancer. In HIV-1-infected women, HPV infection is more prevalent and a higher risk of cervical cancer has been identified. We aimed to calculate the prevalence of infection by HR-HPV, determine the factors associated with this infection and abnormal cytology findings and to describe the history of cervical cancer screening in HIV-1-infected women.We enrolled 479 HIV-1–infected women from the PISCIS cohort. Each patient underwent a gynecological check-up, PAP smear, HPV AND Hybrid capture, HPV genotyping, and colposcopy and biopsy, if necessary. We applied questionnaires to obtain information on sociodemographic, behavioral, clinical, and cervical screening variables. We present a cross-sectional analysis.3 versus >500cells/mm3 , HIV-1 viral load >10,000copies/mL versus <400copies/mL , and use of oral contraceptives . Sixty percent of HIV-1–infected women had had one Pap smear within the last 2 years.Median age was 42 years. The prevalence of HR-HPV infection was 33.2% and that of high-grade squamous intraepithelial lesions (HSIL) was 3.8%. The most common genotypes were 16(23%), 53(20.3%), and 52(16.2%). The factor associated with HR-HPV infection was age <30 years . The factors associated with the presence of HSIL or low-grade squamous intraepithelial lesions (LSIL) were CD4T-lymphocyte count <200cells/mmThe high prevalence of HPV infection and cervical lesions in the HIV-1–infected population in Catalonia, as well as the low coverage and frequency of screening in this group, means that better preventive efforts are necessary and should include vaccination against HPV, better accessibility to screening programs, training of health care professionals, and specific health education for HIV-1–infected women. High-risk human papillomavirus infection (HR-HPV) is a prerequisite for the development of squamous intraepithelial lesions (SIL) and invasive cervical cancer (ICC). Several epidemiological and molecular studies have established a causal association between HPV and ICC ICC accounts for 9.8% of all human cancers, and every year 500,000 new cases are diagnosed throughout the world. Around 280,000 of these women die, and at least 80% of these deaths occur in developing countries Spain has one of the highest prevalences of HIV-1 infection in Western and Central Europe In Spain, the annual incidence of ICC in women of all ages is 6.3 per 100,000 inhabitants and mortality is 1.9 per 100,000 inhabitants. In young women (15 to 44 years), the incidence of ICC is 6.6 per 100,000, making it the second most common cancer after breast cancer HIV-1–infected women are more susceptible to developing HPV infection and SIL Several authors have discussed the question of whether the high prevalence of HPV infection and ICC in HIV-1–infected women varies depending on the effectiveness of cervical screening programs With the aim of studying the association between HIV-1 infection and HR-HPV in our setting, we selected a subcohort of HIV-1–infected women from the PISCIS cohort . The objectives of the present study were to estimate the prevalence of HR-HPV infection and SIL and associated factors, to describe clinical-epidemiologic characteristics, and examine the history of cervical cancer screening in this population.The present study was designed as a cohort of HIV positive women from 9 hospitals included in PISCIS cohort. The presented manuscript only shows a cross-sectional analysis of base line data obtained during the first visit of the participating women to the gynecologist. The recruitment period was from September 2007 to March 2009The study population was an opportunistic sample taken from patients included in the PISCIS cohort Nine centers from Catalonia were responsible for recruiting and following the women who participated in this study. The hospitals were Hospital Clínico-IDIBAPS (Barcelona), Hospital Universitario de Bellvitge , Hospital de Mataró (Mataró), Corporació Sanitària Parc Taulí (Sabadell), Hospital Universitario Germans Trias i Pujol , Hospital General de L'Hospitalet , Hospital de Palamós , Hospital Comarcal del Alt Penedès (Vilafranca del Penedès), and Hospital de la Santa Creu i Sant Pau (Barcelona).With an alpha error of 0.05, a hypothetical prevalence of HPV of 45%, and a power of 80% for an odds ratio greater than or equal to 1.9 in the risk factors study, the necessary sample size was calculated to be approximately 500 women. The sample n = 500 considered an oversizing of 10% for attrition (losses) of the sample, the power of 80% was considered for the original sample without oversizing.The ethics committees of the participating hospitals approved the study and patients gave their signed informed consent to participate. A total of 479 HIV-1–infected patients were recruited during the study period.Recruitment was carried out by internists who invited the patient to participate in the project and obtained the informed consent, they derived the recruited women to the gynecologist participating in the study and communicated to the coordinating center the number of women who had been recruited for the study.The study was very well accepted among the women participating in the PISCIS Cohort and only 2 women refused to participate.At the first visit, a clinical-epidemiologic questionnaire consisting of 40 questions to obtain information on sociodemographic, behavioral, clinical, and cervical screening variables, was administered by the gynecologist. In addition, the questionnaire included 9 questions on screening history; these 9 questions were completed by the gynecologist from medical records.All the patients on the first visit underwent a gynecological check-up and two biological samples were obtained: a first sample was taken for a conventional or liquid cytology test (PAP smear) for subsequent processing at the hospital. A second sample was taken to determine the presence of HPV DNA using a second-generation hybrid capture assay  and to genotype the virus . The second group of samples was processed at the HPV laboratory of the Cancer and Infections Unit . The frequency of the follow-up visits and treatment depended on the results of the diagnostic tests: patients with normal cytology results and negative HPV DNA results had a follow-up visit at 12 months. Patients with an abnormal cytology result or positive HR-HPV, or both underwent colposcopy. Irrespective of the results of colposcopy, patients attended a follow-up visit at 6 months. In cases of an abnormal colposcopy result, a cervical biopsy was performed according to the internal procedures of each hospital. All those patients with cervical lesions were treated according to care protocols.The HC2 technique identifies HPV DNA using in vitro hybridization with specific RNA probes and can detect 13 types of HR-HPV .Genotyping by Linear Array amplifies and detects a fragment of HPV DNA. The PGMY09/11 primer set targets a 450-bp region of the HPV L1 gene and can identify 37 types of HPV with a low, high, and probable oncogenic risk .In order to cross the HIV and HPV co infection data of women participating in the study, data from HIV infection history (CD4 (cells/mm3), CV (copies/mL), treatment, etc.) of each patient were retrieved from the PISCIS Cohort databases this information was taken from the medical records. The data of CD4 and VL measures included in the analysis was the obtained closer to the date of the first visits to the gynecologist in the study of each patient, the latest CD4 and VL that were in medical records.2 test; when the expected frequencies were less than 5, the Fisher exact test was applied. Univariate and multivariate logistic regression models were applied to identify possible factors associated with HR-HPV infection and the presence of abnormal cytology findings . The measure of association used was the odds ratio (OR), and a p-value of <0.05 was considered statistically significant. Data were analyzed using SPSS version 17.0.Quantitative variables were described using the median (interquartile range [IQR]); qualitative variables were described using percentages. Groups were compared using the Pearson χOf the 11,374 patients comprising the PISCIS cohort, 2,519 (22.1%) were women. The population of the present study comprised 479 HIV-1–infected women .3  and the median viral load was 50 copies/mL . Median time on treatment was 90 months , and the median time from diagnosis of HIV-1 infection documented in the medical records was 119 months With regard to history of cervical cancer screening, 11% of the number of women infected by HIV-1 had never had a Pap smear, only 60.0% had had one Pap smear within the last 2 years, and 50.6% reported a frequency of annual screening.Of the 479 patients comprising the study sample, 159 (33.2%) had a positive result by HC2. The prevalence of infection by HR-HPV according to age group is shown in Genotyping was performed on all patients who were HPV (HR) positive by HC2 (n = 159). Genotyping results were obtained for 148 (93.0%) patients, 11 (7%) were other values 0.Thirty two (21.6%) women with genotyping results had only one type of HPV, 116 (78.4%) women presented multiple infections. Most of them had between 2 and 4 different types of HPV. (60.1%) The distribution of genotypes is shown in The most prevalent types of HR-HPV and probable HR-HPV, including single and multiple infections, were HPV 16 , 53 , and 52  . The mosThe results of the Pap smear were as follows: atypical squamous cells of undetermined significance (ASC-US), 7.9% (n = 38); LSIL, 13.8% (n = 66); and HSIL, 3.8% (n = 18). Pap smear revealed that 84.8% and 100% of patients with LSIL and HSIL respectively had HR-HPV. Similarly, 34.2% of patients with ASC-US and 20.2% of patients with a normal cytology result had HR-HPV. The most prevalent HR- HPV and probable HR-HPV types in HSIL, including single and multiple infections, were 16 and 53 (37.5% each) and 52 (31.3%), the most prevalent LR-HPV types in HSIL were Cp6108 (37.5%), HPV 84 and 81 (25% each) and HPV IS36 y 67 (18.8% each)3; most women (64.7%) had a viral load of <400 copies/mL and 48.5% had taken antiretroviral treatment for <60 months.The main differential characteristics in HIV-1–infected patients according to the result of HC2 and cytology are shown in 3 versus >500 cells/mm3 , viral load >10,000 copies/mL versus <400 copies/mL , and use of oral contraceptives   times greater than that of women aged >40 . The fac1.0–3.9) .Our study is the first multicenter study performed in Catalonia to provide data on the prevalence of HR-HPV infection, cervical lesions, and the distribution of high-risk genotypes present in the HIV-1–infected population. We also report data on history of cervical cancer screening.In HIV-1–infected women, viral persistence is greater and clearance of HPV infection is lower than in immunocompetent women; therefore, the prevalence of HPV infection observed in this population is greater than in the general population, and the risk of progression to high-grade lesions and cancer is also greater In the present study, 78.4% of HIV-1–infected women had multiple infections. Irrespective of the technique used to detect HPV DNA, these data are consistent with the results of several studies showing that HIV-1–infected women not only have a greater prevalence of HR-HPV infection, but that they are also infected by a wider variety of types HPV than immunocompetent women As HIV-1 and HPV are both transmitted through sexual relations and share risk factors for acquisition associated with sexual practices, it is difficult to address the infections separately. The diversity of genotypes and the high prevalence of multiple infections in HIV-1–infected women can be explained more by the effects of HIV-1–induced immunosuppression  Consistent with the results of previous studies in Spain Infection by HR-HPV is necessary for the development of SIL and ICC When we analyzed the distribution of HPV in HSIL, we found a high prevalence of multiple infections, together with the presence of HPV genotypes with lower oncogenic potential. This is probably because in HIV-1–infected women, whose immune systems are weakened, these types of HPV are more likely to evade the immune system and persist in preneoplasic lesions According Darwich L et al. Unlike the general population, HIV positive women have a wide range of genotypes in both HSIL and LSIL, probably by the characteristics of sexual behavior and the reactivation of latent infections, which can encourage infection by different HPV types. Taken into account the high prevalence observed of HPV 16 and 18, it is important to consider anti-HPV vaccination as a primary preventive measure in HIV positive women. Studies evaluating the safety and immunogenicity of the quadrivalent human papillomavirus vaccine in HIV-infected women are in progress and are certainly needed.Differences in the capacity to evade the immune system have been observed among different HPV types. It has been shown that the infection by HPV 16 would not depend as much on immune status, whereas infection by other less prevalent types would, as these could be enhanced by suppression of the quality of the immune response Data from the questionnaires and the clinical records show that 75.2% of women participating in the present study became infected by HIV-1 as a result of heterosexual relations. The high level of promiscuity in the study population—most participating women had their first sexual intercourse before age 18 and the proportion of monogamous relationships was low—could explain why the most common variables associated both with HR-HPV infection and with the presence of abnormal cytology findings were not significant in this study.The prevalence of HPV infection decreases with age 3 compared with 6.1% of women with a normal cytology result. A similar association was found for detection of HR-HPV and shorter time of treatment. However, these differences only persisted in the multivariate analysis for abnormal cytology findings but not for detection of HR-HPV—due to the existence of confounders such as age or sexual behavior, also these women might have a poorer follow up in general—and significance disappeared in the multivariate analysis. In any case, longitudinal studies would be necessary to ascertain whether immune reconstitution induced by antiretroviral therapy has an effect on the development and progression of cervical lesions in this group, as well as on the incidence and persistence of HR-HPV infection.The relationship between antiretroviral therapy and the natural history of HPV infection remains controversial. Whereas some studies have not found a clear association between antiretroviral therapy and the reduction in the prevalence of cervical lesions and HPV infection 3 or cytology findings are abnormal. The present study revealed inadequate coverage and frequency of cervical cancer screening in HIV-1–infected women: 60.0% had a Pap smear within the last 2 years and 50.6% reported a frequency of annual screening. The data showed an inadequate screening in HIV positive women. It is necessary to design and to implement specific protocols addressed to vulnerable populations.The Catalonian Cervical Cancer Screening Protocol recommends that HIV-1–infected women undergo 2 Pap smear separated by an interval of 6 months or 1 Pap smear with colposcopy. In addition, women should have an annual Pap smear, or more frequent Pap smears if their CD4 T-lymphocyte count is <500 cells/mmTaking into account an estimated incidence of HSIL in the Spanish general population of 10 per 100,000 inhabitants, we would expect to find 0.047 cases of HSIL in 479 women, thus confirming the finding that 18 cases of HSIL in our study sample (n = 479) is much higher than expected in women from the general population who receive regular gynecological care. These findings are consistent with the high incidence of ICC found in HIV-1–infected women in Catalonia Our study has some limitations. The prevalence of HR-HPV infection was determined based on the sensitivity level set in HC2 for detection of high-risk types, and this limit has been clinically validated in several randomized clinical trials. Higher sensitivity limits or broader-spectrum primers could have revealed more cases of HPV infection. However, these limitations did not affect the objectives of the study: the wide scientific evidence available shows that HR-HPV are the most important types of HPV at an epidemiological level, since they cause more than 99% of cases of ICC in both the general population and the HIV-1–infected population. It has to be taken into account that the cohort included a non-discriminatory sample, according to degree of immunosuppression, evolution of HIV infection and treatment, as a consequence, the population of women with recent infection with HIV was not represented in our study. The sample included only women who had a prolonged history of infection . In addition, many were taking HAART (92.7%) and the median age was high (42 years). These findings could represent an underestimation of HR-HPV infection and of cervical lesions in this population. On the other hand, the sample was not representative of the total HIV infected population in Catalonia, however, when we contrast our data with the AIDS registry of Catalonia, the clinical epidemiological characteristics did not differ, moreover, the sample came from 9 hospitals that account for 80% of HIV patients in Catalonia.Finally, we retrieved the data closer to the date of 1st visit to the gynecologist for each patient (at inclusion of patients in the study), which means, the latest CD4 and VL who were in medical records. The time frame was not defined, however, over 80% of records corresponding to the last six months, both CD4 and VL.In conclusion, we confirmed the high prevalence of HR-HPV infection and cervical lesions in HIV-1–positive patients. HPV16 was the most prevalent type. The most important determinant of HR-HPV infection was age less than 30 years. More abnormal cytology findings were recorded in women with a poorer immune status. Coverage and frequency of cervical cancer screening in HIV-1–infected women in Catalonia is suboptimal, especially considering the high-risk nature of this population. Optimizing prevention of ICC is a priority in these patients.


To determine the potential association between HPV infection and the squamous cell component of urothelial carcinoma (UC) of the bladder and to validate p16 overexpression as a surrogate marker for HPV infection in these cancers among Koreans.We analyzed the presence of HPV infection using an HPV-DNA chip and the expression of p16 using immunohistochemistry in 47 subjects between July 2001 and March 2011. The study group (n = 35) included patients with squamous differentiation of UC of the bladder. The control group (n = 12) included patients with squamous metaplasia of the bladder.Baseline characteristics of control and study groups were similar. HPV DNA detection rates were approximately 2-fold higher in the study than the control group , but the difference was not statistically significant. P16 overexpression was detected in 16/35 (45.7%) study group and 1/12 (8.3%) control group samples (p = 0.034). Both HPV-positivity and p16 overexpression were present in 3/35 (8.8%) study group samples, but none of the control group (p = 0.295). In the study group, the percentage of HPV-positive cases who were non-smokers was 2-fold higher than the percentage of HPV-negative cases who were non-smokers ; however, statistical significance was not achieved due to the small sample size.HPV infection may be associated with UC of the bladder with squamous differentiation, especially in non-smokers. However, p16 expression does not appear to be a strong surrogate marker for evidence of HPV infection in this type of cancer. Bladder cancer (BC) accounts for 3.2–4% of all cancers worldwide and approximately 90% of BC are urothelial carcinoma (UC). Although UC of the bladder often has focal squamous differentiation, it is differentiated from squamous cell carcinoma (SCC) of bladder, which contains solely keratin-forming carcinoma cells. BC composed of mixed urothelial and squamous phenotypes is called UC with squamous differentiation (UC/SCC) Among the known carcinogens of UC of the bladder, cigarette smoking is the most well-characterized risk factor. It is responsible for nearly half of BCs Recently, the role of viruses has received considerable attention as potential carcinogens, especially human papilloma virus (HPV). HPV is a small, circular, double-stranded DNA virus that infects stratified squamous epithelium and has been estimated linked to almost 10% of all cancers worldwide, especially a subset of SCCs of the vulva, penis, anus, and oropharynx A related issue involves the possibility that p16 protein is overexpressed in UC of the bladder. The expression of p16 is well known to be associated with high-risk HPV infection in cervical cancer and head and neck cancer and it has been suggested as a qualified surrogate marker for the identification of biologically active HPV infection Therefore, the aim of the present study was to determine the presence of HPV infection and to validate the possibility of p16 overexpression as a surrogate marker for HPV infection in UC/SCC of the bladder in Koreans smokers and non-smokers.Following approval of the study by our Institutional Review Board, 47 patients were selected from the archives of the hospital from July 2001 to March 2011 (IRB No. NCCNCS-12-643). All patients provided verbal informed consent. The written consent was not obtained because the IRB of National Cancer Center approved to exempt the written consent procedure. We examined paraffin-embedded tissue samples obtained during transurethral resection of the bladder or cystectomy. The study group consisted of 35 patients with mixed UC/SCC of the bladder and the control group consisted of 12 patients with squamous metaplasia of the bladder. Of the study group patients, 33 had evidence of mixed UC/SCC and two had extensive SCC with a history of UC. The UC of the bladder specimens were classified according to the World Health Organization (WHO) and International Society of Urological pathology (ISUP) grading criteria and staged according to the TNM 2010 version. None of the patients had known or suspected histories of Schistosoma heamatobium infection, uterine cervix SCC, or head and neck SCC.A representative SCC component of each paraffin block sample was selected based on the presence of adequate quality and quantity of extracted DNA. DNA was extracted using Qiagen BioRobot M48 workstation  and the quality and quantity of DNA was measured using Nanodrop ND 1000 . The presence of HPV infection was analyzed using an HPV-DNA chip. HPV genotyping was performed as per the manufacturer's protocol using a polymerase chain reaction (PCR)-based DNA microarray system , consisting of multiple probes of the HPV L1 sequence Immunohistochemistry (IHC) staining of p16 protein was performed using specific E6H4 antibodies . IHC p16 expression was scored using a semiquantitative composite scoring system as follows: (1) staining intensity, defined as 0 for negative, 1+ for weak, 2+ for moderate, and 3+ for strong; (2) positive area, defined as the (10 X) fraction of stained tumor cells in the entire tumor; and (3) expression score, defined as the staining intensity multiplied by the positive area. The highest possible score was 30. Overexpression of p16 was defined as a score >20 .The relationships between the presence of HPV infection and clinicopathological parameters were assessed using Fisher's exact tests and Mann-Whitney tests. All statistical analyses were performed using STATA . Results were considered statistically significant if the two-sided p value was <0.05.Clinicopathological characteristics of the 35 UC/SCC study group patients and 12 squamous metaplasia control group patients are shown in HPV DNA was detected in 6 of the 35 (17.1%) study group samples, and 1 of the 12 (8.3%) control group samples . All HPVFor the study group, there were no differences in age, sex, stage, or grade between HPV-negative and HPV-positive cases. Of the 29 HPV-negative cases, 20 (69.0%) were smokers or ex-smokers, whereas of the 6 HPV-positive cases, only 2 (33.3%) were smokers or ex-smokers. For smokers or ex-smokers, the percentage with HPV-negative BC was approximately 2-fold higher than the percentage with HPV-positive BC. By contrast, the BC samples of non-smokers demonstrated an approximately 2-fold higher percentage of HPV-positivity than HPV-negativity .In addition to cigarette smoking, occupational exposure to aromatic amines, and use of specific drugs, such as cyclophosphamide and phenacetin, as known risk factors of BC, HPV infection has also been suggested as a potential causative agent of UC of the bladder Youshya et al. The HPV has an affinity for squamous cells and its association with human SCC of the uterine cervix, head and neck, and anus has been well established Although HPV infection has been associated with BC in many studies, it is important to realize that such an association is not equivalent to causation. None of the previous studies have proven the existence of a direct link between HPV infection and carcinogenesis in BC, although the possibility of a causal role has been suggested because of the similarity to cervical and head and neck cancers, for which a causal relationship has been established HPV encodes two oncoproteins: E6 and E7 Additional findings of interest in this study were the types of HPV and the higher percentage of non-smokers in the HPV-positive group than in the HPV-negative group. Almost all HPV-positive samples in the study group were type 18. The sole exception was a sample with HPV type 35, which has been considered a variant of HPV 16 and which has not been previously isolated from the bladder This study had some limitations. A retrospective study with a relatively small number of samples limited its statistical power and potential bias. The serology to HPV suggested by Badawi could not be performed because of the study design, even though the antibody response (immunoglobulin [Ig]G) to L1 capsids could be used as a marker of cumulative HPV exposures Based on the findings of this study, HPV infection appears to be associated with the development of mixed UC/SCC of the urinary bladder, especially in non-smokers among Koreans. However, the role of HPV infection in the development of this type of cancer may not be as significant as for SCC of the uterine cervix or the head and neck. Additionally, p16 expression does not appear to be a strong surrogate marker for evidence of HPV infection in UC of the bladder with squamous differentiation.


A vaccination programme targeted against human papillomavirus (HPV) types16 and 18 was introduced in the UK in 2008, with the aim of decreasing incidence of cervical disease. Vaccine roll out to 12–13 year old girls with a catch-up programme for girls aged up to 17 years and 364 days was accompanied by a very comprehensive public health information (PHI) campaign which described the role of HPV in the development of cervical cancer.st year medical students (school leavers: 83% in age range 17–20) at a UK university. The study was timed so that the first survey in 2008 immediately followed a summer's intensive PHI campaign and very shortly after vaccine roll-out (150 students). The second survey was exactly one year later over which time there was a sustained PHI campaign (213 students).A brief questionnaire, designed to assess acquisition of knowledge of HPV infection and its association to cervical cancer, was administered to two different cohorts of male and female 1We addressed three research questions: knowledge about three specific details of HPV infection that could be acquired from PHI, whether length of the PHI campaign and/or vaccination of females had any bearing on HPV knowledge, and knowledge differences between men and women regarding HPV. No female student in the 2008 cohort had completed the three-dose vaccine schedule compared to 58.4% of female students in 2009. Overall, participants’ knowledge regarding the sexually transmitted nature of HPV and its association with cervical cancer was high in both year groups. However, in both years, less than 50% of students correctly identified that HPV causes over 90% of cases of cervical cancer. Males gave fewer correct answers for these two details in 2009. In 2008 only around 50% of students recognised that the current vaccine protects against a limited subset of cervical cancer-causing HPV sub-types, although there was a significant increase in correct response among female students in the 2009 cohort compared to the 2008 cohort.This study highlights a lack of understanding regarding the extent of protection against cervical cancer conferred by the HPV vaccine, even among an educated population in the UK who could have a vested interest in acquiring such knowledge. The intensive PHI campaign accompanying the first year of HPV vaccination seemed to have little effect on knowledge over time. This is one of the first studies to assess detailed knowledge of HPV in both males and females. There is scope for continued improvements to PHI regarding the link between HPV infection and cervical cancer. Anogenital human papillomavirus (HPV) infection is the most prevalent viral sexually transmitted infection (STI) in the world today . InfectiSeptember 2008 saw the introduction of a UK nationwide HPV immunisation programme . The vachttp://www.fightcervicalcancer.org.uk/) and associated literature. The website has now been withdrawn and replaced with information on immunisation (http://www.immunisationscotland.org.uk/vaccines-and-diseases/hpv.aspx). Although these efforts were largely targeted to women, young men would also have been expected to have some exposure to the media campaign because of its intensity and range  in 2008 [Vaccine uptake in the UK has been excellent, at > 90% for 12–13 year-old girls. However, uptake rates for the catch-up programme are lower at less than 60% overall . VaccinaCorrect knowledge about HPV infection and cervical cancer could be important to inform decision making regarding uptake of the vaccine. Prior to vaccine roll out many studies revealed a lack of awareness of HPV (i.e. whether respondents had heard of the virus) and a lack of knowledge of the association between HPV and cervical cancer in the UK and a range of other countries -14. ReceThis study was conducted at the University of Glasgow, Scotland. Prospective medical students at this university are admitted to study on the basis of interview where depth of knowledge of current topics in medicine is assessed. The HPV vaccine was one of the main topics for 2008 and 2009 so new medical students at the university could be expected to acquire some knowledge of HPV. HPV knowledge was assessed in two groups of first year medical students, mean age range 17–20 years of age. The timings of the surveys were specifically chosen. The first survey was carried out in November 2008 immediately following the roll-out of the HPV vaccine and a summer’s intensive media campaign that aimed to inform the public about HPV and the HPV vaccine. The second cohort was surveyed one year later when many more females had received the vaccine and associated PHI. The multimedia PHI campaign was sustained over this one year period. Moreover, media interest in the death from cervical cancer of the celebrity Jade Goody ,22, and st, 2010), which is a rise in uptake of around 4.5 percent in comparison to previous years [http://www.cks.nhs.uk/cervical_screening/management/scenario_when_to_offer_cervical_screening/. The final research question was to find out if men and women acquired similar levels of knowledge about HPV from the same or different sources. This study is among the first to compare knowledge of details of HPV of an educated mixed gender cohort at a time of an extremely intensive media campaign then exactly one year later in a similar cohort where most of the females had received at least one dose of the HPV vaccine and following a sustained PHI campaign.We sought to answer three research questions. The first question we addressed was knowledge about three specific details of HPV infection that could be acquired from reading of PHI leaflets accompanying the HPV vaccination programme and from PHI in an intensive media campaign between 2008 and 2009. The second research question was whether vaccination of females in the group and a year’s PHI campaign had any bearing on HPV knowledge. In contract to policy in the rest of the UK where screening starts at age 25, from age 20, Scottish (and Welsh) women are offered three-yearly cervical screening for the detection of HPV-associated cervical disease. 73.7% of eligible women were screened in the past three and a half years . Medical students were chosen as a "best case scenario" of HPV knowledge acquisition among young adults because they are an educated population and may have a greater awareness of current medical issues: entry into medical school at Glasgow is by interview where prospective students are questioned on knowledge of current topics in human health. Moreover, as health professionals of the future, they may have high motivation to pay attention to and acquire current PHI. Student knowledge and opinion was assessed in 2008 in that year’s intake of medical students i.e. immediately following the implementation of HPV vaccine delivery in the UK, and then one year later in the 2009 intake of students. The same questionnaire was administered in both years.An 8-item closed-ended questionnaire, with, for females, a 3-item data gathering section to determine stage of vaccination, intention to attend smear testing and smear testing status, was developed from a literature review and in consultation with a vaccine specialist at Health Protection Scotland and NHS public health information specialists. No studies examining detailed knowledge of HPV in the UK were reported in the literature at the time of this study so three questions relating to HPV were designed to address specific points regarding HPV knowledge discussed in leaflets delivered to vaccinees and in other PHI. The draft questionnaire was piloted on students in the final year honours virology classes of both years. The questionnaire was then reviewed by members with relevant expertise of the University of Glasgow Ethics Committee and modified according to feedback received. The survey was administered at the same time of year, in November, first in 2008 and then in 2009. This timing was chosen because it preceded taught modules on cancer, including cervical cancer. The questionnaire was completed in a ten minute period at the end of a teaching session in a lecture theatre. One of the researchers introduced the study and explained the ethical permissions and voluntary nature of participation. Students made aware of how anonymity would be ensured. Although signed informed consent was given, the consent forms were collected separately to the questionnaires so that no questionnaire answers could be traced back to an individual. Ethical approval was obtained from the University of Glasgow’s Faculty of Biomedical and Life Sciences Ethics Committee.In 2008 the timing of the survey was chosen to be immediately following first roll out of the vaccine (September 2008) after a summer's intensive public health campaign. This included television, radio, internet, media, cinema and billboard adverts and age-directed leaflets available in public places, community and health centres and GP surgeries. Few females in the population were vaccinated in 2008. In contrast, in 2009, many of the female students had been offered the vaccine, delivered in association with appropriate PHI. Moreover, there was a sustained high level PHI campaign throughout 2009. In particular, during this period there was extensive media coverage of the illness and death due to cervical cancer of the celebrity Jade Goody ,22.In 2008 all 150 students (100%) and 213 out of 217 students in 2009 (98.2%) who attended the taught sessions at which the questionnaires were given out responded to the questionnaire. In 2008 the survey population consisted of 100 female and 50 male participants while in 2009 the survey population contained 114 female and 99 male students. In both survey populations the majority of students were between seventeen and twenty years of age   of students (97% of those eligible for screening (i.e. ≥20 years of age)) had been for a smear test while in 2009, 13.3% (n=15) (68% of those eligible for screening) had been tested. In both years the majority of women answered positively when asked if they would go for a smear test when invited. Importantly, in 2009, 94% of females who had been fully vaccinated said they would attend for a smear test when invited.The impact of the HPV vaccine campaign was also examined Table . Most stMany studies in different countries in a range of socio-economic groups have demonstrated an overall lack of awareness of HPV and its link with cervical cancer ,16,24,25The UK PHI leaflets accompanying HPV vaccination note the sexually transmitted nature of HPV. Surprisingly, although most students seemed to appreciate this fact, in agreement with another study , the 200other HR-HPV types. Consequently, anecdotal evidence suggests that the public may believe it is possible to develop cervical cancer without being infected with HPV [PHI leaflets clearly state that the vaccine protects against 70% of cases of cervical cancer. However, it is not made clear that the remaining 30% of cases are caused by with HPV ,32,33. SThe vaccine PHI intended to inform the public that the vaccine protected against two types of HPV. However, in 2008 around half the survey population did not appear to appreciate this fact. Male understanding did not increase by 2009 but females gave a larger number of correct responses in that year. This could be due to the increased percentage of vaccinated female students in 2009 that correlated with an increased number who claimed to have seen or heard HPV information in formal education, as expected due to the PHI delivered to vaccinees. However, as noted above, a lower percentage of the 2009 cohort of women realised that HPV caused most cervical cancers. Therefore our evidence is equivocal that PHI delivered with the vaccine improved HPV knowledge.Although there was some evidence of gender differences in HPV knowledge, especially in the 2009 cohort, gender did not have a significant difference on the sources of information on HPV. However, a much higher percentage of male students reported receiving information from word of mouth and more males in 2009 compared to 2008 were unable to rate PHI on the HPV vaccine. Interestingly, although the males in the study displayed knowledge of HPV association with cervical cancer, the majority answered ‘no’ to a question included in the survey “Do you think males should be offered the vaccine?” This could indicate perception of HPV vaccination as a female issue.The study has some limitations. Prior to administering the questionnaire it is possible that some students had begun to prepare for the oncology portion of their course by reading recommended materials and had thus acquired a greater appreciation of the subject prior to questioning. Some homogenisation of answers could have occurred through students comparing answers in class. The restricted time period (ten minutes) for filling in the questionnaire was designed to avoid this. In itself this may have introduced another limitation because the short time period may have led the students to provide rapid, poorly considered responses.Public knowledge of HPV, cervical cancer and vaccination is central to ensuring good vaccination coverage in the female population and success of future HPV testing strategies that will reduce the burden of disease. This survey indicates that PHI has been somewhat successful in delivering key elements of HPV knowledge for both genders in the young educated population studied, but that there are still important knowledge gaps especially in males. Continued efforts in pressing home the main messages regarding HPV, the HPV vaccine, and cervical screening are required.University of Glasgow Faculty of Biomedical Sciences Ethics Committee project approval number FBLS 0825 17/11/2008 and FBLS 0930 01/10/2009.HPV: Human papillomavirus; PHI: Public health information; STI: Sexually transmitted infectionWe declare no conflict of interest.SMcC compared the data analytically from both cohorts and wrote the first draft of the paper. GL conducted the survey and analysed data in 2008. IM conducted the survey and analysed data in 2009. AMcD carried out the statistical analyses. CC advised on analysis and co-wrote the paper. SG supervised the study and co-wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/264/prepub