Since carotenoids have physiological functions necessary for maintaining health, individuals should be selected to actively seek and develop a specific appetite for these compounds.Parus major in a diet choice experiment, both in captivity and the field, preferred carotenoid-enriched diets to control diets. The food items did not differ in any other aspects measured besides carotenoid content.Great tits Specific appetite for carotenoids is here demonstrated for the first time, placing these compounds on a par with essential nutrients as sodium or calcium. Mineral and nutrient appetite is defined as the motivation to seek or choose specific mineral/nutrient-containing items Parus major, a small passerine bird with carotenoid-based plumage coloration Here we show that great tits Tenebrio molitor) that had been experimentally enriched with carotenoids over control mealworms   , despite p<0.05) .T = 248, P<0.001). While there was no significant difference from the null expectation for the first mealworm chosen , this preference subsequently increased to 88% for mealworms with high carotenoid levels . Thus, there was evidence for an increase in preference over time from the first to all later choices combined . There was no significant effect of sex or age of great tits on the proportion of mealworms taken from the high carotenoid treatment .In field tests, great tits also significantly chose mealworms with experimentally high levels of carotenoids over controls  , that may be related to selection for carotenoid-rich food Although we do not have specific information on the underlying mechanism for the carotenoid preference, the lack of differences in appearance for mealworms used in the different treatments, makes us suggest that the preference may be based on olfaction or taste. There are no known olfactory or gustatory receptors for carotenoids in any animal. However, the volatile degradation products of carotenoids could potentially be readily tasted and smelled. However, we have to emphasize that although in the captivity experiment there was room for a learning process or a post-ingestive physiological feedback mechanism , since birds were trained to feed from the different mealworms previously to our experiment, great tits in the field tests made their choice of carotenoid enriched mealworms with no intervening training process. This strongly suggests a true specific appetite for carotenoids.In many different species, carotenoids are fundamental not only for maintenance of health but also for signaling individual quality The great tit is a small passerine bird broadly distributed in the Paleartic 3 cages with water ad-libitum and a nest box for roosting for a period of 20 days before being tested. Birds were trained for two weeks previously to the experiment, to eat from two feeders; one, containing carotenoid-enriched mealworms (larvae of Tenebrio molitor) and another containing control mealworms. The two feeders were both always present. Mealworms were fed with either corn (carotenoid-rich), or refined wheat flour (control) that differ greatly in carotenoid content, corn flour having a higher content of both lutein and zeaxanthin Great tits  were captured with funnel traps ad libitum. Since animals were housed individually, birds were not subjected to any social stress. Since experimentation consisted only of diet choice trials, and food provided consisted only of mealworms, approval by a special committee was not necessary.Great tits were captured and maintained in captivity with a special permission from the Catalan Government, Direcció General del Medi Natural, Generalitat de Catalunya. Experiments were conducted according to Catalan guidelines for the use of animals in research. The cages used were large enough (see before) to guarantee Great tit welfare. All the cages contained a nest box for roosting and resting, and food and water were available N = 11 great tits; A. P. Møller, J. Erritzøe and F. Karadas unpublished information]. This treatment assured that carotenoid-rich and control mealworms differed only in carotenoid content.Great tits were captured in mist nets near commercial bird feeders consisting of fat and seeds during December 2008– February 2009 in Orsay, France, and they were subsequently provided with color rings for identification. The food preference test was also based on mealworms differing in carotenoid content. Mealworms were kept on a diet of wheat flour with low carotenoid content until use. Following capture of great tits, pairs of 5 cm diameter cups for food with a distance between cups of 10 cm were provided in the same sites as where mist nets were used, with one randomly chosen cup holding either 1). five mealworms each injected in the hemocoel with 10 µl of 1.8% lutein and 0.2% zeaxanthin , or 2). five mealworms injected with 1 µl lutein and zeaxanthin (hereafter carotenoids) and 9 µl sterile water. The concentrations of carotenoids in mealworms were thus 1.43 and 0.14 µg/g, respectively. The amounts injected were so small that they remained inside the mealworms, and great care was taken to ensure that the liquid was not visible on the outside. These two treatments were chosen to assure that carotenoids were present in both treatments, and that a potential preference would relate to the amount of carotenoids rather than mere presence or absence of carotenoids. A total of 10 µl carotenoids in one mealworm amounted to 0.2% of estimated body content (92 µg) Using a pair of binoculars at a distance of 30 m APM recorded for each individual great tit visiting the choice apparatus the number and the order of mealworms taken from each of the two cups during a period of 15 min, or until the bird had ingested all (5) mealworms from one of the cups. A total of 34 great tits were tested during these trials. Each individual was tested only once. All birds ate from both feeders.Cyanistes caeruleus) has λ = 371 nm The coloration of the two types of mealworm was recorded using a Minolta CM-2600d spectrophotometer, that measures from 360 to 700 nm and provides values of brightness, chroma and hue (LCH) using the standard software provided by the instrument We measured coloration on the dorsal area and flanks of the mealworms used in the aviary experiment. Three measurements were obtained from each mealworm on different random areas of the body, and values were averaged. We ensured a good measurement by using a mesh of 3 mm and by gently pressing on the mealworms.Both types of mealworms were ground up to obtain a homogeneous mixture. For carotenoid extraction, one gram of each sample was weighed in triplicate and placed into falcon tubes containing 25 ml acetone. Suspensions were subsequently re-ground in ultra-turrax and vacuum filtered. Extraction procedures were repeated several times until filtrates were completely colourless.Extracts were then combined in decanting flasks and 25 ml ethyl ether were added. We homogenized solutions and added 50 ml of a 10% aqueous NaCl solution. The colorless hypophases were discarded and the epiphases repeatedly washed with the NaCl solution. Epiphases were filtered through beds of anhydrous sodium sulfate and placed in rotary-evaporator flasks. Solvents were evaporated and pigment concentrates recovered from flasks with small additions of HPLC grade acetone to a final volume of 10 ml. Aliquots of 0.5 ml from different pigment solutions were filtered through syringe filters with 0.2 µm of pore diameter for subsequent injection in an HPLC system.−1. The volume of sample injected was 20 µl, and detection was performed at 450 nm using a fixed-wave UV-visible detector.The separation system used was described by Mínguez-Mosquera and Hornero-Mendez Carotenoid pigments were identified by comparing their retention time and spectral data under the elution conditions with those obtained using pure standards One gram of mealworms (in 5 replicates) were placed in pre-weighed extraction thimbles and dried for 48 h at 80°C. They were cooled in an exsiccator and weighed on an analytical balance after which the thimbles were placed in a Soxhlet-apparatus. Neutral fat was extracted using a mixture of petroleum ether and chloroform (95/5 v/v) overnight. The mass decrease after drying at 40°C was taken as the amount of neutral fat. The remaining mass can be taken to consist of protein and ash. Ash content was determined by incinerating a subsample of the residue in a pre-weighed crucible at 600°C for 24 h. The method is the standard according to Blem In the diet choice preference experiments with captive birds, we measured the percentage of times that test individuals had fed from the carotenoid enriched mealworm along 10 tests. This percentage was used as a measure of preference (or avoidance). We analysed the data with a Wilcoxon signed-rank test In the diet choice preference experiments with free ranging wild birds, we compared for each individual (matched-pairs test) the number of carotenoid-enriched and control mealworms that it had ingested. For the analysis of a preference for the first mealworm chosen and for the subsequent selections, we used the log-likelihood ratio test, with a null hypothesis of 50% ingestion of carotenoid enriched mealworms.


Disparities in cervical cancer incidence and mortality rates exist among women of African ancestry . Persistent cervical infection with Human papillomavirus (HPV) is associated with cervical dysplasia and if untreated, could potentially progress to invasive cervical cancer. Very few studies have been conducted to examine the true prevalence of HPV infection in this population. Comparisons of cervical HPV infection and the type-specific distribution of HPV were performed between cancer-free Caribbean and US women.The Caribbean population consisted of 212 women from Tobago and 99 women from Jamaica. The US population tested, consisted of 82 women from Pittsburgh. The majority of the US subjects was Caucasian, 74% (61/82) while 12% (10/82) and 13% (11/82) were African-American or other ethnic groups, respectively. The age-adjusted prevalence of any HPV infection among women from Tobago was 35%, while for Jamaica, it was 81% (p < 0.0001). The age-adjusted prevalence of HPV infection for Caribbean subjects was not statistically significantly different from the US ; no difference was observed between US-Blacks and Jamaicans . However, US-Whites had a lower age-adjusted prevalence of HPV infections compared to Jamaican subjects . Subjects from Jamaica, Tobago, and US-Blacks had a higher proportion of high-risk HPV infections  compared to US-Whites (15%). Similar observations were made for the presence of infections with multiple high-risk HPV types . Although we observed similar prevalence of HPV16 infections among Caribbean and US-White women, there was a distinct distribution of high-risk HPV types when comparisons were made between the ethnic groups.The higher prevalence of cervical HPV infections and multiple high-risk infections in Caribbean and US-Black women may contribute to the high incidence and prevalence of cervical cancer in these populations. Evaluation of a larger sample size is currently ongoing to confirm the distinct distribution of HPV types between ethnic groups. Disparities in cervical cancer incidence and mortality rates exist among all women of African ancestry  ,2. The wAlmost all cases of invasive cancers of the cervix, most other anogenital tract cancers , and appWe have previously reported the HPV type distribution in two cancer-free Caribbean populations, Jamaica  and TobaThe Caribbean populations consisted of 212 women from Tobago  and 99 wAll subjects from Jamaica and Tobago combined are referred to in this study as Caribbean. The majority of subjects were African-Caribbean, although in Tobago, there were 19 subjects who defined their ethnicity as East Indian. The self-reported ethnic groups in the US population were classified as White, Black or other ethnic groups. One subject did not define her ethnicity and was included in the other ethnic group category.Standardized protocols were implemented for sample collection, DNA extraction and HPV testing for all three sub-populations that were included in this analysis. A nurse or clinician collected cervical brush samples and the DNA was extracted from these samples using the Puregene DNA purification kit . The HPV genotyping was performed on these samples using the Linear Array HPV Genotyping kit (Roche Diagnostics). All cervical samples were tested at the University of Pittsburgh. The assay involved amplification of samples by PCR using a master mix which contained biotin-labeled primers for the detection of the 37 most common HPV genotypes as well as the human beta-globin gene. The PCR products were chemically denatured and hybridized for 30 min at 53°C to linear array strips which contained specific and one cross-reactive oligonucleotide probe for the HPV genotypes as well as a high and low concentration of a beta-globin probe. The HPV genotypes were identified when visualized using a streptavidin-horseradish peroxidase conjugate and a substrate solution containing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine which yielded a blue precipitate at the positions where the hybridization occurred.For six samples from the US population, beta-globin was not positive, which indicated either poor quality or low yield DNA samples. These samples were excluded. Therefore, HPV results were available for only 82 US samples. All of the samples from Jamaica were tested with the AMPLICOR HPV method  prior toThe Linear Array protocol does not specifically detect HPV52. The cross-reactive probe in this assay detects HPV 33, 35, 52 and 58 combined. According to the manufacturer's protocol, samples that are negative for HPV 33, 35, and 58 individually, but positive for the cross-reactive probe are classified as HPV 52-positive. Samples that are positive for HPV 33, 35, and/or 58 individually, as well as the cross-reactive probe have an uncertain HPV 52 status. For our study these samples were considered negative for HPV 52.HPV-risk classification was based on the epidemiological classification of HPV types that are associated with cervical cancer . BrieflyAll statistical analyses were performed using STATA SE (version 10), . Age-adjusted prevalence rates and confidence intervals were calculated using logistic regression estimates of infection with any HPV type or any high-risk HPV type, adjusted for age. Observations with missing values of age were dropped from the analysis before estimation. The chi-squared test of proportions was used to calculate p-values for the unadjusted differences in proportions as well as, p-values for the differences in adjusted proportions. Although age and age at first sexual intercourse were both normally distributed, we rejected the hypothesis of equal variances. Therefore, the comparisons of mean age and age at first sexual intercourse were performed using two-sample t-tests on the equality of means adjusting for unequal variances.Table The mean age at first sexual intercourse for each subpopulation ranged from 17 to 19 years old, with an overall mean of 18 ± 3.5 years. The age at first sexual intercourse for Jamaican women was statistically significantly younger than women from Tobago  and US women .Thirty-five percent 75/212) of the women from Tobago, 84% (83/99) of the women from Jamaica and 32% (26/82) of US women, tested positive for HPV infection of any HPV type. Table 12 of theFigure The proportion of high-risk HPV infections appears to be highest among women of African ancestry. Jamaican women had the highest proportion of high-risk HPV infections (58.6%), then US-Black (40%), Tobago 20.3%) and US White (14.5%) women, followed by women from the US-other group (10%),  than in women from the United States (39%), although the age-adjusted prevalence of high-risk HPV infections  was similar in both populations. The inability to achieve statistical significance for high-risk HPV infections may be related to the small sample size of the US population and consequently insufficient statistical power to detect a difference between the two groups. Nevertheless, our findings suggest that the higher rates of HPV infection in the Caribbean compared to the US, are likely to explain the higher incidence of cervical cancer reported in the region [The distribution of HPV types was similar in the two Caribbean islands but was different from that observed in the US. This is not unexpected, since differences in the geographical distribution of HPV infections have previously been reported . We obseThe current HPV vaccine prevents infections from HPV types 6, 11, 16 and 18 . We obseCaribbean and US-Black subjects in comparison with US-Whites have higher prevalence rates of high-risk HPV infections and also a higher proportion of subjects with multiple-high-risk HPV infections. This may be related to sexual behaviour, social class , high parity, lack of barrier contraceptive protection and use of steroidal contraception, ,12-16. HStudies show that younger age is associated with an increased risk of cervical HPV infection -20. TherThe higher prevalence of cervical HPV infections and multiple high-risk infections in Caribbean and US-Black women may contribute to the high incidence and prevalence of cervical cancer in these populations. Further studies of the distribution of HPV types between ethnic groups and the risk of cervical cancer among women with multiple high-risk infections are currently ongoing.The authors declare that they have no competing interests.CR contributed to study design, sample/data collection for Tobago and US populations, performed the data analysis and wrote the manuscript. JBW performed the sample and data collection of the Tobago population. ET, contributed to the overall study design and the writing of the manuscript. AW, contributed to the study design, sample/data collection of the Jamaican population and performed the HPV testing. NM and RE: contributed to study design of the US population. AP, V W, CB and SMG: contributed to study design and sample/data collection for the Tobago population. SE: performed the HPV testing. HF, DG, MJ, MS, NY, NMA: contributed to the study design, sample/data collection of the Jamaican population. All co-authors reviewed and approved the manuscript.


Vaccines, that target human papillomavirus (HPV) high risk genotypes 16 and 18, have recently been developed. This study was aimed at determining genotypes commonly found in high-risk and multiple-HPV infections in Jamaican women. Two hundred and fifty three (253) women were enrolled in the study. Of these, 120 pregnant women, aged 15–44 years, were recruited from the Ante Natal Clinic at the University Hospital of the West Indies and 116 non-pregnant, aged 19–83, from a family practice in Western Jamaica. Cervical cell samples were collected from the women and HPV DNA was detected using Polymerase Chain Reaction and Reverse Line Hybridization. HPV genotypes were assessed in 236 women. Data were collected from January 2003 to October 2006.HPV DNA was detected in 87.7% (207/236) and of these 80.2% were positive for high-risk types. The most common high-risk HPV types were: HPV 45 (21.7%), HPV 58 (18.8%), HPV 16 (18.4%), HPV 35 (15.0%), HPV 18 (14.5%), HPV 52 (12.0%) and HPV 51(11.1%). Other high-risk types were present in frequencies of 1.4% – 7.2%.Multivariate regression analyses showed that bacterial vaginosis predicted the presence of multiple infections  and that alcohol use  and age at first sexual encounter  were significantly associated with high risk infections. Cervical cytology was normal in the majority of women despite the presence of high-risk and multiple infections.HPV genotype distribution in this group of Jamaican women differs from the patterns found in Europe, North America and some parts of Asia. It may be necessary therefore to consider development of other vaccines which target genotypes found in our and similar populations. HPV genotyping as well as Pap smears should be considered. Infection with oncogenic human papillomavirus (HPV) types is the primary cause of cervical cancer, the second most common cancer among women worldwide . In deveTwo hundred and thirty six women were enrolled in the study: 120 pregnant women aged 15 – 44 years (median age 27.5 years) at their first antenatal clinic visit to the University Hospital of the West Indies (UHWI) in Kingston, Jamaica and 116 non-pregnant women (median age 35 years) attending a family practice in Western Jamaica. Selection of the participants was consecutive. Participants were asked to complete a questionnaire that assessed information on socio-demographic factors, sexual practices and history of sexually transmitted infections (STI). Ethical approval was obtained from the University of the West Indies (UWI)/UHWI Ethical Committee. Informed consent was obtained from all participants. Participants were recruited January 2003 – October 2006.At enrollment, a routine gynecological examination was conducted and cervical specimens were collected first for Papanicolaou (Pap) smear and for HPV testing. The cervical cells for HPV testing were taken from the endocervix with one to two cytobrushes. The brushes were then agitated in a 15 ml tube containing 1 ml of specimen transport medium  for several seconds to release the cells. The cervical cells were stored at -20°C until further processing. Cytology results were obtained from Pap smears. Cytological abnormalities were classified according to the Bethesda classification system as within normal limits or reactive cellular changes ; atypical squamous cells of undetermined significance (ASCUS); cervical intraepithelial neoplasia I (CINI); cervical intraepithelial neoplasia II (CINII); cervical intraepithelial neoplasia III (CINIII) and carcinoma.Briefly, 250 μl aliquot of the cervical cell sample collected in the specimen transport medium was lysed in the presence of proteinase K. Isolation and purification of the released nucleic acid were carried out using columns and elution reagents and the presence of HPV DNA was detected using PCR-based methods: AMPLICOR  and the the presence of HPV infection and lifestyle/sexual practices characteristics. Multiple logistic regression analyses were used to examine the association of sexual and lifestyle behaviours with HPV infections. Statistical analyses were performed by the Statistical Package for Social Sciences (SPSS version 12).Chi-square statistics were used in the cross-classification of Table The median age of the pregnant and non-pregnant women was 27.5 years and 35 years respectively. Approximately two-thirds of women reported having attained secondary education and less than 10% were employed in professional occupations. HPV DNA was detected in 87.7% of the sample; the virus was identified in 90.5% of pregnant and 83.8% of non-pregnant women.Cytology results were obtained for 120 pregnant women and 92 non-pregnant women. Women with cervical dysplasia (14/207) had no consistent specific HPV types present, but many had multiple infections consisting of high- and low-risk HPV types, of which HPV types 16 and 58 were most common (data not shown). The majority of women with high-risk infection (93.6%) were diagnosed with normal cytology (data not shown).Eighty percent (80.2%) of HPV-positive women had HR-HPV infections and of these 60.9% had multiple infections. The percentages of low-risk and single infections were 61.8% and 34.3% respectively. Figure There was a wide distribution of genotypes in both groups of women (36 genotypes detected), although certain genotypes occurred more frequently in pregnant women. High-risk types 16, 18, 51, 52, 58, 59, and 68 were found in greater frequencies in pregnant women than non-pregnant women. Pregnant women also had the highest frequency of HR-HPV DNA (83.8%) and multiple infections (66.9%) compared to non-pregnant women . Twice as many pregnant as non-pregnant women had multiple genotypes present  or less than 34 years old  or were diagnosed with bacterial vaginosis (17.1%) were more likely to be infected by multiple genotypes. The patterns of multiple infections by all other variables were similar to those with high-risk HPV-positive infections (data not shown).Univariate regression analyses were used to examine associations of behavioural and reproductive factors with three outcomes: HPV positivity, the presence of multiple infections and high-risk HPV infections. Women who consumed alcohol  or who were 20 years or older at first sexual intercourse  were at decreased risk of high-risk infections. The presence of bacterial vaginosis at screening , being 25 years or younger  and pregnancy  increased the risk of multiple infections. There were no associations of any behavioural or reproductive variables with HPV positivity.In multivariate logistic regression analyses, controlling for age as a potential confounder, we examined significantly associated variables with high-risk HPV infections and multiple infections Table . Two varThis study which used cervical cells, a good medium for detection of HPV DNA [10 ] and the Gravitt et al. detection method , the mosThe most important finding was that unlike the genotype distribution patterns seen in North America, Europe and some parts of Asia ,12 HPV tDespite the predominance of genotypes other than types 45 and 58 in our population, it is not known whether these genotypes are important for the development of cancer. Several authors have suggested that HPV types 51, 52, 56 and 58 may act with HPV 16 in the development of dysplasia -21. MultThe majority of the women (93.2%) including those with high-risk infections had normal cytology. It has been suggested that cytological abnormalities may resolve before clearance of high-risk genotypes , and thiVarious studies have reported prevalence ranging from 14% – 90% . Our ratIn our sample younger women were more likely to be positive with high risk infections or to have multiple infections. However, age, was not a significant predictor of any of the outcome variables. This is in contrast to studies that show that younger women when compared to their older counterparts were at greater risk for HPV infection and that this may be due to older women having acquired greater immunity to HPV infection ,28.Multiple infections were detected in 60.9% of HPV-positive women. It has been suggested that (i) women with multiple HPV genotypes may be more prone to persistent HPV infections, and (ii) there is interaction between genotypes resulting in the development of dysplasia and cancer ,29. AlthAlthough we found no relationship with HPV infection and other STIs (data not shown), a significant association was observed between the presence of bacterial vaginosis and multiple infections. Watts et al reported similar findings and suggested that changes in the vaginal flora may compromise cervical mucosa making it more vulnerable to HPV infections .More pregnant, than non-pregnant women, in our population were found to acquire multiple infections. It is possible that pregnant women are at heightened susceptibility to infections as a result of the immunosuppression that occurs during pregnancy . PregnanOur results indicate that genotype distribution in Jamaica differs from that found in Europe, North America and some parts of Asia. This has implications for the use of the recently developed vaccines, in Jamaica and similar populations. HPV genotyping as well as Pap smears should be considered for additional testing.The authors declare that they have no competing interests.NA conceived of the study, carried out statistical analyses and drafted the manuscript. AW was responsible for recruitment and data collection and laboratory assays. CR provided assistance and guidance with the HPV testing and genotyping. DG was responsible for recruitment and data collection at his practice. MJ and NY participated in design and statistical analyses. MS and HF advised on design and recruitment. All authors contributed to the writing of the manuscript and read and approved the final manuscript.


Women with ACC also had significantly higher HPV DNA copy number per cell compared to the control group (P=0.00007). Higher HPV DNA copy number is associated with risk of developing ACC.The impact of the success of organised cervical screening programme results in a steady decline of the incidence of squamous cell carcinoma of the cervix but a concomitant increase in the incidence of the less common histological subtypes, particularly adenocarcinoma of the cervix (ACC). Although Human papillomavirus (HPV) infection is believed to be a necessary cause of cervical cancer, its role in the pathogenesis of ACC is not well established. Established associations between oncogenic strains of HPV and ACC are based on molecular studies carried out on entire tumour block sections. In this study, the cervical adenocarcinoma cells of a 10-year cohort of women diagnosed with ACC were dissected using the PixCell II Laser Microdissecting System to detect the HPV 16 genome sequence using the real-time quantitative polymerase chain reaction to confirm the presence of HPV DNA within ACC cells. By coupling these two sophisticated techniques, the HPV DNA copy number cell could be calculated to investigate its role. The prevalence of HPV 16 infection in this cohort was 24%, which is significantly higher than the control group ( In countries such as the United Kingdom, organised cervical screening has resulted in a steady decline in the overall incidence of cervical cancer. Cervical screening has enabled identification and treatment of squamous epithelial changes in its pre-malignant state, leading to a noted decline in the incidence of squamous cell carcinoma (SCC) in all age groups. Conversely, there is a concomitant rise in the incidence of ACC. This is largely due to a relative increase, but there is evidence of an absolute increase in the incidence of ACC in more recent birth cohorts ; particuHPV infection is necessary for the development of cervical cancer  system, which selects specific cell of interest for analysis, with minimal loss and destruction of DNA material (M Tris/HCl (pH8.5), 1 mM EDTA. The Nucleon BACC II DNA extraction kit used was from Tepnel Life Sciences, Manchester, UK. The TAQMAN Universal PCR Mastermix, Passive Reference and optimised buffer components was from Applied Biosystems, CA, USA.PCR grade Proteinase K digest is purchased from Roche. The Tween 20 (Polyxyethylene Sorbitane) and Ethylenediamine tetraacetic acid (EDTA) were purchased from Sigma. The DNA extraction buffer comprised a mixture of 50 mWe obtained permission from the Grampian Research Ethics Committee to identify the cases and controls from the Grampian University Hospitals (GUH) pathology database and to use archival paraffin-fixed specimens for analysis.Using the GUH population-based pathology database, a cohort of women diagnosed with ACC between 1991 and 2000 were identified. The archival paraffin-embedded tissue blocks were retrieved from the GUH tissue bank and reviewed by two consultant pathologists to confirm the histopathological diagnoses.Age-matched controls were identified from women who had undergone hysterectomy or amputation of cervix in 1991–2000. Four controls were selected for each case by year of operation and their date of birth were matched to within 24 months of their case.μm sections were cut onto glass slides and dried overnight. The sections were dewaxed and stained with 0.25% Touloudine blue (pH4.5). The cells were microdissected using the Pixcell II Laser Microdissection System  using the following settings: laser pulse spot 15 μm, voltage 100 mW and duration of laser exposure 10.0 milliseconds. Approximately 500 pulses are taken per tumour sample.The tissue sections were prepared for laser microdissection as follows: 5 The adenocarcinoma cells were dissected and used for the cases. For the controls, the columnar epithelium was microdissected to avoid contamination of possible HPV infection in the squamous epithelium. The squamous epithelial cells were laser microdissected and the DNA extracted from these cells to analyse for the HPV 16 gene sequence.The transfer film containing the dissected cells was removed from the vial cap  and incubated in a proteinase K digest  on a heating block at 55°C and then heat inactivated at 98°C. DNA extracted from these cells was stored at 4°C for sequence analysis.The Pixcell II Laser Microdissection System  is an inverted microscope with an infrared dissection laser, which targets the sample from above. The cells are transferred onto a transfer film, which is permanently bonded to the underside of a transparent vial cap (CapSure™ HS).The ABI 7700 PRISM® Sequence Detector  enables real-time quantitative PCR (RTqPCR) analysis for particular gene sequences to allow quantitation of PCR product.Beta-globin (accession number NG 000007.2) gene were entered into the programme to design primer and probe sets optimal for the ABI7700 PRISM® sequence detector . A BLAST search was performed (http://www.ncbi.nlm.nih.gov/BLAST) for each primer and probe oligonucleotide sequence to confirm specificity for the target locus. The probe was labelled with the fluorescent reporter molecule FAM, which emits as the PCR product is formed. The specific primer and probe sets were ordered from Applied Biosystems. The Beta-globin primer and probe sets had been used successfully with the ABI7700 Sequence Detector (The primer and probe sets for RTqPCR were designed using the Primer Express 1.0 software. The complete genome sequences of HPV16 (accession number NC 001526) and Detector .2/95% air with media changes twice weekly. Cultured cells were passaged weekly using PBS (Oxoid), followed by detachment of the cells using a mixture of trypsin/EDTA and then incubated at 37°C for 5 min. The cells are pelleted and seeded into sterile flasks where they were washed with ice-cold PBS and scraped into 10 ml ice-cold PBS. 100 μl of the cell suspension is removed and added to 100 μl of Trypan buffer. Once the cells were counted using a haemocytometer, the suspension was centrifuged to give a pellet.The CaSki cell line was cultured in Dulbecco's medium (Life Technologies) supplemented with 10% (v/v) fetal calf serum (FCS) in a 37°C incubator with 5% COBeta-globin gene sequence was used as a reference gene, the positive PCR product confirmed successful DNA extraction. A no-template triplicate was used as a negative control. DNA extracted from CaSki cell line pellet using the Nucleon BACC II method  was used as positive control. Each CaSki cell contains 600 copies of the HPV16 gene . V16 gene .Beta-globin DNA copy numbers using the average CT generated by quantitative PCR analysis of serially diluted DNA extract of a known numbers of Caski cells .The 2 × 2 A total of 55 cases of ACC were identified from the GUH pathology population-based database. The age distribution showed a bimodal pattern, with peaks in the 36–45 and over 55 years age groups . In all,Beta-globin gene sequence, was found in all the samples. Real-time quantitative PCR analysis showed that 24% (13/55) of the cases were HPV 16 positive. In the control group, 21/208 (10%) were positive for HPV16. HPV 16 infection was 2.7 times more likely to be found in ACC cells than in the control group and this finding is statistically significant  Odds Ratio=2.76 (95% CI 1.19–6.33).Successful DNA extraction, defined by presence of nificant . (χ2P=0χ2P=0.00007). There was no statistical difference in the HPV copy number in the grade of the cancer (P=0.03)  .The squamous epithelium adjacent to the cervical adenocarcinoma cells was analysed separately for HPV 16 infection. In all, 39 specimens had squamous epithelium in the archival specimens; the remaining 16 samples were punch biopsies of the endocervix and had no adjacent squamous epithelium in the archival specimens. In all, 10 of the 39 cases with adjacent squamous epithelium for analysis, had HPV 16 in the adenocarcinoma cells. Seven squamous epithelium specimens were positive for HPV infection. Of this, three had HPV 16 infection in both squamous and columnar epithelium. All three had low HPV 16 DNA copy number in the squamous epithelium. All three women were over 40 years old at the time of their diagnosis. Although the frequency of simultaneous infection is higher, the association is not statistically significant.The key finding in this study is the direct demonstration of HPV 16 DNA within the cervical adenocarcinoma cells. It confirmed the infection of the adenocarcinoma cells and therefore allowed the demonstration of the true prevalence of HPV16 infection in ACC. This added significant strength to the link between HPV 16 infection and the aetiology of adenocarcinoma of the cervix. The demonstration of HPV 16 infection in the adjacent squamous epithelium but not in the adenocarcinoma cells, supports our hypothesis that the previously recorded high prevalence of HPV 16 infection in ACC is due to infection in adjacent squamous epithelium.We have shown that using the LCM technique of tissue selection for analysis, a higher yield of DNA extraction is achieved. The specificity is also increased as we can confirm that the HPV 16 DNA is found within the tumour cells of ACC. Contamination of HPV 16 DNA in concurrent CIN can also be avoided. We also decrease the false negative rates by reducing the loss by dilution effect. By extracting DNA only from the tumour cells, the amplified PCR product has a higher concentration of HPV DNA.Previously published studies were based on a qualitative analysis of presence or absence of the PCR product. By using real-time quantitative PCR, the cycle by cycle detection of accumulated PCR product allows us to generate a standard curve, in order to calculate the gene DNA copy number. In conjunction with the LCM, for the first time, we are able to expand our understanding of HPV infection in ACC by studying the effect of HPV DNA viral load on the cancer cells.Women with ACC are 2.7 times more likely to have HPV 16 infection compared to the control population and have a higher HPV DNA copy number than the controls. The prevalence of HPV 16 infection in our control population is 10%, which is consistent with epidemiological studies and all of these women had a low HPV 16 DNA copy number. This finding strongly supports the theory that most HPV16 infection is transient  and highOver half of the HPV 16 positive samples came from women under 45 years old, which reflects the sexually transmissible nature of the infection. By using real-time quantitative PCR, we have demonstrated the variation in HPV 16 copy number across this population. The DNA copy number, however, is higher in women over 45 years old. One possible explanation for this finding is DNA replication during the longer incubation period of HPV16 infection before transformation to malignancy . The lowDirect infection of columnar cells is the main route of HPV 16 infection, as only 27% of the HPV 16 positive cases have concurrent infected squamous epithelium. This lower prevalence on concurrent HPV infection in the squamous epithelium can be explained by clearance of HPV infection from the squamous epithelium. In Stanley's immunobiological model on HPV infection of squamous epithelium, HPV infection is targeted at the basal epithelial cells. It is possible that the infected basal cells, of Stanley's biological model, could have differentiated into glandular epithelial cells and therefore the squamous cell will bear no evidence of HPV infection.By coupling the two powerful techniques of laser capture microdissection and real-time quantitative polymerase chain reaction, we were able to demonstrate a more accurate picture of the role of HPV infection in the pathogenesis of cervical adenocarcinoma.


Sir,British Journal of Cancer  infection  using different sets of primers, the PCR products were separated on a 2% agarose gel. Consistent with the studies of E6 and E7 (panel D). As expected, we also detected the long and short size variants of E6 mRNA that are generated by RNA splicing. Corresponding to our PCR data that indicated a lack of HPV DNA in the breast cells, we also found no evidence for expression of HPV-18 mRNA (panel D). To validate our RNA purification, we performed RT–PCR for GAPDH mRNA, which demonstrated that the RNA samples were of sufficient quality to detect the expression of a single copy gene.To address the possibility that the copy number of HPV-18 genomes was extremely low in these cells and undetectable by PCR, we also performed more sensitive RT–PCR reactions to detect HPV-18 mRNA. Cellular RNA was isolated by the TRIzol method, followed by one-step RT–PCR . PCR products were separated on a 2% agarose gel. In HeLa cells we detected transcription products for the two major transforming genes of HPV-18, et al. As the E6 and E7 genes of the high-risk HPVs are retained and expressed in all HPV-induced cervical cancers (To conclude, HPV-18 DNA and mRNA are not detectable in the MDA-MB-175VII and SK-Br-3 breast cancer cell lines, contradicting the study of Heng  cancers  and thei cancers , our res


Human papillomavirus (HPV) type-specific distribution was evaluated in genital samples collected from 654 women from the South of Italy undergoing voluntary screening and correlated with cyto-histological abnormalities. HPV DNA was detected in 45.9% of the samples, 41.7% of which had multiple infection and 89.0% had high-risk HPV infection. The prevalence of HPV infection and the rate of multiple infections decreased with age, suggesting natural selection of HPV types with better fitness. In line with other Italian studies, the most common HPV types were HPV-6 and HPV-16, followed by HPV-51, HPV-31, HPV-53, and HPV-66, in women with both normal and abnormal cytology. Cervical intraepithelial lesions grade 2 or 3 were associated with high-risk HPV-16, HPV-18, HPV-31, and HPV-51 infection. These data indicate that prophylactic HPV vaccination is expected to reduce the burden of HPV-related cervical lesions in this population, but also suggest the potential utility of new vaccines with larger type coverage. With the advent of the HPV vaccine, data on genotype-specific prevalence of HPV infection in sexually active female population would be useful to predict the potential benefits of HPV vaccination and to monitor the impact of vaccination on HPV type replacement. A recent meta-analysis estimated that the worldwide HPV prevalence in the cervix of women with normal cytology is about 10.4% . The patThis cross-sectional study describes HPV prevalence and type distribution in cervical samples from women resident in southern areas of Italy, that is, Apulia region and Naples area, who were referred for opportunistic screening and for gynaecological care for evaluation of suspected HPV-related lesions.From 2005 to 2008, a total of 654 genital swabs, which were collected from women aged from 17 to 60 years and living in Apulia region  and Naples city in the South of Italy, were analyzed. Women were referred to laboratory “Dr. Pignatelli  for opportunistic screening and for evaluation of HPV-associated lesions, so this patient population does not represent the general population of women attending public screening programs. The in force Italian privacy law did not allow to collect any data about life-style, sexual partners' number, and so forth. Most samples (87%) were cervical swabs, 3.5% were vulvar swabs, 3% vaginal, and 6.5% other genital swabs. Samples were stored in 1.5 mL saline solution at −20°C until DNA purification.μL cellular suspension by using an ABI PRISM 6100 System  and eluted in a final volume of 120 μL; 10 μL of each DNA sample were used for HPV PCR analysis. HPV PCR was performed using the SPF10 primers , which amplify a 65-bp fragment of the L1 open reading frame and allow detection of at least 28 different HPV types by hybridization on oligonucleotide probes [Total DNA was purified from 150 e probes , 5, as dThe INNO-LiPA HPV genotyping system can identify the following HPV types: HPV-6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69/71, 70, 73, 74 and 82. HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 were defined high-risk (HR) HPV types, whereas the other types were defined low-risk (LR) HPVs [P value of less than .05 was considered statistically significant.The association between HPV infection and clinical variables was assessed through the Chi-square test. A HPV DNA was identified in 300 of 654 samples (45.9%); 175 (58.3%) samples had single infection and 125 (41.7%) had multiple infection. Among HPV-positive samples, 267 (89.0%) had HR-HPV infection . HPV-6 aP < .0001), as well as the prevalence of multiple HPV infections tended to decrease with age, probably due to selection by immunity or HPV type fitness, as suggested by the different distribution of HPV types in different age groups. HPV DNA detection rate decreased with age, placing on 56.1% for women under 40 and on 37.7% in older ones  and ASC-US (n = 11) and in 83.3% of LSIL . Detection of HR-HPVs was common (80%) both in normal and abnormal Pap smears.In the 231 women who performed both HPV typing and PAP test, HPV DNA was found in about 50% of negative PAP tests  detection progressively increases with the worsening of histological pattern, with 100% CIN-2/3 cases positive for HR-HPVs, including HPV-16, HPV-18, HPV-31, and HPV-51 . This study on a female population from the South of Italy undergoing HPV testing for opportunistic screening or for evaluation of genital lesions shows a relatively high prevalence of HPV infection, 40% of which involved multiple HPV types. The most frequent HPV types were HPV-6, HPV-16, HPV-51, HPV-31, HPV-53, and HPV-66, with a different distribution in young versus older women, suggesting the existence of a natural selection of HPV types which preserve a better fitness, such as HPV-16 and HPV-31. HR-HPVs were detected in all CIN-2/3 samples, with HPV-16, HPV-18, HPV-31, and HPV-51 as the most frequent types. However, HR-HPV types were detected also in a high rate in women with a negative Pap test or with a negative histology. These findings are in agreement with other studies on HPV infection and type distribution in Italian women from different regions and in other European countries. In fact, a high prevalence of HPV infection, mostly caused by HR-HPV types, has been observed also in other Italian regions –18. All Similar HPV type distribution has been recently reported from other European countries, such as Denmark  and GermIn the present study, HR-HPVs, including HPV-16, HPV-18, HPV-31, and HPV-51, were detected in 100% of CIN2/3 lesions. The same distribution of HR-HPV types was identified in high-grade cervical lesions investigated in other Italian regions , 18, 21 The results of this paper provide information on HPV type distribution in women from Southern Italy and, by a revision of the recent literature, indicate that the epidemiology of HPV infection in these areas is not different from other Italian regions and from other European countries. Besides HPV-16 and HPV-18, which are targeted by the currently available HPV vaccines, other HR-HPVs, such as HPV-31 and HPV-51, appear to be diffused in the population of sexually active women and to be associated with high-grade cervical lesions. These HPVs should be taken into consideration in the development of enhanced vaccines with larger type coverage.


We earlier demonstrated, in a randomised clinical trial, that the regression time of flat penile lsions in male sexual partners of women with cervical intraepithelial neoplasia (CIN) was shorter in men who used condoms compared to those who did not. To further evaluate this finding, we examined whether the effect of condom use on the regression of flat penile lesions depends on the presence of human papillomavirus (HPV) type concordance in sexual couples, as determined in cervical and penile scrapes by GP5+/6+ PCR testing. A Cox model with time-dependent covariates showed a beneficial effect of condoms on regression of flat penile lesions in concordant couples  but not in those who were nonconcordant. When both partners harboured different HPV types, no effect of condoms was found . Delayed regression of flat penile lesions was associated with either stable lesions or with new penile lesions developing at sites surrounding pre-existing lesions suggesting reinfection of the penile epithelium. We conclude that condom use blocks sexual HPV transmission by preventing reinfection and development of new penile lesions in men who are susceptible to the same type as present in the female partner. Infection with high-risk types of human papillomavirus (HPV) is the major cause of cervical cancer and its precursor lesions . GenitalPartner studies have shown that penile lesions are found in more than half of the male partners of women with cervical intraepithelial neoplasia (CIN) , to identify 14 high-risk  and six low-risk  HPV genotypes with a high-risk and a low-risk HPV probe cocktail, respectively. Additionally, individual typing was performed for HPV 16, 18, 31, 33, 6, and 11, using type-specific probes in an EIA format. Samples, which were inadequate for HPV testing, that is, were negative in both β-globin and HPV PCR assays, were excluded from the analysis.Processing of specimens and testing for HPV was carried out at the Department of Pathology, VU University Medical Center, as described previously , where appropriate.Differences in characteristics of the condom and the noncondom groups were assessed by use of The outcome of interest in this study was regression of flat penile lesions. To study the complex interrelationship between HPV presence, concordance, and condom use on the regression of the penile lesions, we choose an approach in which changes in condom use or presence of female HPV infection during the study could be included. Therefore, to examine whether the effect of condom use on regression of flat penile lesions was related to the presence of HPV type concordance in sexual couples, Cox regression analysis with time-dependent covariates  was performed to compute hazard ratios and 95% confidence intervals (CI). In the modelling process, hazard ratios for the effect of condom use were computed using the noncondom group as the reference group in couples with a comparable HPV status.The HPV status in men was defined as positive or negative when penile scrapes were ever or never HPV positive during the study, respectively  and/or other low-risk HPV (not HPV type 6 and 11) during the study. This definition was used because of practical reasons that individual typing was only performed for HPV 16, 18, 31, 33, 6, and 11, whereas other high-risk HPVs and other low-risk HPVs each typed as a group. A more restrictive definition of concordance was subsequently used including only the couples that were concordant for 16, 18, 31, 33, 6, and/or 11 and excluding the couples that were concordant for other high-risk and/or other low-risk HPV types.For Cox regression analyses with time-dependent covariates, Stata 6.0 was used and for the remaining analyses SPSS version 9.0 software.P=0.388).A total of 139 men had flat penile lesions at baseline. Six of them used condoms at baseline and were excluded from this study. No follow-up results were available from 14 men, and in one female partner the presence of HPV could not be determined due to inadequate sample quality at baseline. The remaining 118 men formed the study population. At baseline, 61 (51.7%) couples started condom use and 57 (48.3%) did not. The median duration of condom use was 5.3 months (range 2.5–53.7). Failures of condom use were reported by six couples with a median of 1.5 times during the follow-up period (range 1–2). The median follow-up time of the study population was 12.5 months (range 2.5–77.5): 12.9 months (range 3.0–68.5) in the noncondom and 12.3 months (range 2.5–77.5) in the condom group (P=0.496 in women and P=0.775 in men). Women with HPV at baseline (n=101) had a median duration of HPV infection of 9.2 months and were not statistically different in the condom and noncondom group (P=0.852).Baseline characteristics of the study population are presented in P=0.350). Human papillomavirus type concordance was found for HPV 16 in 33 (58%), HPV 18 in 0 (0%), HPV 31 in two (4%), HPV 33 in two (4%), other high-risk HPV type in eight (14%), HPV 6 in one (2%), HPV 11 in zero (0%), other low-risk HPV type in three (5%), and multiple HPV types in eight (14%) couples, that is, HPV 16 and 31 (n=1), HPV 16 and other high-risk type (n=4), HPV 16 and 6 (n=1), HPV 33 and other low-risk type (n=1), and other high-risk and other low-risk HPV (n=1). Concordance for HPV 16 was equally prevalent in the condom and the noncondom groups (P=0.952).During the study, at least one concordant HPV type was found in 57 couples: 32 couples of the condom and 25 couples of the noncondom group . The estimated hazard ratio for the effect of condom use when comparing the concordant and the nonconcordant HPV-positive couples was 2.63/0.90=2.93, 95% CI 0.66–13.07 (P=0.159).Using a Cox model with time-dependent covariates, we examined whether the effect of condom use on regression of flat penile lesions was related to the presence of HPV type concordance in sexual partners. This analysis showed that the beneficial effect of condoms on regression of flat penile lesions was present in concordant couples  or the histological CIN grade . Inclusion of these determinants into the Cox model did not markedly change the hazard ratios for condom use.Regression of flat penile lesions was not related to the age of the men (hazard ratio 0.99, 95% CI 0.95–1.03, In case of a delayed regression of flat penile lesions, we observed that either the lesions were stable or that new penile lesions developed at sites surrounding pre-existing lesions suggesting reinfection of the penile epithelium. Hence, a diverse clinical course of flat penile lesions was noticed which was present in both the condom and the noncondom arm.Our data, collected in a randomised clinical trial, earlier demonstrated that the regression time of flat penile lesions was shorter in men who used condoms compared to those who did not (The presence of penile lesions associated with a given HPV type indicates that the man is susceptible for this particular HPV type. In men with penile lesions who harboured a similar HPV type as their female partner, we observed a delayed regression when couples did not use condoms compared to those who did. This finding suggests that failure of penile lesions to cure depends on reinfection by cervical HPV from the female partner for which the man is apparently susceptible. This is substantiated by the observation that new penile lesions often developed next to pre-existing ones. The beneficial effect of condom use in HPV concordant couples can be explained by blocking HPV transmission during sexual intercourse, thereby preventing reinfection and development of new penile lesions. In nonconcordant couples, we found no effect of condom use on regression of penile lesions. Apparently, these men are not susceptible for the development of new lesions by the HPV type, which is present in the female partner. Consequently, the clinical implication of our findings would be that condoms sort only effect in heterosexual couples that are positive for the same HPV type and that, in these couples, condom use should be continued as long as the female partner is HPV positive. In addition, condom use could also be advocated in sexual couples of whom the woman is HPV positive and the male partner does not have penile lesions since we do not know whether the male partner is susceptible for the HPV type of the woman and thus can acquire a penile lesion.So far, studies on the effectiveness of condoms in relation to the prevention of HPV infection and related disorders were mainly focused on women and available data were too inconsistent to provide precise estimates . Only a In this partner study, however, it should be realised that grouping into concordant and nonconcordant may be subject to some degree of underestimation of concordant sexual couples. Nondetection of HPV in penile scrapes does not necessarily reflect absence of HPV (In conclusion, the beneficial effect of condoms on regression of flat penile lesions was only found when sexual couples were HPV concordant. The effect of condoms is probably the result of blocking transmission of female HPV to the penis, thereby preventing reinfection and induction of a penile lesion to which a man is susceptible. As HPV testing becomes more widely used both for the management of women with HPV-related disorders and in cervical screening programs, accurate information on how to protect against HPV is needed. Our data suggest that condom use can reduce the spread of HPV in a sexually active population. Our findings warrant further studies on this topic.


Minimal deviation adenocarcinoma (MDA) of the uterine cervix is defined as an extremely well differentiated variant of cervical adenocarcinoma, with well-formed glands that resemble benign glands but show distinct nuclear anaplasia or evidence of stromal invasion. Thus, MDA is difficult to differentiate from other cervical hyperplastic lesions. Monoclonality is a major characteristic of most tumors, whereas normal tissue and reactive hyperplasia are polyclonal.The clinicopathological features and clonality of MDA were investigated using laser microdissection and a clonality assay based on the polymorphism of androgen receptor (AR) and X-chromosomal inactivation mosaicism in female somatic tissues. The results demonstrated that the glands were positive for CEA, Ki-67, and p53 and negative for estrogen receptor (ER), progesterone receptor (PR), and high-risk human papilloma virus (HPV) DNA. The index of proliferation for Ki-67 was more than 50%. However, the stromal cells were positive for ER, PR, vimentin, and SM-actin. The clonal assay showed that MDA was monoclonal. Thus, our findings indicate that MDA is a true neoplasm but is not associated with high-risk HPV.Diagnosis of MDA depends mainly on its clinical manifestations, the pathological feature that MDA glands are located deeper than the lower level of normal endocervical glands, and immunostaining. Minimal deviation adenocarcinoma (MDA) of the uterine cervix, also known as adenoma malignum, is defined as an extremely well-differentiated variant of cervical adenocarcinoma. One percent to 3% of all cervical glandular malignancies fit this definition . DistortAll seven cases of samples, including 3 cases of MDA, 1 adenomatous hyperplasia, and 3 hysteromyoma (controls), were collected between January 2008 and September 2009 from Tangdu Hospital, the Fourth Military Medical University . The study protocol was approved by the Medical Ethics Commission of the Fourth Military Medical University in Xi'an. All samples were surgically resected, fixed in 4% formaldehyde, and embedded in paraffin. Serial sections were cut and stained with hematoxylin and eosin (HE). Each case was examined by three pathologists and diagnosed according to the morphological criteria for MDA, adenomatous hyperplasia, and hysteromyoma.Immunostaining was carried out using a streptavidin-labeled peroxidase (S-P) kit (KIT9730) according to the manufacturer's instructions. The primary antibodies used in this study included mouse anti-human monoclonal antibodies (mAb) against carcinoembryonic antigen (CEA), cytokeratin (CK)18, cancer antigen (CA)125, estrogen receptor (ER), desmin, Ki-67, p53, and progesterone receptor (PR); rabbit anti-human polyclonal antibodies against S-100 protein and smooth muscle actin (SM-actin); and mouse anti-pig mAb against vimentin. All of the reagents for immunostaining were supplied by Maixin Biotechnology Corporation Limited, Fuzhou, China.In situ hybridization for high-risk human papillomavirus (HPV) was performed using a commercially available biotinylated HPV DNA probe mixture for HPV types 16 and 18  according to the manufacturer's instructions. Briefly, a 4-μm-thick formalin-fixed, paraffin-embedded sample tissue section was deparaffinaged in xylene and pretreated by microwave irradiation and pepsin digestion at 37°C for 30 minutes. Approximately 4 μL of the HPV DNA probe mixture was dropped on a pretreated sample tissue section, denatured at 95°C for 5 minutes, and hybridized at 37°C for 16 hours using a programmable temperature control system . The slide was washed, incubated with streptavidin-alkaline phosphatase solution (Maixin), and then visualized with nitroblue tetrazolium/bromo-4-chloro-3-indolyphosphatase. For the evaluation, samples were considered positive for high-risk HPV when >10% of glandular or cancerous cell nuclei were stained positively.2), obtained from representative paraffin blocks, were placed on a UV-absorbing membrane and underwent laser microdissection by LMD6000 . After HE-staining, the slides were mounted on a microstat, and the distorted glands were then dissected by UV laser using the motorized optical-beam scanning mode  at 37°C for 3 h in a volume of 20 μL containing 2 μL of 10× reaction buffer. The digested DNA samples, 1 μL each, were then subjected to nested PCR. The reaction mixture was 50 μL in volume, containing 4 μL of 10 mM dNTP , primers AR1A and AR1B (20 μM each), and 5 μL of 10× buffer, 1.5 μL of 50 mM MgCl2, and 2.5 U of Taq DNA polymerase (Gibco BRL). The amplification was conducted using a PT-200 thermocycler  for 25 cycles . For the second round, PCR products (1 μL) were used as templates for a second PCR reaction using primers AR2A and AR2B. The amplification procedure was the same as that in the first round. Finally, the amplification efficiency was checked by resolving PCR reaction aliquots on 2% agarose gels. The PCR products, 4 μL for each, were also mixed with the same volume of loading buffer  and resolved on an 8% polyacrylamide gel containing 8 mol/L urea using the Mini-VE system  at 120 V for 4 h. Bands were visualized using silver staining.Nested PCR was used to amplify detection of the length polymorphism of CAG short-tandem repeat (STR) in exon 1 of the AR gene loci as described previously . The genHpa I digestion, was used as indicator of loss of X chromosome inactivation mosaicism [Images of PCR gels were recorded, and the intensities of the PCR bands for both alleles were quantitated using an image-analyzing system . A reduction in fluorescence intensity of at least 50% for either band, as compared with the intensity of the bands obtained in the absence of Microscopically, lesions were characterized by mucinous glands that resembled normal endocervical glands but showed distinct nuclear anaplasia or evidence of stromal invasion. Mitotic figures was found Figure .It showed that CEA, Ki-67 and p53 were expressed in the glandular epithelium of MDA, but not in the normal cervical tissues Figure . StromalAs for in situ hybridization, all 3 MDAs examined were negative for high-risk HPV DNA, but blue grandular nuclei were found in the cervical tissue with positive control for HPV types 16 or 18.Hha I, PCR gels of DNA samples obtained from glandular epithelium showed two AR bands. After Hha I digestion, one band was no longer evident. However, regardless of Hha I digestion, the intensities of the two bands were equal for all the stromal cells, the case of adenomatous hyperplasia, and the 3 cases of normal cervical tissues  of the uterine cervix, also known as adenoma malignum, is a highly differentiated form of adenocarcinoma, representing about 1% to 3% of all endocervical adenocarcinomas . The terThe average age of patients described in the literature ranges from 42.6 to 53.3 years, and the youngest patient reported was 16 years . The aveHuman papillomavirus (HPV) is widely considered to be related to cervical carcinogenesis, and it is always detected in squamous cell carcinoma and its precursors. The oncoproteins E6 and E7, which are encoded in high-risk HPV types 16 and 18, typically are found in these lesions. A high incidence of HPV also has been reported in adenocarcinoma and adenocarcinoma in situ of the uterine cervix . HoweverHha I, the normal and reactive hyperplastic tissues with polymorphism show two alleles with equal intensity. Neoplastic tissues show only one of the two alleles, with obviously reduced intensity [The diagnosis of MDA is based on histopathological characteristics and immunophenotype. Microscopically, the glands are irregular in size and shape and lined predominantly by mucin-containing columnar epithelial cells with basal nuclei. Moreover, they typically exhibit deep invasion of the cervical wall. Immunohistochemically, glands are positive for CEA and Ki-67 but negative for ER, PR, and CA125. In our study, the results of histopathological and immunohistochemical observation were compatible with this description. In addition, we found that ER and PR were expressed in the stromal cells of both MDA and normal cervical tissues, a finding not described in the literature. However, it is almost impossible to histologically differentiate these tumor glands from normal endocervical glands, because the tumor glands of MDA are highly differentiated. Monoclonality is one of the main features of most tumors, whereas normal and reactive hyperplastic lesions are polyclonal . After dIn our study, the result of the clonal assay demonstrated that MDA was a neoplastic lesion, whereas adenomatous hyperplasia and normal cervical tissues were polyclonal, further confirming the nature of MDA. Understanding this distinction will help clinicians to differentiate MDA from benign hyperplastic lesions such as microglandular hyperplasia, adenomatous hyperplasia, diffuse laminar endocervical glandular hyperplasia, and lobular endocervical glandular hyperplasia.2. Although we performed laser microdissection in our study to obtain the pure glands, the sample was composed of numerous different glands, and sample sizes were about 1000 cells. The reason for pseudopolyclonal results is likely contamination caused by a heavy inflammatory cell infiltrate or inadvertent inclusion of stromal cells. We used laser microdissection to avoid this circumstance. Therefore, we believe that our result is reliable. However, additional studies with larger sample sizes are necessary to conclusively prove our hypothesis.PCR-based clonality analysis occasionally produces a pseudomonoclonal or pseudopolyclonal result . The majIn summary, MDA is a highly differentiated form of adenocarcinoma. The results of clonal assay indicate that MDA is a true neoplasm but is not associated with high-risk HPV. Diagnosis of MDA mainly depends on its clinical manifestations and the pathological feature that MDA glands are located deeper than the lower level of normal endocervical glands. Immunostaining is extremely helpful in diagnosing MDA.MDA: minimal deviation adenocarcinoma; AR: androgen receptor; PCR: Polymerase chain reaction; CR: corrected ratio; HPV: human papillomavirus; CEA: carcinoembryonic antigen; CK: cytokeratin; CA (cancer antigen)125; ER: estrogen receptor; SM-actin: smooth muscle actin; PR: progesterone receptorThe authors declare that they have no competing interests.LG selected the research topic, participated in the study, and wrote the manuscript. WDZ participated in writing the manuscript. YHL and WZ provided grant support. XYL and LY participated in the study. SJZ and XJH conducted the pathological examination. ML provided the technique support. All authors have read and approved the final manuscript.


Vulval intraepithelial neoplasia (VIN) is a premalignant condition, which is frequently associated with type HPV16 infection, and multifocal disease has high rates of surgical treatment failure.We report a phase II clinical trial of the topical immunomodulator, imiquimod, for 8 weeks, followed by 3 doses  of therapeutic human papillomavirus (HPV) vaccination  in 19 women with VIN grades 2 and 3. Histology and HPV testing of biopsies were performed at weeks 0, 10, 20 and 52. Intralesional infiltration of T-cell subsets and lymphocyte proliferation for HPV systemic immune responses were also assessed.Lesion response (complete regression of VIN on histology) was observed in 32% (6 out of 19) of women at week 10, increasing to 58% (11 out of 19) at week 20 and 63% (12 out of 19) at week 52. At this time, 36% (5 out of 14) of lesions showed HPV16 clearance and 79% (15 out of 19) of women were symptom free. At week 20, after treatment with imiquimod and vaccination, there was significantly increased local infiltration of CD8 and CD4 T cells in lesion responders; in contrast, non-responders (persistent VIN by histology) showed an increased density of T regulatory cells. After vaccination, only lesion responders had significantly increased lympho-proliferation to the HPV vaccine antigens.The therapeutic effect of treatment depends on the differential immune response of responders and non-responders with affect locally and systemically. Vulval intraepithelial neoplasia (VIN) is increasing in incidence, and more so in younger women. A >400% increase in the incidence of VIN has been reported between 1973 and 2000, whereas the corresponding increase in incidence of vulval cancer has only been 20% . Indeed,VIN, which is often multifocal and extensive, has been traditionally treated by surgical excision or laser ablation, but such therapies are associated with high rates of recurrence , but a rSeveral different clinical studies influencing local and/or systemic immunity to HPV using imiquimod, photodynamic therapy (PDT) and therapeutic HPV vaccination in the management of VIN have been reported. Imiquimod, an immune response modifier that exerts an effect through a Toll-like receptor (TLR),  can stimOptimal therapeutic immunity results from the interaction of nonspecific innate immunity and antigen-specific adaptive immunity. Stimulation of the local innate immunity can have a direct controlling effect on virally infected cells as well as attracting local infiltration of TILs  at weeks 10, 14 and 18. The primary objective was to measure treatment effect on VIN by lesion size and histology and the secondary objectives were to assess lesion HPV status, symptoms, immune responses as well as safety, toxicity and tolerability. Women were reviewed at weeks 0, 10, 14, 18, 20, 26 and 52, the primary end point. Punch biopsies were taken for histology and HPV typing at weeks 0, 10, 20 and 52. Heparinised blood (40 ml) was obtained for immunological assays at weeks 0, 10 and 20.Women aged 18–70 years with biopsy-proven VIN grades 2 and 3 were recruited between March 2006 and May 2007. Exclusion criteria were pregnancy, invasive disease, immunosuppression, history of severe allergy and previous HPV vaccination. Imiquimod 5% cream was self-administered for 8 weeks, escalating from one application in week 1 to two in week 2 and three applications in weeks 3–8. This was followed by three intramuscular doses of TA-CIN (1 ml of 128 Complete regression of VIN on histopathology of an index lesion(s) was designated the lesion response. Histological assessment was performed on formalin-fixed punch biopsies. Biopsies were analysed for HPV typing by reverse line blot assay, which is able to amplify and detect 37 HPV genotypes  at a concentration of 1 : 100 and mouse IgG1 anti-FOXP3  at a concentration of 1 : 50 and respectively detected using goat antimouse IgG2a Alexa Fluor 546 3 μl per 1000 μl and goat antimouse IgG1 Alexa Fluor 488 at 3 μl per 1000 μl . CD8 labelling was performed using rabbit IgG anti-CD8 (Abcam) at a concentration of 1 : 100 and was detected using goat anti rabbit-IgG Alexa Fluor 488 at 3 μl per 1000 μl. Nuclei were stained with DAPI. Five microscopic fields were imaged in a step-wise manner for each cellular phenotype investigated in the VIN biopsies. In most cases, this covered almost the whole specimen, as the sections were small punch biopsies. The TIL counting was then performed using Image J image analysis software version 1.36b (http://rsb.info.nih.gov/ij/), using the cell counting feature of the software. An independent observer reviewed a range of slides and the counts differed by no more than 5%. Results were reported as density per unit area of CD4, CD8 and double-stained FoxP3CD4 cells.Local immune responses were quantified by assessing the TIL density on 7 μg ml–1 recombinant HPV antigens – HPV16 E6E7L2 protein TA-CIN , HPV16 GST E6, HPV16 GST E7  alone or PBMCs with 25 Neutralising antibodies to HPV16 pseudovirions were tested as by the SEAP pseudovirus neutralisation-based assay as previously described (U-test was used to test for differences in proliferative and lesion T-cell density between groups (lesion responders and non-responders). The non-parametric Wilcoxon's signed-ranks test was used to analyse differences in the responses within groups at different time points. All reported P-values are two sided and have not been adjusted for multiple comparisons. A P-value of ⩽0.05 was considered to indicate statistical significance.Analyses were performed using ‘Stats Direct Statistical Software’ . Differences between responder and non-responder categories  were analysed using Fisher's exact test. The non-parametric Mann–Whitney A total of 20 women consented to the trial. One woman, who was found to have early stromal invasion on the first study biopsy, was withdrawn from the study and excluded from the analysis, which left 19 women. Patient demographics are presented in The majority of women experienced local inflammation, ulceration, malaise and flu-like symptoms within the initial few weeks of imiquimod. Side effects were classed as severe in 14 and moderate in 5 women. To improve tolerance, treatment-free intervals of no more than a few days were allowed to achieve the total dose, but to let the side effects subside. Out of 19 women, 16 (84%) completed the prescribed course of imiquimod treatment. Three women noted a marked improvement in facial acne with fewer spots and smoother skin in general while on imiquimod treatment. TA-CIN, which was administered intramuscularly into the deltoid muscle, was well tolerated with no side effects or adverse events.P=0.01). At 0, 10, 20 and 52 weeks, 14, 8, 9 and 10 of the 19 women were HPV16-positive, respectively, and 16 out of 19 were HPV16 positive on at least one occasion. Three women were consistently HPV16 negative but two showed other HPV-type infection at some point. Of the women who showed lesion responses at week 52, 5 out of 10 also cleared their HPV16 infection compared with only 1 out of 6 non-responders; however, this did not reach statistical significance. There were no statistical differences in the lesion responder and non-responder groups with respect to disease duration (P=0.58) or HPV16 detection at baseline (P=0.66). Out of 19 women, 12 were active smokers during the trial period. Current smoking habit was not associated with lesion response (P=0.1).duration  at trialExtended follow-up of these patients for an average of 15 months beyond the primary end point of week 52 showed 84% of patients with a ⩾50% reduction in lesion size consistent with continuing control of VIN . UnfortuPre-existing HPV16 antibodies correlated with documented HPV16 exposure in 12 out of 15 cases. Overall patient pre-existing neutralising antibody levels did not differ or alter significantly after imiquimod or vaccination in either the lesion responders who cleared VIN histologically or the non-responders who failed to clear VIN (data not shown).P=0.4). After vaccination, 10 out of 12 lesion responders compared with 3 out of 7 non-responders showed more than two-fold SI change compared with baseline. P=0.4 and P=0.2 in the group) or when patients were stratified by lesion response (P=0.7 and P=0.5 in responders and P=0.3 and P=0.3 in non-responders) (P=0.01) and this was associated with lesion responders (P=0.008) but not the non-responders (P=0.7) , with this increased proliferation associated with lesion responders  and not non-responders  (P=0.4) or the non-responder (P=0.9) groups  showed pre-existing lymphoproliferative response to TA-CIN (SI of ⩾2), of whom 14 were HPV16 positive on vulval biopsy on at least one occasion. Of these, 11 were lesion responders and 5 non-responders with no significant difference in magnitude of pre-existing response (ponders) . In cont (P=0.7) . Similard P=0.2) . Specifi) groups . No signP=0.03, P=0.01 and P=0.04, respectively); a significant increase in CD8 density was apparent after imiquimod (P=0.04). At week 20, the increased CD4 and CD8 density was significantly associated only with the lesion responders (P=0.03 and P=0.03) whereas increased FoxP3CD4 density was associated with the patients who did not show lesion response (P=0.05). There were no significant pre-treatment differences in the density of CD4 or CD8 T cells in lesion responder and non-responders  but intralesional FoxP3CD4 density was significantly higher in non-responders compared with responders (P=0.05). By week 20, a significant reduction in FoxP3CD4 T-cell population density was apparent in lesion responders, which was significantly different from non-responder densities (P=0.01).To analyse local immune factors in the vulval biopsies before treatment, and after imiquimod and vaccination, the densities per unit area of CD4, CD8 and double-stained FoxP3CD4 (T regulatory) cells were assessed. Imiquimod treatment was expected to enhance the local immune infiltration. The data are presented in This study was designed on the premise that imiquimod and therapeutic HPV vaccination could combine to alter the balance of local immunity through inducing a local inflammatory environment and enhancing T-cell responses to HPV E6 and E7 proteins. Immunotherapies that tip the balance of immune equilibrium in favour of the host effector response and away from regulatory and viral evasion strategies of the HPV may be the key to enhancing the cell-mediated immunity required to eradicate persistent HPV infection and established disease. This study showed that imiquimod followed by vaccination achieved histological clearance of VIN at 52 weeks in almost 60% of a heavily pre-treated cohort of women with high-grade, long-standing VIN.With the proviso that the study size was small, the analyses of lesion-associated T cells showed that higher pre-existing and post-treatment levels of regulatory T cells are associated with a lack of lesion response to treatment. As expected in the group as a whole, imiquimod treatment induced T-cell infiltration, which was most apparent by week 20. However, the increased CD4 and CD8 T-cell density was significant only in the lesion responder group, whereas a significantly higher regulatory T-cell density was only observed in the non-responder group. These observations are consistent with immune control and therapeutic effect reflecting the balance of useful CD4- and CD8-directed effectors against their control by T regulatory activity. Thus, increasing the CD8 and CD4 T-cell density may be able to re-establish local immune control that is lost or suppressed in chronic VIN . T regulThe TA-CIN vaccination component of the protocol was aimed at expanding E6- and E7-specific T effectors with the possibility that after imiquimod their entry and activity in the VIN lesions would not be limited by local immunosuppressive factors. Lymphoproliferation of PBMCs established that all patients were immunocompetent and that the vaccination was immunogenic and HPV16 antigen specific. Importantly, these systemic immune responses to HPV16 antigens were significantly associated with lesion responders and not the non-responders. There was no influence on immunity to HPV as a result of the imiquimod treatment. However, a trend towards higher pre-existing responses to HPV16 early antigens in the lesion responder group was noted in this study, which was significantly stimulated by the vaccination. In contrast, the lesion non-responders showed little pre-existing response and this was not boosted by the vaccine. Previous studies have noted a significant correlation between pre-existing systemic HPV16-specific T cells and regression of HPV16-positive lesions , the response enhanced with time. It is reasonable to argue that an immunologically challenged chronic condition that takes time to establish will equally take time to regress, explaining the continuing response noted long after treatment completion. In this study of a heavily pre-treated cohort of women with long-standing disease, containment of response for 2 years is noteworthy. Patient 5  and patient 19  illustrate particularly effective lesion responses achieved by week 52 in this study, and that have been maintained for a further 2 years thus far. In both these cases, intralesional pre-treatment T regulatory cell density was low, with no enhancement after treatment, contrary to increased density of CD4 and CD8 cells. A pre-existing proliferative response to HPV16 with significant increase in proliferation response after vaccination was also showed. It would be beneficial if the likelihood of response to treatment could be determined using biomarkers either before treatment or, in the case of combined therapy, after imiquimod to avoid subjecting likely non-responders to unnecessary treatment. At face value, pre-treatment assessment of dominant immunological cell types in the lesion microenvironment and analysing pre-existing immunity to HPV could provide the basis for selecting patients most likely to benefit from imiquimod with or without vaccination regimes.Overall, it seems that the natural history of the VIN in the non-responders is related to modulation of both local and systemic immune responses allowing persistence of HPV infection. The mechanisms underlying this chronic VIN state are not known but might include genetic predisposition involving immune and other parameters  showed 60 and 79% lesion size response rate (⩾50% reduction) at 3 and 12 months of follow-up (Following another recently published study of imiquimod followed by PDT (


Prevalence of high risk (HR) human papillomavirus (HPV) types in the states of San Luis Potosí (SLP) and Guanajuato (Gto), Mexico, was determined by restriction fragment length-polymorphism (RFLP) analysis on the E6 ~250 bp (E6-250) HR-HPV products amplified from cervical scrapings of 442 women with cervical intraepithelial neoplasia and invasive carcinoma (280 from SLP and 192 from Gto). Fresh cervical scrapings for HPV detection and typing were obtained from all of them and cytological and/or histological diagnoses were performed on 383.Low grade intraepithelial squamous lesions (LSIL) were diagnosed in 280 cases (73.1%), high grade intraepithelial squamous lesions (HSIL) in 64 cases (16.7%) and invasive carcinoma in 39 cases (10.2%). In the 437 cervical scrapings containing amplifiable DNA, only four (0.9%) were not infected by HPV, whereas 402 (92.0%) were infected HR-HPV and 31 (7.1%) by low-risk HPV. RFLP analysis of the amplifiable samples identified infections by one HR-HPV type in 71.4%, by two types in 25.9% and by three types in 2.7%. The overall prevalence of HR-HPV types was, in descending order: 16 (53.4%) > 31 (15.6%) > 18 (8.9%) > 35 (5.6) > 52 (5.4%) > 33 (1.2%) > 58 (0.7%) = unidentified types (0.7%); in double infections (type 58 absent in Gto) it was 16 (88.5%) > 31 (57.7%) > 35 (19.2%) > 18 (16.3%) = 52 (16.3%) > 33 (2.8%) = 58 (2.8%) > unidentified types (1.0%); in triple infections (types 33 and 58 absent in both states) it was 16 (100.0%) > 35 (54.5%) > 31 (45.5%) = 52 (45.5%) > 18 (27.3%). Overall frequency of cervical lesions was LSIL (73.1%) > HSIL (16.7%) > invasive cancer (10.2%). The ratio of single to multiple infections was inversely proportional to the severity of the lesions: 2.46 for LSIL, 2.37 for HSIL and 2.15 for invasive cancer. The frequency of HR-HPV types in HSIL and invasive cancer lesions was 16 (55.0%) > 31 (18.6%) > 35 (7.9%) > 52 (7.1%) > 18 (4.3%) > unidentified types (3.6%) > 33 (2.9%) > 58 (0.7%).Ninety percent of the women included in this study were infected by HR-HPV, with a prevalence 1.14 higher in Gto. All seven HR-HPV types identifiable with the PCR-RFLP method used circulate in SLP and Gto, and were diagnosed in 99.3% of the cases. Seventy-one percent of HR-HPV infections were due to a single type, 25.9% were double and 2.7% were triple. Overall frequency of lesions was LSIL (73.1%) > HSIL (16.7%) > invasive cancer (10.2%), and the ratio of single to multiple infections was inversely proportional to severity of the lesions: 2.46 for LSIL, 2.37 for HSIL and 2.15 for invasive cancer. The frequency of HR-HPV types found in HSIL and invasive cancer was 16 (55.0%) > 31 (18.6%) > 35 (7.9%) > 52 (7.1%) > 18 (4.3%) > unidentified types (3.6%) > 33 (2.9%) > 58 (0.7%). Since the three predominant types  cause 77.9% of the HR-HPV infections and immunization against type 16 prevents type 31 infections, in this region the efficacy of the prophylactic vaccine against types 16 and 18 would be close to 80%. Cervical cancer (CC) is the second cause of death by cancer among women in the world and the first in most developing countries ,2. In thzur Hausen  proposedThere are 11 major LR-HPV types  and 15 major HR-HPV types  . The twoDuring the year 2000 the incidence of CC in Mexico was 40.5 cases per 100,000 women over 25 years of age and the mortality rate was 17.1 , which mIn the present work we determined the prevalence of the HR-HPV types circulating in the neighboring states of San Luis Potosí and Guanajuato by restriction fragment length polymorphism (RFLP) analysis with the method of Fujinaga et al. , in womeThe 442 women enrolled in this study ranged from 16 to 78 years of age. Fresh cervical scrapings for HPV detection and typing were obtained from all of them whereas cytological and/or histological diagnoses were performed on 383. Low grade intraepithelial squamous lesions (LSIL) were diagnosed in 280 cases (73.1%), high grade intraepithelial squamous lesions (HSIL) in 64 cases (16.7%) and invasive carcinoma in 39 cases (10.2%).Amplification of the HR-HPV E6-250 product by direct and nested PCR was compared. Direct PCR was performed with primer set pU 1M/2R whereas nested PCR was performed in two successive reactions: in the first one ('direct') the E6-650 segment was amplified using primer set LCRS/E7AS, whereas in the second one ('nested') a portion of the first reaction mixture (presumed to contain the E6-650 product) was used as template for the internal primers (pU 1M/2R) to generate E6-250.Amplification of 35 random samples showed that 11 (31.4%) were positive by direct PCR and 24 (68.6%) by nested PCR. Since E6-250 detection was 2.2 times more sensitive with nested PCR, this modality was chosen to analyze all problem samples.To determine the presence of HPV DNA in cervical scrapings the algorithm shown in Fig. In the analysis of samples from women with cervical dysplasia and neoplasia, as is the case of this study, this algorithm saves labor, time and reagents because it first verifies the presence of HPV through nested PCR (expected to be positive in most samples) and then the amplificability of the HPV-negative samples (expected to be rare) by performing a PCR assay for a single copy housekeeping gene such as that of β-globin.Using the algorithm for HPV detection Fig.  the 437 The sizes of E6-250 products and their corresponding restriction fragments for the seven HR-HPV types that can be identified with the PCR-RFLP method used appear in Table Ava II, the first enzyme used on each HR-HPV positive sample, identifies types 16, 18 and 33  > 31 (15.6%) > 18 (8.9%) > 35 (5.6) > 52 (5.4%) > 33 (1.2%) > 58 (0.7%) = unidentified types (0.7%). Types 16, 31 and 18 therefore accounted for 77.9% of all the infections whereas the other four types accounted for only 12.9% of them. HPV type 16 predominated slightly in SLP (53.3%) over Gto (52.9%), type 31 in Gto (22.3%) over SLP (10.1%), and type 18 was equally prevalent in SLP and Gto (8.8%)  than in Gto 67.4%), whereas the overall prevalence of infections by two and three HR-HPV types was respectively 25.9% and 2.7%, of which 22.6% and 2.4% corresponded to SLP and 29.5% and 3.2% to Gto  followed by types 18 (10.2%) and 31 (9.9%) and then by types 52 (3.3%), 33 (1.3%) = 35 (1.3%), and 58 (0.3%)  predominated, followed by types 18 (9.4%), 31 (8.2%), 52 (2.5%), 33 (1.3%), 35 (0.6%) = 58 (0.6%). In Gto the prevalence of type 16 was lower (63.3%) and type 31 was second (13.3%), followed by types 18 (12.5%), 52 (4.7%), 35 (2.3%) and 33 (1.6%)  in triple infections  > HSIL (16.7%) > invasive cancer (10.2%)  was, in decreasing order, 16 (55.0%) > 31 (18.6%) > 35 7.9%) > 52 (7.1%) > 18 (4.3%) > unidentified types (3.6%) > 33 (2.9%) > 58 (0.7%) , expected to be HPV-positive. This prediction was fulfilled because nearly all women were shown to be infected with HPV, and HR types were identified in most of them.To determine HPV infection and to identify the high-risk types involved we used the PCR-RFLP method based on amplification of the E6-250 HR-HPV product followed by digestion with endonucleases  and fragThe E6-250 HR-HPV product was amplified through nested PCR because this modality turned out to be at least twice as sensitive as direct PCR. The universal primer sets for the first ('direct') reaction amplified the E6-650 product ,26 and tEight HR-HPV types  are responsible for 95% of the invasive CC cases in the world . The PCRConsidering all cervical samples included in this work, 98.8% contained amplifiable DNA, 97.8% had HPV infections which in 90.2% of the cases corresponded to HR-HPV types whose prevalence was higher in Gto (99.0%) than in SLP (82.6%) Table .Ava II and then the types 31, 35, 52 and 58 by restriction with Rsa I, Ava I, Bgl II and Acc I.In the 437 samples with amplifiable DNA included in this study, 528 cervical infections were demonstrated by HR-HPV types that were identified in 99.1% of the cases  is almost identical to the world average (53%) calculated by Munoz (2000). In contrast, the regional prevalence of type 18 (8.9%) is substantially lower than the world average (15%) calculated by Munoz . The preSingle and triple infections were more frequent in SLP, whereas double infections were more frequent in Gto. Single infections included all seven HR-HPV identifiable types where the three predominant ones were, in descending order, 16 > 18 > 31 with a cumulative frequency of 92.7%. The prevalence of these types in SLP was, in descending order, 16 > 18 > 31 with a cumulative frequency of 95.0%, whereas in Gto it was 16 > 31 > 18 with a cumulative frequency of 89.1%.The typing method allowed us to identify a considerable proportion of infections by two viral types  and three viral types   is over three times higher than the world average (8.1%) calculated by Molano et al. . This feThe five predominant HR-HPV types  found in the 140 HSIL and invasive cancer lesions accounted for 92.9% of the cases and their frequency was similar to their overall prevalence  circulate in SLP and Gto and were diagnosed in nearly all (99.3%) the cases.Seventy-one percent of the infections were caused by one HR-HPV type, 25.9% by two types and 2.7% by three types.The overall frequency of cervical lesions was LSIL (73.1%) > HSIL (16.7%) > invasive cancer (10.2%) and the ratio of single to multiple infections was inversely proportional to the severity of the lesions: 2.46 for LSIL, 2.37 for HSIL and 2.15 for invasive cancer.The frequency of HR-HPV types found in HSIL and invasive cancer was 16 (55.0%) > 31 (18.6%) > 35 (7.9%) > 52 (7.1%) > 18 (4.3%) > unidentified types (3.6%) > 33 (2.9%) > 58 (0.7%).Since the three predominant HR-HPV types found  cause 77.9% of the HR-HPV infections associated to cervical dysplasia in this region, and immunization against type 16 prevents type 31 infections, in this region the efficacy of the prophylactic vaccine against types 16 and 18 would be close to 80%.The 442 women included in this study were selected for having precancerous or cancerous lesions of the cervical epithelium confirmed by cytologic analysis. Two hundred and eighteen resided in the city of San Luis Potosí, capital of the state of San Luis Potosí (SLP), and 192 in the cities of León, Celaya and Irapuato from the state of Guanajuato (Gto), Mexico. Cervical scrapings from SLP were obtained at the Dysplasia Clinic of the SLP Health Services, the School of Nursing of Universidad Autónoma de San Luis Potosí, and from private patients. Samples from Gto were obtained at the Dysplasia Clinics of the cities of Irapuato, Celaya and León and sent to our laboratory by the Guanajuato Health Secretariat. The study was authorized by the Health Secretariats of SLP and Gto and performed with the informed consent of all participating women.2HPO4, 2 mM KH2HPO2, 25 mM disodium EDTA, pH 7.4). Once detached from the cytobrush and suspended in the PBS-EDTA vehicle, each sample was fixed by addition of 1.5 ml 96% ethanol and processed to extract DNA on the same day or up to 30 days after being kept at room temperature. Reagents were purchased from J.T. Baker  unless other source is specified.Each scraping was taken with an endocervical brush ('cytobrush') that was immediately inserted in a 5 ml polypropylene tube  containing 1 ml phosphate buffered saline supplemented with sterile disodium ethtylene-diamino-tetraacetate . The supernatant was discarded by decantation and to each pellet were added 500 μl of Tris-EDTA-saline , 50 μl of 10% sodium dodecyl sulphate and 20 μl of proteinase K (20 mg/ml). Mixtures were incubated at 55°C for 3 h, at the end of which 150 μl of 5 M NaCl were added and centrifuged again for 15 min. Each supernatant was aspirated and transferred to a tube to which 577 μl cold isopropanol were immediately added and then left stand for 10 min at 4°C to precipitate the nucleic acids. The tubes were centrifuged again for 10 min and supernatants discarded by decantation. Each pellet was washed by vortexing with 1 ml of 70% cold ethanol and centrifuged for 10 min at 10,000 rpm  and room temperature. Supernatants were discarded by aspiration and the pellets dried out by inverting the tubes for 15 min on a paper towel. Each pellet was dissolved with 50 μl TE .To extract the DNA each fixed sample was mixed by vortexing and 1 ml transfered to a 1.5 ml tube and spun in a Hettich Mikro 20 microcentrifuge  for 5 min at 13,000 rpm  was used as marker. After staining for 20 min with ethidium bromide (1 μg/ml) gels were illuminated with ultraviolet light and their fluorescence recorded with the Bio-Rad ChemiDoc EQ  photo documenter.The quality of extracted DNA was verified by electrophoretic analysis in 1% agarose gels with TAE . Two-μl from each sample were applied to gels which were run at 60 V for 90 min. λ-phage? DNA digested with DNA was quantitated by fluorometry with the PicoGreen dsDNA Quantitation kit  by interpolation in a standard curve containing up to 50 ng of λ-phage DNA. To each well of a black FIA 96 well plate  198 μl of the assay solution (PicoGreen diluted 1:400 in TE) and 2 μl of standard DNA or problem samples were added, and their fluorescence determined using a 485 nm excitation filter and a 535 emission filter in the GENios Pro fluorometer  with the Magellan 4 software.Since amplification of the β-globin gene required = 25 ng DNA per PCR mixture, 100 ng was the minimum amount of DNA required from each cervical scraping in order to prepare at least four PCR mixtures.The range of pure DNA obtained from the first 370 cervical scrapings was 53–16,590 ng. The amount of purified DNA was sufficient  in all samples except five of the initial ones.2 concentrations and annealing temperatures were tested. The β-globin primer set was added to PCR mixtures containing double-serial dilutions of DNA from a HPV-positive sample (range: 0.78–50 ng). To optimize conditions for HPV primer sets, 1–5 mM MgCl2 concentrations and annealing temperatures from 55°C to 61.4°C were tested.The oligonucleotide primer sets and the sizes expected for the HPV amplification products are depicted in Table To maximize HPV detection through PCR amplification, three variables were optimized: DNA content, annealing temperature and magnesium concentration.2 concentrations in the 1–5 mM range were tested. Maximum efficiency was attained at 2.5 mM, concentration chosen for the rest of the work.With the PC04/GH20 primer set for β-globin the minimum amount of template DNA required in PCR mixtures was determined with double serial dilutions leading to a maximum of 50 ng DNA per mixture. Fluorescence of the E6-250 product increased with DNA content up to 25 ng, which was the amount selected to amplify HPV DNA from each sample. Annealing temperature in the range of 55°C to 62°C was assessed with each primer set. As major products attained maximum fluorescence at 57°C this annealing temperature was used with all primer sets for the rest of the work. MgClThe primer set LCRS/E7AS amplifies the E6-650 segment and the set pU 1M/2R amplifies the 'nested' E6-250 segment included in the first one. In the HPV16 genome, the set LCRS/E7AS spans nucleotides 26–671 645 bp) and the set pU 1M/2R spans nt 419–656 (237 bp). The set MY 09/11 amplifies the L1-450 segment which spans nt 6584–7035 (451 bp). To explore the feasibility of using duplex PCR to simultaneously amplify E6-250 with primer set pU 1M/2R and L1-450 with primer set MY 09/11, we tested both sets at 0.6 μM concentration. However, our finding that in duplex mixtures the L1-450 band was nearly lost and the E6-250 band was less intense than in uniplex mixtures  and other components were purchased from Invitrogen (Mexico).LCR/E7 ~650 bp (E6-650), E6/E7 ~250 bp (E6-250) and L1 ~450 bp (L1-450) products were amplified with primer sets pU 1M/2R, LCRS/E7AS and MY 09/11, respectively . Before starting they were incubated at 94°C for 4 min. Each cycle consisted of 1 min periods at 94°C (denaturation), 57°C  and 72°C (extension), followed by 10 min final extension at 72°C. Each run included a positive control with 25 ng of HeLa cell DNA known to contain 10–50 copies of the HPV type 18 genome per cell , and a nPCR products (10 μl from each mixture) were subjected to electrophoresis in 1.5% agarose gels with 5 mM sodium borate, pH 8.5  that werTo identify HR-HPV types the method of Fujinaga et al.  was usedAva I, Ava II and Bgl II endonucleases and their buffers were purchased from Biolabs , whereas Rsa I and Acc I and their buffers were obtained from Invitrogen . Digestion mixtures containing 8 μl of PCR amplification mixture, 1.5 μl endonuclease solution (10 U/μl), 2 μl buffer and 8.5 μl water were incubated 3 h at 37°C. The restriction fragments present in 10 μl from each digestion mixture were analyzed by electrophoresis in 2% agarose gels with 5 mM sodium borate, pH 8.5 (Brody and Kern 2004) that were run at 120 V for 90 min.The author(s) declare that they have no competing interests.LAMC performed most of the molecular studies. MSG suggested and set up the PCR-RFLP method. RLR conceived and designed the study, obtained the funds to carry it out and drafted the manuscript. All authors read and approved the final manuscript.