Lactobacillus spp. than HPV-negative women. Fusobacteria, including Sneathia spp., were identified as a possible microbiological marker associated with HPV infection. The vaginal microbiotas of twin pairs were significantly more similar to each other than to those from unrelated individuals. In addition, there were marked significant differences from those of their mother, possibly due to differences in menopausal status. Postmenopausal women had a lower proportion of Lactobacillus spp. and a significantly higher microbiota diversity. This study indicated that HPV infection was associated with the composition of the vaginal microbiota, which is influenced by multiple host factors such as genetics and menopause. The potential biological markers identified in this study could provide insight into HPV pathogenesis and may represent biological targets for diagnostics.Human papillomavirus (HPV) is the most important causative agent of cervical cancers worldwide. However, our understanding of how the vaginal microbiota might be associated with HPV infection is limited. In addition, the influence of human genetic and physiological factors on the vaginal microbiota is unclear. Studies on twins and their families provide the ideal settings to investigate the complicated nature of human microbiota. This study investigated the vaginal microbiota of 68 HPV-infected or uninfected female twins and their families using 454-pyrosequencing analysis targeting the variable region (V2–V3) of the bacterial 16S rRNA gene. Analysis of the vaginal microbiota from both premenopausal women and HPV-discordant twins indicated that HPV-positive women had significantly higher microbial diversity with a lower proportion of  Lactobacillus spp., which are commonly found on the vaginal epithelium, and contribute to women’s health by maintaining a low pH in the vagina through lactic acid production. In addition, the microbiota inhibit competing bacterial taxa by producing bacteriocins, which kill closely related bacterial species, and biosurfactants, which prevent the attachment of other species to the epithelium Vaginal microbiota play a crucial protective role in women’s health, and the host biochemical and immunological response to the microbiota can serve as an indicator of the health indicators of the vaginal environment Cervical cancer is the third most common cancer in women worldwide Studies on twins and their mothers can provide valuable information for discriminating the effects of genetic and various environmental factors on vaginal microbiota FlSTR Identifier Kit  with 16 short tandem repeat (STR) markers  in 67% of the twins. For the remaining 33%, a zygosity-determining questionnaire with a validated accuracy of >90% was applied This study was performed using cervicovaginal samples collected from 912 women who participated in the Healthy Twin Study, a part of the Korean Genome Epidemiology Study vs. low risk). HPV genotypes 16, 18, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 66 were identified as high risk and the others were identified as low risk. Among the 912 subjects, 72 (7.9%) were positive for HPV infection; of these 72 subjects, those who had received antibiotics in the 6-month period prior to specimen sampling were excluded.In the selection of these samples, we examined 912 women who had undergone a Pap smear test between 2005 and 2009 in South Korea as part of the health examination for the Healthy Twin Study. The average age of twins and siblings was 43 and that of their mothers was 65. Each cervicovaginal sample was collected with a separate endocervical brush. The liquid-based preparation method for collecting cervicovaginal samples was performed according to the manufacturer’s instructions (ThinPrep® and Surepath™) and stored until further analysis Trichomonas vaginalis infection and/or atypical cells of undetermined significance (ASCUS), as determined by clinical examination, were also excluded to eliminate possible confounding effects associated with these agents. As a result, we chose 23 HPV-positive and 45 HPV-negative subjects. The 23 HPV-positive subjects included 10 HPV-discordant MZ twins , four HPV-positive concordant MZ twin pairs (both MZ twin pairs were HPV positive), five HPV-positive unpaired MZ or DZ twins , and four siblings (N = 4). The 45 HPV-negative subjects included HPV-discordant MZ twins (N = 10), HPV-negative concordant MZ twins (N = 2), DZ paired twin pair (N = 2), HPV-negative unpaired MZ twin (N = 6), siblings (N = 15), and mothers (N = 10). The epidemiological and clinical information of the study population are summarized in In addition, patients with Among the study subjects (N = 68), 50 and 18 were pre- and postmenopausal women as identified by self reporting, respectively. Postmenopausal women included some of the twins (N = 4), their siblings (N = 4), and all of the twins’ mothers (N = 10). Among the 18 postmenopausal women, three had a history of hormone replacement therapy (HRT). To determine the effect of HPV infection on vaginal microbiota and women’s health, 50 subjects from three subgroups–HPV negative and CIN negative (N = 26), HPV positive and CIN negative (N = 19), and CIN positive –were selected. Postmenopausal women and those with noticeable infection on physical examination were excluded from this portion of the study. We included only 26 women as HPV-negative healthy controls from the 50 premenopausal women after exclusion of subjects with HPV, other noticeable infections, or CIN. Among the five women with CIN, four were identified as HPV positive. For the analysis of HPV-discordant twins, only nine MZ twin pairs (N = 18) without CIN, who were 31−49 years old, were selected. One discordant twin pair with CIN was excluded from this analysis due to possible interference with clinical symptoms. 5′-CCTATCCCCTGTGTGCCTTGGCAGTC-3′), a four-base linker sequence (“TCAG”), and the broadly conserved bacterial primer 8F (5′-AGAGTTTGATCCTGGCTCAG-3′). The reverse primer contained the 454 Life Sciences (Roche) Titanium-compatible adaptor sequences (5′-CCATCTCATCCCTGCGTGTCTCCGAC-3′), a four-base linker sequence (“TCAG”), and a 9- or 10-base multiplex identifier (MID) used to tag each PCR product, the bacterial primer 534R (5′-ATTACCGCGGCTGCTGG-3′)  using an extraction kit. Genomic DNA was extracted from the cytobrush using the chemagic viral DNA/RNA kit  according to the manufacturer’s instructions. The extraction method for total nucleic acids was compared with the conventional bead beating method using 16S rRNA amplification and DGGE analysis. No significant difference was observed. The extracted nucleic acids were stored at −70°C until use. The V2 and V3 regions of the 16S rRNA genes were integrally amplified by polymerase chain reaction (PCR) using the barcoded universal primers presented in CTGG-3′) . PCR reahttp://qiime.sourceforge.net) Lactobacillus species level was further identified using the Rtax method with a pre-clustered Greengene database (gg_97_otus_4feb2011.fasta) http://www.tm4.org) Data were analyzed using Quantitative Insights Into Microbial Ecology (QIIME) 1.2.0  in normal premenopausal women Lactobacillus spp., was much lower (<57%), and Proteobacteria were significantly more abundant, including species in the families Enterobacteriaceae and Caulobacteraceae, in addition to Streptococcus spp. and Anaerococcus spp.  in postmenopausal women than premenopausal women In this study, we investigated the compositional differences between the vaginal microbiota of healthy and HPV-positive individuals using 68 Korean twins and their family members. Vaginal microbiota composition varied significantly among individual subjects . The vascus spp. . These rLactobacillus spp. was the most abundant microorganism. Of the Lactobacillus species identified in vaginal microbiotas, L. iners and L. crispatus were most abundant  in this study  and cluster I (L. crispatus). The prevalence of HPV infection did not differ significantly based on the type of vaginal microbiota (36.6% for cluster III (L. iners), and 21.5% for cluster I (L. crispatus) or menopausal status . The prevalence of HPV infection did not differ between clusters (cluster III and cluster I) of Lactobacillus spp. (p = 0.17). Regardless of the Lactobacillus spp. cluster, the observed decrease in the prevalence of Lactobacillus spp. in subjects that had undergone menopause was consistent with the results of a previous study As described above, is study . In addiSneathia spp. were identified as microbiological markers of HPV infection  to identify a potential microbiological predictor of HPV infection  and highnfection . Additionfection . SneathiLactobacillus spp. abundance was strongly associated with premenopausal women  (Prevotella spp. (phylum Bacteroidetes), Sneathia spp. (phylum Fusobacteria), and Clostridiales (phylum Firmicutes), significantly increased among the group with HPV infection. These results indicate that HPV infection was significantly associated with the composition of the vaginal microbiota.Because menopause status was strongly-associated with the vaginal microbiota, samples from postmenopausal women were excluded from the investigation of the association between the vaginal microbiota and HPV infection. After excluding the 18 postmenopausal women, we compared the vaginal microbiota of 23 HPV-positive and 27 HPV-negative premenopausal women. Among the 23 HPV-positive premenopausal women, four had CIN, which is a potential precursor of cervical cancer. In contrast, only one had CIN among the 27 HPV-negative premenopausal women. The average abundance of e higher . Instead t-test) . Other mLactobacillus spp. and other facultative or anaerobic species. The percentage of Lactobacillus spp. was significantly lower in the HPV-infected group (mean 47%) than in the uninfected group (mean 77%). In particular, a decrease in the abundance of L. iners in the vaginal microbiota was associated with HPV infection among discordant MZ twins (P = 0.03) (L. crispatus was not significantly associated with HPV infection (P = 0.76). Lactobacillus spp. are prevalent in the vagina, where they play a role in maintaining a low pH through their metabolic activities. Thus, Lactobacillus spp. might confer resistance to HPV infection in addition to protecting against colonization of overt pathogens or against dominance by potentially pathogenic species To adjust for potential genetic confounding factors, we characterized the association between HPV infection and the vaginal microbiota using nine HPV infection-discordant MZ twin pairs without CIN (N = 18) . Despite = 0.03) . HoweverPrevotella, Sneathia, Dialister, and Bacillus species were detected in the HPV-infected than in HPV-negative discordant MZ twin pairs  and did not detect the presence or absence of Sneathia spp.  et al. reported the association of Fusobacterium with colorectal carcinoma We performed LEfSe analysis using HPV-discordant twin pairs (N = 18) . Sneathihia spp. . TherefoLactobacillus spp. could render the vaginal environment more susceptible to HPV infection. Previous studies have indicated that BV is significantly associated with sexually transmitted viral diseases, including HPV Lactobacillus spp. in the vaginal environment, the reduction in glycogen level is at least partially responsible for the changes in vaginal pH, which lead to changes in the composition of the microbiota It should be noted that our study demonstrates only an association between the composition of the vaginal microbiota and HPV infection. The possibility exists that a lower proportion of P<0.05). Both average weighted and unweighted UniFrac distances of vaginal microbiotas were most similar between MZ and MZ, followed by MZ-sister, MZ-mother, and then among unrelated subjects. These results suggest associations between genetics and the vaginal microbiota. The similarity of the vaginal microbiotas of twins was due to both shared OTUs (unweighted) and shared OTU population distributions (weighted). In addition, the vaginal microbiotas of MZ twins were much more similar than to those of their sisters and mothers (P<0.05). The distributions of shared OTUs of the vaginal microbiotas of twins were significantly different from those of their sisters (P<10−4). However, shared OTUs did not differ significantly according to unweighted UniFrac results (P>0.05). The microbiotas of MZ twins were significantly different from those of their mothers when analyzed by both weighted (P<0.05) and unweighted UniFrac (P<10−4). All mothers were 55−73 years old and menopausal Click here for additional data file.Figure S2(A) Rarefaction curves for postmenopausal women receiving HRT (N = 3) and not receiving HRT (N = 15). (B) Microbiological markers of postmenopausal (N = 18) and premenopausal women without either HPV infection or CIN (N = 26) by LEfSe. (C) LDA scores and markers of postmenopausal women receiving (N = 3) and not receiving HRT (N = 15) by LEfSe.(TIF)Click here for additional data file.Table S1Summary of the epidemiological and clinical information of this study population (N = 68).(DOC)Click here for additional data file.Table S2Barcoded universal primers for amplification of 16S rRNA.(DOC)Click here for additional data file.Table S3Summary of vaginal microbiota phyla (A) and the relative abundance of the top 50 most abundant genera (B) and species (C) (Average ± SD).(DOC)Click here for additional data file.


Cervical cancer is a major public health problem worldwide. While Romania has the highest incidence of cervical cancer in Europe, the prevalence of HPV has not been evaluated. We report the first data on HPV prevalence and type distribution in Northeast Romania.HPV prevalence and genotype distribution was investigated in 514 consecutively women with or without cervical lesions in Northeast Romania. Genotyping was performed with Linear Array Genotyping/Roche kit.In our study group, 192/514 (37.4%) patients were positive for HPV (infected with single and with multiple HPV types). Most frequent types were: 16 (10.5%), 53 (5.44%), 51 (5.05%), 52 (4.08%) 18 (2.91%) and 31 (2.73%).Infection with high risk types of HPV is common in Northeast Romania. Enhanced and systematic screening for cervical cancer is needed. Our results call for the implementation of a National HPV vaccine program in Romania. Human Papilloma Virus (HPV) is the necessary cause of cervical cancer  and seveRomania has the highest incidence of cancer in Europe, with an incidence of 23.9/100,000 . In RomaThe goal of this study was to establish HPV prevalence and genotype distribution in women with and without cervical lesions in Northeast Romania (a population of 4.5 million people) in an attempt to gather sufficient information to support future vaccination program.Neisseria gonorrhoeae, Candida spp, Trichomonas vaginalis, Treponema pallidum); 85/514 (16.5%) reported cigarette smoking, while and 53/514 (10.3%) declared having more than three sexual partners. Papanicolau smear test results were as follows: ASCUS - 71/514 (13.8%), ASCH - 49/514 (9.5%), LGSIL - 107/514 (20.8%), HGSIL - 41/514 (8.0%), inflammatory - 31/514 (6.0%) while a normal Pat test was reported for 164/514 (31.9%) patients. Note that 51/514 (9.9%) did not had any cytology examination prior to the study, while presenting with macroscopic and colposcopic suspicion for HPV infection. These results are comparable to those reported in other regions of Romania [After 2 years of systematic testing the following results were obtained in our study group with regards to classical risk factors for developing cervical cancer: 71/514 (13.8%) of interviewed subjects declared use of oral contraceptives; 146/514 (28.4%) had other gynecologic infections  samples were positive for HPV/DNA, 120 of which (23.3%) were single HPV type infections and 72 (14%) tested positive for multiple HPV types  ASC-H, 19/108 (17.6%) LGSI, 21/108 (19.4%) HGSIL and 7/108 (6.5%) having "inflammatory" cytological result. In 22/108 (20.4%) cases positive for HR HPV, the result of conventional smear was reported as "normal"  of women with no Pap test and in different percent for patients with Pap smear scores: 15/108 (13.9%) Since the relative prevalence of different HPV type may inform the composition of the polyvalent vaccine in a given region, we focused on the eight first HPV types detected that were HPV 16, 53, 51, 52, 18, 31, CP6108, 6 and HPV 42. The highest prevalence for multiple HPV type infection was observed in the age group of 21-30 years: 19/40 (47.5%), while women in the age group of 31-40 years had the highest prevalence of HR HPV types: 40/108 (37%). Of all the HR HPV positive women detected, 20/71 (18.5%) declared use of oral contraceptives, 32/146 (29.6%) had associated gynecological pathology, 31/85 (28.7%) reported cigarette smoking and 16/53 (14.8%) reported more than 3 sexual partners. Of all the DNA/HPV negative women, 11.3% declared use of oral contraceptives, 27% had associated gynecological pathology, 12.6% reported cigarette smoking and 5.5% reported more than 3 sexual partners.Multiple HPV types infections were found in 8/72 (11.1%) persons who were never tested for Papanicolau cytology and 20/72 (27.8%) women with normal result. For patients that received other scores, these percents varied between 5.6 and 20.8%. One explanation for detecting DNA/HPV in women with normal cytology is that the majority of HPV infections are transient.In the group of patients that were tested for cytologic examination, we have detected 18/51 (35.3%) positive with HR HPV (high risk oncogenic types), pHR HPV (probable high risk types), LR HPV (low risk oncogenic types) and multiple HPV type infections [The prevalence of HPV genotypes included in the quadrivalent vaccine was as follows: HPV type 16-10.5% (54/514), HPV type 18-2.91% (15/514), HPV type 6-2.33% (12/514) and HPV type 11-0.19% (1/514).Women identified to carry with HR HPV infection will follow the European Guidelines for quality assurance in cervical cancer screening (2008): if the cytology is normal, they will repeat the HPV DNA test after 6-12 month; if they present with an abnormal Pap smear, they will be monitored by repetition of the smear, and referral for colposcopy. Women with HR HPV and HGSIL usually are candidate for excisional procedures, but the choice of treatment for women with HSIL will depend on the suspected diagnosis, the size and type of transformation zone, age and fertility aspirations .INNO LiPA, Reverse Line Blot) [The relative prevalence of HPV types was similar in patients infected with single genotypes and in those with multiple infections. Multiple infections were produced by two to eight genotypes per sample. Note that these results may be due to either a numerous reexposure rate or to a high sensitivity of the assay used in our study in comparison to other HPV genotyping techniques (ne Blot) . In ordene Blot) .Thus, our study identified a high prevalence of HPV infection associated with cervical cancer in our area and therefore supports implementation of primary prevention of cervical cancer by vaccination in Northeast Romania, and in all country as well.In our region, screening of cervical cancer is not systematic and the quality control guidelines for conventional smears are not implemented . Additioversus 96%) [It is considered that full spectrum genotyping is useful for evaluation HPV epidemiology, and to the study of the impact of HPV vaccination and for clinical practice, to assess the persistence of HPV infection: stratification of risk for CIN 2+, ASCUS follow-up or treatment follow up. However, in Romania, similar to numerous other nations, screening for cervical cancer is not systematic and even conventional smear is not done on a regular basis . In addisus 96%) . This casus 96%) ,16 folloThere is currently an open national cervical cancer screening pilot study undergoing in Romania. However, this study is still regional and aims to a 10% coverage only. These numbers are clearly different from those observed in other EU countries . This demonstrate that in order to significantly improve the efficacy of cervical cancer screening there is an important need for education of women as well as of medical professionals too . At the Being a new member of EU, in Romania there are still challenges regarding starting of one organised programmes for cervical cancer: the main reason is the lack of the infrastructure necessary to implement it .Even though our pilot study was carried out on a limited number of patients, it covered a large number of patients and generated results that may permit the policy makers to initiate cervical cancer screening and vaccination programs for teenagers.In developing countries as Romania, HPV testing can be used in primary cervical cancer screening. This will be beneficial for patients who will thus avoid excessive referrals to colposcopy and overtreatment ,14,22.In the future, screening via HPV/DNA tests and vaccination must become coordinated, with programs providing the most cost-effective cervical cancer prevention strategies.Our results regarding epidemiological issues are comparable with WHO data at global and European level . Also, tThe efficiency of prevention programs  is highest if the aim of these programs is focused on community needs. In this respect, the high prevalence of HR HPV documented by our study justifies entirely the necessity of cervical cancer prevention programs in Romania.The study was carried out between September 2009 and November 2011, and included 514/1896 consecutive women  from the "Cuza-Vodă" Gynecology Clinical Hospital. All patients were monitored by clinical gynecological examination, cytology exam and some of them, by colposcopic examination.Atypical Squamous Cells of Undetermined Significance, ASC-H--Atypical Squamous Cells for which a High-grade lesion cannot be excluded, LGSIL--Low Grade Squamous Intraepithelial Lesion, HGSIL--High Grade Squamous Intraepithelial Lesion or inflammatory result) or with only suggestive colposcopy exam were genotyped for HPV infection.Women with abnormal Pap smears , and provided cytological results of previous Papanicolau tests.Cobas PCR Cell Collection Media (Roche). DNA purification was done using the High Pure PCR Template (Roche); HPV detection was performed using the Linear Array HPV Detection kit (Roche), and then DNA was genotyped with the Linear Array HPV Genotyping Test . Genotyping via this technique involves one step of PCR amplification (ABI 9700): the final volume contained 50 μl of purified DNA and 50 μl of master mix (Roche). The thermal profile included the next steps: HOLD program: 2 min/50°C; an initial denaturation of 9 min at 95°C was followed by 40 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min, with a final extension for 7 min at 72°C. The amplicons have been denatured at the end of PCR amplification.Cervical cells were sampled with a cervical brush and they were transported in TwinCubator  using Working Hybridization Buffer, Working Ambient Wash Buffer, Working Stringent Wash Buffer, Working Conjugate, Working Citrate Buffer and Working Substrate (Roche). The technique was validated through the use of positive and negative controls at each shift; the strips contain two bands for beta globin, which checks if the sample was correctly processed.HPV types were detected by hybridizations of the amplicons. Hybridization was performed in a Linear Array HPV Genotyping test (Roche) is detecting 14 HR HPV (high risk) types  and 24 pHR HPV (probably high risk) and LR HPV (low risk) types . We validated our HPV genotyping technique by obtaining proficiency for all the HPV types included in WHO HPV Proficiency Study, 2010.Stratagene manufacturers) detection using quantitative evaluation.For the main HR HPV types  we confirmed the results of LA genotyping result by qPCR/MX3005P (HPV: Human papilloma virus; HR HPV types: High Risk HPV types; pHR HPV: Probably high risk HPV types; LR HPV types: Low risk HPV types.The authors declare that they have no competing interests.URG: HPV/DNA purification, PCR amplification, hybridization, results analysis, manuscript preparation. OM and ND: selection of patients, sampling the cervical cell. ILS: director of grant research, searching for founding, study design and manuscript preparation. All authors read and approved the final manuscript.


The female genital tract is an important bacterial habitat of the human body, and vaginal microbiota plays a crucial role in vaginal health. The alteration of vaginal microbiota affects millions of women annually, and is associated with numerous adverse health outcomes, including human papillomavirus (HPV) infection. However, previous studies have primarily focused on the association between bacterial vaginosis and HPV infection. Little is known about the composition of vaginal microbial communities involved in HPV acquisition. The present study was performed to investigate whether HPV infection was associated with the diversity and composition of vaginal microbiota.A total of 70 healthy women (32 HPV-negative and 38 HPV-positive) with normal cervical cytology were enrolled in this study. Culture-independent polymerase chain reaction-denaturing gradient gel electrophoresis was used to measure the diversity and composition of vaginal microbiota of all subjects.p < 0.001). Lactobacillus, including L. gallinarum, L. iners and L. gasseri, was the predominant genus and was detected in all women. No significant difference between HPV-positive and HPV-negative women was found for the frequency of detection of L. gallinarum (p = 0.775) or L. iners (p = 0.717), but L. gasseri was found at a significantly higher frequency in HPV-positive women (p = 0.005). Gardnerella vaginalis was also found at a significantly higher frequency in HPV-positive women (p = 0.031). Dendrograms revealed that vaginal microbiota from the two groups had different profiles.We found significantly greater biological diversity in the vaginal microbiota of HPV-positive women (Our study is the first systematic evaluation of an association between vaginal microbiota and HPV infection, and we have demonstrated that compared with HPV-negative women, the bacterial diversity of HPV-positive women is more complex and the composition of vaginal microbiota is different. Microbes inhabit virtually all sites of the human body and play an important role in human health, yet we know relatively little about them. The Human Microbiome Project (HMP), funded Lactobacillus species as the predominance genus in the vagina, which helps to promote a healthy vaginal milieu[Lactobacillus species and a concurrent overgrowth of anaerobic bacteria, has been associated with vaginal discharge syndrome, poor pregnancy outcomes, pelvic inflammatory disease, post-operative wound infections, and endometritis after elective abortions[L. crispatus was significantly reduced and there was a shift in the composition of the Lactobacillus microbiota in HPV infection.The female genital tract is an important habitat for human microbiota. Investigation of “normal” vaginal microbiota typically reveals l milieu. Bacteribortions-11. Addibortions. Guo et bortions reportedbortions resistedPersistent HPV infection is the central factor in the development of cervical cancer, and is a prerequisite for progression to high-grade cervical lesions. HoweverIt is difficult to assess the microbial community in an environment where more than 80% of microbiota is nonculturable (such as the vagina),20. HoweTo examine the relationship between the diversity and composition of vaginal microbiota and HPV positivity, we used PCR-DGGE to examine the vaginal microbiota of 38 HPV-negative and 32 HPV-positive healthy women with no BV (using Amsel criteria). To our knowledge, this is the first study of the association between HPV infection and vaginal microbiota to use a molecular biological technique to examine the microbiota.A total of 100 healthy women (50 HPV-negative and 50 HPV-positive) with normal cervical cytology, who accepted a routine gynecological examination and Thinprep Cytology Test (TCT) in Beijing Cancer Hospital from January 2012 to June 2012, were initially recruited for this study. Subsequently, TCTs were reexamined by two cytologists and HPV infection was reexamined by PCR. If there was a difference between the first and second TCT reports, or between the results from the two cytologists performing the second TCT, the woman was excluded from the study. In addition, if a recruited woman was infected with more than one HPV type, or there was a difference between the initial HPV report using Hybrid Capture II and the second HPV report using PCR, the woman was excluded from the study. A total of 30 women were excluded from the study based on these four criteria. Inclusion criteria were age <50, no BV by the Amsel method, no use of antibiotics or vaginal antimicrobials  in the previous month, and no vaginal intercourse or vaginal lavage within the last 3 days. All subjects were free of systemic diseases such as diabetes, autoimmune disease, and malignant tumors. Informed written consent was obtained from all participants prior to enrollment. This study was approved by the ethical committee of Peking University Cancer Hospital and Institute, Beijing, China.When women underwent genital examination, a sterile swab sample was taken from near the vaginal fornix and cervix from each participant. The swab was placed into 1 ml sterile saline, placed on ice packs immediately, and transferred to the laboratory within 30 min. The sample was pelleted by centrifugation at ≥10,000 × g (25°C) for 10 min and stored at −80°C until further analysis, as previously published.Bacterial DNA was extracted according to the procedures described by Signoretto and ZijnThe hypervariable region chosen for amplification can influence the PCR-DGGE profiles. The V2–V3 region of the 16S rRNA gene is reported to be the most reliable,31. The 2, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 0.4 mM dATP and 0.4 mM dTTP)  and RNase-free H2O in a final volume of 50 μl. The cycling parameters were 95°C for 5 min; 25 cycles of 95°C for 2 min, 42°C for 30 s, 72°C for 4 min; and a final cycle of 72°C for 20 min. The temperature was held at 4°C following the final cycle[The first PCR mixture contained 100 ng of DNA template, 5 pmol of each primer, 25 μl of 2× PCR Master Mix  and RNase free H2O in a final volume of 50 μl. The cycling parameters were 95°C for 5 min; 30 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 1 min; and a final cycle of 72°C for 8 min. The temperature was held at 4°C following the final cycle[The second PCR mixture contained 5 μl of PCR product from the above reaction, 10 pmol of each primer, 25 μl of 2× PCR Master Mix  or high (70%) concentrations of urea and formamide that increased in the direction of electrophoresis.PCR products were mixed with a loading buffer containing bromophenol blue, xylene cyanol, and 70% glycerol in TAE buffer  and loaded into the gels. About 20 μl of the reference mixture was combined with loading buffer, and the mixture was loaded so that it flanked the vaginal samples. Electrophoresis was performed for 16 h at 60 V and 58°C. The gels were stained with SYBR Green I  for 30 min.+Gel Imaging System  and analyzed by Gel Compare® . Each gel was normalized according to a DGGE standard marker (10 markers according to the GC base-pair percentage).DGGE images were digitally captured and recorded by the BIO-RAD Gel Doc™ XREscherichia coli Top10, and the extracted plasmid was sequenced by the Beijing Genomics Institute. Sequence identification of the plasmid was performed by searching the NCBI BLAST database.The main DGGE bands were excised, and the DNA fragments were amplified with primers HDA1 (without the CG clamp) and HDA2. Amplified material was cloned into the pGM®-T vector using the pGM®-T cloning Kit , according to the manufacturer’s instructions. The vector was then cloned into HPV DNA was extracted from the remnant TCT sample by TIANamp Virus DNA/RNA Kit (Tiangen) according to the manufacturer’s instructions. HPV DNA was first amplified using the HPV L1 consensus MY09/MY11 primer pair, followed by nested PCR with GP5+/GP6+ primers. The PCR amplification was performed as described previously,37.PCR products were subjected to direct DNA sequencing by the Beijing Genomics Institute. The obtained sequences were compared with documented virus sequences available in the GenBank database using the BLAST program. The subjects whose results were different to the previous results from Hybrid Capture or who were infected with multiple HPV genotypes were eliminated from this study. HPV-negative women and women infected with one HPV (single high-risk) subtype were included in this study. The HPV types are not listed in this paper.The similarities of PCR-DGGE DNA profiles were analyzed with Gel Compare® software (Applied Maths) using Dice’s similarity coefficient. The clustering algorithm, unweighted pair-group method with arithmetic means (UPGMA), was used to calculate the dendrogram.H’ = −Σpilnpi. For morphological analysis, pi is the proportion of individuals in the ith taxon. For PCR-DGGE analysis, pi is the importance probability of the bands in a gel lane and measured as pi = ni/N, where ni is the intensity of a band and N is the sum of all band intensities in the densitometry profile. The Mann–Whitney U test and Kruskal-Wallis tests were performed to compare the diversity indices, and p < 0.05 was interpreted to be statistically significant.Vaginal microbiota diversity was expressed by the Shannon–Weiner diversity index-40 and cp = 0.57).A total of 38 HPV-negative women (mean age 37.0) and 32 HPV-positive women (mean age 37.8) were enrolled in this study. There was no significant difference in age between the two groups .The number of bands in the DGGE profile from each woman is shown in Table p < 0.001).Vaginal microbiota diversity was expressed using the Shannon-Weiner diversity index and compared using the Mann–Whitney U test. The diversity index for subjects with and without HPV infection is displayed in Figure The UPGMA clustering algorithm was used to construct a dendrogram of the DGGE profiles of vaginal microbiota from all 70 women enrolled in this study. Hierarchical cluster analysis of the dendrogram indicated that six clusters were formed, and revealed differences in the composition of vaginal microbiota between HPV-negative women and HPV-positive women Figure . Most saThe discriminative character analysis of DGGE profiles showed that the bands in box A are present in both HPV-positive and HPV-negative women Figure . Howeverp values <0.001). The Kruskal-Wallis test was used to compare the Shannon-Weiner diversity indices from each of the six clusters, and we found that the diversity of at least one of the clusters was significantly different from the other clusters (p < 0.001).We compared the number of bands, Shannon–Weiner diversity index and the proportion of samples from HPV-positive and HPV-negative women in each of the six clusters Table . There wExcised DGGE gel bands were sequenced and BLAST database searches were used to identify 18 bacterial species present in vaginal samples from the 70 women studied. The name of each microorganism, sequence identities and NCBI Sequence ID are shown in Table Lactobacillus was the most predominant genus, and was detected in all women included in this study. The Lactobacillus members are grouped into three species: L. gallinarum was the most abundant , followed by L. iners  and L. gasseri  , followed by Atopobium vaginae . A Chi-squared analysis revealed that there is no difference in the frequency of identification of L. gallinarum between HPV-negative and HPV-positive women (p = 0.775). Likewise, no significant difference existed in the frequency of identification of L. iners (p = 0.717) or Atopobium vaginae (p = 0.150). However, L. gasseri and Gardnerella vaginalis were isolated more frequently in HPV-positive women than in HPV-negative women .abundant 5/70, 64%L. gallinarum and L. iners are the two bands in box A of Figure Gardnerella vaginalis is the predominant genus of the bands in box B, and other bands in box B include Escherichia fergusonii, Atopobium vaginae, Streptococcus australis, Streptococcus intermedius and Alloscardovia omnicolens. L. gasseri is the predominant genus of the bands in box C, and another band in box C is ureaplasma parvum serovar 3 str.Biologic susceptibility to HPV acquisition and immune competence for clearance of an HPV infection can be affected by vaginal bacterial infection, which disrupts the balance of vaginal microbiota,43. PrevIf the target 16S rRNA gene copy number in the sample is low, it is likely that it cannot be amplified sufficiently to be visualized as a band. Therefore, the bands generated in DGGE reflect the most abundant genus from each vaginal sample. In our p <0.001). Other studies have also demonstrated a difference in the abundance of bacterial species present in the vagina when comparing healthy women to those with BV or cervicitis[The Shannon-Weiner diversity index uses the total number and relative intensities of DGGE bands, which makes it more suitable to estimate bacterial diversity than the number of bands alone. A Mann–rvicitis,46. Thisrvicitis.The UPGMA clustering algorithm has been used previously, and in Lactobacillus have been identified, and more than 20 species have been detected in the vagina. However, vaginal microbiota is not reported to contain very many different species of Lactobacillus[Lactobacilli species are predominant. For example, L. crispatus and L. jensenii were the most common genera for white women[L. crispatus and L. gasseri were more common in Japanese women[L. crispatus, L. iners and L. gasseri were the most common genera in Chinese women[Over 120 species of obacillus. Typicalte women, while Lse women. Recentlse women.Lactobacillus members: L. gallinarum, L. iners and L. gasseri, all of which are obligately homofermentative species. L. gallinarum and L. iners are the two bands in box A . Cherpes et al. reported that L. gasseri was vaginal colonization from the rectum[L. gasseri and BV[L. crispatus was significantly reduced and there was a shift in the composition of the Lactobacillus microbiota in women with HPV infection.In our study, sequence analysis identified three  Figure , and thee rectum, and a si and BV. And Doli and BV resistedL. gallinarum from the other Lactobacillus spp., because the BLAST result indicated that the similarity score of L. gallinarum was the same as that of other Lactobacillus spp., such as L. crispatus, L. acidophilus, L. amylovorus, L. rhamnosus, L. fermentum, L. helveticus, L. kitasatonis and L. ultunensis. One possible explanation for this is that the genera of Lactobacillus spp. mentioned above have the same DNA sequence as L. gallinarum in the V2–V3 region of 16S rDNA. Future research will seek to resolve this issue.But we were not able to distinguish Gardnerella vaginalis and Atopobium vaginae. Gardnerella vaginalis is a facultative anaerobic bacterium of the Bifidobacteriaceae family[Atopobium lies within the family Coriobacteriaceae and forms a distinct branch within the phylum Actinomycetes[Gardnerella vaginalis and Atopobium vaginae were frequently detected in association with BV, Menard deemed the combination of the two bacterial genera as predictive criteria for the diagnosis of BV[Gardnerella vaginalis  and Atopobium vaginae  were more frequently detected in HPV-infected women than in HPV-negative women, though the difference of Atopobium vaginae in the two groups was not statistically significant.The bands in box B are the primary bands that distinguish cluster 2 from other clusters. The main microorganisms in box B are e family, while tnomycetes. Becauseis of BV. In our Because this was a cross-sectional study, we were unable to determine whether a change in vaginal microbiota preceded HPV infection, or whether HPV infection preceded a change in vaginal microbiota. Furthermore, we initially recruited 100 women with normal cervical cytology and excluded 30 from the study, which led to a relatively small sample size. However, because this is the first study to use PCR-DGGE to examine the association between vaginal microbiota and HPV infection, even with the limitations mentioned above we believe it has substantial importance.Gardnerella vaginalis and L. gasseri were detected significantly more frequently in HPV-positive women. Abnormal vaginal microbiota might act as a co-factor for the acquisition of HPV. Continued exploration of the interplay between vaginal bacterial communities and HPV may shed light on biologic susceptibility to HPV infection, as well as immune competence for clearance of HPV, which may provide new insights into the early steps in the development of cervical cancer.In conclusion, we found that the bacterial diversity and composition in HPV-positive women were more complex than in HPV-negative women. BV: Bacterial Vaginosis; DGGE: Denaturing Gradient Gel Electrophoresis; HPV: Human Papillomavirus; PCR: Polymerase Chain Reaction; HMP: the Human Microbiome Project; TCT: Thinprep Cytology Test; UPGMA: Unweighted Pair-Group Method with Arithmetic Means.The authors declare that they have no competing interests.WJ Gao conducted the PCR-DGGE studies, performed the statistical analysis, participated in clinical sample collection and drafted the manuscript. JL Weng conducted the cloning of DNA fragments and participated in the statistical analysis. YN Gao and XC Chen conceived the study, participated in its design and coordination and helped to draft the manuscript. YN Gao carried out clinical sample collection and was responsible for clinical data. XC Chen helped conduct the molecular biological experiments and was responsible for the experimental data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/271/prepub


Human papillomaviruses (HPVs) are small, non-enveloped DNA viruses that infect stratified squamous mucosal and cutaneous epithelia, causing diseases ranging from benign warts to invasive tumors. Failure of the immune system to detect and clear persistent HPV infections frequently leads to the development of oral warts and cancer. HPV infection has been etiologically linked with oral warts and a subset of oral squamous cell carcinoma, particularly in HIV infected patients. The incidence of HPV-related oral lesions is increased in HIV+ subjects on highly active antiretroviral therapy (HAART). We previously showed that tumor cells in oral carcinoma in situ (CIS) lesions overexpress human beta-defensin-3 (hBD-3), an antimicrobial peptide with immunomodulatory capabilities. Expression of hBD-3 in CIS contributes to the local pro-tumor immune response by selectively chemoattracting tumor-associated macrophages and by enhancing tumor development and progression.To elucidate mechanisms by which high-risk HPV could evade immune detection and clearing via infected epithelial cells, we investigated if oncoproteins E6 and E7 derived from high-risk HPV-16 modulate the innate immune response of infected epithelial cells and the role of HPV-induced gene expression in orchestrating local immunity. We have found that cancer cells of HPV-related oral and oropharyngeal squamous cell carcinoma biopsies overproduce hBD-3. Introduction of an expression vector producing HPV-16 E6 or E7 oncogene into oral squamous cancer cell lines or primary oral epithelial cells increases the levels of hBD-3 mRNA and peptide. However, E6 derived from the low-risk HPV-11 is significantly less potent in promoting hBD-3 expression. Combination of oncogenic E6 and E7 in oral epithelial cells also shows reduced induction of hBD-3. Furthermore, the transactivity of an hBD-3 luciferase promoter construct is differentially stimulated by oncogenic E6 and E7 compared with MEKK1, a known inducer of hBD-3 expression. Although the pharmacological inhibitors for MAPK and PI3K reduce the transactivity of a 2.5 kb hBD-3 promoter reporter, they do not exhibit the inhibitory effect on the promoter reporter containing a 450 bp 3’-regulatory region. These data suggest that high-risk and low-risk early genes of HPV differentially modulate hBD-3 expression in oral epithelial cells.Our results suggest that oncoproteins of high-risk HPV strains induce higher levels of hBD-3 expression compared with early genes of low-risk HPV. The oncogenic E6 and E7 genes may contribute to overexpression of hBD-3 in the early oral lesion, which then leads to recruitment of tumor-associated macrophages to further develop and promote the progression of cancer.


Background: At least one-fourth of U.S. veterans who served in the 1990–1991 Gulf War are affected by the chronic symptomatic illness known as Gulf War illness (GWI). Clear determination of the causes of GWI has been hindered by many factors, including limitations in how epidemiologic studies have assessed the impact of the complex deployment environment on veterans’ health.Objective: We sought to address GWI etiologic questions by evaluating the association of symptomatic illness with characteristics of veterans’ deployment.Methods: We compared veteran-reported wartime experiences in a population-based sample of 304 Gulf War veterans: 144 cases who met preestablished criteria for GWI and 160 controls. Veteran subgroups and confounding among deployment variables were considered in the analyses.Results: Deployment experiences and the prevalence of GWI differed significantly by veterans’ location in theater. Among personnel who were in Iraq or Kuwait, where all battles took place, GWI was most strongly associated with using pyridostigmine bromide pills  and being within 1 mile of an exploding SCUD missile . For veterans who remained in support areas, GWI was significantly associated only with personal pesticide use, with increased prevalence  in the relatively small subgroup that wore pesticide-treated uniforms, nearly all of whom also used skin pesticides. Combat service was not significantly associated with GWI.Conclusions: Findings support a role for a limited number of wartime exposures in the etiology of GWI, which differed in importance with the deployment milieu in which veterans served. Twenty years after the 1990–1991 Gulf War, military personnel who served in that conflict continue to report health problems that are not adequately explained by established medical or psychiatric diagnoses . Studies of diverse Gulf War veteran populations consistently describe a symptomatic illness that affects Gulf War veterans at significantly elevated rates compared with other military populations . SymptomStudies suggest that at least 25% of the nearly 700,000 U.S. veterans of the 1990–1991 Gulf War are affected by GWI  pills taken by U.S. and some coalition forces to protect against acute effects of nerve agents, excessive use of pesticides and insect repellants, munitions containing depleted uranium, receipt of numerous vaccines, and diverse other potential hazards.Determining the specific causes of GWI has been hindered by a lack of measured data indicating who was exposed to what during the war, and at what levels. Epidemiologic studies have therefore evaluated risk factors for GWI based on veterans’ own reports of their exposures during deployment. Some studies have suggested that nearly all of the many experiences and exposures queried appear to be linked to poor health outcomes . Others In 2008, the RAC-GWVI, a congressionally mandated federal advisory panel, released a comprehensive review of scientific literature and government-issued reports pertaining to the health of Gulf War veterans (RAC-GWVI 2008). The report described limitations in existing Gulf War epidemiologic studies, specifically pointing out that studies have often failed to assess risk factors for GWI using analytic methods appropriate for the complex Gulf War exposure scenario. The panel recommended that studies of Gulf War veterans evaluate GWI risk factors in veteran subgroups that may be relevant to the outcomes of interest and use analytic methods that consider possible confounding effects of concurrent exposures.a) veterans’ experiences and exposures in theater, b) variability in enzymes that metabolize a number of exposures linked with Gulf War service, and c) function of the autonomic nervous system. Results of laboratory and physiologic assessments will be provided elsewhere. Here we focus on evaluation of associations between GWI and characteristics of veterans’ military service during the Gulf War. In particular, we attempt to clarify some of the ambiguities and apparent inconsistencies from previous studies by identifying veteran subgroups of importance and controlling for spurious effects that can result from confounding errors introduced by multiple concurrent exposures.We report here results of a case–control study initiated in 2000 as part of a multipart project to evaluate possible associations between GWI and Study population and recruitment. This study had a case–control design involving a population-based sample of 304 veterans who served in the 1990–1991 Gulf War. In 2000, a random sample of Gulf War veterans, stratified by sex, was identified from among veterans residing in the greater Kansas City metropolitan area, including both Kansas and Missouri residents. The sampling pool for Kansas residents was drawn from a database of all Kansas Gulf War–era veterans maintained by the Kansas Commission on Veterans Affairs . Gulf War veteran residents of Missouri were sampled from among area veterans identified through the U.S. Department of Defense’s (DOD) Defense Manpower Data Center .a) fatigue, b) mood/cognition, and c) musculoskeletal pain. sMSI– veterans reported fewer or no symptoms.Potential study participants were contacted by telephone and invited to complete a brief screening interview to determine study eligibility. Eligible veterans were required to have deployed to the Gulf War theater of operations for any period between 1 August 1990 and 31 July 1991. To optimize the proportions of cases and controls enrolled, the screening interview queried veterans about 10 symptoms to determine if they screened positive or negative for multisymptom illness (sMSI+ or sMSI–), as defined by In accordance with the GWI case definition used for the study, potential participants were excluded if they reported being diagnosed by a physician with chronic medical conditions that might account for their symptoms  or had persistent health problems due to chronic infection or serious injury. Veterans were also excluded if they reported being diagnosed with schizophrenia or bipolar disorder or if they had been hospitalized since the Gulf War for alcohol or drug dependence, depression, or posttraumatic stress disorder (PTSD).Veterans determined to be eligible were invited to come to the study site to provide blood samples for genetic/enzyme activity testing and to complete a self-administered questionnaire providing information on their health, military, and deployment characteristics. Recruitment continued until a final sample of at least 300 veterans completed appointments at the study site, including similar numbers of sMSI+ and sMSI– veterans, with similar proportions of women in each group.Among the 906 households initially contacted, informants indicated that 76 veterans could not participate ; 86 (9%) refused to provide information or be interviewed. Of the remaining 744 veterans, 98 (13%) reported they had not deployed to the Persian Gulf region during the required period, and 646 completed the screening interview. Overall, 288 (45%) of the 646 veterans screened sMSI+ and 358 (55%) screened sMSI–. A total of 121 veterans were ineligible for the study based on health exclusions, 450 were eligible and were invited to participate, and 75 qualified but were not recruited based on their sMSI status or sex. Of the 450 recruited veterans, 385 (86%) agreed to participate, and 304 (68%) completed appointments at the study site.n = 27), chronic infection (n = 25), heart disease (n = 21), cancer (n = 18), diabetes (n = 18), and liver disease (n = 12). Of the 74 veterans who reported physician-diagnosed psychiatric conditions, 19 were excluded for one or more of the following: diagnosed schizophrenia (n = 3) or bipolar disorder (n = 8), postwar hospitalization for PTSD (n = 2), drug or alcohol dependence (n = 8), or depression (n = 6). The remaining 56 veterans who reported being diagnosed, but not hospitalized, for psychiatric conditions were eligible for the study and remained in the sampling pool.Among the 121 veterans excluded for health reasons, 102 were excluded for one or more medical conditions, 12 were excluded based on self-reported psychiatric hospitalizations or diagnoses, and 7 were excluded for both medical and psychiatric reasons. Leading reasons for medical exclusions included chronic problems resulting from serious injury  fatigue/sleep problems, b) somatic pain, c) neurologic/cognitive/mood symptoms, d) gastrointestinal symptoms, e) respiratory symptoms, and f ) skin abnormalities. Kansas GWI case criteria also exclude veterans who report being diagnosed with medical or psychiatric conditions that could explain their symptoms or interfere with their ability to report them, as detailed above in the exclusionary criteria for this study. Controls had insufficient symptoms to meet GWI case criteria and also reported no exclusionary diagnoses.veterans . This caThe Fukuda/CDC MSI criteria, described above , provideData analyses. We compared demographic and general health characteristics of GWI cases and controls using chi-square statistics. Bivariate (unadjusted) associations between case status and deployment and military characteristics were determined by calculating crude prevalence odds ratios (ORs) and 95% confidence intervals (CIs).a) veterans who reported ever being in Iraq or Kuwait; or veterans who had not been in Iraq or Kuwait who were b) primarily at sea while in theater, c) primarily in eastern Saudi Arabia, or d) primarily on land in other support areas. The distribution of deployment experiences and exposures reported by veterans in each location subgroup was assessed using chi-square analyses.Preliminary analyses indicated that GWI case status was strongly associated with veterans’ reports of having been in Iraq, Kuwait, or both during deployment, regardless of the duration of time spent in those areas. To further evaluate associations between location in theater and deployment characteristics, four mutually exclusive veteran subgroups were defined: p < 0.05) with GWI in bivariate analyses were entered into a single logistic regression model to identify independent associations between these variables and GWI. Variables that had the weakest association with GWI  were dropped from the model sequentially until only those significantly associated with GWI remained. In the second step of the process, logistic regression analyses were used to determine multivariable associations for each individual exposure queried, controlling only for effects of the variables that were significantly associated with GWI in the backward elimination process.Because the number of veterans in some location subgroups did not accommodate multivariable modeling of exposures in relation to case status, veterans who had not entered Iraq or Kuwait were combined into a single subgroup. We used a two-step process to identify independent associations between exposures and GWI for all veterans combined and for the two location subgroups of interest. First, ORs and 95% CIs were calculated to determine bivariate associations between case status and each exposure. All exposure variables that were significantly associated . However, the number was considerably higher for several variables: exposure to chemical agent resistant coating (CARC) paint , receipt of shots (injections) in the buttocks (19 missing) or arm (17 missing), and proximity to an exploded SCUD missile (12 missing). All analyses were conducted using SAS statistical software .Study population. The final study population consisted of 304 Gulf War veterans: 144 GWI cases and 160 controls. By design, a similar proportion of women veterans were in each group. Cases were also similar to controls in terms of age, but significantly lower proportions of cases were white and had earned college degrees (p = 0.46), 4 with alcohol or drug dependence , and 5 with PTSD . Cases and controls reported similar general health status before Gulf War deployment, but cases reported significantly worse general health at the time of the study. degrees . Twenty-Bivariate (unadjusted) associations between GWI and deployment characteristics. As detailed in p < 0.05, Breslow-Day test for homogeneity of ORs). Specifically, the increased prevalence of GWI among Army veterans was limited to personnel who remained in support areas . Army troops who were in Iraq or Kuwait did not have a greater prevalence of GWI than did personnel from other branches in those areas .A large proportion of Army veterans  indicated they had been in Iraq and/or Kuwait during deployment. Stratified analyses to assess possible overlap between branch of service and deployment location in relation to GWI identified a significant interaction between these two variables . To further examine the implications of these interrelationships, we determined the extent to which exposures were differentially reported by veterans who had been located in different geographic areas. As shown in Multivariable (adjusted) associations between GWI and deployment characteristics. Multivariable analyses assessed the independent association of each deployment experience/exposure with GWI for all veterans combined, and for subgroups defined by veterans’ locations —the only significant risk factor identified in this subgroup. This association was not straightforward, however. All but one veteran who reported wearing pesticide-treated uniforms also reported using pesticides on their skin, so it was not possible to estimate the independent effect of wearing pesticide-treated uniforms among veterans in this subgroup. In addition, the association was based on a limited number of observations: 10 exposed cases  and 2 exposed controls .Among veterans who did not enter Iraq or Kuwait, those who reported wearing uniforms treated with pesticides had a substantially increased risk for GWI , GWI was significantly elevated only in the relatively small subgroup of veterans who wore pesticide-treated uniforms, most of whom also had used skin pesticides.Most veteran-reported deployment experiences and exposures were not significantly associated with GWI, when analyses took into account effects of other exposures. Experiences associated with a high degree of psychological stress  were not significantly associated with GWI in our study. Widespread military use of the anthrax vaccine first occurred during the Gulf War, with most anthrax shots given in theater (DOD 2000). However, in our study, veterans who reported receiving shots in theater did not have a significantly increased prevalence of GWI when other exposures were taken into account. In addition, our results do not support a role for pesticides used in area fogging, contact with dead animals, or exposure to depleted uranium through contact with destroyed enemy vehicles as risk factors for GWI.Our findings are consistent with those of previous studies of Gulf War veterans that assessed individual risk factors for symptomatic illness using statistical methods that controlled for confounding effects of concurrent exposures . Those sIt is important to emphasize that identified differences among risk factors for veterans in different areas of theater most likely relate to differences in patterns or degree of exposure by location. For example, personnel in forward areas, where nerve agent exposure was a prominent concern, likely used PB more frequently and for longer periods of time than did personnel in support areas. Similarly, effects of oil smoke exposure would be expected to differ by location. Personnel nearer the burning oil well fires in Kuwait would have experienced, on average, greater and more sustained exposure to the dense smoke compared with personnel in areas farther away.N,N-diethyl-m-toluamide) and lindane powder, an organochlorine used in delousing enemy prisoners of war and provided to some troops (predominantly Army personnel) for personal use, intended for application to clothing , but government reports also indicate that thousands of troops were overexposed to pesticides during the 1990–1991 deployment. Among 64 pesticide products used during the Gulf War, the 15 “pesticides of concern” identified by the DOD (2003) include permethrin, a synthetic pyrethroid that impregnates fabrics and persists through multiple launderings. Permethrin was recommended to be sprayed onto uniforms once every 6 weeks. Some pesticides of concern are no longer used by the military, including a 70% solution of the insect repellant DEET (a) use of pesticides on uniforms may not have been limited to permethrin, b) use of pesticides applied to uniforms was likely to have been excessively high in some individuals, and c) adverse effects of pesticides sprayed onto uniforms may have been enhanced by synergistic interactions with other pesticides and repellants.According to detailed government investigations of pesticide use during the Gulf War, U.S. military personnel were sometimes poorly informed about appropriate use of pesticides and repellants in theater  and those obtained after multivariable adjustments indicates that confounding was likely a much more pervasive problem than recognized in those studies.Because of the many exposures of interest, epidemiologic studies of Gulf War veterans such as ours have generally reported results from multiple tests of association in a single population, raising the possibility that some significant findings occurred by chance. This cannot be ruled out entirely. However, our major findings regarding the prominence of PB and pesticides as risk factors for GWI are consistent with other Gulf War studies that used multivariable analytic methods, suggesting that these associations did not randomly occur as a result of chance.The size of our study population was sufficient only to detect significant risk factors associated with an OR ≥ 2 in the two veteran subgroups evaluated and did not allow us to evaluate more precisely defined subgroups. It is also possible that other exposures, not identified by our data collection, occurred differentially by location in theater or within veteran subgroups and may account for differences in GWI prevalence. For example, we did not ask veterans if they had been exposed to chemical nerve agents because of limitations in their ability to know the answer with any certainty. Two studies have reported brain structure alterations in Gulf War veterans potentially exposed to low-level chemical nerve agents, as estimated by DOD models, in connection with weapons demolition operations at Khamisiyah, Iraq (DOD 2002), after the Gulf War cease-fire .In addition to our analytic approach, major strengths of our study include our sample, selected randomly from among eligible veterans in a defined geographical area, providing distinct advantages over clinical and registry-based samples. We believe our use of the Kansas GWI case definition was also an important asset. We performed a series of exploratory analyses similar to those reported here but assigning case status based on the CDC MSI criteria . ResultsImproved understanding of the causes of GWI remains essential, 20 years after Desert Storm, to clarify the biological nature of this problem and prevent its occurrence in future wars. Our results implicate Gulf War exposures, most prominently the use of pesticides and PB pills, as significant risk factors for GWI. More generally, our findings suggest that the etiology of GWI is complex and likely involves several deployment-related exposures whose relative contributions to the GWI problem differ in identifiable veteran subgroups.


Betapapillomavirus genus and are most closely related to HPV-96 and HPV-22, respectively. As in other members of this genus, the intergenic E2-L2 region is very short and does not encode for an E5 gene. Both genotypes contain typical zinc binding domains in their E6 and E7 proteins, but HPV-151 lacks the regular pRb-binding core sequence within its E7 protein. In order to assess the tissue predilection and clinical significance of the novel genotypes, quantitative type-specific real-time PCR assays were developed. The 95% detection limits of the HPV-150 and HPV-151 assays were 7.3 copies/reaction (range 5.6 to 11.4) and 3.4 copies/reaction (range 2.5 to 6.0), respectively. Testing of a representative collection of HPV-associated mucosal and cutaneous benign and malignant neoplasms and hair follicles  revealed that HPV-150 and HPV-151 are relatively rare genotypes with a cutaneous tropism. Both genotypes were found in sporadic cases of common warts and SCC and BCC of the skin as single or multiple infections usually with low viral loads. HPV-150 can establish persistent infection of hair follicles in immuno-competent individuals. A partial L1 sequence of a putative novel HPV genotype, related to HPV-150, was identified in a squamous cell carcinoma of the skin obtained from a 64-year old immuno-compromised male patient.DNA from two novel HPV genotypes, HPV-150 and HPV-151, isolated from hair follicles of immuno-competent individuals, was fully cloned, sequenced and characterized. The complete genomes of HPV-150 and HPV-151 are 7,436-bp and 7,386-bp in length, respectively. Both contain genes for at least six proteins, namely E6, E7, E1, E2, L2, L1, as well as a non-coding upstream regulatory region located between the L1 and E6 genes: spanning 416-bp in HPV-150  and 322-bp in HPV-151 . HPV-150 and HPV-151 are phylogenetically placed within the  Alpha-, Beta-, Gamma-, Mu- and NupapillomavirusPapilomaviruses (PVs) are a diverse family of small viruses with a circular double stranded DNA genome, which are etiologically linked with several skin and mucosal epithelial lesions of animals and humans. They are hierarchically classified into categories designated as genera, species and genotypes According to the 2004 guidelines for PV nomenclature, published by the Study Group of Papillomaviruses of the International Committee on Taxonomy of Viruses (ICTV), in order to be officially recognized as a unique genotype, a candidate PV isolate must differ by at least 10% of its complete gene coding for the major capsid protein (L1 gene) from all other known genotypes, and its complete genome must be sequenced and deposited in the form of clones to the Reference Centre for Papillomaviruses in Heidelberg, Germany verrucae vulgarisBetapapillomaviruses - can cause serious clinical manifestations, such as numerous benign and malignant tumors in patients with the hereditary disease epidermodysplasia verruciformisBetapapillomavirus HPVs, such as HPV-22, HPV-38, HPV-92 or HPV-96, have been etiologically linked with the development of squamous cell carcinoma of the skin Cutaneous HPV genotypes are found within all five PV genera that contain HPVs. They are ubiquitously present in human skin and in the hair follicles of immuno-competent individuals In the last decade, several studies have shown that HPV DNA can be recovered from healthy skin in humans from very early stages of life In this study, two novel HPV genotypes: HPV-150 and HPV-151, isolated originally from eyebrow hair follicles  and initially characterized by our group in 2005 The original isolates of HPV-150 (SIBX1) and HPV-151 (SIBX2) were obtained from eyebrow hair follicles of two immuno-competent males, aged 28 and 25 years, respectively, during our previous study of HPV distribution in eyebrow hair follicles of patients with genital warts 2O.Whole genome amplification (WGA) of both HPV-150 and HPV-151 was performed prior to PCR amplification, by using an Illustra™ TempliPhi 100 Amplification Kit , according to the manufacturer's instructions. Following the amplification, the WGA product was diluted 200x fold with PCR-grade HPrimers for the reverse long template PCR of HPV-150 were constructed manually on the basis of the previously obtained 373-bp sequence of the HPV-150 L1 gene (GenBank Acc. No AJ606880). A 7,367-bp PCR product of HPV-150 was generated using primer pair X1-longF and X1-longR  and the 2, 500 µM of dNTPs, 0.75 U of Expand Long Template Enzyme Mix, and 3 pmol of each primer. The thermal cycler program was set to 2 min at 94°C, followed by 40 cycles consisting of 10s at 94°C, 30 s at 53°C, and 8 min at 68°C. The final extension step was performed at 68°C for 10 min and the reaction mixtures were then cooled to 4°C.PCR was carried out in a 25 µl reaction volume containing 5 µl of diluted WGA product, 2.5 µl of 10× Expand Long Template buffer 2 with 27.5 mM MgClInitial sequencing of the 7,367-bp HPV-150 PCR fragment was done by primer walking  of dNTPs, 0.02 U of KOD Xtreme™ Hot Start DNA Polymerase, and 0.6 pmol of each primer. The thermal cycler program was set to 2 min at 94°C, followed by 40 cycles consisting of 10 s at 94°C, 30 s at 60°C, and 3 to 6 min at 68°C. Following amplification, the reaction mixtures were cooled to 4°C.E.coli strain JM107 with the HPV-150 clones, according to the manufacturer's instructions.Before cloning, PCR products were excised and purified from 1% agarose gel using a GeneJET™ Gel Extraction Kit . Plasmids containing overlapping PCR products of HPV-150 were prepared using a CloneJET™ PCR Cloning Kit (Fermentas), according to the manufacturer's instructions. A TransformAid™ Bacterial Transformation Kit (Fermentas) was used to transform Plasmid clones isolated from the transformed overnight bacterial culture with a GeneJET™ Plasmid Miniprep Kit (Fermentas) were checked for correct HPV inserts by size analysis and sequencing with pJET1.2 sequencing primers, according to the manufacturer's instructions.Sequencing by primer walking was carried out for both clones, confirming the full genome sequence of HPV-150. The obtained sequences were additionally confirmed by commercial sequencing of the selected clones at Macrogen Inc.  and at Microsynth AG .All primers used in the study were designed manually and checked for Tm and secondary structures using NetPrimer  and TIB-Molbiol  free web-based software. The ‘Primer Pair Specificity Checking Parameters’ function of Primer-BLAST (NCBI) was used in order to check for the specificity of the primers.In the case of HPV-151, the primers initially constructed on the basis of the previously obtained 367-bp sequence of the HPV-151 L1 gene (GenBank Acc. No AJ606879) failed to amplify the remainder of the genome. In order to solve this problem, additional degenerate primers were designed, based on the genome of HPV-22, the genotype which was most closely related to HPV-151. An initial assessment of the primers was done by short PCR using the DreamTaq™ Green PCR Master Mix (2X) , following the manufacturer's instructions. The amplified DNA was examined by gel electrophoresis and several degenerate primer pairs produced PCR products of the expected size, ranging from 100 to 1,328-bp. Following sequencing of these short amplicons, several additional primers, specific for the novel genotype HPV-151, were designed . Finally2, 500 µM of dNTPs, 0.75 U of Expand Long Template Enzyme Mix and 3 pmol of each primer. The thermal cycler program was set to 2 min at 94°C, followed by 40 cycles consisting of 10 s at 94°C, 30 s at 55°C, and 7 min at 68°C. The final extension step was performed at 68°C for 7 min and the reaction mixtures were then cooled to 4°C.PCR was carried out in a 25 µl reaction volume containing 1 µl of diluted WGA product, 2.5 µl of 10× Expand Long Template buffer 2 with 27.5 mM MgClInitial sequencing of the 6,256-bp HPV-151 PCR fragment was done by primer walking with the primers listed in For cloning HPV-151, two overlapping blunt-end PCR products of sizes 2,785-bp and 5,481-bp were generated using primer pairs X2F17 and X2R17; and X2F3 and X2R3, respectively  by usingPCR conditions and the entire further cloning and sequencing procedure for HPV-151 were the same as described above for HPV-150.All partial sequences obtained for HPV-150 and HPV-151 were assembled using ContigExpress of Vector NTI Advance™ v11.0 . The open reading frames (ORFs) of HPV-150 and HPV-151 were determined using the ORF finder function of Vector NTI Advance™ v11.0 and were additionally confirmed by genome organization analysis and comparison with closely related HPV genotypes using the BioEdit Sequence Alignment Editor http://mafft.cbrc.jp/alignment/software) http://www.bork.embl.de/pal2nal/) http://molevol.cmima.csic.es/castresana/Gblocks.html) The phylogenetic relationships of HPV-150 and HPV-151 within the PV family were determined based on the L1 gene. Briefly, L1 nucleic acid sequences were converted to protein sequences and aligned using MAFFT v6.832b  and RT-150-F , together with FAM-labeled Universal Probe #50 (Roche Applied Science) were used for HPV-150 specific real-time PCR.Primers and a probe specific for HPV-150 were chosen within the L1 gene by using the on-line ProbeFinder software (Roche Applied Science), resulting in a PCR product of 60-bp in length. Primers RT-150-F  and RT-151-R  for HPV-151 specific real-time PCR.Primers and a probe specific for HPV-151 were chosen within the L1 gene by using the on-line ProbeFinder software (Roche Applied Science), resulting in a PCR product of 60-bp in length. FAM-labeled Universal Probe #127 (Roche Applied Science) was used together with primers RT-151-F  and the number of plasmid copies/µl was calculated using the equation of Whelan et al. The optimized real-time PCR reaction mixture for both type-specific real-time PCRs contained 10 µl of LightCycler® Probes Master 2x conc. (Roche Applied Science), 0.2 µM of each primer, 0.05 µM of Universal Probe #50 or #127 (Roche Applied Science), 1U of LightCycler® Uracil-N-Glycosylase (Roche Applied Science), sample DNA and water up to a final volume of 20 µl. The assays were performed on a LightCycler® 480 (Roche) under the following conditions: Uracil-N-Glycosylase activation at 40°C and pre-incubation at 95°C for 10 min each, followed by 45 cycles of 95°C for 10 s (denaturation) and 59°C for 30 s  and 72°C for 1 s (extension). Acquisition of the fluorescence signal was performed in single mode during the annealing step of each cycle. The final cooling step consisted of a 10 s hold at 40°C. The conditions of the real-time PCR for HPV-150 and HPV-151 were intentionally identical, since this allowed simultaneous testing of samples on the LightCycler® 480 (Roche).To evaluate the linearity of the assays, triplicate ten-fold serial dilutions of the reference plasmids of HPV-150 and HPV-151 were prepared in 1.5 ml DNA LoBind tubes  using a water solution with carrier RNA (1 µg/ml). The limits of detection of the assays were determined by testing 15 replicates of the reference plasmid dilutions corresponding to an input of 25, 13, 6, 3, 1.5, 0.8 and 0.4 copies per reaction.7 to 108 copies of synthetic partial L1 sequences  of the following Betapapillomaviruses: HPV-9, HPV-15, HPV-17, HPV-22, HPV-23, HPV-37, HPV-38, HPV-80, HPV-100, HPV-111, HPV-113, HPV-120, HPV-122, HPV-92 and HPV-96.The specificity and cross-reactivity of the assays were tested using 10The tissue predilection and clinical significance of HPV-150 and HPV-151 were assessed on a representative collection of HPV-associated benign and malignant neoplasms and hair follicles. The DNA was isolated from a total of 540 samples obtained from the same number of patients, including tissue samples of histologically confirmed genital warts (71), laryngeal papillomas (78), cervical squamous cell carcinomas (100), common warts (101), squamous cell carcinoma (SCC) of the skin (52), basal cell carcinoma (BCC) of the skin (49) and hair follicles (89), as described previously Alphapapillomavirus genotypes; RHA skin (beta) HPV , which allows identification of 26 different Betapapillomavirus genotypes, and additionally by consensus GP5+/6+ in-house PCR and sequencing of PCR products The presence of additional HPV genotypes in HPV-150 and/or HPV-151 positive samples was determined using two commercial line-probe assays: INNO-LiPA HPV Genotyping Extra , which allows identification of 28 different None of the 540 clinical samples tested were collected solely for the purpose of this study. All samples included in the study were collected in compliance with the Helsinki Declaration. Samples of cervical cancer, hair follicles, genital warts and common warts were collected prospectively in our previous studies. These studies were approved by the Ethics Committee of the Ministry of Health of the Republic of Slovenia  and written informed consent was obtained from each patient. Samples of laryngeal papillomas, squamous cell carcinoma and basal cell carcinoma of the skin were retrieved from the tissue collection of paraffin-embedded samples of the Institute of Pathology, Faculty of Medicine, University of Ljubljana. Approval from the Institutional Review Board of the Institute of Pathology, Faculty of Medicine, University of Ljubljana was obtained prior to starting work on samples included in this study. In accordance with the national legislation of the Republic of Slovenia, no informed consent is needed for research on archival clinical samples. In order to protect the identity of the patient, all archival clinical samples used in the study were coded and tested anonymously. The only available data were patient gender, age and immune status .Partial L1 sequences of HPV-150  and HPV-151  were deposited in GenBank in March 2005 under Acc. Nos. AJ606880 and AJ606879, respectively. The four reference clones, covering the full genomes of HPV-150 and HPV-151, were deposited in March 2010 in the Reference Centre for Papillomaviruses, Heidelberg, Germany, where their sequences were independently confirmed and the two genotypes were officially named in April 2010.The genome of HPV-150 has a total length of 7,436-bp, with a GC ratio of 39.70%, and is deposited in GenBank under Acc. No. FN677755. The genome of HPV-151 has a total length of 7,386-bp, with a GC ratio of 39.79%, and is deposited in GenBank under Acc. No. FN677756.Betapapillomavirus species 1 Betapapillomavirus species Betapapillomaviruses, the intergenic region located between E2 and L2 genes is very short  and 84-bp in HPV-151 ) and does not encode for an E5 gene.ORF analysis and sequence alignment comparisons showed both HPV-150 and HPV-151 to have the typical genomic organization of HPVs and to contain at least six viral genes: E6, E7, E1, E2, L2 and L1, as well as possibly a gene for the E4 protein . In addi29CxxC) within their E6 protein, at amino-acid positions 24 and 98; and 27 and 101, respectively (29CxxC) within their E7 protein, at amino-acid positions 50 and 53, respectively. However only HPV-150 also contains the regular pRb-binding core sequence (LxCxE) at amino-acid position 24 of E7  within its E7 protein (HPV-150 and HPV-151 contain two regular zinc binding domains (CxxC(x)ectively . Both ge24 of E7 . Unlike  protein .Betapapillomavirus genus . HPV-22 was originally isolated from chest macules Betapapillomavirus species 2 encompasses 17 HPV genotypes isolated from humans, making it one of the largest species among PVs. The generation of such viral diversity has probably occurred through intra-host duplication events, which may reflect multiple ways of succeeding in adaptation to the human host Betapapillomavirus species 2 have been found in lesions ranging from macules and other benign skin lesions, to actinic keratosis, BCC and SCC of the skin, and even head and neck and esophageal carcinoma, as in the case of HPV-100 As shown in 2 = 1.00) between the Ct (threshold) and the log of starting viral copy number across at least seven orders of magnitude. The 95% limits of detection determined by Probit analysis using the PASW Statistics 18 program  were 7.3 copies/reaction (range 5.6 to 11.4) and 3.4 copies/reaction (range 2.5 to 6.0), for the HPV-150 and HPV-151 assays, respectively. For HPV-150 assay, the intra- and inter-assay coefficient of variation (CV (%)  =  (st.dev/mean Ct)×100) were 0.55 and 1.43 for 100 copies per reaction, respectively, and 1.73 and 1.81 for 10 copies per reaction, respectively, and corresponding values for HPV-151 assay were 0.81 and 1.54 for 100 copies per reaction, respectively, and 1.02 and 1.98 for 10 copies per reaction, respectively. Both assays showed no cross-reactivity with any of the tested non-targeted HPV genotypes.In order to assess the biological and clinical importance of the novel HPV genotypes, two quantitative HPV-150 and HPV-151 type-specific PCR assays were developed. The standard curves of both assays showed an excellent correlation coefficient  and possible outcomes of HPV infection . The panAs shown in Alphapapillomavirus species 4, a well known genotype capable of causing common warts; HPV-5 and HPV-14, cutaneotropic members of Betapapillomavirus species 1; and HPV-15 belonging to Betapapillomavirus species 2 – all three later being genotypes associated with the development of cutaneous tumors in immuno-suppressed patients or patients with epidermodysplasia verruciformis. Due to the presence of more than one HPV genotype in these lesions, the development of common warts could not be conclusively attributed to HPV-150. In contrast, in the fourth HPV-150 positive common wart  our finding is in agreement with recent studies which showed that viral load of Betapapillomaviruses associated with cutaneous warts is substantially lower than that of Alphapapillomaviruses  associated with similar lesions  Of the 101 samples of common warts tested, four samples (4.0%) contained HPV-150, indicating the cutaneous tropism of this genotype . Of HPV-Betapapillomavirus genus were present in three out of four HPV-150 positive samples of malignant skin tumors included in the study  contained HPV-151, indicating its cutaneous tropism . Of HPV-As shown in Betapapillomaviruses in non-melanoma skin cancer In the single HPV-151 positive BCC, where no other HPV genotypes were present  showed 90.68% nt identity with the corresponding part of the L1 sequence of HPV-150 but its other characteristics remain to be determined in future studies.In addition to full characterization of two novel HPV genotypes, a putative novel HPV genotype, phylogenetically related to HPV-150, was identified in a tissue sample of SCC of the skin obtained from a 64-year old male immuno-compromised patient, together with HPV-9, the type species genotype of Betapapillomaviruses HPV-150 and HPV-151, as well as the partial identification of a putative novel HPV genotype closely related to HPV-150 (isolate SI-HPV-Beta5), improves our knowledge of the diversity of Betapapillomaviruses infecting humans. HPV-150 and HPV-151 belong phylogenetically within the Betapapillomavirus genus and are most closely related to HPV-96 and HPV-22, respectively. Both novel genotypes are relatively rare, most likely lack mucosal tropism and have clear cutaneous tropism. HPV-150 can establish persistent infection of hair follicles in immuno-competent individuals. HPV-150 and HPV-151 have been found in sporadic cases of common warts, SCC and BCC of the skin preferably in elderly immuno-competent individuals, as both single and multiple infections with viral loads ranging from 1/12 to 1/57,950 viral genomes/No. of human cells.The cloning and full characterization of two novel Table S1Primers used for initial amplification, primer walking and preparation of HPV-150 and HPV-151 reference clones. Nt – Nucleotide position. N – Random nucleotide. Cw – Clock-wise. Ccw – Counter clock-wise.(DOC)Click here for additional data file.


There were errors in affiliations 1 and 7. The correct affiliations are:1 Department of Radiology, Kurume University School of Medicine, Kurume, Fukuoka, Japan,7 Department of Surgery, Kurume University School of Medicine, Kurume, Fukuoka, Japan.


Metaplastic carcinoma, an uncommon subtype of breast cancer, is part of the spectrum of basal-like, triple receptor-negative breast carcinomas. The present study examined 20 surgical specimens of metaplastic breast carcinomas, for the presence of high-risk Human papillomavirus (HPV), which is suspected to be a potential carcinogenic agent for breast carcinoma.Mastectomy specimens from patients harboring metaplastic breast carcinoma, as defined by the World Health Organization (WHO), and who attended the Instituto Nacional de Cancerologia in Mexico City, were retrieved from the files of the Department of Pathology accumulated during a 16-year period (1995–2008). Demographic and clinical information was obtained from patients’ medical records. DNA was extracted from formalin-fixed, paraffin-embedded tumors and HPV type-specific amplification was performed by means of Polymerase chain reaction (PCR). Quantitative Real-time (RT) PCR was conducted in HPV positive cases. Statistically, the association of continuous or categorical variables with HPV status was tested by the Student t, the Chi square, or Fisher’s exact tests, as appropriate.High-risk HPV DNA was detected in eight (40%) of 20 metaplastic breast carcinomas: seven (87.5%) HPV-16 and one (12.5%) HPV-18. Mean age of patients with HPV-positive cases was 49 years (range 24–72 years), the same as for HPV-negative cases . There were not striking differences between HPV + and HPV– metaplastic carcinomas regarding clinical findings. Nearly all cases were negative for estrogen, progesterone and Human epidermal growth factor receptor 2 (HER2), but positive for Epidermal growth factor receptor (EGFR).High-risk HPV has been strongly associated with conventional breast carcinomas, although the subtle mechanism of neoplastic transformation is poorly understood. In Mexican patients, the prevalence of HPV infection among metaplastic breast carcinomas is higher than in non-metaplastic ones, as so the HPV viral loads; notwithstanding, HPV viral loads show wide variation and remain even lower than cervical and other non-cervical carcinomas, making it difficult to assume that HPV could play a key role in breast carcinogenesis. Further studies are warranted to elucidate the meaning of the presence of high-risk HPVDNA in breast carcinomas. Accordiet al. , who rep [et al.  found HPThis is an observational and descriptive study considered by the Mexican regulation in health research as a safely study that does not need informed consent . The stuFormalin-fixed, paraffin-embedded metaplastic breast carcinomas obtained from Mexican patients attending the Instituto Nacional de Cancerología, were retrieved from the files of the Department of Pathology accumulated during a 16-year period (1995–2008). Demographic and clinical information was obtained from patients’ medical records. Histologic classification was assessed as proposed by the World Health Organization (WHO) classification of breast tumors [et al. [globin gene (PCO4/GH2O) under conditions described by Resnick et al. [L1 gene: L1C1/L1C2, MY09/MY11, and GP5/GP6 [E6 gene of HPV types-16 and −18 as described by Lizano et al. [Twenty-μm sections of formalin-fixed, paraffin-embedded tumors were dewaxed through incubation with N-octane and washings with 100% ethanol. This process was repeated twice, after which the pellet was dried. The deparaffinized sample was digested with 1 ml of lysis buffer  at 55°C for 3 hr. DNA was extracted with phenol/chloroform precipitations as described by Sambrook et al.  To test k et al.  Samples  GP5/GP6 -17. HPV HPV PCR products were electrophoresed in a 1.2% agarose gels and visualized by ethidium bromide staining. HPV typing was performed through direct sequencing of PCR products by means of BigDyeTM Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). The resulting sequences were analyzed in the Basic Local Alignment Search Tool (BLAST) data bank for comparison with known HPV sequences. HPV- 16 and-18 DNA amplification was conducted for each sample, using specific primers as previously described . DNA ext3 to 1 × 109 copy number, employing the program created by Andrew Staroscik (2004) (http://cels.uri.edu/gsc/cndna.html). As positive control, the SiHa cell line containing 1–2 copies of HPV-16 per cell was used. To generate the standard curve GAPDH gene fragment cloned into pJET1.2/blunt plasmid was used, with 300 nM of each primer: GAPDH-Reverse: ATGGGTGGAATCATATTGGAAC, and GAPDH-Forward: GAAGGTCGGAGTCAACGGATTT. PCR conditions were 95°C for 30 sec and 60°C for 1 min for 40 cycles. The amount of GAPDH DNA present in each sample was divided by the weight of one genome equivalent (6.6 pg per cell) and a factor of 2  to obtain the number of genome equivalents per cell [per 104 cells.As previously indicated, DNA was isolated just from neoplastic tissue. HPV physical status was not determined. To estimate the copy numbers of HPV-16 genomes in biopsy samples, the primers utilized to amplify the E6 oncogene fragment were the following: E6-HPV16-648-Reverse: GAACCGAAACCGGTTAGTAT, and E6-HPV16- 419-Forward: GGACACAGTGGCTTTTGACA. Real-time PCR assays were performed using SBYR GREEN (Applied Biosystems). PCR conditions were optimized to 300 nM E6-HPV16-648-Reverse primer and 300 nM E6-HPV16-419- Forward primer. PCR reactions were performed in a Rotor-Gene 6000 (Corbett Life Science) with the following PCR conditions: 95°C for 30 sec and 59°C for 1 min for 40 cycles. Quantification was performed using a standard curve from pBR322-HPV16 plasmid that contains the entire genome of HPV-16 with a dilution series from 1 × 10taroscik 04  following the manufacturer’s instructions. Control cell lines provided by the manufacturer (Dako) were used as negative and positive controls.Immunohistochemical studies were performed on 4 μm paraffin sections employing a Ventana automated immunostainer , according to the company’s protocol with minor modifications Table . Estroget test or the Chi square test, as appropriate. Two-tailed statistics were considered in all cases, and a probability value of 0.05 or lower was considered as significant. The SPSS version 20 software  for MAC was employed for all computations.After descriptive statistics, the association of continuous or categorical variables with HPV status was tested by the Student in situ hybridization (FISH) for detecting the gene amplification was not performed. On the other hand, all cases were positive for EGFR 1  . Regardless of its relationship with breast tumors, nine women had previous medical history of at least one cervical smear for screening purposes, as part of the National campaign against carcinoma of the cervix uteri; typical changes of HPV infection appeared in a case of HPV-positive adenosquamous carcinoma, and in one HPV- negative squamous cell/sarcomatoid carcinoma, whereas in the remaining seven cases, the result was reported according to the Bethesda System as negative for intraepithelial lesion or malignancy. Viral loads were constantly low, ranging from 0.02040313 (metaplastic carcinoma with chondroid differentiation)–1.015210939 (metaplastic carcinoma with squamous differentiation) copies/cell , when compared with number of HPV copies/cell in the cell line SiHa (3.985 copies/cell) . Mean age of the patients was 49 years . HPV DNA was detected in eight of 20 (40%) metaplastic breast carcinomas: HPV-16 in seven (87.5%) cases, and HPV-18 in one (12.5%) case of matrix-producing bone carcinoma. Distribution of histological subtypes according to HPV status, is summarized in Table –2008. MeIn this study, high-risk HPV DNA was detected in eight of 20 40%) metaplastic carcinomas of the mammary gland. HPV-16 was found in seven of these (87.5%), whereas HPV-18 was present in the remaining case (12.5%). Distribution of the HPV genotype is in accordance with previous Mexican studies in which HPV-16 was the commonest HPV detected among breast carcinoma . Accordi% metaplaneu but positive for EGFR, as in nearly all of our cases. It is noteworthy that the majority of HPV-associated carcinomas reported in the literature are closely related with the “triple-negative” profile. In the study of Kroupis et al. [On the other hand, it was also suggested that metaplastic carcinomas, together with less differentiated breast carcinomas, represent the progenitor/stem-cell end of the spectrum in which breast carcinomas could be arranged, according to different protein expression patterns and (cyto)genetic alteration patterns . Within s et al.  HPV-posis et al.  and the High-risk HPV has been strongly associated with conventional breast carcinomas, although the subtle mechanism of neoplastic transformation is poorly understood. In Mexican patients, the prevalence of HPV infection among metaplastic breast carcinomas is higher than in non-metaplastic ones, as so the HPV viral loads; notwithstanding, HPV viral loads show wide variation and remain even lower than cervical and other non-cervical carcinomas, making it difficult to assume that HPV could play a key role in breast carcinogenesis. Further studies are warranted to elucidate the meaning of the presence of high-risk HPVDNA in breast carcinomas.HPV: Human papillomavirus; DNA: Deoxyribonucleic acid; ER: Estrogen receptors; PgR: Progesterone receptors; HER2: Human epidermal growth factor receptor 2; EGFR: Epidermal growth factor receptor; PCR: Polymerase chain reaction.The authors declare that they have no competing interest.RHG, study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript; critical revision of the manuscript for important intellectual content; study supervision. TVC, acquisition of data; analysis and interpretation of immunohistochemical studies; critical revision of the manuscript for important intellectual content. ACG, carried out the molecular studies; critical revision of the manuscript for important intellectual content. MLS, analysis and interpretation of molecular data; critical revision of the manuscript for important intellectual content. AAM, carried out the molecular studies; critical revision of the manuscript for important intellectual content. LFOO, analysis and interpretation of data; statistical analysis; critical revision of the manuscript for important intellectual content. RSRH, acquisition of data; analysis and interpretation of cytopathologic data; critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/445/prepub