Human papillomavirus-associated oropharyngeal carcinoma (HPV-OPC) is clinicopathologically distinct entity from the HPV-unassociated one (nHPV-OPC). This study aimed to determine the relationship between histological subtypes of OPC and HPV status for Japanese cases and to identify histological structures of HPV-OPC.66 OPC cases were categorized into conventional squamous cell carcinoma (SCC) and the variants. Conventional SCC was subcategorized into keratinizing (KSCC), non-keratinizing (NKSCC), and hybrid SCC (HSCC). HPV status of all cases was determined using p16-immunohistochemistry and HPV-DNA ISH.Two histological subtypes, NKSCC and HSCC, tended to be HPV-OPC and KSCC tended to be nHPV-OPC with statistical significance. Two histological structures, abrupt keratinization, defined in the text, and comedo-necrosis among non-maturing tumor island, were observed for 58.1% and 38.7% of HPV-OPC, and tended to exist for HPV-OPC with statistical significance.This study showed the association of NKSCC/HSCC with HPV-OPC in Japanese cases, and two histological structures, abrupt keratinization and comedo-necrosis among non-maturing island, were considered characteristic histological features of HPV-OPC.http://www.diagnosticpathology.diagnomx.eu/vs/1816432541113073.The virtual slide(s) for this article can be found here: Human papillomavirus (HPV)-associated oropharyngeal carcinoma (HPV-OPC) has been recognized as a distinct clinicopathologic entity of head and neck cancer. HPV-OPCIn terms of histopathology, there have been several papers on some histological subtypes/variants that are more likely to be HPV-associated-15. For In this study, we classified our HPV-OPC and nHPV-OPC cases by histological subtypes and determined if there are some histological features of concern, in order to determine the specificity/sensitivity of NKSCC, KSCC, HSCC for HPV-OPC with Japanese OPC cases and to identify histological structures characteristic for HPV-OPC.In this study, HPV status for each case was judged by p16 immunohistochemistry (IHC)/in situ hybridization (ISH). Although there have been studies which propose genomic amplification of human telomerase gene or C-MYC as helpful screening methods for HPV-associated cancer/high-grade lesions in cases of uterine cervix, a gold 66 pathological specimens of previously untreated oropharyngeal carcinoma (OPC), of which 39 were biopsy and 27 were surgical specimens, were studied. All patients were treated in the Department of Otorhinolaryngology, Head and Neck Surgery, Juntendo University, between 2004 and 2012. All materials were obtained before chemo- or radiotherapy. The clinical data, including tobacco/alcohol exposure, tumor site, clinical stage, and HPV status of cases, are shown in Table The histopathological classification and assessment of the tumors were conducted using light microscopy by three of the authors , in a blinded manner and without knowledge of HPV status. Formalin-fixed, paraffin-embedded samples were cut into 4-um sections and stained with hematoxylin and eosin (H&E). One to 5 sections for each were reviewed. For the histological classification, we followed the definitions in the WHO classification/previous articles-15,22, bSince two histopathological features, (i) ‘abrupt keratinization’ and (ii) ‘comedo-necrosis among non-maturing tumor island’, were often seen in our OPC cases, but were usually not observed in our daily pathological practice for squamous cell carcinoma of any other organ/tissue, we carefully evaluated the existence of these two histological structures. Here, we defined (i) ‘abrupt keratinization’ as cancer pearl formation or evident squamous maturation among or peripheral to the non-maturing tumor cell nest Figure A and iiB. The exIHC for p16 was performed using 4-μm-thick tissue sections from formalin-fixed paraffin-embedded tissues and with DAKO EnVision™ + Kit  following the manufacturer’s instructions.For ISH, a catalyzed signal amplification method for biotinylated probes  was used. Briefly, 5-μm tissue sections underwent deparaffinization, heat-induced target retrieval in citrate buffer , and digestion using Proteinase K . Slides were subsequently hybridized with a biotinylated HPV16/18-type-specific probe . Signal amplification was performed by consecutive application of streptavidin-HRP complex and biotinyl tyramide. Visualization of positive hybridization signals was performed by incubation with the chromogenic substrate diaminobenzidine. Interpretation of staining was performed without knowledge of p16 IHC positivity or tumor origin. In some cases where there was discordance between p16 IHC and HPV 16/18 status , we performed analysis with a more extended panel of ISH probes that included types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68  in order to check for other high-risk HPV associations.As controls, squamous cell carcinoma of the tonsil was hybridized with a positive control  biotinylated DNA probe, and with a negative control  biotinylated DNA probe.To determine the association of each of the histological subtypes/histological structures with HPV status, and to see the relationship of patients’ age (43-59/60-87) and sex (F/M) to histological subtypes of OPC, cross-tabulation analyses were performed using Fisher’s exact test. The statistical analyses were performed using PASW Statistics 18.0 .This study was approved by the ethics committee of Juntendo University School of Medicine.Among all the 66 OPC cases examined, 54 (81.8%) were categorized as conventional SCC, being subcategorized into 22 (33.3%), 21 (31.8%), and 11 (16.7%) cases of KSCC, NKSCC and HSCC [Table Abrupt keratinization, as defined above, was observed in 18/31 HPV-OPC cases (58.1%) and in 2/35 nHPV-OPC cases (5.7%). Comedo-necrosis among non-maturing tumor island was observed in 12/31 nHPV-OPC cases (38.7%) and in 1/35 of nHPV-OPC cases (2.9%). The existence of abrupt keratinization/comedo-necrosis among non-maturing tumor island was both statistically associated with HPV-OPC  Tables .The present study showed that most  OPC cases were conventional SCC, and were subcategorized into 36.7% KSCC, 31.7% NKSCC, and 18.3% HSCC, where NKSCC and HSCC were more likely to be HPV-OPC, and most KSCC was nHPV-OPC with statistical significance. These results for Japanese OPC cases are in accordance with those of Chernock et al.[In the present Japanese study, no significant difference in clinical data, including risk factors, was seen between HPV-OPC and nHPV-OPC groups. In Western countries, patients with HPV-OPC have different epidemiological data including less tobacco exposure, more marijuana exposure, and lower age than those with nHPV-OPC,5. It seA few researchers have reported the association of papillary, basaloid, and undifferentiated SCC  with HPV-OPC-15. In tWhen investigating the histopathological features of HPV-OPC, some histological structures were noteworthy. Abrupt keratinization, cancer pearl formation/squamous maturation in an abrupt fashion among non-mature tumor island, or at a peripheral site of tumor nest, were not usually observed in our daily pathological practice for squamous cell carcinoma of any other organ/tissue. Although comedo-necrosis is not rare in many carcinomas including keratinizing SCC, comedo-necrosis among non-maturing SCC cells is unusual. Hence, we analyzed whether these two structures are characteristic histological structures for HPV-OPC or not. This study suggested that these two histological structures, abrupt keratinization and comedo-necrosis among non-maturing tumor island, are very specific for HPV-OPC.Our study showed that NKSCC and HSCC are statistically more likely to be HPV-associated carcinoma, while KSCC is statistically more likely to be nHPV-OPC, which was also true for Japanese patients. Two histological structures, abrupt keratinization and comedo-necrosis among non-maturing island, were considered to be very specific pathological structures for HPV-OPC.Written informed consent was obtained from the patient for the publication of this report and any accompanying images.The authors declare that they have no competing interests.JY and KI conceived the study idea and designed the study. MF, YF, and AK reviewed the literature, performed in situ hybridization and statistical analyses. KM performed immunohistochemistry. MF and YF extracted data and drafted the manuscript. TY reviewed and edited the manuscript. All authors read and approved the final manuscript.


Persistent high-risk (HR) human papillomavirus (HPV) infection and increased HR-HPV viral load are associated with the development of cancer. This study investigated the effect of human immunodeficiency virus (HIV) co-infection, HIV viral load and CD4 count on the HR-HPV viral load; and also investigated the predictors of cervical abnormalities.Participants were 292 HIV-negative and 258 HIV-positive women. HR-HPV viral loads in cervical cells were determined by the real-time polymerase chain reaction.HIV-positive women had a significantly higher viral load for combined alpha-9 HPV species compared to HIV-negative women . This was not observed for individual HPV types. HIV-positive women with CD4 counts >350/μl had significantly lower viral loads for alpha-7 HPV species (median 0.12 copies per cell) than HIV-positive women with CD4 ≤350/μl , but low CD4 count was not significantly associated with increased viral load for other HPV species. High viral loads for alpha-6, alpha-7 and alpha-9 HPV species were significant predictors of abnormal cytology in women.HIV co-infection significantly increased the combined alpha-9 HPV viral load in women but not viral loads for individual HPV types. High HR-HPV viral load was associated with cervical abnormal cytology. Persistent cervical infection with high-risk (HR) human papillomavirus (HPV) leads to the development of cervical lesions and this is accelerated in human immunodeficiency virus (HIV)-positive women-3. HIV cThe increased HPV viral load and low CD4 counts among HIV-positive women increases the probability of developing cervical cancer-9. The HIn the present study we detected and quantified genital HPV types in women using the in-house real-time polymerase chain reaction (RT-PCR). The objectives of the study were (i) to detect and quantify the respective HR-HPV types in HIV-positive and HIV-negative women; (ii) to investigate the effect of HIV co-infection on HR-HPV viral load; and (iii) to investigate predictors of cervical abnormality.Study participants were recruited through clinics, bus stops and taxi ranks. Eligible individuals were asked to come to Manyanani clinic  with their sexual partner between 2006 and 2009. The Research Ethics Committee of the University of Cape Town approved all aspects of the investigation (reference: 258/2006) and informed consent was obtained. A total of 258 HIV-positive women and 292 HIV-negative women were enrolled for this study. For enrolment we did not select certain participants; however, as they were coming in they were enrolled. SamplesDNA was extracted from cervical cells using the MagNA Pure Compact Nucleic Acid Isolation Kit  and automated Roche MagNA Pure Compact machine. DNA was stored at -20°C and shipped to University of Uppsala, Sweden for detection and quantification of HR-HPV DNA. The real-time PCR assay used in this study has been reported to have similar sensitivity and specificity to Hybrid Capture 2 , a United States Food and Drug Administration approved assay. HR-HPV The assay used was based on four parallel real-time PCRs from each DNA sample, one reaction to quantify the amount of a human single-copy gene ; GenBank accession no. M95623.1) and the three other reactions to detect and quantify HR-HPVs . The results are presented as individual types, except for HPV-18 and -45, which are detected as a phylogenetically-related group, and HPV-33, -52, and -58, which are similarly detected. The real-time PCR assay was carried-out in a final volume of 25 μl, containing 3 μl template DNA from cervical cells, Taqman® Universal PCR master mix with no AmpErase® UNG , 3.1 μg of bovine serum albumin , 200 nM of each primer  and probe . Primers and probes are listed in Table 2 to 105 copies were established for each HPV type or group of HPV types to be detected Gustavsson et al.. Similarly, in women infected with α9 HPV species, the odds of abnormal cervical cytology were significantly associated with α9 HPV viral load [OR 1.66 (95% CI: 1.28-2.15) per unit increase in log viral load] Table .In this study we investigated the HR-HPV viral load in HIV-positive and HIV-negative women. To our knowledge this is the first study to report individual HR-HPV type viral loads in HIV-positive and HIV-negative women detected by this kind of RT-PCR assay. HIV co-infection significantly increased cervical HR-HPV prevalence in HIV-positive women. The prevalence of HR-HPV in HIV-positive women (73.3%) and HIV-negative women (45.5%) was lower than that reported by Luchters et al.,. This diHIV-positive women were found to have increased α9 HPV viral load compared to HIV-negative women, similar to findings reported in other studies,17,19,20The association of the immune system with HPV viral load is reflected in the increasing α7 HPV viral load with decreasing CD4 count among HIV-positive women. Levi et al. reported that women with low CD4 counts were at increased risk of high HPV viral load and cervical abnormal cytology compared to women with higher CD4 counts. We did HIV infection and high HPV viral load were found to be predictors of abnormal cytology, however; in multivariate analysis after controlling for HPV viral load, the effect of HIV status on this association was not statistically significant-24. InfeAlthough the current study is a cross-sectional study, we observed that HIV co-infection significantly influenced HR-HPV prevalence in women and α9 HPV viral load in women. High HR-HPV viral loads were found to be associated with cervical abnormal cytology. Data from this study will add to the growing HPV viral load information in South African HIV-positive and HIV-negative women.Ethical approval was given by the Research Ethics Committee of the University of Cape Town (reference: 258/2006) and informed consent was obtained.th International Papillomavirus Conference and Clinical Workshop .Some of the results from this manuscript were presented at the 25ASCUS: Atypical squamous cell of undetermined significance; CI: Confidence interval; HIV: Human immunodeficiency virus; HMBS: Hydroxymethylbilane synthase; HPV: Human papillomavirus; HSIL: High-grade squamous intraepithelial lesion; HR: High-risk; LSIL: Low-grade squamous intraepithelial lesion; RT-PCR: Real time polymerase chain reaction.The authors declare that they have no competing interests.DC, DJM and ALW were responsible for study set-up and specimen collection. ZZAM was responsible for specimen storage. IG, UG and ZZAM were responsible for generating HPV viral load data. The first draft was written by ZZAM, with contributions from DJM, IG, UG, LFJ, DC, and ALW. Statistical analysis was conducted by LFJ. All authors were actively involved in the interpretation of the data, creation and revision of the manuscript; and approval of the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/51/prepubDifferences in HPV viral load in HIV-infected women, by CD4 count.Click here for file


Then, 16 types of HPV DNA sequences were assessed with the PapiPlex method using genomic DNA samples extracted from each bladder tumor as well as urethral condyloma. HPV-11 was detected in DNA extracted from the urethral condyloma, while no HPV DNA sequences were positive in any of the genomic DNA samples extracted from the bladder tumors.Human papillomaviruses (HPVs) are associated with proliferative lesions in a variety of human epithelial types. A 38-year-old female presented with a diagnosis of urethral condyloma acuminatum. She underwent transurethral resection of the urethral condyloma. At that time, multiple (five) bladder tumors were simultaneously found and also removed by transurethral resection. Four of the bladder tumors were diagnosed as squamous papilloma, and the other was urothelial inverted papilloma. Postoperative course was uneventful. Genomic DNA was extracted from 10  With oplasias . AssociaA 38-year-old female presented with a diagnosis of urethral condyloma acuminatum. There were no condylomatous lesions in the gynecological area. She underwent transurethral resection of the urethral condyloma. At that time, multiple (five) bladder tumors of less than 1 cm in diameter were simultaneously found and also removed by transurethral resection. Tissues were fixed in 10% formalin, then embedded in paraffin. Sections were stained with hematoxylin and eosin (HE). Four of the bladder tumors were diagnosed as squamous papilloma , and theμm thick sections of each bladder tumor as well as urethral condyloma. Then, HPV-6, 11, 16, 18, 30, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 66 DNA sequences were assessed at one time with the PapiPlex method [Genomic DNA was extracted from 10 Schistosoma haematobium may be unique. HPV-16 was detected in all of 27 bladder cancers due to schistosomiasis [In the urinary tract, linkage of urothelial cancer and HPV infection is still controversial. Saltzstein et al. did not detect HPV-6, 11, 16, 18, 31, and 33 in any of 33 urothelial cancer cases regardless of their invasiveness . In addisomiasis . For othsomiasis .In the present case, HPV-11 was positive in urethral condyloma acuminatum, but none of the HPVs was detected in urothelial and squamous papillomas in the bladder. Those papillomas in the patient may not be caused by HPV infection and were concomitant with urethral condyloma by chance, although the multiplicity of the simultaneous papillomas elicits the possibility of the same genesis. Similarly to this case, Olsen et al. detected HPV-6 and 11 in a male patient with urethral condyloma, while no HPVs were demonstrable in recurring papillomatous urothelial cancers in the same patient . When pa


Pancreatic mucinous cystic neoplasms (PMCN) predominantly affect women in the reproductive age, are located in the body and tail of the pancreas, and share morphological features with similar tumors of the ovary. We report the detection of human papillomavirus (HPV) using several different PCR protocols in a borderline PMCN from a female patient. Type-specific PCR demonstrated the HPV to be type 16. If confirmed by others, this group of neoplasms might become preventable by HPV vaccination. Pancreatic cancers are a heterogeneous group of tumors with significantly different clinical features, anatomic locations, histopathological features, molecular pathogenesis, and prognosis. The majority of these tumors are highly malignant adenocarcinomas, occur in the head of the pancreas (PANCA), and have a higher incidence in men. Pancreatic mucinous cystic neoplasms (PMCN) and solid-pseudopapillary tumor are rare tumors which occur mostly in women, the latter nearly exclusively so. Early detection and complete resection of these tumors offer a much better clinical outcome than PANCA. These differences suggest that different etiologic agents and intracellular pathways may be involved in tumorigenesis.Recently, a series of ovarian adenocarcinomas was reported in which oncogenic HPV were found. Synchronous and metachronous endocervical adenocarcinomas positive for the same HPV types were identified . We posiThe patient is a previously healthy 45 year-old woman who experienced epigastric pain for several days before presentation. Physical examination showed a firm left upper quadrant abdominal mass. There was no tenderness or ascites. No lymphadenopathy was found. Rectal and vaginal examinations were negative for pelvic mass. Serum amylase level was normal. Abdominal ultrasonography revealed a large cystic lesion at the tail of the pancreas. Computerized axial tomography confirmed the presence of a thin walled cystic lesion with peripheral septations arising from the pancreatic tail, measuring 12 × 11 × 9 cm Fig. . The strTissue blocks with tumor and non-neoplastic pancreatic tissue were selected for HPV detection by general primer and type-specific PCR as previously reported -4. In brHPV was identified in tumor blocks by general primer PCR reactions employing GP5+/6+ Fig.  and SPF1Pancreatic mucinous cystic neoplasms are of unknown etiology and are much more common in women than in men. Its location in the body and tail of the pancreas is in contrast to conventional pancreatic adenocarcinoma of the head of the pancreas (PANCA), which has a higher incidence in men . The evoIn terms of molecular pathways involved, PMCN differs from PANCA in being mostly negative for immunohistochemical expression of p53 . In uterRecently, a series of ten ovarian adenocarcinomas was reported, having synchronous and metachronous HPV-induced endocervical adenocarcinomas, some masquerading as pre-invasive lesions . PancreaThe hypothesis of PMCN being caused by HPV is novel, as we were not able to find similar case report in the English language medical literature. How the pancreas is infected by HPV is unknown. It is more likely to be the seat of metastasis from an occult primary elsewhere that is caused by HPV. A search for occult tumors, for example in the endocervix, and testing for HPV in PMCN will help to confirm or refute an etiologic link of oncogenic HPV and PMCN. On June 8, 2006, the U.S. Food and Drug Administration licenses the recombinant HPV vaccine Gardasil® as a vaccine for the prevention of cervical cancer and other diseases in females caused by HPV. The vaccine was developed against HPV types 6, 11, 16 and 18. Clinical trials have demonstrated near 100% efficacy in protection against precancerous lesions caused by types 16 and 18, which are oncogenic. If PMCN is indeed caused by high-risk HPVs, vaccination against these epitheliotropic viruses might also prevent these tumors.In summary, employing different PCR protocols, we consistently detected human papillomavirus, typed as the oncogenic HPV16, in a pancreatic mucinous cystic neoplasm. Further studies of these tumors are warranted.


Background. A significant number of non-small-cell lung cancers (NSCLC) have human papillomavirus (HPV) DNA integrated in their genome. This study sought to further establish HPV's possible etiologic link to NSCLC by evaluating an immune response to HPV's oncogene, E7, in patients with NSCLC. Patients and Methods. Antibodies (IgG) in serum against E7 for HPV 16 and 18 in 100 patients with NSCLC were examined by enzyme-linked immunosorbent assay (ELISA). Results. Sixteen NSCLC patients were found to have a high titration of IgG for HPV oncogenic E7 protein. 23.5% of adenocarcinomas  and 15.4% of squamous cell carcinomas (SCC) were positive for IgG against HPV E7. HPV-18 (11%) had a slightly higher frequency than HPV-16 (6%). Of the six positive cases for HPV-16, 3 were AC, 2 SCC, and 1 NOS (not otherwise specified). For the 11 HPV-18 positives, 7 were AC, and 4 SCC. The one case with IgG against HPV 16 and 18 was AC. One case had high cross-reactive levels against E7 of HPV 16 and 18. Two (28%) of 7 patients who reported never smoking were positive for HPV, and 12 (13.6%) of 88 smokers were HPV positive. Conclusions. The study detected high levels of IgG against E7 in 16% of NSCLC patients. This adds evidence to a potential role of HPV in the pathogenesis of NSCLC. Lung cancer remains the leading cause of cancer-related deaths worldwide, causing over a million deaths per year worldwide, with an estimated 221,130 new cases and 156,940 deaths in 2011 in the United States [The genome of HPV is a circular double-stranded DNA molecule of approximately 8000 bp. The open reading frames (ORF) include E1, E2, E4, E5, E6, and E7, which are expressed at different stages during epithelial differentiation. The high-risk HPV E6 and E7 ORF encode the viral oncoproteins that are invariably expressed in HPV-positive human cancers. E6 and E7 joint expression is necessary and sufficient for the immortalization of primary human keratinocytes in vitro . PreviouE6 and E7 oncoprotein activity also explains the difference in potential carcinogenesis after infection with high-risk HPV versus low-risk HPV . Muller In cervical cancer patients that Ghim and Jenson have followed with this serological test, a predominant IgG response against E7 was associated with periods of tumor stability (dormancy), especially when the cancer was confined to the lymph nodes. An IgG response was usually associated with various rates of progression of tumors, particularly in extranodal sites . A recent study using PCR confirmed the presence of 16.7 percent HPV DNA integrated in the DNA of NSCLC . ConsideThe study utilized 100 unidentified serum samples of NSCLC patients and controls obtained from the Cancer Database and Specimen Repository (CDSR) at the James Graham Brown Cancer Center. Sera from HPV-16 and -18 positive cervical cancer patients were collected as the controls at 4 and 6 different consecutive phases of clinical progression, respectively. However, no detailed clinical histories were available. The sera were used to measure the titration of total IgG against E7 proteins of HPV-16 and -18 by ELISA using recombinant E7 proteins as antigen. E. coli and purified as recommended by the manufacturers . The expression and quality of these recombinant proteins were examined by SDS-PAGE, immunoblot, and ELISA using standardized molecular biological techniques. The concentration of produced proteins was determined by protein titration using Bio-Rad Protein assay (BioRad). Previously, we successfully generated E7 fusion proteins of HPV16 and 18 using molecular biological techniques. They were fused to either maltose binding protein (MBP) fusion or histidine. E7 genes of HPV16 or 18 were cloned into prokaryotic expression vectors, pMalC2 (New England BioLabs) or pQE30 vector (Qiagen) by PCR cloning technology. The E7 proteins were expressed in μL bicarbonate buffer (pH 9.6)  was coated onto ELISA microplates  for 1 hr at 37°C. After three washings with 200 μL of PBS, 200 mL PBS containing 5% nonfat skimmed milk (5% PBS-SM) was added for 1 hr at 37°C. Then sera diluted to 1/100 in PBS containing 1% bovine serum albumin (1% PBSA) was added for 1 hr at RT followed by the alkaline-phosphatase-(AP-) conjugated goat anti-IgG (H&L chains) of human at a 1/1000 dilution in 1% PBSA for an incubation of 1 hr at 37°C. The reaction was revealed with AP-chromogenic substrate . The adsorption was measured at 405 nm. Purified MBP proteins were used as control negative. Positive control human sera, which had been previously tested as positive for these HPV oncoproteins, were employed for each experiment. Direct ELISA method was used to measure IgG titration against E7 of HPV16 and 18. Briefly, approximately 100 ng protein/well in 50 According to the relationship of anti-E7 IgG levels of HPV16 and HPV18, the cut-off points for the positives of HPV16 and/or 18 were selected at the slope changes using the nonlinear least square method. Based on the distribution of ELISA values, the cut-off values of E7 IgG were found at 0.59 (0.045 standard deviation) and 0.48 (0.036 standard deviation) in HPV-16 and -18, respectively. The cut-off points were used to group the patients into the positive and the negative based on whether their immune responses are beyond the cut-off points or not. The samples that showed higher values for both HPV-16 and 18 E7 proteins were selected, and the sample, which showed same distance to both cut-off lines, was decided as the case of double infections of HPV-16 and 18. Statistically significant association of the HPV positive patients with gender, disease stages, smoking status, and age were evaluated by the binary logistic regression method. As shown on The control sera from HPV-16 and -18 positive cervical cancer patients were tested to see if the IgG response to the E7 reflects the prognosis of the cancer. Both sera showed low IgG titrations in the early phase of the illness but showed instant and high immune responses to the oncoprotein in the course of the disease. The titrations increased over time with disease progression except during a short period within the HPV-18 positive group . BecauseWith the ELISA values of lung cancer patients, the cut-off values of the E7 at 0.59 with 0.045 standard deviations and 0.48 with 0.036 standard deviations in HPV-16 and -18, respectively, were obtained in the statistical distribution . At the 23.5% and 15.4% of AC and SCC were positive for HPV E7, respectively . HPV-18 Twenty-three of the patients had a previous history of at least one other cancer (HPV status at time of recent lung cancer diagnosis in parentheses): uterine (HPV16), rectal, two NSCLC, three bladder (one HPV16), three cervical, laryngeal (HPV16), endometrial (HPV 16) palate, basal cell (HPV16), skin, trigone molar, renal cell (HPV16), prostate (HPV18), tongue (HPV 16/18), melanoma (HPV16), vulva, CLL, and three breast cancers. The seropositivity against E7-IgG was not correlated with other HPV associated cancers.P values of 0.02 and 0.08. However, the other did not show any highly statistically significant association at under α = 0.05 with the sample group. Using binary logistic regression any association of HPV infection with gender, disease stages, smoking status, and age of lung cancer patients was examined. The age of the patients showed significant effect on the anti-E7 levels . Both, HThis study detected IgG against E7 for high risk HPV (type 16 and 18) in 16% of patients with NSCLC. The presence of antibodies against E7 for HPV 16/18 complements previous studies detecting DNA of HPV within NSCLC's genome. The present study found that the age of the patients had a significant effect on the anti-E7 levels. The reason for this is not obvious, but may be due to reinfection or prolonged exposure to the HPV. Never smoking did not correlate clearly statistically with the presence of IgG directed towards HPV-E7, although the number of never smokers was low in this study. IgG against E7 was detected in squamous and nonsquamous histology of NSCLC, which complements the fact that HPV DNA is found in the DNA of both histologies , 10. PatThe presence of HPV DNA alone is not enough to support an active role of the virus in the development of lung cancer, but synthesis of E6 and E7 oncoproteins is a mandatory condition for cell transformation and development of cancer. The early oncoprotein E6 binds and degrades the p53 protein, while the E7 protein binds and inactivates the protein Rb, resulting in abnormal cell proliferation and tumor growth , 12. The prevalence of high IgG levels against E7 in lung cancer patients, as well as the fact that the titers increased with age and during the course of the disease, may point to a role of HPV in the pathogenesis of this epidemic disease, that affects also a significant number of never smokers.In conclusion, the immune response to HPV's oncogene E7 found in NSCLC patients adds to the evidence linking HPV to NSCLC carcinogenesis. In the last years HPV vaccination has been implemented as public policy. The use of these vaccines may also have impact on the incidence of lung cancer. Further studies should be pursued in the future to effects of HPV and vaccines against HPV on NSCLC.


Vaccine implementation for vulnerable girls and women faces multiple barriers that include high vaccine costs, inadequate delivery infrastructure, and lack of community engagement to generate awareness about cervical cancer and early screening tools. For Human Papillomavirus vaccines to work as a public health solution, the quality-assured delivery of cheaper vaccines must be integrated with strengthened capacity for community-based health education and screening.Human Papillomavirus vaccines are widely hailed as a sweeping pharmaceutical innovation for the universal benefit of all women. The implementation of the vaccines, however, is far from universal or equitable. Socio-economically marginalized women in emerging and developing, and many advanced economies alike, suffer a disproportionately large burden of cervical cancer. Despite the marketing of Human Papillomavirus vaccines as  On occasion, the scientific literature tends to equate HPV immunization with the 'end of cervical cancer' [HPV vaccines are marketed as a women's right, choice and duty for health security, exemplified in the Gardasil 'I choose' commercials ,66. Soci cancer' ,70.Pharmaceutical advertising of the so-called 'cervical cancer vaccine' resonates with an operative dilemma for public health 'social marketing': Is the acceptability of the vaccine improved by presenting it as 'anti-cancer', instead of 'anti-STI' ? On the The high cost of the vaccine has compelled public payers to ration its subsidization to unexposed adolescent females with the expectation that high uptake will build herd immunity. This has resulted in HPV, HPV-related disease, and HPV vaccine being marked as 'women-only,' despite the fact that men also get and transmit the virus. The prescription of 'HPV immunization for girls' to ensure 'herd immunity' suggests that HPV is an exclusively heterosexual concern. HPV also affects lesbian, gay, bisexual and transgender (LGBT) people. HPV vaccine commercials and public health messages focus on cervical cancer, to the exclusion of throat, anal and penile cancer . DespiteFocus on school-based immunization of preadolescent girls removes attention from the risk faced by older women from invasive disease due to persistent HPV infection . In CanaIt will take at least 20 years of targeted immunization of adolescent cohorts before there is a measurable reduction in cervical cancer incidence . In the The clinical efficacy of both HBV and HPV vaccines is in their ability to block the acquisition of viruses that are transmitted through non-casual intimate physical contact  ,68,86. RAt the time of introduction, both vaccines have raised concerns about cost, cost-effectiveness, public acceptability, and priority for public health ,87. A coGAVI may be able to subsidize HPV vaccines for delivery in the poorest countries, provided vaccine manufacturers sell the vaccine to GAVI at lower prices . This prIn the US, HBV vaccine mandates have been passed, delivering an otherwise expensive vaccine to children prior to their school entry. A comparison of the differing histories of HBV and HPV vaccines in the US illustrates the importance of institutional credibility, dialogue, and regulatory decisions related to licensing and implementation.It took seven years for the HPV vaccine to go from FDA approval in 1986, to the US Advisory Committee on Immunisation Practices (ACIP) recommendations in 1991, to mandated requirement for day care attendance, in 1993. This time, along with its cost-reducing inclusion in the Vaccines for Children (VFC) program in 1994, provided the opportunity for the public to become aware of HBV immunization and accept its mandate . In contImmunization against HPV, unlike that against HBV, specifically and conspicuously targets female adolescents . SociallThe successful development and market authorization of vaccines do not in themselves realize the public health purposes of immunization. A vaccine, no matter how innovative and efficacious, serves public health only if it is a safe, affordable, accessible and cost-effective intervention. There are multiple barriers to making HPV vaccines available for use in medically-underserved developing, economies. In general, the delivery of vaccines in developing countries faces a wide range of socio-technical challenges that include lack of institutional capacity for monitoring of pre-market vaccine quality and safety and post-market surveillance for adverse events following immunization (AEFI), cold chain difficulties in vaccine delivery to populations in remote rural areas, risk of infection during needle-administration, and fear of needles . With HP®) also remains contentious. Despite clinical data that supports targeting unexposed recipients, parental beliefs about the appropriateness of pre-adolescent immunization limit the uptake of HPV vaccines among pre-teens. Vaccine decisions are made not only by designated policy-makers but also by the public, whose knowledge, attitudes and beliefs (KAB) are key factors in vaccine acceptance. Consistently, across a wide range of countries and ethnicities, KAB studies report socio-cultural barriers to HPV vaccines. These include lack of awareness about HPV effects and protections, parental and physician unwillingness to vaccinate or to discuss sex and STI with preadolescent females, concerns about vaccine costs and uncertain duration of effectiveness, fear of unethical motives and practices in vaccine trials, fear of needle pain, adverse effects , socially unacceptable vaccine ingredients , stigma and sexual disinhibition [As discussed, the availability of HPV vaccines in developing countries is particularly hampered by high vaccine costs, bottlenecks of technology transfer for developing cheaper subsequent entry vaccines, low acceptability of vaccines against STI for pre-adolescents, and the problems of integrating HPV vaccines into the formularies of country EPIs -21. Theshibition -110.Uncertainties and fears associated with unknown substances in the vaccine can discourage some. Rumors of adverse (or rumoured deliberate) effects, such as sterilization, led to the failure of polio vaccine initiatives in northern Nigeria . A 3-dosEven if inexpensive HPV vaccines could be developed, manufactured, subsidized and supplied with quality assurance, parents may not always accept immunization of their pre-adolescent children for an STI . ChallenThe integration of screening and immunization into public health education is critical to efforts to control cervical cancer . The eviInternationally, the implementation of HPV immunization has had a variety of outcomes, associated with a constellation of factors affecting health practicalities, policies and priorities.Australia, Canada and the UK have publicly funded programs that provide HPV immunization for peri-adolescent girls through school based and catch-up programs ,118,119.In the US, HPV immunization is available outside the school system. Uninsured adolescents in the age-eligible cohort can obtain the vaccine free of cost through the VFC program . For othIn developing countries, HPV vaccines are not offered as part of national immunization schedules. Immunization costs are not covered and vaccine uptake may be determined by the market (and marketing). Although school-based delivery platforms have a good performance record and are relatively equitable, they may have less success in many developing countries. Even where schools are available, young girls are often unable to attend due to obligations both in and outside the home. These programs also bypass street-dwelling youth in developing and advanced economies . Catch-uAlthough HPV vaccines represent a promising biological innovation, they will yield the greatest public health benefits only when introduced at affordable prices into a context of robust healthcare infrastructure. Determining which vaccines to include in a public health program requires capacity-intensive infrastructure for the regulation, production, and post-market surveillance of vaccines . In the HPV vaccine remains unavailable to poor and vulnerable women. Those most able to access HPV immunization may be also those more able to access cervical screening . The resACIP: Advisory Committee on Immunization Practices; ACOG: American Congress of Obstetricians and Gynecologists; AEFI: Adverse Events Following Immunization; ASCUS: Atypical Squamous Cells of Undisclosed Significance; EPI: Expanded Program in Immunization; EU: European Union; FDA: Food and Drugs Administration; GAVI: Global Alliance for Vaccines Initiative; HBV: Hepatitis B Virus; HIV: Human Immunodeficiency Virus; HPV: Human Papillomavirus; HSV: Herpes Simplex Virus; KAB: Knowledge, Attitudes and Behavior; LBC: Liquid-Based Cytology; LGBT: Lesbian, Gay, Bisexual and Transgender; STI: Sexually transmitted infections; VIA: Visual Inspection with Acetic Acid; VILI: Visual Inspection with Lugol's Iodine; WHO: World Health Organization.The authors declare that they have no competing interests.AM and JG conceptualized and designed the study. AM selected the literature and drafted the manuscript. JG revised the manuscript. AM and JG read and approved the final manuscript.JG is Professor and Canada Research Chair in Bioethics, and Professor of Pediatrics (Division of Infectious Diseases) in the Faculty of Medicine at Dalhousie University. She is a medical anthropologist who draws upon epidemiology and technology assessment to study cultural, technical and moral issues related to health and the regulation of biotechnologies. She currently studies new and emerging vaccines, including vaccines against HPV, pandemic influenza, and the conjugate meningitis A vaccine for sub-Saharan Africa.AM is a sociologist and a Postdoctoral Research Fellow at the Technoscience and Regulation Research Unit, Faculty of Medicine, Dalhousie University, Canada. Her current project 'The Administration of a Quadrivalent HPV Vaccine for Minors' is funded by the Canadian Institutes of Health Research (CIHR) and examines knowledge, attitudes and practices in HPV immunization in Nova Scotia, Canada.Appendix. The data file is a word file containing an explanatory appendix with a statement regarding the use of a proprietary term.Click here for file


Human papillomavirus (HPV) has been associated with several disorders of the genital tract, skin and oropharynx. The aims of our study were to evaluate the prevalence of HPV infection in women between 15 and 54 years of age in North Sardinia, Italy, to identify the prevalence of High Risk - Human papillomaviruses (HR-HPV) genotypes and to establish a correlation between molecular and cytological results.From 2007 to 2009 we consecutively enrolled women aged 15-54 years admitted to public and private outpatient settings. All the participants filled in a questionnaire about the socio-cultural state, sexual activity and awareness about HPV. 323 cervical specimens were tested for HPV-DNA and HPV genotypes with INNO-LiPA HPV Genotyping CE Amp kit. Samples showing positivity to some HPV genotypes were re-tested using "in house" quantitative Real-Time PCR assays.Overall HPV-DNA positivity was detected in 35.9% of the women. The prevalence of HR-HPV infection among HPV positive samples was 93.1% with a specific prevalence of HPV 16, 51, 31, 53 and 18 of 54.3%, 37.9%, 10.3%, 6.9% and 5.2%, respectively. Co-infection with any HPV, HR-HPV, LR-HPV and HR/LR-HPV type was 18.3%, 14.9%, 0.9% and 2.5%, respectively; HPV 16/51 co-infection was detected in 64.6% of the HR-HPV co-infection group. The most frequent HPV-genotypes detected were 16 (32.5%) and 51 (22.7%). Among the 57 patients harboring mono-infection the most prevalent HPV genotypes were 16 (38.6%) and 31(10.5%). A multivariate analysis identified a statistical significant association between HPV infection and age and between HPV infection and previous sexual transmitted diseases. A statistically significant association between cytological cervical lesions and generic HPV exposure was identified.To our knowledge, this is the first survey evaluating the prevalence of HPV infection in Northern Sardinia and drawing attention to the unusual high proportion of genotype HPV 51. Given the recent implementation of a widespread immunization program with vaccines not containing HPV 51, it has been relevant to prove the high prevalence of this HPV genotype from the start of the vaccination campaign, in order to avoid in the future attributing to the vaccination program a possible selection effect (HPV replacement). IThe aims of our study were to evaluate the prevalence of HPV infection in women between 15 and 54 years of age in North Sardinia, Italy, to identify the prevalence of HR-HPV genotypes and to establish a correlation between molecular and cytological results.In Sardinia, Italy, the average coverage of PAP-test in women aged 25-64 years is 49.3%, of which 24.9% on voluntary basis ,14. DuriPersonal data have been treated in compliance with the Law Decree No. 196/2003, article 24 . This study, for which a written consent from each enrolled patient or her relatives was obtained, was formerly approved by the Ethical Committee of the Azienda Sanitaria Locale n°1 of Sassari (PN-132) on June 18, 2007.A power of 80% and a level of statistical significance of 5% was necessary to determine the sample size in order to detect a difference between proportions: on the basis of the female population aged 15-54 in Sassari in 2006 , the eligible  population was 9,301 women. Since the prevalences of HPV-positivity and of HPV-16, estimated in an Italian city, , the ovDifference between proportions were compared via z-test. Categorical variables were compared by Chi-squared test and continuous variables by t-test or Mann-Whitney test. Logistic regression analysis of the association between HPV infection in cervical lesions with potential covariates was performed.A p-value of ≤0.05 was considered statistically significant. Data were collected on standardised e-forms and analysed using Stata 9.0 .Cervical specimens were collected with Cervex-brush and suspended in a 20 ml preservation solution, PreservCyt transport medium .The DNA extraction was performed following the protocol described in the High Pure Viral Nucleic Acid kit from Roche Diagnostics. HPV DNA amplification was carried out with INNO-LiPA HPV Genotyping CE Amp kit , which tThe sample mixture is heated in order to separate the two strands of the DNA helix and expose the target sequences to the primers. After 40 cycles, a multi-amplified biotinylated target sequence is obtained.HPV genotyping was performed using reverse hybridization by the INNO-line probe assay (INNO-LiPA HPV Genotyping Extra). The diagnostic test contains probes for 28 anogenital HPV genotypes . The hybridization steps were carried out following the INNO-LiPA kit instruction. The oligonucleotide probes, together with 3 controls, were immobilized in parallel lines on nitrocellulose membrane strips by the supplier. After hybridization of the PCR product to the probes on the strip under stringent conditions, followed by stringent washing, the hybrids were detected by alkaline phosphatase/streptavidin conjugate; reaction with a substrate (5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium) results in a purple precipitate at the positive probe lines. After drying, the strips were interpreted visually by using the INNO-LiPA HPV genotyping v2 interpretation chart.4 cells.Samples found to be positive for HR-HPV genotypes 16, 18, 31, 45, 51 and 52 by INNO-LiPA were further confirmed by the previously described "in-house" Real-Time quantitative TaqMan PCR assays -21. AmplThe characteristics of the population studied are described in table More than half of the enrolled women (57.9%) were married (165 currently married while 22 in the recent past), 50% were smokers and/or ex-smokers, 55.0% (177/323) were nulliparous, most of them taking oral contraceptives. Mean (SD) age of first sexual intercourse was 18.7 (3.3) years.Number of lifetime sexual partners was at least 2 in more than 50%: 37.6% (121/323) and 19.3% (62/323) had had 2-3 and 4-9 sexual partners, respectively; 10.5% (34/323) had had a new sexual partner in the last three months.Twenty-eight percent (89/318) of the enrolled individuals stated that they had never used condom during their sexual relationships; 17.8% (56/315) had been affected by sexually transmitted diseases: about half of them being infected by HR-HPV genotypes  versus about one-third of those without a history of sexually transmitted diseases .The proportion of women , who had had a Pap test in the last three years, was significantly higher than the national average (39.8%) -17.Table From the 116 HPV positive specimens, 194 HPV genotypes were detected , 51 , 6 , 31  and 53 .Out of 323 women, 108 (33.4%) were positive for at least one HR-HPV genotype. The prevalence of HR-HPV infection among the HPV positive samples was 93.1% (108/116) with a specific prevalence of HPV 16, 51, 31, 53 and 18 of 54.3% (63/116), 37.9% (44/116), 10.3% (12/116), 6.9% (8/116) and 5.2% (6/116), respectively. The most frequent LR-HPV genotypes among the HPV positive samples were type 6  and type 11 .i.e., HPV16 and 51) was 19.8% and 13.8%, respectively. The observed proportion of HP16/51 co-infection was 9.7%, whereas the expected one was 2.7% (p < 0.001). No HPV16/18 co-infection was detected.Co-infection with any HPV, HR-HPV, LR-HPV and HR/LR-HPV type was detected in 59/323 (18.3%), 48/323 (14.9%), 3/323 (0.9%) and 8/323 (2.5%), respectively; HPV 16/51 co-infection was detected in 64.6% of the HR-HPV co-infection group. Overall, the prevalence of the two most frequent types , 31 (10.5%), 53 (8.8%) and 51 (7.0%).4 cells with median values for the two most prevalent HPV genotypes 16 and 51 being 962 and 2055 copies/104 cells respectively.The specificity of INNO-LiPA positive samples for HR-HPV genotypes 16, 18, 31, 45, 51 and 52 was further confirmed by previously described "in-house" Real-Time quantitative TaqMan PCR assays -21. All Out of 303 women with cytological assessment, 242 (79.9%) showed negative cytology while 32 (10.6%), 25 (8.3%) and 4 (1.3%) showed a pattern of atypical cells of undetermined significance (ASCUS), a low-grade of squamous intraepithelial lesion (LSIL) and a high-grade of squamous intraepithelial lesion (HSIL), respectively , number of sexual partners , number of sexual partners in the last three months , age  and onset of sexual activity , whereas, the multivariate analysis identified a statistical significance only for age and previous sexually transmitted diseases.Although a statistically significant association between cytological cervical lesions and generic HPV exposure  was identified, this was not confirmed for infections due to HPV 16, 18 and 51.Our results highlight that the prevalence of HPV cervical infection in Northern Sardinia 35.9%) is almost similar to that estimated in other Italian geographical areas  % is almo, but higOne of the striking findings is the high prevalence of HPV 16/51 coinfections; our study was aimed at studying HPV infection's prevalence, although it would have been interesting to follow-up co-infected individuals in order to assess the association between cervical neoplasm and multiple infections.A high frequency of HPV-16 infection, followed by HPV-51 infection, was observed in our survey, whereas the prevalence of HPV-18 infection is significantly lower than that identified in the Southern Sardinian areas .It is remarkable the observed high relative frequency of HPV-51 infection together with other HR- and LR-HPV types: this could be an epidemiological feature of Northern Sardinia, confirming data described in several recent Italian cross-sectional studies (12% -15.3%) where it is frequently detected with other HR-HPV types and in patients affected by CIN 2 .Logistic regression analysis showed a statistically significant association between cytological cervical lesions and HPV infection , but such association was not confirmed with HPV 16, 18 and 51 infections, probably due to the low statistical power of the study, that was set up using the estimated prevalence of HPV-16 infection.Several variables have been found to be significant associated with HPV infection both positively  and negatively , confirming the validity of the immunization strategy, mainly aimed at girls aged 12 years, before starting of sexual activity.The successful nationwide implementation of the HPV vaccination program in Italy will probably change the frequency of the most prevalent genotypes, decreasing the prevalence of the current genotypes and increasing the possibility of HPV replacement following vaccination, as already described for several bacterial infections . Even ifTo our knowledge, this is the first survey addressing the prevalence of HPV infection in Northern Sardinian population and it draws attention to the unusual high prevalence of genotype HPV 51 in this territory. Even if some studies in other regions have already described a high frequency of this HR HPV type, HPV 51 continues to remain a rare genotype almost everywhere. Given the recent implementation in Italy of a widespread vaccination program with prophylactic HPV vaccines not containing HPV 51, it is important to emphasize the high prevalence of this "atypical" HPV genotype from the start of the vaccination campaign, in order to avoid attributing to the vaccination program in the future a possible selection effect with consequent HPV replacement. Therefore, our results confirm the need of pre and post-vaccine extensive surveillance in order to better understand the effectiveness of vaccination.However, the precision of our study, as displayed by the wide confidence intervals, may be limited by the small sample size. Therefore, for some low prevalent genotypes the statistical power was low and the possibility that the true prevalence may be different than the observed one cannot be excluded. Moreover, we know that the results of the study may be limited because of the characteristics of the screening adopted in Northern Sardinia so far. In fact, a voluntary based access instead of a systematic one may have selected those patients with higher prevalence of symptoms and it may explain the higher HPV prevalence observed. However, the potential selection bias might be not so influent, since no significant differences between positive and negative women were detected in terms of compliance to the PAP test in the last 3 years, as reported in Tab. In conclusion, this study has confirmed the high prevalence of HPV infection in our region, as previously reported for Southern Sardinia, moreover highlighted the possible role of uncommon genotypes such as HPV-51.HPVs: Human papillomaviruses; HR-HPV: High Risk - Human papillomaviruses; LR-HPV: Low Risk - Human papillomaviruses; dNTPs: Deoxynucleotide triphosphates; ASCUS: Atypical Cells of Undetermined Significance; LSIL: Low-grade of Squamous Intraepithelial Lesion; HSIL: High-grade of Squamous Intraepithelial Lesion; OR: Odds ratio; CI: Confidence Intervals; CIN: Cervical Intraepitelial Neoplasia; PAP test: Papanicolau testThe authors declares that he has no competing interests.All of the authors participated in planning and design of the study, and all read and approved the manuscript. AP, SP and CC participated to define protocols for molecular analysis. SD and GC selected the patients; VM carried out the histological examination. GS and PC performed the statistical analysis.AP, GS, PC and EM conceived the study, participated in its design and wrote the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/785/prepub


Pathological brain states that result in [K+]o and pH dysregulation have been shown to cause astrocyte swelling. However, whether astrocyte volume changes occur under physiological conditions is not known. In this study we used two-photon imaging to visualize real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were observed to swell by 19.0±0.9% in response to a small physiological increase in the concentration of [K+]o (3 mM). Astrocyte swelling was mediated by the influx of bicarbonate (HCO3−) ions as swelling was significantly decreased when the influx of HCO3− was reduced. We found: 1) in HCO3− free extracellular solution astrocytes swelled by 5.4±0.7%, 2) when the activity of the sodium-bicarbonate cotransporter (NBC) was blocked the astrocytes swelled by 8.3±0.7%, and 3) in the presence of an extracellular carbonic anhydrase (CA) inhibitor astrocytes swelled by 11.4±0.6%. Because a significant HCO3− efflux is known to occur through the γ-amino-butyric acid (GABA) channel, we performed a series of experiments to determine if astrocytes were capable of HCO3− mediated volume shrinkage with GABA channel activation. Astrocytes were found to shrink −7.7±0.5% of control in response to the GABAA channel agonist muscimol. Astrocyte shrinkage from GABAA channel activation was significantly decreased to −5.0±0.6% of control in the presence of the membrane-permeant CA inhibitor acetazolamide (ACTZ). These dynamic astrocyte volume changes may represent a previously unappreciated yet fundamental mechanism by which astrocytes regulate physiological brain functioning.Astrocytes, the major type of non-neuronal cells in the brain, play an important functional role in extracellular potassium ([K Astrocytes play an essential role in maintaining optimal physiological brain functioning by tightly regulating the concentrations of K+ and pH in the ECS, as alterations can affect a wide range of cellular processes that include neuronal excitability, synaptic transmission, metabolism, and the activity of many enzymes, channels, and transporters + buffering +, well outside physiological range. As a result, the currently available literature reflects only what might be occurring during pathological states Neuronal firing of action potentials leads to the release of potassium ions , and can be blocked with the removal of extracellular HCO3−, or by inhibition of the main astrocyte HCO3− transporter, the sodium-bicarbonate cotransporter (NBC) Under physiological conditions even moderate amounts of neuronal activity can lead to a build up of extracellular K+]o, and to shrink in response to GABAA receptor activation. Both the astrocyte swelling and shrinking were mediated by the flux of HCO3−. These dynamic astrocyte volume changes may represent a previously unappreciated yet fundamental mechanism by which astrocytes regulate physiological brain functioning.In the present study we performed two-photon imaging in the brain slice preparation to directly examine real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were found to undergo robust swelling in response to a physiological increase in o from an initial control ACSF containing 2.5 mM [K+]o. +]o ACSF (A green) and after maximal swelling has occurred with the 3 mM increase of [K+]o (B red). The overlayed image  = 22.094, p<0.05] -1-piperazineethanesulfonic acid (HEPES) buffered ACSF. HCO3− free ACSF significantly decreased the +3 mM [K+]o –induced astrocyte swelling compared to the swelling observed under HCO3− buffered ACSF, conditions; 5.4±0.7%, n = 31 astrocytes from 7 slices, p<0.05 (3− was reduced using a membrane impermeant extracellular carbonic anhydrase (CA) inhibitor to decrease the production of extracellular HCO3−. The membrane impermeant CA inhibitor 1-(4-sulfamoylphenylethyl)-2,4,6-trimethyl-pyridinium perchlorate (STPP) (100 µM) was used to selectively inhibit the formation HCO3− in the extracellular solution. +3 mM [K+]o –induced astrocyte swelling was significantly reduced in the presence of STPP; 11.4±0.6%, n = 50 cells from 7 slices, p<0.05 (3− influx through the sodium-bicarbonate cotransporter (NBC) cotransporter. Inhibition of NBC activity with the NBC inhibitor DIDS (500 µM) significantly decreased the +3 mM [K+]o –induced astrocyte swelling; 8.3± 0.7%, n = 40 astrocyte from 6 slices, p<0.05  was found to cause significant astrocyte shrinking; −7.7± 0.5% of control, n = 42 astrocyte from 8 slices, p<0.05)  (100 µM) was used to block the formation of intracellular HCO3− during muscimol applications. ACTZ significantly decreased the amount of GABAA channel mediated astrocyte shrinkage by muscimol; −5.0±0.6% of control, n = 34 astrocytes from 7 slices, p<0.05 (+]o for 40 minutes and astrocytes that were incubated with ACSF containing ACTZ . A one-way ANOVA confirmed significance between the control, muscimol, and muscimol with ACTZ groups  = 46.28, p<0.05).The selective GABA p<0.05) . To dete, p<0.05 . A Stude+]o, a concentration that is well within physiological range, and to shrink in response to GABAA receptor activation. These dynamic volume changes were regulated in a HCO3− dependent manner.In the present study, two-photon imaging was used to directly examine real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were found to swell in response to a 3 mM increase in [K+]o caused a 19% increase and GABAA channel activation an 8% decrease for the same time period. A number of recent elegant studies have used comparable high-resolution imaging approaches to investigate dynamic astrocyte volume changes in the brain slice preparation, and thus offer the opportunity for quantitative comparisons to be made between the magnitude of the astrocyte volume changes we report here and those that resulted from pathological conditions, such as osmotic stress, ischemia, and cortical spreading depression (SD) events et al. (2010) induced spreading depression in the brain slice preparation by perfusing ACSF containing a +40 mM increase in [K+]o and found that astrocytes swelled 40% +]o, which suggests that astrocytes are not the primary contributor to SD propagation. As in the Zhou et al. (2010) study, Risher et al. (2009) found that brain slices perfused with ACSF containing high [K+]o (+26 mM) also caused a doubling in the magnitude of astrocyte swelling compared to our findings, showing that astrocytes swelled 38.9% +]o -induced astrocyte swelling was greater than the astrocyte swelling that resulted from −40mOsm osmotic challenge (17%) and oxygen/glucose deprivation (29%) et al. found that astrocytes swelled to a maximum of 40% in vivo to examine osmotic- and ischemia-induced volume changes in astrocytes. Astrocyte somas were shown to swell by 21% as a result of hypoosmotic stress induced by IP distilled water injection and 33% from induced global ischemia induced from cardiac arrest et al. (2012) showed that astrocyte somas in the metabolically compromised ischemic penumbra-like area swelled by 31% as a result from the initial induced spreading depolarizations and by 30% as a result of spreading depolarization induced by a transient bilateral common carotid artery occlusion model of global ischemia To the best of our knowledge this is the first study to report on astrocyte volume changes in the brain slice preparation that resulted from non-pathological induction methods. We were able to achieve high image resolution and stable measurements of astrocyte area during long-term imaging experiments and determined that astrocyte area changed less than 1% over 40 minutes in control ACSF, while a modest +3 mM increase in [K+]o and muscimol once applied, were perfused for the remainder of imaging experiments. While this model allowed us to very precisely quantify astrocyte volume changes and unmask a previously underestimated contribution of HCO3− flux, and certainly does comment on the astonishing propensity for astrocytes to change their volumes, it in no way predicts the likely magnitude of astrocyte volume changes that are to occur during normal physiological brain functioning. Indeed, currently available in vivo data indicates that [K+]o changes in a fluctuating manner in the ECS and that modest astrocyte volume changes may occur in this manner as well under physiological brain functioning, such as during slow (∼1 Hz) sleep oscillations The robust magnitude of the astrocyte volume changes found in the present study that occurred from the modest induction methods compared to the studies above, likely result from the experimental paradigm used here; that is, the +3 mM [K+]o that occur as a result of neural activity must be tightly regulated. Astrocytes are thought to play a key role in this regulation by taking up excess K+ from the ECS +]o astrocytes are known to rapidly undergo an intracellular alkalinization 3− in [K+]o -induced astrocyte swelling under physiological conditions was only recently speculated +]o and that the removal of HCO3− from the extracellular solution, inhibition of the extracellular CA activity, and inhibition of the NBC activity all resulted in significant decreases in the magnitude of the [K+]o -induced swelling.For normal neuronal functioning to be maintained, the local changes in o as a result of neuronal activity causes a net driving force for K+ influx into astrocytes, while at the same time causes a depolarization of the astrocyte membrane. This depolarization is believed to activate the uptake of Na+ and HCO3− through the NBC, which is highly expressed in astrocytes +, Na+, and HCO3− in the astrocyte cytoplasm would then generate an osmotic gradient and drive water influx through aquaporin 4 water channels (AQP4) that are highly expressed in astrocytes +, and the metabolic bi-product, CO2, of neuronal activity from the ECS at the same time. Recent work from the Nagelhus group suggests that activity-dependent astrocyte water uptake may be partly offset by an AQP4-mediated water efflux that may serve to moderate the activity-dependent astrocyte volume changes and offset the resulting ECS shrinkage Our findings support a mechanism for K3− from the extracellular solution, inhibition of the extracellular CA activity, and inhibition of the NBC activity all resulted in significant decreases in the magnitude of the [K+]o -induced swelling, but each of the individual conditions did not fully prevent the astrocyte swelling. A wide variety of other transporters are known to contribute to the regulation of astrocyte volume. These include the sodium-potassium chloride cotransporter (NKCC), the K+-Cl- cotransporter (KCC), the Cl–HCO3− anion exchanger (AE), the Na+ dependent Cl–HCO3− exchanger (NDCBE) and the Na+-H+ exchangers (NHE)  used moderate astrocyte swelling to test for VRAC activity under conditions resembling those to which astrocytes may be exposed to upon physiological stimulation and elegantly demonstrated Ca2+-dependent VRAC glutamate release Finally, in an attempt to counteract swelling and to recover normal volume, astrocytes initiate volume regulatory mechanisms by modifying their concentration of osmotically active solutes through a process termed regulatory volume decrease (RVD). Volume-regulated anion channels (VRACs) are activated by increased cell volume and are an important conduit through which glutamate release from astrocytes occurs during pathological brain states The results of this study demonstrate the existence of dynamic astrocyte volume changes that may be an intrinsic phenomenon that occur under physiological conditions. These volume changes may play a fundamental role in the control of extracellular brain signaling and in the regulation of synaptic plasticity.Movie S1This file contains a movie showing astrocyte swelling and recovery in a series of two-photon z-stack projection images of SR101 labeled astrocytes in the CA1 stratum radiatum region of the hippocampus. Images were acquired every 10 minutes over the course of 100 minutes. At time zero the slice was switched from 2.5 mM [K+]o ACSF to 5.5 mM [K+]o ACSF. After the 40-minute time point the perfusate was switched back to 2.5 mM [K+]o.(MOV)Click here for additional data file.Movie S2A agonist muscimol.This file contains a movie showing astrocyte shrinking from the selective GABA At time zero muscimol was added to the ACSF and images were acquired every 10 minutes for 40 minutes.(MOV)Click here for additional data file.


Men who have sex with men (MSM) are at high risk of having anal cancer. Anal high-grade squamous intraepithelial lesion (HSIL) is the precursor of anal cancer. We explored the use of different biomarkers associated with human papillomavirus (HPV) infection and HPV-mediated cell transformation to detect and predict HSIL among HIV-positive and HIV-negative MSM.A total of 123 HIV-positive and 123 HIV-negative MSM were enrolled and followed for 12 months. High-resolution anoscopy (HRA) with biopsies were performed at every visit along with anal sample collection for cytology, high-risk HPV DNA genotyping, HPV E6/E7 mRNA, and p16 immunocytochemistry. Performance characteristics and area under the receiver operator characteristics curve were calculated for these biomarkers at baseline, and Cox regression compared the usefulness of these biomarkers in predicting incident HSIL. High-risk HPV DNA, E6/E7 mRNA, and p16 immunocytochemistry each identified 43–46% of MSM whose baseline test positivity would trigger HRA referral. E6/E7 mRNA had the highest sensitivity (64.7%) and correctly classified the highest number of prevalent HSIL cases. With the exception of p16 immunochemistry, most tests showed significant increases in sensitivity but decreases specificity versus anal cytology, while the overall number of correctly classified cases was not significantly different. Baseline or persistent type 16 and/or 18 HPV DNA was the only test significantly predicting incident histologic HSIL within 12 months in models adjusted for HIV status and low-grade squamous intraepithelial lesions at baseline.Countries with a high HIV prevalence among MSM and limited HRA resources may consider using biomarkers to identify individuals at high risk of HSIL. E6/E7 mRNA had the highest sensitivity for prevalent HSIL detection regardless of HIV status, whereas type 16 and/or 18 HPV DNA performed best in predicting development of incident HSIL within 12 months. Men who have sex with men (MSM) are at high risk of having anal cancer, and HIV-positive MSM have 5 times higher risk than HIV-negative MSM Anal cytology has generally been used first in the anal squamous intraepithelial lesion (SIL) screening algorithm Similar to cervical cytology, low sensitivity and specificity for identifying those with biopsy-proven high-grade disease are commonly reported for anal cytology All participants gave written informed consent. The study was approved by the institutional review board of Chulalongkorn University in Bangkok, Thailand .Thai men aged ≥18 years with a history of anal sex with men and documented HIV status were enrolled into the study at the Thai Red Cross AIDS Research Centre. MSM who had prior treatment for anal cancer, previous anal cytology or HRA were excluded from the study. Infrared coagulation ≤12 months before enrollment, intra-anal application of trichloroacetic acid or podophyllin ≤1 month before enrollment, or evidence of current intra-anal or perianal bacterial or herpes simplex virus anal infection at enrollment, were also exclusion criteria.Participants were followed at month-12 after baseline except for the first 120 participants who were also scheduled for month-6 follow-up. Anal sample collection, HRA and HRA-guided biopsy of visible lesions were performed at all visits by the same study physician (NT).® EndoCervex-Brush®, Rovers Medical Devices B.V., The Netherlands, or FLOQSwabsTM, Copan Italia S.p.A., Italy) was used to collect anal samples. Swabs were placed into liquid-based cytology fluid  and stored at 4°C for ≤7 days before processing for anal cytology and p16 immunocytochemistry. Remaining cytology fluid was stored at −80°C until processing for HPV DNA and HPV E6/E7 mRNA. HRA was performed immediately after anal sample collection, using 5% acetic acid and Lugol's solution to aid visualization of abnormal anal tissue for biopsy.A moistened, non-lubricated swab , which amplifies target DNA within the polymorphic L1 region of the HPV genome, and subsequently hybridizes this product to probes for 37 anogenital HPV DNA genotypes , 81, 82 (MM4), 83 (MM7), 84 (MM8), IS39 and CP6108). Primers for human β-globin gene were used for quality control.HPV genotyping was done using the LINEAR ARRAYin situ hybridization for E6/E7 mRNA was performed using a cocktail of 5′- and 3′-labeled oligonucleotide probes . The kit covers the detection of E6/E7 mRNA from HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 69. Three-color flow cytometry was performed on Beckman Coulter Cytomics FC500. Cells were included in the analysis if they exhibited green fluorescence  and blue fluorescence  but lacked red fluorescence (polymorphonuclear cells). Samples with ≥2% of cells exhibiting E6/E7 mRNA were considered positive.An aliquot of liquid-based cytology fluid was used for intracellular HPV E6/E7 mRNA flow cytometry. Cell pellets were prepared, fixed and permeabilized. Fluorescence Slides were prepared for p16 immunocytochemistry only from samples obtained at the baseline visit by the Immunohistochemistry and Immunocytochemistry Laboratory, Department of Pathology, Chulalongkorn University. The monoclonal antibody p16INK4a was used as a primary reagent and staining was performed using Bench Mark XT Instrument . p16 immunocytochemistry was scored by one anatomic pathologist (SK) unaware of the clinico-pathologic diagnosis and slides were considered p16-positive if cells with cytoplasmic and/or nuclear staining were present.Statistical analysis was conducted with Stata version 12.1 . The prevalence and 12-month incidence of histologic HSIL in individuals without HSIL at baseline and 95% confidence intervals (95% CI) were calculated. The histologic diagnosis was used as the gold standard for comparing performance characteristics, and we assumed all participants with HSIL were identified.Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), negative likelihood ratio (LR), and positive LR and 95% CI were calculated for the use of anal cytology, high-risk HPV DNA, intracellular HPV E6/E7 mRNA, and p16 immunocytochemistry to detect HSIL at baseline, for MSM overall and by HIV status. Receiver operator characteristics (ROC) plots were made to demonstrate sensitivity and specificity of these tests in detecting HSIL at baseline. Combinations of anal cytology, high-risk HPV DNA and E6/E7 mRNA to detect baseline and incident HSIL were also evaluated. Baseline differences in sensitivity and specificity between biomarkers were compared in subjects with HSIL and without HSIL respectively A total of 246 MSM (123 each HIV-positive and HIV-negative) were enrolled from December 2009– December 2010. Among the first 120 MSM (91 HIV-positive and 29 HIV-negative) scheduled for month-6 follow-up, 90 MSM (73 HIV-positive and 17 HIV-negative) attended the clinic. 167 MSM (89 HIV-positive and 78 HIV-negative) completed month-12 visit.3 and 10% had baseline plasma HIV RNA <40 copies/mL. HAART use increased from 13% at baseline to 47% at month 12. Mean (SD) CD4 count at month 12 was 388 (130) cells/mm3 and 33% had plasma HIV RNA <40 copies/mL.The mean  age at enrollment was 28.8 (6.9) years for HIV-positive and 28.9 (7.4) years for HIV-negative MSM. The mean (SD) age of sexual debut was 18.0 (3.7) years for HIV-positive and 18.8 (3.7) years for HIV-negative MSM (p = 0.11). In HIV-positive MSM, the mean (SD) baseline CD4 count was 353 (146) cells/mmHRA identified anal lesions in 55% (N = 136/246) of participants at baseline, 66% (N = 61/92) at month 6 and 56% (N = 94/167) at month 12 . AbnormaBaseline prevalence of HSIL was 18.9% in HIV-positive and 8.9% in HIV-negative MSM (p = 0.03). Over the study period, 28.8% of HIV-positive and 4.1% of HIV-negative MSM with no HSIL at baseline, developed HSIL.Abnormal anal cytology defined as ASC-US or worse was found in 12.7% (11.2% of HIV-positive and 14.1% of HIV-negative) MSM at baseline . HIV-posHigh-risk HPV types were detected in 46.3% of MSM at baseline (56.1% of HIV-positive and 36.5% of HIV-negative MSM) . HPV 16 Baseline E6/E7 mRNA was positive in 45.5% of MSM (49.2% of HIV-positive and 41.8% of HIV-negative MSM) . E6/E7 mPositive p16 immunocytochemistry was found among 43.4% of MSM at baseline (41.9% of HIV-positive and 45% of HIV-negative MSM) . PositivAmong all MSM at baseline, anal cytology had a sensitivity of 18.8% and a specificity of 88.2% to detect HSIL. E6/E7 mRNA was the most sensitive test (64.7%), followed by high-risk HPV DNA (50%), p16 immunocytochemistry (31.3%), and HPV 16 and/or 18 (23.5%) . SpecifiWe developed proportional hazards models for the usefulness of baseline positive biomarkers, and persistent positive biomarkers test results at two consecutive visits, in predicting progression to HSIL within 12 months. Based on our previous work where the only subject demographic, disease related and behavioral characteristics associated with development of HSIL were HIV-status and LSIL at baseline Among MSM without HSIL at baseline, a baseline positive high-risk HPV DNA or positive type 16 and/or 18 HPV DNA were the only biomarkers associated with an increased risk of developing HSIL in the following year in univariate models. When adjusted for HIV status and baseline LSIL, only baseline positive type 16 and/or 18 HPV DNA was associated with an increased risk of developing HSIL within 12 months  .Persistent positive abnormal anal cytology, high-risk HPV DNA, type 16 and/or 18 HPV DNA, and a combination of persistent abnormal anal cytology and/or high-risk HPV DNA were predicted progression to HSIL in univariate models. In adjusted models, only persistent type 16 and/or 18 HPV DNA predicted development of HSIL within 12 months  .The high prevalence (18.9%) and 12-month incidence (28.8%) of histologic HSIL observed among HIV-positive MSM in our study were within the range reported in Western studies Performance characteristics of biomarkers associated with anal HPV infection and HPV-mediated cell transformation demonstrated that high-risk HPV DNA, E6/E7 mRNA, and p16 immunocytochemistry, when used as a primary screening test to trigger referral of MSM at high risk for HSIL to HRA, identified comparable proportions (ranging from 43–46%) of men with baseline positivity. E6/E7 mRNA had higher sensitivity, higher PPV, and higher AROC than the other tests, and identified a significantly higher proportion of subjects with HSIL versus LSIL or no SIL than anal cytology (p = 0.02). Anal cytology only identified 12.7% of MSM for HRA referral with a lower sensitivity and lower AROC than E6/E7 mRNA, although with comparable PPV. Use of E6/E7 mRNA in combination with anal cytology provided a higher sensitivity and a higher proportion of correctly classified cases than when either test was used alone, and showed a significant difference in the proportion of subjects with HSIL versus LSIL or no SIL (p = 0.004).Recent studies have compared the performances of E6/E7 mRNA and high-risk HPV DNA to detect HSIL among HIV-positive MSM. A US study demonstrated that E6/E7 mRNA provided a lower sensitivity than high-risk HPV DNA (79.8% vs. 100%) but with a higher PPV (50.9% vs. 40.3%) to detect HSIL, and a higher proportion of correctly classified cases Among those with no HSIL at baseline, baseline or persistent type 16 and/or 18 HPV DNA was the only biomarker which predicted development of HSIL within 12 months. E6/E7 mRNA and p16 immunocytochemistry, which are considered biomarkers that identify transforming HPV infections and precancer, did not show a significant association. Types of high-risk HPV covered by these tests are unlikely to explain this finding as the high-risk HPV types covered by the HPV DNA genotyping test and the E6/E7 mRNA kit used in our study are almost identical and p16 immunocytochemistry is not type-restricted. Nevertheless, because of our small number of incident cases these data should be interpreted with caution.® HPV Genotyping Test which is less readily available in most countries than the other HPV DNA tests such as Hybrid Capture® 2. The LINEAR ARRAY® HPV Genotyping Test was selected for its ability to identify each of the 37 HPV types, as one of our study cohort's objectives was to characterize HPV types distribution in the anal canal of Thai MSM. Lastly, the data presented in this study may not be applicable to other settings with different anal HPV infection and HSIL epidemics than described here.HRA performance on all study participants and rigorous anal sampling for different biomarkers by the same study physician is a major strength of our study. Nevertheless, our study has several limitations. First, while a recent study demonstrated that random biopsies during HRA significantly increased the rate of HSIL detection, only 55% of participants who underwent HRA at baseline in our study had abnormal findings which were biopsied In summary, we demonstrated that the use of E6/E7 mRNA seemed to correlate best with prevalent histologic HSIL detection both among HIV-positive and HIV-negative MSM, and a significantly higher proportion of subjects with HSIL than LSIL or no SIL showed baseline E6/E7 mRNA. For predicting development of incident histologic HSIL at 12-months, baseline and persistent type 16 and/or 18 HPV DNA were significantly better than other biomarkers studied. Countries with high prevalence of HIV infection among MSM and limited resources for HRA may benefit from using biomarkers in their HSIL screening programs for MSM. As none of the biomarkers evaluated in our study performed at high level, additional studies are in need to identify new biomarkers or combinations of biomarkers with better performances to correctly trigger referral for HRA.


HPV infection is a worldwide problem strictly linked to the development of cervical cancer. Persistence of the infection is one of the main factors responsible for the invasive progression and women diagnosed with intraepithelial squamous lesions are referred for further assessment and surgical treatments which are prone to complications. Despite this, there are several reports on the spontaneous regression of the infection.This study was carried out to evaluate the effectiveness of a long term polyhexamethylene biguanide (PHMB)-based local treatment in improving the viral clearance, reducing the time exposure to the infection and avoiding the complications associated with the invasive treatments currently available.100 women diagnosed with HPV infection were randomly assigned to receive six months of treatment with a PHMB-based gynecological solution  or to remain untreated for the same period of time.vs 70% control group) was observed at the end of the study highlighting the time-dependent ability of PHMB to interact with the infection progression.A greater number of patients, who received the treatment were cleared of the infection at the two time points of the study (three and six months) compared to that of the control group. A significant difference in the regression rate (90% Monogin group The topic treatment with PHMB is a preliminary safe and promising approach for patients with detected HPV infection increasing the chance of clearance and avoiding the use of invasive treatments when not strictly necessary.ClinicalTrials.gov Identifier NCT01571141 Human papilloma virus (HPV) is a worldwide genital infection and it is considered the most common cause of sexual transmitted infections (STIs). It is possible to count more than 100 HPV types known to infect humans and among these 13 have been classified as “high-risk” (HR) by the WHO International Agency for Research on Cancer. In particular, types 16 and 18 have been linked to more than 70% of cervical cancer .Currently, the only tool that medicine has to identify the HPV infection early is cervical cancer cytological screening and women diagnosed with high-grade intraepithelial squamous lesions (H-SIL) are referred for further assessment with colposcopy and biopsy to prevent the invasive progression.On the other hand, a follow up check is carried out on women with detected low-grade intraepithelial squamous lesions (L-SIL) due the increased risk of developing H-SIL ,4.HPV transmission depends on several factors such as the infectivity of the organisms, sexual behavior and the effectiveness of control interventions.The majority of the treatments currently available  may reduce the symptoms but are relatively ineffective in eradicating the infection and/or decreasing the likelihood of disease transmission .2) are associated with several complications including cervical stenosis, bleeding and infection. Furthermore, there are a few reports on the spontaneous regression of low- and medium-grade lesions which offer the possibility to avoid the treatment [So far, the surgical removal of the lesions followed by the local application of therapeutic agents is considered the most effective therapy, avoiding the progression to pre-cancer lesions which are more likely to occur when the infection persists over time. Despite this, all the invasive treatments currently performed -based compounds has been mainly reported for the treatment of bacterial vaginosis and wounds. PHMB is a strong base which interacts with acidic phospholipids leading to increased fluidity and permeability of the bacterial cellular membrane and consequently to the death of the pathogen. Neutral phospholipids are not affected by PHMB thus justifying the low toxicity against the human cells . FurtherIn this study, a long-term treatment with a PHMB-based gynecological solution  has been performed on patients diagnosed with HPV infection to evaluate the effectiveness in increasing the spontaneous regression rate.From January to December 2010, 100 women were enrolled at the “Consultorio Familiare Terme Vigliatore, A.S.P. 5” of Messina, Italy. Subjects aged between 30 and 45 years and diagnosed with the HPV infection after the pathologic Pap smear , a positive “high risk” HPV DNA test and a positive colposcopy examination  were included in the study. Exclusion criteria were wart therapy in the previous six months, pregnancy, invasive disease, immunosuppression and previous HPV vaccination. All the patients gave their written informed consent before entering the study which was approved by the SIFIOG  ethical committee.This hyphotesis-generating study was performed to investigate whether a long treatment with a PHMB based gynecological solution, which has been reported to have biocidal activity, may improve the regression rate of HPV infection.A pre-enrolment screening questionnaire was used to record the social and medical history of the patients and no differences were observed. Patients were randomly assigned to two groups using a computer based program.Group A consisted of 50 women who received treatment with a polyhexamethylenbiguanide (PHMB) gynecological solution  every three days for fifteen days and then every fifteen days for the subsequent six months. Monogin® is a mono-dose gynecological liquid-gel constituted by PHMB, EDTA, glycerol, hydroxymethyl cellulose, potassium chloride, lactic acid and water; the administration did not require any particular procedure and it was self-administered by the patients before bedtime, following the information reported in the leaflet.Group B, consisting of other 50 women, did not receive any treatment and was considered as control group.Evaluation of PHMB effectiveness in increasing the regression rate of the infection was performed by taking cervical scrapings for HPV analysis, Pap smears and a colposcopy examination. This procedure was performed at the enrollment and repeated after three (T1) and six months (T2), following the initial diagnosis of HPV infection.-1 threshold was adopted.HPV detection was performed using the HPV high-risk test  which detects 13 high risk oncogenic HPV types . An HPV sample was considered positive according to the Food Drug Administration (FDA) guidelines and the standard 1.0 pg HPVDNA mlCervical scrape samples were collected in ThinPrep vials containing PreservCyt  transport medium or in Specimens Transport Medium (STM)  and the results were classified following the 2001 Bethesda System .Colposcopy examination was performed following the general guidelines described elsewhere .P values less than 0.05 were considered statistically significant.To compare the regression rate of HPV infection in the two groups the Fischer’s exact test has been used and The analysis has been conducted using GraphPad Prism software .All the enrolled patients completed the study. The evaluation of Pap smear, HPV DNA test and colposcopy were repeated after three months and at the end of the study and the infection was considered cleared when all the features were found negative.vs 70% control group, P value = 0.023; RR 1.29, CI95 1.05-1.58; Table Results have shown that after three months T1) a regression rate of the infection was observed in the 66% (33/50 patients) of the patients treated with Monogin® gynaecological solution compared to the 56% (28/50 patients) of the control group  increases the regression rate of the HPV infection. In particular, the treatment already exerts beneficial effects at three months compared to an untreated control group.The presence of cervical HPV DNA is often associated with cytological and histologic changes of cervical intraepithelial neoplasia (CIN) . DespiteOn the other hand, since the time exposure with high risk HPV types is directly connected with the risk of development of cervical cancer, the development of non-invasive therapeutic agents able to improve the viral clearance is necessary.Up to now, only a few therapeutic agents able to interact with the infective process are known and currently prescribed. In particular, Imiquimod (Aldara®) which entered in the market in 1997, is an immune response modifier which induces the activation of congenital and acquired T-cell immunity stimulating the production of host interferon and other endogenous cytokines with antiviral properties. Another therapy currently available is represented by interferon (IFN) which are endogenous intracellular proteins possessing not only anti-cancer, but also antiviral immunomodulating effects . NeverthPHMB is a well-known antimicrobial agent and a number of scientific evidences on its antiviral activity have also been reported. Specific activity against cell-free viruses may be attributable to interactions between PHMB and components of the virion membrane. It has been suggested that PHMB is effective against HIV-1 in-vitro since the cellular infection is mediated by components in both the cellular membrane and the viral envelope. These interactions cause a decrease of the virulence disrupting the integrity of the viral particle .Further studies confirmed the antiviral activity of biguanide-based molecules suggesting a specific mechanism that has a co-receptor dependency . AdditioMarelli et al. first investigated the clinical effectiveness of a PHMB-based cream in the treatment of genital warts and the same authors suggested that PHMB can be considered an effective therapeutic option in controlling HPV infection . Our stu2+ and Ca2+) which are necessary for the adhesive process [Additionally, several cell components such as integrin α6β1, integrin α6β4 and glycosaminoglycans (GAGs) have been proposed as primary receptors of HPV in the cellular adhesion process ,19. Both process ,21. Furt process . The syn process .In has been reported that in the in vivo infection process the first HPV binding site is represented by the basal membrane (BM) through an heparan sulphate proteoglycan (HSPG)-dependent binding mechanism. HSPG is a linear, highly charged, anionic polysaccharide which belongs to the GAG family whom interaction with macromolecules appears to be based mainly on its anionic properties . BecauseWe speculate that on the basis of combined antimicrobial, protective, immunomodulatory, anti-inflammatory and chelating effects, PHMB and EDTA interact with HPV increasing the chance to clear the infection. In our study, this regression has been reported after three months of treatment in 66% of patients compared to the 56% in the untreated control group. The difference between the two groups became significant after six months suggesting a promising effect of the treatment in the reduction of the infection persistence overtime.The spontaneous regression rate observed in the control group is in line to the data reported in several clinical studies .On the basis of these preliminary results, further studies to evaluate the effects of PHMB both on cervical and on other types of HPV infected lesions such as common warts in the skin, oral squamous cell carcinoma and verrucous carcinoma are necessary and could provide additional information on the efficacy of this molecule in reducing the viral adhesion process.In conclusion, the results obtained from this pilot study indicate that the long term local treatment with PHMB may represent a safe and effective approach for patients with low- and medium-grade lesions enhancing the regression rate of HPV infection. In particular, this study suggests that a PHMB-based therapy may represent an alternative preliminary treatment for patients with detected HPV infection, increasing the chance to clear the infection, avoiding the complications associated with the conventional invasive treatments and reducing the time exposure to HP. In order to confirm these preliminary results further studies are necessary.The authors declare that they have no competing interests.AG designed and coordinated the study, carried out the colposcopy examination, collected the data and wrote the initial draft of the manuscript. SG and GDR were responsible for the laboratory analyses, participated in the evaluation and interpretation of results and substantially contributed to the content of the manuscript. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6890/12/17/prepub


Focal epithelial hyperplasia is a rare contagious disease caused by human papilloma virus. Usually HPV involves either cutaneous or mucosal surfaces, whereas concomitant mucocutaneous involvement is extremely rare. We report such a unique case of multifocal epithelial hyperplasia involving multiple sites of oral cavity along with skin lesions in a 65-year-old female. We also discuss the probable multifactorial etiology and variable clinical presentations of the lesions, including evidence of HPV 16 expression, as detected by polymerase chain reaction. The present report illustrates the need for careful examination and prompt diagnosis of the disease, as it might be associated with high risk genotypes such as HPV 16 and 18. Human papilloma virus (HPV) infection is known to induce proliferative lesions on the skin and mucosa which may be benign or malignant. Over 120 genotypes have been identified in the HPV family, among which HPV 16 and 18 are considered to be high risk genotypes owing to their malignant potential. Benign oral lesions associated with HPV include squamous papilloma, verruca vulgaris, condyloma acuminatum, focal epithelial hyperplasia, and malignant variants like verrucous carcinoma and squamous cell carcinoma [Focal epithelial hyperplasia of the oral cavity is a benign infectious disease caused by HPV, clinically presenting as multiple, well circumscribed papules on the oral mucosa, primarily involving gingiva, buccal, or labial mucosa . The lacWe hereby report a unique case that presented clinically as multifocal epithelial hyperplasia of the oral mucosa in concurrence with the skin lesions.A 65-year-old female patient presented with complaint of sensitivity of teeth since 2 months. Her past medical, dental, and personal histories were noncontributory. On extra oral examination multiple warts were seen involving face, scalp Figures  and 2, aFocal epithelial hyperplasia of the oral cavity was first reported among Navajo Indians as multiple nodular elevations, similar to those described in South American Indians and Eskimos of Greenland and Alaska . The firOver the past few years HPV 13, 32, 1, 6, 11, and 16 were the common subtypes studied in association with the disease. Although HPV is known to be the etiological agent, possibility of genetic predisposition, nutritional deficiencies and environmental factors like poverty and lack of hygiene, role of immunosuppression have been suggested as the other risk factors , 5. In tFocal epithelial hyperplasia predominantly affects patients between the 1st and 2nd decades of life with a female predilection. The most frequent site was the lower lip . In thisThe diagnosis of Heck's disease is mainly based on the clinical picture and histopathological findings. The prominent microscopic features consist of parakeratin layering, extensive acanthosis, epithelial cells of the spinous layer with enlarged nucleus, degeneration of koilocytes, and mitosoid cells . Our casRecent advancements in molecular biology techniques for HPV testing include light microscopy; electron microscopy; nonamplified molecular techniques like in situ hybridization, southern blot, and dot blot hybridization; target amplification using PCR; signal amplified techniques like hybrid capture technology; and gene expression using DNA microarrays . In the Lesions that do not remit or cause functional and/or aesthetic problems can be removed by various modalities like surgery, cryotherapy, electrocoagulation, laser, chemical agents , or immunostimulants  . Harris The present case highlighted a rare presentation of HPV 16 infection as multifocal epithelial hyperplasia of the oral cavity with associated skin lesions. Clinicians need to be aware of such rare presentations to facilitate a prompt diagnosis and better treatment outcome, as certain strains like HPV 16 and 18 are known to have high malignant potential.