Oncoproteins from high-risk human papillomaviruses (HPV) downregulate the transcription of the class I major histocompatibility complex (MHC-I) antigen presentation apparatus in tissue culture model systems. This could allow infected or transformed cells to evade the adaptive immune response. Using data from over 800 human cervical and head & neck tumors from The Cancer Genome Atlas (TCGA), we determined the impact of HPV status on the mRNA expression of all six MHC-I heavy chain genes, and the β2 microglobulin light chain. Unexpectedly, these genes were all expressed at high levels in HPV positive (HPV+) cancers compared with normal control tissues. Indeed, many of these genes were expressed at significantly enhanced levels in HPV+ tumors. Similarly, the transcript levels of several other components of the MHC-I peptide-loading complex were also high in HPV+ cancers. The coordinated expression of high mRNA levels of the MHC-I antigen presentation apparatus could be a consequence of the higher intratumoral levels of interferon γ in HPV+ carcinomas, which correlate with signatures of increased infiltration by T- and NK-cells. These data, which were obtained from both cervical and oral tumors in large human cohorts, indicates that HPV oncoproteins do not efficiently suppress the transcription of the antigen presentation apparatus in human tumors. Anti-viral immunity is comprised of multiple levels of defenses that cooperatively block, control, and eliminate infection. Intrinsic and innate immunity function as pre-existing defenses against viral infection, and serve as the first levels of response ,2. In thDespite the effectiveness of the immune system, many viruses have acquired sophisticated methods to evade anti-viral defenses, including the adaptive CTL response. For example, several viral proteins have been identified that block MHC-I antigen presentation by downregulating the loading of antigen onto MHC-I, the expression of MHC-I itself, and the transport of MHC-I to the cell surface. Viral proteins have also been found to trigger MHC-I internalization and/or degradation . CollectHuman papillomaviruses (HPV) are small, non-enveloped, double-stranded DNA viruses that infect mucosal or cutaneous epithelia and induce cellular proliferation . HPV infHPV encodes two main oncogenes, E6 and E7 , which aMultiple studies have suggested that HPV oncoproteins contribute to the evasion of the adaptive immune response, which supports a model by which the establishment of a persistent HPV infection contributes to the ability of HPV+ cancers to evade anti-tumor CTL responses . AlthougWhile the majority of published studies using tissue culture models indicate that HPV oncoproteins such as E7 repress the expression of the antigen presentation apparatus, cognate studies on the effects of HPV in human carcinomas are less consistent. Conflicting reports suggest that HPV+ carcinomas/dysplastic lesions display increased ,36, decrIn this study, we used data from over 800 human cervical  and headhttps://gdac.broadinstitute.org/). For all of the genes, the gene level Firehose dataset was used. Normalized expression data was extracted into Microsoft Excel, and the HPV status was manually curated based on published datasets [p-values were assigned using a one-tailed non-parametric Mann–Whitney U test. Post-hoc power calculations were performed with G*Power software version 3.1.9.2 [t-test family calculations, with effect size selected as 0.8 and α = 0.05. All comparisons achieved a power value >0.8, or demonstrated significant differences, unless otherwise noted in the text.Level 3 RNA-Seq by Expectation Maximization (RSEM) normalized Illumina HiSeq RNA expression data for the TCGA head & neck cancer (HNSC) and cervical carcinoma (CESC) cohorts was downloaded from the Broad Genome Data Analysis Center’s Firehose server , and manually annotated for HPV status. This resulted in 67 HPV+ and 431 HPV− samples for the HNSC cohort, and 181 HPV+ and 10 HPV– samples for the CESC cohort. Differences in the frequencies of DNA mutations in selected genes were calculated between HPV+ and HPV− samples using the maftools package [p < 0.05) was determined using Fisher’s exact test.Somatic mutations present in selected genes of the tumor samples in the TCGA, HNSC, and CESC cohorts were downloaded as the DNA Mutation Packager Calls dataset from the Broad Genome Data Analysis Center’s Firehose server  or non-classical  genes and the invariant β2 microglobulin light chain . We analSimilarly, higher or comparable levels of expression of the non-classical genes, HLA-E, HLA-F, and HLA-G, were observed in HPV+ cancers with respect to normal control tissues in the HNSC cohort . Again, The MHC-I heavy chain folds and assembles with the β2 microglobulin light chain within the endoplasmic reticulum, in a process dependent on binding to a peptide antigen . The MHCIFN-γ, the product of the IFNG locus, can coordinately induce higher levels of transcription in many of the genes involved in antigen processing and presentation by MHC-I, and provides enhanced immune surveillance under inflammatory conditions . Given oWe first assessed the relative proportion of T- and NK-cells in these samples, which are the primary producers of IFN-γ. Similarly to what has been done by others , we analWe next looked for IFN-γ mRNA to determine whether these lymphocytes were producing this proinflammatory cytokine . AlthougPrecancerous cells undergo strong selection for properties that are advantageous for growth and survival, leading to clonal evolution and cancer formation. As such, tumor cells frequently acquire somatic mutations that activate oncogenes or inactivate growth inhibitory genes. In HPV-dependent cancers, the expression of virally-derived oncogenes should eliminate the need for somatic mutations in the key cellular regulatory pathways they target. Thus, if high-risk HPV E7 repressed MHC-I expression and function, the frequency of mutations in MHC-I that would allow tumor escape from the immune system should be reduced in HPV+ versus HPV− carcinomas.To validate this concept, we first analyzed the frequency of mutations in TP53 and CDKN2A (We next compared the frequency of somatic mutations in all of the genes encoding MHC-I and the antigen-loading complex in HPV+ samples versus HPV− samples in the TCGA HNSC and CESC cohorts (The stable surface expression of MHC-I requires loading of the heavy chain/light chain dimer with an antigenic peptide in the endoplasmic reticulum. The loading complex consists of multiple essential factors, including the peptide transporter TAP, the bridging factor tapasin, and the chaperones: calreticulin, calnexin, and ERp57. Reduced expression or mutation of any of these components can compromise MHC-I dependent antigen presentation, and allow the infected or cancerous cell to escape T-cell recognition and clearance [HPV infection and subsequent expression of the viral oncoproteins leads to a large-scale reprogramming of gene expression. Multiple studies have reported that the high-risk HPV E7 oncoprotein contributes to the evasion of the adaptive immune response; this process occurs through transcriptional downregulation of the MHC-I heavy chain genes or other components necessary for antigen loading and presentation ,25,26. TWhile cell culture models have allowed detailed studies on the mechanism of E7-mediated transcriptional downregulation of components in the MHC-I antigen presentation apparatus, far less is known about how similar these effects are to actual HPV+ tumors. In this study, our goal was to determine whether the transcriptional reduction in MHC-I components mediated by E7 is recapitulated in primary HPV+ tumors from different anatomical locations.Using data from over 800 primary human tumors, we provide evidence that HPV+ tumors from both the head & neck and cervical sites display high mRNA levels for MHC-I components, including heavy and light chains, as well as all factors required for MHC-I loading. Indeed, HPV+ head & neck carcinomas exhibit significant increases, rather than decreases, in mRNA levels for many MHC-I components compared with normal control tissue, and this trend continues in the cervical carcinoma cohort . These uAs our results specifically addressed the question of whether HPV oncoproteins repress expression of the MHC-I genes at the transcriptional level, they do not eliminate the possibility that other post-transcriptional mechanisms, such as those mediated by high-risk HPV E5, can reduce antigen presentation. However, a comprehensive analysis of MHC-I protein expression by immunohistochemical detection indicated that only a subset of about 40% of HPV+ cervical carcinomas have reduced expression of one or more components at the protein level . As onlyOur results also do not rule out the possibility that E7 could mediate a reduction in MHC-I transcription in non-cancerous or pre-cancerous lesions, as all samples in this study are carcinomas. However, a purpose for the reacquisition of expression of MHC-I during the progression of a non-neoplastic lesion to a carcinoma is difficult to reconcile with current models of tumorigenesis.Further evidence supporting our conclusion that HPV oncoproteins are unable to functionally antagonize MHC-I dependent antigen presentation in human carcinomas is provided by our comparison of somatic mutation frequencies between HPV+ and HPV− samples . The antFurthermore, HPV utilizes multiple other strategies to evade the adaptive immune response, which suggests that a simple reduction in MHC-I transcription is unlikely to be sufficient to escape surveillance by the immune system. These include the ability of E5 to block MHC-I transport to the cell surface , the preA simple mechanism to explain the lack of MHC-I repression would be a failure of these tumors to express the HPV E7 oncogene, which has been reported to be necessary and sufficient for the downregulation of transcription from at least some of these genes ,26,27,28An alternative method of activating expression of MHC-I and components of the antigen-loading complex could be via the activation of the JAK/STAT/IRF-1 signal transduction pathway by exposure to IFN-γ. However, multiple studies done in tissue culture models have shown that HPV E7 abrogates the IRF-1 response, thus inhibiting the IFN-γ induction of MHC-I expression, antigen presentation, and CTL-induced cell death ,32,33,34Our analysis of over 350 HPV+ head & neck or cervical carcinomas in comparison with over 450 HPV− cancers demonstrates subtle differences in the effect of HPV on MHC-I transcription between the two anatomical sites. Specifically, HPV does not repress the transcription of MHC-I heavy chain genes in cervical carcinomas, which exhibit high levels with respect to both HPV− carcinomas and normal control tissue. In contrast, a statistically significant but modest reduction in MHC-I heavy chain mRNA levels was observed in HPV+ vs HPV− head & neck cancers. However, transcription of these genes was nevertheless substantially elevated with respect to normal control tissues. Interestingly, our observations with the MHC-I heavy chain genes extend to many of the other genes involved in MHC-I loading. These results are in sharp contrast to much of the existing literature derived from tissue culture-based experiments, possibly because of the complex interplay between the MHC-I antigen presentation apparatus and cytokines produced by infiltrating lymphocytes present in actual tumors. This differs markedly from our previous study on the effects of HPV on numerous regulators of H3K27 methylation and the expression of multiple downstream targets of these regulators, in which we showed that tissue culture models faithfully recapitulate what is observed in human tumors .Importantly, MHC-I dependent antigen presentation is critical for CD8+ T-cell responses, which are essential for the control and clearance of virally infected or cancerous cells. This is particularly important as recent advances in cancer immunotherapy using immune checkpoint blockades rely on reactivating antigen-dependent clearance of tumor cells . Althoug


Adenocarcinoma was more frequent in RJ than in PA . The frequency of HPV 16 in PA was higher in younger women (p < 0.05). The success of a cervical cancer control program should consider HPV types, local health system organization, and sociodemographic diversity of Brazilian regions.To evaluate the impact of HPV immunization and possible changes in virus type-specific prevalence associated with cervical cancer, it is important to obtain baseline information based on socioeconomic, educational, and environmental characteristics in human populations. We describe these characteristics and the type-specific HPV distribution in 1,183 women diagnosed with cervical cancer in two Brazilian healthcare institutions located at the Southeastern (Rio de Janeiro/RJ) and the Amazonian (Belém/PA) regions. Large differences were observed between women in these regions regarding economic, educational, and reproductive characteristics. The eight most frequent HPV types found in tumor samples were the following: 16, 18, 31, 33, 35, 45, 52, and 58. Some HPV types classified as unknown or low risk were found in tumor samples with single infections, HPV 83 in RJ and HPV 11, 61, and 69 in PA. The proportion of squamous cervical cancer was lower in RJ than in PA (76.3% versus 87.3%,  Cervical cancer is the fourth most common neoplasm among women worldwide, with the majority of cases occurring in developing countries, and it is responsible for more than 260,000 yearly deaths . Human pEarly detection of precancerous lesions using the Pap test is the main population-based screening strategy. As the Pap test is only one of the steps in the screening process, this strategy requires the existence of an efficient health system and, thus, its implementation has been more successful in developed than in developing countries. More specifically, after the detection of a cytological cervical precursor lesion  or adenocarcinoma in situ (AIS)), it is necessary to confirm the diagnosis and to deliver treatment, which involves full access to different facilities of the health system . The absWith the advent of first- and second-generation type-specific HPV vaccines targeting the most prevalent carcinogenic types , a new preventive paradigm has emerged. However, the impact of this strategy on mortality rates will be observed only in the future, and its effectiveness depends on the coverage of these vaccines in the target population [In Brazil, in 2016, cervical cancer incidence rate was estimated at 15.85/100,000 women, representing the 3rd most incident cancer for this group. Incidence rates, however, vary markedly by region. Cervical cancer is the most common female cancer in the Northern (Amazonian) region, with an incidence of about 23.97/100,000 women, which is about twice as high as the incidence in the Southeastern region  [Age-adjusted cervical cancer mortality in Brazil in 2014 was 4.88/100,000 women, representing the 4th most lethal cancer. The variation in mortality for region is even more striking than that for incidence: it was 11.75/100,000 in the Northern region  and 3.47/100,000 for Southeast region (sixth cause), that is, greater than a 3-fold difference , 10. VarBrazil started to implement the Cervical Cancer Control Program in its Public Health System in 1996, based on the Pap test. This program has increased its coverage in the whole country, since 2002. In 2013, a population-wide survey showed that 79.4% of women between the ages of 25 and 64 years (target population) reported at least one Pap's smear test in the past three years . In the In the present paper we describe the sociodemographic profile and type-specific HPV distribution by histological tumor type of women diagnosed with cervical cancer in reference public hospitals in two Brazilian capitals: Rio de Janeiro city (Rio de Janeiro State), in the Southeastern region, and Belém city (Pará State), in the Northern (Amazonian) region. These institutions receive the majority of the cervical cancer cases diagnosed in their respective States. The areas have distinct socioeconomic characteristics expressed by the Human Developmental Index (HDI): 0.761 for Rio de Janeiro and 0.646 for Pará. The principal aim of this study is to provide baseline information of type-specific HPV frequencies in women with cervical cancer in these distinct scenarios, for future evaluation after the implementation of the HPV immunization program in the country.Participants were patients with newly diagnosed cervical cancer at the Brazilian National Cancer Institute  in Rio de Janeiro (RJ) and Hospital Ophir Loyola (HOL) in Belém (PA). Between August 2012 and March 2014 in INCA and from April 2013 to September 2015, all the patients who attended the hospitals for the first clinical visit were invited to participate of the study.Included in the study were women with histopathological diagnosis of cervical cancer, aged ≥ 18 years, with no previous oncological treatment for cervical cancer  and those who had performed a biopsy during this first clinical visit. Women with cognitive or physical abnormalities that prevented them from answering the questionnaire were excluded from the study.ca. 5 mm in all dimensions) during the clinical visit, using a forceps, and stored in RNA-Latter until nucleic acid isolation.A pretested questionnaire was used to collect data on socioeconomic variables, knowledge about cervical cancer prevention, access to diagnosis and treatment, hormonal and reproductive histories, and tobacco use. The criteria for definitions of sociodemographic variables in this study were those used by the Brazilian Geographic and Statistics Institute (IBGE). Information on histological diagnosis and tumor stage was collected from medical records. The sample for type identification was collected by a gynecologist . If problems were noted, the group met with the data collectors and technicians for problem solving.The Institutional Ethic Committees approved all the procedures and all patients signed an informed consent form (INCA (RJ): CEP 156/10 and CAAE: 53398416.0.0000.5274, in 02/25/2011 and, in HOL (PA): CAAE: 03288212.0.1001.0018, in 11/28/2012).β-globin, and positive reactions to β-globin and negative ones for nested PCR were considered negative for HPV. For HPV identification, PCR products were purified with Illustra GFX PCR DNA and Gel Band Purification Kit  and subjected to DNA sequencing in both directions, using the Big Dye Terminator Cycle Sequencing Ready Reaction V3.1 Kit (Life technologies®), following the supplier's instructions.DNA was isolated from biopsies using the QIAamp DNA Mini Kit (Qiagen®). HPV detection was carried out by PCR amplification using primer sets PGMY07 and PGMY09  and reacThe electropherograms of each sample were checked by eye and a consensus sequence of the bidirectional sequencing was subjected to HPV type identification using the Blast software . SamplesEpidemiological data were stored using Epi-Info software and then linked to both the clinical data and the DNA HPV data. The final database was analyzed using Stata v.12.0. Categorical variables of women's characteristics were reported as counts and percentages and compared between cities using Chi-squared or Fisher's exact tests.A total of 1,702 women (968 in RJ and 734 in PA) with cervical cancer diagnosis signed the informed consent and were interviewed, but 1,198 (70.4%) were biopsied. The reasons for not being biopsied were technical difficulties related to sample collection (271), clinical conditions of the patient (86), and refusal (147). Of the 1,198 samples, 11 were excluded due technical problems in the handling of the sample, and the HPV DNA was not detected in four samples by nested PCR using the primers PGMY and GP5+/6+. Thus, the final sample of this study is 1,183 women (590 from RJ and 593 from PA).Characteristics of participants are shown in The majority of women initiated sexual activity between 16 and 18 years old (about 40% in both sites), and the proportions of first sexual intercourse before age 16 years were lower in RJ than in PA. The proportion of women who reported more than five lifetime sexual partners was much higher in RJ than in PA. Women with one or two childbirths predominated in RJ, while in PA women with seven or more childbirths were most often represented .Knowledge of the Pap test's purpose was lower in PA (31.9%) than in RJ (60.1%). Around 80% of women in RJ and 75% in PA had had at least one Pap test before the current health problem. Frequency of Pap smear test was also different between States, and about half of patients from RJ reported to never being tested before or having the test with an interval longer than 3 years, but in PA the proportions were around 80% .Besides, the frequency of never smokers was about the same in both locations, current smoker proportion was higher in Rio de Janeiro than in Pará .We identified 23 HPV types in Pará and 19 HPV types in Rio de Janeiro, considering tumors with both single and multiple HPV infection. The seven oncogenic HPV types with the highest frequencies in RJ were 16, 18, 45, 35, 31/33, and 58, while in PA they were 16, 18, 33/45, 31, 52, and 35.Of the 1183 women, the sequence analysis identified 1114 women with single infection (554 from RJ and 558 from PA), corresponding to 96.6% and 95.4%, respectively. HPV genotype was not identified in 22 cases (HPV-X). In tumor samples with single infections, from those HPV types considered as “unknown or low risk group,” we found only one type in RJ (HPV 83) and three types in PA  .p < 0.001). Adenocarcinoma (ADC) represented 13.5% of tumors in RJ and 6.9% in PA (p < 0.001)  was lower in RJ than in PA  .https://doi.org/10.1155/2017/1645074).Multiple infections were identified in 47 women (20 from RJ and 27 from PA). HPV 16 was found in 40 of the 41 tumors and HPV 18 in 25 of the 41 tumors. In only one case of multiple infections HPV 16 was not present. In six samples it was not possible to identify the HPV type due to the small amount of DNA. The “unknown or low risk” HPV 42 and 54 types appeared in association with high-risk types , these proportions were fairly similar to the other types in both States.The proportions of HPV 16, 18, Alpha-7, and Alpha-9 by age and location are shown in Supplementary Table  2. With the exception of Pará, where HPV 16 seems to be more common in the younger age groups (2 (estimated for 2010) and the household monthly nominal income per capita in 2015 was R$1,285 (~US$390). Pará has the third-worst Human Development Index (HDI) among the Brazilian States [2. The nominal household monthly income per capita of the resident population was R$672 (~US$202) [The two Brazilian States where the study was carried out have distinct socioeconomic characteristics. Rio de Janeiro has the second economy in Brazil. With an estimated population for 2015 of 16,550,024 distributed in 92 municipalities, the population density is 365.23 per kmn States , with a These characteristics are reflected in our findings with regard to the economic, educational, and behavioral/sexual differences observed between the two groups of women. These distinct characteristics may also reflect the differences observed in cervical cancer incidence rate for 2016  and in the age-adjusted cervical cancer mortality rate for 2014  [Alpha-7 HPV types  were more frequent in RJ than in PA. As they are more strongly associated with adenocarcinoma, it may explain the difference observed in distribution of histological types of tumors between the Brazilian States. A growing body of evidence suggests that cervical cancer screening is less effective against adenocarcinoma than squamous carcinoma [Of the probably or possibly carcinogenic types, the most common type in our study was HPV 73, thus suggesting that its carcinogenic potential should be further evaluated in future studies.The lower diversity of HPV types, in RJ than in PA , was also observed in another Southeastern State, São Paulo –24.A comparison of our data on HPV type frequency with those from de Oliveira et al.  shows thThe frequency of multiple HPV infections detected here (4.1%) was lower than that reported in other studies in Brazil, 9%, by Fernandes et al., 2010  and 24%, High + Low Papillomastrip Kit (OPERON) only allows the identification of specific HPV types for which there are probes in the Kit.One of the limitations of our study is that the methodology to detect multiple infections, based on DNA sequencing (using the Sanger method), does not detect less frequent HPV DNA in a tumor sample (<20%) . In addiConsidering the results obtained in this study, the 4-valent HPV vaccine, adopted by the Public Health System in Brazil since 2014, can potentially reduce at least 70% of cervical cancer incidence in RJ and PA. Implementation of the new 9-valent HPV vaccine has the potential of increasing the impact of vaccination on cervical cancer incidence by 85%. However, one of the nine most frequent carcinogenic types, HPV 35, has not been included in the current available vaccines, a limitation that may impact on HPV primary prevention in Brazil.In addition to vaccination, the policy for Cervical Cancer Control in Brazil recommends the maintenance of screening programs for all the women at the age of 25 through 64 years old. The success of this strategy to control cervical cancer has to take into account the distinct demographic and sociocultural characteristics of the different Brazilian regions. Given its lower population density and transportation problems, the Amazonian Region poses a special challenge with regard to access of its population to the healthcare system.Multiple infections were identified in 47 women (20 from RJ and 27 from PA). HPV 16 was found in 40 of the 41 tumors and HPV 18 in 25 of the 41 tumors. In only one case of multiple infections HPV 16 was not present. In six samples it was not possible to identify the HPV type due to the small amount of DNA. The “unknown or low risk” HPV 42 and 54 types appeared in association with high-risk types.


We have previously demonstrated the immunogenicity of VGX-3100, a multicomponent DNA immunotherapy for the treatment of Human Papillomavirus (HPV)16/18-positive CIN2/3 in a phase 1 clinical trial. Here, we report on the ability to boost immune responses with an additional dose of VGX-3100. Patients completing our initial phase 1 trial were offered enrollment into a follow on trial consisting of a single boost dose of VGX-3100. Data show both cellular and humoral immune responses could be augmented above pre-boost levels, including the induction of interferon (IFN)γ production, tumor necrosis factor (TNF)α production, CD8+ T cell activation and the synthesis of lytic proteins. Moreover, observation of antigen-specific regulation of immune-related gene transcripts suggests the induction of a proinflammatory response following the boost. Analysis of T cell receptor (TCR) sequencing suggests the localization of putative HPV-specific T cell clones to the cervical mucosa, which underscores the putative mechanism of action of lesion regression and HPV16/18 elimination noted in our double-blind placebo-controlled phase 2B trial. Taken together, these data indicate that VGX-3100 drives the induction of robust cellular and humoral immune responses that can be augmented by a fourth “booster” dose. These data could be important in the scope of increasing the clinical efficacy rate of VGX-3100. As immune-mediated control and elimination of chronic viral infections are associated with CD8+ T cell responses,20–23 an immunotherapy able to generate HPV-specific T cells that show long-term persistence and can migrate to cervical tissues and mediate clearance of virus would be a useful tool for prevention of disease recurrence in patients with persistent infection. Moreover, the ability to benefit from additional treatments of such an immunotherapy would prove useful in this endeavor.In the cervix, the intraepithelial lesion that precedes virtually all squamous cervical cancers, cervical intraepithelial neoplasia 2/3 (CIN2/3), is treated with surgical excision.in vivo electroporation (EP), in women who had undergone an excisional procedure for intraepithelial HPV disease.24 We have also reported the results from our phase 2 double-blind randomized placebo-controlled efficacy study of VGX-3100 in which we noted statistically significant rates of regression of HPV16/18-positive CIN2/3 and clearance of HPV16/18, which were statistically associated with an immunological response.25 Here, we describe a follow-on Phase I trial in which thirteen of the eighteen subjects enrolled in the original Phase I study were administered a single boost of 6 mg VGX-3100 followed by EP in order to study the ability of previously generated HPV16- and HPV18-specific immune responses to be further boosted. Immune responses measured after the boost revealed an augmented humoral response, an increase in cytokine expression from both the CD4+ and CD8+ T cell compartments and an increase in the expression of lytic proteins within HPV-specific CD8+ T cells. These responses were similar in magnitude and quality to those observed in our phase II double-blind placebo-controlled efficacy study of VGX-3100  injection followed by Screening evaluations were performed within 30 days prior to study enrollment on Day 0. If the screening assessments were performed within 7 days of dosing on Day 0, a single assessment could count as both the screening and Day 0 evaluations, however, pregnancy testing had to be repeated if screening and Day 0 assessments were performed greater than 48 hours apart.The Month 9 visit of the HPV-001 study could count as the screening visit for the HPV-002 study, provided that all of the necessary evaluations for both visits were performed. A total of 18 subjects were identified for screening from the HPV-001 study, of which 13 were eligible for HPV-002 and 13 provided written informed consent, and were enrolled to receive 6 mg of VGX-3100 followed by EP, regardless of the dose that they received in the HPV-001 study. Three subjects were not eligible due to pregnancy, one was not given the option to participate in HPV-002 due to protocol noncompliance, and one subject declined due to shoulder pain. All 13 subjects received 6 mg of VGX-3100, and all except one completed the follow-up visit (Month 6). The mean age of the study population was 30.5 years (range: 20–42 years).Injection site reactions were evaluated within 30 minutes after EP and on Day 7 postdose. Overall, injection of 6.0 mg of VGX-3100 followed by EP was well-tolerated. No severe or potentially life-threatening reactions were noted during the course of the study. Local postadministration injection site reactions were evaluated for severity by “Grade” on Day 0 at 15 minutes post-EP and on Day 7 postdose. “Grade” was defined as Grade 1 = “mild”, Grade 2 = “moderate”, Grade 3 = “severe,” and Grade 4 = “potentially life threatening.” One or more of the local postadministration injection site reactions  were experienced by each of the 13 subjects. No grade 3 or 4 adverse events (AEs) or serious adverse events (SAEs) were observed during the study .No deaths or serious adverse events were reported and no adverse events led to withdrawal from the study. There were also no Grade 3 or Grade 4 adverse events reported. Overall, eight subjects (62%) reported any treatment emergent AEs with the highest severity as “moderate” for one subject, and “mild” for five subjects. All were determined to be not related or not likely related to VGX-3100.de novo cellular immune responses generated after immunization that persisted out to 6 months following the third dose.24 Upon establishing this, we began analysis of the impact of a single 6.0 mg boost with VGX-3100 had on that established HPV-specific immune response. To that end, we performed ELISA and IFNγ ELISpot in order to assess the humoral and cellular compartments, respectively. Observation of humoral responses following the boost revealed that the median endpoint titer against HPV16 E6 and HPV18 E7 proteins at entry of HPV002  were boosted by the administration of one dose of VGX-3100 to a median titer of 1:1,350 and 1:4,050, respectively  consisting of a three-dose regimen of 0.6 to 6 mg VGX-3100, we identified robust ectively . Responsectively . In totaectively .6 Peripheral Blood Mononuclear Cells (PBMCs)) was boosted by 1.5-fold after the administration of one dose of VGX-3100 to a median magnitude of 297 SFU/106 PBMCs  and the latter increasing more than 2.5-fold from pre- to postboost  those that co-expressed granzyme B and perforin concurrent with a cytokine  in the absence of CD107a (termed GrzB+Prf+/CD107a-/Cytokine+), (ii) those that coexpressed granzyme B and perforin concurrent with CD107a but in the absence of a cytokine (termed GrzB+Prf+/CD107a+/Cytokine-), and (iii) those that were positive for granzyme B, perforin, CD107a and at least one cytokine (termed GrzB+Prf+/CD107a+/Cytokine+). The first described population would constitute CD8+ T cells that were HPV specific and had lytic potential but did not degranulate within the 6-hour window of the assay (as evidenced by being CD107a-). Such cells would likely not be capable of direct lysis within that narrow timeframe, but would harbor the correct lytic proteins to mediate this type of cell death if prolonged stimulation allowed for them to degranulate. Moreover, expression of TNFα from these cells could mediate receptor/ligand-mediated lysis and the production of IFNγ could aid in sensitizing tumor cells to receptor-mediated lysis.34–36 The second population would constitute those CD8+ T cells that were HPV specific and harbored lytic potential as well as the ability to degranulate as evidenced by being positive for CD107a. The final population expresses all markers consistent with a high-functioning population of CTLs, which have the capability of killing tumor cells by multiple mechanisms including direct lysis via lytic proteins, receptor/ligand-mediated lysis and sensitizing tumors to lysis by lytic granules. All three populations require concomitant expression of at least three antigen-specific regulated markers, thereby enforcing a requirement for polyfunctionality in order to be considered for analysis. Examination of these phenotype’s across the study population revealed that total HPV16 and HPV18-specific activity was augmented by administration of the boost over two-fold as compared with preboost activity  and is a well established proinflammatory molecule.38 IL-15 (interleukin 15) drives the expansion and activation of CD8+ T cells, including the induction of the synthesis of lytic proteins necessary for an effective cytotoxic response.39 Additionally, TBX21  is a transcription factor known to directly activate transcription of Interferon-γ and is associated with increased antigen-specific CD8+ T cell cytotoxicity.40 Moreover, three transcripts were found to be downregulated in patient PBMCs in an antigen-specific manner: CCR7, IL-10, and HMOX1. CCR7 is a chemokine receptor that is well established as identifying cells that will home to and remain in the lymph nodes in a Central Memory or Naive phenotype. Thus its downregulation is suggestive of an antigen-specific shift towards an effector phenotype. IL-10 (interleukin 10) is a known suppressive factor for the induction of an inflammatory and cytotoxic response, thus its downregulation after antigenic stimulation aids in ensuring these responses are not turned off. HMOX1 encodes for heme oxygenase 1, which has been shown to mediate the anti-inflammatory effect of IL-10  affords the opportunity to examine various parameters of an inflammatory response in an exploratory manner, broadening the possibility of identifying immune signatures from cells that had either been unstimulated or stimulated with HPV antigens encoded by VGX-3100. Comparison of these samples allowed us to identify a set of genes that were differentially regulated based on HPV16/18-specific activation. Results of this analysis are presented in a heatmap in -10 ref. , thus it42–44 Although mechanisms behind these phenomena vary, a heavy and prolonged antigenic burden on the cellular arm of the immune system is thought to play a role in this immune incompetence. Study of cellular immune responses from our previous phase 1 dose-ranging trial demonstrated that HPV-specific CD8+ T cells generated by VGX-3100 were able to activate, remain active, and synthesize lytic proteins during a prolonged antigenic stimulation (120 hours).24 We also noted this ability in our phase 2 study and established a statistical association between this phenomenon and clinical efficacy.25 Thus, this type of profile is believed to be key for an effective CD8+ T cell response capable of eliminating a chronic viral infection and is an important hallmark of an appropriate immune response generated by VGX-3100. In the current study, we sought to determine whether a single booster dose of VGX-3100 augmented the frequency of HPV-specific activated CD8+ T cells as well as whether or not any notable increase in cosynthesis of granzyme B and perforin occurred. To that end, we performed a prolonged antigenic stimulation (120 hours) using HPV peptide and irrelevant control peptide  on subject PBMCs that were isolated prior to and after the boost and assessed activation status and lytic protein synthesis by flow cytometry (46) was used as a marker of CD8+ T cell activation. CD137 has been established by our group and others as an accurate marker of antigen-specific activation46 and its expression is linked with a functional tumor-infiltrating lymphocyte profile,47 making it particularly relevant in the context of advanced dyplasia and cancer driven by HPV infection. Analysis of the data generated by this assay reveals that the boost did not appreciably increase the frequency of total HPV-specific activated CD8+ T cells as determined by CD137 expression  of the TCR β chain50 has previously been utilized to identify HPV-specific clonal expansions in the cervical tissue.48 In the current study, samples from the cervix were collected in the form of Digene swabs both before and after the boost was administered. We thus performed TCR sequencing from digene samples in order to determine if a single boost of VGX-3100 influenced putative TCR signatures in cervical tissue. To that end, paired cervical specimens and PBMC samples from a subset of immunized patients (n = 6) both pre- and postboost were obtained and allocated for TCR sequencing.Following our observation of increased HPV-specific T cell responses in the peripheral blood after a boost with VGX-3100, we were next interested in determining if HPV-specific T cells were able to be detected in the cervical mucosa and if the frequencies of these sequences increased after the boost. Previous studies in humans have shown the ability of intramuscular immunization to elicit cellular responses at cervical sites during HPV infectionex vivo expansion. Initial analysis revealed that the frequency of numerous TCR sequences was increased in the postboost cervical swab when compared to the same patient’s preboost sample . While these procedures are often effective, they are not a targeted approach to elimination of HPV infection. Indeed, analysis of patients at varying intervals after LEEP procedures reveal that many remain HPV positive and the mean recurrence rate of CIN2/3 after LEEP is roughly 10%.17–19 The development of a method of treatment of high-grade dysplasia that leads to the complete resolution of infection would not only treat and regress dysplastic lesions caused by HPV infection, but would also prevent recurrence of dysplasia. The patient’s own immune system presents itself as the natural choice for the development of such a treatment, as it has been previously established that the induction of an HPV-specific immune response, particularly a cellular response, is often identified in the context of resolution of HPV-driven pathology and elimination of viral infection.23 Moreover, the establishment of immunological memory presents itself as an attractive concept for this purpose as a durable cellular immune response that was capable of mediating regression of an initial infection should be able to control and eliminate future HPV infections of the same type. The ability to boost this response would aid in further ensuring that immune system was capable of controlling HPV-driven pathology and eliminating infection. To this end, we have designed an immunotherapy based on the induction of HPV-specific T cell responses generated via the use of plasmid DNA delivered by in vivo electroporation.Persistent infection with a high risk HPV types such as HPV16 or HPV18 is strongly correlated with an increased risk of adenocarcinoma of the cervix, making elimination of infection of paramount importance in the prevention of this cancer.24 The immunological data reported here are the first data generated in regards to the ability to boost responses driven by VGX-3100 with subsequent dosing. Analyses conducted after administration of a single 6 mg boost of VGX-3100 revealed a therapy-driven increase in the production of HPV-specific antibodies as well as markedly increased cytokine production from both the CD4+ and CD8+ T cell compartments in the form of IFNγ and TNFα  in adult women who have been previously treated with three doses of VGX-3100 containing 0.6, 2, or 6 mg of DNA per dose. The study was conducted at three centers in the United States . The study protocol was approved by a central Institutional Review Board (Integreview) and the protocol was adhered to by following the guidelines of Good Clinical Practices and the Declaration of Helsinki. Written informed consent was obtained prior to participation in the HPV-001 study and again, prior to participation in the HPV-002 study. The subjects had to have successfully enrolled and completed all study procedures and follow-up in the HPV-001 study. Adult women deemed eligible for participation were between 18–46 years of age, inclusive, had a diagnosis of CIN 2 or 3 resulting in a postsurgical (including LEEP and conization) or ablative treatment while under a physician’s care . Women of child bearing potential could not be pregnant or nursing and agreed to remain sexually abstinent, use medically effective contraception, or have a partner who is sterile from time of enrollment to study discharge. Each subject was required to have a serum pregnancy test during screening with a negative result prior to enrollment. A negative spot urine pregnancy test was required prior to each dose.HPV-002 was a phase 1, open-label study to evaluate the safety, tolerability, and immunogenicity of a fourth, 6 mg dose of VGX-3100 (DNA plasmid encoding E6 and E7 proteins of HPV types 16 and 18) followed by Major exclusionary criteria included the subject not being able to participate in a study with an investigational compound or device (other than VGX-3100) within 30 days of signing informed consent, and would be excluded if she had an active infection with herpes simplex virus.Subjects that were previously enrolled in the HPV-001 study and received three doses  of VGX-3100 , and who consented to participate in the HPV-002 study were given a 1.0 ml intramuscular injection containing 6 mg of VGX-3100 in the deltoid followed immediately by IM EP with the CELLECTRA-5P device. The boost was administered no earlier than 6 months following the third dose  in the HPV-001 study, and subjects were followed for 6 months after their fourth dose.Subjects were monitored for fever, local (injection site reactions) and clinical AEs, and SAEs. The injection site was examined prior to and within 30 minutes after EP on the day of treatment and again at approximately 7 days later. PAP smears were also performed at Day 0 (prior to dosing) and at the 6-month follow-up visit. Additional safety assessments included physical examination at screening and discharge, vital signs, pre- and postdose ECG, and evaluation of clinical laboratory tests (data not shown). Samples for virologic assessments will were obtained using ThinPrep test kits. The ThinPrep sample was sent to a central laboratory to be analyzed for the presence of HPV high-risk genotypes and if positive, were reflexed to HPV genotyping via polymerase chain reaction (PCR) assay. Cervical cytology samples were collected to test for the presence of HPV and genotyping in subjects before and after administration of VGX-3100. Samples were obtained on Day 0, but could be performed up to 7 days prior to the first vaccination, and at the 6-month visit.All immunological assays were performed based on sample availability. For each assay, all available samples were used for analysis assuming as sample integrity allowed. For cellular assessments, the ELISpot assay was given priority and remaining cells were then allocated to ICS and/or Lytic Granule Loading assays, followed by the Gene Array assay. TCR sequencing was performed on DNA isolated from Digene brushes so long as the yield from the DNA extraction was sufficient for sequencing purposes.A standardized binding ELISA was performed to measure the anti–HPV16/18 E6 or E7 antibody response induced by VGX-3100. Endpoint titers of antibodies were determined by coating 96-well enzyme immunoassay plates with 1 mg/ml HPV16 or HPV18 E6 or E7 proteins . Samples were considered positive if the average optical density (OD) of a sample was greater than 0.15 absorbance units and greater than the average OD at baseline (prior to receiving any doses of VGX-3100) plus 2.5 times SD of OD at baseline at the same dilution. For graphing purposes, values of 0 were changed 1 in order to be displayed on a log scale.1 The standard ELISpot protocol with 24 hours peptide stimulation was previously cross-validated across different laboratories using HIV-specific peptide pools. Differences between the above-referenced protocol and the protocol used in the current study relate only to the use of HPV peptides as the stimulating antigen (in place of HIV peptides) and not to the length of PBMC stimulation, which remained at 18 to 24 hours per protocol. Specifically, the current protocol used two sets of pooled peptides, each containing 2.5 μg/ml of 15–amino acid residues overlapping by 11 amino acids spanning the full-length consensus sequence of HPV16 E6 and HPV16 E7 or HPV18 E6 and HPV18 E7 antigens. The average number of spot forming units (SFU) counted in R10 wells  was subtracted from the average in HPV peptide pool stimulated wells and then adjusted to 1 × 106 PBMCs for each HPV peptide pool.The ELISpot assay was performed by the University of Pennsylvania Human Immunology Core Facility using a qualified protocol as previously described.Intracellular cytokine staining was performed as previously described(1) using the following markers: CD3-APCH7, CD107a PECy7, CD14-Pacific Blue, CD16-Pacific Blue, CD4-PECy5.5, IFN-γ-PerCPCy5.5, CD45RO-AF700, CD19-Pacific Blue , TNFα-AF647, CD8-BV570, CCR7-BV711 , granzyme B-PE Texas Red (Invitrogen), CD27-PECy5  perforin-FITC. Prepared cells were acquired using an LSR II flow cytometer equipped with BD FACSDiva software (BD Biosciences). Acquired data was analyzed using the FlowJo software version 7.6.3 (Tree Star).6 PBMCs were seeded into a 96-well plate. For antigen-specific responses, cells were stimulated 120 hours with HPV16/18 E6 and E7 antigens, while R10 media alone was used as a negative control, OVA was used as an irrelevant peptide control and concanavalin A was used as a positive control (Sigma-Aldrich). At the end of the 5-day incubation period, all samples were washed with phosphate buffered saline and subjected to staining for CD3-APCH7, CD4-PerCPCy5.5, CD8-FITC, CD137-APC (BD Biosciences), Granzyme B-PETR and Perforin-PE using the same protocol as listed above. Staining was performed once per time point per sample listed.The lytic granule loading assay was performed as previously described1. Briefly, 10PBMCs isolated after the VGX-3100 boost were thawed and rested overnight. After recovery, cells were counted and plated at 200,000 cells per well in a 96-well flat, clear bottom plate. Cells were stimulated with HPV16 E6/E7 and HPV18 E6/E7 pooled peptides (2 µg/ml per well) or received no stimulation (R10 media only). After 21 hours, cells were collected and the total RNA was purified using the PureLink RNA Mini Kit and reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher). The NanoPhotometer was used to calculate cDNA sample concentrations. TaqMan Array, Human Immune (Thermo Fisher), fast 96-well plates were used to perform RT-qPCR on the QuantStudio 7 Flex Real-Time PCR System using a thermal cycle of 50 °C for 2 minutes, 95 °C for 20 seconds, 40 × 95 °C for 3 seconds, 60 °C for 30 seconds. Data was analyzed using the QuantStudio RT qPCR software. The gene expression heat map was generated using the program software R i386 3.2.0.PMBC and cervical Digene swab samples used for DNA isolation were obtained from subjects pre- and postboost. PBMC samples from pre- and postboost were stimulated with pooled HPV16 E6/E7 and HPV18 E6/E7 peptides or irrelevant OVA peptides (preboost only) for 5 days prior to isolating DNA for TCR sequence analysis . DNA was isolated directly from cervical samples without any stimulation and from negative control cord blood samples after 5 days of stimulation with either HPV or OVA peptides.


While the role of genus alpha human papillomaviruses in the tumorigenesis and tumor maintenance of anogenital and oropharyngeal cancers is well-established, the role of genus beta human papilloviruses (β-HPVs) in non-melanoma skin cancers (NMSCs) is less certain. Persistent β-HPV infections cause NMSCs in sun-exposed skin of people with a rare genetic disorder, epidermodysplasia verruciformis. However, β-HPV infections in people without epidermodysplasia verruciformis are typically transient. Further, β-HPV gene expression is not necessary for tumor maintenance in the general population as on average there is fewer than one copy of the β-HPV genome per cell in NMSC tumor biopsies. Cell culture, epidemiological, and mouse model experiments support a role for β-HPV infections in the initiation of NMSCs through a “hit and run” mechanism. The virus is hypothesized to act as a cofactor, augmenting the genome destabilizing effects of UV. Supporting this idea, two β-HPV proteins (β-HPV E6 and E7) disrupt the cellular response to UV exposure and other genome destabilizing events by abrogating DNA repair and deregulating cell cycle progression. The aberrant damage response increases the likelihood of oncogenic mutations capable of driving tumorigenesis independent of a sustained β-HPV infection or continued viral protein expression. This review summarizes what is currently known about the deleterious effects of β-HPV on genome maintenance in the context of the virus's putative role in NMSC initiation. Human papillomavirus (HPV) is a family of small, non-enveloped double-stranded DNA viruses that infect mucosal and cutaneous epithelia. This family is comprised of five genera  spanning across over 396 different HPV types potentially inhabiting human skin  and presented the first link between β-HPV infections and skin carcinogenesis Orth, . β-HPV+ β-HPVs inhabit the cutaneous epithelium and are found in abundance throughout the population , BRCA1 and BRCA2 are similarly important for a myriad of DDRs  pathway  or the inhibition of p53 activation (β-HPV 23 through HIPK2 interaction). β-HPV 17, 38, and 92 E6 interact with p53 directly, leading to its stabilization but altering or restricting p53's transcriptional activity. β-HPV 5, 8, and 38 E6 interfere with DDR and checkpoint signaling through interaction with p300. A combination of inhibited apoptosis, impaired DNA damage signaling and attenuated DSB response bears a potential to destabilize the genome over time or cause carcinogenesis in combination with DNA-damaging agents that is consistent with β-HPV's proposed contribution to skin cancer. Notably, although through markedly different mechanisms, high risk α-HPV oncogenes similarly contribute to tumorigenesis by impairing the cellular response to genome destabilizing events.A canonical function of the high risk α-HPV E7 oncogene is the degradation of the tumor suppressor pRb  mouse model that expresses can individual and groups of β-HPV genes  or the entire early region of the virus under a keratin promoter. Placing the expression under a keratin promoter restricts expression to keratinocytes, the cell type infected by β-HPV. The result is that genes are only expressed at anatomical sites relevant in the context of β-HPV infections. Our review of the literature shows that β-HPV 8 is the most frequently investigated β-HPV type, followed by β-HPV 38, β-HPV 49, and β-HPV 20. Additionally, one study has investigated β-HPV 5 E7 in a nude mouse/artificial skin graft model  promoter to express the entire early gene region of β-HPV 8. Transgene expression was the highest for E2, followed by E6 and E7  bind p300 strongly enough to destabilize the histone acetyltransferase, causing delayed repair of thymine dimers and double strand breaks  impair pRB function. The sole mechanism of pRB interference by β-HPV 5 and 8 E7 is through binding and induction of hyperphosphorylation of pRB. In addition to promoting the phosphorylation of pRB, β-HPV 38 and 49 E7 also decrease the half-life of pRB. β-HPV 24 E7 is only known to reduce pRB's half-life. Further, β-HPV 38 E7 has the ability to interfere with p53 activity by stabilizing the p53 antagonist, ΔNp73α, and increase inhibitory post-translational modifications of p53.in vivo transgenic mouse models of β-HPV associated tumorigenesis reflect the varied ability of β-HPV to degrade the fidelity of host cells  that recapitulates some but not all of the characteristics of β-HPV . These viruses are expected to promote carcinogenesis via the “hit and run” model that is dependent on their amplification of UV's genome destabilizing effects and otherwise decreasing genome fidelity. Since the mechanisms of abrogating genome stability vary among β-HPVs, it is reasonable to expect that the carcinogenic potential of individual β-HPVs depends on their cellular environment during infection. As a result, not only is the clear delineation of pathways and proteins impacted by β-HPV necessary to understand their oncogenic potential, but also the relative ability among β-HPVs to disrupt these pathways. A major knowledge gap regarding β-HPV induced oncogenesis is the extent that the reduced availability of ATM, ATR, BRCA1, and BRCA2 affects the activity of these proteins in each of the repair pathways that they participate in. This could expand the known ability of β-HPV proteins to disrupt DNA repair to include nearly every cellular response to damaged DNA. Alternatively, if the impairment of these repair proteins was context specific, it could highlight particularly deleterious situations for a β-HPV infection to occur. Further, there is a diversity of genome destabilizing events beyond DNA damage  that if disrupted would fit the ascribed “hit and run” model of tumorigenesis. Defining the mutagenic potential of β-HPV infections is essential to take next steps in preventing disease associated with these viruses. Table Wrote the manuscript: SW and NW. Reviewed and edited the manuscript: SW and NW.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PIK3CA) and phosphatase and tensin homolog (PTEN), human leukocyte antigen A and B (HLA-A and HLA-B)-A/B, and the transforming growth factor beta (TGFβ) pathway, and rarely have mutations in the tumor protein p53 (TP53) and RB transcriptional corepressor 1 (RB1) tumor suppressor genes. There are some variations by tumor site, such as NOTCH1 mutations which are primarily found in head and neck cancers. Understanding the somatic events following HPV infection and persistence can aid the development of early detection biomarkers, particularly when mutations in precancers are characterized. Somatic mutations may also influence prognosis and treatment decisions. High-risk human papilloma virus (HPV) infections cause cancers in different organ sites, most commonly cervical and head and neck cancers. While carcinogenesis is initiated by two viral oncoproteins, E6 and E7, increasing evidence shows the importance of specific somatic events in host cells for malignant transformation. HPV-driven cancers share characteristic somatic changes, including apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-driven mutations and genomic instability leading to copy number variations and large chromosomal rearrangements. HPV-associated cancers have recurrent somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( High-risk human papilloma virus (HPV) infections cause cancers at many sites. It is estimated that almost all cervical cancers , 20–70% HPV infection alone is an insufficient cause of carcinogenesis. Most HPV infections become undetectable after a few months and never result in malignancies, with 91% becoming undetectable after two years, although it has been proposed that there may be some level of persistent latent infection that is undetectable by PCR ,11. HighHPV-derived cancers share many carcinogenic features across cancer sites, suggesting that the viral oncoproteins E6 and E7 work similarly at different sites. A previous review on this topic  predatesINK4A, a cyclin-dependent kinase inhibitor, through a disrupted feedback loop  mutation signature in particular is very common in HPV-positive cancers, likely triggered by the host response to HPV infection . TP63), altered in 77% of samples), 3q24.1 (transforming growth factor beta receptor 2 (TGFBR2), 36%), 10q23.31 (phosphatase and tensin homolog (PTEN), 31%), 18q21.2 (SMAD family member 4 (SMAD4), 28%), and 7p11.2 , 17%) . P. P3  . PIK3CA is also commonly mutated in endometrial and some ovarian cancers [Overall, more than 50% of cancers of the cervix and anus have at least one mutation in the PI3K/AKT pathway . Similar cancers ,112, whiHuman leukocyte antigen (HLA) alleles are important components of host cell-mediated immune responses to viral infections and are essential to the major histocompatibility complex (MHC) immune response pathway. HLA-A and HLA-B are MHC class I molecules that present viral antigens on the cell surface to alert the immune system to infection [HLA-A in 8% and HLA-B in 6–9% of cervical squamous cell carcinomas [HLA gene alterations including gene mutations, loss of heterozygosity, and other genetic changes [HLA alterations are found frequently in cervical precancers as well, suggesting that it is an early event in cervical carcinogenesis [HLA-A/B mutations are somewhat more common in HPV-positive (11%) than HPV-negative (7%) head and neck cancers [HLA-A or HLA-B could lead to loss of presentation of tumor antigens and immune cell recognition. One small study reported frequent mutations in the HLA pathway-associated transporter associated with antigen processing (TAP) gene (52%) in cervical carcinomas [nfection  , CREB binding protein (CREBBP) and E1A binding protein p300 (EP300) (activators), and SMAD4 (a transcription factor and tumor suppressor), and mutations in at least one of these genes have been reported in 30% of cervical squamous cell carcinomas [EP300 was in the top 30 mutated genes in head and neck squamous cell carcinomas [TGFBR2, CREBBP, and SMAD4 may be cervical squamous cell carcinoma-specific, although E7-driven expression effects in the TGFβ pathway may still play a role in carcinogenesis in other HPV-positive cancers. SMAD4 downregulation is also associated with HPV-negative head and neck cancers [The transforming growth factor beta (TGFβ) pathway inhibits DNA synthesis and plays a tumor suppressor role, although it can also promote cancer progression once carcinogenesis has been initiated ,118,119.rcinomas . This im cancers , and SMA cancers . NOTCH1 receptor are found in both HPV-negative (12–26%) and in HPV-positive (6–17%) head and neck cancers, albeit somewhat more frequently in HPV-negative tumors, and are not commonly reported in cervix or other HPV-driven cancer sites [NOTCH1 has indeed been reported as a driver gene in oral tumorigenesis independent of HPV status [FBXW7) is involved in angiogenesis through regulation of the Notch pathway [The Notch signaling pathway is responsible for cellular differentiation. Mutations in the egative 1–26% and er sites ,37,76,77V status . F-box a pathway  and is m pathway ,35,36.KRAS) is an oncogene in which mutations are found in 8–23% in cervical adenocarcinomas but rarely in cervical squamous cell carcinomas [KRAS in head and neck cancers is 6% [EGFR is a tumor suppressor in the same pathway in which mutations are found in 3–33% of cervical squamous cell carcinomas but rarely in cervical adenocarcinomas [FGFR2 and FGFR3, which have combined mutation rates of 10–17% in HPV-positive head and neck cancers [FGFR genes may potentially be therapeutic targets [The RAS/EGFR/ERK  pathway is involved in cellular proliferation and survival . It consrcinomas ,35,62,75rs is 6% . In contrcinomas ,62,70,71 cancers ,35,36,37TRAF3) is involved in viral immune responses [E2F1, a cell cycle related gene more commonly mutated in HPV-positive cancers (19% versus 2%), and FAT atypical cadherin 1 (FAT1) and ajuba LIM protein (AJUBA), two genes involved in differentiation that are more commonly mutated in HPV-negative cancers  [The tumor necrosis factor (TNF) receptor associated factor 3  compared with mutations at other sites [BCAR4, in which amplifications and gene fusions have been found in cervical cancer and which is targeted by lapatinib [CD274 and PDCD1LG2 are immunotherapy targets with amplifications reported in cervical cancer [ERBB2; HER2) and erb-b2 receptor tyrosine kinase 3  are mutated in a subset of cervical adenocarcinomas and these tumors may be susceptible to targeted therapies, and PTEN and AT-rich interaction domain 1A (ARID1A) alterations are also potential targets [In addition to early detection, somatic characterization can be important for prognosis and targeted treatment strategies. For example, e PIK3CA . Site-sper sites . Anotherapatinib ,134. CD2l cancer . Erb-b2  targets . The PI3 targets . The dev


Hydra vulgaris and its dominant colonizer Curvibacter as a model system that enables the visualization and quantification of colonizer population size at the single cell resolution, in vivo, in real time. We estimate the carrying capacity of a single Hydra polyp as 2 × 105Curvibacter cells, which is robust among individuals and time. Colonization experiments reveal a clear priority effect of first colonizers that depends on arrival time and colonization history. First arriving colonizers achieve a numerical advantage over secondary colonizers within a short time lag of 24 h. Furthermore, colonizers primed for the Hydra habitat achieve a numerical advantage in the absence of a time lag. These results follow the theoretical expectations for any bacterial habitat with a finite carrying capacity. Thus, Hydra colonization and succession processes are largely determined by the habitat occupancy over time and Curvibacter colonization history. Our experiments provide empirical data on the basic steps of host-associated microbiota establishment – the colonization stage. The presented approach supplies a framework for studying habitat characteristics and colonization dynamics within the host–microbe setting.Most eukaryotic species are colonized by a microbial community – the microbiota – that is acquired during early life stages and is critical to host development and health. Much research has focused on the microbiota biodiversity during the host life, however, empirical data on the basic ecological principles that govern microbiota assembly is lacking. Here we quantify the contribution of colonizer order, arrival time and colonization history to microbiota assembly on a host. We established the freshwater polyp  Most eukaryotic organisms are colonized by a microbial community that is known to have diverse functions in health and development of their host . Yet, thHydra vulgaris strain AEP (hereafter Hydra) and its dominant bacterial colonizer. Under constant laboratory conditions Hydra reproduces clonally; hence, individual polyps are assumed isogenic. Hydra stably maintains a highly specific microbiota, including three main families, whose composition under laboratory conditions is similar to that in nature . Furthermore, we test for priority effects in host colonization, and whether those are directly dependent on the time lag between competing colonizers.The quantification of host carrying capacity provides an estimate for the total host-associated microbiota population size that in turn provides information about ecological and evolutionary trajectories of the inhabiting bacteria. Much less is known about the colonizer population size dynamics during the initial colonization phase from single colonizer cells to maximum population size . If the host, here the g., soil  or partig., soil ]. Here wHydra vulgaris (strain AEP) was routinely cultured according to standard procedures at 18°C , 3 or 5 μg ml-1 and gentamicin (Gm), 2 μg ml-1. The Curvibacter sp. strains described in Table S1, Supplementary File E. coli MFDpir ) was constructed using a transposon vector developed by The bacterial strains and plasmids used in this study are provided in Table S1, Supplementary File i MFDpir  as plasmi MFDpir  was usedHydra polyps were divided in multiple pools with a defined number of animals in each group and kept in 50 ml Hydra medium  populations. Experiments were carried out either for 168, 288 h or until a maximum of 312 h.Germ-free a medium  throughoHydra polyps colonized with their natural resident microbiota, polyps were incubated with approximately 100 cells per polyp of Curvibacter carrying pRL153-GFP and treated as described above.For experiments with Curvibacter on-host population size , non-linear least-squares minimization was used to fit a logistic growth model to the results and estimate the carrying capacity using the R package growthcurver . Single polyps were transferred on concave microscopy glass slides and subsequently cooled down on ice for 10 min prior to imaging.Curvibacter carrying plasmid pRL153-GFP were independently incubated on Hydra polyp pools for 168 h. After homogenization of the polyps, Curvibacter cells were carefully pelleted by centrifugation, recovered and serially diluted in Hydra medium. In parallel, two populations of Curvibacter wild type were incubated in R2A medium with daily transfers (1:100) for 168 h. Host-primed Curvibacter cells (obtained from the homogenized Hydra pools) and Curvibacter grown in R2A medium were used to initiate on-host-competition experiments. The experiments were conducted with approximately 100 cells per polyp of both Curvibacter strains. The number of both competitors per polyp was determined as described for the colonization assay.Two replicate populations of Hydra we equipped Curvibacter with the mobilizable RSF1010 derivative (IncQ) pRL153-GFP  . For expression of the second fluorophore, we replaced GFP with mCherry (pTW1-mCherry). Infecting germ-free Hydra with marked Curvibacter lines enabled us to visualize Curvibacter populations on Hydra in real time at a single cell resolution . We observed no impact of plasmid carriage on the fitness of Curvibacter compared to the plasmid-free wild type in pure culture  . In bipaCurvibacter . The Curvibacter cells pattern on the polyp revealed a gradual increase of the Curvibacter population size on Hydra over time. Furthermore, our results reveal that Curvibacter settles in neat lines that follow the structure of the epithelium, which is determined by the shape of individual epithelium muscle cells . This distinct spatial pattern suggests that the continuous constriction movement of Hydra has an impact on the Curvibacter spatial distribution. After 72 h the highest density of Curvibacter was observed in the Hydra tentacles region, hence Curvibacter distribution on Hydra is not homogeneous rather it is biased toward specific body parts , as recently shown . We began by infecting two independent large germ-free Hydra polyp pools with Curvibacter . Batches of three polyps from each animal pool were sampled at consecutive time points and homogenized. The homogenate was plated on non-selective R2A plates. The number of colony forming units (CFUs) served as a direct estimate of the total bacterial population size on Hydra . The quantification of CFUs per polyp for Curvibacter carrying pRL153-GFP or pTW1-mCherry was conducted by plating on non-selective R2A plates and R2A plates supplemented with kanamycin in parallel. No reduction in the plating efficiency of plasmid-carrying Curvibacter was observed. Hence, the CFU proxy was not affected by the marker presence. Furthermore, the plasmids pRL153-GFP and pTW1-mCherry were stable in Curvibacter under non-selective conditions. Only fluorescent colonies were found on non-selective R2A plates, hence the wild type CFU counts were not biased by the emergence of plasmid-free segregants.To further study Hydra pools. At 24 h post-infection the Curvibacter population attached to Hydra was smaller than the inoculum size and reached a stable maximum after 96–120 h . The gradual increase in CFU counts conforms to the increasing density of bacteria visually observed on Hydra over 72 h . The wild type and the marked lines reached a similar stable maximum around 2 × 105 cells per polyp 168 h post-infection. We note that the CFU per polyp dynamics were similar among the marked and non-marked lines, hence there is no difference in their relative fitness. Repeating the experiment for a longer time of 312 h showed that a maximum of about 2 × 105 CFU per polyp was reached after 96 h and remained stable 312 h post-infection . Conducting the colonization experiment with an inoculum size of 104 cells per polyp showed that a similar stable maximum was rapidly reached 48–72 h post-infection , indicating that the Curvibacter colonies originate only from Hydra polyps. In addition, we incubated Curvibacter in Hydra medium supplemented with glucose and plated aliquots of the medium after 24 h. No colonies were observed on non-selective plates in this experiment, indicating that Curvibacter was not persisting in the Hydra medium. Consequently, we conclude that the colony counts in our pipeline truly reflect the abundance of Hydra-associated bacteria.Using the described pipeline, we followed the colonization dynamics of three lines, including the wild type, GFP, and mCherry lines in independent Curvibacter is highly motile during early and mid-logarithmic growth phase and the presence of a full flagellum gene repertoire indicates that Curvibacter motility is flagellum-driven. For the purpose of this experiment we constructed a deletion mutant where a putative flgC gene was replaced by a marker cassette . Starting with an inoculum of about 100 cells per polyp revealed no colonies 24–168 h post-infection. Increasing the initial inoculum to 104 cells per polyp improved the colonization success. About 500 cells per polyp were observed 72 h post-infection but only a maximum abundance of about 5 × 103 cells was reached 168 h post-infection . A comparison of these results to the wild type colonization experiment, which was done in parallel , indicates that Curvibacter motility is an important factor for the colonization initiation and maintenance.We further tested our approach to document colonization dynamics by investigating whether swimming motility plays a role in the early colonization process. motility . Using oCurvibacter in Hydra host colonization, namely, consistent growth kinetics of Curvibacter among hosts that are characterized by a simple logistic growth model . We show that the Hydra carrying capacity amounts to about 2 × 105Curvibacter cells. This capacity is reached rapidly within 24–72 h, it is stable over long time, and it has robust characteristics among Hydra individuals.Overall, we find robust growth characteristics of Hydra colonization and its carrying capacity we studied the colonization dynamics after an artificial disturbance of the colonizing Curvibacter population. Curvibacter is sensitive to gentamicin [adding 2 μg ml-1 gentamicin to a pure culture (108 cells) resulted in no detectable CFU after 2 h of incubation]. Thus, we disturbed the host-inhabiting Curvibacter population by dosing the colonized Hydra polyps with gentamicin. In this experiment, we disturbed the Curvibacter population by treating the Hydra pool with a dose of 2 μg ml-1 gentamicin. The first treatment was applied at 96 h post-infection and was repeated every 24 h (without changing the medium) until no colonies could be observed. Overall, four gentamicin treatments were required to eliminate the total Curvibacter population . To test the Hydra carrying capacity robustness, we re-infected the Hydra pool with a gentamicin resistant Curvibacter . Within 72 h the Curvibacter population size of the re-colonization phase reached the typical Hydra carrying capacity (∼2 × 105). This experiment demonstrates that the Hydra carrying capacity is a robust characteristic of the host habitat that is independent of disturbance events. Additionally, we show that a decline in the existing colonizer population opens up ecological opportunities for new incoming colonizer populations.To test the robustness of Curvibacter–Hydra model we tested the consequences of preemptive competition in the Hydra habitat during colonization. To quantify the impact of colonization order and time, we manipulated the time lag between two consecutive colonizer populations. Using our pipeline , Curvibacter wild type served as the primary colonizer while Curvibacter carrying plasmid pRL153-GFP was used as the secondary colonizer. The secondary colonizer (100 cells per polyp) was added at different time lags of 24, 48, or 72 h post-primary infection. We evaluated the colonizer population size by plating simultaneously on non-selective and selective media. The growth characteristics and Hydra carrying capacity of both colonizers were estimated using a logistic growth model.Using the Hydra carrying capacity in this experiment was similar to the observed for a single colonizer population  and the carrying capacity of both colonizers remained stable over time . Nevertheless, the secondary colonizer reached a slightly higher carrying capacity in comparison to the primary colonizer and constituted about 60% of the final colonizer population . This suggests that the secondary colonizer had a slight advantage over the primary colonizer on Hydra under these conditions despite the later arrival.In the first time-lag experiment the secondary colonizer population was added 24 h post-primary infection. The secondary colonizer population size was slightly fluctuating over time but resulted in a similar density as the primary colonizer 288 h post-infection. The total Hydra with the secondary colonizer. Shortly after the re-infection, secondary colonizers were observed inhabiting the host while their population size slightly fluctuated around a maximum of 2.5 × 104 cells per polyp. Together, the two colonizers reached a stable population size after 96 h with a total population size (∼2 × 105) similar to the estimates obtained from previous experiments. However, the secondary colonizer contributed to around 30% of the total colonizer population size only . At this time lag, the primary colonizer had a significant numerical advantage over the secondary colonizer that remained stable over time. Thus, in contrast to a time lag of 24 h, a time lag of 48 h between primary and secondary colonization results in different carrying capacity for the two colonizers .In the next experiment we applied a time lag of 48 h prior to infection of Hydra (Table 1). This means that the carrying capacity for the consecutive colonizer was strongly reduced due to the high numerical advantage of the primary colonizer . Thus, with 48 and 72 h time lag the Hydra carrying capacity of the secondary colonizer is significantly lower in comparison to the primary colonizer, whereas the total carrying capacity is similar to the previous experiments and is stable over time .The third experiment was performed with an extended time lag of 72 h between primary and secondary infection. Here, the secondary colonizer contributed about 10% to the total bacterial population size on Hydra individuals that were fully colonized by their natural microbiota. Our results revealed no CFU of the incoming Curvibacter colonizer over 168 h, hence the incoming Curvibacter population completely failed to colonize a previously populated Hydra.To further test the consequences of host occupancy on the success of an incoming colonizer we performed a colonization experiment with Hydra. Since the Hydra carrying capacity is finite, the carrying capacity of an incoming colonizer largely depends on the Hydra habitat occupancy. Host occupancy is thus an important determinant of the establishment success of incoming colonizers.Our results demonstrate that the time lag between consecutive colonization is a decisive determinant of their population size on Curvibacter populations that were cultivated on Hydra prior to the experiment, while naïve colonizers (wild type) were sampled from Curvibacter populations maintained in liquid R2A medium (as in previous colonization experiments). A pool of germ-free Hydra polyps was inoculated with primed and naïve colonizers simultaneously at an equal density of approximately 100 cells per polyp. Using our pipeline, we documented changes in the primed and naïve colonizer population size by plating on non-selective and selective media. Our results reveal that 24 h post-infection both colonizers reach similar cell densities on the Hydra polyps. After 48 h the primed colonizer population size was slightly larger than that of the naïve colonizer, whereas at 72 h post-infection the primed colonizer largely outcompeted the naïve colonizer. Throughout the remaining experiment time, the naïve colonizer remained at low cell density of about 100 cells per polyp with a ratio of approximately 1:100 naïve to primed colonizer cells . To test whether this result was due to a general fitness benefit of the primed Curvibacter strain, we compared the fitness of the primed relative to the naïve strain in the absence of the host. A competition experiment between these two strains in liquid culture revealed no advantage of the primed strain after 24 or 48 h  were sampled from Hydra host colonization process over time and to test whether basic ecological concepts of habitat occupation  hold true in a reductionist one host – one colonizer system. The ability to genetically modify Curvibacter enables the quantification of host colonization dynamics from single colonizer cells to growth-limited populations in real time. Our finding that IncQ plasmids are stably maintained in Curvibacter enables us to visualize Curvibacter colonization on Hydra and follow the emergence of colonization patterns over time. Our results confirm the hypothesis that the carrying capacity of Hydra as a habitat is finite, stable over time and has little variation among individual polyps . The carrying capacity we observed for Curvibacter is comparable to the total microbiota load on Hydra  showed that leaf colonization by epiphytic bacteria occurs at multiple colonization sites having different carrying capacities (Curvibacter on Hydra may reveal a similar effect highlighting that a host can be comprised of different habitat niches.The robust characteristics of host habitat carrying capacity can be studied by a comparison of habitat characteristics among isogenic host individuals. In contrast to our results on ividuals . This coividuals . Furtherpacities . The patpacities . The visVibrio symbiosis where the number of bacterial cells entering the light organ is limited and the community diversification occurs after the initial colonization (Our results provide insights into the basic principles that govern host-associated bacterial colonization. As such, our data supports the idea that the microbiota is an ecological community utilizing the finite resources of a host habitat . As it hnization .Hydra–Curvibacter model we demonstrate the importance of colonization history, which may depend on specific host-mediated modifications of Curvibacter quorum-sensing signals (A priority effect that is further enhanced by adaptation can lead to intraspecific genetic diversification of colonizers in distinct habitats e.g., , where l signals , in hostHydra polyps and wild type Curvibacter sp. AEP1.3 strain. TW, TD, SF, TB, TR, and NH wrote the manuscript.TW, TD, and NH designed the study. TW and NH performed the experiments. TW, TD, TR, and NH analyzed the data. SF and TB contributed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer TM and handling Editor declared their shared affiliation.


Blue corn is a cereal rich in phenolic compounds used to make blue tortillas. Tortillas are an important part of the Mexican diet. Blue corn and tortilla represent an important source of the natural antioxidants anthocyanins. However, studies on their biological activity on cancer cell lines are limited. The goal of this study was to evaluate the antioxidant and antiproliferative activity of blue corn and tortilla on different cancer cell lines.Total polyphenol content, monomeric anthocyanins, and antioxidant activity by the DPPH and TBARS methods of blue corn and tortilla were determined. The anthocyanin profile of tortilla was obtained by means of HPLC–ESI-MS. The antiproliferative activity of blue corn and tortilla extract on HepG2, H-460, Hela, MCF-7 and PC-3 was evaluated by the MTT assay.Blue corn had higher content of total polyphenols and monomeric anthocyanins as well as lower percentage of polymeric color than tortilla; however, both showed similar antioxidant activity by DPPH. In addition, although a higher degradation of anthocyanins was observed on tortilla extract, both extracts inhibited lipid peroxidation (IC50) at a similar concentration. The anthocyanin profile showed 28 compounds which are primarily derived from cyanidin, including acylated anthocyanins and proanthocyanidins. Blue corn and tortilla extracts showed antiproliferative effects against HepG2, H-460, MCF-7 and PC-3 cells at 1000 μg/mL, however Hela cells were more sensitive at this concentration.This is the first report to demonstrate anticancer properties in vitro of tortilla derived from blue corn, suggesting that this product has beneficial health effects. In addition, blue corn could be a potential source of nutraceuticals with anticancer activity. Epidemiological studies from several countries point out that consumption of fruits, vegetables and cereals reduce the risk of chronic degenerative diseases due to the presence of bioactive compounds such as phenolic compounds , 2. In rZea mays L.). Species have an extensive genetic diversity, with 59 different races described with different shapes and colors ranging from white to yellow, red, purple and blue [Zea mays L.) is the most important cereal in Mexico from which tortilla is produced. Tortillas are a staple food for Mexicans, consumed by 94% of the Mexican population, with a 335 g/day consumption per capita, equivalent to 122 kg/year [Mexico is the center of origin and biodiversity of maize , folin reagent, gallic acid, ethanol, potassium acetate, quercetin, iron(III) chloride, hydrochloric acid, trolox, 2,2-difenil-1-picril-hidrazil (DPPH), amberlite XAD-7, acetic acid, 3--2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich. Trypsin, fetal bovine serum (FBS), GlutaMAX (100×) were purchased from Gibco. Dulbecco’s Modified Eagle’s medium (DMEM), Roswell Park Memorial Institute Medium (RPMI-1640) and phosphate buffer saline (PBS) tablet were supplied by Lonza.Traditional nixtamalization was done by boiling blue corn in a solution of 1% calcium hydroxide at 92°C for 35 min. After standing for 16 h, the cooked blue corn nixtamal was rinsed three times with 1 L of purified water. Then it was grounded in a manual grinder to obtain dough. A domestic press was used to make tortillas with a thickness of 1 ± 0.5 mm, 12 ± 0.5 cm in diameter and 17.5 ± 0.5 g in weight. Discs of dough were put into a pan at 240 ± 2 °C for 30 s on the side A, followed by 65 s on side B, and 30 s again on the side A.Ground blue corn or tortilla (1:5 p:v) were homogenized with ethanol acidified with citric acid 1M. This was performed using an ultrasonic homogenizer . The sample was placed under refrigeration for 24 h and centrifuged at 4000 rpm for 15 min at a temperature of 5°C. The process was repeated twice and the extract was concentrated using a rotary evaporator under vacuum. Extensive extractions were performed in order to obtain the highest content of polyphenols and anthocyanins. The conditions of extraction have been included in a patent request, MX/A/20131011202.The Folin–Ciocalteau method modified by Singleton and Rossi , was use−1 mg−1 and a molecular weight of 449.2 g/L.Monomeric anthocyanins were quantified using the pH differential method previously described by Giusti and Wrolstad . Samples−1 of the sample (ET).Antioxidant activity was determined by the DPPH method . A standThe evaluation of lipid peroxidation was performed by the TBARS method following the methodology described by Ohkawa . For thi−1 and heated capillary temperature of 180 °C. Under these conditions an electrospray ionization-mass spectrometry (ESI-MS) analysis of the isolated compounds was performed.A 45 × 1.5 cm column was packed with amberlite XAD-7 pre-conditioned with acidified water (5% acetic acid) . The resl-glutamine and 0.1% piruvate. HepG2, Hela, H460 and MCF-7 cells were maintained in DMEM medium which contained 10% FBS. The antiproliferative action of ethanolic extracts of tortilla and blue maize of the Mixteco variety was evaluated in HepG2, H460, Hela, MCF-7 and PC-3 cancer cell lines. For this, cells in 96-well plates were grown to 80% confluency. The cell count was performed using a Neubauer chamber, 5000 cells were seeded per well for PC-3 and 10,000 cells per well for HepG2, H-460, Hela and MCF-7. The blue corn and tortilla extracts were applied in concentrations of 125, 250, 500 and 1000 µg/mL, 100 µL per well. Cell culture in medium was used as a negative control, R (−), and quercetin 50 µM as a positive control, R (+). Extracts and quercetin were solubilized in culture medium. Treatments were allowed to incubate for 48 h. Post incubation time cells were treated with 5 mg/mL of MTT (10 µL per well) and incubated at 37 °C, 5% CO2 for 2 h. Finally, 100 µL of DMSO were added, the absorbance was determined at 595 nm [Hepatocellular carcinoma (HepG2), lung carcinoma (H-460), cervix adenocarcinoma (Hela), breast adenocarcinoma (MCF-7), and androgen-independent prostate adenocarcinoma (PC-3) human cancer cell lines were obtained from the American Type Culture Collection, United States of America . PC-3 cells were maintained in RPMI-1640 medium which contained 10% FBS, 1% t 595 nm .Data analysis was performed using the GraphPad Prism 6.0 statistics software. Analysis of variance (ANOVA) and Tukey’s test comparison with a 95% confidence interval were performed. The results of cell viability are presented as mean ± SD of independent experiments.In the present study, the content of total polyphenols, monomeric anthocyanins and percent polymeric color in the grain of blue corn and the tortilla derived from it were evaluated, since it is relevant to know how the tortilla preparation process affects the concentration of phenolic compounds such as anthocyanins. The total phenolics value for blue corn was of 287.3, and 70.3 mg GAE/100 g for tortilla; while the monomeric anthocyanins content was 70.50 and 27.8 mg C3G/100 g in blue corn and tortilla, respectively , lung carcinoma (H-460), cervix adenocarcinoma (Hela), mammary adenocarcinoma (MCF-7) and prostate cancer androgen dependent PC-3) as shown in Figs. , lung ca as shownFor HepG2, H-460, MCF-7and PC-3 cancer cell lines, the blue corn extract significantly inhibited cell proliferation at 1000 µg/mL,  showed slightly lower cell viability than blue corn extract (68.69 ± 2.6%) for the concentration of 1000 μg/mL. It could be attributed to changes on anthocyanin profile during nixtamalization, which probably favors the antiproliferative activity of tortilla extract in the cell line Hela. This evidence highlights once again the importance of the traditional nixtamalization process to make tortilla, particularly its effect on anthocyanins, compounds with anticancer properties.A study done by Bagchi et al.  found thThe proanthocyanidins are phenolic compounds that have shown chemoprotective properties against oxygen free radicals and oxidative stress; however, the mechanisms behind their anti-cancer activity have not been completely elucidated. Some authors suggest that the complexity of proanthocyanidins allows them to interfere in signaling systems .Nevertheless, several molecules present in foods, such as quercetin, can inhibit the growth of cancer cells . TherefoOur study is the first report showing that traditional tortilla-making process from blue corn favors antiproliferative activity in several cancer cell lines. This evidence highlights the importance of the traditional nixtamalization process, particularly its effect on anthocyanins. The findings suggest that consumption of blue corn and tortilla could have a positive effect on health. Further investigation is required to clarify the molecular mechanism(s) involved in the anticancer activity observed in blue corn and tortilla from maize of the Mixteco race.


Adolescent HPV vaccination rates remain suboptimal. The purpose of the study was to investigate attitudes about HPV vaccine relative to other adolescent vaccines among clinical staff from primary care offices and school based clinics.We interviewed clinicians in primary care offices and school-based clinics regarding their attitudes about HPV vaccine relative to Tdap and MCV4.Respondents (n = 36) included clinical staff in family medicine (47%), pediatrics (25%), obstetrics/gynecology (19%) and school-based health clinics (8%). Only 3% strongly agreed and 17% agreed that completion of HPV vaccine was more important than completion of pertussis vaccine (Tdap), while 6% strongly agreed and 33% agreed that completion of HPV vaccine was more important than completion of meningitis vaccine (MCV4).Providing clinicians with additional information about the cancer prevention benefits of the HPV vaccine and the greater risk for HPV infection/disease relative to other vaccine preventable adolescent diseases may help to increase HPV vaccination rates among adolescents. According to national estimates, at least 79 million people in the United States (US) are presently infected with human papillomavirus (HPV), making HPV the most common sexually transmitted infection (STI) . HPV incOver 33,000 HPV-associated cancers are diagnosed annually in the US, with about 60% occurring among females; the proportion attributable to HPV is substantial including 90% of cervical and anal cancers, 70% - 75% of vaginal and oropharyngeal (base of tongue/tonsil) and 60% - 70% of vulvar and penile cancers  [5]. In ®  was initially approved for use among females ages 9 - 26 years of age in 2006 to prevent cervical, vulvar and vaginal cancers/precancers and genital warts; the label was expanded in 2009 to include genital wart and anal cancer prevention in males ages 9 - 26. Cervarix®  was approved by the FDA in 2009 for protection against cancers and precancers in females . All. All9]. National estimates of HPV vaccination in the United States (US) (with either HPV2 or HPV4) reveal suboptimal coverage rates, which are substantially below the 80% target established by Healthy People 2020 and represent unrealized cancer prevention opportunities . Among fIn a national survey, fewer US physicians report HPV vaccination as important when compared to tetanus, diphtheria and acellular pertussis (Tdap) and meningococcal vaccines in an adolescent population. These clinicians also cited a need for longer patient counseling pertaining to HPV vaccine in contrast to other vaccines . AnotherThis report investigates attitudes about HPV vaccine relative to other adolescent vaccines  among clinical staff from primary care offices and school based clinics.Design: A mixed methods approach used structured interviews to understand/identify potential barriers and facilitators to adolescent HPV vaccination.Population: Participants for the structured interviews were recruited through emails to primary care clinicians  and members of a regional vaccine coalition across the western region of New York State (NYS). This analysis includes only respondents affiliated with primary care offices or non-college school-based health clinics.Measures: Interviewer administered surveys lasting 25 - 30 minutes included closed-and open-ended items as well as self-reported demographics were conducted in 2015. A 5 point Likert scale (ranging from strongly agree to strongly disagree) was used to explore attitudes regarding 1) whether completion of HPV vaccine is more important than completion of MCV4, 2) whether completion of HPV vaccine is more important than completion of Tdap, and 3) whether completion of HPV should be required as is Tdap. Institutional Review Board approval was received for this project.Statistical analyses: Descriptive analyses were conducted using SPSS 21.0 .As shown in As presented in As it relates to results of this study, two recent reviews have examined barriers to HPV vaccination. A US based study noted that clinicians expressed concerns about parent attitudes regarding vaccination while parents reported knowledge gaps and lacked a valid understanding of disease risk versus benefits of vaccination; parents however, did endorse the importance of clinician recommendation in having their child vaccinated . A seconAlthough physician recommendation is considered to be an essential component of getting vaccinated , cliniciOur findings suggest that another contributor to the lack of urgency for HPV vaccination may be misperceptions among clinicians regarding both the risk of HPV infection and severity of HPV-related disease relative to both pertussis (prevented by Tdap vaccine) and bacterial meningitis (prevented by MCV4 vaccine). It could be argued that the array of cancers and pre-cancers associated with HPV infections  is more et al. (2015), used an online survey of pediatricians and family physicians who were recruited through a survey research company; the response rate was 43% (1022/2368) and 76% of respondents (n = 776) were eligible and completed the survey. Nearly 2/3 of respondents described a standardized approach to presenting vaccines to their 11 - 12 year old patients with 73% presenting Tdap vaccine first, compared to meningococcal vaccine (15% first) and HPV vaccine (12% first); conversely, 70% of the time HPV vaccine was discussed last. This suggests that clinicians approach HPV vaccine differently from other recommended adolescent vaccines and it is possible that parents may perceive this hierarchical approach as ascribing less importance to HPV vaccine. Relatedly if physicians are perceived as strongly endorsing completion of Tdap and/or meningococcal vaccines [Similar to the theme identified in our study, a recent report noted comparable findings based upon a different research design . The stu43% 1022/68 and 76vaccines , parentsvaccines .th grade while meningitis vaccine will be required starting in September 2016. While a school mandate would be expected to increase rates of HPV vaccination [School vaccine mandates have been successful in decreasing disease incidence and increasing rates of vaccination . Despitecination , the levThese findings have strong clinical relevance since they are based on the engagement of a group of participants providing clinical care to adolescents. Limitations of this study include its focus on a single geographic region and enrollment of a limited number of participants using a non-random recruitment process. This approach could have resulted in respondent bias favoring the delivery of HPV vaccination. However study results do not support such an enrollment bias, and if present, the true results would likely be even less enthusiastic regarding the importance of HPV vaccination relative to other adolescent vaccines.In conclusion, only 20% and 39% of participants agree or strongly agree that completion of HPV vaccine is more important than completion of Tdap or MCV4 vaccine, respectively. This observation may explain in part reasons why many clinicians seem to prioritize completion of adolescent vaccines other than HPV. These results suggest a potential opportunity to review with clinicians the epidemiology and disease burden attributable to HPV, relative to pertussis and meningitis, as an additional strategy to correct misperceptions and to systematically recommend HPV vaccines to 11 - 12 year olds.