Browsing by Subject "HPV"

Human papillomavirus (HPV) infection of mucosal epithelium by `high-risk' HPV types has a prominent role in the development of anogenital intraepithelial neoplasias and carcinomas. Human epithelial cells transformed with the HPV E6 oncoprotein survive even under conditions that normally lead to cell apoptosis. This phenomenon has been attributed to HPV E6's ability to promote the degradation of the tumor suppressor protein p53. More recently, it has been demonstrated that HPV E6 contributes to activation of the NF-kB pathway. NF-kB is a transcription factor involved in the regulation of genes associated with cellular proliferation, apoptosis and inflammatory responses. In addition to p53 suppression, HPV E6 modulation of NF-kB activation presents another mechanism for HPV-driven tumorigenesis. However, it was not known how HPV E6 promotes NF-kB pathway activation. To address how HPV E6 leads to NF-kB activation, we identified an association between HPV E6 and the human cylindromatosis gene product (CYLD). CYLD is an endogenous inhibitor of canonical NF-kB activation. We showed that HPV E6 proteins could precipitate CYLD in vitro using a co-immunoprecipitation assay. Demonstrating that HPV E6 and CYLD proteins bind each other raised the possibility that this binding relationship would have a functional effect upon the NF-kB pathway by altering CYLD-mediated suppression of NF-kB activation. To identify HPV E6 functional relationship with CYLD and to determine how HPV E6 activates the NF-kB pathway, we transfected cells with either HPV E6 expression vectors containing the high-risk HPV type 16 E6 or the low-risk HPV type 11 E6 along with a CYLD expression vector. We showed HPV16 E6 expression in 293 cells blocked the ability of CYLD to inhibit CD40 ligand-stimulated NF-kB activation. Interestingly, HPV11 E6 was unable to inhibit CYLD mediated suppression of NF-kB in our system. CYLD had previously been shown to suppress NF-kB activation by removing stimulatory lysine 63-linked ubiquitin chains from TRAF2. We found CYLD expression in 293 cells leads to dose-dependent reduction in TRAF2 levels. This CYLD-mediated loss of TRAF2 is inhibited by co-expression of high-, but not low-risk E6 proteins. It was known that CYLD phosphorylation in vivo suppresses CYLD deubiquitination actions of canonical pathway proteins; we therefore tested the extent of CYLD phosphorylation when co-expressed with HPV E6, and discovered that CYLD phosphorylation was increased in the presence of HPV E6. This compilation of experiments suggests that with HPV16 E6 binding to CYLD, the E6 protein blocks CYLD-mediated TRAF2 loss and thereby TRAF2 is available to activate the canonical NF-κB pathway. Blocking HPV E6-mediated NF-kB activation may prove beneficial as a means for designing therapies that inhibit HPV-mediated tumorigenesis. The differences we detected between HPV11 E6 and HPV16 E6 are supported by other studies that showed E6 protein variations account for molecular and clinical differences among HPV infection outcomes. Similarly, there exist intratype E6 variations in HPV16. We obtained cervical specimens from patients with cytopathogenic changes consistent with the onset of cervical dysplasia infected with HPV16 E6 and the human immunodeficiency virus. We hypothesized the immunocompromised individual may harbor unique HPV16 E6 variants. Using PCR detection methods to amplify the HPV16 E6 DNA and sequencing technology, we identified that some of the samples indeed had nucleotide polymorphisms, resulting in amino acid sequence changes. However, the HPV E6 variants we detected were previously described, and fit know geographic HPV clades. Some of the HPV E6 variants we observed are suggested to be associated with progression to cervical cancer, but further evaluation is required.

This project is predicated on the ability of the boranophosphate modification of siRNA to increase its therapeutic applicability for gene silencing in in vitro and in vivo systems. It has been shown that the boranophosphate (BH3-PO3) can overcome many of the limitations that are traditionally found when using RNAi, namely nuclease stability. The synthesis of siRNA modified with 5'-(alpha-P-borano)-nucleoside triphosphates (NTP) analogs alone and in combination with 2'-deoxy-2'-fluoro nucleoside triphosphate analogs were performed and optimized. It was found that normal RNA transcriptions showed the highest yield with higher NTP concentrations and shorter incubation times. Boranophosphate modified RNA and 2'F/borano modified RNA transcription yield was optimal at lower NTP concentrations and extended incubations. The boranophosphate NTPs and RNA were characterized with high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance, indicating successful synthesis of NTPalphaB and 2'F NTPs. PAGE and mass spectrometry analysis were performed to ensure full-length transcription of the modified siRNA molecules. The effects of these modifications were explored with respect to the biophysical properties of the modified homoduplex and heteroduplex siRNA. The techniques used in this work included hybridization affinity assays (melting temperature), secondary structure determination (circular dichroism), nuclease stability assays, and assessment of the lipophilicity of the modified siRNA by determining partition coefficients.

Modification of siRNA with boranophosphate and 2'fluoro/borano modified NTPs appears to have caused the homoduplexes and heteroduplexes to adopt a more B form-like helix that had lower Tm compared to unmodified RNA. The stability of the siRNA transcript to enzymatic hydrolysis by Exonuclease T was on the order of 2'fluoro/borano> normal = boranophosphate. Boranophosphate modification increased the stability of the transcript to enzymatic hydrolysis by the endonuclease RNase A, compared to both normal and 2' fluoro modified siRNA. Overall, the 2' fluoro/borano modified siRNA showed the greatest biological stability. Modification of the siRNA with increasing percentages of boranophosphates resulted in increasing lipophilicity of the molecule up to 60-fold, compared to both normal and 2' fluoro RNA.

A method to site-specifically modify the boranophosphate siRNA using T4 RNA ligase was also investigated. Finally, the siRNA in this work was tested in several in vitro systems, yielding promising results for the usage of boranophosphate siRNA for use against human viruses and cancers. It was shown that in for in vitro systems for human papillomavirus gene expression (HeLa, SiHa, and W12E) and luciferase expression (B16F10 cells), boranophosphate modified siRNA can specifically downregulate gene expression, and in the case of human papillomavirus, can downregulate cell growth.

Introduction: Cervical cancer is one of the leading causes of death for women in Haiti. The purpose of this study was to evaluate the feasibility of HPV self-sampling using CareHPV™ and subsequent treatment with cryotherapy in urban and rural areas of Haiti. CareHPV™ is a vaginal self-sample HPV testing kit used to detect 14 types of high-risk HPV and cryotherapy is a form of treatment that freezes precancerous lesions with CO2 or nitrous oxide. Methods: The study took place in Port-au-Prince and three rural communities within the suburban commune of Leogane. Screening took place at clinics, community centers, and churches. Participants were given consent forms to sign, as well as a demographic questionnaire and an acceptability survey. If their HPV test result was positive, they were called up to three times to go the community clinic for treatment. The number of women that returned for treatment following a positive HPV test result were compared in the urban and rural communities with a chi square test of association and a prevalence rate ratio. Acceptability was measured quantitatively on the Likert Scale. Results: Feasibility was defined as 80% acceptability and 80% treatment uptake. Other factors related to feasibility such as screening numbers and geographical barriers were discussed. Eighty percent acceptability was reached in both rural and urban communities. Eighty percent treatment uptake was only reached in the rural communities, with a treatment uptake of 83.3%. Eighty percent treatment uptake was not reached in the urban communities, with a treatment uptake of 42.1%. The prevalence rate ratio of 1.98 indicates that rural participants were found to be nearly twice as likely to return for treatment than urban participants. The chi square test of association shows that this difference in treatment uptake is significant with an estimated p-value of 0.01 at an alpha of 0.05. Further research is needed to investigate the reasons for higher loss to follow-up for treatment in urban communities to further efforts to establish a national HPV screening program in Haiti.

Abstract

Background: While highly preventable cervical cancer remains a leading cause of

cancer in women globally. Sub-Saharan Africa is disproportionately affected, and in

Kenya specifically, over 4,800 new cervical cancer cases are diagnosed and over 2,000

deaths occur each year. While screening for human papillomavirus (HPV) is a more

cost-effective screening strategy with the potential to increase screening uptake, there is

substantial lost to follow-up (LTFU) for treatment following a positive HPV screen. This

study aimed to identify the predictors of successful treatment acquisition and explore

the barriers and facilitators to seeking treatment among HPV positive women.

Methods: This mixed-methods study was integrated into an ongoing clusterrandomized

trial of implementation strategies in rural western Kenya. This study

randomly selected 100 HPV positive women from the original study database and

conducted a treatment acquisition behavior survey. The study sought a 50/50 ratio of

women who were treated and LTFU, but obtained data from 61 treated women and 39

LTFU women. A subset of 10 women in each group were then selected for in-depth

interviews. Analysis included descriptive statistics to compare treated and LTFU

women’s responses to the survey questionnaire. Interview transcripts were coded and

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analyzed through code-by-code comparisons of women who were treated and women

who were LTFU.

Results: Cost of transportation and distance to the health facility were the most

common challenges in seeking treatment among both treated and LTFU women. Among

women who sought treatment, 67% (n=41) reported that their peers knew their HPV test

result, whereas among LTFU women only 38% (n=15) reported that their peers knew

their HPV test result (p=0.007). There was a significant difference in knowing their peers’

HPV result between treated and LTFU women (p=0.03). Partner support was described

by treated and LTFU women similarly, in that most women reported that they relied on

their partners for transportation money, and that men not understanding the disease

may prevent them from supporting their wives in seeking treatment. Additional barriers

included fear of the treatment process, stigma within their community, logistical

barriers, and lack of information on the disease and treatment. Facilitators to treatment

seeking included peer encouragement, support and encouragement of their children,

involving men in educational sessions, bringing facilities closer and providing

transportation to the health facility.

Conclusions: Cost of transportation, distance to the treatment facility, support of

partners and children, feelings of fear and stress, stigma within the community and

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logistical barriers were reported similarly across treated and LTFU groups. The greatest

disparity between the two groups was a lack of social support among LTFU women.

Given the potential impact of involving men and women in the community in

educational sessions, and promoting treatment seeking in groups, interventions that use

these treatment facilitators are needed.