Human Papillomavirus (HPV) is a key factor in the development of cervical cancer. Out of the more than 100 known HPV types 13 are considered oncogenic. In addition to presence of the virus several other factors have been proposed to influence risk of cervical cancer. This thesis focuses on viral load and HLA class II alleles as risk factors for cervical cancer. To enable quantification of the most common oncogenic HPV types, a real-time PCR-based assay was developed and evaluated in terms of technical sensitivity and specificity. This assay was then employed on archival smears from 457 cases and 552 controls to assess associations between viral load and cervical carcinoma in situ (CIS). Whereas the data indicate a pronounced dose dependent effect of HPV 16 load on the risk of CIS, other HPV types only seem to increase CIS risk at higher viral loads. These effects were observed even when cytology indicated that cells were normal. We then investigated viral load as a risk factor for invasive cervical carcinoma (ICC) in a retrospective study comprising 139 cases and 550 controls. Viral load contributed similarly to the risk of ICC as to the risk of CIS. Finally, associations between HLA class II alleles, viral load and CIS were investigated. Carriers of the DRB1*1301 allele were less prone to infections and high viral loads of HPV 31 and -18/45. Moreover, DRB1*1301 had a protective effect against CIS among women infected by HPV 31 or -18/45. In contrast, carriers of DRB1*1501 and DQB1*0602 were more susceptible to infections and high viral loads of HPV 16. These results indicate that HPV load may have HPV-type specific effects on cervical cancer risk. Furthermore, HLA class II alleles may confer either susceptibility or protection against cervical cancer by acting on the HPV infections preceding tumor development.

Cervical cancer is the second most common cancer among women worldwide and human papillomavirus (HPV) is a prerequisit for the development of this cancer. HPV belongs to the Papillomaviridae family and infects the basal layer of epithelial cells where it generally progresses into warts or condylomas. HPV can only reproduce in differentiating epithelia and it is therefore difficult to study the natural infection of HPV. More than 100 HPV types exist and they are divided into different genera based on their L1 open reading frame sequence. Most of the HPV types in the alpha-papillomavirus genus infect the mucosal epithelium while HPVs from the beta-papillomavirus genus usually infect cutaneous epithelial cells. Presently, it is not known what decides the anatomical tropism and our aim was to study determinants of this tropism. By using HPV virus like particles (VLP) and pseudovirus we found that VLPs from the two alpha-papillomaviruses HPV-6 and HPV-16 interacted with cell-surface heparan sulfate (HS) for initial attachment. When we labelled HPV VLPs with a fluorescent dye to study internalization HPV-6 was more strongly inhibited than HPV-16. Furthermore, a pseudovirus infection assay demonstrated that the beta-papillomavirus HPV-5 was less dependent on HS for infection than HPV-16. By analyzing the isoelectric point (p1) of the HPV L1 capsid protein we found that alpha HPV types were more positively charged than beta HPV types. Also, HPV-6 had a higher positive charge than HPV-16. Thus, the inhibition of the negatively charged heparin against HPV infection was clearly related to the charge of the HPV L1 capsid. This suggested that the initial interaction could be one of the determinants of tropism although not the sole factor. Lactoferrin is a protein found in milk, saliva, semen, tear fluid and endocervical secretions that has antiviral activities. Both human and bovine lactoferrin inhibited HPV infection but we found no significant differences in inhibition of alpha- and beta-papillomavirus infection. We could however demonstrate that different lactoferricins, small peptide derivates from the N-terminal part of lactoferrin, were able to inhibit HPV infection. This antiviral activity depended on lactoferricin peptide, HPV type and cell origin. The regulation of HPV gene expression in the host cell could also determine HPV tropism. The HPV long control region (LCR) contains cis-responsive elements that regulate HPV transcription and the epithelial tropism of HPV is determined by epithelial specific constitutive enhancers in the LCR. It has been hypothesized that the combination of transcription factors in the host cell determines the cell-type-specific expression. In cells with a skin origin the HPV-5 LCR was twice as efficient in transcriptional activation compared to HPV-16 LCR, while in cervical cells the HPV-16 LCR was almost twice as effective in activating transcription compared to HPV-5 LCR. To conclude, alpha- and beta-papillomaviruses differed regarding their ability to infect cells and regulate viral gene expression. These abilities corresponded with their natural host cells and suggested that HPV anatomical tropism could be determined at several steps in the HPV life cycle.

Head and neck squamous cell carcinomas (HNSCCs) most commonly develop in older patients (≥60 years of age) with a history of tobacco and alcohol use. However, young individuals (≤45 years of age) can also develop HNSCC, often without common risk factors. Increasing evidence shows that Human Papillomavirus (HPV) infection is associated with particular HNSCC sites (e.g. oropharynx). We assessed the Roche Linear Array HPV Genotyping Test in several lesions and then examined the prevalence of HPV in HNSCCs from young and older patients. HPV infection was most prevalent in oropharyngeal cancers (16/22, 73%), rarely found in oral cavity cancers (2/53, 4%), and other head and neck sites (1/17, 6%). HPV positive tumors were associated with patients that were >40 and <60 years old (p=0.02). The absence or shortened time of carcinogen exposure from common risk factors and the development of oral squamous cell carcinoma (OSCC) at an early age suggest aberrant genetic events that are different than those in OSSCs from older patients. We used Affymetrix SNP 6.0 arrays to genomically profile oral tumors from young and older patients. Tumors from young patients showed different regions/genes of copy number alterations than those from older patient tumors. An increase of regions of loss of heterozygosity (LOH) in tumors from older patients was observed, and there was a high prevalence of copy number neutral LOH on chromosome 9 in tumors from young and older patients. These data suggest different genetic mechanisms in these patient groups. We have previously shown that HNSCCs from younger patients exhibited a high incidence of microsatellite instability (MSI), a marker of defective mismatch repair (MMR). Deregulated mRNA levels of hPMS1, hPMS2 and hMLH1 were observed and absent/low expression of hPMS1, hPMS2 and hMLH1 protein levels were observed in >50% of OSCCs. No mutations were observed in hPMS1 and hPMS2 and no significant differences of MSI or LOH were observed across genomic loci between tumors of young and older patients. The role of these genetic mechanisms in oral cancer appears complex; studies such as ours should further improve our knowledge of the molecular mechanisms leading to early-onset oral carcinomas.

Head and neck squamous cell carcinomas (HNSCCs) most commonly develop in older patients (≥60 years of age) with a history of tobacco and alcohol use. However, young individuals (≤45 years of age) can also develop HNSCC, often without common risk factors. Increasing evidence shows that Human Papillomavirus (HPV) infection is associated with particular HNSCC sites (e.g. oropharynx). We assessed the Roche Linear Array HPV Genotyping Test in several lesions and then examined the prevalence of HPV in HNSCCs from young and older patients. HPV infection was most prevalent in oropharyngeal cancers (16/22, 73%), rarely found in oral cavity cancers (2/53, 4%), and other head and neck sites (1/17, 6%). HPV positive tumors were associated with patients that were >40 and <60 years old (p=0.02). The absence or shortened time of carcinogen exposure from common risk factors and the development of oral squamous cell carcinoma (OSCC) at an early age suggest aberrant genetic events that are different than those in OSSCs from older patients. We used Affymetrix SNP 6.0 arrays to genomically profile oral tumors from young and older patients. Tumors from young patients showed different regions/genes of copy number alterations than those from older patient tumors. An increase of regions of loss of heterozygosity (LOH) in tumors from older patients was observed, and there was a high prevalence of copy number neutral LOH on chromosome 9 in tumors from young and older patients. These data suggest different genetic mechanisms in these patient groups. We have previously shown that HNSCCs from younger patients exhibited a high incidence of microsatellite instability (MSI), a marker of defective mismatch repair (MMR). Deregulated mRNA levels of hPMS1, hPMS2 and hMLH1 were observed and absent/low expression of hPMS1, hPMS2 and hMLH1 protein levels were observed in >50% of OSCCs. No mutations were observed in hPMS1 and hPMS2 and no significant differences of MSI or LOH were observed across genomic loci between tumors of young and older patients. The role of these genetic mechanisms in oral cancer appears complex; studies such as ours should further improve our knowledge of the molecular mechanisms leading to early-onset oral carcinomas.

Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. The life cycle of this oncogenic DNA tumour virus is strictly associated with the differentiation program of the infected epithelial cells. Expression of the viral capsid genes L1 and L2 can only be detected in the terminally differentiated epithelial cells. The studies here focus on the regulation of HPV-16 late gene expression, which is under tight regulation. Our experimental system consisted of almost the full length HPV-16 genome driven by a strong CMV promoter. This plasmid and mutants thereof could be transfected into HeLa cells and RNA levels monitored. Using this system, we identified an hnRNP A1-dependent splicing silencer between positions 178 and 226 of the L1 gene. This silencer inhibited the use of the 3' splice site, located immediately upstream of the L1 AUG. We speculate that this splicing silencer plays an essential role in preventing late gene expression at an early stage of the viral life cycle. We subsequently identified a splicing enhancer located in the first 17 nucleotides of L1 that may be needed to counteract the multiple hnRNP A1 dependent splicing silencers in the L1 coding region. A 55kDa protein specifically bound to this splicing enhancer. We also demonstrated that binding of the cellular factors to the splicing silencer in the L1 coding region had an inhibitory effect on expression from L1 cDNA expression plasmids. The HPV-16 genome is divided into the early region and the late region, separated by the early poly(A) signal (pAE). pAE is used preferentially early in infection, thereby efficiently blocking late gene expression. We demonstrated that a 57 nucleotide U-rich region of the early 3’untranslated region (3’eUTR) acted as an enhancing upstream element on the usage of pAE. We demonstrated that this U-rich region specifically interacts with hFip1, CstF-64, hnRNP C1/C2 and PTB, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 pAE. In conclusion, two regulatory RNA elements that both act to prevent late gene expression at an early stage in the viral life cycle and in proliferating cells were identified: a splicing silencer in the late region and an upstream u-rich element at the pAE.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 71-78).

To learn how to address health issues specific to Hispanic cultures in an effort to address health disparities, learning from cultural aspects that affect health from the countries of origin would be most useful. Community programs built on rigorous and systematic research prove to be more powerful than ad-hoc programs. Qualitative research techniques offer powerful alternatives for public health professionals to develop adequate and directed programs at the community level, especially among underserved communities and those represented by oral/spoken traditions. The study was conducted among 132 working class single and married Mestizo women between the ages of 20-40 living in Panama City, Panama. This group of women has the highest incidence of HPV in Panama so are at the highest risk of cervical cancer. Using social construction as the theoretical framework, this study uses four different qualitative research techniques: free listing, pile sorting, individual semi-structured, and group interviews. Key findings include the importance of religion and family, women's understandings of the relationship between sexuality and health, influence of media, other women, and husbands help construct screening knowledge among women in the study. Culturally relevant health education interventions and programs delivered in a group format at the community level in a participatory mode would be most effective in reaching women in Panama and other Hispanic populations. Future quantitative studies and influences of social networks are suggested.

Background: Coinfections with multiple types of human papillomavirus (HPV) are a common occurrence among HPV-infected individuals, but the clinical significance and etiology of these infections remain unclear. Though current evidence suggests that women with coinfections have increased HPV exposure (i.e. more sexual partners), it is also hypothesized that these women may represent a subgroup with increased HPV susceptibility, though this has been rarely studied to date. Purpose: The purpose of this project was to examine the occurrence, determinants and dynamics of HPV coinfections in a cohort of university students in order to explore the relationship(s) between coinfections, lifestyle factors and immunological susceptibility. Methods: This project is based on a secondary analysis of data from the McGill-Concordia Cohort, a longitudinal study of the natural history of HPV infection in 621 female university students in Montreal, Quebec. Participants were followed for 2 years at 6-month intervals. At each visit, cervical specimens were collected for cytology and HPV testing, and women completed a questionnaire about lifestyle and risk behaviours. Two definitions of coinfections were used: cumulative coinfection over follow-up and concurrent coinfection at each visit. Kaplan-Meier techniques were used to estimate incidence and duration of coinfections and multiple logistic regression was used to identify determinants of coinfections and associations between coinfections and squamous intraepithelial lesions (SIL). Results: More than half of the cohort became infected with HPV and of those, over 60% acquired multiple HPV types over follow-up. Incidence of coinfections was significantly increased among HPV-infected women at enrollment. The most important determinant of coinfection occurrence was number of sexual partners (both lifetime and new), though some genes of the immune response (HLA-DQB1*06:02, HLA-G*01:01:03 and HLA-G*01:01:05) were also significant predictors. Women with coinfections, particularly those with 4+ HPV types, also had longer infection durations and greatly increased odds of SIL. Conclusions: Women with coinfections acquire new HPV types at an increased rate and have greater HPV persistence and occurrence of SIL, which may indicate immunological susceptibility. HPV coinfections mainly occur due to increased sexual activity but a decreased immune response to the virus may also be involved in a subset of women. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2011-04-21 01:04:26.265

The overall aim of this thesis is to provide a better understanding of knowledge, attitudes, consent, and decision-making regarding Human papillomavirus (HPV) vaccination, seen from the perspectives of concerned parties – high school students, school nurses, and parents. Two quantitative studies were performed: one descriptive cross-sectional study and one quasi-experimental intervention study. Qualitative studies using focus group interviews and individual interviews were also performed. High school students’ knowledge about HPV and HPV prevention was low but their attitudes toward HPV vaccination were positive. An educational intervention significantly increased the students’ knowledge regarding HPV and HPV prevention. Their already positive attitudes toward condom use and HPV vaccination remained unchanged. The students wanted to receive more information about HPV from school nurses. The school nurses were also positive to HPV vaccination but identified many challenges concerning e.g. priorities, obtaining informed consent, culture, and gender. They saw an ethical dilemma in conflicting values such as the child’s right to self-determination, the parents’ right to make autonomous choices on behalf of their children, and the nurse’s obligation to promote health. They were also unsure of how, what, and to whom information about HPV should be given. Parents, who had consented to vaccination of their young daughters, reasoned as follows: A vaccine recommended by the authorities is likely to be safe and effective, and the parents were willing to do what they could to decrease the risk of a serious disease for their daughter. Fear of unknown adverse events was overweighed by the benefits of vaccination. Parents also saw the school nurse as an important source of HPV information. Conclusions: Positive attitudes toward HPV vaccination despite limited knowledge about HPV, are overarching themes in this thesis. School nurses have a crucial role to inform about HPV prevention. It is important that the concerned parties are adequately informed about HPV and its preventive methods, so that they can make an informed decision about vaccination. A short school-based intervention can increase knowledge about HPV among students. From a public health perspective, high vaccination coverage is important as it can lead to a reduced number of HPV-related disease cases.

Cervical cancer is the forth most commonly diagnosed cancer among women worldwide. It is caused by persistent infection with an oncogenic type of Human Papillomavirus (HPV). The HPV is a necessary but not sufficient cause of cervical cancer. Environmental factors such as smoking, high parity and long-term use of oral contraceptives increases the risk of cervical cancer. Genetic factors also affect the risk of developing the disease. The aim of this thesis is to search for and evaluate genetic risk factors for cervical cancer using both a candidate gene approach and a genome-wide association study (GWAS). Paper I examined the association of genetic variation in three Fanconi Anemia (FA) genes (FANCA, FANCC and FANCL), involved in DNA repair, with cervical cancer susceptibility in the Swedish population. No association was observed. Paper II evaluated the association of genetic variation in the TMC6 and TMC8 genes with susceptibility to cervical cancer in the Swedish population and an association of two SNPs (rs2290907 and rs16970849) with cervical cancer was observed.  In paper III the first GWAS performed in cervical cancer was reported. Three independent loci in the major histocompatibility complex (MHC) region at 6p21.3 were found to affect the susceptibility to cervical cancer. Paper IV examined the sequence variation in the TMC6 and TMC8 region and its association with cervical cancer. A highly polymorphic 21 bp sequence was identified and found to be repeated 5 to 42 times in both cases and controls. Lack of this repeat was associated with increased risk of cervical cancer. An intronic SNP (rs2926778) located in between the TNRC6C and TMC6 genes was also found to be associated with cervical cancer. The thesis provides evidence for the importance of genes in the immune system for cervical cancer susceptibility. The genetic risk factors identified explain only a part of the genetic susceptibility, implying that other risk factors remains to be identified