Objective: To review evidence on the efficacy of HPV vaccines in the prevention of non-cancer lesions (anogenital warts [AGW], recurrent laryngeal papillomatosis and oral papillomatosis). Materials and methods: We conducted a systematic review of randomized trials. We performed random effect models and effects were reported as relative risks (RR) and their confidence intervals (95%CI) following both intention to treat (ITT) and per protocol (PP) analyses. Results: We included six studies (n=27 078). One study was rated as high risk of bias. One study could not be included in the meta-analysis because it provided combined results. We found that quadrivalent vaccine reduced the risk of AGW by 62% (RR: 0.38, 95%CI:0.32-0.45, I2:0%) in the ITT analysis and by 95% (RR: 0.05, 95%CI:0.01-0.25, I2:66%) in the PP analysis. Subgroup analyses of studies in women or with low-risk of bias provided similar results. Conclusion: HPV quadrivalent vaccine is efficacious in preventing AGW in men and women. / HPV quadrivalent vaccine is efficacious in preventing AGW in men and women. / Revisión por pares

Indiana University-Purdue University Indianapolis (IUPUI) / Women who are medically underserved may be less likely to obtain provider-based cervical cancer screening because of structural and intrapersonal barriers. Self-collection for human papillomavirus (HPV) testing, a method for women to collect their own sample through a vaginal swab or urine collection, has accuracy comparable to provider-based cervico-vaginal HPV testing and may be useful in overcoming barriers to provider-based cervical cancer screening. The purpose of this dissertation study is to examine factors associated with self-collection for HPV testing, psychometrically test Diffusion of Innovations (DOI) instruments, and identify preferences for self-collection for HPV testing. Three distinct aims were developed: 1) factors (sociodemographic, health-related, and theoretical variables) associated with mailed return of vaginal self-collection for HPV testing, 2) psychometric examination of DOI ÷instruments (relative advantages and complexity), and 3) dimensions of self-collection and characteristic preferences to self-collection for HPV testing stratified by age cohorts. This dissertation involved two cross-sectional studies. In Chapters 2-3, data were collected from women (n=168) at food pantries and online. Women were eligible if they were: 1) female, 2) between the ages of 30-65, 3) could read and speak English, and 4) at 2019 federal poverty guidelines for income and family size as defined by the Department of Health and Human Services. Women were not eligible if they had a history of hysterectomy or were adherent to cervical cancer screening guidelines. Logistic regression analyses, item analysis, Cronbach’s alpha, exploratory factor analysis, and tests were used to analyze data. Chapter 4 involved collection of data from an online survey with a sample (n=878) provided by Dynata. Participants evaluated 9 scenarios that varied along 4 attributes: HPV self-collection kit type (vaginal swab or urine collection), HPV self-collection kit delivery (mail, pharmacy pick-up, or clinic pick-up), HPV self-collection kit return (mail, pharmacy drop-off, or clinic drop-off), and HPV test result communication (mail, phone call, or text message). Ratings-based conjoint analysis (RBCA) determined how each attribute influenced the ratings of each scenario.

No description available.

Human papillomavirus (HPV) is a sexually transmitted disease that often presents as genital warts, but may also lead to cancers, including those of the vagina, penis, mouth and tonsils. Despite three vaccines being currently available to prevent HPV, the HPV vaccine retains a low national average vaccination rate when compared to the Tetanus-Diptheria- Pertussis (Tdap) vaccine. Considering the need for improvement it is important to identify factors that may be contributing to this low national immunization rate, one of them being parental race. The purpose of this literature review is to identify whether race affects parents' intent to vaccinate their children against HPV. A database search of CINAHL Plus with Full Text, MEDLINE, and PsycINFO was conducted and a total of 13 articles were reviewed based on the relevance to the purpose of the literature review. While racial differences were noted, there were other factors that also affect a parent's intent to vaccinate their children against HPV. There is more research to be done when looking at how race may independently affect a parent's intent to vaccinate their children against HPV.

Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The link between infection with Human Papillomavirus (HPV) and the development of cervical cancer has been established by several epidemiology studies. Cervical cancer is the second most common cancer among women and it occurs at a rate of 22.8 cases per 100 000 women in South Africa. Approximately 86% of newly reported cases of cervical cancer occur in developing countries where limited access to medical facilities hampers efforts to prevent and screen for HPV infection. Two commercial virus-like particle (VLP) vaccines consisting of HPV major structural protein L1, which protect against the most common high-risk HPV-types, are currently available. The high cost and type specificity of these commercially available vaccines have necessitated the development of a low cost, broad-spectrum HPV vaccine. Inclusion of the minor structural protein L2 has been shown to induce broadly cross-neutralizing antibodies and therefore a chimera was constructed that contains an epitope of L2 inserted within the L1 sequence. This construct, renamed SAF, was shown to be highly immunogenic and thus has the potential to be used as a prophylactic cervical cancer vaccine. Methylotrophic yeasts are known to be excellent producers of recombinant proteins due to their strongly inducible promoters that allow culturing of these yeasts to very high cell densities. Pichia pastoris and Hansenula polymorpha have been employed in several studies for heterologous protein production and levels of protein higher than 1 g/L have been reported. These yeasts also have GRAS status and can therefore be used to manufacture products for use in humans. In this study, the potential of H. polymorpha and P. pastoris to produce SAF intracellularly was evaluated. The effect of increased gene dosage and peroxisomal targeting on SAF production was examined as possible strategies to increase the yield of SAF. Peroxisomal targeting was achieved by fusing the SAF gene at the C-terminal end with the Peroxisomal Targeting Sequence 1 (PTS1) which consists of a short tri-peptide: –SKL. The functionality of PTS1 was confirmed using green fluorescent protein (GFP), fluorescence microscopy and peroxisome isolation. Peroxisomal targeting was shown to have a negative effect on SAF production levels in both H. polymorpha and P. pastoris. An increase in gene dosage had no discernable effect on SAF yield in H. polymorpha which is in contrast to previous research. The highest production levels were achieved by P. pastoris KM71 (24.86 mg/L) which compares well to levels of L1 achieved by other research groups. The most significant insight emerging from this work was that all the strains that produced SAF at detectable levels were equally efficient at the production of SAF. Increased biomass was therefore the biggest contributor to high SAF levels (mg/L) in the P. pastoris strains as significantly higher cell densities were achieved during culturing of these strains. With the necessary optimisation, the methylotrophic yeasts have the potential to be used as hosts for the production of a broad-spectrum HPV vaccine. / AFRIKAANSE OPSOMMING: Die skakel tussen infeksie met Mens Papilloomvirus (HPV) en die ontwikkeling van servikale kanker is deur verskeie epidemiologiese studies bevestig. Servikale kanker is die tweede mees algemene kanker onder vroue en dit kom voor teen ‘n tempo van 22.8 gevalle per 100 000 vroue in Suid Afrika. Ongeveer 86% van alle nuwe gevalle kom voor in ontwikkelende lande waar beperkte toegang tot mediese fasiliteite pogings om HPV infeksie te voorkom en te behandel, belemmer. Twee pseudovirale-partikel (VLP) entstowwe teen HPV is tans op die mark beskikbaar en hierdie entstowwe verleen immuniteit teen die mees algemene hoë-risiko HPV tipes. Die hoë koste en nou spektrum van hierdie entstowwe het dit nodig gemaak om ‘n goedkoop, wye-spektrum HPV entstof te ontwikkel. Navorsing het bewys dat die insluiting van die strukturele L2 proteïen in die VLP entstof, lei tot die indusering van neutraliserende teenliggame, wat wye spektrum antigenisiteit tot gevolg het. ‘n Chimeriese proteïen wat ‘n epitoop van L2 binne die L1 volgorde bevat is gekonstrueer, en hierdie proteïen is benoem SAF. SAF het hoë immunogenisiteit en kan dus potensieel as ‘n voorkomende servikale kanker entstof gebruik word. Metielotrofiese giste is bekend vir hulle vermoë om hoë vlakke rekombinante proteïene te produseer as gevolg van hulle induseerbare promotors wat groei tot baie hoë sel digthede toelaat. Pichia pastoris en Hansenula polymorpha is in menigte studies gebruik om heteroloë proteïene te produseer tot vlakke bo 1 g/L. Hierdie giste en die proteïen produkte wat hulle vorm word algemeen aanvaar as veilig vir menslike gebruik. In hierdie studie het ons die potensiaal van H. polymorpha en P. pastoris om SAF intrasellulêr te produseer, geevalueer. Die effek op SAF produksie van verhoogde geen dosering asook die teiken van SAF na die peroksisoom was ondersoek as moontlike strategieë om die opbrengs van SAF te verhoog. Die teiken van SAF na die peroksisoom is behaal deur die Peroksisomale Teiken Volgorde 1 (PTS1) aan die C-terminaal van SAF te heg. Die funksionaliteit van PTS1 was bevestig deur gebruik te maak van groen fluoroserende proteïen (GFP), fluoressensie mikroskopie en isolering van peroksisome. Teiken van SAF na die peroksisoom het ‘n negatiewe uitwerking gehad op SAF uitdrukking in beide H. polymorpha en P. pastoris. ‘n Verhoging in geen dosering het geen onderskeibare effek gehad op SAF opbrengs in H. polymorpha nie wat in teenstelling is met vorige navorsing. Die hoogste produksie vlakke is opgelewer deur P. pastoris KM71 (24.86 mg/L) wat goed vergelyk met vlakke van L1 wat deur ander navorsings groepe behaal is. Die belangrikste gevolgtrekking wat gemaak kan word uit hierdie studie is dat al die rasse wat SAF geproduseer het in meetbare hoeveelhede ewe effektief was. Verhoogde biomassa was dus die grootste bydraende faktor tot hoë SAF vlakke (mg/L) in die P. pastoris rasse as gevolg van die hoë sel digthede wat hierdie rasse kan bereik. Dit is duidelik dat metielotrofiese giste, met die nodige optimisering, oor die potensiaal beskik om as gasheer sisteme te dien vir die produksie van ‘n wye spektrum HPV entstof. / The NRF and the Department of Microbiology for financial support

In addition, we investigated the cytotoxic effect of a widely used chemotherapeutic agent 5Fu on laryngeal squamous cell cancer cell lines and evaluated the role of p53 in 5Fu treatment. We found that the apoptosis and G1/S cell arrest mediated by 5Fu in laryngeal cancers is p53-independent but p21 WAF1/CIP1-dependent. We further demonstrated the effect of 5Fu on HPV16-associated laryngeal cancer cells. Using cytotoxicity assay and Annexin V staining, we proved that 5Fu induces apoptosis in all of the transfected cells in a dose- and time-dependent manner, suggesting that the process was not prevented by HPV16 E6 or E7. 5Fu induced the accumulation of active pRb and cyclin dependent kinase inhibitor p21WAF1/CIP1 together with an increase in Bak and Bax expression and a decrease in Bcl-2 levels in all the transfected cells. In addition, G1/S phase cell cycle arrest was associated with the antiproliferation activity of 5Fu in all cell lines. Through RT-PCR, 5Fu also presented some effects on the E6 and E7 oncoproteins of HPV16 in transfected UMSCC 12 cells. / Our results suggest that HPV16 E6 and E7 oncoproteins do not prevent 5Fu medicated apoptosis and G1/S cell arrest in laryngeal cancers. The anti-cancer effect of 5Fu is probably decided by the level of p21 WAF1/CIP1 while the sensitivity of laryngeal cancer cells responded to 5Fu treatment is associated with the increase of Bak or/and the decrease in Bcl-2, not with the HPV16 viral proteins and p53 status. 5Fu also presented some effects on the E6 and E7 oncoproteins of HPV16 in laryngeal cancer. However, the anti-viral effect of 5Fu still needs further investigation. / Our study indicated that (1) the evasion of apoptosis mediated by HPV16 E6 and E7 plays a critical role in laryngeal carcinogenesis; (2) HPV16 E6 or E7 plays an important role in regulating the expression of Bak, Bax and Bcl-2; (3) The degradation of Bak by HPV16 E6 is not caused by interacting with the promoter of Bak; (4) The induction of Bcl-2 is mediated through HPV16 E7; (5) HPV16 transfection does not interfere with the apoptosis and cell cycle arrest mediated by 5Fu in human laryngeal squamous cancer cells. / There is a growing body of evidence that human papillomavirus type 16 (HPV16) is involved in the development of human laryngeal cancer, especially in Chinese population. The two oncoproteins, HPV16 E6 and E7 that target host cell tumor suppressor proteins p53 and Rb respectively, may generate antiapoptotic effects and induce cell immortalization. However, the effect of both oncoproteins on apoptosis in laryngeal cancers is not completely clear. In this study, we demonstrated the possible mechanism of high risk HPV16 in laryngeal carcinogenesis and evaluated the effect of 5Fu on HPV16-positive laryngeal cancer cells. / We employed two human laryngeal cancer cell lines---UMSCC12 (with truncated p53) and UMSCC11A (with mutant but functional p53) in this study. These two cell lines were stably transfected with HPV16 E6, E7 or empty vector, pcDNA3.1, which provided a good foundation for further study on the carcinogenic mechanism of HPV16 E6 or E7 in human laryngeal cancers. Through Annexin V staining and protein stability assay, we found that the transfection of HPV16 E6 and E7 induced fewer spontaneous apoptosis in both UMSCC11A and UMSCC12 cells accompanied with enhanced protein stability of Bcl-2 and increased protein degradation of Bak. Similar results were obtained when E6- and E7-transfected cells exposed to apoptosis stimuli---TNF-alpha/CHX. These results indicate that stable transfection of E6 and E7 in human laryngeal cancer cells on one hand shortened the half-life of Bak protein, and on the other hand, enhanced the steady-state levels of Bcl-2 protein. In order to gain insight into the role of Bak and Bcl-2 in regulating apoptosis in HPV-associated laryngeal cancer cells, we performed transient transfection of Bcl-2 into E6- and E7-transfected cells. It is found that HPV16 E7 statistically enhanced the expression of Bcl-2 in laryngeal cancer, indicating that the induction of Bcl-2 require the transfection of HPV16 E7. Furthermore, Luciferase assay was performed to investigate whether the viral proteins E6 and E7 altered the stability of Bak through interaction with the promoter of Bak. Negative results were obtained, suggesting that E6 or E7 do not alter the transcription activity of Bak, indicating the degradation of Bak by E6 or E7 may be mediated through other mechanisms. / Liu Han-ching. / "August 2006." / Advisers: C. A. van Hasselt; George G. Chen. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1569. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 245-274). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Les papillomavirus humains (HPV), dont on dénombre plus de 300 types différents, infectent spécifiquement les épithéliums. Ces infections sont communes et généralement asymptomatiques. Cependant, lorsqu’elles persistent, elles peuvent donner lieu à des lésions bénignes, telles que les verrues, ou cancéreuses, telles que le cancer du col de l’utérus. Les facteurs de l’hôte impliqués dans la persistance et la pathogénie des infections par les HPV restent largement méconnus. Les premières évidences du rôle de l’axe de signalisation CXCL12/CXCR4 dans la pathogénie virale proviennent d’observations faites dans le contexte d’un déficit immunitaire rare, le syndrome WHIM. En effet, ce syndrome est dû à des dysfonctions de l’axe CXCL12/CXCR4 − causées par des mutations de CXCR4 conduisant à un gain de fonction de l’axe CXCL12/CXCR4 − et est caractérisé par une susceptibilité sélective des patients à des infections sévères, persistantes et parfois malignes par les HPV. Au vu de cette susceptibilité, l’objectif de ma thèse a été d’approfondir cet éventuel lien causal entre les dysfonctions de l’axe CXCL12/CXCR4 et la pathogenèse associée aux infections par les HPV et de caractériser les mécanismes moléculaires en jeu.Afin de répondre à cette problématique, je me suis intéressée dans la première partie de mes travaux de thèse aux conséquences des dysfonctions de l’axe CXCL12/CXCR4 − à travers le gain de fonction de CXCR4 associé au syndrome WHIM − sur le cycle biologique d’HPV18 étudié dans des cultures organotypiques épithéliales tridimensionnelles. Ces travaux nous ont permis de mettre en évidence que les dysfonctions de CXCR4 limitaient la production virale au profit de la mise en place d’un processus de transformation cellulaire. Les mécanismes en jeu impliquent une augmentation de la prolifération cellulaire et un changement du profil d’expression des protéines virales en faveur des oncoprotéines et au détriment de celles impliquées dans la réplication virale.Dans la seconde partie de mes travaux, je me suis attachée à déterminer les effets du blocage de l’axe CXCL12/CXCR4 dans un modèle murin de néoplasie épithéliale induite par HPV16 (souris K14-HPV16). Le traitement de ces souris par l’AMD3100, un antagoniste sélectif de CXCR4, induit une tendance à la normalisation se manifestant par une diminution significative de l’hyperplasie induite par HPV16. Cet effet est associé à une réduction de l’hyperprolifération des kératinocytes et de l’infiltrat de cellules immunitaires dans le derme.En conclusion, ce travail de thèse identifie l’axe CXCL12/CXCR4 comme un facteur de l’hôte impliqué dans la carcinogenèse induite par les HPV, et révèle le bénéfice de stratégies thérapeutiques basées sur le blocage de cet axe. / Human papillomaviruses (HPVs), which encompass almost 300 different types identified so far, specifically infect epitheliums. Most of the time, HPVs are associated with asymptomatic infections suggesting an efficient control by the host immune system. However, when these infections persist, HPVs can cause cutaneous warts but also mucosal lesions that can progress to dysplasia and cancer (e.g. cervical cancers). The host factors involved in HPV persistence and derived-pathogenesis remain quite obscure. The first evidence for a role of the CXCL12/CXCR4 signaling axis in HPV pathogenesis came from observations made in the context of a rare immunodeficiency disorder, the WHIM syndrome. This syndrome is caused by dysfunctions of the axis formed by the chemokine CXCL12 and its receptor CXCR4 – caused by inherited heterozygous mutations in CXCR4 leading to a gain-of-function of the CXCL12/CXCR4 axis – and featured by a high susceptibility to severe, persistent and sometimes malignant HPV infections. In light of this susceptibility, the aim of my thesis was to characterise the molecular mechanisms involved and to find out whether it extend to a more general interplay between the CXCL12/CXCR4 axis and HPV biological cycle and pathogenesis.In the first part of my work, I investigated the consequences of CXCL12/CXCR4 dysfunctions – through the CXCR4 gain-of-function – on the HPV18 life cycle in three-dimensional organotypic epithelial cultures. We found that CXCR4 dysfunctions limited the viral replication at the benefit of cell transformation. The mechanisms included an increased in cell proliferation and a change in viral protein expression profile in favour of oncoproteins and at the expense of proteins involved in viral replication.In the second part of my work, I determined the impact of the CXCL12/CXCR4 blockade on a murin model of HPV16-induced neoplasia (K14-HPV16 mice). Treatment of these mice by AMD3100, a selective antagonist of CXCR4, results in a normalisation of HPV-induced lesions manifested by a significant decrease of skin hyperplasia. This effect is associated with a reduction in keratinocyte hyperproliferation and immune cell infiltration in dermis.To conclude, this thesis work identifies the CXCL12/CXCR4 axis as a new host factor involved in human papillomavirus-induced carcinogenesis, and reveals the benefit of therapeutic strategies based on the blockade of this axis.

La persistance d'une infection par un même papillomavirus humain à haut risque oncogène (HPV-HR) est le principal facteur de risque d'apparition et de progression des lésions précancéreuses et cancéreuses du col de l'utérus. La recherche des lésions précancéreuses et l'introduction des tests basés sur la détection des HPV-HR dans le dépistage ont permis de réduire l'incidence du cancer du col de l'utérus dans les pays développés. Un des enjeux actuels réside dans l'identification de nouveaux marqueurs qui permettraient d'améliorer le dépistage. Dans ce contexte, les objectifs de cette thèse étaient d'explorer l'histoire naturelle de l'infection par HPV-HR au travers de facteurs tels que la charge virale, l'intégration, l'expression des oncogènes viraux ou encore la méthylation du promoteur viral à partir de prélèvements biologiques et d'évaluer l'intérêt de ces facteurs en pratique clinique. Après optimisation et validation d'outils de biologie moléculaire, l'analyse de frottis cervico-utérins représentatifs de la progression des lésions du col de l'utérus a permis de mieux comprendre l'histoire naturelle de l'infection transformante par HPV16. Nos données confirment la valeur diagnostique de la charge virale, indiquent une méthylation tardive du promoteur viral et suggèrent que les niveaux d'expression des gènes viraux ne sont pas suffisamment robustes pour présenter un intérêt clinique. D'autre part, nos travaux ont mis en évidence de potentielles analogies de comportement virologique entre génotypes apparentés. Enfin, la caractérisation de tumeurs du canal anal à l'aide de nos marqueurs viraux a permis d'identifier une signature moléculaire des cancers associés à HPV16. / The main risk factor of precancerous and cancerous cervical lesions is a persistent infection with the same high-risk HPV (HR-HPV). Screening based on precancerous lesions and HR-HPV detection considerably reduced cervical cancer incidence in developed countries. A current challenge remains the identification of new biomarkers in order to improve cervical cancer screening. This work was conducted with the purpose of (i) exploring HR-HPV infection natural history through viral factors such as viral load, integration, viral oncogene expression and viral promoter methylation and (ii) evaluating the clinical value of these potential markers. After optimization and validation of several molecular biology methods, the analysis of cervical smears representative of the full spectrum of cervical diseases allowed a better knowledge of transforming HPVI6-infection natural history. Our data confirm the potential diagnostic value of viral load. show that HPVI6 promoter methylation seems to be a late event in cervical carcinogenesis, and suggest that viral oncogene expression levels are not robust enough to be transferred to clinical practice. Moreover, our data highlight similar molecular behavior between closely related HR-HPV genotypes. Finally, the characterization of anal tumors with markers identified a molecular signature pattern of HPVI6-associated tumors

Fung, Man See Joyce. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 111-120). / Abstracts in English and Chinese. / Table of Contents / Acknowledgements --- p.I / Abstract --- p.II / 論文摘要 --- p.VII / Table of Contents --- p.X / List of Figures --- p.XIV / List of Tables --- p.XVI / Abbreviations --- p.XVII / Chapter Chapter 1 - --- Introduction --- p.l / Chapter 1.1 --- Biology of HPV --- p.2 / Chapter 1.1.1 --- History --- p.2 / Chapter 1.1.2 --- Classification --- p.2 / Chapter 1.1.3 --- Genome structure --- p.3 / Chapter 1.2 --- HPV and cervical cancer --- p.8 / Chapter 1.2.1 --- Classification of cervical lesions --- p.8 / Chapter 1.2.2 --- Natural history of development of cervical cancer --- p.9 / Chapter 1.2.3 --- Risk factors --- p.11 / Chapter 1.3 --- Prevention of cervical cancer --- p.12 / Chapter 1.3.1 --- Vaccination --- p.12 / Chapter 1.3.2 --- Screening --- p.12 / Chapter 1.3.2.1 --- Pap test --- p.12 / Chapter 1.3.2.2 --- HPV DNA test --- p.13 / Chapter 1.3.2.3 --- Methylation pattern as a novel marker --- p.13 / Chapter 1.4 --- Biology of Methylation --- p.14 / Chapter 1.4.1 --- Definition --- p.14 / Chapter 1.4.2 --- Silencing effect --- p.18 / Chapter 1.4.3 --- Roles in normal development --- p.20 / Chapter 1.5 --- Methylation and human diseases --- p.20 / Chapter 1.5.1 --- Genetic diseases --- p.20 / Chapter 1.5.2 --- Cancers --- p.21 / Chapter 1.5.3 --- Methylation and oncogenic viruses --- p.23 / Chapter 1.5.4 --- Potential of methylation pattern as a novel biomarker of cancer --- p.24 / Chapter 1.5.5 --- Epigenetic therapy --- p.25 / Chapter 1.6 --- Methylation and HPV --- p.25 / Chapter 1.6.1 --- History --- p.25 / Chapter 1.6.2 --- Potential roles in transcription regulation of HPV --- p.26 / Chapter 1.6.3 --- Viral gene methylation --- p.27 / Chapter Chapter 2 - --- "Hypotheses, Objectives and Study Design" --- p.28 / Chapter 2.1 --- Hypotheses --- p.29 / Chapter 2.2 --- Objectives --- p.30 / Chapter 2.3 --- Study Design --- p.30 / Chapter Chapter 3 - --- Materials and Methods --- p.34 / Chapter 3.1 --- Work flow --- p.35 / Chapter 3.2 --- Study subjects --- p.37 / Chapter 3.2.1 --- Invasive cervical cancer group --- p.37 / Chapter 3.2.2 --- Low-grade group --- p.37 / Chapter 3.2.3 --- Cell lines --- p.38 / Chapter 3.3 --- DNA extraction --- p.38 / Chapter 3.4 --- HPV genotyping --- p.39 / Chapter 3.5 --- PCR of HPV16 LCR --- p.39 / Chapter 3.6 --- Sequencing of HPV 16 LCR --- p.42 / Chapter 3.6.1 --- Purification of PCR products --- p.42 / Chapter 3.6.2 --- Cycle sequencing reaction --- p.42 / Chapter 3.6.3 --- Purification of cycle sequencing products --- p.43 / Chapter 3.6.4 --- Sequencer and data analysis --- p.43 / Chapter 3.7 --- Bisulfite modification --- p.43 / Chapter 3.8 --- PCR of bisulfite modified LCR --- p.45 / Chapter 3.9 --- Cloning --- p.48 / Chapter 3.9.1 --- Ligation --- p.48 / Chapter 3.9.2 --- Transformation --- p.48 / Chapter 3.9.3 --- Colony PCR --- p.49 / Chapter 3.10 --- Sequencing of clones --- p.51 / Chapter 3.10.1 --- Purification of PCR products --- p.51 / Chapter 3.10.2 --- Cycle sequencing reaction --- p.51 / Chapter 3.10.3 --- Purification of cycle sequencing products --- p.52 / Chapter 3.10.4 --- Sequencer and data analysis --- p.52 / Chapter 3.11 --- Statistical methods --- p.52 / Chapter Chapter 4 - --- Results --- p.54 / Chapter 4.1 --- Sample selection --- p.55 / Chapter 4.2 --- HPV16 LCR PCR and sequencing --- p.57 / Chapter 4.3 --- Methylation patterns --- p.61 / Chapter 4.3.1 --- Cell lines --- p.61 / Chapter 4.3.2 --- Cancer group --- p.63 / Chapter 4.3.2.1 --- Overview --- p.63 / Chapter 4.3.2.2 --- Methylation pattern of the cancer samples --- p.66 / Chapter 4.3.2.3 --- Methylation pattern of the promoter region --- p.74 / Chapter 4.3.3 --- Low-grade group --- p.76 / Chapter 4.3.3.1 --- Overview --- p.76 / Chapter 4.3.3.2 --- Methylation pattern of the low-grade samples --- p.79 / Chapter 4.3.4 --- Comparison of the methylation patterns of the cancer samples and the low-grade samples --- p.84 / Chapter Chapter 5 - --- Discussion --- p.95 / Chapter 5.1 --- Sequence variations of HPV 16 LCR --- p.96 / Chapter 5.2 --- Methylation patterns of CaSki and SiHa cell lines --- p.98 / Chapter 5.3 --- Methylation pattern of the cancer samples --- p.99 / Chapter 5.4 --- Methylation pattern of the low-grade samples --- p.100 / Chapter 5.5 --- Comparison of methylation patterns of the cancer samples and the low-grade samples --- p.101 / Chapter 5.5.1 --- Promoter region in 3' LCR --- p.102 / Chapter 5.5.1.1 --- SP1 binding site --- p.102 / Chapter 5.5.1.2 --- E2BS3 and E2BS4 --- p.103 / Chapter 5.5.2 --- Silencer region --- p.104 / Chapter 5.5.3 --- Enhancer region in central LCR --- p.105 / Chapter 5.5.4 --- CpG sites within 5' LCR --- p.106 / Chapter 5.6 --- Role of methylation in HPV 16 --- p.107 / Chapter 5.7 --- Potential as novel biomarker --- p.108 / Chapter 5.8 --- Conclusions --- p.109 / References --- p.111 / Appendix A

Le cancer du col utérin est précédé par des lésions prénéoplasiques. Celles-ci sont associées dans plus de 95% des cas à une infection par un papillomavirus (HPV). Un phénomène fréquent durant la cancérogenèse cervicale est l'intégration du génome dun HPV oncogène dans lADN cellulaire. Celle-ci entraîne une expression sélective de gènes codant pour des oncoprotéines virales (appelées E6 et E7) capables d'inactiver les produits de certains gènes suppresseurs de tumeurs (p53, p21, pRb) ou dinteragir avec dautres protéines cellulaires impliquées dans le contrôle du cycle cellulaire. Des travaux antérieurs du laboratoire daccueil suggèrent que le développement du cancer du col utérin est associé à une faible capacité de présentation dantigènes au système immunitaire, comme le démontre la rareté et le déficit fonctionnel des cellules de Langerhans (LC, cellules dendritiques ayant une fonction professionnelle de présentation antigénique au niveau de la peau et des muqueuses) dans les lésions (pré)cancéreuses cervicales. Ces altérations pourraient empêcher une réponse immunitaire efficace et faciliter la persistance du virus ainsi que la progression tumorale. Il est actuellement bien admis que les kératinocytes (cellules cibles de linfection par HPV) sont susceptibles dinfluencer les réactions immunitaires au niveau de la peau et des muqueuses épidermoïdes par lintermédiaire de facteurs solubles, les chémokines (CCL20, contrôlant linfiltration des LC immatures au sein de lépithélium) ou de contacts membranaires (E-cadhérine). Les kératinocytes infectés par HPV pourraient se différencier des cellules normales pour la production de ces facteurs, ce qui pourrait contribuer aux altérations des cellules de Langerhans/cellules dendritiques (LC/DC) observées dans les lésions (pré)cancéreuses cervicales. Le fait que la molécule dadhésion E-cadhérine intervienne dans lattachement des LC aux kératinocytes suggère limportance de cette molécule dadhésion dans la rétention des CL au sein de lépithélium cervical. Les objectifs de ce travail ont été détudier linfluence des oncogènes viraux sur lexpression de facteurs immunitaires et dexaminer les conséquences de linhibition de E6 et de E7 sur lexpression de la E-cadhérine et de CCL20, qui jouent un rôle important dans limmunosurveillance au niveau des épithélia via leur action sur les cellules de Langerhans. En accord avec notre hypothèse, nous avons montré une diminution de lexpression de la E-cadhérine dans les lésions (pré)néoplasiques du col par rapport à lépithélium exocervical normal (Hubert et coll. 2005). Par des expériences dARN interférence (siRNA), nous avons également démontré limplication de loncoprotéine virale E7 dans linhibition de lexpression de la E-cadhérine membranaire (Caberg et coll. 2008) et limplication des oncoprotéines virales E6 et E7 dans la diminution de la sécrétion de la chémokine CCL20 dans des kératinocytes transformés par HPV16 (Caberg et coll. 2008).