De récents essais cliniques ont montré les bénéfices thérapeutiques des nouvelles immunothérapies (Sipoleucel T pour le cancer de la prostate…). Mais jusqu’ici la majorité des essais cliniques de vaccins contre le cancer n’ont montré que de faibles effets sur les patients, ce qui contraste avec les résultats obtenus dans les modèles pré cliniques. Ceux-ci, comprennent la greffe sous cutanée de cellules tumorales ce qui ne mime pas la vrai localisation anatomique de la lésion tumorale. De plus, dans la plupart des cas les vaccins anti-cancéreux sont administrés par voie systémique et induisent une réponse antitumorale avec un effet thérapeutique au niveau du compartiment systémique. La réponse antitumorale induite par ces vaccins au niveau du microenvironnement tumoral et ses effets antitumoraux sur des modèles orthotopiques n’ont jamais été décrits sur des modèles pré cliniques. Comme la majorité des tumeurs humaines se développe dans des localisations muqueuses, nous avons étudié l’effet de la voie d’immunisation dans l’induction des réponses antitumorales au niveau du site anatomique de la tumeur en comparant l’induction de LT CD8+ spécifiques de l’antigène tumoral après une vaccination par voie systémique (intramusculaire) ou muqueuse (intranasale). Cette stratégie de vaccination repose sur l’utilisation d’un vecteur non réplicatif qui cible les antigènes in vivo aux cellules dendritiques et qui a été développé au sein de notre unité. Il s’agit de la sous unité B de la toxine de Shiga (STxB) associée à un antigène tumoral (protéine E7 de l’HPV16) nous avons également analysé l’effet antitumoral de cette vaccination sur deux modèles de tumeurs orthotopiques à localisations ORL et pulmonaires exprimant l’antigène E7. Nous avons montré que la vaccination i.n. induisait une plus forte réponse LT-CD8+ spécifique et des effets antitumoraux au niveau des localisations muqueuses que la vaccination par voie systémique, et que cette immunisation par voie i.n. induisait un phénotype particulier sur ces LT-CD8+ spécifiques et en particulier une augmentation de l’expression des intégrines CD103 et CD49a en opposition à la voie systémique. L’inhibition de CD49a réduit l’effet thérapeutique de la vaccination par voie i.n. et le nombre de LT-CD8+ infiltrant les tumeurs orthotopiques. Nos résultats montrent que la réponse LT-CD8+ systémique ne sert pas comme marqueur prédictif de la qualité de la réponse immunitaire antitumorale au niveau local. Nos observations mettent en évidence l’existence d’une compartimentalisation de la réponse muqueuse antitumorale, une découverte capitale pour le développement rationnel des vaccins anti cancer / Recent clinical trials have shown the therapeutic benefits of new promising immunotherapies (Sipoleucel T for prostate cancer, Ipilimumab in melanoma…). But by far, the majority of cancer vaccine clinical trials have shown modest clinical effects on cancer patients, contrasting with results found in preclinical models. Those preclinical models of cancer rely on subcutaneous grafts of tumor cells which do not mimic the true anatomic location of tumor lesions. In addition, in most cases cancer vaccines are administrated by systemic route, eliciting systemic antitumor responses and therapeutic effects. The antitumor response elicited by those vaccine strategies at the local environment of tumor location and their antitumor effect on orthotopic tumor models has not yet been addressed in preclinical cancer models. Since the majority of human tumors develop at mucosal surfaces, we addressed the question of the effect of the immunization route in the induction of local mucosal antitumor CD8+T cell responses by comparing a systemic intramuscular (i.m.) and intranasal (i.n.) route of administration of cancer vaccine. This vaccine consists of a non-replicative vaccine strategy that targets tumor antigen in vivo to dendritic cells developed at our laboratory and composed of the B subunit of the Shiga toxin (STxB) associated to a tumor antigen (E7 protein of HPV16). We also analyzed the antitumor effect of these vaccinations on two mucosal orthotopic tumor models of head and neck and lung cancer expressing the E7 antigen. We found that intranasal vaccination induced stronger specific CD8+T cell responses and antitumor effects at mucosal sites than systemic immunization, and, that mucosal vaccination induced a mucosal imprinting phenotype on mucosal derived antigen specific T cells as they expressed the mucosal integrins CD103 and CD49a, as opposed to systemic specific CD8+T cells or tumor infiltrating T cells in subcutaneous tumors. Inhibition of CD49a reduced the antitumor efficacy of the nasal vaccine and the number of tumor infiltrating CD8+T cells on orthotopic mucosal tumors. Our results showed that systemic antigen-specific T cell responses as typically assessed did not predict the quality of local mucosal immune response. Our observations provide direct evidence for the compartmentalization of mucosal tumor immunity, a critical finding for the rational design of better cancer vaccines

Background: Although subclinical persistent infections with the human polyomaviruses are ubiquitous worldwide, they are known to vary in relation to geographical location, diseases present and may associate with different human tumours, especially in immunocompromised patients. The current study hypothesised that there may be co-operativity between HPV and polyomaviruses, particularly in HIV positive women, that could influence the rate of progression to invasive cervical carcinoma. Patients and Methods: Novel PCR methods were developed for the detection of SV40, MCV, JCV and BKV polyomavirus DNA. These were used to test DNAs extracted from 220 cervical smears and 77 invasive cervical carcinomas (ICCs) from HIV positive and negative Kenyan women of known HPV status. An expression plasmid was constructed containing JCV Large T (LT) antigen and this, in addition to empty vector control, used to stably transfect HPV16 E6/E7 immortalised human keratinocytes. Expression of LT was analysed in transfected cell lines by PCR, immunocytology and Western blotting. These cells were then used to test for changes in Cell contact growth inhibition; Growth rate and Epithelial to Mesenchymal Transition (EMT). Screening of full transcriptome microarrays was carried out on vector and LT transfected cells and their sensitivity to the drug mefloquine tested by comparison of growth rates and live/dead cell assays. Results: PCR accurately detected ~18 copies of SV40, MCV, JCV and BKV DNA in addition to simultaneous detection of JCV and BKV. None of the clinical samples tested were positive for SV40, MCV, or BKV DNA. However, JCV DNA was detected in 24/297 (8%) of cervical specimens. Comparison of the incidence of JCV in cervical smears and ICCs showed a ~3-fold increase in samples from HIV positive women with ICC (P=0.025) whereas no significant difference was found between smears and ICCs from HIV negative women (P=0.553). Analysis of the consequences of ectopic expression of JCV LT in E6/E7 immortalised human keratinocytes showed no difference in either growth rates or contact inhibition and changes in the EMT marker vimentin were found to be related to cellular clonality. Microarray analysis showed LT related alterations in gene expression which could have bearing on its carcinogenic potential in addition to changes related to clonality. JCV LT expressing monoclonal cell were the most sensitive to mefloquine treatment. Conclusion: The simultaneous JCV/BKV detection method, described herein, is unique and has been evaluated by the WHO for this purpose. The results indicate the prevalence JCV and BKV with respect to the African geographical location and suggest that JCV may combine with high-risk HPV in a sub-set of HIV positive women to influence the rate of progression to invasive cervical carcinoma. In vitro JCV LT was found not to be an overt oncogene in the cell system used although cell cloning procedures clearly affected the assays. LT induced changes in total gene expression were consistent with neoplastic progression although a high proportion of genes with unknown function were dsyregulated with respect to clonality. The anti JCV drug mefloquine showed some selectivity for LT expressing cells and further investigation of this indication is warranted.

PROJECT ONE Introduction HPV is detected in about 99.7% of cervical cancers. However, the HPV type distribution in South African women is unknown. Objectives To determine HPV-type distribution among women with cervical dysplasia in relation to oral contraceptive usage. Methods Prospective cross-sectional study of four groups of patients according to oral contraceptive usage: non-users, users of less than five years duration, users of between five years and ten years and users of more than ten years duration. Swabs of the cervix were analysed for HPV DNA using polymerase chain reaction method. Results A total of 124 women were recruited for the study. There were 75 HIV-infected patients (seroprevalence 61%). Of the 102(82%) HPV-positive patients, 79 patients had high-risk HPV DNA (78%). In terms of the four oral contraceptive groups, high-risk HPV DNA was detected in 70% (n=21), 79% (n=22), 90% (n=21) and 71% (n=15) of patients, respectively. The odds of having HPV DNA was six times higher for the combination of contraceptive users of less than 5 years duration/non-users (OR 5.9, 95% CI: 1.87 - 18.77). There was no change when adjustment was made for age (OR 6.1, 95% CI: 1.9 - 19.4). HPV DNA types 16 and or 18 was present in a total of 21 patients (49%) (non-contraceptive users and users < 5years duration) versus 15 patients (42%) who used oral contraceptives of more than 5 years duration (p=0.524). HPV type 16 was the commonest HPV type detected (20.2%) and HPV type 58 was the next commonest high-risk HPV type (16.1%). HPV types 58 and 33 was detected in a much greater percentage of our population and HPV 16 in a much smaller percentage of our population compared with a non-South African population. Conclusion The findings of this study demonstrate an interesting distribution of HPV types in a South African population. PROJECT TWO Introduction Various risk factors have been implicated in the causation of cervical cancer including human papillomavirus (HPV), the early genes (E6 and E7) of which encode the main transforming proteins. Studies have suggested that steroid hormones may enhance the expression of these genes leading to loss of p53 gene-mediated cell apoptosis. Methods A total of 120 cervical tissue samples were obtained from patients with proven cervical cancer. Patients who used depo-medroxyprogesterone acetate steroid contraception were recruited as part of the study arm. Only HPV DNA type 16 samples were used for the study. Controls included three cell lines (CaSki, SiHa,&C33A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal housekeeping gene. Of 120 patients, there were 111 patients with HPV type 16 identified. Of this number, RNA was present in 63 samples. There were 30 women (30/63) who used steroid contraception. In relation to patients who used contraception, HPV 16 E6 gene expression was present in 79% (n = 23) and 88% (n = 30) of steroid users compared to nonusers, respectively. In total there were 25 patients (40%) with expression of the HPV 16 E6*I gene and 30 patients with expression of the E6*II gene. There were 57% of steroid users (n = 17) who had expression of the E6*I/E6*II gene, compared to 52% (n = 17) of nonusers (P = 0.800). Conclusion From a molecular level, this study reflects almost similar distribution of the HPV 16 E6/E6*1 and E6*11 and does not confirm the role of injectable progesterones in cervical carcinogenesis. Further studies with larger patient numbers are needed. / Thesis (PhD)--University of Pretoria, 2011. / Obstetrics and Gynaecology / unrestricted

In the United States, the South has a disproportionate burden of cervical cancer, yet research reporting regional prevalence of human papillomavirus (HPV) infection is scarce. Since 2008, Virginia has passed a HPV vaccine mandate and Louisiana a HPV education bill. This dissertation estimated the prevalence of HPV infection among females and assessed the impact of Virginia’s and Louisiana’s HPV vaccination policy on vaccination among adolescent females. The first manuscript estimated the prevalence of HPV infection using data from 4,250 females collected during the 2007–2010 National Health and Nutrition Examination Survey. Among 14–26 year-olds, the prevalence of high-risk oncogenic HPV was 25.6% (95% CI: 22.4 ̶ 33.3) in the South and 29.1% (95% CI: 24.8 ̶ 33.8) in the rest of the country (p= 0.15). Among 27–59 year-olds, infection rates were 20.9% (95% CI: 17.4 ̶ 24.9) for the South and 14.5% (95% CI: 12.9 ̶ 16.3) for the rest of the country (p=0.0001). The second manuscript assessed the impact of Virginia’s HPV vaccine mandate on vaccination using National Immunization Survey-Teen 2008-2012 data (n=3,203). A difference-in-differences estimation and logistic regression analysis were performed with South Carolina and Tennessee serving as comparison states. Virginia’s mandate was not associated with an increase in vaccination rates. Physician recommendation was strongly associated with vaccination in the Virginia-South Carolina (aOR=10.3; p=0.0001) and Virginia-Tennessee analyses (aOR=9.33; 95%CI: 6.11 ̶ 14.3). The third manuscript assessed the impact of Louisiana’s HPV education policy on vaccination using difference-in-differences estimation and logistic regression analysis, with Alabama and Mississippi as comparison states (n=2,327). There was no evidence that the policy increased vaccination rates. Physician recommendation was associated with vaccination in the Louisiana-Alabama (aOR=7.74; 95% CI: 5.22 ̶ 11.5) and Louisiana-Mississippi comparison (aOR=7.05; 95% CI: 4.6 ̶ 10.5). This study found a higher prevalence of HPV infection among females aged 27 ̶ 59 years in the South compared to the rest of the country. Additionally, physician recommendation was strongly associated with vaccination despite HPV policy implementation. These findings highlight the importance of physician recommendation for HPV vaccination and the need for recommended cervical cancer screening, particularly in the South.

Human papillomavirus (HPV) related cervical cancer is still the most common gynaecological malignancy in developing countries and, as yet, there is no alternative to surgery for the treatment of HPV-associated pre-malignant lesions. HPV 'hijacks' the host-cell ubiquitin-proteasome system to degrade the p53 and Rb tumour suppressor proteins which in turn, leads to the development of cancer. Previous studies have shown that the HIV protease inhibitor lopinavir selectively inhibits the chymotryptic-like activity of the 26S proteasome which stabilises p53 and induces the apoptosis of HPV positive cervical carcinoma cells. Based on this it was hypothesised that lopinavir treatment of HPV positive cervical carcinoma cells would produce changes in the levels of a wide range of cellular proteins that are dis-regulated by HPV-related activation of the proteasome. In order to address this, antibody microarray screening was carried out on lopinavir treated and control untreated HPV positive SiHa cervical carcinoma cells. This showed lopinavir induced alterations in 51 proteins including the cellular antiviral defence protein RNase L. Lopinavir induced both a dose and time dependent increase in RNase L which was subsequently confirmed by western blotting. Transient siRNA silencing of RNase L expression reduced the lopinavir-dependent toxicity in SiHa cells, suggesting an important role for this protein in the toxicity of lopinavir in HPV infected cells. SiHa cells were much more sensitive to lopinavir than CaSKi cervical carcinoma cells which had much higher levels of the E6 protein and did not up regulate RNase L. Furthermore, lopinavir treated HPV16 E6/E7 immortalised keratinocytes were also shown to up regulate RNase L protein expression and these cells were much more sensitive to lopinavir induced apoptosis than mortal control keratinocytes. In addition, transient expression of RNase L in RNase L-deficient C33A cells and the same cells stably transfected with HPV16 E6 (C33AE6) demonstrated that E6 protected these cells from RNaseL-induced cell death. Surprisingly, analysis of RNase L protein levels in these cells demonstrated that E6 did not induce the degradation of the RNase L protein. Instead it was found that E6 stabilised the interaction between RNase L and its endogenous inhibitor protein, ABCE1, and that lopinavir de-stabilised this interaction. Given that C33A tumour cells, E6/E7 immortalised keratinocytes and hTert immortalised keratinocytes are all sensitive to lopinavir, this implies that this compound does not specifically target HPV immortalised cells but rather targets immortalised cells in general, regardless of how this was achieved. The optimum concentration of lopinavir for all these effects was 25 μM, which is 15-fold higher than is observed in cervico-vaginal secretions following oral dosing with the drug Kaletra. In conclusion these results have confirmed the potential of lopinavir to treat HPV related pre-cancerous cervical lesions and provided at least part of the mode-of-action. Indeed they strongly support the use of lopinavir as a low-cost, self-applied topical alternative to surgery for this disease which will be of particular benefit in low-resource countries. Finally, the ability of lopinavir to induce apoptosis of non-HPV related immortalised cells merits further investigation since this indicates this drug may be useful for the treatment of other non HPV related pre-malignant conditions.

Background: The overall good prognosis for Human Papillomavirus (HPV) driven base of the tongue cancer has prompted an increasing interest in whether this group could benefit from a less aggressive treatment regime. Different studies have drawn different conclusions as to which laboratory test should be used to identify these patients, using the surrogate marker p16, analyzing for HPV presence or both. Aim: The main purpose of this study was to investigate the presence of HPV-DNA and p16 in base of tongue cancer patients and their respective prognostic value, both used individually as well as combined. Material and methods: This was a retrospective cohort study consisting of 40 patients diagnosed with base of tongue cancer. The follow-up period was 5 years. Survival analysis was performed both depending on the combined results from the p16 immunohistochemistry analysis and the HPV DNA PCR, as well as separately. Results: Five-year survival rates were 73.9% for p16(+) and 17,6% for p16(-) subjects (p&lt;0.001), 60.7% for HPV-DNA(+) and 25.0% for HPV-DNA(-) subjects (p=0.025). Five-year survival rates when combining p16 and HPV-DNA were 73.9% for p16(+)/HPV DNA(+), 25.0% for p16(-)/HPV DNA(-) and 0.0% for p16(-)/HPV DNA(+) (p&lt;0.001). Conclusion: Our results add to previous research that p16 is a strong predictor of prognosis for base of tongue cancer patients, and could have the clinical implication of serving as a reliable tool for clinicians when determining prognosis and identifying patients who could benefit from treatment de-escalation in the future.

Objetivo: Describir la prevalencia de genotipos del VPH con PCR y secuenciación del ADN en 397 mujeres que acudieron a consulta externa del Hospital Regional Docente de Cajamarca desde Marzo hasta Septiembre del 2017. Materiales y métodos: las muestras del endocérvix y ectocérvix fueron almacenadas y luego se procedió a la detección de tipos del alto y bajo riesgo con PCR. Resultados: un PCR positivo para VPH se observó en 121 muestras cervicales. Se encontró 63.6% (77/121) de genotipos de alto riesgo, 23.1% (28/121) genotipos probablemente oncogénicos y 7.4% genotipos de bajo riesgo. De los genotipos de alto riesgo, VPH-31 fue el más común en un 20% (21/77), seguido de VPH-16 en un 11.4% (12/77). Coinfección entre dos o más genotipos se observó en 12 casos. / Objective: Describe the prevalence of HPV genotypes via PCR and DNA sequencing in 397 women who attended to the gynecological outpatient clinic in the Hospital Regional Docente de Cajamarca from March to September 2017. Methods and materials: The samples from the endocervix and ectocervix were stored and then we proceeded to do detection with genotyping, high and low risk types, by PCR. Results: A positive PCR result for HPV was observed in 121 cervical samples. A high-risk genotype was found in 63.6% (77/121) of patients, a probably oncogenic type in 23.1% (28/121) and a low-risk type in 7.4%. Among the high-risk genotypes, HPV-31 was the most common one present in 20% (21/77), followed by HPV-16 in 11.4% (12/77). Coinfections between two or more genotypes were observed in 12 cases. / Tesis

This thesis deals with human powered vehicles. It contains history, division, examples of these vehicles, but especially design of quite new vehicle with an unique driving mechanism.

Objective: To characterize the cervicovaginal microbiota of HPV-positive and HPV-negative asymptomatic Peruvian women, by identifying the presence of 13 representative bacteria genus. Results: A total of 100 HPV-positive and 100 HPV-negative women were matched by age for comparison of microbiota. The following bacteria were more frequently identified in HPV-positive patients compared to HPV-negative: Eubacterium (68 vs 32%), Actinobacteria (46 vs 33%), Fusobacterium (11 vs 6%) and Bacteroides (20 vs 13%). A comparison between high-risk and low-risk genotypes was performed and differences were found in the detection of Actinobacteria (50 vs 33.33%), Bifidobacterium (50 vs 20.83%) and Enterococcus (50 vs 29.17%). / Revisión por pares

No description available.