abstract: CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self/nonself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2017

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cervical cancer is the second most common cancer among women worldwide and one of the most prevalent female cancers in Brazil. In premalignant and malignant lesions of the uterine cervix the p16INK4a protein, which participates in cell cycle control, exhibits a dramatic increase in its expression possibly due to the presence of human papillomavirus (HPV) oncoproteins. Two polymorphisms in the p16 gene, p16INK4a 540C>G and p16 580C> T, are located in the 3' untranslated region (3'UTR), which is involved in the post-transcriptional regulation of gene expression. The aim of this study was to investigate possible associations between the p16 500C>G and p16 540C>T polymorphisms and the developing of cervical neoplasias and/or lesion severity, considering the expression levels of the p16INK4a protein in cervical lesions, and some classic risk factors for cervical cancer, including HPV infection. A total of 567 women residents in Rio de Janeiro was selected, 319 with abnormal cervical cytology results (case group), and 248 with no previous history of cervical cytological changes (comparison group). Peripheral blood samples from all participants were used for molecular analysis of the polymorphisms p16 500C>G and p16 540C>T, which was performed by PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) using the restriction enzymes MspI and HaeIII, respectively. The expression of p16 in 137 biopsies of women belonging to the group of cases was assessed by immunohistochemistry. The detection of HPV DNA in cervical cells was performed in all samples of the comparison group and in 194 samples of the case group by a PCR-based method using two primers pairs, MY09/MY11 and GP05+/GP06+. TheINK4a two study groups are in Hardy-Weinberg equilibrium. The genotype distributions for p16 500C>G and p16 540C>T and the distributions of haplotype combinations in the two groups were not statistically different. The analysis of the subgroup HSIL+cancer (cases with high-grade intraepithelial lesion or invasive carcinoma) compared to the subgroup HSIL (cases with low-grade squamous intraepithelial lesions) revealed a significant difference in the distribution of haplotype combinations (p = 0.036) and marginal differences in the genotype distributions for p16 500C>G (p = 0.071) and p16 540C>T (p = 0.051). The p16 540G allele, in heterozygosis or homozygosis (OR = 1.91, 95% CI = 1.08-3.37), and the haplotype combination p16 500C-540C 500G-540C (OR = 2.34, 95% CI = 1.202-4.555) showed to be associated with the severity of cervical lesions. On the other hand the p16 580T/T genotype (OR = 0.25, 95% CI = 0.08-0.79), and the haplotype combination p16 500C-540T 500C-540T (OR=0.27, 95% CI = 0.088-0.827) exhibited a protective effect against the development of higher grade cervical lesions. Interaction analyses between the p16 polymorphisms and the p16 protein expression or the HPV infection were compromised by the reduced number of analyzed samples. No interaction was observed between the studied polymorphisms and the classical risk factor for cervical cancer. Our data point out the importance of the p16 gene polymorphisms as severity markers in cervical neoplasia. INK4aINK4aINK4a. / O câncer de colo do útero é o segundo carcinoma mais frequente em mulheres no mundo e um dos cânceres femininos mais incidentes no Brasil. Em lesões pré-malignas e malignas do colo uterino, a proteína p16INK4a, que participa do controle do ciclo celular, apresenta um aumento considerável de sua expressão, devido possivelmente à presença de oncoproteínas do papilomavírus humano (HPV). Dois polimorfismos no gene p16INK4a, p16 500C>G e p16 540C>T, estão localizados na região 3 não traduzida (3UTR), que está envolvida na regulação pós-transcricional da expressão gênica. O objetivo deste estudo foi avaliar possíveis associações entre os polimorfismos p16 500C>G e p16 540C>T e o desenvolvimento de neoplasias cervicais e/ou a severidade das lesões, considerando os níveis de expressão da proteína p16INK4a nas lesões cervicais e certos fatores de risco clássicos para o câncer cervical, incluindo a infecção pelo HPV. Para isso, foram selecionadas 567 mulheres residentes no Rio de Janeiro, 319 com citologia cervical alterada (grupo de casos) e 248 sem história prévia de alteração citológica do colo uterino (grupo de comparação). Amostras de sangue periférico de todas as participantes foram utilizadas na análise molecular dos polimorfismos p16 500C>G e p16 540C>T através da técnica de PCR-RFLP (reação em cadeia da polimerase - polimorfismo de comprimento de fragmento de restrição), usando as enzimas de restrição MspI e HaeIII, respectivamente. A expressão da proteína p16INK4a em 137 biópsias de mulheres pertencentes ao grupo de casos foi avaliada por imunohistoquímica. A detecção de DNA do HPV em células cervicais foi feita em todas as amostras do grupo de comparação e em 194 amostras do grupo de casos pela técnica de PCR, usando dois pares de oligonucleotídeos, MY09/MY11 e GP05+/GP06+. Os dois grupos de estudo se encontram em equilíbrio de Hardy-Weinberg. As distribuições genotípicas para p16 500C>G e p16 540C>T e as distribuições de combinações haplotípicas nos dois grupos não apresentaram diferenças significativas. A análise do subgrupo HSIL+câncer (casos com lesão intraepitelial de alto grau ou carcinoma invasivo) em comparação com o subgrupo LSIL (casos com lesão intraepitelial de baixo grau) revelou diferença significativa entre as distribuições das combinações haplotípicas (p = 0,036) e diferenças marginais entre as distribuições genotípicas para p16 500C>G (p = 0,071) e p16 540C>T (p = 0,051). O alelo p16 540G, em heterozigose ou homozigose (OR = 1,91, IC 95% = 1,08-3,37), e a combinação haplotípica p16 500C-540C 500G-540C (OR = 2,34, IC 95% = 1,202-4,555) mostraram-se associados com a severidade da lesões cervicais. Já o genótipo p16 540T/T (OR = 0,25, IC 95% = 0,08-0,79), e a combinação haplotípica p16 500C-540T 500C-540T (OR = 0,27, IC 95% = 0,088-0,827) exibiram papel protetor contra o desenvolvimento de lesões mais severas. As análises de interação entre os polimorfismos de p16INK4a e a expressão de p16 ou a infecção pelo HPV foram comprometidas pelo número reduzido de amostras analisadas. Não se observou qualquer interação entre os polimorfismos estudados e os fatores de risco clássicos para o câncer de colo uterino. Nossos resultados apontam para a importância dos polimorfismos do gene p16INK4a como marcadores de severidade da neoplasia cervical.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-10-05T00:28:19Z No. of bitstreams: 1 AnaKarinaSilvaCavalcantiFontela_DISSERT.pdf: 1824901 bytes, checksum: a254de7c0021428a7bf8f8b15aebb282 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-10-16T23:46:51Z (GMT) No. of bitstreams: 1 AnaKarinaSilvaCavalcantiFontela_DISSERT.pdf: 1824901 bytes, checksum: a254de7c0021428a7bf8f8b15aebb282 (MD5) / Made available in DSpace on 2017-10-16T23:46:51Z (GMT). No. of bitstreams: 1 AnaKarinaSilvaCavalcantiFontela_DISSERT.pdf: 1824901 bytes, checksum: a254de7c0021428a7bf8f8b15aebb282 (MD5) Previous issue date: 2016-08-12 / Diante da necessidade de diminuir a incid?ncia do c?ncer de colo de ?tero e as les?es causadas pelo v?rus HPV, o Minist?rio da Sa?de iniciou, em mar?o de 2014, a campanha de vacina??o contra o HPV nas meninas de 11 a 13 anos em todo o territ?rio nacional. Em virtude da procura dessas jovens adolescentes pelo servi?o da aten??o prim?ria, considerou-se importante desenvolver um estudo, cujo objetivo foi o de analisar como as usu?rias vacinadas contra o HPV, em uma Unidade de Sa?de de Recife, percebem a preven??o do c?ncer de colo uterino. Tratou-se de uma pesquisa-a??o, para cujo desenvolvimento foram constitu?dos dois grupos focais e duas oficinas. Cada grupo contou com um moderador, que conduziu a discuss?o, por meio de um roteiro-guia, com os seguintes temas: conhecimento e compreens?o sobre a infec??o pelo HPV e sua liga??o com o c?ncer do colo de ?tero; entendimentos e preocupa??es sobre a vacina??o contra o HPV; experi?ncia de vacina??o; entendimentos sobre a import?ncia do rastreio do c?ncer do colo de ?tero e conhecimento sobre as estrat?gias de preven??o do c?ncer uterino na aten??o b?sica, e com um anotador/registrador, respons?vel por gravar o ?udio e transcrever as discuss?es. A amostra foi composta de vinte meninas na faixa et?ria entre 11 e 14 anos, que j? haviam tomado a primeira dose da vacina, frequentavam a Unidade de Sa?de Professor M?rio Ramos h?, pelo menos, seis meses e eram acompanhadas pelo Programa de Agentes Comunit?rios de Sa?de. As reuni?es ocorreram em um local confort?vel e neutro, o encontro foi quinzenal, em dia e hora combinados com as adolescentes, e as participantes foram convidadas pelos agentes de sa?de que as visitaram no dia anterior para confirmar sua presen?a. Os dados foram interpretados ? luz da An?lise de Conte?do de Bardin. Conclui-se que a maioria das adolescentes sente necessidade de mais esclarecimentos sobre o HPV e sua rela??o com o c?ncer uterino. Ficou evidente que os conhecimentos a respeito do HPV e suas formas de preven??o e a vacina dispon?vel para essa faixa et?ria s?o limitados. Este estudo aponta para a import?ncia de se discutir sobre a preven??o do c?ncer uterino nas escolas p?blicas, nas privadas e nos servi?os de aten??o ? sa?de, e que ? preciso explorar com mais frequ?ncia o assunto na esfera da sexualidade. H? que se ressaltar que ? imprescind?vel conhecer e redimensionar as dificuldades das jovens e propor uma reflex?o sobre o autoconhecimento, esclarecendo d?vidas sobre os procedimentos cl?nicos, compartilhar experi?ncias com outras jovens, criar v?nculos com os profissionais de sa?de e aderir ?s pr?ticas participativas. Espera-se que, atrav?s dessas a??es, diminua-se o n?mero de casos e de ?bitos causados pelo HPV. / Faced with the need to reduce the incidence of cervical cancer and lesions caused by the HPV virus the Ministry of Health began in March 2014 the vaccination campaign against HPV in girls 11-13 years throughout the country, because that the demand for such young adolescents at the service of primary care has become important to develop a study, which the research objective to analyze the perception on the prevention of cervical cancer in vaccinated users against HPV in a Recife Health Unit. This is an action research in which there will be two focus groups and a workshop. Each group will consist of 01 moderator leading the discussion by a road map to guide the following themes: knowledge and understanding of HPV infection and its link to cervical cancer; understandings and concerns about HPV vaccination; vaccination experiments; understandings about the importance of cervical cancer screening and knowledge on strategies to prevent cancer of the cervix in primary care, 01 recorder / registrar responsible for recording with audio and transcribe the discussions minimum of five and maximum number 10 girls aged between 11 and 14 years, who have already taken the first dose of the vaccine, attending the Health Unit Professor Mario Ramos for at least six months and is accompanied by the Community Health Agents Program. The meetings will take place in a comfortable and neutral location, the meetings will be held monthly on day and time combined with the teenagers, and participants will be invited by CHWs who had visited the day before confirming their presence. Data analysis will be conducted through Bardin Content Analysis. It is concluded that the majority of adolescents feel the need for further clarification on HPV and its relation to uterine cancer. It was clear that knowledge about HPV and its forms of prevention and available vaccine for this age range are limited. This study points to the importance of discussing the prevention of uterine cancer in public schools, private schools and health care services, and that the issue of sexuality must be explored more frequently. It is necessary to emphasize that it is essential to know and resize the difficulties of young people and to propose a reflection on self-knowledge, clarifying doubts about clinical procedures, sharing experiences with other young people, creating links with health professionals and adhering to participatory practices. It is hoped that, through these actions, the number of cases and deaths caused by HPV will decrease.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Introduction: HPV infection is the most common sexually transmitted disease in humans, which can cause benign diseases (warty lesion) or malignant as the anogenital cancer, oral cancer and, with the highest incidence, cervical cancer. Overall, the main HPV detection method is based on cytological evaluation of Papanicolaou; however, false-negative results and unsatisfactory sampling complicate the disease diagnosis and prevention. In this context, the investigation of new diagnosis platforms and prophylaxys has great interest. Objectives: The aim of this study was to conduct an epidemiological study of HPV infection in the women population of southeastern Brazil and then propose new diagnostic and vaccine approaches. Methodology: Genital samples of 5,223 women were evaluated by a new molecular tool based on PCR (Nested Multiplex PCR - NMPCR), using a cocktail of primers for simultaneous detection of 38 different HPV types, in single tube. Another molecular approach was proposed by Phage Display technique to select the IgA binding peptides from cervical samples of patients with HPV infection. In silico (Linear and 3D bioinformatics) and in vitro (ELISA, Slot blot and Bioelectrode) analysis were performed to validate the selected phages and synthetic peptides in cervical (n=91) and salivary specimens (n=66). The sensitivity and specificity parameters of HPV diagnosis were determined by ROC curve. To test the vaccine potential of synthetic peptides, in silico (3D bioinformatic), in vivo (female BALB/c mice immunization) or in vitro analyzes (MTT, ELISA, splenocytes culture, CBA and Neutralization assay) were carried out. Results: Among the 5,223 women evaluated, there was a prevalence of 58.9% of HPV infection, especially the types 53, 52 and 06, showing the relevance to propose new alternatives for HPV detection and prevention. In molecular approach of Phage Display, thirty-two distinct phage clones were selected against IgA from positive HPV cervical samples. Phage C.B1 showed higher reactivity against HPV samples compared to the negative control group by ELISA. This peptide is a putative epitope of HPV major capsid protein (L1) and has distinguished efficiently infection from HPV controls in cervical and saliva samples (p <0.0001), with high sensitivity (above 95%) and specificity (above 71%) in both fluids. The C.B1 peptide was also successfully incorporated (p<0.05) onto a graphite bioelectrode for HPV direct diagnosis in saliva. In the vaccine approach, three peptides (PEP1, PEP3 and PEP4) showed no toxicity in murine macrophages. Immunizations showed potent induction of serum IgG antibodies production by PEP3, and increase in IgA titer in the vaginal mucosa under PEP1 and PEP3 stimuli. The evaluation of cellular immune response indicated that the PEP1 and, particularly, the PEP3 chimeric peptide were able to polarize the immune response towards to Th1 profile and sensitize the cells to a pro-inflammatory \"status\", suitable for antiviral activity. The HPV-16 PsVs neutralization assay showed that PEP3 was able to reduce the infection by 49%. Conclusions: Overall, the studies presented here showed a high prevalence of HPV among Brazilian women by viral types still little explored in the literature. We selected mimotopes by Phage Display tool capable of detecting HPV in both saliva samples as in the cervical secretion, providing a low cost, simple and non-invasive diagnostics for population screening. In addition, the mimotopes selected have attractive applicability in vaccine formulations, but further studies are still needed. / Introdução: A infecção por HPV é a doença sexualmente transmissível mais comum em humanos, que pode causar doenças benignas (lesões verrucosas) ou malignas como o câncer anogenital, câncer oral e, com maior incidência, o câncer do colo uterino. Globalmente, o principal método de detecção do HPV baseia-se na avaliação citopatológica de Papanicolaou, entretanto, resultados falsonegativos e obtenção de amostras insatisfatórias dificultam o diagnóstico e a prevenção da doença. Neste contexto, a busca por novas plataformas diagnósticas e profiláticas para o HPV é de grande interesse. Objetivos: O objetivo do presente estudo foi realizar um levantamento epidemiológico da infecção por HPV na populacão feminina da região Sudeste do Brasil e, a partir de então, propor novas abordagens diagnósticas e vacinais. Metodologia: Amostras genitais de 5.223 mulheres foram avaliadas por uma nova ferramenta molecular baseada em PCR (Nested Multiplex PCR - NMPCR), utilizando-se um coquetel de primers para detecção simultânea de 38 diferentes tipos virais de HPV, em um único tubo. Outra abordagem molecular foi proposta através da técnica de Phage Display, a fim de selecionar peptídeos ligantes a anticorpos IgA oriundos de amostras cervicas de pacientes com infecção por HPV. Análises in silico (Bioinformática linear e 3D) e in vitro (ELISA, Slot blot e Bioeletrodo) foram realizadas para a validação dos fagos selecionados e dos peptídeos sintetizados em espécimes cervicais (n=91) e salivares (n=66). Os parâmetros de sensibilidade e especificidade do diagnóstico do HPV foram determinados pela curva ROC. Para testar o potencial vacinal dos peptídeos sintéticos, análises in silico (Bioinformática 3D), in vivo (imunização em camundongos fêmeas BALB/c) e in vitro (MTT, ELISA, Cultura de esplenócitos, CBA e Ensaio de Neutralização) foram conduzidas. Resultados: Dentre as 5.223 mulheres avaliadas, houve uma prevalência de 58,9% de infecção por HPV, com predominância dos tipos 53, 52 e 06, evidenciando a relevância de se propor novas alternativas de detecção e profilaxia do HPV. Na abordagem molecular de Phage Display trinta e dois clones de fagos distintos foram selecionados contra a IgA de amostras cervicais positivas para o HPV. O fago C.B1 apresentou maior reatividade contra amostras de HPV em comparação ao grupo controle negativo, por ELISA. Este peptídeo é um epítopo putativo da proteína principal do capsídeo do HPV (L1) e tem discriminado eficientemente amostras HPV de controles, em amostras cervicais e salivares (p<0,0001); com alta sensibilidade (acima de 95%) e especificidade (acima de 71%) em ambos fluidos. O peptídeo C.B1 também foi incorporado com sucesso (p<0,05) em um bioeletrodo de grafite para o diagnóstico do HPV direto na saliva. Na abordagem vacinal, três peptídeos (PEP1, PEP3 e PEP4) não apresentaram toxicidade em macrófagos murinos. As imunizações revelaram potente indução na produção de anticorpos séricos IgG pelo PEP3, e aumento nos títulos de IgA na mucosa vaginal sob estímulo dos PEP1 e PEP3. A avaliação da resposta imune celular indicou que o PEP1 e, sobretudo o peptídeo quimérico PEP3, foram capazes de polarizar a resposta imune para o perfil Th1 e sensibilizar as células a um status pró-inflamatório, propício para atuação antiviral. O ensaio de neutralização de PsVs do HPV-16 mostrou que o PEP3 foi capaz de reduzir a infecção em 49%. Conclusões: De maneira geral, os estudos aqui apresentados evidenciaram uma alta prevalência de HPV em mulheres brasileiras por tipos virais ainda pouco explorados na literatura mundial. Nós selecionamos mimotopos pela ferramenta de Phage Display capazes de detectar o HPV tanto em amostras de saliva quanto em secreção cervical, possibilitando um diagnóstico simples, de baixo custo, e não invasivo para rastreios populacionais. Além disso, os mimotopos aqui selecionados possuem atraente aplicabilidade em formulações vacinais, porém estudos adicionais ainda são necessários. / Doutor em Genética e Bioquímica

Head and neck squamous cell carcinomas (HNSCC) remain a significant cause of morbidity worldwide, with approximately 550,000 new cases diagnosed each year. The main etiological factors include smoking and alcohol consumption. The incidence of non-oropharyngeal HNSCC is gradually decreasing while the incidence of squamous cell oropharyngeal carcinoma (OPSCC) is still on the rise. This increasing incidence can be most likely attributed to an increasing prevalence of human papillomavirus (HPV) infection. From the clinical point of view the most significant fact is that patients with HPV positive OPSCC have better prognosis. HNSCC is linked to an alteration in the immune system. Only a limited number of studies have correlated both the immunological parameters and HPV status with patient prognosis. Therefore, we focused on the research of the immunological profile of patients with HNSCC of viral and non-viral etiology. In our study, 110 patients with HNSCC were enrolled. They were divided into HPV-positive and HPV-negative groups based on the expression of HPV 16 E6 mRNA detected in the tumor tissue. Basic lymphocyte subpopulations (CD3+, CD4+ CD25+ Treg, CD4+ CD25+ FoxP3 Treg, CD4+, CD8+, CD19, and CD3- CD16+ CD56+ cells) were determined by flow cytometry in the peripheral blood (PB). We observed...

Submitted by Isaac Francisco de Souza Dias (isaac.souzadias@ufpe.br) on 2016-03-04T17:16:30Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) MICHELINE OLIVEIRA LOBO PEREIRA DA COSTA- TESE DOUTORADO -23-10-2015.pdf: 15371820 bytes, checksum: 4afd4b560150641ba2dc59721bbb563e (MD5) / Made available in DSpace on 2016-03-04T17:16:30Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) MICHELINE OLIVEIRA LOBO PEREIRA DA COSTA- TESE DOUTORADO -23-10-2015.pdf: 15371820 bytes, checksum: 4afd4b560150641ba2dc59721bbb563e (MD5) Previous issue date: 2015-01-23 / CAPES / O presente estudo avaliou o desempenho da metodologia citológica de base líquida (ThinPrep-TP) com o da citologia convencional de Papanicolaou (CC) no diagnóstico de alterações citopatológicas e de resultados insatisfatórios, sob a visão de um serviço público no estado de Pernambuco (LACEN-PE) e da Fundação Oncocentro de São Paulo (FOSP-SP). Também foram avaliadas as associações entre a presença de coinfecções genitais dos subtipos de Human papillomavirus (HPV) com Chlamydia trachomatis (CT) e/ou outras microfloras, com os diferentes estágios de alterações cervicais das pacientes. A população do estudo foi de 525 mulheres na faixa etária dos 18-65 anos, atendidas por demanda espontânea, pelas Unidades Básicas de Saúde no Estado de Pernambuco, no período de abril a novembro de 2011. Um questionário padronizado com informações sobre características sociodemográficas, sexuais, reprodutivas e de hábitos (tabagismo, consumo de bebidas alcoólicas e uso de drogas) foram obtidos de todas as pacientes do estudo. A presença de DNA do HPV e da CT foram diagnósticadas através da reação em cadeia da polimerase (PCR) e o exame citológico foi realizado para detecção das demais infecções. Para avaliar a relação das infecções genitais com a presença de alterações intraepiteliais cervicais, foi utilizado o teste exato de Fisher. Os resultados mostraram que 11,05% das pacientes tinham menos de 25 anos, 30,86% eram solteiras, 6,86% tiveram mais de 5 parceiros sexuais, 44% não faziam uso de métodos contraceptivos, 38,85% eram usuárias de álcool, 24,38% eram fumantes e 3,24% haviam consumido drogas. Além disso, 42,01% tinham queixas ginecológicas; e 12,19% história pregressa de doenças sexualmente transmissíveis (DST). Quando comparadas a eficiência das técnicas TP e CC sob a visão do LACEN-PE, observou-se que as duas metodologias avaliadas apresentaram fraca concordância entre os métodos (k=0,19;95%IC(0,11-0,26); p<<0,001). A metodologia TP reduziu a taxa de resultados insatisfatórios de 4,38% para 1,71% (×2= 5,28; p=0,02), e o número de alterações citopatológicas diagnosticadas aumentaram de 2,47% para 3,04%. Porém sob a avaliação da FOSP-SP, as duas metodologias apresentaram concordância (k=0,39;95%IC(0,29-0,50); p<<0,001). A metodologia TP apresentou taxas insatisfatórias e praticamente semelhantes de 3,20% para 3,60% (×2= 5,00; p=0,17), e de alterações citopatológicas de 5,60% para 4,20%. Em 87 casos, foram observadas alterações cervicais por uma das metodologias utilizadas; destas, em 83,91% foram detectados CT e 82,76% foram positivas para HPV, ocorrendo coinfecção em 65 casos (74,7%). Em 93,1% dos casos houveram alterações colposcópicas, observando-se uma associação estatisticamente significativa entre coinfecção HPV-CT e presença de lesão (p=0.037). Outras microfloras encontradas foram Gardnerella vaginalis (35,6%), cocos (18,4%), Candida sp (9,2%), Trichomonas vaginalis (6,9%), Lactobacillus sp (4,6%) e herpesvírus (1,15%). Flora mista (coinfecção por várias microfloras – com exceção de CT simultaneamente) ocorreram em 41,38% dos casos. No entanto, nenhuma associação da presença desses microrganismos não-virais e os herpesvírus com a gravidade das lesões intraepiteliais foram encontradas. Os subtipos de HPV mais frequentes foram 16 e 31 (34,3% e 17,15%, respectivamente). Porém nas lesões de maior gravidade, os mais prevalentes foram 16 e 18. Desta forma, concluimos que este estudo demonstra a superioridade da metodologia TP no diagnóstico citológico das amostras cervicais o que poderá contribuir na diminuição de possíveis perdas por repetição citológica e seguimento das pacientes. É importante chamar atenção para as infecções genitais, em especial a CT, que devem sem investigadas e tratadas adequadamente, haja vista, que coinfecções com o HPV estão associadas ao favorecimento de lesões cervicais, e podem evoluir a graus mais avançados. / This study evaluated the performance of cytological methodology net basis (ThinPrep-TP) with the conventional Pap cytology (CC) in the diagnosis of cytopathological findings and unsatisfactory results under the vision of a public service in the state of Pernambuco (LACEN -PE) and Oncocentro Foundation of São Paulo (FOSP-SP). We also evaluated whether there is an association between the presence of genital co-infections of human papillomavirus subtypes (HPV) with Chlamydia trachomatis (CT) and or other microflora, with the different stages of cervical abnormalities of patients. The study population of 525 women between the ages of 18-65 years, assisted by spontaneous demand, the Basic Health Units in the State of Pernambuco, in the period from April to November 2011. A standardized questionnaire with information on sociodemographic characteristics, sexual, reproductive habits (such as smoking, alcohol consumption and drug use) were obtained from all study patients. The presence of HPV DNA and CT were both diagnosed by polymerase chain reaction (PCR) and cytological examination was performed to detect other infections. To evaluate the relationship of genital infections with the presence of cervical intraepithelial changes, Fisher's exact test was used. The results showed that 11.05% of patients were under 25yrs, 30.86% were single, 6.86% had more than five sexual partners, 44% did not use contraception, 38.85% were users of alcohol, 24.38% smokers and 3.24% had used drugs before. Moreover, gynecological complaints were 42.01%; and 12.19% history of STD. When comparing the efficiency of two techniques used in diagóstico of cervical abnormalities in the view of LACEN-PE, it was observed that the two methodologies evaluated showed poor agreement between the methods (k = 0.19; 95% CI (0,11- 0.26), p << 0.001). TP method reduced the rate of unsatisfactory 4.38% to 1.71% (5.28 = 2 × p = 0.02) and the number of cytopathological changes diagnosed increased 2.47% to 3 04%. But in the assessment of FOSP-SP, the two methodologies showed reasonable agreement (k = 0.39; 95% CI (0.29 to 0.50); p << 0.001) .The TP methodology showed unsatisfactory results virtually rates similar 3.20% to 3.60% (× 2 = 5.00; p = 0.17), and cytological changes from 5.60% to 4.20%. In 87 cases, cervical abnormalities were observed by any of the methods used, and of these, 83.91% were detected CT, and 82.76% were positive for HPV, occurring co-infection in 65 cases (74.7%). In 93.1% of cases there were colposcopic changes, observing a statistically significant association between co-infection HPV-CT and presence of lesions (p = 0.037). Other microflora were found Gardnerella vaginalis (35.6%), coconut (18.4%), Candida sp (9.2%), Trichomonas vaginalis (6.9%), Lactobacillus sp (4.6%) and herpesviruses (1.15%). Mixed flora (co-infection with various microflora - CT exception with both) occurred in 41.38% of cases. However, no association between the presence of non-viral organisms and herpesviruses with the severity of intraepithelial lesions were found. The most common HPV subtypes there were 16 and 31 (34.3% and 17.15%, respectively). But in more severe injuries, the most prevalent were 16 and 18. Thus, we conclude that this study demonstrates the superiority of TP methodology in the cytological diagnosis of cervical samples which could contribute to decrease possible losses by repetition and cytologic follow-up of patients . It is important to draw attention to the genital infections, especially CT, which are not investigated and dealt with appropriately, given that co-infections with HPV are associated with the favoring of cervical lesions, and can progress to more advanced degrees.

Infection with Human Papillomavirus (HPV) is the sexually transmitted viral disease most prevalent in world. The infections can range from asymptomatic establishment to induction of squamous cell carcinomas. It has been discussed the correlation of HPV infection and the development and/or aggravation of lesions in the oral mucosa. The aim of this study was to evaluate the occurrence of HPV and its genotypes in patients with oral lesions and in healthy oral mucosa of users and non-users of drugs in the state of Sergipe, Brazil. Thirty-nine patients aged 2 to 83 years with clinically detectable lesions in the oral mucosa and 106 patients with healthy oral mucosa between 11 and 79 years were evaluated. Samples were collected by exfoliating the oral mucosa. For quality control of DNA extraction beta-globin PCR was performed. HPV DNA was detected using primers MY09/MY11 and GP5+/GP6+. Genotyping was performed by multiplex-PCR with specific primers for HPV types 6, 16 and 18. Our study detected the virus in all types of lesions evaluated. The occurrence of HPV was 76.92% (30/39) in patients with oral lesions. The most common virus type was HPV-6 in 56.67% (17/30), followed by HPV-18 in 26.67% (8/30) and HPV-16 in 6.67% (2/30). Positive results were found in 83.02% (88/106) of patients with healthy oral mucosa. The most common virus type was HPV-6 in 45.45% (40/88), followed by HPV-18 in 35.23% (31/88) and HPV-16 in 4.54% (4/88). Between multiple drug users 86.67% (52/60) were positive and multiple HPV infections were identified in 23.08% (12/52). At |non-users| the occurrence was 78.26% (36/46). A high occurrence of HPV was found in the study, both in oral lesions and in healthy mucosa. Rates of HPV detection in the oral cavity vary markedly in the world and make the relationship between HPV and oral carcinogenesis still controversial. Additional studies to evaluate the role of human papillomavirus in the development of lesions in the oral mucosa are necessary. There are few data available on the frequency of oral HPV infection in Brazilian population and especially among drug users. Other studies on HPV prevalence among drug users are needed for a better understanding of their exposure to the virus and for the development of prevention strategies. / A infecção pelo Papilomavírus Humano (HPV) é a doença viral sexualmente transmissível mais prevalente no mundo. Suas infecções podem variar de assintomáticas à indução de Carcinomas de Células Escamosas. Entre os agentes infecciosos associados ao câncer oral, tem-se discutido a correlação da infecção por HPV em mucosa oral e o desenvolvimento e/ou agravamento das lesões. O objetivo deste estudo foi avaliar a ocorrência do HPV e seus genótipos em pacientes com lesão oral e em mucosa oral saudável de usuários e não-usuários de drogas no estado de Sergipe, Brasil. Foram avaliados 39 pacientes com idade entre 2 e 83 anos, com lesões clinicamente detectáveis na mucosa oral e 106 pacientes com mucosa oral saudável entre 11 e 79 anos. As amostras foram coletadas por esfoliação da mucosa oral. Para o controle de qualidade da extração de DNA foi utilizado PCR para beta-globina. DNA-HPV foi detectado utilizando primers MY09/MY11 e GP5+/GP6+. A genotipagem foi realizada através de multiplex-PCR com primers específicos para os tipos virais 6, 16 e 18. Nosso estudo detectou DNA-HPV em todos os tipos de lesões avaliadas. A prevalência do HPV foi de 76,92% (30/39) nos pacientes com lesões orais. O tipo viral mais frequente foi o HPV-6, presente em 56,67% (17/30), seguido do HPV-18 em 26,67% (8/30) e do HPV-16 em 6,67% (2/30). DNA-HPV foi encontrado em 83,02% (88/106) dos pacientes com mucosa oral sadia. O tipo viral mais frequente foi o HPV-6, presente em 45,45% (40/88), seguido do HPV-18 em 35,23% (31/88) e do HPV-16 em 4,54% (4/88). Entre usuários de múltiplas drogas 86,67% (52/60) foram positivos e infecções múltiplas por mais de um tipo viral foram identificadas em 23,08% (12/52) dos indivíduos. Entre os "não usuários" a taxa de infecção pelo HPV foi de 78,26% (36/46). Desta forma, foi verificada uma alta ocorrência do HPV em nosso estudo, tanto em lesões orais quanto em mucosas saudáveis. As taxas de detecção do vírus em cavidade oral variam acentuadamente no mundo e tornam a relação do HPV com o processo de carcinogênese oral ainda controversa. Isso faz necessária a realização de estudos adicionais que avaliem o papel do Papilomavírus Humano no desenvolvimento de lesões na mucosa oral. Há poucos dados disponíveis sobre a frequência de infecção oral por HPV na população brasileira e especialmente entre usuários de drogas. Novos estudos sobre a prevalência do HPV entre usuários de drogas são necessários para melhor compreensão da sua exposição ao vírus e o desenvolvimento de estratégias de prevenção.

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No. of bitstreams: 2 Dissertação - Rodrigo Cesar Assis Caixeta - 2014.pdf: 3427166 bytes, checksum: 4e0cd6ad305c26afada6419c7fc8663c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-06-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / HPV, BV and CI are common conditions in adolescents and young women. Studies have reported that BV and CI can be cofactors for the acquisition and persistence of HPV, enabling the development of cytological abnormalities precursor of cervical cancer. The aim of this study was to estimate the prevalence of HPV, BV and IC in adolescents and young women and to verify that these conditions are associated with the detection of cytological abnormalities in cervical smears in adolescents and young women. Were included samples of sexually active patients and users of the Unified Health System (SUS) who underwent cervical screening by the conventional method. The cervical specimens were analyzed at the Center for Clinical Analysis Rômulo Rocha, Faculty of Pharmacy/UFG, by qualified professionals following strict quality control. The detection of HPV-DNA was made by PCR using pools of primers PGMY09/PGMY11. The diagnosis of BV was established from the observation of the presence of 20% or more of clue cells in cervical smears. The IC was assessed by counting the number of leukocytes in cervical smears in five noncontiguous with increased microscopic fields 1000X average, according Castle et al. (2001). Statistical analyzes with hierarchical logistic and control of confounding variables regression model were performed. The magnitude of association was estimated by calculating the odds ratio (OR) with confidence intervals (CI*) of 95%. 251 samples were included, with 54,9% (138/251) of adolescents aged between 15 and 19 years and 45,1% (113/251) of young women aged between 20 and 25 years. Positive results for cytological abnormalities accounted for 9,5% (24/251) of all cervical smears. The diagnosis of atypical squamous cells of undetermined significance (atypical squamous cells of undetermined significance - ASCUS) (50,0% 12/24) and squamous intraepithelial lesions (low grade squamous intraepithelial lesion - LSIL) (29,1 % 7/24) were more frequent. The overall prevalence of HPV, BV, and CI 44,2% (111/251), 41,0% (103/251) and 83,2% (209/251), respectively. Of the variables investigated, VB (OR = 2,46 CI*: 1,26 to 3,80; p: 0,006) and the detection of abnormal cytological diagnosis (OR = 1.36 CI*: 1.07 to 1.74; p: 0.013) were associated with HPV positivity in multivariate analysis. Whereas the detection of cytological abnormalities there was a significant association with HPV and BV detected in the same patient (OR = 2,59 CI*: 1,09 to 6,20; p: 0,032) and for the detection of HPV, BV and CI together (OR = 3,58 CI*: 1,06 to 12,15; p: 0,040), however, they didn’t remain independently associated in bivariate analysis. BV and cytological abnormalities were independently associated with HPV infection in adolescents and young women. HPV, BV and CI in cervical smears can indicate high possibility to detect cytological abnormalities in adolescents and young women. / HPV, VB e IC são condições frequentes em adolescentes e mulheres jovens. Estudos têm relatado que VB e IC podem ser cofatores para a aquisição e persistência do HPV, possibilitando o desenvolvimento de anormalidades citológicas precursoras do câncer do colo uterino. Assim, o objetivo desse estudo foi estimar a prevalência de HPV, VB e IC em adolescentes e mulheres jovens e verificar se estas condições estão associadas à detecção de anormalidades citológicas em esfregaços cervicais em adolescentes e mulheres. Foram incluídas amostras de pacientes sexualmente ativas e usuárias do Sistema Único de Saúde (SUS) que realizaram exame citopatológico através do método convencional. Os esfregaços cervicais foram analisados no Centro de Análises Clínicas Rômulo Rocha, da Faculdade de Farmácia/UFG, por profissionais qualificados e seguindo rigoroso controle de qualidade. A detecção de DNA do HPV foi feita por PCR, utilizando os pools de primers PGMY09/PGMY11. O diagnóstico de VB foi realizado a partir da observação da presença de 20% de células indicadoras nos esfregaços cervicais. A IC foi avaliada através da média da contagem do número de leucócitos nos esfregaços cervicais, em cinco campos microscópicos não adjacentes com aumento de 1000X, segundo Castle et al. (2001). Foram realizadas análises estatísticas com modelo de regressão logística hierárquica e controle de variáveis de confusão. A magnitude de associação foi estimada através do cálculo de odds ratio (OR) com intervalos de confiança (IC*) de 95%. Foram incluídas 251 amostras, sendo 54,9% (138/251) de adolescentes com faixa etária entre 15 e 19 anos e 45,1% (113/251) de mulheres jovens com idades entre 20 e 25 anos. Resultados positivos para anormalidades citológicas representaram 9,5% (24/251) do total de esfregaços cervicais. Os diagnósticos de células escamosas atípicas de significado indeterminado (Atypical Squamous Cells of Undetermined Significance – ASC-US) (50,0% 12/24) e lesão intraepitelial escamosa de baixo grau (Low grade Squamous Intraepithelial Lesion - LSIL) (29,1% 7/24) foram mais frequentes. As prevalências totais de HPV, VB e IC foram de 44,2% (111/251), 41,0% (103/251) e 83,2% (209/251), respectivamente. Das variáveis investigadas, VB (OR = 2,46 IC*: 1,26-3,80, p: 0,006) e a detecção de diagnóstico citológico anormal (OR = 1,36 IC*: 1,07-1,74, p: 0,013) foram associadas com a positividade para HPV pela análise multivariada. Considerando a detecção de anormalidades citológicas, houve uma associação significativa com o HPV e VB detectado no mesmo paciente (OR = 2,59 IC*: 1,09-6,20, p: 0,032), bem como para a detecção de HPV, VB e IC em conjunto (OR = 3,58 IC*: 1,06-12,15, p: 0,040), no entanto, eles não permaneceram independentemente associadas na análise bivariada. VB e esfregaço cervical anormal foram independentemente associados a infecção por HPV em adolescentes e mulheres jovens. HPV, VB e IC em esfregaços cervicais pode indicar uma maior possibilidade de detectar anormalidades citológicas em adolescentes e mulheres jovens.

Introduction: Human papillomavirus (HPV) is changing the prevalence, survival and treatment paradigms in oropharyngeal squamous cell carcinoma (OPSCC). Improved survival of patients with HPV positive compared to HPV negative OPSCC has led to trials of treatment de-escalation. Current HPV detection methods are imprecise, therefore standardised assessment of transcriptionally active HPV in OPSCC is required. Furthermore, the differences in immune characteristics and/or the hypoxia response/effects could explain observed differences in prognosis between HPV positive and negative OPSCC. Rigorous HPV detection and subsequent biomarker evaluation should provide additional information required before introduction of treatment de-escalation in broad patient groupings. Methods: The study cohort was 218 patients with OPSCC who received radiotherapy with curative intent. HPV status was determined on pre-treatment, formalin-fixed paraffin-embedded blocks using: 1) polymerase chain reaction (PCR); 2) in-situ hybridisation (ISH) and 3) immuno-histochemistry (IHC). QuantiGene multiplex assay was designed to detect mRNA of reference sequences of the common high-risk HPV types (16, 18, 33, 35, 45, 52 and 58). HPV detection methods were compared with mRNA quantification. Multimarker IHC of immune cell markers using chromogenic and fluorescent staining was performed, analysed and compared with single marker IHC using automated multispectral image analysis. A validated multiplex IHC method was used for a) chromogenic (CD3, CD4, CD8, and FoxP3) and b) fluorescent (CD8, CD68 and PD1/PD-L1) evaluation in tumour and stroma compartments. Single marker IHC was used to investigate tumour hypoxia markers (HIF-1α and CA-IX) in HPV positive and negative OPSCC. Results: p16 IHC and ISH were the most sensitive and specific, respectively, for classifying HPV status. The combination of the three tests had the highest positive/negative predictive values compared with QuantiGene mRNA detection. Multiplex validation showed that, for serial sections up to 6 μm apart, there were highly significant correlations (P<0.0001) between single and multiplex counts for both chromogenic and fluorescent IHC. Overall there was less variation in cell counts with fluorescent staining when compared to chromogenic staining. Multiplex IHC of TILs in HPV positive and negative OPSCC showed higher infiltration in both tumour and stromal areas of CD3+CD4+ and CD3+CD8+ T cells but not CD4+FoxP3 Tregs in HPV positive compared with HPV negative OPSCC. Only CD3+CD8+ stromal and not tumour area infiltration was associated with increased survival (P=0.02). PD-L1 expression was higher in HPV negative OPSCC and this was related to macrophage (CD68) expression of PD-L1. In HPV negative tumours infiltration with CD68+PD-L1 was associated with a good prognosis. HPV negative patients had higher expression of HIF-1α but not CA-IX. High expression of both markers was associated with a poor prognosis irrespective of HPV status. Conclusions: There are other prognostic factors operating in the larger subdivision of HPV positive and negative OPSCC. Precise HPV detection and inclusion of other prognostic factors is required before treatment de-escalation is used. Expression of immune inhibitory factors (PD1/PD-L1) alone without contextualisation with immune cell density is insufficient for patient prognostication and potential selection for therapy using immune checkpoint inhibitors. Hypoxia modification of radiotherapy should be explored in both HPV positive and negative OPSCC.

This dissertation explores how the sociocultural experiences of migration and acquisition of health knowledge influence the beliefs and behaviors related to human papillomavirus (HPV) risks and cervical cancer prevention among women who have emigrated from English-speaking Caribbean nations and now live in the Tampa Bay metropolitan area. Genital human papillomavirus is very common, and cervical cancer is the most common HPV-associated cancer. Additionally, all cervical cancers are caused by the HPV infection. More women of color, including Black and Hispanic women, are diagnosed with cervical cancer and at a later stage of the disease than women of other races or ethnicities. Black women have lower levels of knowledge and awareness of HPV and related preventive measures compared to Whites. The incidence of cervical cancer is higher among African American/Black women and Latina women than among White women. Globally, Caribbean countries have some of the highest incidence and mortality rates of cervical cancer. It is unclear how knowledge, perceptions and behaviors surrounding HPV risks and cervical cancer influence prevention practices among immigrant women from English-speaking Caribbean countries residing in the United States. Existing literature highlights factors which influence cervical cancer prevention behaviors and HPV knowledge among immigrants in the United States, including educational barriers, HPV tests and vaccine costs, duration of time within the United States, in addition to the beliefs, myths and stigma surrounding cervical cancer originating in the birth country. But there is a dearth of information on immigrant women from the Caribbean. Ethnographic methods were employed in this study, including participant observation, key-informant interviewing, focus groups, and semi-structured in-depth interviewing to assess attitudes, available knowledge, culturally specific perceptions, and behavioral practices of the study participants. This dissertation develops a modified approach in the Critical Medical Anthropology (CMA) genre that links political economy with an interpretive approach. It also utilizes the theoretical approaches of transnationalism and embodiment to analyze the phenomena under consideration. Some key outcomes of this research are as follows: Many women were very aware of HPV, and most women were familiar with cervical cancer. However, the majority of women were not confident regarding how HPV and cervical cancer were connected. They did not know how a virus causes a chronic disease. Even with some of the study participants having the HPV vaccine, they were still not aware of the link between the two. This lead the researcher to inquire what HPV or a sexually transmitted disease meant to the women, resulting in a mixture of responses ranging from never thinking about HPV or acquiring an infection to placing blame on being “loose” or “promiscuous” as a woman. Their narratives provided insights into how their childhood and familial experiences as young Caribbean women contributed to how they act upon knowledge about being sick, having an infection, or living a healthy lifestyle since migrating to the United States. This research contributes to works applying anthropological perspectives and ethnographic methodology to narrow the gap in available literature relevant to migration, Black Caribbean immigrant health and cancer health disparities.