Saúde sexual e reprodutiva de mulheres vivendo com HIV/AIDS atendidas em Hospital Dia

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo clínico e genético de uma família portadora de carcinoma medular de tireóide

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expressão das proteínas p53, Ki-67 e CD31 no tumor e nas margens vaginais após histerectomia radical em pacientes com carcinoma invasor do colo uterino

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção molecular do Papilomavirus humano (HPV) em amostras teciduais de tumores da mama

A neoplasia maligna da mama é uma das principais causa de mortalidade feminina, considerada como problema de saúde pública. Neste trabalho pesquisamos a presença do Papilomavírus Humano (HPV) nos tumores mamários benignos e malignos e em amostras de tecido mamário normal. Foi utilizada a técnica da Reação em Cadeia de Polimerase (PCR) para detecção molecular do DNA HPV em 63 pacientes, assim distribuídas: 28 tumores malignos, 17 tumores benignos e 18 amostras de tecido retro areolar de mamas normais. Os nossos resultados revelaram positividade para a seqüência do DNA HPV em 11 amostras, todas pertencentes às portadoras de tumores malignos: 17,4% de todas as amostras e 39,2% dos tumores malignos. Todos os tumores positivos revelaram DNA HPV para os tipos oncogênicos 16 e/ou 18, não foi detectado DNA HPV 06 e 11. Os resultados demonstraram elevada positividade para os receptores hormonais nas amostras positivas examinadas e apresentaram um seguimento com prevalência de eventos desfavoráveis como recidivas loco-regionais, metástases e óbito nas portadoras de DNA HPV. Os achados ratificam os dados encontrados na literatura, mostrando uma possível participação deste vírus no desenvolvimento do câncer de mama e possível contribuição desfavorável na evolução clínica.

Epidemiologia da infecção genital pelo Papilomavírus humano (HPV) em população feminina geral e população carcerária

A infecção genital pelo Papilomavírus humano (HPV) é considerada uma das doenças sexualmente transmissíveis (DSTs) mais comum, representando um importante problema na Saúde Pública, além de estar diretamente relacionado à promoção do câncer de colo uterino. Este estudo teve o intuito de investigar os aspectos epidemiológicos da infecção genital pelo HPV em dois grupos distintos: mulheres de população geral e mulheres encarceradas. Para tanto foi conduzido um estudo transversal analítico com 423 mulheres a partir dos 18 anos que se submeteram ao exame preventivo do câncer do colo uterino, sendo 233 mulheres da população geral oriundas de uma unidade básica de saúde da cidade de Belém do Pará e 190 provenientes do Centro de Reeducação Feminino em Ananindeua no mesmo Estado, no período de janeiro de 2008 a março de 2010. Amostras da cérvice uterina foram coletadas para a realização da colpocitologia convencional e para a detecção do DNA do HPV através da reação em cadeia da polimerase (PCR) mediada pelos oligonucleotídeos iniciadores universais MY9/11. Todas as mulheres responderam a um formulário clínico e epidemiológico. Entre as 423 mulheres analisadas, a prevalência geral de infecção genital pelo HPV foi de 13,0% com variação entre 15,0% para a amostra geral e 10,5% para a carcerária. A faixa etária mais acometida foi a de 13 a 25 anos (19%) na amostra geral; e em mulheres com 45 anos ou mais (21,1%), nas carcerárias. Anormalidades Colpocitológicas, situação conjugal, número de parceiros sexuais novos, o uso de anticoncepcionais orais, história de DST e de sintomas genitais, além de tabagismo atual, foram fatores que se mostraram associados à infecção genital pelo HPV de maneira diferenciada entre amostras da população geral e carcerária.

Correlação entre o câncer bucal e de laringe e a presença do Papilomavírus Humano (HPV) e do vírus Epstein-Barr (EBV) no estado do Pará

O câncer bucal e de laringe representam um problema crescente de saúde pública no Brasil. O tabagismo e o álcool são considerados os principais causadores do câncer bucal e de laringe, porém uma parte da população desenvolve a doença sem estar exposta a estes fatores de risco, sugerindo a existência de outras causas como: predisposição genética, alteração de genes supressores tumorais, dieta e agentes virais, em particular o Papilomavírus humano (HPV) e o vírus Epstein-Barr (EBV). Este estudo teve como proposição verificar a prevalência do HPV e do EBV na mucosa oral normal e no câncer bucal e de laringe, e quais os tipos mais prevalentes nestas duas situações. Para este estudo foram estabelecidos dois grupos: um composto por 70 espécimes emblocados em parafina, com diagnóstico confirmado de câncer bucal e laringe e outro com 166 indivíduos sem presença de lesões na cavidade bucal. A análise laboratorial para detecção viral do HPV e a detecção e tipagem do EBV (EBV 1 ou tipo 2) foram realizadas através da técnica de PCR (Reação em Cadeia de Polimerase) convencional. Já as tipagens das amostras positivas para o HPV (tipos 6, 11, 16, 18, 33, 35, 38, 52 e 58) foram realizadas por PCR em tempo real, utilizando sondas específicas para cada tipo. A prevalência do HPV e EBV encontrada nas neoplasias orais e de laringe foi de 78,6% para HPV e de 84,3% para EBV e de 24,1% e 45,8% para HPV e EBV, respectivamente, em indivíduos sem lesões orais. Os tipos mais prevalentes de HPV foram HPV 58 (50,9%), HPV 6 (9,1%) e HPV 16 (9,1%) nas neoplasias e HPV 18 (12,5%), HPV 6 (7,5%) e HPV 58 (2,5%) no grupo com ausência de lesões. O EBV 2 foi mais prevalente tanto nas lesões neoplásicas quanto nos indivíduos sem lesões, com frequência de 94,9% e 82,9%, respectivamente. Não houve associação da infecção por HPV e EBV com o sexo, sendo a prevalência semelhante para homens e mulheres. Foi observada associação entre as prevalências de HPV e EBV e suas co-infecções com o grupo que desenvolveu câncer. A prevalência de infecção por HPV e EBV e a razão de chances na ocorrência do câncer foi de 8,86 (p<0,0001) nos indivíduos infectados pelo HPV e de 4,08 (p=0,0004) nos infectados pelo EBV. O valor probabilístico estimado para prevalência de HPV e EBV e co-infecção e a ocorrência de câncer, demonstrou que o indivíduo infectado pelos dois vírus tem 65,72% de probabilidade de desenvolver câncer, enquanto o infectado pelo HPV tem 31,94% e o infectado pelo EBV 17,79%. Os resultados encontrados neste estudo permitem sugerir que os agentes virais (HPV e EBV) são fatores de risco importantes para o desenvolvimento da carcinogênese, sendo o HPV mais efetivo que o EBV no desencadeamento da doença.

Indications for the HPV vaccine in adolescents: A review of the literature

Background: The vaccine against human papillomavirus (HPV) was created to abrogate the risk that the virus presents for the development of cervical cancers. The prevalence of HPV infection among healthy individuals is significant (20%). We performed a review of the literature published in the period from 2008 to 2012 regarding the use of the vaccine against HPV specifically in adolescents. Methods: The articles were selected from a search of the PubMed database with the key words "vaccine", "HPV" and "adolescent". This search identified 576 articles; based on readings of the titles and abstracts, the list of included article was reduced to 42. Results: We observed that the majority of authors are in favor of the vaccine for adolescents particularly females. Conclusion: Recommending the use of the HPV vaccine and other vaccines represents an attempt to broaden the reach of these vaccines among both sexes of the adolescent population. Vaccination is a strategy for the prevention of pre-cancerous Lesions in the genital and oropharyngeal regions. (C) 2014 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Limited. All rights reserved.

Avaliação de atividades biológicas dos extratos de Rosmarinus officinalis L. (alecrim) e Thymus vulgaris L. (tomilho)

Pós-graduação em Biopatologia Bucal - ICT

Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines

The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells. (C) 2012 International Society for Advancement of Cytometry

Angiotensin II Facilitates Breast Cancer Cell Migration and Metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pretreatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

TGF-beta 1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Background: Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-beta 1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. Methods: The mRNA expression levels of TGF-beta isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-beta 1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. Results: In general, TGF-beta 2, T beta RI and T beta RII are over-expressed in more aggressive cells, except for T beta RI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-beta 1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-beta 1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-beta 1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-beta 1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-beta 1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-beta 1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Conclusion: Altogether, our results support that TGF-beta 1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-beta 1 still remains a promising target for breast cancer treatment.

HPV type infection in different anogenital sites among HIV-positive Brazilian women

Abstract Objectives To evaluate the prevalence of human papillomavirus (HPV) types, and risk factors for HPV positivity across cervix, vagina and anus, we conducted a study among 138 women with human immunodeficiency virus (HIV). Goal Compare the prevalence of different HPV types and the risk factors for HPV positivity in three sites. Results The most frequently detected HPV types in all sites were, in decreasing order, HPV16, 53, 18, 61 and 81. Agreement between the cervix and vagina was good (kappa 0.60 – 0.80) for HPV16 and 53 and excellent (Kappa > 0.80) for HPV18 and 61. HPV positivity was inversely associated with age for all combinations including the anal site. Conclusion In HIV positive women, HPV18 is the most spread HPV type found in combinations of anal and genital sites. The relationship of anal to genital infection has implications for the development of anal malignancies. Thus, the efficacy of the current HPV vaccine may be considered not only for the cervix, but also for prevention of HPV18 anal infection among immunossuppressed individuals.

TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Abstract Background Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. Methods The mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. Results In general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Conclusion Altogether, our results support that TGF-β1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-β1 still remains a promising target for breast cancer treatment.

Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.

RNA Interference and cyclooxygenase-2 (COX-2) regulation in colon cancer cells

Despite new methods and combined strategies, conventional cancer chemotherapy still lacks specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or specifically silencing any given target gene. Concerning gene silencing, attention has recently shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced, respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the research community new important perspectives for the comprehension of the physiological and, above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs expression have been identified in several types of neoplasia and it has also been proposed that the overexpression of genes in cancer cells may be due to the disruption of a control network in which relevant miRNA are implicated. For these reasons, I focused my research on a possible link between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC), since it has been established that the transition adenoma-adenocarcinoma and the progression of CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell invasion, tumour growth and metastatization. On the basis of data reported in the literature, the first aim of my research was to develop an innovative and effective tool, based on the RNAi mechanism, able to silence strongly and specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2 gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2 previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2 deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, results reported here indicate an easy-to-use, powerful and high selective virus-based method to knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore, they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as therapeutic agent for human cancers overexpressing COX-2. In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2 expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but also the most specific system to downregulate COX-2 in colon cancer cells. Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes, which encode for two bacterial factors needed for successful transfer of shRNA in mammalian cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29 and HCA-7 cell lines were in high agreement with data previously collected after the transfection of pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool for the treatment of colorectal cancer. Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer. In particular, it is known that components of the miRNA/RNAi pathway may be altered during the progressive development of colorectal cancer (CRC), and it has been already demonstrated that some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or more tumor suppressor miRNAs. In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR- 101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I observed that COX-2 overexpression induced by hypoxia is always coupled to a significant decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation could be involved in the adaption of cancer cells to the hypoxic environment that strongly characterize CRC tissues.