Infecção pelo Papilomavírus Humano em Adolescentes: Relação com o Método Anticoncepcional, Gravidez, Fumo e Achados Citológicos

Objetivo: avaliar a influência da gravidez, do hábito de fumar, do método anticoncepcional e quais os achados citológicos mais freqüentes em adolescentes com infecção pelo HPV. Métodos: foram analisadas retrospectivamente 54.985 citologias de pacientes atendidas entre julho de 1993 e dezembro de 1998. Deste total, 6.498 exames (11,8%) eram de pacientes com idade inferior a 20 anos, sendo que 326 (5,9%) apresentavam sinais citológicos de infecção por HPV, associada ou não a neoplasia intra-epitelial cervical (NIC) grau I. O grupo controle foi composto por 333 pacientes na mesma faixa etária, sem sinais citológicos de infecção por HPV. Resultados: a infecção pelo HPV foi mais freqüente nas adolescentes que fazem uso de anticoncepcionais orais (16,9% versus 13,8%, p<0,01) e que apresentam o achado citológico de clue cells (22,4% versus 14,7%, p<0,001). Os casais usuários de condom tiveram menor freqüência de infecção pelo HPV (0% versus 2,1%, p<0,01). A diferença do número de gestantes (41,1% versus 44,1%) e de fumantes (21,8% versus 16,5%) não foi estatisticamente significante. Conclusões: a infecção pelo HPV é mais freqüente em mulheres adolescentes que fazem uso de anticoncepcionais orais e que apresentam achado citológico de "clue cells". Os casais usuários de condom tiveram menor incidência de infecção pelo HPV. A gestação e o hábito de fumar não influenciaram na incidência da infecção pelo HPV.

Infecção pelo papilomavírus humano durante a gravidez: relação com achados citológicos

Objetivo: estudar um grupo de mulheres grávidas com infecção pelo papilomavírus humano (HPV), analisando a faixa etária, a idade gestacional, o número de gestações e os achados citológicos. Métodos: no período de julho de 1993 a dezembro de 1998, 245 gestantes atendidas em nosso serviço apresentavam alterações citológicas compatíveis com infecção pelo HPV, associados ou não à neoplasia intra-epitelial cervical (NIC) grau I. Foram colhidos dados clínicos relacionados à idade, período gestacional (primeira ou segunda metade), número de gestações e achado citológico de Trichomonas vaginalis, Candida sp. e clue cells. O grupo controle foi constituído por 386 pacientes gestantes atendidas no mesmo período e sem achados citológicos de infecção pelo HPV. Na análise estatística utilizou-se o teste do c² (qui-quadrado) com correção de Yates com nível de significância menor que 0,05. Resultados: a infecção pelo HPV foi mais freqüente nas grávidas com idade inferior a 20 anos (45,3% versus 28,2%, p<0,001). O achado citológico mais freqüente nas grávidas com infecção pelo HPV, comparando-se com o controle, foi o de clue cells (21,6% versus 12,4%, p<0,02). O achado de clue cells foi mais freqüente nas gestantes com HPV com idade superior a 20 anos e na segunda metade da gestação (27% versus 12,2%, p<0,01). A diferença quanto ao número de gestações anteriores não foi significativa. Conclusões: para o grupo estudado, a infecção pelo HPV foi mais freqüente em grávidas com idade inferior a 20 anos. Os achados citológicos de clue cells e de HPV foram mais freqüentes nas mulheres grávidas com idade superior a 20 anos e na segunda metade da gestação.

Perfil clínico e microbiológico de mulheres com vaginose bacteriana

OBJETIVO: estudar o perfil clínico e microbiológico de mulheres portadoras de vaginose bacteriana participantes de um ensaio clínico randomizado, duplamente mascarado, que comparou aroeira e metronidazol, em uso vaginal, para tratamento do corrimento genital. MÉTODOS: o estudo constitui-se em uma série de casos de 277 mulheres portadoras de vaginose bacteriana diagnosticada, concomitantemente, pelos critérios de Amsel e Nugent, selecionadas a partir de um total de 462 recrutadas, utilizando as informações colhidas antes da intervenção. A análise dos dados foi efetuada utilizando-se o programa Epi-Info 3.32. Para comparar as frequências dos desfechos entre os grupos de intervenção, foi utilizado o teste do χ2 e foi calculada a razão de risco e o intervalo de confiança a 95%. Foi feita análise por intenção de tratar. Além dos parâmetros de diagnósticos, foram também colhidas cultura do conteúdo vaginal e uma citologia de Papanicolaou. RESULTADOS: entre as queixas clínicas, as mais frequentes foram o corrimento genital, observado em 206 participantes (74,4%) e o odor de peixe da secreção vaginal, que ocorreu em 68,6% dos casos (190 pacientes). Dentre os critérios clínicos de diagnósticos, a presença de clue-cells foi positiva em 275 mulheres (99,3%), o teste de Whiff positivo apareceu em 266 participantes (96,0%), seguido do pH >4,5, que ocorreu em 92,8% dos casos e da presença de corrimento fluido e acinzentado, citado por 206 participantes (74,4%). Com relação ao critério de Nugent, a mediana dos escores foi o valor 8,0. As culturas de conteúdo vaginal permitiram a identificação de Gardnerella vaginalis em 96,8% e de Mobiluncus, em 53,1% dos casos. Apenas uma terça parte dos exames mostrou a presença de Lactobacillus (89 mulheres - 32,1%). Houve crescimento de fungos em culturas de 14 participantes (5,1%). Na maior parte dos casos, os resultados das culturas demonstraram a presença de Corynebacterium (94,2%), Cocos Gram-positivos (98,2%), além de bacilos Gram-positivos (99,3%) e negativos (91,0%). As colpocitologias oncóticas mostraram presença muito escassa de lactobacilos, que estiveram presentes em apenas 8 citologias (2,9%) do total de 273 exames realizados. CONCLUSÕES: os resultados do estudo não mostraram diferença em relação à literatura quanto aos sintomas referidos pelas mulheres, os critérios clínicos mais observados no diagnóstico, ou as espécies bacterianas demonstradas nas culturas de conteúdo vaginal. Os achados demonstram serem necessários novos estudos que melhor elucidem as inter-relações entre os achados microbiológicos e a expressão clínica da vaginose bacteriana. Registro do ensaio clínico: ISRCTN18987156

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

The study reported here is a classical bottom-up proteomic approach where proteins from wasp venom were extracted and separated by 2-DE; the individual protein spots were proteolytically digested and subsequently identified by using tandem mass spectrometry and database query with the protein search engine MASCOT. Eighty-four venom proteins belonging to 12 different molecular functions were identified. These proteins were classified into three groups; the first is constituted of typical venom proteins: antigens-5, hyaluronidases, phospholipases, heat shock proteins, metalloproteinases, metalloproteinase-desintegrin like proteins, serine proteinases, proteinase inhibitors, vascular endothelial growth factor-related protein, arginine kinases, Sol i-II and -II like proteins, alpha-glucosidase, and superoxide dismutases. The second contained proteins structurally related to the muscles that involves the venom reservoir. The third group, associated with the housekeeping of cells from venom glands, was composed of enzymes, membrane proteins of different types, and transcriptional factors. The composition of P. paulista venom permits us to hypothesize about a general envenoming mechanism based on five actions: (i) diffusion of venom through the tissues and to the blood, (ii) tissue, (iii) hemolysis, (iv) inflammation, and (v) allergy-played by antigen-5, PLA1, hyaluronidase, HSP 60, HSP 90, and arginine kinases.

Does the correlation between EBNA-1 and p63 expression in breast carcinomas provide a clue to tumorigenesis in Epstein-Barr virus-related breast malignancies?

Several investigators have identified Epstein-Barr virus (EBV) particles in breast carcinomas, a fact that supports a role for EBV in mammary tumorigenesis. The possible mechanism involved in this process is not clear. The present study was carried out in an attempt to determine whether there is a relationship between latent infection with EBV and p53 and p63 expression in breast carcinomas. Immunohistochemistry developed with 3.3-diaminobenzidine tetrahydrochloride was performed in 85 formalin-fixed paraffin-embedded breast carcinomas using anti-EBV EBNA-1, anti-p63, anti-p53, anti-estrogen receptor (ER) and anti-progesterone receptor (PR) antibodies. The cases were selected to represent each of the various histologic types: intraductal carcinoma (N = 12), grade I invasive ductal carcinoma (N = 15), grade II invasive ductal carcinoma (N = 15), grade III invasive ductal carcinoma (N = 15), tubular carcinoma (N = 8), lobular carcinoma (N = 10), and medullary carcinoma (N = 10). The ductal breast carcinomas were graded I, II and III based on the Scarff-Bloom and Richardson grading system modified by Elston and Ellis. One slide containing at least 1000 neoplastic cells was examined in each case. ER, PR, p63, p53 and EBNA-1 were positive in 60, 40, 11.8, 21.2 and 37.6% of carcinomas, respectively. There was a correlation between EBNA-1 and p63 expression (P < 0.001), but not between EBNA-1 and p53 (P = 0.10). These data suggest a possible role for p63 in the mammary tumorigenesis associated with Epstein-Barr virus infection.

Adenosine receptor type 2a is differently modulated by nicotine in dorsal brainstem cells of Wistar Kyoto and spontaneously hypertensive rats

Hypertension can result from neuronal network imbalance in areas of central nervous system that control blood pressure, such as the nucleus tractus solitarius (NTS). There are several neurotransmitters and neuromodulatory substances within the NTS, such as adenosine, which acts on purinoreceptors A(2a) (A(2a)R). The A(2a)R modulates neurotransmission in the NTS where its activation may induce decrease in blood pressure by different mechanisms. Nicotine is a molecule that crosses the hematoencephalic barrier and acts in several areas of central nervous system including the NTS, where it may interact with some neurotransmitter systems and contributes to the development of hypertension in subjects with genetic predisposition to this disease. In this study we first determined A(2a)R binding, protein, and mRNA expression in dorsomedial medulla oblongata of neonate normotensive (WKY) and spontaneously hypertensive rats (SHR). Subsequently, we analyzed the modulatory effects of nicotine on A(2a)R in cell culture in order to evaluate its possible involvement in the development of hypertension. Data showed a decreased A(2a)R binding and increased protein and mRNA expression in tissue sample and culture of dorsal brainstem from SHR compared with those from WKY rats at basal conditions. Moreover, nicotine modulated A(2a)R binding, protein, and mRNA expression in cells from both strains. Interestingly, nicotine decreased A(2a)R binding and increased protein levels, as well as, induced a differential modulation in A(2a)R mRNA expression. Results give us a clue about the mechanisms involved in the modulatory effects of nicotine on A(2a)R as well as hypothesize its possible contribution to the development of hypertension. In conclusion, we demonstrated that A(2a)R of SHR cells which differ from WKY and nicotine differentially modulates A(2a)R in dorsal brainstem cells of SHR and WKY.

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pipkiiα Is Widely Expressed In Hematopoietic-derived Cells And May Play A Role In The Expression Of Alpha- And Gamma-globins In K562 Cells.

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

An Efficient Protocol For The Generation Of Monocyte Derived Dendritic Cells Using Serum-free Media For Clinical Applications In Post Remission Aml Patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on good manufacture practices (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Up-regulation Of Dnam-1 And Nkp30, Associated With Improvement Of Nk Cells Activation After Long-term Culture Of Mononuclear Cells From Patients With Ovarian Neoplasia.

This study aimed at evaluating the functional activation and activating receptors expression on resting, short- and long-term NK and NK-like T cells from blood of ovarian neoplasia patients. Blood from patients with adnexal benign alterations (n = 10) and ovarian cancer (grade I-IV n = 14) were collected after signed consent. Effector cells activation was evaluated by the expression of the CD107a molecule. Short-term culture was conducted overnight with IL-2 and long-term culture for 21 days, by a method designed to expand CD56(+) lymphocytes. Short-term culture significantly increased NK cells activation compared to resting NK cells (p<0.05), however, the long-term procedure supported an even higher increase (p<0.001). Resting NK-like T cells showed poor activation, which was not altered by the culture procedures. The long-term culture effectively increased the expression of the activating receptors on NK and NK-like T cells, either by increasing the number of cells expressing a given receptor and/or by up-regulating their expression intensity. As a conclusion, the long-term culture system employed, resulted in a high number of functional NK cells. The culture system was particularly efficient on the up-regulation of NKp30 and DNAM-1 receptors on NK cells.

Nanomolar Levels Of Pahs In Extracts From Urban Air Induce Mapk Signaling In Hepg2 Cells.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that occur naturally in complex mixtures. Many of the adverse health effects of PAHs including cancer are linked to the activation of intracellular stress response signaling. This study has investigated intracellular MAPK signaling in response to PAHs in extracts from urban air collected in Stockholm, Sweden and Limeira, Brazil, in comparison to BP in HepG2 cells. Nanomolar concentrations of PAHs in the extracts induced activation of MEK4 signaling with down-stream increased gene expression of several important stress response mediators. Involvement of the MEK4/JNK pathway was confirmed using siRNA and an inhibitor of JNK signaling resulting in significantly reduced MAPK signaling transactivated by the AP-1 transcription factors ATF2 and c-Jun. ATF2 was also identified as a sensitive stress responsive protein with activation observed at extract concentrations equivalent to 0.1 nM BP. We show that exposure to low levels of environmental PAH mixtures more strongly activates these signaling pathways compared to BP alone suggesting effects due to interactions. Taken together, this is the first study showing the involvement of MEK4/JNK/AP-1 pathway in regulating the intracellular stress response after exposure to nanomolar levels of PAHs in environmental mixtures.

Changes In Chromatin Structure In Nih 3t3 Cells Induced By Valproic Acid And Trichostatin A.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA.

Metabolic Memory Of ß-cells Controls Insulin Secretion And Is Mediated By Camkii.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in calcium pulses as a form of metabolic memory, just as neurons store cognitive information. To test this hypothesis, we developed a novel paradigm of pulsed exposure of ß-cells to intervals of high glucose, followed by a 24-h consolidation period to eliminate any acute metabolic effects. Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. Metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and Ca(2+)-dependent vesicle secretion, and by elevated levels of the key ß-cell transcription factor MAFA. In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information.

Fatty Acid Synthase Inhibitors Induce Apoptosis In Non-tumorigenic Melan-a Cells Associated With Inhibition Of Mitochondrial Respiration.

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.