Hepatitis B virus X protein differentially regulates the angiogenesis of Hepatocellular Carcinoma through p53-VEGF axis according to glucose levels.

INTRODUCTION AND OBJECTIVES: Blood glucose fluctuates severely in the diabetes (DM) and tumor microenvironment. Our previous works have found Hepatitis B virus X protein (HBx) differentially regulated metastasis and apoptosis of hepatoma cells depending on glucose concentration. We here aimed to explore whether HBx played dual roles in the angiogenesis of hepatocellular carcinoma varying on different glucose levels. MATERIALS AND METHODS: We collected conditioned medium from HBx-overexpressing cells cultured with two solubilities of glucose, and then applied to EA.hy926 cells. Alternatively, a co-culture cell system was established with hepatoma cells and EA.hy926 cells. We analyzed the angiogenesis of EA.hy926 cells with CCK8, wound-healing, transwell-migartion and tube formation experiment. ELISA was conducted to detect the secretion levels of angiogenesis-related factors. siRNAs were used to detect the P53-VEGF axis. RESULTS: HBx expressed in hepatoma cells suppressed VEGF secretion, and subsequently inhibited the proliferation, migration and tube formation of EA.hy926 cells in a high glucose condition, while attenuating these in the lower glucose condition. Furthermore, the p53-VEGF axis was required for the dual role of HBx in angiogenesis. Additionally, HBx mainly regulated the nuclear p53. CONCLUSIONS: These data suggest that the dual roles of HBx confer hepatoma cells to remain in a glucose-rich environment and escape from the glucose-low milieu through tumor vessels, promoting liver tumor progression overall. We exclusively revealed the dual role of HBx on the angiogenesis of liver tumors, which may shed new light on the mechanism and management strategy of HBV- and DM-related hepatocellular carcinoma.

Interleukin-27 Promotes Divergent Effects on HIV-1 Infection in Peripheral Blood Mononuclear Cells through BST-2/Tetherin.

Interleukin-27 (IL-27) is able to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), macrophages, and dendritic cells. Here, we identify that IL-27 can produce opposing effects on HIV-1 replication in PBMCs and that the HIV-1 restriction factor BST-2/Tetherin is involved in both inhibitory and enhancing effects on HIV-1 infection induced by IL-27. IL-27 inhibited HIV-1 replication when added to cells 2 h after infection, promoting the prototypical BST-2/Tetherin-induced virion accumulation at the cell membrane of HIV-1-infected PBMCs. BST-2/Tetherin gene expression was significantly upregulated in the IL-27-treated PBMCs, with a simultaneous increase in the number of BST-2/Tetherin+ cells. The silencing of BST-2/Tetherin diminished the anti-HIV-1 effect of IL-27. In contrast, IL-27 increased HIV-1 production when added to infected cells 4 days after infection. This enhancing effect was prevented by BST-2/Tetherin gene knockdown, which also permitted IL-27 to function again as an HIV-1 inhibitory factor. These contrasting roles of IL-27 were associated with the dynamic of viral production, since the IL-27-mediated enhancement of virus replication was prevented by antiretroviral treatment of infected cells, as well as by keeping cells under agitation to avoid cell-to-cell contact. Likewise, inhibition of CD11a, an integrin associated with HIV-1 cell-to-cell transmission, abrogated the IL-27 enhancement of HIV-1 production. Our findings illustrate the complexity of the HIV-1-host interactions and may impact the potential therapeutic use of IL-27 and other soluble mediators that induce BST-2/Tetherin expression for HIV-1 infection. IMPORTANCE Here, we describe new findings related to the ability of the cytokine IL-27 to regulate the growth of HIV-1 in CD4+ T lymphocytes. IL-27 has long been considered a potent inhibitor of HIV-1 replication, a notion based on several reports showing that this cytokine controls HIV-1 infection in peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages, and dendritic cells. However, our present results are contrary to the current knowledge that IL-27 acts only as a powerful downregulator of HIV-1 replication. We observed that IL-27 can either prevent or enhance viral growth in PBMCs, an outcome dependent on when this cytokine is added to the infected cells. We detected that the increase of HIV-1 dissemination is due to enhanced cell-to-cell transmission with the involvement of the interferon-induced HIV-1 restriction factor BST-2/Tetherin and CD11a (LFA-1), an integrin that participates in formation of virological synapse.

HBX suppresses PTEN to promote the malignant progression of hepatocellular carcinoma through mi-R155 activation.

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most common and fatal tumors in the world, ranking third in cancer-related mortality. Chronic HBV infection is one of the major risk factors for hepatocellular carcinoma in China, Korea, and Sub-Saharan Africa. The HBx protein encoded by the X gene of HBV is a broadly regulated protein involved in transcriptional activation, epigenetics, apoptosis, DNA repair, and other regulatory processes. This study aimed to investigate the mechanism of HBx regulation of miR-155 and PTEN (Phosphatase and tensin homolog deleted on chromosome ten) in HBV-HCC. METHODS: Exosomal miR-155 quantity was analyzed by sampling serum exosomes of patients with hepatocellular carcinoma and normal subjects. The analysis was divided into different subgroups according to HBV positivity or negativity. At the cellular level, the biological roles of HBX, microRNA-155 and PTEN on hepatocellular carcinoma cells and their regulatory relationships with each other were verified. RESULTS: MicroRNA-155 and PTEN expression in HBV-positive HCC liver cancer tissues were negatively correlated, and HBX and miR-155 expression were positively correlated; microRNA-155 could target and inhibit PTEN expression, thereby promoting hepatocellular carcinoma cell activity, inhibiting apoptosis, and promoting invasion and migration; HBX could upregulate microRNA-155 thereby inhibit PTEN to promote malignant transformation of hepatocellular carcinoma. CONCLUSIONS: HBX could promote malignant transformation of hepatocellular carcinoma cells by upregulating microRNA-155 expression and thereby inhibiting the PTEN/PI3K-AKT pathway. Blocking miR-155 expression could attenuate the proliferation-promoting and invasive effects of HBX.

Phosphorylation of Phylogenetically Conserved Amino Acid Residues Confines HBx within Different Cell Compartments of Human Hepatocarcinoma Cells.

Hepatitis B virus (HBV) is a circular, and partially double-stranded DNA virus. Upon infection, the viral genome is translocated into the cell nucleus, generating the covalently closed circular DNA (cccDNA) intermediate, and forming a mini chromosome. HBV HBx is a small protein displaying multiple roles in HBV-infected cells, and in different subcellular locations. In the nucleus, the HBx protein is required to initiate and maintain viral transcription from the viral mini chromosome. In contrast, HBx also functions in the cytoplasm, where it is able to alter multiple cellular functions such as mitochondria metabolism, apoptosis and signal transduction pathways. It has been reported that in cultured cells, at low expression levels, the HBx protein is localized in the nucleus, whereas at high expression levels, it accumulates in the cytoplasm. This dynamic subcellular distribution of HBx might be essential to exert its multiple roles during viral infection. However, the mechanism that regulates different subcellular localizations of the HBx protein is unknown. We have previously taken a bioinformatics approach to investigate whether HBx might be regulated via post-translational modification, and we have proposed that the multiple nucleocytoplasmic functions of HBx might be regulated by an evolutionarily conserved mechanism via phosphorylation. In the current study, phylogenetically conserved amino acids of HBx with a high potential of phosphorylation were targeted for site-directed mutagenesis. Two conserved serine (Ser25 and Ser41), and one conserved threonine (Thr81) amino acids were replaced by either alanine or aspartic acid residues to simulate an unphosphorylated or phosphorylated state, respectively. Human hepatoma cells were transfected with increasing amounts of the HBx DNA constructs, and the cells were analyzed by fluorescence microscopy. Together, our results show that the nucleocytoplasmic distribution of the HBx protein could be regulated by phosphorylation since some of the modified proteins were mainly confined to distinct subcellular compartments. Remarkably, both HBx Ser41A, and HBx Thr81D proteins were predominantly localized within the nuclear compartment throughout the different expression levels of HBx mutants.

Exploring the Catalytic Mechanism of the RNA Cap Modification by nsp16-nsp10 Complex of SARS-CoV-2 through a QM/MM Approach.

The inhibition of key enzymes that may contain the viral replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have assumed central importance in drug discovery projects. Nonstructural proteins (nsps) are essential for RNA capping and coronavirus replication since it protects the virus from host innate immune restriction. In particular, nonstructural protein 16 (nsp16) in complex with nsp10 is a Cap-0 binding enzyme. The heterodimer formed by nsp16-nsp10 methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs and thus it is one of the enzymes that is a potential target for antiviral therapy. In this study, we have evaluated the mechanism of the 2'-O methylation of the viral mRNA cap using hybrid quantum mechanics/molecular mechanics (QM/MM) approach. It was found that the calculated free energy barriers obtained at M062X/6-31+G(d,p) is in agreement with experimental observations. Overall, we provide a detailed molecular analysis of the catalytic mechanism involving the 2'-O methylation of the viral mRNA cap and, as expected, the results demonstrate that the TS stabilization is critical for the catalysis.

Feline coronavirus isolates from a part of Brazil: insights into molecular epidemiology and phylogeny inferred from the 7b gene.

The Feline coronavirus (FCoV) can lead to Feline infectious peritonitis (FIP), which the precise cause is still unknown. The theory of internal mutation suggests that a less virulent biotype of FCoV (FECV) would lead to another more pathogenic biotype (FIPV) capable of causing FIP. In this work, the 7b gene was amplified from 51 domestic cat plasma samples by semi-nested PCR and tested through phylogenetic and phylogeographical approaches. The 7b gene of Brazilian isolates displayed high conservation, a strong correlation between the geographic origin of the viral isolates and their genealogy, and its evolution was possibly shaped by a combination of high rates of nucleotide substitution and purifying selection.

Hepatitis B Virus X Gene Differentially Modulates Subgenotype F1b and F4 Replication.

Hepatitis B virus (HBV) is classified into ten genotypes and numerous subgenotypes (sgt). In particular, sgt F1b and sgt F4, native of Latin America, have been associated with differences in clinical and virological characteristics. Hepatitis B virus X protein (HBx) is a multifunctional regulatory protein associated with the modulation of viral transcription and replication. In this work, we analyzed the role of the X gene and the encoded X protein in sgtF1b and sgtF4 replication. Transfection with HBx deficient genomes revealed remarkable differences in the replicative capacity of sgtF1b and sgtF4 mutants. The silencing of HBx increased sgtF1b X(-) transcription and replication by more than 2.5 fold compared to the wild type variant, while it decreased sgtF4 X(-) transcription and replication by more than 3 fold. Trans-complementation of HBx restore sgtF1b and sgtF4 wild type transcription and replication levels. In addition, transfection with chimeric variants, carrying wild type (F1b/XF4 and F4/XF1b) or mutated (F1b/X(-)F4 and F4/X(-)F1b) X gene of one sgt in the backbone of the other sgt, showed that the nucleotide sequence of the X gene, that includes regulatory elements that modulate pgRNA transcription, was responsible for the disparity observed between sgtF1b X(-) and sgtF4 X(-). These results showed that sgtF1b and sgtF4 X gene play a central role in regulating HBV transcription and replication, which eventually lead to a common purpose, to reach wild type replication levels of sgtF1b and sgtF4 viruses.

Assessment of Genetic Diversity of HTLV-1 ORF-I Sequences Collected from Patients with Different Clinical Profiles.

The human T cell lymphotropic virus type 1 (HTLV-1) infects 5 to 10 million individuals and remains without specific treatment. This retrovirus genome is composed of the genes gag, pol, env, and a region known as pX. This region contains four open reading frames (ORFs) that encode specific proteins. The ORF-I produces the protein p12 and its cleavage product, p8. In this study, we analyzed the genetic diversity of 32 ORF-I sequences from patients with different clinical profiles. Seven amino acid changes with frequency over 5% were identified: G29S, P34L, L55F, F61L, S63P, F78L, and S91P. The identification of regions where the posttranslational sites were identified showed a high identity among the sequences and the amino acid changes exclusive of specific clinical profile were found in less than 5% of the samples. We compare the findings with 2.406 sequences available in GenBank. The low overall genetic diversity found suggested that this region could be used in the HTLV-1 vaccine development.

Complete sequence of human T cell leukemia virus type 1 in ATLL patients from Northeast Iran, Mashhad revealed a prematurely terminated protease and an elongated pX open reading frame III.

To gain insight into the origin, evolution, dissemination and viral factors affecting HTLV-1-associated diseases, knowing the complete viral genome sequences is important. So far, no full-length HTLV-1 genome sequence has been reported from Iran. Here we report the complete nucleotide sequence of HTLV-1 viruses isolated from adult T cell leukemia/lymphoma (ATLL) patients from this region. The genome size of HTLV-1-MhD (Mashhad) was found to be 9036 bp and sequence analysis of the LTR region showed that it belongs to cosmopolitan subtype A. Comparing the sequences with isolates from another endemic area (HTLV-1ATK) revealed variations in the U3 region (~3.4%), while there was 99.1% and 97.0% similarity in R and U5 regions, respectively. The nucleotide sequences of HTLV-1 gag, pro and pol genes had a difference of 1.1% compared with HTLV-1 ATK with 16 nucleotides replaced in the gag and 27 in the pol regions. There was no variability in the amino acid sequences in the p24gag, however three residues were different in the p19gag and one in the p15gag. The nucleotide sequence of env showed a divergence of 1.5% compared to ATK with 22-nucleotide variation. The HTLV-1-MhD Tax, p13, p30, and p12 had 99.1, 100, 98.8, and 98%, respectively similarity with the prototype strain. Four amino acid changes were detected in ORF1 and ORF2 products p12 and p30, respectively, while the p13 region showed 100% conservation. The nucleotide identity between the isolates of Mashhad and those isolated from France, Germany, China, Canada and Brazil was 99.1%, 99.2%, 97.9%, 99% and 99.3%, respectively. Four amino acid changes compared with HTLV-1ATK from Japan were detected in ORF1 and ORF2 products p12 and p30, respectively, while the p13 region showed 100% conservation. This data could provide information regarding the evolutionary history, phylogeny, origin of the virus and vaccine design.

Hepatitis B virus in Mar del Plata, Argentina: Genomic characterization and evolutionary analysis of subgenotype F1b.

The aim is to describe the molecular epidemiology and perform a genomic characterization of hepatitis B virus (HBV) circulating in Mar del Plata and to identify the origin and diversification patterns of the most prevalent genotype. The S gene and the region encompassing the X gene, basal core promoter (BCP), and precore (preC) was analyzed in 56 samples. They were genotyped as: 80% F1b, 9% A2, 7% D3, and 2% D1. A recombinant F4/D2 genome was detected. The double substitution G1764A/A1762T at the BCP (reduced HBeAg expression) was found in 20% F1b, 2% A2, 2% D1, and 2% D3 samples. A unique D3 presented the G1896A substitution at the preC (HBeAg negative phenotype). A 13% of the samples showed mutations at the HBsAg "a" immunodeterminant (escape from neutralizing antibodies). Mutations at the polymerase (antiviral resistance) were found in 52% of the samples. Coalescent analysis of subgenotype F1b, the most prevalent in the city, showed that viral diversification in Mar del Plata started by year 2000. F1b was the most prevalent genotype detected, being a characteristic of actual HBV infections in Mar del Plata. Local HBV exhibit clinically relevant mutations, but a minority of them was shown to be associated to potential vaccination escape or antiviral resistance. Nevertheless, further studies are needed to determine whether any of these mutants could pose a threat to prevention, diagnosis, or treatment.

X protein variants of the autochthonous Latin American hepatitis B virus F genotype promotes human hepatocyte death by the induction of apoptosis and autophagy.

The hepatitis B virus X protein (HBV-X) is a multifunctional regulatory protein associated with the pathogenesis of liver disease in chronic HBV infection. Basal core promoter mutations (BCP), associated with the clinical course of chronic HBV infection, affect HBV-X at 130-131 positions. The role of these mutations on HBV-X biological activity remains largely unknown. The aim of this study was to analyze the impact of the presence of different amino acids at 130-131 positions of HBV-X on the biological activity of the protein. Transient expression of wild type and mutant F1b and F4 HBV-X increased cell mortality by the induction of apoptosis in human hepatoma cells. The wild type and mutant HBV-X differentially modulate the expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2 and Bcl-X) regulatory proteins of the Bcl-2 family. Furthermore, the expression of HBV-X variants of both subgenotypes induced autophagy of human tumoral hepatocytes. In conclusion, HBV-X variants of the Latin American HBV F genotype promotes human hepatocytes death by the induction of apoptosis and autophagy. The results of this work describe some of the molecular mechanisms by which HBV-X variants contribute to the pathogenesis of liver diseases in the infected liver and help to the biological characterization of genotype F, responsible of the majority of HBV infections in Argentina.

Farnesoid X receptor ablation sensitizes mice to hepatitis b virus X protein-induced hepatocarcinogenesis.

Chronic hepatitis B virus infection is a major risk factor for hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) is a hepatitis B virus protein that has multiple cellular functions, but its role in HCC pathogenesis has been controversial. Farnesoid X receptor (FXR) is a nuclear receptor with activities in anti-inflammation and inhibition of hepatocarcinogenesis. However, whether or how FXR can impact hepatitis B virus/HBx-induced hepatocarcinogenesis remains unclear. In this study, we showed that HBx can interact with FXR and function as a coactivator of FXR. Expression of HBx in vivo enhanced FXR-responsive gene regulation. HBx also increased the transcriptional activity of FXR in a luciferase reporter gene assay. The HBx-FXR interaction was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays, and the FXR activation function 1 domain was mapped to bind to the third α helix in the C terminus of HBx. We also found that the C-terminally truncated variants of HBx, which were found in clinical HCC, were not effective at transactivating FXR. Interestingly, recruitment of the full-length HBx, but not the C-terminally truncated HBx, enhanced the binding of FXR to its response element. In vivo, FXR ablation markedly sensitized mice to HBx-induced hepatocarcinogenesis. CONCLUSIONS: We propose that transactivation of FXR by full-length HBx may represent a protective mechanism to inhibit HCC and that this inhibition may be compromised upon the appearance of C-terminally truncated HBx or when the expression and/or activity of FXR is decreased. (Hepatology 2017;65:893-906).

The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state.

With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients.

HTLV-1 ORF-I Encoded Proteins and the Regulation of Host Immune Response: Viral Induced Dysregulation of Intracellular Signaling.

The human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus associated with both proliferative and inflammatory disorders. This virus causes a persistent infection, mainly in CD4+ T lymphocyte. The ability to persist in the host is associated with the virus capacity to evade the immune response and to induce infected T-cell proliferation, once the HTLV-1 maintains the infection mainly by clonal expansion of infected cells. There are several evidences that ORF-I encoded proteins, such as p12 and p8, play an important role in this context. The present study will review the molecular mechanisms that HTLV-1 ORF-I encoded proteins have to induce dysregulation of intracellular signaling, in order to escape from immune response and to increase the infected T-cell proliferation rate. The work will also address the impact of ORF-I mutations on the human host and perspectives in this study field.

Hepatitis B virus X protein activates human hepatic stellate cells through upregulating TGFß1.

We investigated the effects of the hepatitis B virus X gene (HBV X) on the activation of human hepatic stellate cells (HSCs) and the possible mechanisms underlying the pathway. Recombinant plasmid pHBV-X-IRES2-EGFP was constructed and transfected into HL-7702 cells using a lipid-mediated method. Transfected cells were screened by G418, which detected stable expression of the X gene by reverse transcription (RT)-PCR and Western blot analysis, and named L02/x. Cells not subjected to G418-selection were analyzed to confirm the transient expression of the X gene and named L02/48x. Subsequently, L02/x and L02/48x, together with non-HBx-expressing cells, were co-cultured with HSCs in a non-contact transwell system. After 36 h of co-culture, the proliferation and migration of HSCs was detected using different cell counting methods. Finally, the mRNA and protein levels of α-SMA, Col I, and TGFß1 in HSCs were detected by real-time PCR and western blot analysis. RT-PCR and Western blot analysis showed that L02/x and L02/48x cells can express HBV X gene mRNA and protein. Additionally, HSCs co-cultured with L02/x or L02/48x cells showed significantly higher proliferation and migration levels than control groups. Real-time PCR and Western blot analysis showed that the mRNA and protein expressions of α-SMA, Col I, and TGFß1 in HSCs co-cultured with HBx-expressing liver cells were higher than those in control groups. HBx protein activated HSCs in vitro, leading to increased proliferation and migration of HSCs and upregulation of α-SMA and Col I. The TGFß1 gene may be involved in this pathway.

Determinants of emergency contraception non-use among women in unplanned or ambivalent pregnancies / Determinantes del no uso de la anticoncepción de emergencia entre mujeres con embarazo no planeado u ambivalente / Determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente

Objective To analyze the determinants of emergency contraception non-use among women in unplanned and ambivalent pregnancies. Method Cross-sectional study with a probabilistic sample of 366 pregnant women from 12 primary health care units in the city of São Paulo, Brazil. A multinomial logistic regression was performed, comparing three groups: women who used emergency contraception to prevent ongoing pregnancies (reference); women who made no use of emergency contraception, but used other contraceptive methods; and women who made no use of any contraceptive methods at all. Results Cohabitation with a partner was the common determinant of emergency contraception non-use. No pregnancy risk awareness, ambivalent pregnancies and no previous use of emergency contraception also contributed to emergency contraception non-use. Conclusion Apart from what is pointed out in the literature, knowledge of emergency contraception and the fertile period were not associated to its use. .

Objetivo Analizar los determinantes del no uso de la anticoncepción de emergencia entre las mujeres con embarazo no planeado o ambivalente. Método Estudio transversal en una muestra probabilística de 366 mujeres embarazadas de 12 Unidades Básicas de Salud de São Paulo. Mediante regresión logística multinomial, se comparó tres grupos de mujeres: aquellas que usaron la anticoncepción de emergencia para prevenir el embarazo en curso (referencia), aquellas que usaron algún método anticonceptivo, pero no la anticoncepción de emergência; y aquellas que no usaron ningún método. Resultados Los hallazgos mostraron que vivir com la pareja fue el determinante común del no uso de la anticoncepción de emergencia. No tener conciencia del riesgo de embarazo, estar en un embarazo ambivalente y nunca tener utilizado la anticoncepción de emergencia también fueron associados con su no uso para prevenir el embarazo en curso. Conclusión Contrariamente a lo que reporta la literatura, el conocimiento de la anticoncepción de emergencia y el período fértil no mostró asociación con el no uso. .

Objetivo Analisar os determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente. Método Estudo transversal com amostra probabilística de 366 gestantes de 12 Unidades Básicas de Saúde da cidade de São Paulo. Por meio de regressão logística multinomial, compararam-se três grupos de mulheres: as que usaram anticoncepção de emergência para prevenir a gravidez em curso (referência); as que usaram algum método contraceptivo, mas não anticoncepção de emergência; e as que não usaram nenhum método. Resultados Os achados mostraram que morar com o parceiro foi o determinante comum do não uso da anticoncepção de emergência. Não ter consciência do risco de engravidar, estar em uma gravidez ambivalente e nunca ter usado anticoncepção de emergência também foram associados ao seu não uso para prevenir a gravidez em curso. Conclusão Diferentemente do que relata a literatura, o conhecimento sobre anticoncepção de emergência e sobre o período fértil não mostrou qualquer associação ao não uso. .

Differential expression of transforming growth factor-ß1 and HBx enhances hepatitis B virus replication and augments host immune cytokines and chemokines.

BACKGROUND/AIMS: This study investigated how HBV replication and host immune response are effected by reduced expression of TGF-ß1 and HBx. MATERIAL AND METHODS: Short interfering RNA (siRNA) knockdown technology has been used to examine the role of TGF-ß1 in hepatitis B virus replication. The siTGF-ß1 has been transfected along with 1.3mer HBV x-null to investigate the knockdown effect of TGF-ß1 on HBV replication and host immune factors. RESULTS: In this study, we found that diminished expression of TGF-ß1 and increased expression of HBx enhances HBV replication several folds. The differential expression of TGF-ß1 and HBx also stimulated transcriptional viral replicative intermediate (pgRNA) and secretion of core and 'e' antigen at translational level. Consequently, several cytokines such as IL-2, IL-8 and chemokine monocyte- chemoattractant protein (MCP-1) were increased significantly in response to stimulation of HBV replication. In contrast, TNF-α and RANTES mRNA expression increased insignificantly in response to enhanced HBV replication. CONCLUSIONS: We concluded that reduced expression of TGF-ß1 together with HBx expression stimulate HBV replication and immune response, although the underlying mechanism of stimulation most likely differs.