Análise da expressão das proteínas celulares p53, p16 e ciclina D1 em amostras de tumores penianos

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

Detection of serum antibodies to parainfluenza type 3 virus, respiratory syncytial virus, bovine viral diarrhea virus, and herpes virus type 1 in sheep in the region of Botucatu, São Paulo - Brazil

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

HPV16 Oncoproteins Induce MMPs/RECK-TIMP-2 Imbalance in Primary Keratinocytes: Possible Implications in Cervical Carcinogenesis

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/ RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome.

Targets of emerging therapies for viral hepatitis B and C

Viral hepatitis B and C, structurally two completely different viruses, commonly infect human hepatocytes and cause similar clinical manifestations. Since their discovery, IFN has been a pillar in the treatment. However, because of the different natures of the viruses, therapeutic approaches diverge and new treatment targets are tailored specifically for each virus. Herein, the authors analyse therapeutic approaches for hepatitis B virus (HBV) and hepatitis C virus (HCV) and focus on emerging concepts that are under clinical evaluation. In particular, promising viral inhibitors for HBV and HCV are reviewed and the current status of research for gene therapy for HCV is described. Immune therapy is a fast-moving field with fascinating results which include therapeutic vaccines and toll-like receptor agonists that could improve tomorrow's treatment approaches.

Time-dependent interactions of the two porphyrinic compounds chlorin e6 and mono-L-aspartyl-chlorin e6 with phospholipid vesicles probed by NMR spectroscopy

The distribution processes of chlorin e6 (CE) and monoaspartyl-chlorin e6 (MACE) between the outer and inner phospholipid monolayers of 1,2-dioleoyl-phosphatidylcholine (DOPC) vesicles were monitored by 1H NMR spectroscopy through analysis of chemical shifts and line widths of the DOPC vesicle resonances. Chlorin adsorption to the outer vesicle monolayer induced changes in the DOPC 1H NMR spectrum. Most pronounced was a split of the N-methyl choline resonance, allowing for separate analysis of inner and outer vesicle layers. Transbilayer distribution of the chlorin compounds was indicated by time-dependent characteristic spectral changes of the DOPC resonances. Kinetic parameters for the flip-flop processes, that is, half-lives and rate constants, were obtained from the experimental data points. In comparison to CE, MACE transbilayer movement was significantly reduced, with MACE remaining more or less attached to the outer membrane layer. The distribution coefficients for CE and MACE between the vesicular and aqueous phase were determined. Both CE and MACE exhibited a high affinity for the vesicular phase. For CE, a positive correlation was found between transfer rate and increasing molar ratio CE/DOPC. Enhanced membrane rigidity induced by increasing amounts of cholesterol into the model membrane was accompanied by a decrease of CE flip-flop rates across the membrane. The present study shows that the movement of porphyrins across membranes can efficiently be investigated by 1H NMR spectroscopy and that small changes in porphyrin structure can have large effects on membrane kinetics.

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

CHARACTERIZATION OF TOBACCO MOSAIC VIRUS DIRECTED REPROGRAMMING OF PHLOEM GENE EXPRESSION TO PROMOTE VIRAL PHLOEM LOADING AND SYSTMEIC MOVEMENT

Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. Yet little is known about this process and the mechanisms that control it. In this study, an interaction between the replication protein of Tobacco mosaic virus (TMV) and phloem specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading. Promoter expression studies show TMV 126/183 kDa interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CC). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus but not during infection with a non-interacting virus. In situ analysis of virus spread shows the inability of TMV variants to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at systemic movement than a non-interacting virus. Similarly, CC expression and over-accumulation of a degradation-resistant-interacting Aux/IAA protein was found to selectively inhibit TMV accumulation and phloem loading. Transcriptional expression studies demonstrate a role for interacting Aux/IAA proteins in the regulation of salicylic acid and jasmonic acid dependent host defense responses as well as virus specific movement factors including pectin methylesterase that are involved in regulating plasmodesmata size exclusion limits and promoting virus cell-to-cell movement. Further characterization of the phloem environment was done using two phloem specific promoters (pSUC2 and pSULTR2;2) to generate epitope-tagged polysomal-RNA complexes. Immuno-purification using the epitope tag allowed us to obtain mRNAs bound to polysomes (the translatome) specifically in phloem tissue. We found the phloem translatome is uniquely altered during TMV infection with 90% and 88% of genes down regulated in the pSUC2 and pSULTR2;2 phloem translatomes, compared to 31% of genes down regulated in the whole plant p35S translatome. Transcripts down regulated in phloem include genes involved in callose deposition at plasmodesmata, host defense responses, and RNA silencing. Combined, these findings indicate TMV reprograms gene expression within the vascular phloem as a means to enhance phloem loading and systemic spread.

Sulphated polysaccharides from Ulva clathrata and Cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion, in NDV infection

Sulphated polysaccharides (SP) extracted from seaweeds have antiviral properties and are much less cytotoxic than conventional drugs, but little is known about their mode of action. Combination antiviral chemotherapy may offer advantages over single agent therapy, increasing efficiency, potency and delaying the emergence of resistant virus. The paramyxoviridae family includes pathogens causing morbidity and mortality worldwide in humans and animals, such as the Newcastle Disease Virus (NDV) in poultry. This study aims at determining the antiviral activity and mechanism of action in vitro of an ulvan (SP from the green seaweed Ulva clathrata), and of its mixture with a fucoidan (SP from Cladosiphon okamuranus), against La Sota NDV strain. The ulvan antiviral activity was tested using syncytia formation, exhibiting an IC50 of 0.1 μg/mL; ulvan had a better anti cell-cell spread effect than that previously shown for fucoidan, and inhibited cell-cell fusion via a direct effect on the F0 protein, but did not show any virucidal effect. The mixture of ulvan and fucoidan showed a greater anti-spread effect than SPs alone, but ulvan antagonizes the effect of fucoidan on the viral attachment/entry. Both SPs may be promising antivirals against paramyxovirus infection but their mixture has no clear synergistic advantage

Low frequency of p53 mutations in cervical carcinomas among Brazilian women

Human papillomavirus (HPV) infections of the high-risk types are strongly linked to the development of cervical carcinoma. The HPV oncoproteins E6 and E7 are thought to play a crucial role in this process through their interactions with the p53 protein and the retinoblastoma susceptibility gene product pRb, respectively. E6 binds to p53 protein promoting its degradation. This is considered to contribute to the oncogenesis of HPV-associated anogenital cancer. On the other hand, in HPV-negative cervical carcinoma, p53 mutations are thought to have a role in the transformation process. A total of 122 HPV-positive cervical carcinoma tissue samples were evaluated for the presence of mutations in exons 5-8 of the p53 gene by single-stranded conformation polymorphism analysis and DNA sequencing. Only four missense point mutations were detected. These findings suggest that other mechanisms independent of p53 inactivation may play a role in the genesis of cervical carcinomas.

HPV16 Tumor Associated Macrophages Suppress Antitumor T Cell Responses

Purpose: High-risk human papillomavirus (HPV) is the main etiologic factor for cervical cancer. The severity of HPV-associated cervical lesions has been correlated to the number of infiltrating macrophages. The objective of this work is to characterize the role of tumor-associated macrophages (TAM) on the immune cellular response against the tumor. Experimental Design: We used the HPV16 E6- and E7-expressing TC-1 mouse tumor model to study the effect of TAM on T-cell function in vitro, and depleted TAM, using clodronate-containing liposomes, to characterize its role in vivo. Results: TAM, characterized by the positive expression of CD45, F4/80, and CD11b, formed the major population of infiltrating tumor cells. TAM displayed high basal Arginase I activity, producing interleukin-10 (IL-10); they were resistant to iNOSll activity induction, therefore reversion to M1 phenotype, when stimulated in vitro with lipopolysaccharide/IFN gamma, indicating an M2 phentoype. In cultures of isolated TAM, TAM induced regulatory phenotype, characterized by IL-10 and Foxp3 expression, and inhibited proliferation of CD8 lymphocytes. In vivo, depletion of TAM inhibited tumor growth and stimulated the infiltration of tumors by HPV16 E7(49-57)-specific CD8 lymphocytes, whereas depletion of Gr1(+) tumor-associated cells had no effect. Conclusions: M2-like macrophages infiltrate HPV16-associated tumors causing suppression of antitumor T-cell response, thus facilitating tumor growth. Depletion or phenotype alteration of this population should be considered in immunotherapy strategies.

Papilomavírus humano e polimorfismo do gene TP53 no carcinoma espinocelular de cabeça e pescoço

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

Caracterização funcional de HTRA1 em linhagens celulares HPV positiva e HPV negativa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)