Caracterização e infecção de células tronco mesenquimais e células semelhantes a neurônios, derivadas de cordão umbilical bovino pelo herpesvírus bovino Tipo 5 (BoHV-5)

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

Monitoração dos parâmetros reprodutivos e perfil sorológico do herpesvírus caprino tipo 1 e do herpesvírus bovino tipo 1 em rebanhos caprinos dos estados de São Paulo e Minas Gerais

Pós-graduação em Medicina Veterinária - FCAV

Apoptose relacionada à infecção in vitro por herpesvírus bovinos tipo 1 e 5

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

Sarcoid-derived fibroblasts: links between genomic instability, energy metabolism and senescence.

Bovine papillomavirus 1 (BPV-1) is a well recognized etiopathogenetic factor in a cancer-like state in horses, namely equine sarcoid disease. Nevertheless, little is known about BPV-1-mediated cell transforming effects. It was shown that BPV-1 triggers genomic instability through DNA hypomethylation and oxidative stress. In the present study, we further characterized BPV-1-positive fibroblasts derived from sarcoid tumors. The focus was on cancer-like features of sarcoid-derived fibroblasts, including cell cycle perturbation, comprehensive DNA damage analysis, end-replication problem, energy metabolism and oncogene-induced premature senescence. The S phase of the cell cycle, polyploidy events, DNA double strand breaks (DSBs) and DNA single strand breaks (SSBs) were increased in BPV-1-positive cells compared to control fibroblasts. BPV-1-mediated oxidative stress may contribute to telomere dysfunction in sarcoid-derived fibroblasts. Loss of mitochondrial membrane potential and concurrent elevation in intracellular ATP production may be a consequence of changes in energy-supplying pathways in BPV-1-positive cells which is also typical for cancer cells. Shifts in energy metabolism may support rapid proliferation in cells infected by BPV-1. Nevertheless, sarcoid-derived fibroblasts representing a heterogeneous cell fraction vary in some aspects of metabolic phenotype due to a dual role of BPV-1 in cell transformation and oncogene-induced premature senescence. This was shown with increased senescence-associated β-galactosidase (SA-β-gal) activity. Taken together, metabolic phenotypes in sarcoid-derived fibroblasts are plastic, which are similar to greater plasticity of cancer tissues than normal tissues.

DNA hypomethylation and oxidative stress-mediated increase in genomic instability in equine sarcoid-derived fibroblasts

It is widely accepted that equine sarcoid disease, the most common skin associated neoplasm in equids, is induced by bovine papillomavirus (BPV-1). Although BPV-1 DNA has been found in almost all examined sarcoids so far, its detailed impact on the horse's host cell metabolism is largely unknown. We used equine fibroblast cell lines originating from sarcoid biopsies to study BPV-1-associated changes on DNA methylation status and oxidative stress parameters. Sarcoid-derived fibroblasts manifested increased proliferation in vitro, transcriptional rDNA activity (NORs expression) and DNA hypomethylation compared to control cells. Cells isolated from equine sarcoids suffered from oxidative stress: the expression of antioxidant enzymes was decreased and the superoxide production was increased. Moreover, increased ploidy, oxidative DNA damage and micronuclei formation was monitored in sarcoid cells. We postulate that both altered DNA methylation status and redox milieu may affect genomic stability in BPV-1-infected cells and in turn contribute to sarcoid pathology.

Factors associated with HPV16 status and survival among patients with squamous cell carcinoma of the oropharynx

Background: Squamous cell carcinoma of the oropharynx (SCCOP) is characterized by local tumor aggressiveness, high recurrence rates, a high incidence of second primary tumors, and medical comorbidities. Significant trends in demographic and clinical characteristics as well as survival among SCCOP patients have been observed over time, likely owing to the changing etiology of the disease. Human papillomavirus type 16 (HPV16) infection is associated with a significant proportion of these cancers. Biomarkers that may aid in identifying patients that are at higher risk of recurrence and death are important so that these patients may be followed more closely to improve their quality of life. ^ Study population and methods: The retrospective review (Specific Aim 2) included 3891 newly diagnosed, previously untreated patients presenting to our institution between 1955 and 2004. A total of 2299 patients treated at our institution were included in survival and recursive partitioning analysis. The prospective cohort study (Specific Aim 3) included 266 patients presenting to our institution between January 2006 and September 2009. ^ Results: The results from the retrospective review showed that over time, patients presented at younger ages and were more likely to have base of tongue/tonsil tumors and to be never/former smokers and moreover survival improved significantly over time. In survival and recursive partitioning analyses, the TNM staging system was efficient in prognosticating patients prior to 1995. However, in the recent decade, the TNM staging system was completely inadequate. The factors having the greatest positive effect on overall survival since 1995 were those common to HPV-associated SCCOP. The results from the prospective cohort study indicate that patients with high nodal stage and those with late stage disease have increased levels of pretreatment serum HPV DNA. ^ Conclusions: We saw a distinct improvement in survival among SCCOP patients over the past 50 years at our institution. The main factors contributing to this were changes in clinical characteristics, in particular surrogates for HPV status. The current TNM staging system for SCCOP is inadequate and incorporation of HPV status (and perhaps smoking status) is encouraged. Furthermore, although pretreatment circulating levels of HPV DNA was associated with higher N category and overall disease stage, it has limited utility as a marker for recurrence among SCCOP patients.^

Late Quateranry dinoflagellate cysts of ODP Site 202-1233

The late Quaternary organic-walled dinoflagellate cyst record of Site 1233 (41°S, offshore Chile) was studied with a ?200 year resolution spanning the last 25,000 years. The study provides the first continuous record of sub-recent and recent dinoflagellate cysts in the Southeast (SE) Pacific. Major changes in the composition of the cyst association, cyst concentration and morphology of Operculodinium centrocarpum reflect changes in sea surface temperature (SST), sea surface salinity (SSS), palaeoproductivity and upwelling intensity. These changes can be associated with latitudinal shifts of the circumpolar frontal systems. The high cyst concentration, high Brigantedinium spp. abundances, low species diversity and the occurrence of certain cold water species are supportive for a 7-10° equatorward shift of the Antarctic Circumpolar Current (ACC) during the coldest phase of the Last Glacial Maximum (LGM) between 25 and 21.1 cal ka BP. Deglacial warming initiated at ~18.6 cal ka BP. Termination I (18.6-11.1 cal ka BP) is interrupted by an unstable period of extreme seasonality, rather than a cooling event, between 14.4 and 13.2 cal ka BP, synchronous with the Antarctic Cold Reversal (ACR). The Holocene Maximum is observed between 11.6 and 9.8 cal ka BP and is typified by the most southward position of the northern margin of the ACC. A cooling phase occurred during the early Holocene (until ~7 cal ka BP) and during the last ~0.8 ka. Our data indicates that the SE Pacific (41°S) climate has been influenced over the whole record by changes in the Southern Hemisphere (SH) high-latitudes, while during the mid to late Holocene, also a tropical forcing mechanism was involved, including the El Niño Southern Oscillation and the variable Hadley cell intensity. Furthermore, this study showed a relationship between the variable morphology of the spines/processes of O. centrocarpum and the combined variation of sea surface salinity and temperature (SSS/SST-ratio).

Rapid induction of senescence in human cervical carcinoma cells

Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers.

Morphological classification of microtidal sand and gravel beaches

A new classification of microtidal sand and gravel beaches with very different morphologies is presented below. In 557 studied transects, 14 variables were used. Among the variables to be emphasized is the depth of the Posidonia oceanica. The classification was performed for 9 types of beaches: Type 1: Sand and gravel beaches, Type 2: Sand and gravel separated beaches, Type 3: Gravel and sand beaches, Type 4: Gravel and sand separated beaches, Type 5: Pure gravel beaches, Type 6: Open sand beaches, Type 7: Supported sand beaches, Type 8: Bisupported sand beaches and Type 9: Enclosed beaches. For the classification, several tools were used: discriminant analysis, neural networks and Support Vector Machines (SVM), the results were then compared. As there is no theory for deciding which is the most convenient neural network architecture to deal with a particular data set, an experimental study was performed with different numbers of neuron in the hidden layer. Finally, an architecture with 30 neurons was chosen. Different kernels were employed for SVM (Linear, Polynomial, Radial basis function and Sigmoid). The results obtained for the discriminant analysis were not as good as those obtained for the other two methods (ANN and SVM) which showed similar success.

Impaired Antigen Presentation and Effectiveness of Combined Active/Passive Immunotherapy for Epithelial Tumors

Background: Although immunization with tumor antigens can eliminate many transplantable tumors in animal models, immune effector mechanisms associated with successful immunotherapy of epithelial cancers remain undefined. Methods: Skin from transgenic mice expressing the cervical cancer-associated tumor antigen human papillornavirus type 16 (HPV16) E6 or E7 proteins from a keratin 14 promoter was grafted onto syngeneic, non-transgenic mice. Skin graft rejection was measured after active immunization with HPV16 E7 and adoptive transfer of antigen-specific T cells. Cytokine secretion of lymphocytes from mice receiving skin grafts and immunotherapy was detected by enzyme-linked immunosorbent assay, and HPV16 E7-specific memory CD8(+) T cells were detected by flow cytometry and ELISPOT. Results: Skin grafts containing HPV16 E6- or E7-expressing keratinocytes were not rejected spontaneously or following immunization with E7 protein and adjuvant. Adoptive transfer of E7-specific T-cell receptor transgenic CD8(+) T cells combined with immunization resulted in induction of antigen-specific interferon gamma-secreting CD8(+) T cells and rejection of HPV16 E7-expressing grafts. Specific memory CD8(+) T cells were generated by immunotherapy. However, a further HPV16 E7 graft was rejected from animals with memory T cells only after a second E7 immunization. Conclusions: Antigen-specific CD8(+) T cells can destroy epithelium expressing HPV16 E7 tumor antigen, but presentation of E7 antigen from skin is insufficient to reactivate memory CD8(+) T cells induced by immunotherapy. Thus, effective cancer immunotherapy in humans may need to invoke sufficient effector as well as memory T cells.

Functional memory CD8+ T cells can be generated in vivo without evident T help

Synthetic cytotoxic T cell (CTL) epitope peptides provide an effective and safe means of vaccination against cancers and viruses, as these peptides can induce specific CD8+ effector T cells in vivo. However, the effector CD8+ T cells induced by the minimal CTL epitope peptides do not last past about 3 weeks after the induction and no functional memory CD8+ T cells are generated. It is held that simultaneous induction of CD4+ T cells by incorporating peptides containing T-helper epitopes in the vaccine at the time of primary vaccination are necessary for the induction of long-lived functional memory CD8+ T cells. We now report that, surprisingly, incorporation of medium length (>20 AA) peptides devoid of detectable T-helper epitopes in a minimal CTL epitope-based vaccine can also induce long-lasting! functional rumour antigen specific memory CD8+ T cells that are capable of promoting protection against tumour challenge. This observation may have implications for the formulation of therapeutic anti-cancer and anti-virus peptide vaccines where a strong induction of CD4 T help would be undesirable. (C) 2004 Elsevier Ltd. All rights reserved.

Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX (TM) adjuvant in women with cervical intraepithelial neoplasia

Purpose: Persistent infection of cervical epithelium with high risk human papillomavirus (HPV) results in cervical intraepithelial neoplasia (CIN) from which squamous cancer of the cervix can arise. A study was undertaken to evaluate the safety and immunogenicity of an HPV 16 immunotherapeutic consisting of a mixture of HPV16 E6E7 fusion protein and ISCOMATRIX(TM) adjuvant (HPV16 Immunotherapeutic) for patients with CIN. Experimental design: Patients with CIN (n = 3 1) were recruited to a randomised blinded placebo controlled dose ranging study of immunotherapy. Results: Immunotherapy was well tolerated. Immunised subjects developed HPV16 E6E7 specific immunity. Antibody, delayed type hypersensitivity, in vitro cytokine release, and CD8 T cell responses to E6 and E7 proteins were each significantly greater in the immunised subjects than in placebo recipients. Loss of HPV16 DNA from the cervix was observed in some vaccine and placebo recipients. Conclusions : The HPV16 Immunotherapeutic comprising HPV16E6E7 fusion protein and ISCOMATRIX(TM) adjuvant is safe and induces vaccine antigen specific cell mediated immunity. (C) 2004 Elsevier Ltd. All rights reserved.

ISCOMATRIX (TM) adjuvant: An adjuvant suitable for use in anticancer vaccines

Human Papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are associated with cervical cancer development and progression and can therefore be used as target antigens for cancer immunotherapy. In this study we evaluated the immunogenicity in mice, of different vaccine formulations using recombinant HPV16 derived E6E7 or E7GST fusion proteins. When co-administered with ISCOMATRIX(TM) adjuvant, these E6E7 proteins consistently induced E7 specific CTL, in vivo tumor protection, antibody and DTH responses. ISCOMATRIX(TM) adjuvant has been developed for use in the formulation of novel human vaccines and has been evaluated for safety and toxicity in human trials. A formulation containing aluminum hydroxide (Al(OH)(3)) gave a lesser degree of E7 specific antibody, and no local E7 specific CTL response but similar DTH and tumor protection. These findings demonstrate the potential of ISCOMATRIX(TM) adjuvant to stimulate both cellular and humoral immune responses to endogenously processed target antigens, and hence is the preferred adjuvant when CTL responses are desirable. (C) 2004 Elsevier Ltd. All rights reserved.

Gene codon composition determines differentiation-dependent expression of a viral capsid gene in keratinocytes in vitro and in vivo

By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism

Methyl chloride purification for the silicone industry

This study experimentally investigated methyl chloride (MeCl) purification method using an inhouse designed and built volumetric adsorption/desorption rig. MeCl is an essential raw material in the manufacture of silicone however all technical grades of MeCl contain concentrations (0.2 - 1.0 % wt) of dimethyl ether (DME) which poison the process. The project industrial partner had previously exhausted numerous separation methods, which all have been deemed not suitable for various reasons. Therefore, adsorption/desorption separation was proposed in this study as a potential solution with less economic and environmental impact. Pure component adsorption/desorption was carried out for DME and MeCl on six different adsorbents namely: zeolite molecular sieves (types 4 Å and 5 Å); silica gels (35-70 mesh, amorphous precipitated, and 35-60 mesh) and granular activated carbon (type 8-12 mesh). Subsequent binary gas mixture adsorption in batch and continuous mode was carried out on both zeolites and all three silica gels following thermal pre-treatment in vacuum. The adsorbents were tested as received and after being subjected to different thermal and vacuum pre-treatment conditions. The various adsorption studies were carried out at low pressure and temperature ranges of 0.5 - 3.5 atm and 20 - 100 °C. All adsorbents were characterised using Brunauer Emmett Teller (BET), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDXA) to investigate their physical and chemical properties. The well-known helium (He) expansion method was used to determine the empty manifold and adsorption cell (AC) regions and respective void volumes for the different adsorbents. The amounts adsorbed were determined using Ideal gas laws via the differential pressure method. The heat of adsorption for the various adsorbate-adsorbent (A-S) interactions was calculated using a new calorimetric method based on direct temperature measurements inside the AC. Further adsorption analysis included use of various empirical and kinetic models to determine and understand the behaviour of the respective interactions. The gas purification behaviour was investigated using gas chromatography and mass spectroscopy (GC-MC) analysis. Binary gas mixture samples were syringed from the manifold iii and AC outlet before and after adsorption/desorption analysis through manual sample injections into the GC-MS to detect and quantify the presence of DME and ultimately observe for methyl chloride purification. Convincing gas purification behaviour was confirmed using two different GC columns, thus giving more confidence on the measurement reliability. From the single pure component adsorption of DME and MeCl on the as received zeolite 4A subjected to 1 h vacuum pre-treatment, both gases exhibited pseudo second order adsorption kinetics with DME exhibiting a rate constant nearly double that of MeCl thus suggesting a faster rate of adsorption. From the adsorption isotherm classification both DME and MeCl exhibited Type II and I adsorption isotherm classifications, respectively. The strength of bonding was confirmed by the differential heat of adsorption measurement, which was found to be 23.30 and 10.21 kJ mol-1 for DME and MeCl, respectively. The former is believed to adsorb heterogeneously through hydrogen bonding whilst MeCl adsorbs homogenously via van der Waal’s (VDW) forces. Single pure component adsorption on as received zeolite 5A, silica gels (35-70, amorphous precipitated and 35-60) resulted in similar adsorption/desorption behaviour in similar quantities (mol kg-1). The adsorption isotherms for DME and MeCl on zeolite 5A, silica gels (35-70, amorphous precipitated and 35-60) and activated carbon 8-12 exhibited Type I classifications, respectively. Experiments on zeolite 5A indicated that DME adsorbed stronger, faster and with a slightly stronger strength of interaction than MeCl but in lesser quantities. On the silica gels adsorbents, DME exhibited a slightly greater adsorption capacity whilst adsorbing at a similar rate and strength of interaction compared to MeCl. On the activated carbon adsorbent, MeCl exhibited the greater adsorption capacity at a faster rate but with similar heats of adsorption. The effect of prolonged vacuum (15 h), thermal pre-treatment (150 °C) and extended equilibrium time (15 min) were investigated for the adsorption behaviour of DME and MeCl on both zeolites 4A and 5A, respectively. Compared to adsorption on as received adsorbents subjected to 1 h vacuum the adsorption capacities for DME and MeCl were found to increase by 1.95 % and 20.37 % on zeolite 4A and by 4.52 % and 6.69 % on zeolite 5A, respectively. In addition the empirical and kinetic models and differential heats of adsorption resulted in more definitive fitting curves and trends due to the true equilibrium position of the adsorbate with the adsorbent. Batch binary mixture adsorption on thermally and vacuum pre-treated zeolite 4A demonstrated purification behaviour of all adsorbents used for MeCl streams containing DME impurities, with a concentration as low as 0.66 vol. %. The GC-MS analysis showed no DME detection for the tested concentration mixtures at the AC outlet after 15 or 30 min, whereas MeCl was detectable in measurable amounts. Similar behaviour was also observed when carrying out adsorption in continuous mode. On the other hand, similar studies on the other adsorbents did not show such favourable MeCl purification behaviour. Overall this study investigated a wide range of adsorbents (zeolites, silica gels and activated carbon) and demonstrated for the first time potential to purify MeCl streams containing DME impurities using adsorption/desorption separation under different adsorbent pre-treatment and adsorption operating conditions. The study also revealed for the first time the adsorption isotherms, empirical and kinetic models and heats of adsorption for the respective adsorbentsurface (A-S) interactions. In conclusion, this study has shown strong evidence to propose zeolite 4A for adsorptive purification of MeCl. It is believed that with a technical grade MeCl stream competitive yet simultaneous co-adsorption of DME and MeCl occurs with evidence of molecular sieiving effects whereby the larger DME molecules are unable to penetrate through the adsorbent bed whereas the smaller MeCl molecules diffuse through resulting in a purified MeCl stream at the AC outlet. Ultimately, further studies are recommended for increased adsorption capacities by considering wider operating conditions, e.g. different adsorbent thermal and vacuum pre-treatment and adsorbing at temperatures closer to the boiling point of the gases and different conditions of pressure and temperature.