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Biomedical subjects

R F Silva

Publications and source records attributed to R F Silva.

At least 19 recordsLinked to original sources
PubMed2006

Oncogenic Marek's disease viruses lacking the 132 base pair repeats can still be attenuated by serial in vitro cell culture passages.

Marek's disease virus (MDV) can be attenuated by serially passing the virus in cell culture. During cell culture passage, two copies of a 132 bp repeat are expanded to over 30 copies. We deleted the two copies of the 132 bp repeat region in a pathogenic MDV and demonstrated that the virus was still pathogenic. The pattern and frequency of tumors in the parental and mutant virus were the same. Early virus replication, and the appearance of persistent neurological disease were also similar between the parental and deleted virus. Nevertheless, wild-type MDV and the deletion virus could be attenuated by serial in vitro cell culture passages. Based upon analyzing the passage 40 viruses, attenuation of the MDV lacking the 132 bp repeats appears to occur in a manner that is analogous to the process occurring wild-type MDV attenuation. Whatever process is involved in the cell culture attenuation of MDV, the mechanism does not involve the 132 bp repeat region.

Animals↗
PubMed2005

Novel criteria for the diagnosis of Marek's disease virus-induced lymphomas.

Several novel criteria have been tested to assist in the differential diagnosis of tumours induced by Marek's disease virus (MDV) from those induced by avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). A collection of tumours induced by inoculation of specific strains of MDV, ALV and REV, alone or in combination, were tested for quantification of MDV DNA by real-time polymerase chain reaction, expression of the MDV oncogene Meq, expression of several cell markers associated with transformation (CD30, Marek's disease-associated surface antigen, and p53), and level of DNA methylation in the tumour cells. In addition, tissues latently infected with MDV and non-infected tissues were tested as controls. Tumours induced by MDV had about 10(2)-fold more copies of MDV DNA than either tissues latently infected by MDV or tumours induced by retrovirus in MDV-vaccinated chickens. Moreover, the MDV antigen Meq was consistently expressed in all MDV tumours but it could not be detected in tissues latently infected with MDV or in tumours induced by retrovirus in MDV-vaccinated chickens. Other markers studied were not specific for MDV and therefore had limited value for diagnosis. Nonetheless, some of these markers might have potential value in research as they will help to identify transformed cells.

Animals↗
PubMed2005

The pp38 gene of Marek's disease virus (MDV) is necessary for cytolytic infection of B cells and maintenance of the transformed state but not for cytolytic infection of the feather follicle epithelium and horizontal spread of MDV.

Marek's disease virus has a unique phosphoprotein, pp38, which is suspected to play an important role in Marek's disease pathogenesis. The objective of the present study was to utilize a mutant virus lacking the pp38 gene (rMd5Deltapp38) to better characterize the biological function of pp38. This work shows that the pp38 gene is necessary to establish cytolytic infection in B cells but not in feather follicle epithelium, to produce an adequate level of latently infected T cells, and to maintain the transformed status in vivo.

Animals↗
PubMed2004

Complications following transjugular intrahepatic portosystemic shunt: a retrospective analysis.

Transjugular intrahepatic portosystemic shunt (TIPS) has been the therapeutic option for severe decompensation of chronic liver disease and as a bridge to liver transplantation. The aim of this study was to analyze the complications of this procedure. The records of 47 patients (39 men) of mean age 48 years underwent TIPS procedures from 1998 to 2003 were reviewed. Forty-one patients received 45 successful TIPS; it failed in six patients. Improvement was observed in 20 of 28 patients with upper gastrointestinal bleeding (71%); 9 of 11 with ascites (82%); and 5 of 8 with impaired renal function (62%). The Child-Pugh scores improved in 6 of the 47 patients (13%). Transplantation was performed in 11 patients (23%). The complications were: encephalopathy (49%); infection (19%); renal failure (17%); TIPS migration to the portal vein (4%) and to the right atrium (4%). Mortality was 32% (15/47) over 3 months. Eight patients developed active bleeding during TIPS installation requiring mechanical ventilation and intensive care, and died within the first week. Other causes of death were sepsis (n = 2), liver failure (n = 1), accidental puncture of the Glisson's capsule leading to intra-abdominal bleeding (n = 1) and refractory upper gastrointestinal bleeding (n = 3). The latter four patients had TIPS placement failure. In conclusion, TIPS produced clinical improvement among 51% of patients with complications in 49%. The main complications were encephalopathy (49%), infection (19%), and renal failure (17%). The 3-month mortality rate after TIPS placement was 32%.

Cause of Death↗
PubMed2004

Expansion of a unique region in the Marek's disease virus genome occurs concomitantly with attenuation but is not sufficient to cause attenuation.

Pathogenic Marek's disease viruses (MDVs) have two head-to-tail copies of a 132-bp repeat. As MDV is serially passaged in cell culture, the virus becomes attenuated and the number of copies of the 132-bp repeat increases from 2 to often more than 20 copies. To determine the role of the repeats in attenuation, we used five overlapping cosmid clones that spanned the MDV genome to reconstitute infectious virus (rMd5). By mutating the appropriate cosmids, we generated clones of infectious MDVs that contained zero copies of the 132-bp repeats, rMd5(Delta132); nine copies of the 132-bp repeats, rMd5(9-132); and nine copies of the 132-bp repeats inserted in the reverse orientation, rMd5(rev9-132). After two passages in cell culture, wild-type Md5, rMd5, and rMd5(Delta132) were stable. However, rMd5(9-132) and rMd5(rev9-132) contained a population of viruses that contained from 3 to over 20 copies of the repeats. A major 1.8-kb mRNA, containing two copies of the 132-bp repeat, was present in wild-type Md5 and rMd5 but was not present in rMd5(Delta132), rMd5(9-132), rMd5(rev9-132), or an attenuated MDV. Instead, the RNAs transcribed from the 132-bp repeat region in rMd5(9-132) and rMd5(rev9-132) closely resembled the pattern of RNAs transcribed in attenuated MDVs. When inoculated into susceptible day-old chicks, all viruses produced various lesions. Thus, expansion of the number of copies of 132-bp repeats, which accompanies attenuation, is not sufficient in itself to attenuate pathogenic MDVs.

Animals↗
PubMed2002

Densification route and mechanical properties of Si3N4-bioglass biocomposites.

The processing route and the final microstructural and mechanical characteristics of a novel biomaterial composite are described. This new material is composed of 70 wt% Si3N4 ceramic phase and 30 wt% bioglass, the later performing as a liquid sintering aid system and simultaneously providing bioactivity characteristics to the composite. The conditions for fabrication of an almost fully dense material (approximately 98% of relative density) were pursued. Optimised parameters were 1350 degrees C-40 min-30 MPa by hot-pressing technique. The very fast densification rate of the process avoided the crystallisation of the bioglass intergranular phase and therefore its intrinsic properties were maintained. Also, the large amount of glassy phase assured the densification by liquid phase assisted grain rearrangement without Si3N4 phase transformation. The final mechanical properties of the Si3N4 bioglass were as follows: fracture toughness, K(IC) = 4.4 MPa m(1/2); Vickers hardness, Hv = 10.3 GPa; Young's modulus, E = 197 GPa; bending strength, sigma(g) = 383 MPa; Weibull modulus, m = 8.3. These values provide an attractive set of properties among other bioactive materials, namely by upgrading the main drawback of bioceramcs and bioglasses for high-load medical applications, which is the lack of satisfactory fracture toughness.

Biocompatible Materials↗
PubMed2002

Wettability and surface charge of Si3N4-bioglass composites in contact with simulated physiological liquids.

Wettability and surface charge studies were performed on a novel Si3N4-30wt% bioglass biocomposite. Contact angle and surface tension variation with time were determined at 25 degrees C, respectively, by the sessile and pendant drop techniques, for distinct testing liquids: water, diiodomethane, simulated body fluid (SBF) and bovine serum albumin (BSA) dissolved in SBF solution. This biocomposite revealed a hydrophilic character (theta = 26.6 +/- 2.0 degrees) and a surface tension value (66.6 mJ m(-2)) comparatively higher than those of the most common bioceramics. An important characteristic is the high work of adhesion towards SBF + BSA (96.4 +/- 0.2mJ m(-2)) that was measured. The Si3N4-bioglass material is negatively surface charged above the pH(IEP) = 2.5 in aqueous SBF + BSA solution, as a result of the presence at the surface of unsaturated Si-O bonds and Si-OH groups. The very high negative zeta potential at pH approximately 7 (-58.6 +/- 5.5mV) influenced albumin adsorption and mechanisms are discussed in terms of entropy and enthalpy gains from conformational unfolding and cations coadsorption.

Glass↗
PubMed2002

Si(3)N(4)-bioglass composites stimulate the proliferation of MG63 osteoblast-like cells and support the osteogenic differentiation of human bone marrow cells.

The in vitro osteocompatibility of a novel Si(3)N(4)-bioglass composite (70-30% weight proportion) with improved mechanical properties (fracture toughness = 4.4 M Pa m(1/2); bending strength = 383 +/- 47 MPa) is reported. Immersion of the composite samples in culture medium (30 min to 7 days) resulted in rapid protein adsorption to the surface and, also, dissolution of the intergranular phase of bioglass (time-dependent process) with the formation of different size cavities. "As-received" and pre-treated material samples presented a similar behaviour concerning the proliferation of MG63 osteoblast-like cells, evaluated during a 5-day culture period. Seeded materials showed a higher cell growth rate as compared to cultures performed on the standard plastic culture plates. To assess the osteogenic potential of the composite, "as-received" material samples were seeded with human bone marrow cells and cultured for 35 days in experimental conditions that favour the development of the osteoblastic phenotype. The cell adhesion process was similar to that observed in control cultures. Cells successfully adapted to the irregularities of the surface and were able to grow towards inside the cavities; in addition, osteogenic differentiation occurred with the formation of abundant cell-mediated mineralised deposits. Results suggest that this Si(3)N(4)-bioglass composite seems to be a promising candidate for high-stress medical applications.

Adsorption↗
PubMed2001

Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxycholic acid but prevented by ursodeoxycholic acid.

BACKGROUND/AIMS: Unconjugated bilirubin (UCB) can be neurotoxic in jaundiced neonates and in patients with Crigler-Najjar syndrome. UCB toxicity may culminate in cell death, however, the occurrence of apoptosis has never been investigated. Ursodeoxycholic acid (UDCA) is a strong modulator of the apoptotic threshold in both hepatic and nonhepatic cells. The aims of this study were to determine whether apoptosis plays a role in neural cell death induced by UCB, and to investigate the ability of UDCA to prevent cell death. METHODS: Cultured rat astrocytes were incubated with UCB (17 and 86 microM) plus albumin (5.7 and 28.7 microM) for 4-22 h. In addition, astrocytes and neurones were treated with either UCB, 50 microM UDCA, or their combination for 4 h. Cultures were scored for nonviable cells by trypan blue dye exclusion. Apoptosis was assessed by Hoechst staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling assay. RESULTS: UCB induced a concentration- and time-dependent decrease in astrocyte viability. Apoptosis was 4- and 7-fold increased after 4 h exposure to 17 and 86 microM UCB, respectively (P < 0.01). UDCA reduced apoptosis to <7%, which represents a appoximately 60% protection (P < 0.01). Cholic acid was not protective, and chenodeoxyholic acid aggravated UCB toxicity (P < 0.05). Finally, neurones showed a 1.5-fold greater sensitivity than astrocytes to UCB, while UDCA was still protective. CONCLUSIONS: UCB is toxic to both astrocytes and neurones, causing cell death through an apoptotic process. Moreover, UDCA inhibits UCB-induced apoptosis in neural cells and this could not be mimicked by other bile acids.

Animals↗
PubMed2001

Characterization of a recombinant fowlpox virus expressing the native hexon of hemorrhagic enteritis virus.

The structure of the icosahedral adenovirus capsid is highly conserved among Adenoviridae. In its native form, the hexon is the major capsid protein. The nascent hexon requires the 100 kDa folding protein to fold into its native, trimeric form. The hexon and 100 kDa folding protein were co-expressed in a fowlpox virus (FPV) vector and in the recombinant FPVs (rFPVs) in which the hexon and 100 kDa folding protein genes are cloned head to tail, the native hexon could be detected with indirect immunofluorescence and immunoprecipitation using a native hexon monoclonal antibody. The FPV-@X100 construct, in which the 100kDa folding protein gene follows the hexon gene in a head to tail fashion, elicited the best humoral response in chickens. An attenuated HEV commercial vaccine elicited higher and longer lasting anti-HEV titers than FPV-@X100. Humoral immunity was also compared in turkeys inoculated with rFPVs expressing the hexon alone, the 100 kDa folding protein alone, or expressing both genes in different configurations. No anti-HEV humoral immune response was detected in turkeys inoculated with the rFPVs expressing the hexon alone or the 100 kDa folding protein alone.

Adenoviridae↗
PubMed2001

Endocytosis in rat cultured astrocytes is inhibited by unconjugated bilirubin.

Excessive hyperbilirubinemia can cause irreversible neurological damage in the neonatal period. However, the complete understanding of the pathogenesis of unconjugated bilirubin (UCB) encephalopathy remains a matter of debate. This study investigates whether UCB inhibits the endocytosis of cationized ferritin (CF) by cultured rat astrocytes. The relationship between endocytosis and MTT reduction, as well as changes on tubulin and glial fibrillary acidic protein (GFAP) assembly, were also evaluated. Inhibition of endocytosis was complete in the presence of 171 microM UCB, while a marked decrease of CF labeling was noticed for 86 microM UCB. In addition, MTT reduction was inhibited by 60 to 76% as UCB concentrations changed from 17 to 171 microM, while alterations on both GFAP and microtubule morphology were only achieved by cell exposure to 171 microM UCB. These findings indicate that inhibition of CF endocytosis in rat cortical astrocytes by UCB is a concentration-dependent process that appears to be primarily related to a direct effect on the cell membrane and not to any alteration of cytoskeletal microtubules and intermediate filaments.

Animals↗
PubMed2000

Hypervariability in the envelope genes of subgroup J avian leukosis viruses obtained from different farms in the United States.

Avian leukosis virus, subgroup J (ALV-J), has a wide host range, preferentially infecting meat-type birds, and produces a high incidence of myelocytomatosis and nephromas. Using the published sequences from HPRS-103 (ALV-J isolated in 1989 in Great Britain), we designed a set of PCR primers that amplified proviral DNA from nine U.S. field samples. The primers were specific for ALV-J, not amplifying DNA from uninfected cells or cells infected with ALV subgroups A-E. These primers expanded a 2.4-kb fragment that encompasses gp85, gp37, the E element, and most of the 3' LTR. We also developed a set of PCR primers that amplified a 2.1-kb fragment from ALV-J-infected cells and a 1.6-kb fragment from uninfected ev- chicken embryo fibroblasts (Line 0). Upon cloning and DNA sequencing, we determined that the 2.1- and 1.6-kb fragments contained ALV-J gp85- and gp37-like sequences. Comparison of the amino acid sequences demonstrated that the Line 0 sequences were 97.5% identical with the gp85 and gp37 of HPRS-103 and somewhat less identical with the other nine U.S. isolates. This suggests that the envelope genes of ALV-J may have arisen as a result of a recombination event between exogenous ALV and Line 0-like sequences in the chicken. Phylogenetic analysis also showed that the U.S. field isolates were closely related to one another and more distantly related to the European HPRS-103. The pattern of mutations in the U.S. field isolates suggests that the U.S. strains are slowly drifting away from their progenitor Line 0-like sequences. The development of effective vaccines and diagnostic tests is likely to become more problematic as the viruses continue to mutate.

Amino Acid Sequence↗
PubMed2000

Microstructural dependence of Young's and shear moduli of P2O5 glass reinforced hydroxyapatite for biomedical applications.

P2O5 glass reinforced hydroxyapatite composite materials were prepared through a liquid-phase sintering process. Secondary phases, beta- and alpha-tricalcium phosphates (beta-TCP and alpha-TCP), were formed in the microstructure of the composites, due to the reaction between the liquid glassy phase and the hydroxyapatite matrix. The dynamic Young's modulus (E) and shear modulus (G) of these composites were determined using an impulse excitation method. By applying the Duckworth-Knudsen equation, the elastic property results were correlated with the relative proportion of beta-TCP and alpha-TCP phases and with the porosity percentage present in the microstructure. Glass reinforced hydroxyapatite composites showed lower Young's and shear moduli than unmodified hydroxyapatite, mainly due to the presence of beta-TCP phase. The Duckworth-Knudsen model demonstrated an exponential dependence of E and G modulus with porosity and mathematical equations were derived for composite materials with porosity correction factors (b) of 4.04 and 4.11, respectively, indicating that porosity largely decreased both E and G moduli.

Biocompatible Materials↗
PubMed1999

A genetically engineered cell line resistant to subgroup J avian leukosis virus infection (C/J).

A cell line (DF-1¿J) expressing the envelope protein isolated from the ADOL-Hc1 strain of the avian leukosis virus subgroup J (ALV-J) was used to analyze receptor interference to six different isolates of ALV-J as well as ALV subgroups A-D. The traditional gag-specific enzyme-linked immunosorbent assay (ELISA) as well as flow cytometry was used to evaluate viral infection. The parental cell line (DF-1) was susceptible to all ALV subgroups tested while the DF-1¿J cell line was selectively resistant to the subgroup J isolates. The DF-1¿J cell line was resistant to infection by all six ALV-J isolates as determined using the gag-specific ELISA. There was no interference with the other ALV subgroups (A-D) induced by the expression of the ADOL-Hcl envelope. The ALV-J isolates used in this analysis are serologically distinct when analyzed by flow cytometry. Convalescent sera to ADOL-Hcl cross-reacts with all of the ALV-J isolates tested; however, sera to HPRS-103 did not bind to four of the six isolates. Based on the intensity and differential binding of these antisera using flow cytometry, the six ALV-J isolates used can be grouped into four categories. Thus the DF-1¿J cell line is resistant to infection by a serologically and genetically diverse group of ALV-J isolates and should be useful as a diagnostic tool.

Amino Acid Sequence↗
PubMed1996

Construction of portable intron cassettes for the delivery and expression of foreign genes.

The use of viral vectors to deliver foreign genes offers some promise of generating new and more efficacious vaccines. However, the insertion of foreign genes into viral genomes often results in the insertional mutagenesis of one or more genes that adversely affect replication. In an attempt to overcome this problem, we constructed two portable intron cassettes. The cassettes were derived from the adenovirus late leader 1 intron and were cloned into either the chloramphenicol acetyltransferase (CAT) gene or the LacZ gene of Escherichia coli. The intron cassettes were transfected into chicken embryo fibroblasts (CEFs) and the cell lysates were later assayed for either beta-galactosidase (beta-Gal) or CAT activity. The first intron cassette (type A) contained flanking adenovirus exon sequences. Consequently, the flanking adenovirus exon sequences remained in the spliced transcript. With the type A intron inserted in the correct orientation for splicing, CAT activity was not diminished. However, in the reverse orientation, no CAT activity could be detected. The second intron cassette (type B) had the splice donor and splice acceptor sites converted to the blunt-end restriction endonuclease sites Pml I and Pvu II, respectively. The blunt-end restriction endonuclease sites enabled the portable intron to be removed from the flanking adenovirus exon sequences and inserted into any blunt-end restriction endonuclease site in the recipient gene. After splicing, no adenovirus exon sequences remained in the recipient gene's RNA transcript. To demonstrate its usefulness, an insertion cassette was made by cloning the E. coli LacZ gene into a multiple cloning site within the type B intron. The insertion cassette was then cloned into a Pvu II site in the middle of the CAT gene. Following transfection in CEFs, high levels of both CAT and beta-Gal were detected, demonstrating that both genes were properly transcribed and translated.

Adenoviridae↗
PubMed1996

A comparative study of histological conditions suitable for both immunofluorescence and in situ hybridization in the detection of Herpesvirus and its antigens in chicken tissues.

Our objective was to identify an optimal single set of conditions for use in both indirect immunofluorescence assays (IFA) and in situ hybridization (ISH) to detect viral proteins and nucleic acids in avian lymphoid and neural tissues. Various fixatives were evaluated for use with IFA to detect turkey Herpesvirus (HVT) glycoprotein B (gB) and ISH to identify HVT mRNA in chicken tissues. A precipitating fixative (acetone) was compared to crosslinking fixatives [buffered glutaraldehyde-picric acid (BGPA), 10% formalin, and 4% paraformaldehyde] for both IFA and ISH using spleen, thymus, bursa, sciatic plexus, and brachial plexus of 28-day-old chickens. Four percent paraformaldehyde was found to be the optimal fixative for preservation of all chicken tissues examined with both IFA and ISH. Glass slide preparation, incubation temperatures, and tissue processing were each individually evaluated for ISH and IFA. Silylated slides provided the best retention of tissue sections for both procedures. For IFA, 37 degrees C was the ideal incubation temperature tested, whereas the optimal incubation temperature tested for ISH was 47 degrees C. Of the blocking agents compared, Evans blue dye prevented background fluorescence to a greater extent than either calf serum or bovine serum albumin. These findings provide a technical basis for investigations into various aspects of the molecular pathology of avian diseases.

Animals↗
PubMed1996

Effect of dipyrone, L-NAME and L-arginine on endotoxin-induced rat paw edema.

Paw edema was induced in male Wistar rats (200-250 g) by intraplantar (ipl) administration of 2.5 micrograms endotoxin (Etx). Etx, like carrageenin, produced two distinct edema formation phases, an early phase (75 min) followed by a late phase (7 h). We showed that the edema formation in the early phase was antagonized by dipyrone (80 mg/kg, i.p.) and indomethacin (1 mg/kg, i.p.) by 52% and 55%, respectively, and that the late phase was resistant to these drugs. These results suggest that in the early phase prostaglandins appear to be involved in the process. However, the activation of the kinin cascade leading to the release of other mediators may be involved in the increase of edema in the late phase. To test this hypothesis, we investigated whether the release of nitric oxide (NO) is involved in the mechanism of endotoxin-induced rat paw edema during the late phase, using N omega-nitro-L-arginine methyl ester (L-NAME) (50 micrograms, ipl) as inhibitor of NO synthase and L-arginine (1 mg, ipl) as substrate of NO synthase. The paw edema induced by Etx was inhibited by L-NAME by 56% and increased by L-arginine by 81%. Furthermore, L-arginine given in combination with L-NAME completely reversed the inhibition of Etx-induced edema produced by L-NAME. These results support the hypothesis that in the late phase NO production is associated with the edema evoked by Etx.

Animals↗