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R Silva

Publications and source records attributed to R Silva.

At least 37 records · Page 2Linked to original sources
PubMed2001

Effect of P64k presensitization on its efficacy as an immunological carrier in mice.

Recently, we have successfully employed the meningococcal P64k protein as a carrier for weak immunogens. Here, we study if presensitization with it can affect the murine antibody response against the hapten chemically coupled to P64k. We found that priming with 10 microg of P64k did not induce epitope-specific suppression against two out of three synthetic peptides, from viral proteins, conjugated to this carrier. Depending on the anti-carrier antibody titers elicited in the presensitized mice, we observed or not a suppressed immune response against the third peptide. Presensitization with 100 microg of P64k resulted in epitope-specific suppression when lower doses of conjugate were administered. In summary, as described for other protein carriers, P64k could induce epitope-specific suppression in mice, but it depends on the hapten and the extent of carrier-specific immunity. Furthermore, this suppression can be overcome by increasing the amount of conjugate administered per dose in the presensitized animals.

Amino Acid Sequence↗
PubMed2001

The costimulatory molecule ICOS plays an important role in the immunopathogenesis of EAE.

The inducible costimulatory molecule (ICOS) is expressed on activated T cells and participates in a variety of important immunoregulatory functions. After the induction of experimental allergic encephalomyelitis in SJL mice with proteolipid protein (PLP), brain ICOS mRNA and protein were up-regulated on infiltrating CD3+ T cells before disease onset. ICOS blockade during the efferent immune response (9-20 days after immunization) abrogated disease, but blockade during antigen priming (1-10 days after immunization) exacerbated disease. Upon culture with PLP and compared with immunized controls, splenocytes produced either decreased interferon-gamma (IFN-gamma, in efferent blockade) or excessive IFN-gamma (in priming blockade). PLP-specific immunoglobulin G1 was decreased in animals treated with anti-ICOS during antigen priming, but not in other groups.

Animals↗
PubMed2001

Safety and preliminary immunogenicity of the recombinant outer membrane protein P64k of Neisseria meningitidis in human volunteers.

P64k is a meningococcal protein from Neisseria meningitidis that has been obtained by recombinant DNA technology. Recombinant P64k has been extensively characterized by physicochemical and immunological methods. Lately this protein has been found to act as a versatile immunological carrier for weak antigens in mice. In the present work, a Phase I clinical trial was carried out in healthy volunteers who received three inoculations of either placebo or recombinant P64k (20 or 50 microg). No severe adverse events occurred during the trial. Only mild adverse events in ten volunteers were observed. At 1 month after the third dose, 15 out of 18 volunteers (83.3%) who received the recombinant antigen had a P64k-specific antibody titre > or =1:100, as detected by ELISA. A fourth dose, given 9 months after the third one, elicited a potent booster immune response in P64k vaccinees. Accordingly, these P64k formulations were considered safe and immunogenic in healthy human volunteers.

Adult↗
PubMed2001

Palpebral twitching in a depressed adolescent on citalopram.

Current estimates suggest that between 0.4% and 8.3% of children and adolescents are affected by major depression. We report a favorable response to treatment with citalopram by a 15-year-old boy with major depression who exhibited palpebral twitching during his first 2 weeks of treatment. This may have been a side effect of citalopram as it remitted with redistribution of doses.

Adolescent↗
PubMed2001

Mapping of monoclonal antibodies specific to P64k: a common antigen of several isolates of Neisseria meningitidis.

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.

Amino Acid Sequence↗
PubMed2000

Anti-PorA antibodies elicited by immunization with peptides conjugated to P64k.

To increase the humoral immune response against two cyclic synthetic peptides, derived from variable regions within the outer membrane meningococcal protein PorA (subtypes 19 and 15), we conjugated the peptides to P64k, a novel carrier protein from the same bacterium expressed in Escherichia coli. In addition, one of these peptides was restricted to a linear conformation before it was chemically coupled to the carrier. The conjugates were administered to mice in a three-dose immunization schedule, resulting in a potent anti-peptide immune response, which suggested that chemical conjugation to this carrier provided T-cell help. Antisera directed to the three conjugates reacted with Neisseria meningitidis outer membrane PorA upon immunoblot analysis. Moreover, in two out of three conjugates, the anti-peptide sera reacted with native meningococcal outer membrane vesicles in ELISA.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of recombinant subgroup J avian leukosis viruses (ALV) expressing subgroup A ALV envelope.

Three recombinant avian leukosis subgroup J viruses, ADOL 5701A, ADOL 5701ADelta, and ADOL 6803A, carrying a subgroup A envelope have been isolated and characterized. These viruses were identified by their ability to replicate in DF-1/J, a recombinant chicken embryo fibroblast (CEF) cell line expressing the subgroup J envelope that is resistant to subgroup J replication. Flow cytometric analysis of DF-1/J cells infected with ADOL 5701 and ADOL 6803, two subgroup J isolates, indicated cross-reactivity with subgroup A chicken polyclonal serum. Based on published sequences of subgroups A and J isolates, we designed a series of primers to PCR amplify the envelope and LTR of these viruses. PCR products were obtained when the forward primer was specific for subgroup A gp85 envelope protein gene and the reverse primer was specific for subgroup J LTR. Sequence analysis of the PCR products indicated that these viruses had a subgroup A gp85, a subgroup E gp37, and a subgroup J LTR. Interestingly, these viruses had previously been propagated in CEF from the alv6 chicken line, a line that carries a replication defective recombinant endogenous virus expressing a subgroup A envelope (RAV 0-A(1)). Sequence analysis of RAV 0-A(1) gp85 and gp37 envelope proteins indicated that they were almost identical to those of the recombinants ADOL 5701A and ADOL 6803A. These results indicate that these three recombinant viruses arose by recombination between exogenous subgroup J isolates and a recombinant defective endogenous virus with subgroup A envelope.

Animals↗
PubMed2000

Cognitive factors and stress-induced changes in catecholamine biochemistry.

The purpose of the present research was to determine whether dysfunctional attitudes, a cognitive attribute, predicted changes in catecholamine biochemistry. A cognitive task was used to induce stress in female subjects (n=21), and levels of plasma norepinephrine (NE) and homovanillic acid (HVA) were measured at three time points: at baseline (T1); immediately after stress exposure (T2); and 40 min later (T3). Dysfunctional attitudes were significantly and positively related to levels of plasma NE at T3, controlling for baseline levels. Dysfunctional attitudes were not significantly related to plasma HVA levels at any time point. Our findings provide initial support for the idea that dysfunctional attitudes, an attribute shown to play an important role in some forms of unipolar depression, predict stress-induced alterations in noradrenergic output.

Adult↗
PubMed2000

Evaluation of dentin root canal permeability after instrumentation and Er:YAG laser application.

BACKGROUND AND OBJECTIVES: Smear layer removal with EDTA from root canal walls allows greater cleaning and disinfection of root canals. However, because Er:YAG laser acts on the removal of the smear layer, the objective of investigation was to analyze in vitro the effect of Er:YAG laser on dentin root canal wall permeability after endodontic instrumentation and irrigation with water or sodium hypochlorite and Er:YAG laser application. STUDY DESIGN/MATERIALS AND METHODS: A total of 25 extracted human maxillary incisors were divided into five groups: Group I, instrumentation with deionized distilled water as the irrigating solution; Group II, instrumentation with 1% sodium hypochlorite as the irrigating solution; Group III, instrumentation with deionized distilled water as the irrigating solution and Er:YAG laser application; Group IV, instrumentation with 1% sodium hypochlorite solution as the irrigating solution and Er:YAG laser application; Group V, instrumentation only up to #20 file with deionized distilled water as the irrigating solution and Er:YAG laser irradiation. The laser parameters were 15 Hz, 140 mJ, total energy 42 J, 300 pulses (Kavo Key Laser). Copper sulfate (10%) was used to evaluate dentin permeability. The penetration of copper ions into the dentinal tubules was observed using 1% rubeanic acid, which reveals copper ions, forming a stained compound ranging in color from deep blue to black. Transverse sections (500-microm thick) were obtained with a diamond disk from the cervical, middle, and apical thirds. RESULTS: The instrumentation of the root canal that used water as the irrigating solution followed by Er:YAG laser irradiation promoted the greatest increase in dentin permeability. The use of Er:YAG laser, 1% sodium hypochlorite + Er:YAG, and 1% sodium hypochlorite used alone showed an intermediate capacity of increasing dentin permeability. The use of water as the irrigating solution without Er:YAG laser promoted the least dentin permeability. CONCLUSIONS: The use of water as the irrigating solution after instrumentation and Er:YAG laser irradiation was an effective procedure for increasing dentin permeability.

Dental Pulp Cavity↗
PubMed2000

Leukocyte recruitment during onset of experimental allergic encephalomyelitis is CCR1 dependent.

We have shown that macrophages and microglia present within demyelinating plaques of patients with multiple sclerosis (MS) are immunoreactive for the chemokine receptor CCR1 and its ligand, macrophage inflammatory protein-1alpha. To test the importance of CCR1 to the pathogenesis of MS, we studied the progression of experimental allergic encephalomyelitis (EAE) in CCR1(+/+) vs. CCR1(-/-) mice. After immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide, nearly all CCR1(+/+) mice developed EAE (95% incidence, severity 2.5+/-0.1), whereas CCR1(-/-) mice had less severe disease (55% incidence, p<0.001; severity 1. 2+/-0.2, p<0.001). CCR1(+/+) mice showed elevated brain mRNA for the chemokines immune protein (IP)-10, RANTES and monocyte chemoattractant protein-1 prior to disease onset, whereas only IP-10 mRNA was elevated in CCR1(-/-) mice. Both groups of mice had comparable in vitro lymphocyte proliferation and cytokine production upon stimulation with MOG peptide, and similar cutaneous hypersensitivity responses to 2,4-dinitrofluorobenzene, suggesting that CCR1(-/-) mice were not systemically immunosuppressed. These data demonstrate that deletion of a chemokine receptor is at least partially protective in EAE, and suggest that targeting of CCR1 may be of therapeutic significance clinically.

Animals↗
PubMed2000

Assessment of bilirubin toxicity to erythrocytes. Implication in neonatal jaundice management.

BACKGROUND: Neonatal hyperbilirubinaemia remains one of the most common clinical conditions requiring therapeutic intervention. Nevertheless, reliable indicators of bilirubin toxicity are still missing. This prompted us to investigate (a) the progression of cytotoxic events produced by increasing concentrations of bilirubin; (b) the relevance of the membrane lipid package on bilirubin binding to erythrocytes; and (c) the reliability of chloroform extraction compared with albumin extraction to evaluate erythrocyte-bound bilirubin and cytotoxicity. MATERIALS AND METHODS: Morphological alterations, free bilirubin, erythrocyte-bound bilirubin (albumin- and chloroform-extractable), haemolysis and membrane-released lipids, were determined in human erythrocytes at 4 degrees C or 37 degrees C, after 4 h incubation at pH 7.4, with increasing molar ratios of bilirubin to albumin (0.5-5). The reversibility of cytotoxicity by albumin washing was assessed by morphological analysis. RESULTS: Decreased free bilirubin, lower erythrocyte-bound bilirubin concentration by albumin extraction (superficial/non-aggregated bilirubin) and higher values by chloroform extraction (deep/aggregated bilirubin) were observed for 37 degrees C vs. 4 degrees C, at molar ratios > 1. Echinocytosis increased with bilirubin concentration and temperature and was not fully reversed by albumin washing. Haemolysis was already significant at a molar ratio of 1, and was enhanced by temperature at molar ratios 3 and 5 (P < 0.01). The loss of membrane lipids was remarkable at molar ratios > or = 0.5, both at 4 degrees C and 37 degrees C (P < 0.01), although correlation with bilirubin concentration was only significant at 37 degrees C (r = 0.971; P < 0.01). CONCLUSIONS: These results suggest that increased lipid fluidity and high bilirubin concentrations promote membrane bilirubin translocation and toxicity. They also show that albumin is not able to displace the bilirubin located deeply or aggregated within the membrane, which in turn is removed by chloroform. Accordingly, chloroform-extractable rather than albumin-extractable bilirubin is a more accurate parameter to assess erythrocyte-bound bilirubin during severe hyperbilirubinaemia.

Bilirubin↗
PubMed2000

P64k meningococcal protein as immunological carrier for weak immunogens.

Previously, the P64k meningococcal protein, an antigen of 64 kDa expressed in Escherichia coli, has been extensively characterized. We have successfully conjugated several synthetic peptides and meningococcal group C polysaccharide to P64k. In three out of four model peptides, the murine humoral immune response against the homologous peptide, evaluated after three doses of conjugate, was higher in the animals immunized with the coupled peptide than in those that received free peptide. The fourth and largest was immunogenic by itself. Similarly, the antigroup C polysaccharide levels reached by conjugated polysaccharide were significantly higher than those produced against unconjugated polysaccharide. As a carrier for one of the peptides, P64k was compared with bovine serum albumin (BSA) and tetanus toxoid (TT), being able to induce slightly higher or similar antipeptide antibody levels than these well-establish protein carriers. Our results suggest that recombinant P64k protein could be a readily available immunological carrier, as efficient as other commonly used large carrier molecules.

Amino Acid Sequence↗
PubMed2000

Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization.

BACKGROUND: The pathogenesis of bilirubin encephalopathy and Alzheimer's disease appears to result from accumulation of unconjugated bilirubin (UCB) and amyloid-beta (Abeta) peptide, respectively, which may cause apoptosis. Permeabilization of the mitochondrial membrane, with release of intermembrane proteins, has been strongly implicated in cell death. Inhibition of the mitochondrial permeability is one pathway by which ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC) protect against apoptosis in hepatic and nonhepatic cells. In this study, we further characterize UCB- and Abeta-induced cytotoxicty in isolated neural cells, and investigate membrane perturbation during incubation of isolated mitochondria with both agents. In addition, we evaluate whether the anti-apoptotic drugs UDC and TUDC prevent any changes from occurring. MATERIALS AND METHODS: Primary rat neuron and astrocyte cultures were incubated with UCB or Abeta peptide, either alone or in the presence of UDC. Apoptosis was assessed by DNA fragmentation and nuclear morphological changes. Isolated mitochondria were treated with each toxic, either alone or in combination with UDC, TUDC, or cyclosporine A. Mitochondrial swelling was measured spectrophotometrically and cytochrome c protein levels determined by Western blot. RESULTS: Incubation of neural cells with both UCB and Abeta induced apoptosis (p < 0.01). Coincubation with UDC reduced apoptosis by > 50% (p < 0.05). Both toxins caused membrane permeabilization in isolated mitochondria (p < 0.001); whereas, pretreatment with UDC was protective (p < 0.05). TUDC was even more effective at preventing matrix swelling mediated by Abeta (p < 0.01). UDC and TUDC markedly reduced cytochrome c release associated with mitochondrial permeabilization induced by UCB and Abeta, respectively (p < 0.05). Moreover, cyclosporine A significantly inhibited mitochondrial swelling and cytochrome c efflux mediated by UCB (p < 0.05). CONCLUSION: UCB and Abeta peptide activate the apoptotic machinery in neural cells. Toxicity occurs through a mitochondrial-dependent pathway, which in part involves opening of the permeability transition pore. Furthermore, membrane permeabilization is required for cytochrome c release from mitochondria and can be prevented by UDC or TUDC. These data suggest that the mitochondria is a pharmacological target for cytoprotection during unconjugated hyperbilirubinemia and neurodegenerative disorders, and that UDC or TUDC may be potential therapeutic agents.

Amyloid beta-Peptides↗
PubMed2000

Soft tissue surgery in the oral and maxillofacial region.

The practice of dentistry is most often perceived as the treatment of the hard tissues of the oral region, specifically the teeth and jaws. However, there are many disorders and conditions involving surgical treatment of the soft tissues that extend to the adjacent and associated structures of the oral and maxillofacial surgery region.

Adolescent↗
PubMed1999

Selective alterations of glycosaminoglycans synthesis and proteoglycan expression in rat cortex and hippocampus in pilocarpine-induced epilepsy.

Proteoglycans and glycosaminoglycans are elements of matrix. In the nervous system, glycosaminoglycans modulate neurite outgrowth and are co-receptors for growth factors playing a crucial role in cell differentiation and synaptogenesis. The receptor of protein tyrosine phosphatase beta (RPTPbeta) is a chondroitin sulphate proteoglycan which plays an important role in neural morphogenesis and axon guidance mechanisms. Pilocarpine-treated rats present status epilepticus, which is followed by a seizure-free period (silent), by a period of spontaneous recurrent seizures (chronic), and the hippocampus of these animals exhibits cell loss and mossy fiber sprouting. Thus, the synthesis of heparan sulphate and chondroitin sulphate and the time course of RPTPbeta immunoreactivity were studied in the hippocampus and cerebral cortex during these phases of pilocarpine-induced epilepsy. The results showed decreased synthesis of heparan sulphate during the acute phase and an increased synthesis of chondroitin sulphate during the silent period in the cortex and hippocampus. In control rats RPTPbeta immunoreactivity was detected only in glial cells. After 6 h of status epilepticus the RPTPbeta immunoreactivity was no longer detectable in the glial cells in both tissues and intense staining became evident in the matrix, surrounding CA3 and dentate gyrus and piriform cortex neurones. In the silent and chronic periods RPTPbeta immunoreactivity was mainly detected in neuronal somata and fibers of neurones of hippocampus and cortex. These changes show a selective variation of synthesis and expression of glycosaminoglycans and RPTPbeta in relation to epilepsy suggesting a molecular interplay between glia and neurones during seizures.

Animals↗
PubMed1999

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