Biomedical subjects

S C Oliveira

Publications and source records attributed to S C Oliveira.

At least 19 recordsLinked to original sources

PubMed2006

Construction and partial characterization of a Corynebacterium pseudotuberculosis bacterial artificial chromosome library through genomic survey sequencing.

Corynebacterium pseudotuberculosis is a gram-positive bacterium that causes caseous lymphadenitis in sheep and goats. However, despite the economic losses caused by caseous lymphadenitis, there is little information about the molecular mechanisms of pathogenesis of this bacterium. Genomic libraries constructed in bacterial artificial chromosome (BAC) vectors have become the method of choice for clone development in high-throughput genomic-sequencing projects. Large-insert DNA libraries are useful for isolation and characterization of important genomic regions and genes. In order to identify targets that might be useful for genome sequencing, we constructed a C. pseudotuberculosis BAC library in the vector pBeloBAC11. This library contains about 18,000 BAC clones, with inserts ranging in size from 25 to 120 kb, theoretically representing a 390-fold coverage of the C. pseudotuberculosis genome (estimated to be 2.5-3.1 Mb). Many genomic survey sequences (GSSs) with homology to C. diphtheriae, C. glutamicum, C. efficiens, and C. jeikeium proteins were observed within a sample of 215 sequenced clones, confirming their close phylogenetic relationship. Computer analyses of GSSs did not detect chimeric, deleted, or rearranged BAC clones, showing that this library has low redundancy. This GSSs collection is now available for further genetic and physical analysis of the C. pseudotuberculosis genome. The GSS strategy that we used to develop our library proved to be efficient for the identification of genes and will be an important tool for mapping, assembly, comparative, and functional genomic studies in a C. pseudotuberculosis genome sequencing project that will begin this year.

Chromosomes, Artificial, Bacterial↗

PubMed2006

Identification of a new Schistosoma mansoni membrane-bound protein through bioinformatic analysis.

Progress in schistosome genome research has enabled investigators to move rapidly from genome sequences to vaccine development. Proteins bound to the surface of parasites are potential vaccine candidates, or they can be used for diagnosis. We analyzed 4342 proteins deduced from the Schistosoma mansoni transcriptome with bioinformatic computer programs. Thirty-four proteins had membrane-bound motifs. Within this group, we selected the Sm29 protein to be further characterized by in silico analysis. Sm29 was found to have a signal peptide made up of 26 amino acids, with a cleavage site between Ser26 and Val27. The glycosylation site search revealed three threonines (39, 132 and 133) with high probability of O-glycosylation and two asparagines (58 and 115) with high probability of N-glycosylation. Only one transmembrane helix was found in the C-terminal region of the protein from Leu169 to Lis191. The search for similarities and conserved motifs show that Sm29 is a protein with high identity to proteins present in S. japonicum (53, 52, 49, and 37% of identity) and it possesses disulfide-rich conserved domains. Apparently, Sm29 is a membrane bound protein, and it may be an important molecule in host-parasite interactions.

Amino Acid Sequence↗

PubMed2006

An improved protocol for electrotransformation of Corynebacterium pseudotuberculosis.

We developed an improved protocol for the electrotransformation of Corynebacterium pseudotuberculosis, testing variations of parameters in the procedures that are routinely used for the preparation of electrocompetent cells of this species, including (i) culture conditions, (ii) cell growth phase, (iii) electroporation solutions and (iv) quantity of plasmid DNA. We obtained the greatest efficiency of transformation when the cells were grown until the stationary phase and then washed with 10% glycerol electroporation solution. The transformation efficiency was inversely proportional to the quantity of plasmid DNA. The transformation efficiency reached 10(5) colony-forming units (cfu)/mug plasmid DNA. This protocol would be useful for genetic studies of C. pseudotuberculosis.

Corynebacterium pseudotuberculosis↗

PubMed2006

Concentrations of CXCL8, CXCL9 and sTNFR1 in plasma of patients with pulmonary tuberculosis undergoing treatment.

BACKGROUND: Chemokines are a class of cytokines with chemotactic properties shown to be induced by M. tuberculosis or its antigens in vitro and in experimental infection in vivo. A few studies have also demonstrated the expression of chemokines in clinical samples of patients with active tuberculosis (TB). In the present work, we measured the concentration of chemokines in plasma samples of HIV-negative patients with pulmonary tuberculosis at different stages of chemotherapy. For comparison, we also evaluated the levels of sTNFR1 and TNF-alpha. METHODS: Cytokines and chemokines were measured by ELISA in healthy individuals and patients with active pulmonary TB at different stages of treatment. RESULTS: The concentrations of CXCL8, CXCL9 and sTNFR1 were elevated in patients with active pulmonary TB but returned to background levels at 4-6 months of chemotherapy. The concentration of CCL11 was elevated in patients with active pulmonary tuberculosis when compared to control and remained elevated throughout the specific therapy. There was no difference in the plasma concentration of CCL2 and CXCL10 between pulmonary TB patients and control subjects. CONCLUSION: Measurement of the CXCL8, CXCL9 and sTNFR1 may be useful to assess response to treatment in pulmonary TB patients.

Cytokines↗

PubMed2006

Human antibody responses of patients living in endemic areas for schistosomiasis to the tegumental protein Sm29 identified through genomic studies.

Surface proteins of schistosomes are exposed to host tissues and thus present as potential candidate molecules for the development of new intervention strategies. Herein, we have identified a new tegumental protein of Schistosoma mansoni, termed Sm29. In silico analysis revealed a signal peptide, three glycosylation sites and a transmembrane region on Sm29 amino acid sequence. Sm29 transcription in mammalian developmental stages cDNA libraries of S. mansoni was verified by PCR using specific primers for Sm29 nucleotide sequence and it revealed the presence of transcripts in schistosomula and adult worm stages of the parasite. Sm29 (40-169) fragment was produced in Escherichia coli and purified by affinity chromatography to be used in the immunological assays. Confocal microscopy confirmed bioinformatic studies, revealing that Sm29 is a membrane-bound protein localized on the tegument of S. mansoni adult worm. ELISA was performed using rSm29 protein to investigate the antibody isotype profile to Sm29 in sera of patients living in endemic areas for schistosomiasis. IgG1 and IgG3 subclass antibodies to rSm29 were predominant in sera of individuals naturally resistant to infection and resistant to re-infection whereas low levels of IgM, IgA or IgE were measured. Since, IgG1 and IgG3 are involved in parasite killing and in protective immunity the findings reported here suggest the use of Sm29 as a potential candidate vaccine against schistosomiasis.

Adolescent↗

PubMed2005

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis.

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.

Antifungal Agents↗

PubMed2005

Identification of paramyosin T cell epitopes associated with human resistance to Schistosoma mansoni reinfection.

Paramyosin, a Schistosoma mansoni myoprotein associated with human resistance to infection and reinfection, is a candidate antigen to compose a subunit vaccine against schistosomiasis. In this study, 11 paramyosin peptides selected by TEPITOPE algorithm as promiscuous epitopes were produced synthetically and tested in proliferation and in vitro human leucocyte antigen (HLA)-DR binding assays. A differential proliferative response was observed in individuals resistant to reinfection compared to individuals susceptible to reinfection in response to Para (210-226) peptide stimulation. In addition, this peptide was able to bind to all HLA-DR molecules tested in HLA-DR binding assays, confirming its promiscuity. Para (6-22) and Para (355-371) were also shown to be promiscuous peptides, because they were able to bind to the six and eight most prevalent HLA-DR alleles used in HLA-DR binding assays, respectively, and were also recognized by T cells of the individuals studied. These results suggest that these paramyosin peptides are promising antigens to compose an anti-schistosomiasis vaccine.

Adolescent↗

PubMed2004

Identification of immunodominant epitopes of Schistosoma mansoni vaccine candidate antigens using human T cells.

Paramyosin and Sm14 are two of the six antigens selected by the World Health Organization as candidates to compose a subunit vaccine against schistosomiasis. Both antigens are recognized by individuals naturally resistant to Schistosoma mansoni infection and induced protective immunity in the murine model. Three Sm14 epitopes and eleven paramyosin epitopes were selected by their ability to bind to different HLA-DR molecules using the TEPITOPE computer program, and these peptides were synthetically produced. The cellular recognition of Sm14 and paramyosin epitopes by peripheral blood mononuclear cells of individuals living in endemic area for schistosomiasis was tested by T cell proliferation assay. Among all Sm14 and paramyosin epitopes studied, Sm14-3 was preferentially recognized by individuals naturally resistant to S. mansoni infection while Para-5 was preferentially recognized by individuals resistant to reinfection. These two peptides represent promising antigens to be used in an experimental vaccine against schistosomiasis, since their preferential recognition by resistant individuals suggest their involvement in the induction of protective immunity.

Algorithms↗

PubMed2002

Controlled production of stable heterologous proteins in Lactococcus lactis.

The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P(nisA) and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.

Antigens, Bacterial↗

PubMed2002

Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice.

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.

ATP-Binding Cassette Transporters↗

PubMed2002

Sm14 gene expression in different stages of the Schistosoma mansoni life cycle and immunolocalization of the Sm14 protein within the adult worm.

Sm14 is a 14-kDa vaccine candidate antigen from Schistosoma mansoni that seems to be involved in cytoplasmic trafficking of fatty acids. Although schistosomes have a high requirement for lipids, they are not able to synthesize fatty acids and sterols de novo. Thus, they must acquire host lipids. In order to determine whether Sm14 is present in different stages of the life cycle of the parasite, we performed RT-PCR. Sm14 mRNA was identified in all stages of the life cycle studied, mainly schistosomulum, adult worm and egg. Additionally, we used a rabbit anti-Sm14 polyclonal antibody in an indirect immunofluorescence assay to localize Sm14 in adult worm sections. The basal lamella of the tegument and the gut epithelium were strongly labeled. These tissues have a high flow of and demand for lipids, a finding that supports the putative role of Sm14 as an intracellular transporter of fatty acids from host cells.

Animals↗

PubMed2001

A genetic immunization adjuvant system based on BVP22-antigen fusion.

DNA vaccines must induce a greater immune response to be effective in the biomedical industry. Therefore, we tested the trafficking trait of the bovine herpesvirus 1 (BHV-1) protein VP22 (BVP22) fused to an antigen and applied this unique trait to genetic immunization. DNA immunization with BVP22-antigen stimulates immune responses superior to that of standard DNA immunization. Mice were injected intramuscularly with gene constructs expressing the antigen yellow fluorescent protein (YFP), YFP fused to BVP22, or YFP fused to BHV-1 tegument protein VP16 (BVP16). The results revealed a significantly enhanced YFP antibody response with BVP22-YFP DNA immunization compared with either YFP or BVP16-YFP gene immunization. Notably, the BVP22-YFP DNA construct induced a stronger T helper 1 (Th1) response, based on IFN-gamma and IL-4 cytokine levels, and IgG2a/IgG1 ratios. Furthermore, BVP22-YFP genetic immunization induced a greater cytotoxic T lymphocyte response. The genetic adjuvant properties of BVP22 can make DNA vaccines much more effective clinically.

Animals↗

PubMed2001

Mathematical modeling of controlled-release kinetics of herbicides in a dynamic-water-bath system.

Release of herbicides from lignin-based formulations follows a diffusion-controlled mechanism. For mathematical modeling of diffusive transport, the conventional approach is to assume sink conditions at both surfaces of polymeric matrix. This boundary condition proved to be inadequate to describe experimental data obtained in a water dynamic bath system. However, satisfactory descriptions for this system were obtained when a stagnant unstirred layer of herbicide solution was used as the boundary condition. The adequacy of the model incorporating this new boundary condition was statistically tested using the Fisher test at a confidence level of 95% and plotting the residual distribution.

Biomass↗

PubMed2000

The role of IL-10 and IgG1 in the protection and granulomatous response in Schistosoma mansoni P24-immunized mice.

Previous work by our laboratory identified a fraction of Schistosoma mansoni soluble adult worm antigenic preparation, designated PIII, able to elicit significant in vitro cell proliferation, and lower in vitro and in vivo granuloma formation. In the present work, we investigated some biological activities of P24, an antigenic component of PIII. Immunization of mice with this antigen induced a significant protection degree against challenge infection and significant decrease in the hepatic granuloma formation. Pre-incubation of spleen cells from P24-immunized mice with S. mansoni antigens induced a significant increase of interleukin (IL)-10 levels, but not interferon-gamma, in the cell supernatants. In addition, mice immunized with different S. mansoni antigens and P24 displayed indistinguishable levels of IgG2a in response to anti-S. mansoni antigens, while IgG1 levels were significantly increased. Collectively, our results indicate that P24 might mediate protective anti-parasite immunity and downregulate granulomatous hypersensitivity to S. mansoni eggs in part by its ability to induce a higher production of IgG1 and IL-10.

Animals↗

PubMed2000

Biolistic-mediated gene transfer using the bovine herpesvirus-1 glycoprotein D is an effective delivery system to induce neutralizing antibodies in its natural host.

A genetic vaccine consisting of the bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) gene was constructed and administered to cattle using the biolistic (gene-gun) process. Results were compared to standard intramuscular injection of an inactivated whole BHV-1 commercial vaccine. Cattle genetically immunized by the gene-gun-delivered gD subunit vaccine developed high titers of IgG antibodies specific to gD demonstrating that this immunization method is a potent humoral response inducer. Further, gene-gun vaccinated cattle produced high neutralizing antibody titers to BHV-1 similar to levels induced in the commercial vaccine immunized animals. Additionally, cellular immunity was measured by an increased level of IFN-gamma mRNA detected in PBMC of cattle immunized with the gD gene or with the commercial vaccine, whereas augmented levels of IL-4 were not detected following vaccination. Because of its simplicity and effectiveness in inducing an immune response in cattle similar to a commercial vaccine, gene-gun delivery of a subunit BHV-1 gD vaccine would be a viable alternative to current immunization protocols.

Animals↗

PubMed2000

CD4+ T cells of schistosomiasis naturally resistant individuals living in an endemic area produce interferon-gamma and tumour necrosis factor-alpha in response to the recombinant 14KDA Schistosoma mansoni fatty acid-binding protein.

Cellular immune responses to recombinant (r) Sm14 were examined in chronic, treated patients and uninfected individuals living in an endemic area for schistosomiasis. The lymphocyte proliferative responses and cytokine profile to this antigen were evaluated. Peripheral blood mononuclear cells (PBMC) of all groups studied proliferated to rSm14. However, the highest proliferation index to rSm14 was detected in uninfected endemic normal (EN) individuals who are naturally resistant to schistosomiasis. Regarding the cytokines produced, the levels of interleukin (IL)-5 and IL-10, known as Th2 cytokines, were not statistically different among all groups studied. In contrast, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were produced in significantly higher amounts by PBMC of EN individuals following rSm14 stimulation. Additionally, we have determined by flow cytometry that CD4+ T cells from these individuals are the main lymphocyte subpopulation producing IFN-gamma and TNF-alpha. Moreover, we have used rIL-10 or rIFN-gamma, or monoclonal antibodies (MoAb) against these two cytokines to determine their role on cellular reactivity to rSm14. Exogenous IL-10 suppressed T-cell proliferation and neutralization of endogenous IL-10 restored lymphocyte activation and enhanced IFN-gamma and TNF-alpha production in chronically infected patients. In contrast, the addition of anti-IFN-gamma totally abrogated the PBMC proliferation within the EN group. This study demonstrated that IL-10 is an important cytokine down-regulating T-cell responses in chronic schistosomiasis, whereas lymphocyte proliferation in the uninfected resistant group is dependent on IFN-gamma. Taken together these results suggest that Th1 type of immune response induced in EN individuals to a specific schistosome antigen might be associated with resistance to infection and also highlighted the importance of Sm14 as a potential vaccine candidate.

Adjuvants, Immunologic↗

PubMed2000

Distinctions between bovine herpesvirus 1 and herpes simplex virus type 1 VP22 tegument protein subcellular associations.

The alphaherpesvirus tegument protein VP22 has been characterized with multiple traits including microtubule reorganization, nuclear localization, and nonclassical intercellular trafficking. However, all these data were derived from studies using herpes simplex virus type 1 (HSV-1) and may not apply to VP22 homologs of other alphaherpesviruses. We compared subcellular attributes of HSV-1 VP22 (HVP22) with bovine herpesvirus 1 (BHV-1) VP22 (BVP22) using green fluorescent protein (GFP)-fused VP22 expression vectors. Fluorescence microscopy of cell lines transfected with these constructs revealed differences as well as similarities between the two VP22 homologs. Compared to that of HVP22, the BVP22 microtubule interaction was much less pronounced. The VP22 nuclear interaction varied, with a marbled or halo appearance for BVP22 and a speckled or nucleolus-bound appearance for HVP22. Both VP22 homologs associated with chromatin at various stages of mitosis and could traffic from expressing cells to the nuclei of nonexpressing cells. However, distinct qualitative differences in microtubule, nuclear, and chromatin association as well as trafficking were observed. The differences in VP22 homolog characteristics revealed in this study will help define VP22 function within HSV-1 and BHV-1 infection.

Amino Acid Sequence↗

PubMed2000

Mathematical modeling of controlled-release systems of herbicides using lignins as matrices. A review.

The herbicides applied in soils can be easily lost, owing to leaching, volatilization, and bio- and photodegradation. Controlled-release systems using polymeric matrices claim to solve these problems. The movement of the herbicides in the soil is also an important phenomenon to be studied in order to evaluate the loss processes. The development of mathematical models is a relevant requirement for simulation and optimization of such systems. This study reviews mathematical models as an initial step for modeling data obtained for controlled-release systems of herbicides (diuron, 2,4-dichlorophenoxyacetic acid, and ametryn) using sugarcane bagasse lignin as a polymeric matrix. The release kinetic studies were carried out using several acceptor systems including a water bath, soil, and soil-packed columns. Generally, these models take into account phenomena such as unsteady-state mass transfer by diffusion (Fick's law) and convection, consumption by several processes, and partitioning processes, resulting in partial differential equations with respect to time and space variables.

Delayed-Action Preparations↗