Biomedical subjects

V Azevedo

Publications and source records attributed to V Azevedo.

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

Construction and characterization of a Lactococcus lactis strain deficient in intracellular ClpP and extracellular HtrA proteases.

A Lactococcus lactis strain deficient in both its major proteases, intracellular (ClpP) and extracellular (HtrA), was constructed and characterized. This strain, hereafter called clpP-htrA, could be obtained only by conjugation between a clpP donor strain and an htrA recipient strain in the NZ9000 context, allowing heterologous gene expression under the control of the NICE (nisin-controlled expression) system. The clpP-htrA double mutant showed both higher stress tolerance (e.g. high temperature and ethanol resistance) and higher viability than single clpP or htrA mutant strains. In addition, the secretion rate of two heterologous proteins (staphylococcal nuclease Nuc and Nuc-E7) was also higher in clpP-htrA than in the wild-type strain. This strain should be a useful host for high-level production and quality of stable heterologous proteins.

Conjugation, Genetic↗

PubMed2005

Improvement of bovine beta-lactoglobulin production and secretion by Lactococcus lactis.

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine beta-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (approximately 10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at approximately 8 microg/ml (approximately 2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Animals↗

PubMed2005

High-level resistance to oxidative stress in Lactococcus lactis conferred by Bacillus subtilis catalase KatE.

Lactococcus lactis, a lactic acid bacterium widely used for food fermentations, is often exposed to damaging stress conditions. In particular, oxidative stress leads to DNA, protein and membrane damages that can be lethal. As L. lactis has no catalase, the impact of production of the Bacillus subtilis haem catalase KatE on its oxidative stress resistance was tested. This cytoplasmic catalase was engineered for extracellular expression in L. lactis with an optimization strategy based on fusion to the nisin-inducible promoter and a lactococcal signal peptide (SP(Usp45)). The production of KatE by L. lactis conferred an 800-fold increase in survival after 1 h exposure to 4 mM hydrogen peroxide, and a 160-fold greater survival in long-term (3 days) survival of aerated cultures in a cydA mutant, which is unable to respire. The presence of KatE protected DNA from oxidative damage and limited its degradation after long-term aeration in a cydA/recA mutant, defective in DNA repair. L. lactis is thus able to produce active catalase that can provide efficient antioxidant activity.

Bacillus subtilis↗

PubMed2004

Three-phase partitioning as an efficient method for extraction/concentration of immunoreactive excreted-secreted proteins of Corynebacterium pseudotuberculosis.

The three-phase partitioning (TPP) technique was used upstream to isolate/concentrate secreted proteins from Corynebacterium pseudotuberculosis cultured in a complex liquid medium. Several parameters of the TPP technique (15, 30, or 60% ammonium sulfate concentration; 4.0, 5.5, or 7.0 pH; and primary (n) or tertiary (t)-butanol solvent isomer) were varied to determine the optimal recovery of serologically and cellularly immunoreactive extracted proteins. A TPP extraction made with 30% ammonium sulfate and an initial pH of 4.0 gave the best humoral and cellular immunoreactivity of caseous lymphadenitis infected goats. In particular, two immunogenic secreted (16 and 125 kDa) proteins, which had not been found by other extraction methods, were identified.

Ammonium Sulfate↗

PubMed2003

Experimental Corynebacterium pseudotuberculosis primary infection in goats: kinetics of IgG and interferon-gamma production, IgG avidity and antigen recognition by Western blotting.

Corynebacterium pseudotuberculosis is the cause of caseous lymphadenitis (CLA) in small ruminants, a chronic granulomatous disease that provokes significant zootechnics losses to ovine and goat breeders in northern Brazil. The present work was conducted to analyse aspects of humoral and cellular immune responses after experimental infection. Eight goats were infected intradermally with a single dose of virulent C. pseudotuberculosis strain and specific IgG, interferon-gamma (IFN-gamma) production as well as IgG avidity and antigens pattern recognition dynamics against an excreted-secreted antigen were recorded during 20 weeks. At the end of the follow-up, animals were slaughtered and necropsied. Although no animals showed apparent clinical signs of infection at the end of the trial, IFN-gamma response, even more so than the humoral response, differentiated animals into two groups of high or medium/low response. The time-course of IFN-gamma production presented a short-lived primary response on day 5 after infection of animals of both groups, and a strong and long lasting secondary response starting on day 16 after infection in the high response group. The indirect ELISA used was able to detect a positive antibody titre between 6 and 11 days after infection in the two groups. IgG avidity index oscillated initially between 15 and 45%, and showed approximately 5% units increment during the 20 follow-up weeks. With only one individual exception, the qualitative antigens pattern recognition showed on day 11 after infection remained constant through the experiment. IgG avidity is highly correlated with IgG production, but could not be related with specific immunodominant bands. Both humoral and cellular responses kinetics presented a similar pattern of activation/deactivation but necropsy results suggested that the IFN-gamma test would be a very specific marker of CLA status.

Animals↗

PubMed2003

Kinetics of eosinophil and IgE-mast cell changes following infection with Angiostrongylus costaricensis in Wistar rats.

Human abdominal angiostrongyliasis is a severe eosinophilic disease caused by Angiostrongylus costaricensis. Previous studies have demonstrated that wild rodents are critically involved as definitive hosts to this nematode in nature. In this study, we have evaluated the susceptibility of Wistar rats (Rattus norvegicus) to A. costaricensis infection. Kinetics of parasitological and pathological changes, including the number of adult worms recovered from mesenteric arteries, and of IgE, mast cell and eosinophil levels in several compartments have been assessed. The oral inoculation of third-stage larvae (L3) into adult Wistar rats led to a marked accumulation of worms in the branches of the mesenteric arteries 25 and 50 days post-inoculation. Intense bone marrow eosinophilia ranging from 7 to 50 days was accompanied by marked accumulation of eosinophils in the blood, peritoneal and bronchoalveolar spaces. Eosinophilic periarteritis, oedema and granuloma in the intestinal and lung tissues were also histologically evident. Total serum IgE and specific anti-parasite IgE peaked at 25 days post-infection, as measured by ELISA and by the passive cutaneous anaphylaxis test, respectively. At that time point, there was a drastic reduction in the number of intact mast cells in the peritoneal effluent. These findings indicate that Wistar rats are permissive to A. costaricensis infection. IgE-mast cell activation and massive tissue eosinophil infiltration are marked features in the process and are likely to play a crucial role in the immune-response evoked by this parasite.

Angiostrongylus↗

PubMed2003

Evaluation of community contribution to tuberculosis control in Cape Town, South Africa.

SETTING: Low income, high density, urban settlements in Cape Town, South Africa. OBJECTIVES: To evaluate community treatment supervision as part of tuberculosis (TB) programme implementation. DESIGN: A non-randomised prospective study was conducted under programme conditions from January 1998 to December 1999, comparing TB programme performance in an area with (Guguletu), and an area without (Nyanga) the option of TB treatment supervision by community health workers. RESULTS: Data were collected for 2873 adult TB patients. For smear-positive TB patients, treatment cure rates were higher in the intervention area (Guguletu) than in the control area (Nyanga) (58% vs. 50%, P = 0.0378) and for retreatment cases (47% vs. 35%, P = 0.0791), treatment success rates were similar. CONCLUSIONS: Community health worker support contributed to better TB control programme performance than an approach based exclusively on health facilities. Clear government policy and support for lay health worker programmes in TB control is needed.

Adolescent↗

PubMed2003

Cost and cost-effectiveness of community-based care for tuberculosis in Cape Town, South Africa.

SETTING: Guguletu and Nyanga areas of Cape Town, South Africa. OBJECTIVE: To evaluate the affordability and cost-effectiveness of community involvement in tuberculosis (TB) care. DESIGN: A cost-effectiveness analysis comparing treatment for new smear-positive pulmonary and retreatment TB patients in two similar townships, one providing clinic-based-care with community-based observation options available for its TB patients (Guguletu) and one providing clinic-based care only, with no community-based observation of treatment (Nyanga). Costs were assessed from a societal perspective in 1997 US dollars, and cost-effectiveness was calculated as the cost per patient successfully treated. RESULTS: TB treatment in Guguletu was more cost-effective than TB treatment in Nyanga for both new and retreatment patients (dollars 726 vs. dollars 1201 and dollars 1419 vs. dollars 2058, respectively). This reflected both lower costs (dollars 495 vs. dollars 769 per patient treated for new cases; dollars 823 vs. dollars 1070 per patient treated for retreatment cases) and better treatment outcomes (successful treatment rate 68% vs. 64% and 58% vs. 52% for new and retreatment patients, respectively). Within Guguletu, community-based care was more than twice as cost-effective as clinic-based care (dollars 392 vs. dollars 1302 per patient successfully treated for new patients, and dollars 766 vs. dollars 2008 for retreatment patients), for similar reasons (e.g., for new cases, dollars 314 vs. dollars 703 per patient treated, successful treatment rate 80% vs. 54%). CONCLUSION: Community involvement in TB care can improve the affordability and cost-effectiveness of TB treatment in urban South Africa. Expansion in the Western Cape and in similar areas of the country is worthy of serious consideration by planners and policy-makers.

Community Health Services↗

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

Performance of a rapid phage-based test, FASTPlaqueTB, to diagnose pulmonary tuberculosis from sputum specimens in South Africa.

SETTING: Twelve primary health care clinics in the South Peninsula Administration, Cape Town, Western Cape Province, South Africa. OBJECTIVE: To evaluate the performance of FAST-PlaqueTB, a new phage-based test, for the rapid diagnosis of TB in individuals with no previous history of TB treatment presenting at primary health care clinics in Cape Town, South Africa. DESIGN: A comparative study of FASTPlaqueTB, auramine smear microscopy and Lowenstein-Jensen culture of 1692 decontaminated sputum specimens from 853 patients suspected of having TB. Resolution of discrepant results was undertaken by review of clinical information, chest X-ray and follow-up of treatment outcomes. RESULTS: FASTPlaqueTB detected TB in 75.2% of culture-confirmed cases and 70.3% of all cases with a clinical diagnosis of TB, with a specificity of 98.7% and 99.0%, respectively. The performance parameters of FASTPlaqueTB were significantly superior to those of concentrated auramine smear microscopy (63.4% and 61.3% sensitivity, and 97.4% and 97.3% specificity in culture-confirmed and all cases, respectively). Of those patients with two negative sputum smears, FAST-PlaqueTB detected TB in 54.1% of TB cases confirmed by culture and 48.8% of all cases with a clinical diagnosis of TB. A combination of smear microscopy and FASTPlaqueTB enabled 81.2% of culture-confirmed cases and 78.4% of total TB cases to be detected within 2 days of presentation. CONCLUSION: FASTPlaqueTBTM is a rapid, manual test for the diagnosis of TB. The test has significantly higher sensitivity overall compared with auramine sputum smear microscopy in individuals with no previous history of TB treatment, although smear microscopy did detect the most infectious of the TB cases. The FAST-PlaqueTB test is easy to perform, requires no dedicated equipment, and results are read by eye within 48 hours. This test can be useful for the diagnosis of TB in developing countries with a high burden of TB where other rapid diagnostic tests may not be appropriate. The test shows promising performance, particularly in the diagnosis of smear-negative disease, and could be used in conjunction with smear microscopy to aid in the diagnosis of additional cases of TB.

Adult↗

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

Capacity of mercury volatilization by mer (from Escherichia coli) and glutathione S-transferase (from Schistosoma mansoni) genes cloned in Escherichia coli.

A study was carried out to evaluate the capacity for mercury volatilization by genetically engineered strains that express the mer and glutathione S-transferase genes from Escherichia coli and Schistosoma mansoni, respectively. This method enabled strains containing simultaneously mer and glutathione S-transferase genes to grow in high concentrations of mercuric chloride (30 microg/ml) and to volatilize part of the mercury (248 microg/g cell dry wt.) present in the culture medium, while strains bearing only a single gene, did not have the same behavior. Up to 70% of the total mercury of bacterial volatilization occurred in the first 4 h. Although the findings were preliminary, the genetically engineered strain containing simultaneously the mer and glutathione S-transferase genes show a great potential for bioremediation. It may be used in a closed system to remove by volatilization, and recover mercury (Hg0) from contaminated effluents, such as industrial effluent, for instance.

Animals↗

PubMed2000

The Schistosoma gene discovery program: state of the art.

Schistosoma are dioecious digenetic trematodes carrying a large (270 Mb) genome. Gaining knowledge about the genome of these parasites is of importance for the understanding of their biology, mechanisms of drug resistance and antigenic variation that determine escape from the host's immune system. This review will provide an update on the Schistosoma Gene Discovery Program, which is part of the Schistosoma Genome Project created in 1992. One of the main objectives of this program is the discovery and characterisation of new genes of Schistosoma mansoni and Schistosoma japonicum in an attempt to search for new targets for drugs and vaccine development. The success of the Schistosoma Gene Discovery Program is demonstrated by the number of catalogued genes, that now reaches 15 to 20% of the full gene complement of its genome.

Animals↗

PubMed2000

Human IgG1 and IgG3 recognition of Schistosoma mansoni 14kDa fatty acid-binding recombinant protein.

The Schistosoma mansoni gene coding for a 14-kDa fatty acid-binding protein was amplified by PCR and subcloned into the prokaryotic expression vector pMAL-c2. Escherichia coli DH5alpha was transformed with the pMAL-Sm14 construct, and gene expression was induced hr isopropyl-beta-D-thiogalactopyranoside. The resulting recombinant (r) fusion protein was purified by affinity chromatography and confirmed by immunoblot analysis using antimaltose-binding protein or anti-Sm14 antibodies. Additionally, an antibody isotype profile was determined in sera of schistosomiasis patients to rSm14 or soluble adult worm antigen preparation. IgG1 and IgG3 subclass antibodies to rSm14 were predominant in sera of all patients studied whereas low levels of IgM, IgA or IgE were measured. Expression of a S. mansoni gene encoding a vaccine candidate is an important step to better study human immune responses to defined antigens.

Adolescent↗

PubMed1999

Analysis of the gene expression profile of Schistosoma mansoni cercariae using the expressed sequence tag approach.

ESTs constitute rapid and informative tools with which to study gene-expression profiles of the diverse stages of the schistosome life cycle. Following a comprehensive EST study of adult worms, analysis has now targeted the cercaria, the parasite larval form responsible for infection of the vertebrate host. Two Schistosoma mansoni cercarial cDNA libraries were examined and partial sequence obtained from 957 randomly selected clones. On the basis of database searches, 551 (57.6%) ESTs generated had no homologs in the public databases whilst 308 (32.2%) were putatively identified, totaling 859 informative ESTs. The remaining 98 (10.2%) were uninformative ESTs (ribosomal RNA and non-coding mitochondrial sequences). By clustering analysis we have identified 453 different genes. The most common sequences in both libraries represented Sm8 calcium binding protein (8% of ESTs), fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, cytochrome oxidase subunit 1, ATP guanidine kinase and triose phosphate isomerase. One hundred and nineteen identified genes were sorted into 11 functional categories, with genes associated with energy metabolism being the most abundant (13%) and diverse. The diversity and abundance of genes associated with the transcription/translation machinery and with regulatory/signaling functions were also marked. A paramyosin transcript was identified, indicating that this gene is not exclusively expressed in adult worms and sporocysts (as had been suggested previously). The possible physiological relevance to cercariae of the presence of transcripts with homology to calcium binding proteins of the EF-hand superfamily, Gq-coupled rhodopsin photoreceptor, rod phosphodiesterase 8 subunit and peripheral-type benzodiazepine receptor is discussed.

Animals↗