Biomedical subjects

Augusto Silva

Publications and source records attributed to Augusto Silva.

17 recordsLinked to original sources

PubMed2006

Double bolt regulation of Rad51 by p53: a role for transcriptional repression.

The regulation of homologous recombination (HR) by p53 has been currently associated with its nontransactivating function; now the transcriptional repression of the Rad51 gene by p53 has been elucidated. This extra control by p53 involves the transcriptional downregulation of Rad51 protein, which in turn accounts for the reduction of functional Rad51 foci. Down regulation of Rad51 protein might be relevant, considering that Rad51 is a rate-limiting factor in HR. Moreover, impaired Rad51-repression by p53 mutant proteins may contribute to malignant transformation.

Animals↗

PubMed2006

p53 modulates homologous recombination by transcriptional regulation of the RAD51 gene.

DNA repair by homologous recombination is involved in maintaining genome stability. Previous data report that wild-type p53 suppresses homologous recombination and physically interacts with Rad51. Here, we show the in vivo binding of wild-type p53 to a p53 response element in the promoter of Rad51 and the downregulation of Rad51 messenger RNA and protein by wild-type p53, favoured by DNA damage. Moreover, wild-type p53 inhibits Rad51 foci formation in response to double-strand breaks, whereas p53 contact mutant R280K fails to repress Rad51 mRNA and protein expression and Rad51 foci formation. We propose that transcriptional repression of Rad51 by p53 participates in regulating homologous recombination, and impaired Rad51 repression by p53 mutants may contribute to malignant transformation.

Blotting, Western↗

PubMed2006

High expression levels of receptor activator of nuclear factor-kappa B ligand associated with human chronic periodontitis are mainly secreted by CD4+ T lymphocytes.

BACKGROUND: Chronic periodontitis is an infectious disease characterized by alveolar bone destruction and teeth loss. Receptor activator of nuclear factor-kappa B ligand (RANKL) is an osteoclastogenic cytokine, a central regulatory factor in the osteoclast's lifespan, and a participant in physiological and pathological bone resorption. Gingival T cells synthesize RANKL, contributing to molecular local imbalance that entails the alveolar bone resorption seen in periodontitis. Our study was aimed at associating the levels of RANKL with the CD4(+) T-cell activity present in gingival tissues of chronic periodontitis patients. METHODS: Gingival biopsies were obtained from 33 chronic periodontitis patients and 20 healthy controls. Specimens were either formalin fixed and paraffin embedded for real-time reverse transcription-polymerase chain reaction (RT-PCR) and histologic analysis or tissue digestion processed for cell culture and flow-cytometry analysis. RANKL mRNA and protein levels were determined by quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) in gingival-cell culture supernatants. Gingival leukocytes were quantified by flow cytometry. RANKL and CD4 immunoreactivity were analyzed by flow cytometry and confocal microscopy. RESULTS: RANKL mRNA levels were higher in patients with periodontitis than in healthy subjects, and spontaneous and lipopolysaccharide (LPS)- and phytohemagglutinin (PHA)-stimulated RANKL synthesis were higher also in patients than controls. CD4(+) T lymphocytes were the predominant infiltrate cell subset present in gingival tissues of periodontitis patients. Furthermore, an association between RANKL and CD4(+) T cells was determined by double-staining flow cytometry and confocal microscopy. CONCLUSION: Taken together, these data demonstrate that gingival CD4(+) T cells are the main cells responsible for higher levels of RANKL observed in human chronic periodontitis patients.

Adult↗

PubMed2005

Isolation of a heat-resistant allergen from the fish parasite Anisakis simplex.

The thermal stability of allergenic peptides from the fish parasite Anisakis simplex has not been fully elucidated. This is of special relevance for physicians who should clearly indicate if sensitized patients should avoid ingestion of raw fish only or whether well-cooked fish should also be avoided, if allergenic peptides derived from the parasite remain immunologically detectable. An allergen was purified after heating a crude parasite extract for 30 min. The allergen was further purified by an ethanol fractionation procedure followed by a reversed-phase HPLC. The N-terminal amino acid sequence was obtained. This allergen was detected by 27% of sensitized subjects. The N-terminal amino acid sequence of the 9 kDa allergen showed no similarities to other known proteins. A minor low molecular weight allergen from A. simplex is highly resistant to heating and it could therefore have significant clinical relevance.

Allergens↗

PubMed2004

Bcl-2 family gene modulation during spontaneous apoptosis of B-chronic lymphocytic leukemia cells.

Malignant cell accumulation in B-cell chronic lymphocytic leukemia (B-CLL) is primarily caused by defective apoptosis rather than increased proliferation. To further understand the role of Bcl-2 family members, known regulators of apoptosis, in the abnormal B-CLL survival, we have measured their mRNA levels in fresh B-CLL cells and in cultures undergoing spontaneous apoptosis. Using RNA protection assays we found constitutive expression of most bcl-2 members with high levels of bcl2, bcl-w, bad, bak, bax, and the bcl-2/bax ratio, compared to normal PBL. Spontaneous apoptosis of B-CLL cells by in vitro culture resulted in decreased bcl-2, bcl-w, bfl-1, mcl-1, bak, bax, and bcl-2/bax expression. The pro-apoptotic member bik was only expressed in 5/19 cases and was not modulated during apoptosis, suggesting that bik is not involved in this process. Thus, several Bcl-2 family genes are regulated during B-CLL spontaneous apoptosis and their relative levels may contribute to in vivo progression of the disease.

Apoptosis↗

PubMed2004

Quantitative evaluation of a pulmonary contour segmentation algorithm in X-ray computed tomography images.

RATIONALE AND OBJECTIVES: Pulmonary contour extraction from thoracic x-ray computed tomography images is a mandatory preprocessing step in many automated or semiautomated analysis tasks. This study was conducted to quantitatively assess the performance of a method for pulmonary contour extraction and region identification. MATERIALS AND METHODS: The automatically extracted contours were statistically compared with manually drawn pulmonary contours detected by six radiologists on a set of 30 images. Exploratory data analysis, nonparametric statistical tests, and multivariate analysis were used, on the data obtained using several figures of merit, to perform a study of the interobserver variability among the six radiologists and the contour extraction method. The intraobserver variability of two human observers was also studied. RESULTS: In addition to a strong consistency among all of the quality indexes used, a wider interobserver variability was found among the radiologists than the variability of the contour extraction method when compared with each radiologist. The extraction method exhibits a similar behavior (as a pulmonary contour detector), to the six radiologists, for the used image set. CONCLUSION: As an overall result of the application of this evaluation methodology, the consistency and accuracy of the contour extraction method was confirmed to be adequate for most of the quantitative requirements of radiologists. This evaluation methodology could be applied to other scenarios.

Algorithms↗

PubMed2004

Quantitative real-time polymerase chain reaction based on single copy gene sequence for detection of periodontal pathogens.

OBJECTIVE: To develop a method for quantification of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf) from subgingival plaque samples based on TaqMan real-time polymerase chain reaction (PCR) technology. MATERIAL AND METHODS: Bacterial cells from these species were obtained after culturing reference strains and were counted microscopically. Cellular suspensions in Tris-EDTA buffer were used for DNA extraction after boiling for 20 min. Primers for PCR were selected from sequences of the LktC (Aa), Arg-gingipain (Pg) and BspA antigen (Tf) genes in order to yield amplicons below 100 bp. TaqMan-based real-time PCR was adjusted to quantify each species separately. Cycle threshold (C(T)) values were calculated for each species according to the initial number of copies. A reliability analysis was carried out using intra-class correlation coefficients (ICCs) with a two-way random effects model. RESULTS: A high sensitivity and specificity was obtained for the detection of the three bacterial species. The TaqMan real-time PCR technology yielded a good repeatability in the obtained cycle threshold (C(T)) values for each initial number of copies, demonstrating coefficients of variation below 5% for each bacteria. The reproducibility of the technique was also demonstrated by the high ICCs (>0.98; p<0.00001) obtained for each bacteria with and without the addition of subgingival plaque. CONCLUSION: A novel diagnostic method based on TaqMan real-time PCR was developed for the quantification of Aa, Pg and Tf. It has demonstrated good sensitivity and repeatability on pure cultures. Its diagnostic utility should be demonstrated in subgingival plaque samples.

Aggregatibacter actinomycetemcomitans↗

PubMed2004

Methods of detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in periodontal microbiology, with special emphasis on advanced molecular techniques: a review.

BACKGROUND: Certain specific bacterial species from the subgingival biofilm have demonstrated aetiological relevance in the initiation and progression of periodontitis. Among all the bacteria studied, three have shown the highest association with destructive periodontal diseases: Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf). Therefore, the relevance of having accurate microbiological diagnostic techniques for their identification and quantification is clearly justified. AIM: To evaluate critically all scientific information on the currently available microbial diagnostic techniques aimed for the identification and quantification of Aa, Pg and Tf. SUMMARY: Bacterial culturing has been the reference diagnostic technique for many years and, in fact, most of our current knowledge on periodontal microbiology derives from cultural data. However, the advent of new microbial diagnostics, mostly based on immune and molecular technologies, has not only highlighted some of the shortcomings of cultural techniques but has also allowed their introduction as easy and available adjunct diagnostic tools to be used in clinical research and practice. These technologies, mostly polymerase chain reaction (PCR), represent a field of continuous development; however, we still lack the ideal diagnostic to study the subgingival microflora. Qualitative PCR is still hampered by the limited information provided. Quantitative PCR is still in development; however, the promising early results reported are still hampered by the high cost and the equipment necessary for the processing. CONCLUSION: Quantitative PCR technology may have a major role in the near future as an adjunctive diagnostic tool in both epidemiological and clinical studies in periodontology. However, culture techniques still hold some inherent capabilities, which makes this diagnostic tool the current reference standard in periodontal microbiology.

Aggregatibacter actinomycetemcomitans↗

PubMed2004

Quantitative real-time polymerase chain reaction versus culture: a comparison between two methods for the detection and quantification of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in subgingival plaque samples.

OBJECTIVE: The purpose of this investigation was to validate a real-time quantitative polymerase chain reaction (PCR) assay in identifying and quantifying Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis from subgingival plaque samples taken from subjects with different periodontal conditions, when compared with conventional cultural procedures. PATIENTS AND METHODS: Ninety-two adult subjects participated in this study, 32 with periodontitis, 30 with gingivitis and 30 healthy. A pooled subgingival sample was obtained from every patient. Culturing procedures were carried out using standard techniques. For real-time PCR analysis, primers were selected from sequences of the LktC (A. actinomycetemcomitans), Arg-gingipain (P. gingivalis) and BspA antigen (T. forsythensis) genes. Contingency tables were constructed to compare the qualitative results, while quantitative data were evaluated by paired t-test. RESULTS: A. actinomycetemcomitans was the least frequently recovered species with both techniques. Prevalence of P. gingivalis was low in healthy patients, increased in gingivitis and peaked in periodontitis patients. The frequency of detection of T. forsythensis showed marked differences between culture and PCR, although the same tendency of an increase in prevalence from health to gingivitis and to periodontitis was observed with both methods. Contingency tables demonstrated a good level of agreement between PCR and culture procedures for A. actinomycetemcomitans and P. gingivalis, especially in periodontitis patients. P. gingivalis culture counts were significantly higher than those obtained by PCR. The opposite was true for T. forsythensis, and statistically significant higher counts were obtained by PCR for gingivitis and periodontitis patients. CONCLUSION: This study demonstrated a good agreement between the quantitative PCR technology and the culture procedure. The high sensitivity and specificity of the quantitative PCR technology justify its use in epidemiological studies and as an adjunct in clinical diagnosis of periodontal patients.

Adult↗

PubMed2004

New knowledge of the pathogenesis of periodontal disease.

The aim of this study was to evaluate the relationship between the accumulation of interleukins IL-1beta, TNF-alpha, IL-8, and chemokine RANTES (Regulated upon Activation Normal T-cell Expressed and Secreted) in gingival fluid and periodontal support tissues in patients with periodontitis. A review is also provided of apoptotic processes as events of major importance, highlighting the presence of TUNEL cells and ultrastructural morphologic changes associated with cell apoptosis. There appears to be further evidence to support the important role of inflammation control. Cytokines may be considered as markers of the progression and severity of periodontitis as well as indicators of an appropiate response to treatment. However, further studies are needed to support and characterize this concept.

Antigens, Bacterial↗

PubMed2003

Involvement of p53 in alpha4beta1 integrin-mediated resistance of B-CLL cells to fludarabine.

We recently showed that alpha4beta1 integrin induces B-cell chronic lymphocytic leukemia (B-CLL) cell resistance to fludarabine-induced apoptosis via upregulation of Bcl-xL. We have now studied whether p53 was involved in this response. Cells from five B-CLL patients with wild-type p53 determined by DNA sequencing, or from the EHEB cell line, cultured on the alpha4beta1 ligand H/89 during fludarabine treatment, showed significantly higher viability (P<or=0.001) than cells cultured on poly-D-lysine (pLys). Parallel to this different viability, fludarabine increased p53 expression on pLys-cultured cells and this increase was significantly reduced (P<or=0.05) on cells cultured on H/89. Thus, p53 appears to be part of the survival pathway induced by alpha4beta1 ligation and may contribute to the progressive drug resistance in B-CLL.

Antineoplastic Agents↗

PubMed2003

Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

OBJECTIVE: To establish a method for quantification of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis from subgingival plaque by real-time polymerase chain reaction (PCR) technique. MATERIAL AND METHODS: Bacterial cells from both species were obtained from type culture and counted microscopically. Cellular suspension in sterile distilled water was used for DNA extraction by boiling for 20 min, with a mineral oil cover. Primers for PCR were selected from sequences of LktC gene (A. actinomycetemcomitans) and Arg-gingipain (P. gingivalis) to yield amplicons below 100 bp. SYBR Green I based real-time PCR was adjusted to quantify separately both species. RESULTS: A good sensitivity and specificity were obtained for both species, although the yield was better for A. actinomycetemcomitans. A good repeatability of cycle threshold (CT) was encountered, so coefficient of variation was below 6% at every initial copy number. CONCLUSION: A new method of quantification of A. actinomycetemcomitans and P. gingivalis based on SYBR Green real-time PCR is presented. Its good sensibility and repeatability will allow its application to analysis of subgingival plaque samples.

Aggregatibacter actinomycetemcomitans↗

PubMed2003

Usefulness of liposomes loaded with cytostatic bile acid derivatives to circumvent chemotherapy resistance of enterohepatic tumors.

We have investigated the sensitivity of the cisplatin-resistant enterohepatic tumor cell lines LS174T/R (human colon adenocarcinoma), WIF-B9/R (rat hepatoma-human fibroblast hybrid), and Hepa 1-6/R (mouse hepatoma) to free and liposome-encapsulated cytostatic bile acid derivatives Bamet-R2 and bamet-UD2. Expression of resistance associated genes was measured by quantitative reverse transcription-polymerase chain reaction or Western blotting. Drug uptake was determined by atomic absorption spectrophotometry. In resistant cells, overexpression of MRP1 and MRP2 was accompanied by reduced accumulation of cisplatin. The expression of MDR1 and GST-P was only enhanced in LS 174T/R. A higher expression of p53 was seen in LS 174T/R and Hepa 1-6/R cell lines but not in WIF-B9/R cells. In wild-type counterparts, uptake and cytostatic ability of Bamets were markedly higher (UD2 > R2) than that of cisplatin. Both effects were further enhanced by liposome formulation. Bamets were able to overcome cisplatin resistance in all cell lines. Cisplatin prolonged the survival time of nude mice in whose livers a Hepa 1-6 tumor had been implanted, but failed to exert a beneficial effect when the tumor was Hepa 1-6/R. In both cases, tissue distribution of cisplatin was: kidney >> liver > tumor. Survival was markedly longer in animals receiving Bamet-UD2, even if the implanted tumor was resistant. The accumulation of Bamet-UD2 in tissues was: liver > tumor > kidney. Liposome formulation further enhanced the beneficial properties of Bamet-UD2. Thus, the amount of drug in the tumor was increased and that in liver and kidney was reduced (tumor > liver > kidney), and life span was prolonged. In conclusion, liposomal Bamet-UD2 may be a useful tool to circumvent resistance to chemotherapy, particularly in tumors of the enterohepatic circuit.

Animals↗

PubMed2003

A new concept for an integrated Healthcare Access Model.

The increase of population mobility has been promoting a crescent dispersion of patient clinical records in Healthcare Information Systems. In this scenario, it is mandatory that new services will be available for healthcare practitioners, namely web-based interfaces with strong control access mechanisms providing effective authentication and identification of persons, and the establishment of new access models to the disperse patient information. This paper proposes and describes a Healthcare Access Model that integrates a new set of functionalities coping with patient mobility and implements an innovative concept of a virtual unique Electronic Patient Record--EPR.

Access to Information↗

PubMed2002

Human securin interacts with p53 and modulates p53-mediated transcriptional activity and apoptosis.

The gene PTTG1 (encoding the pituitary tumor-transforming 1 protein) is overexpressed in several different tumor types, is tumorigenic in vivo and shows transcriptional activity. The PTTG1 protein is cell-cycle regulated and was identified as the human securin (a category of proteins involved in the regulation of sister-chromatid separation) on the basis of biochemical similarities with the Pds1p protein of budding yeast and the Cut2p protein of fission yeast. To unravel the function of human securin in oncogenesis, we carried out a phage-display screening to identify proteins that interact with securin. Notably, we isolated the p53 tumor suppressor. Pull-down and co-immunoprecipitation assays demonstrated that p53 interacts specifically with securin both in vitro and in vivo. This interaction blocks the specific binding of p53 to DNA and inhibits its transcriptional activity. Securin also inhibits the ability of p53 to induce cell death. Moreover, we observed that transfection of H1299 cells with securin induced an accumulation of G2 cells that compensated for the loss of G2 cells caused by transfection with p53. We demonstrated the physiological relevance of this interaction in PTTG1-deficient human tumor cells (PTTG1(-/-)): both apoptotic and transactivating functions of p53 were potentiated in these cells compared to parental cells. We propose that the oncogenic effect of increased expression of securin may result from modulation of p53 functions.

Apoptosis↗

PubMed2002

Haematopoietic progenitor cells from adult bone marrow differentiate into cells that express oligodendroglial antigens in the neonatal mouse brain.

Stem cells are self-renewable, pluripotent cells that, in adult life, proliferate by a characteristic asymmetric division in which one daughter cell is committed to differentiation whereas the other remains a stem cell. These cells are also characterized by their ability to differentiate into various cell types under heterotopic environmental influences. In the present study, we have explored the potential of adult haematopoietic bone marrow cells to differentiate into cells of oligodendroglial lineage under physiological, active myelinating conditions. We present evidence of generation of cells expressing oligodendroglial specific markers from a bone marrow subpopulation enriched on adult haematopoietic progenitor cells (CD117+) in vivo after intracerebral transplantation into the neonatal mouse brain. Our results suggest that adult bone marrow cells have the capacity to undergo differentiation from haematopoietic to oligodendroglial cells and add support the validity of bone marrow transplants as an alternative treatment for demyelinating diseases of the CNS including Multiple Sclerosis.

Animals↗

PubMed2002

Engagement of alpha4beta1 integrin by fibronectin induces in vitro resistance of B chronic lymphocytic leukemia cells to fludarabine.

B-cell chronic lymphocytic leukemia is characterized by the accumulation of malignant B lymphocytes as a result of abnormal survival signals operating in vivo. Previously, we showed that adhesion of B-CLL cells to the fibronectin fragment H89, a ligand for alpha4beta1 integrin, prevents their spontaneous apoptosis in vitro. We have now studied whether alpha4beta1/H89 interaction affected the response of B-CLL cells to the therapeutic drug fludarabine. B-CLL cells cultured on H89 during treatment with fludarabine showed significantly higher mean viability (P<0.05) than cells cultured on the control polylysine for all doses of drug tested. Similar results were obtained with the EHEB cell line. Analysis of the expression of Bcl-2-family proteins after 48 h of fludarabine treatment revealed that Bcl-xL levels were significantly higher (P<0.05) for cells cultured on H89 than on polylysine and correlated (r=0.56, P<0.05) with the increased cell viability observed on H89 cultures. These results indicate that Bcl-xL is involved in the survival signals induced by alpha4beta1 ligation and may contribute to the progressive drug resistance observed in B-CLL.

Aged↗