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Biomedical subjects

D T Covas

Publications and source records attributed to D T Covas.

At least 19 recordsLinked to original sources
PubMed2007

Mesenchymal stem cells from patients with chronic myeloid leukemia do not express BCR-ABL and have absence of chimerism after allogeneic bone marrow transplant.

Bone marrow is a heterogeneous cell population which includes hematopoietic and mesenchymal progenitor cells. Dysregulated hematopoiesis occurs in chronic myelogenous leukemia (CML), being caused at least in part by abnormalities in the hematopoietic progenitors. However, the role of mesenchymal stem cells (MSCs) in CML has not been well characterized. The objectives of the present study were to observe the biological characteristics of MSCs from CML patients and to determine if MSCs originate in part from donors in CML patients after bone marrow transplantation (BMT). We analyzed MSCs from 5 untreated patients and from 3 CML patients after sex-mismatched allogeneic BMT. Flow cytometry analysis revealed the typical MSC phenotype and in vitro assays showed ability to differentiate into adipocytes and osteoblasts. Moreover, although some RT-PCR data were contradictory, combined fluorescence in situ hybridization analysis showed that MSCs from CML patients do not express the bcr-abl gene. Regarding MSCs of donor origin, although it is possible to detect Y target sequence by nested PCR, the low frequency (0.14 and 0.34%) of XY cells in 2 MSC CML patients by fluorescence in situ hybridization analysis suggests the presence of contaminant hematopoietic cells and the absence of host-derived MSCs in CML patients. Therefore, we conclude that MSCs from CML patients express the typical MSC phenotype, can differentiate into osteogenic and adipogenic lineages and do not express the bcr-abl gene. MSCs cannot be found in recipients 12 to 20 months after BMT. The influence of MSCs on the dysregulation of hematopoiesis in CML patients deserves further investigation.

Adolescent↗
PubMed2006

Quality control of blood irradiation: determination T cells radiosensitivity to cobalt-60 gamma rays.

BACKGROUND: To identify the most appropriate dose for the prevention of transfusion-associated graft-versus-host disease, the radiosensitivity of T cells has been determined in blood bags irradiated with X-rays produced by a linear accelerator and gamma rays derived from the cesium-137 source of a specific irradiator. In this study, the influence of doses ranging from 500 to 2500 cGy was investigated on T cells isolated from red blood cell (RBC) units preserved with ADSOL and irradiated with a cobalt teletherapy unit. STUDY DESIGN AND METHODS: A thermal device consisting of acrylic and foam was constructed to store the blood bags during irradiation. Blood temperature was monitored with an automated data acquisition system. Dose distribution in the blood bags was analyzed based on isodose curves obtained with a polystyrene phantom constructed for this purpose. The influence of cobalt-60 gamma radiation on T cells was determined by limiting-dilution analysis, which measures clonable T cells. T-cell content of the mononuclear cell population plated was assessed by flow cytometry with a monoclonal antibody specific for CD3. RESULTS: Blood temperature ranged from 2 to 4.5 degrees C during irradiation. Dosimetry performed on the phantom showed a homogenous dose distribution when the phantom was irradiated with a parallel-opposite field. A radiation dose of 1500 cGy led to the inactivation of T cells by 4 log, but T-cell growth was observed in all experiments. At 2500 cGy, no T-cell growth was detected in any of the experiments and a greater than 5 log reduction in functional T cells was noted. CONCLUSION: The results showed that a dose of 2500 cGy completely inactivates T cells in RBC units irradiated with cobalt-60 source.

Adenine↗
PubMed2005

Variation in the FcgammaR3B gene among distinct Brazilian populations.

The FCGR3B gene codes for the FcgammaR3b receptor, which occurs in three polymorphic forms representing the human neutrophil antigens (HNA)-1a, HNA-1b, and HNA-1c. The alleles that code for these antigens are FCGR3B*1, FCGR3B*2, and FCGR3B*3, respectively. New variants of these alleles have been recently described. In order to study the frequency of these alleles and the occurrence of variant forms, we sequenced part of the FCGR3B gene in 149 individuals belonging to four distinct Brazilian populations, i.e., 60 Amerindians, 30 Whites of European descent, 30 Afro-Brazilians, and 30 Japanese. The FCGR3B*1 allele showed high frequency among Amerindians (0.850), with the value detected representing the highest frequency described thus far for this allele in population studies. Its frequency was 0.660 in the Japanese population studied, a value equal to that observed in Afro-Brazilians (0.600) and higher than that observed in Whites (0.480). The FCGR3B*3 allele was only found among Afro-Brazilians, where it occurred at a frequency of 0.080, which was lower than the frequency observed among Afro-North Americans (0.207) and Ugandans (0.166). Two variant haplotypes were detected among Amerindians and Afro-Brazilians, occurring in six individuals (four Amerindians and two Afro-Brazilians). The variant haplotype FCGR3B*1 A227G, which occurred in homozygosis in two Amerindians and in heterozygosis in two Afro-Brazilians, is described for the first time in the present report. In general, these data reveal variability in the frequency of alleles of the FCGR3B gene compared to other populations of the same genetic background in other regions of the world.

Alleles↗
PubMed2004

In vivo tissue characterization using magnetic techniques.

Among the few non-invasive methods to quantify liver iron deposits, magnetic resonance imaging (MRI) and biomagnetic liver susceptometry (BLS) have been considered the best to evaluate iron overload in the body. This diagnosis is necessary for patients who regularly receive red blood cells transfusion and that have a genetic disorder known as hemochromatosis. In this work, we present the evaluation of the clinical usefulness of MRI and BLS of hepatic tissue to quantify iron deposits in non-transfused and transfused patients. Liver iron evaluation by MRI and BLS were performed in a group of 48 patients. The MRI images weighted in T2 were acquired using multi-slice single-spin-echo (SSE) and single-slice multi-spin-echo (MSE), conducted on a 1.5 T whole body scanner. BLS measurements were performed using an ac superconducting susceptometer based on SQUID. Typically MRI is able to evaluate iron overload in liver as high as 30 mg/g(dry tissue) when using MRI scanners provided with specially designed pulse sequences. For higher iron concentrations susceptibility measurement works better than MRI to evaluate higher iron overloads in the liver, because in this case there is saturation of MRI signal.

Adolescent↗
PubMed2004

Quality control system for blood irradiation using a teletherapy unit.

BACKGROUND AND OBJECTIVES: Irradiation of whole blood and blood components before transfusion is currently the only accepted methodology to prevent transfusion-associated graft-vs.-host disease. In the present work, we developed an automated system for blood bag storage during irradiation, using a teletherapy unit. MATERIALS AND METHODS: A device with two thermal compartments was constructed in acrylic and foam, for the storage of blood bags during irradiation. An automatic acquisition system, coupled with an amplifier and a thermal-sensitive probe, were developed to check blood temperature during irradiation. A polystyrene phantom was constructed to simulate the volume of blood routinely irradiated. The dose distribution was measured in the phantom using thermoluminescent dosimeters and represented in terms of isodose curves. RESULTS: The thermal device kept the blood temperature below 6 degrees C for more than 2 h. Our system allowed the simultaneous irradiation of two different blood components while maintaining a constant temperature. The temperature monitoring system remained invariant (0.2 degrees C) over the whole irradiation interval. Phantom dosimetric results showed a homogeneous dose distribution when the phantom was irradiated, using rotational fields with a 2 r.p.m. frequency. CONCLUSIONS: The methodology developed in the present work provides appropriate storage conditions during irradiation of both red blood cells and platelet blood components using a teletherapy unit.

Blood↗
PubMed2003

Isolation and culture of umbilical vein mesenchymal stem cells.

Bone marrow contains a population of stem cells that can support hematopoiesis and can differentiate into different cell lines including adipocytes, osteocytes, chondrocytes, myocytes, astrocytes, and tenocytes. These cells have been denoted mesenchymal stem cells. In the present study we isolated a cell population derived from the endothelium and subendothelium of the umbilical cord vein which possesses morphological, immunophenotypical and cell differentiation characteristics similar to those of mesenchymal stem cells isolated from bone marrow. The cells were isolated from three umbilical cords after treatment of the umbilical vein lumen with collagenase. The cell population isolated consisted of adherent cells with fibroblastoid morphology which, when properly stimulated, gave origin to adipocytes and osteocytes in culture. Immunophenotypically, this cell population was found to be positive for the CD29, CD13, CD44, CD49e, CD54, CD90 and HLA-class 1 markers and negative for CD45, CD14, glycophorin A, HLA-DR, CD51/61, CD106, and CD49d. The characteristics described are the same as those presented by bone marrow mesenchymal stem cells. Taken together, these findings indicate that the umbilical cord obtained from term deliveries is an important source of mesenchymal stem cells that could be used in cell therapy protocols.

Cell Culture Techniques↗
PubMed2001

Dosimetry of blood irradiation using an alanine/ESR dosimeter.

A batch of 80 DL-alanine dosimeters was supplied to Hemocentro of the Hospital and Clinics of Faculdade de Medicina de Ribeirão Preto (HC-FM RP) SP, Brazil for the purpose of quality control of the radiation dose delivered to blood bags. The irradiation was made using two (40 x 40) cm2 parallel opposed radiation fields each with 80 cm of source to surface distance in the Radiotherapy Section of HC-FMRP with the 60Co teletherapy unit. The calculated radiation absorbed dose at the center of the box was 20 Gy. The dosimeter readings were performed using a Varian E-4 ESR Spectrometer operating in X-band. For the 80 dosimeters and over the irradiation volume throughout a blood bag, the minimum and maximum doses were 14 and 23 Gy, respectively. The mean dose was (18 +/- 2) Gy (1sigma), and the coefficient of variability was 11.1%. Alanine dosimeters demonstrated easy handling, good precision and adequate sensitivity for this application.

Alanine↗
PubMed2000

Analysis of the p53 gene by PCR-SSCP in ten cases of Wilms' tumor.

CONTEXT: Mutations of the p53 tumor suppressor gene are the most frequent alterations observed in human neoplasias affecting adults. In pediatric oncology, however, they have seldom been identified. Wilms' tumor is a renal neoplasia commonly occurring in children and is associated with mutations of the WT1 gene. The correlation between Wilms' tumor and alterations of the p53 gene has not been well established, with a low frequency of mutations having been reported in this type of tumor. Mutation may be associated with advanced stage disease and unfavorable histology. OBJECTIVE: To screen for mutations of the p53 gene by the PCR-SSCP method and DNA sequencing in cases of Wilms' tumor sug-gestive of mutation. DESIGN: Case Report. CASE REPORT: Evaluations of exons 5-9 of the p53 gene in DNA samples extracted by PCR-SSCP from 10 Wilms' tumors in children at different stages, and DNA sequencing. Changes in SSCP analy-sis were observed in exon 8 in two samples. The probable muta-tions were not confirmed by DNA sequencing. The absence of point mutations in p53 gene observed in the 10 samples of Wilms' tumor studied agrees with literature data, with DNA sequencing being of fundamental importance for the confirmation of possible mutations.

Child↗
PubMed2000

Gene frequencies of the HPA-3 and HPA-5 platelet antigen alleles among the Amerindians.

Platelet antigens are of importance in several clinical situations and in population genetics. Data are scarce on allele frequencies in ethnic groups other than whites, Asians and African Americans. The frequencies of the alleles of the systems HPA-3 and HPA-5 were determined using the allele-specific restriction enzyme for five South American Amerindian populations and compared with those obtained for Afro-Americans, Japanese and whites from Brazil. The frequency of the HPA-3a allele among the Amerindians as a group did not differ from the values obtained for the other populations. However, differences were observed among the Amerindians, varying from 0.27 to 0.75, the highest frequency thus far observed for a population of Asian origin. Only the HPA-5a allele was found among 130 Amerindian chromosomes. The determination of gene frequencies of the HPA systems in different populations allows inference of gene flows and genetic constitution of populations and the estimation of the risk of platelet-specific alloimmunization.

Alleles↗
PubMed1999

Abnormal uroporphyrin levels in chronic hepatitis C virus infection.

A strong association between hepatitis C virus (HCV) infection and porphyria cutanea tarda (PCT) has been observed, but the implications of the viral infection in the metabolism of porphyrins in patients without clinical manifestations of PCT are not known. The levels of porphyrin in plasma and uroporphyrin (URO) and coproporphyrin (COPRO) in 24-hour urine were measured in 156 patients with chronic HCV infection showing no clinical evidence of PCT. Levels of URO higher than the upper limit were observed in 35 of 156 patients (22.4%). The range and the mean values +/- standard deviation were 26-1,196 microg/24 hours and 82 +/- 204 microg/24 hours. Increased levels of COPRO and plasma porphyrin were observed in 12 of 156 patients (7.7%) and 2 of 156 patients (1.3%) respectively. There were no differences between patients with increased URO levels and patients with normal URO levels in terms of gender, age, risk factors for HCV infection, alcohol abuse, or hepatitis B viral infection. Transferrin saturation (p = 0.040), gamma glutamyl transpeptidase (p < 0.0001), aspartate aminotransferase (p = 0.006), and alanine aminotransferase (p = 0.040) were significantly higher in patients with abnormal URO than in patients with normal URO. The frequency of cirrhosis was higher, but not significantly different, in patients with increased URO (16.7%) compared with patients with normal URO (3.8%). The authors demonstrated that even without a clinical manifestation of PCT it is possible to detect abnormalities in the metabolism of porphyrins in patients with chronic HCV infection. The implications of these findings deserve additional investigation.

Adolescent↗
PubMed1998

High frequency of the GWG (Pro Trp) envelope variant of HIV-1 in Southeast Brazil.

The HIV-1 variant that contains the GWG amino acid sequence in the crown of the principal neutralizing determinant (PND) has been detected in a few patients in Japan, France, and Brazil by direct sequencing. We describe for the first time the use of restriction fragment length polymorphism (RFLP) and limited DNA sequencing of the C2-V3 region of the HIV-1 envelope (env) gene to determine the prevalence of the variant in 75 HIV-1-infected Brazilian patients. Overall prevalence of the GWG sequence as indicated by RFLP was 57% (43 of 75). The prevalence in females (72%) was higher than that in males (32%) and newborns (40%). Two GFG sequences and 1 GLG sequence were also detected. This finding is relevant for the planning of vaccines and for studies of the epidemiology of HIV-1 in Brazil.

Adult↗
PubMed1998

A polymorphism in exon b2 of the major breakpoint cluster region (M-bcr) identified in chronic myeloid leukaemia patients.

The BCR/ABL junctional region and the b3 exon from chronic myeloid leukaemia (CML) patients were sequenced. In all 21 samples analysed the junctional region, as well as the b3 exon of 8 b3a2 mRNA molecules, presented no differences to the already described sequences. However, we identified a polymorphic base in the b2 exon in two out of seven b3a2 samples, four out of 10 b2a2 samples and all four b3a2/b2a2 samples analysed. In the eighth position before the junctional region of BCR/ABL cDNA, a cytosine replaces thymine in these cases. The polymorphism described here could be a useful marker for the differentiation of normal and rearranged BCR alleles in heterozygotes.

Exons↗
PubMed1997

Gene frequencies of the HPA-1 and HPA-2 platelet antigen alleles among the Amerindians.

BACKGROUND AND OBJECTIVES: Platelet-specific alloantigens are important in neonatal alloimmune thrombocytopenia, posttransfusion purpura, refractoriness to platelet transfusions, and population genetics. Data are scarce on allele frequencies in ethnic groups other than whites and Asians. MATERIALS AND METHODS: Using allele-specific restriction enzyme analysis, we studied the distribution of HPA-1 and HPA-2 alleles in six Brazilian Amazon tribes of Amerindians, belonging to five different language stocks. We compared these with the values obtained for blacks and whites. RESULTS: Only the HPA-1a allele was found among 132 Amerindian chromosomes, compared with a gene frequency of HPA-1b of 0.115 and 0.113, respectively, among blacks and whites. The frequency of HPA-2b among the Amerindians (0.042) is lower than that obtained for blacks and whites (0.148 and 0.100, respectively), and the lowest thus far observed in a population of Asian origin. CONCLUSION: Differences in DNA polymorphisms in Amerindian populations have not only anthropological and genetic interest, but also practical applications when they involve coding regions that may change the functional or immunologic features of the protein.

Alleles↗
PubMed1997

A highly sensitive and specific chemiluminescent enzyme-linked immunosorbent assay for diagnosis of active Trypanosoma cruzi infection.

BACKGROUND: Chagas' disease is transmitted to man either by the bite of insects harboring Trypanosoma cruzi or by the transfusion of blood from infected donors. The conventional serologic testing as presently used in blood banks in South America is unsatisfactory, because of a high number of inconclusive and false-positive results. Other methods such as polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens have been proposed, but inherent difficulties have so far precluded their adoption in the large-scale screening required by blood banks. STUDY DESIGN AND METHODS: A highly sensitive and specific chemiluminescent ELISA using a purified trypomastigote glycoconjugate antigen and a complex epimastigote antigen was devised for the diagnosis of active T. cruzi infection. RESULTS: Chemiluminescent ELISA was 100-percent sensitive in the diagnosis of 100 cases of confirmed Chagas' disease. Inconclusive results and false-positive reactions were eliminated in a panel of 115 sera. The specificity of the chemiluminescent ELISA was 100 percent with a purified trypomastigote glycoconjugate antigen and 99.7 percent with a complex epimastigote antigen when applied to 1000 normal human sera and 288 heterologous sera from patients with other infections, including leishmaniasis, and vaccinated individuals. CONCLUSION: The chemiluminescent ELISAs provide a test that is highly sensitive (purified trypomastigote glycoconjugate and complex epimastigote antigens) and specific (purified trypomastigote glycoconjugate antigen) for Chagas' disease diagnosis. It can be used in blood bank screening and to monitor the treatment of patients undergoing chemotherapy.

Animals↗
PubMed1987

Splenic function in chronic myelogenous leukemia.

Spleen function was evaluated by measurement of the clearance of autologous heat-damaged 99mTc-labelled erythrocytes from the circulation and into the spleen and the enumeration of pitted erythrocytes by interference contrast microscopy, and the spleen area was determined by scintillation scanning. All measurements were performed on 12 patients with chronic myelogenous leukemia and compared with 10 controls with apparently normal spleens, 6 splenectomized subjects and 9 patients with a reactive splenomegaly. Patients with CML had spleen function test results similar to normal controls in spite of having enlarged spleens whose projection area did not differ from that of the patients with reactive splenomegaly. Thus, patients with CML have a decreased spleen function per unit volume and signs of splenic hypofunction in the peripheral blood. The reduction of spleen function per unit volume in CML is the result of a severe decrease of the splenic blood perfusion which could result from the combined effects of the myeloid metaplasia and the increased whole-blood viscosity due to high white-cell counts.

Adolescent↗
PubMed1987

Splenic function in haemophilia.

Studies of splenic function were carried out on 17 haemophilic patients over 9 years of age, and 20 control patients. The clearance of autologous heat-damaged 99mTc-labelled erythrocytes from circulation and into the spleen was measured: the spleen area by scintillation scanning, and the enumeration of pitted erythrocytes by direct interference microscopy. Splenic enlargement was observed in 10 patients (59%). On the basis of the clearance half-time, splenic function was normal in 3/13 (23%) and hyperactive in 9/13 (69%) patients. One heavily transfused patient had a hypoactive spleen with long clearance half-time, slow splenic uptake of radioactivity and high pit counts. These results demonstrate that the spleen of haemophiliacs is usually enlarged and functionally abnormal.

Adolescent↗