PubMed Health⌕ Search

Biomedical subjects

J M Silva

Publications and source records attributed to J M Silva.

At least 19 recordsLinked to original sources
PubMed1999

Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations.

Using different molecular techniques, DNA has been detected in the plasma of cancer patients with various types of tumors. We undertook the present study to investigate the presence of plasma DNA, before mastectomy, in patients with breast cancer at diagnosis and to analyze the clinicopathological spectrum of this subgroup of patients with respect to patients without DNA with tumor characteristics. We studied 62 patients with breast cancer, who were selected sequentially after mastectomy and diagnosis of breast carcinomas. Genomic DNA extracted from tumor and normal tissues, normal blood cells, and plasma was used for molecular studies. Alterations in polymorphic markers selected because they had been found to show a high rate of alterations in breast cancer in previous studies (D17S855, D17S654, D16S421, TH2, D10S197, and D9S161), as well as mutations in the p53 gene and aberrant methylation at the first exon of p16INK4a, were used to identify and characterize tumor and plasma DNA. Thirteen clinicopathological parameters were analyzed in each patient. We identified 56 cases (90%) with at least one molecular event in tumor DNA, and 41 cases (66%) with a similar alteration in plasma DNA. Comparison of the clinicopathological parameters between patients with and without plasma DNA revealed significant differences in the axillary involvement, rate of invasive ductal carcinoma, high proliferative index, and the parameter comprised of lymph node metastases, histological grade II, and peritumoral vessel involvement. A high proportion of breast cancer patients exhibited plasma DNA at diagnosis similar to tumor DNA, and its presence correlated significantly with pathological parameters associated with a poor prognosis.

Breast↗
PubMed1999

Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel.

Successful cryopreservation of freshly isolated hepatocytes would significantly decrease the need for freshly-procured livers for the preparation of hepatocytes for experimentation. Hepatocytes can be prepared, cryopreserved, and used for experimentation as needed at different times after isolation. Cryopreservation is especially important for research with human hepatocytes because of the limited availability of fresh human livers. Based on the cumulative experience of this international expert panel, a consensus was reached on the various aspects of hepatocyte cryopreservation, including cryopreservation and thawingprocedures and applications of the cryopreserved hepatocytes. Key to successful cryopreservation includes slow addition of cryopreservants, controlled-rate freezing with adjustment for the heat of crystallization, storage at -150 degrees C, and rapid thawing. There is a general consensus that cryopreserved hepatocytes are useful for short-term xenobiotic metabolism and cytotoxicity evaluation.

Animals↗
PubMed1999

Induction of cytochrome-P450 in cryopreserved rat and human hepatocytes.

Our laboratory has been routinely using suspended and cultured human hepatocytes for predicting drug metabolism and enzyme induction by drug candidates to aid drug discovery. Increasing limitation and irregular availability of human tissue has indicated the need for maximizing the use of this valuable resource. Cryopreservation of surplus hepatocytes after isolation would greatly increase the potential of this model. However, cryopreservation of hepatocytes by various methods has resulted in cells with poor metabolic activity and unacceptably low survival rates in culture. Recently, Zaleski et al. (Biochem. Pharmacol. 46 (1993) 111-116) reported that cryopreserved rat hepatocytes retained metabolic capacity similar to fresh hepatocytes when the cells were preincubated for 30 min at 37 degrees C in Krebs Ringer bicarbonate buffer prior to freezing. To further explore this methodology, both the functional capacity of the cells in culture as well as their ability to retain CYP inducibility were investigated with thawed cryopreserved hepatocytes. Although human hepatocytes were used in this study the initial work focused on rat hepatocytes as a cell model. Our results showed that while the preincubation step did not appear to effect the initial viability of cryopreserved hepatocytes, survival of the cells in culture was greatly enhanced. Plating efficiencies for nonpreincubated cryopreserved hepatocytes were decreased to approximately 15% of fresh cells after 48 h in culture. In contrast, cells that had been preincubated prior to freezing had an excellent plating efficiency (approximately 60%) and responded to classical CYP inducers dexamethasone, beta-naphthoflavone and phenobarbital in a manner indistinguishable from that of fresh hepatocytes. Experiments with human hepatocytes have also demonstrated similar results. This is the first time to our knowledge that cryopreserved hepatocytes from both rat and human have been shown to reproducibly respond to CYP inducers in culture.

Animals↗
PubMed1999

Abnormal frequencies of alleles in polymorphic markers of the 17q21 region is associated with breast cancer.

This study was carried out to evaluate the frequencies of alleles of four polymorphic markers in the 17q21 region in breast cancer patients, and their relation to seven pathological parameters. One hundred and sixty-four patients with breast cancer and 102 controls were analyzed. D17S856, D17S855, D17S1323 and D17S1327 polymorphic markers were studied, and used to investigate loss of heterozygosity in this region. The frequencies of alleles at marker D17S856 differed significantly in breast cancer patients and controls, and were related to histologic features considered to indicate a poor prognosis. When present, the pathophenotype of tumors associated with LOH in the 17q21 region is modified.

Alleles↗
PubMed1999

Cryopreservation of rat and human liver slices by rapid freezing.

The cryopreservation of human liver slices is a promising way to enhance the ability to test the metabolism of drug candidates. This study demonstrates the use of a novel technique for the cryopreservation of both rat and human liver slices. In this technique the slices are treated with Me2SO and sandwiched between aluminum plates separated by a thin gasket. The device is then submerged in liquid nitrogen to freeze the slices, which can then be stored until use. To thaw the slices, the apparatus is submerged in a water bath at 37 degrees C. Slices frozen and thawed in this manner were compared to those frozen in conventional cryovials. The viability of the slices was determined by incubating them in 12-well plates and measuring urea synthesis, ethoxycoumarin metabolism, and cytosolic enzyme leakage (LDH and ALT). The viability of rat slices frozen between plates approached that of fresh slices and was consistently higher than slices frozen in cryovials. Slices from two human samples gave similar results. The technique was found to work over a wide range of Me2SO concentrations (4.5 to 22% was tested) with an optimal concentration between 10 and 15%.

Aged↗
PubMed1999

Loss of heterozygosity in BRCA1 and BRCA2 markers and high-grade malignancy in breast cancer.

Loss of heterozygosity (LOH) in loci of the 17q21 and 13q12-13 regions can collaborate in the inactivation of BRCA1, BRCA2, and possibly other genes implicated in the pathogenesis of breast carcinomas. We investigate allelic losses in microsatellites of the BRCA1 and BRCA2 regions, and their correlations with seven pathologic parameters in 140 breast carcinomas. Those cases showing LOH in the region of the RB gene, 13q14, were excluded from the study. The LOH analysis was performed by amplifying DNA by PCR, using four markers of the 17q21 region (D17S856, D17S855, D17S1323, and D17S1327) and four markers of the 13q12-13 region (D13S290, D13S260, D13S310, and D13S267). LOH in the BRCA1 region was found in 47% of tumors, correlating significantly with estrogen receptor content (p = 0.025), progesterone receptors (p = 0.004), higher grade (p = 0.0008), peritumoral vessel invasion (p = 0.001), and lymph node metastases (p = 0.002). When we excluded the cases with LOH in the BRCA2 region and those not informative for it, the significance disappeared. In the BRCA2 region, a rate of LOH of 51% was found; it correlated significantly with estrogen receptor content (p = 0.002), progesterone receptors (p = 0.03), peritumoral vessel invasion (p = 0.005), higher grade (p = 0.002), and lymph node metastases (p = 0.001). When cases with BRCA1 losses and those not informative were excluded, again the significance disappeared. Concomitant losses in the BRCA1 and BRCA2 regions were found in 32% of cases, correlating significantly with lymph node metastases (p = 0.0002), estrogen receptor content (p = 0.003), progesterone receptors (p = 0.001), histologic grade (p = 0.01), and peritumoral vessel invasion (p = 0.0004). These results suggest that concomitant losses in both regions could have a functional effect, influencing the presence of a poor tumor pathophenotype in breast carcinomas.

Adenocarcinoma↗
PubMed1999

Aberrant DNA methylation of the p16INK4a gene in plasma DNA of breast cancer patients.

Hypermethylation of exon 1 of p16INK4a was examined in tumour and plasma DNA of a series of breast cancer patients. De novo methylation was observed in the tumours of eight patients (23%), and in plasma DNA in five (14%) of these eight patients. Our data show that de novo methylation of exon 1 of p16INK4a can be demonstrated in plasma DNA of breast cancer patients, a fact that provides additional evidence of the tumour-related origin of free plasma DNA in cancer patients.

Amino Acid Sequence↗
PubMed1999

Oxidative bioactivation of the lactol prodrug of a lactone cyclooxygenase-2 inhibitor.

The lactol derivative of a lactone cyclooxygenase-2 inhibitor (DFU) was evaluated in vivo and in vitro for its potential suitability as a prodrug. DFU-lactol was found to be 10 to 20 times more soluble than DFU in a variety of aqueous vehicles. After administration of DFU-lactol at 20 mg kg-1 p.o. in rats, a Cmax of 7.5 microM DFU was reached in the plasma. After oral administration, the ED50s of DFU-lactol in the carrageenan-induced paw edema and lipopolysaccharide-induced pyresis assays in rats are comparable with the ED50s observed when dosing with DFU. Incubations of DFU-lactol with rat and human hepatocytes demonstrated that the oxidation of DFU-lactol can be mediated by liver enzymes and that a competing pathway is direct glucuronidation of the DFU-lactol hydroxyl group. Assays with subcellular fractions from rat liver indicated that most of the oxidation of DFU-lactol occurs in the cytosolic fraction and requires NAD(P)+. Human liver cytosol can also support the oxidation of DFU-lactol to DFU when NAD(P)+ is added to the incubations. Fractionation of human liver cytosolic proteins showed that at least three enzymes are capable of efficiently effecting the oxidation of DFU-lactol to DFU. Incubations with commercially available dehydrogenases suggest that alcohol and hydroxysteroid dehydrogenases are involved in this oxidative process. These data together suggest that lactols may represent useful prodrugs for lactone-containing drugs.

Animals↗
PubMed1999

[Peripheral revascularization by cardiologists].

OBJECTIVE: To analyse the initial experience in peripheral arterial revascularization, using percutaneous techniques, by interventional cardiologists. DESIGN: Retrospective clinical study. SETTING: Cardiology department of a specialised public non-university hospital. PATIENTS: Twenty patients, 18 male and 2 female, with ages between 37 and 84 years, who underwent peripheral interventions between May 5 1989 and February 20 1998. INTERVENTIONS: Patients were subdivided into two groups: Group I, 9 patients, 8 male, average age of 56 +/- 13 years, undergoing renal artery angioplasty; Group II, 11 patients, 10 male, average age of 63 +/- 10 years, undergoing angioplasty of the distal aorta (1), of the right subclavian artery (1), of the iliac arteries (6) and of the femoral arteries (3). In Group I, 11 arteries were dilated, 6 by balloon angioplasty and 5 with stent implantation (Palmaz-Schatz--4 and Symphony--1). In Group II, 12 arteries were dilated, 5 by balloon angioplasty and 7 with 12 stent implantations (Palmaz-Schatz--6, Symphony--5 and NIRTM--1). In 8 patients, 1 of Group I and 7 of Group II, coronary angioplasty was also performed in 14 arteries and 18 segments, exclusively by balloon (3 patients) or with 10 stent implantations (5 patients). RESULTS: There was angiographic success in all patients (100%) and clinical success in 85% (17/20) of patients. All clinical complications occurred in Group I patients: retroperitoneal bleeding in one; right femoral artery pseudoaneurysm requiring surgery in one patient: acute renal failure in one patient. There were no cardiac complications in patients undergoing peripheral and cardiac angioplasties at the same stage. In the mean follow-up period of 26 months, one patient required reangioplasty of a right iliac artery and another underwent iliac-femoral bypass, both of Group II and before peripheral stents were available. CONCLUSIONS: Percutaneous peripheral arterial revascularization performed by experienced interventional cardiologists seems safe and efficient, being perfectly justified in patients requiring simultaneous coronary angioplasty.

Adult↗
PubMed1999

[Hypertriglyceridemias].

Triglyceride atherogenicity is still a matter of controversy for reasons related to their biological and epidemiological characteristics. The authors discuss and review several of these problems, as well as the metabolic relationship of triglycerides and HDL-cholesterol, data indicating a strong influence of triglycerides on cardiovascular risk, mechanisms mediating the pathogenicity of triglycerides, and some questions of drug treatment of hypertriglyceridemias.

Arteriosclerosis↗
PubMed1999

TP53 gene mutations in plasma DNA of cancer patients.

Using different molecular techniques, DNA has been shown to be present in plasma of patients with several types of tumors. Mutations of TP53 are the most common genetic changes in human cancers. We investigated the presence of TP53 gene mutations in plasma DNA of breast and small cell lung cancer patients. Tumor and plasma DNA of 25 patients were studied by PCR-SSCP and direct sequencing, through exons 5, 6, 7, and 8, of TP53. Six cases of mutations in tumor DNA were observed that, in 3 cases (50%), were also identified in plasma of the same patients. Mutations of the TP53 gene are seen in plasma DNA of cancer patients, and may prove to be a useful new tool in the management of these patients.

Breast Neoplasms↗
PubMed1998

Methods for the determination of organochlorine pesticide residues in human serum.

The effectiveness of solid-phase extraction with Florisil for the determination of 12 organochlorine pesticide residues from human serum was examined. Recoveries greater than 84% and coefficients of variation better than 19% were obtained. Others methods, such as column partition and matrix solid-phase dispersion, were compared. The better method provides quantification limits ranging from 1.08 microg/l for gamma-HCH and 37.5 microg/l for p,p'-DDT when capillary gas-liquid chromatography with electron-capture detection is used for the final determination.

Acetone↗
PubMed1998

Loss of heterozygosity in the region including the BRCA1 gene on 17q in colon cancer.

Colon cancer has been proved to be an excellent model to identify and to study the different genetic alterations taking part in its development. BRCA1, a susceptibility gene for breast cancer, has been identified. Evidence of a significant risk for colon cancer in BRCA1-linked families has been reported. We undertook the present study to investigate the possible relation of BRCA1 and other genes on chromosome 17q21 to colon cancer. Eighty-five tumors were examined for loss of heterozygosity at seven microsatellite loci spanning the 17q21 region. Loss of heterozygosity was present in 39 (49%) of the 79 informative cases, with at least one marker. Two loci, D17S855 and D17S579, showed higher rates of allelic loss (16% and 22%, respectively) than the remaining markers. Tumors on the right side of the colon exhibited allelic loss more frequently than those on the left side. Our data suggest that BRCA1 and other genes in the 17q21 region may play an important role in the development of colon cancer.

Base Sequence↗
PubMed1998

Features of primary vesicoureteric reflux detected by investigation of foetal hydronephrosis.

Primary vesicoureteric reflux (VUR) diagnosed on investigation of foetal hydronephrosis accounts for many antenatally detected uropathies. In order to study foetal VUR and its consequences, newborns with foetal hydronephrosis were investigated by ultrasound, micturating cystourethrogram and 99mTechnetium-dimercapto-succinic acid (DMSA), after beginning of chemoprophylaxis. Twenty-eight infants with VUR (bilateral in 15 cases) were identified giving a total 43 renal units for study. There was a predominance of males (86%), moderate/severe reflux (84%) and renal damage (51%). Presence of renal damage was correlated with the severity of reflux. VUR should be investigated in cases of foetal hydronephrosis and our results support that renal damage is frequently congenital and not secondary to urinary tract infection.

Female↗
PubMed1998

Germ-line BRCA1 mutations in women with sporadic breast cancer: clinical correlations.

PURPOSE: Sporadic nonhereditary breast cancer is recognized as the most common form of this malignancy. Presence of germ-line mutations in the BRCA1 gene of these tumors is an infrequent event. We undertook the present study to evaluate the prevalence of germ-line mutations in patients diagnosed with sporadic breast cancer, and to delimit the clinical spectrum of this subgroup of patients with germ-line mutations and their differences with respect to patients with no evidence of BRCA1 gene mutations. METHODS: We studied 105 patients diagnosed with breast cancer, selected from among our living patients; those with carcinoma-in-situ and those with a definite family history of breast or ovarian cancer were excluded. Genomic DNA, obtained from peripheral-blood lymphocytes, was studied for BRCA1 mutations by polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) and direct DNA sequencing. Fourteen clinicopathologic parameters were analyzed in each patient. RESULTS: Six (5.7%) frameshift mutations that corresponded to truncating proteins and three missense mutations, the functional meaning of which remains speculative, were identified. The patients with germ-line mutations were found to have a more advanced age at diagnosis, as well as a longer median survival (51 months). CONCLUSION: Women with sporadic breast cancer of late onset may display a significant incidence of germline BRCA1 mutations, which occur at a rate not previously determined in this group of patients. The presence of variations in the sequence of the BRCA1 gene could influence the longer survival observed in these patients.

Adult↗
PubMed1998

Detection of BRCA1 gene mutations in families with breast cancer patients and their healthy relatives.

Eighty-three families with two or more cases of breast and ovarian cancer were selected to evaluate the prevalence rate of BRCA1 gene mutations, and identify healthy carriers. Blood samples from patients and healthy relatives were obtained. Haplotype study of the 17q21 region using 7 polymorphic markers was performed. Mutational analysis of the coding exons of the BRCA1 gene was performed by the PCR-SSCP method and direct sequencing. We detected germline mutations (frameshift and missense) in 6 families (9.1%). Combining haplotype analysis and PCR-SSCP screening, 18 (15%) healthy female carriers were identified. The prevalence rate of germline BRCA1 gene mutations among our families with breast cancer syndrome is low, and relatives having the same haplotype for the 17q21 region as mutation-carrying patients usually display the same genomic sequence as the patients.

Breast Neoplasms↗