Biomedical subjects

J S Burgos

Publications and source records attributed to J S Burgos.

15 recordsLinked to original sources

PubMed2003

Time course of bioluminescent signal in orthotopic and heterotopic brain tumors in nude mice.

In vivo bioluminescence imaging is becoming increasingly popular. Quantification of bioluminescence signals requires knowledge of the variability and reproducibility of this technique. The objective of this study was to analyze the time course of luminescent signal emitted from firefly luciferase-expressing tumors in two locations, following luciferin injection and at different times after tumor cell implantation. Knowledge of the kinetics of the bioluminescent signals is required for the reliable quantification and comparison of signal during longitudinal studies. The kinetics of bioluminescence was evaluated in orthotopic and heterotopic brain tumors in mice using a human brain tumor cell line constitutively expressing luciferase. Tumor cells were implanted in the brains and flanks of the animals, and whole-body images revealing tumor location were obtained. Tumor burden was monitored over time by the quantitation of photon emission. The magnitude of bioluminescence measured in vivo varied with time after the injection of luciferin, as well as with dose, which necessitated that the comparison of the quantitative results take into consideration the time after injection. Heterotopic and orthotopic tumors exhibited significantly different time courses; however, time after implantation as characterized by kinetic studies performed on days 4 and 14 after cell implantation revealed no significant differences in orthotopic tumors. Future quantitative longitudinal studies must take into account the differences in the kinetics of different models.

Animals↗

PubMed2002

Influence of reagents formulation on real-time PCR parameters.

Real-time polymerase chain reaction (PCR) techniques are increasingly used to quantify target sequences for diagnostic and research purposes. Due to its 'quantitative' character, it is very important to determine the variability of this technique correlating with several experimental conditions. The objective of this study was to analyse the effect of manufacturing lots of PCR reagents on two main PCR parameters, specificity and sensitivity. For this study, we used four different amplicons, using either mouse genomic DNA or viral DNA. Although a PCR product could be obtained in any of the conditions, we observed that there are relevant variations in sensitivity depending on the reagents formulation. We conclude that different lots of reagents may determine the analytical performance of PCR assays indicating that reagents testing are of special importance when the PCR protocol is used for quantitative purposes.

Actins↗

PubMed2002

Expression of p53 protein and tumor angiogenesis as prognostic factors in nasopharyngeal carcinoma patients.

The objective of this study was to evaluate the possible prognostic significance of p53 protein overexpression and tumor angiogenesis (TA) in nasopharyngeal carcinoma (NPC) patients, together with other clinicopathological variables. Forty-two NPC patients were evaluated in relation to survival. Nuclear p53 overexpression in neoplastic and endothelial cells was detected by immunohistochemistry (IHC) with the monoclonal antibody DO-7 and the polyclonal antibody against factor VIII-related antigen, respectively. Thereafter, we evaluated p53 cases in order to determine their nuclear immunoreactivity from negative (-) to positive (+, ++, +++). In addition, microvessels were counted in the most active areas of tumor neovascularization or hotspots using an image computer analyzer (MicroImage). A Cox multiple regression survival analysis was used to determine the best prognostic indicators in NPC patients. As a result, tumor microvessel count, considered as a continuous variable, was the most important independent prognostic indicator in relation to survival (p = 0.0273), with a relative risk of death of 2,4399 [95% confidence interval = 1.1051 ; 5.3871] associated with the highest microvessel counts. Moreover, the only clinicopathological variable that demonstrated prognostic value in a Cox multiple regression survival analysis was histological type (p = 0.05). In addition, we did not observe any statistical association between intratumoral microvessel density (IMD), clinicopathological variables and p53 protein expression.

Biomarkers, Tumor↗

PubMed2001

Effect of orally administered guanosine on seizures and death induced by glutamatergic agents.

Intraperitoneal guanosine has been shown to prevent quinolinic acid-induced seizures in mice. In this study, we investigated the effect of orally administered guanosine on seizures induced by the glutamate agonists quinolinic acid and kainate, and the endogenous glutamate releaser alpha-dendrotoxin. Guanosine (7.5 mg/kg, per os), administered 75 min in advance, prevented 70% of seizures induced by i.c.v. quinolinic acid, being as efficient as the NMDA channel blocker MK-801 administered intraperitoneally. Guanosine was ineffective against kainate-induced seizures, but significantly reversed the potentiation of seizures and death caused by the concomitant injection of MK-801. Guanosine also significantly prevented seizures and death induced by i.c.v. alpha-dendrotoxin, whereas MK-801 and phenobarbital only prevented death. Altogether, our findings underscore the therapeutic potential of oral administration of guanosine for treating diseases involving glutamatergic excitotoxicity, including epilepsy.

Animals↗

PubMed2000

Cl(-)-dependent excitotoxicity is associated with 3H2O influx in chick embryonic retina.

The aim of this study was to show that Cl(-)-dependent excitotoxicity, with its characteristic cell swelling, involves actual water influx into the intracellular compartment. Taking advantage of the Ca2+ omission paradigm of Cl(-)-dependent excitotoxicity, in the chick embryonic neural retina ex vivo, which is associated with toxicity levels (lactate dehydrogenase (LDH) release) considerably higher than those seen after simple exposure of the retinas to glutamate agonists, we have demonstrated that an intracellular water intake of 4.2 microl into retinal cells is associated with 13.3% total retinal LDH release. The fact that mannitol blocks both water inflow and LDH release appears to link both events from a pathogenic point of view.

Animals↗

PubMed2000

Ca2+-dependent kainate excitotoxicity in the chick embryonic neural retina ex vivo.

The chick embryonic neural retina ex vivo has been singled out as a unique example of Cl(-)-dependent/Ca2+-independent excitotoxicity. However, after continuous incubation with 100 microM kainate, we have demonstrated the susceptibility of the chick retina to Ca2+-mediated damage, which becomes apparent after 12 h of exposure to the agonist in the absence of Cl-. Of the 20.8% lactate dehydrogenase released after 24 h incubation with kainate, some 11% is Cl(-)-dependent and the rest (9.8%) is presumably Ca2+-dependent. Upon omission of both Cl- and Ca2+, a 5% residual toxicity can still be detected after 24 h. This can be overcome by inclusion of EGTA in the incubation medium to neutralize Ca2+ released during incubation. A Ca2+-dependent toxicity mechanism is then operative in the embryonic chick retina ex vivo.

Animals↗

PubMed2000

Guanine nucleotides block agonist-driven 45Ca2+ influx in chick embryo retinal explants.

AMPA (EC50 = 1.0 x 10(-6) M) and NMDA (EC50 = 1.3 x 10(-4) M) trigger 45Ca2+ influx in 13-day chick embryonic retinal explants. This agonist-driven cationic flux is specifically inhibited by typical competitive antagonists, such as 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 2-amino-7-phosphonoheptanoate (AP7), respectively. Guanine nucleotides, with different degrees of phosphorylation, namely 5'-GMP, guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and 5'-guanylyl-imidodiphosphate (GppNHp), are also efficient blockers of 45Ca2+ influx. These results confirm the antagonistic behavior of guanine nucleotides towards ionotropic glutamate receptors and suggest a convenient experimental approach for screening of novel agonists and antagonists.

2-Amino-5-phosphonovalerate↗

PubMed2000

Morphometric study of tumor angiogenesis as a new prognostic factor in nasopharyngeal carcinoma patients.

The aim of this study was to determine the possible prognostic significance of tumor angiogenesis (TA) in nasopharyngeal carcinoma (NPC) patients. Fifty-five NPC patients were evaluated in relation to survival. Endothelial cells were immunohistochemically stained with anti-von Willebrand factor (F-VIII), CD-31 and CD-34 antibodies, and microvessels counted in the most active areas of tumor neovascularization or hotspotsusing both a manual and an automatic method. Overall survival analysis calculated by the Kaplan Meiertest revealed that both methods were correlated with a statistical significance between intratumoral microvessel density (IMD) and overall survival, using either manual (p=0.0141) or automatic counting (p=0.0117). Other angiogenic parameters studied were perimeter, roundness and accumulative area of the microvessels using a morphometric analyzer. Moreover, our results show that cases with high IMD demonstrated a prognostic significance in relation to the accumulative area (p=0.0072).

Adult↗

PubMed2000

Immunohistochemical absence of CD21 membrane receptor in nasopharyngeal carcinoma cells infected by Epstein-Barr virus in Spanish patients.

OBJECTIVES/HYPOTHESIS: The aim of this study was to analyze the relevance of the CD21 membrane receptor in nasopharyngeal carcinoma (NPC). CD21 is implicated in the introduction of the Epstein-Barr virus (EBV) genome into epithelial cells and B lymphocytes. STUDY DESIGN: Immunohistochemical analysis of CD21 in NPC. METHODS: Paraffin-embedded samples of NPC of different histological types with demonstrated presence of EBV were analyzed for CD21 expression using immunohistochemistry. RESULTS: We detected EBV by non-isotopic in situ hybridization (NISH) and nested polymerase chain reaction (PCR) in 100% of samples, regardless of histological type, supporting the previous view that all the types of NPC are variants of an EBV-associated malignancy. CD21 was not detected in NPC, and this absence was a typical feature in our data group. CONCLUSIONS: The loss of the CD21 membrane receptor could be one of the immunophenotypical changes of the neoplastic cells that occur in the evolution of the NPC malignancy.

Adolescent↗

PubMed1999

Chick kainate binding protein lacks GTPase activity.

Chick kainate binding protein was solubilized from cerebellar membranes and purified (x19) by use of two chromatographic steps. Measurements of [3H]kainate binding and GTPase activity in the different fractions reveal a consistent decrease of GTPase activity as the purification proceeds so that no GTPase is detectable after the final purification step. This fact, in the context of the differential involvement in nucleotide recognition of some critical amino acid residues in the p-loop motif of GTPases and in the guanine nucleotide-binding sequence of ionotropic glutamate receptors, together with significant discrepancies concerning the activity of individual nucleotides, suggests that both guanine nucleotide-recognizing sequences are unlikely to be alternative expressions of the same functional domain.

Animals↗

PubMed1999

Guanine nucleotides, including GMP, antagonize kainate responses in Xenopus oocytes injected with chick cerebellar membranes.

Injection of chick cerebellar membranes, rich in kainate binding sites, into Xenopus oocytes resulted in the structural integration of chick membrane patches into the oocyte plasma membrane that could be easily identified by specific immunofluorescent staining. Application of kainate to the oocyte perfusion medium, under voltage-clamp conditions, induced dose-dependent (EC50 = 87+/-14 microM) inward currents, confirming the functional incorporation to the oocyte of kainate-driven channels. Responses to kainate were consistently nondesensitizing and strongly potentiated by cyclothiazide, suggesting the selective involvement of alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA)-preferring receptors. Binding experiments with (S)-[3H]AMPA confirmed the presence in the chick membrane preparation of low-affinity AMPA receptors (K(D) = 278 nM) amounting to <2% of the total population of kainate binding sites. A tenfold concentration of guanine nucleotides, with different degrees of phosphorylation, blocked the responses to 100 microM kainate by approximately 90%. In the case of GMP, additional concentration-inhibition studies yielded an IC50 of 180+/-11 microM. Our results illustrate the apparent failure of kainate-binding proteins to form functional channels, even when maintaining their own native membrane environment, and confirm the antagonistic behavior of guanine nucleotides, including GMP, toward glutamate receptors, in agreement with previous results of ligand-binding experiments and, more interestingly, with the marked neuroprotective effects of some guanine nucleotides in different excitotoxicity experimental paradigms.

Animals↗

PubMed1998

Guanine nucleotides protect against kainate toxicity in an ex vivo chick retinal preparation.

Ex vivo preparations of chick neural retina have been successfully used in the assessment of excitotoxicity and in the evaluation of the protective effects of glutamate antagonists. Using a variation of this approach, and measuring the acute and delayed toxic effects of kainate (KA) in terms of lactate dehydrogenase release, we have shown that guanine nucleotides behave as effective neuroprotecting agents. The anti-excitotoxic potency of guanine nucleotides (in the case of GMP and GDPbetaS it is about 100 times lower than that of DNQX, a powerful kainate antagonist) correlates well with their ability to displace KA from retinal KA receptors.

Animals↗

PubMed1997

Immunohistochemical expression of Bcl-2 oncoprotein in EBV-associated nasopharyngeal carcinoma correlated to histological type and survival.

Expression of Bcl-2 is associated with inhibition of apoptosis and extension of cell survival. In vitro Bcl-2 protein expression is up-regulated by the EBV-latency associated antigen latent membrane protein (LMP-1). We have investigated the relationship between the presence of EBV-DNA screened by means of sensitive nested-PCR, nasopharyngeal carcinoma (NPC) histological types according to two different schemata (WHO and Micheau classifications) and Bcl-2-124 immunohistochemical expression in 55 biopsy samples of NPC. EBV genome was detected in 100% of samples with sufficient DNA quality to support the previous view that all types of NPC are variants of EBV-infected neoplasia. Bcl-2 was observed in the basal layer of normal nasopharyngeal mucosa and also at cytoplasmic level in 42 of 55 (76.4%) NPC cases. Mitotic neoplastic cells usually showed strong cytoplasmic and chromosomal staining, a finding not well referred to previously. Bcl-2 expression was significantly associated (p<0.05) to undifferentiated NPC (UNPC) when a histological classification with only two major microscopical types was applied. No close correlations were found between the presence of EBV-DNA, NPC location, clinical stage and age or sex of the patients in relation to Bcl-2 positive expression. However, when comparing Bcl-2 expression and known survival mean of the patients, significant differences were observed (p<0.001) so that mean survivals were 31.1, 24.4, 52.2 and 54.1 months respectively for NPC patients with -, +, ++ and Bcl-2 immunoreactivity. Nevertheless this better clinical outcome in Bcl-2 NPC positive cases may depend on the histological type due to close relationship with UNPC. Only studies of larger series with long-term follow-up and multivariate analyses may document whether Bcl-2 expression is an independent prognostic marker in the evolution of NPC patients.

Adolescent↗

PubMed1996

Immunohistochemical expression of Epstein-Barr virus-encoded latent membrane protein (LMP-1) in paraffin sections of EBV-associated nasopharyngeal carcinoma in Spanish patients.

Nasopharyngeal carcinoma (NPC) paraffin samples, from Spanish patients, of distinct histological types, including squamous cell carcinoma (10 cases), nonkeratinising carcinoma (12 cases) and undifferentiated carcinoma (29 cases) were analysed for Epstein-Barr virus (EBV) detection and EBV-encoded latent membrane protein (LMP-1) expression using a sensitive nested-polymerase chain reaction with four oligonucleotide primers specific for EBV genome (EB-1, 2, 3, 4) and immunohistochemistry by means of CS1-4 pool monoclonal antibody. EBV genome was detected regardless of histological type in 100% of samples with sufficient DNA quality to permit viral diagnosis (50 out of 51 cases), supporting the previous view that all types of NPC are variants of an EBV-associated malignancy. However LMP-1, an EBV-encoded oncogenic protein, was detected in 40 out of 51 samples (78.4%) and LMP-1 immunohistochemical expression was not apparently influenced by histological type, primary or metastatic site, clinical stage, age or sex. This high percentage of detection of LMP-1 in our cases supports a role for EBV in the pathogenesis of different types of NPC, but the lack of constant expression of LMP-1 in NPC remains unclear and various reasons are postulated to explain the absence of this oncogenic protein in some EBV-associated NPCs.

Adolescent↗

PubMedDate not supplied

Analysis of EBV latency by EBER in situ hybridization in nasopharyngeal carcinoma Spanish patients.

BACKGROUND: The aim of this study was to analyse the Epstein-Barr virus (EBV) latency by detecting the EBV-associated latent small nuclear RNAs (EBER), in a group of biopsies from Spanish patients with diagnosed nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: NPC paraffin samples with the presence of EBV demonstrated by non-isotopic in situ hybridization (NISH) and nested-PCR, were analysed for EBV latency using EBER in situ hybridization (EBER-ISH). RESULTS: We detected EBER in 83.3% of samples (10 out of 12 cases), demonstrating the relationships between EBV genome presence with the latent viral infection. We correlated these results of EBV-DNA and -RNA presence with the immunoexpression of latent membrane protein-1 (LMP-1), a viral oncogenic protein (8 out of 12 cases or 66.6%). CONCLUSION: These results indicate that all the types of NPC are variants of an EBV-associated malignancy and that viral latency is a critical phenomenon in the development of this neoplasia.

Adolescent↗