Biomedical subjects
Publications and source records attributed to A Rodriguez.
Two epithelial-myoepithelial carcinoma of parotid gland from two female patients of 48 and 80 years old, and normal gland tissue, are studied with Del Río Hortega-Polak silver impregnation technique. It is a selective and alternative method to mark myoepithelial cells in sections of paraffin embedding material or in frozen sections.
Traumatic brain injury (TBI) is an epigenetic risk factor for Alzheimer's disease (AD). To test the hypothesis that TBI contributes to the onset and/or progression of AD-like beta-amyloid peptide (Abeta) deposits, we studied the long-term effects of TBI in transgenic mice that overexpress human Abeta from a mutant Abeta precursor protein (APP) minigene driven by a platelet derived (PD) growth factor promoter (PDAPP mice). TBI was induced in 4-month-old PDAPP and wild type (WT) mice by controlled cortical impact (CCI). Because Abeta begins to deposit progressively in the PDAPP brain by 6 months, we examined WT and PDAPP mice at 2, 5, and 8 months after TBI or sham treatment (i.e., at 6, 9, and 12 months of age). Hippocampal atrophy in the PDAPP mice was more severe ipsilateral versus contralateral to TBI, and immunohistochemical studies with antibodies to different Abeta peptides demonstrated a statistically significant reduction in hippocampus and cingulate cortex Abeta deposits ipsilateral versus contralateral to CCI in 9-12 month-old PDAPP mice. Hippocampal atrophy and reduced Abeta deposits were not seen in hippocampus or cingulate cortex of sham-injured PDAPP mice or in any WT mice. These data suggest that the vulnerability of brain cells to Abeta toxicity increases and that the accumulation of Abeta deposits decrease in the penumbra of CCI months after TBI. Thus, in addition to providing unique opportunities for elucidating genetic mechanisms of AD, transgenic mice that recapitulate AD pathology also may be relevant animal models for investigating the poorly understood role that TBI and other epigenetic risk factors play in the onset and/or progression of AD.
We have developed a novel fluorescence-based homogeneous binding assay for high-throughput screening of chemical compounds. In this assay, a Cy5- or Cy5.5-labeled ligand binds to receptor immobilized on a particle, either a bead or a cell. The resulting localized signal can be detected by a modified microvolume fluorimeter (MVF). When a molecule which competes with the labeled ligand is present, the localized fluorescence on cells or beads is reduced. Image processing software enumerates events and analyzes fluorescence intensity. We describe MVF assays for the IL-1 and IL-5 receptors. Using synthetic peptides with a range of affinities for the IL-1 receptor, we obtained IC(50) data consistent with those determined by radioligand binding assays. Because the image processing software can discriminate among events with different diameters, we were able to develop a multiplex assay, in which the IL-1R and IL-5R assays were carried out in the same well with each receptor immobilized on a different size of bead. IC(50) values generated in the multiplex assay for ligands specific to each receptor were comparable to those determined independently. Finally, similar IC(50) values were obtained in a 16-microl volume in an 864-well plate. This homogeneous, nonradioactive, miniaturizable, and multiplex-capable assay holds much promise for screening of combinatorial libraries and compound collections.
Transient transfection of recombinant genes into cells is a commonly used approach for analyzing cell-cycle- and/or apoptotic-related activities of cell-cycle control proteins. In this approach, information regarding the functional consequence of expressing a recombinant protein transiently is garnered by comparing against results obtained from cells which are transfected with either a control expression plasmid and/or with mutant expression plasmids. In general however, little attention is paid to whether the transfection procedure itself influences these experiments. Using the calcium phosphate transfection method, we show that the introduction of DNA into cells induces signaling of the cell-cycle control machinery. In Hela cells, a transient increase in G0/G1 cells is observed 8 h after transfection. Furthermore, the introduction of DNA into several cell lines induces apoptosis. Transfection-mediated apoptosis can be elicited through a p53-independent mechanism, suggesting the possible extrapolation to many tumor cell lines. Last, we show that due to a likely cell-cycle-specific entry of marker genes into the nucleus, a highly biased cell-cycle distribution is observed in successfully transfected cells at early times following transfection. The importance of these issues in the interpretation as well as the design of transient transfection-based cell-cycle experiments is discussed.
Multivessel percutaneous transluminal coronary angioplasty (PTCA) is associated with a high requirement for further revascularization procedures. Although stenting can reduce restenosis and clinical events after 1-vessel intervention, little information is available after multivessel coronary stenting. We followed up 136 patients (9% of 1,481 undergoing stenting in our center) who had had stent implantation in at least 2 different major native coronary arteries and were followed-up for >6 months. Each patient had received a mean of 2.3 +/- 0.6 stents (1.13 +/- 0.4 stents per lesion) and procedural success was 95%. In-hospital complications included 1 death, 1 Q-wave infarction, 5 non-Q-wave myocardial infarctions, and 1 repeat PTCA. After a mean of 18 +/- 13 months, 7 patients died (3 of heart failure, 4 of noncardiac causes), 2 required coronary bypass surgery, 1 had a myocardial infarction, 13 target vessel repeat PTCA, and 4 non-target vessel PTCA. Survival free of major cardiac events was 75% at 3 years. A history of heart failure, dilation of a restenotic lesion, and 3-vessel dilation were independent negative predictors of event-free survival. Angiographic follow-up was available in 86 patients: 56 (65%) were restenosis free, 23 (27%) had 1-vessel restenosis, and 6 (7%) had 2-vessel and 1 patient 3-vessel restenosis. Restenosis per vessel was 23% (41 of 177). Reference diameter, past-PTCA minimal luminal diameter, and length of the stent were independent predictors of restenosis. We conclude that multivessel stenting provides good midterm results in selected patients with multivessel coronary artery disease. Midterm events are less frequent than previously reported after balloon PTCA.
Ozone exposure induces increased production of free radicals which may result in oxidative stress. The objectives of this study were to determine the antioxidant effects of vitamin E on memory deficits and lipid peroxidation due to oxidative stress caused by acute ozone exposure. Rats were exposed to 0.7 p.p.m. ozone for 4 h and 50 mg/kg vitamin E was administered either before or after exposure. Experiment 1 evaluated alterations in short-term and long-term memory in a passive avoidance task. Experiment 2 quantified lipid peroxidation levels of the striatum, hippocampus and frontal cortex. Vitamin E administered before or after ozone exposure blocked memory deterioration and increases in lipid peroxidation levels associated with oxidative stress.
Some T lymphocytes express the CD94 Ag, which is known to form heterodimers with members of the NKG2 family. We have studied the expression pattern and function of CD94 heterodimers in different alphabeta or gammadelta T cell clones. Most of the CD94+NKG2A- T cells have a low to intermediate expression of CD94 Ag. The cross-linking of the CD94/NKG2 heterodimer in one of these CD8 alphabeta CD94+NKG2A- T cell clones (K14B06) was able to: 1) increase the intracellular concentration of Ca2+, 2) induce the up-regulation of CD25 Ag expression and the secretion of IFN-gamma, and 3) trigger redirected cytotoxicity in a TCR-independent manner. This activatory property was not shared by any other costimulatory molecule expressed by the K14B06 T cell clone, including CD8, CD28, CD45, CD69, or CD2 Ags. The immunoprecipitation of CD94 heterodimer showed a 39-kDa band with a similar m.w. to the activatory heterodimer found on some NK clones. A novel form of the NKG2 family (NKG2H) was identified in K14B06. NKG2H protein represents an alternative spliced form of the NKG2E gene, displaying a charged residue in the transmembrane portion and a cytoplasmic tail that lacks immunoreceptor tyrosine-based inhibitory motifs. The expression of NKG2H in the cell membrane through its association to CD94 and DAP-12 molecules supports that it could form part of the activatory CD94/Kp39 heterodimer present on K14B06 cells.
The signals and the molecular machinery mediating release of dense matrix granules from pathogenic protozoan parasites are unknown. We compared the secretion of the endogenous dense granule marker GRA3 in Toxoplasma gondii with the release of a stably transfected foreign reporter, beta-lactamase, that localizes to parasite dense granules. Both proteins were released constitutively in a calcium-independent fashion, as shown using both intact and streptolysin O-permeabilized parasites. N-Ethylmaleimide and recombinant bovine Rab-guanine dissociation inhibitor inhibited beta-lactamase secretion in permeabilized parasites, whereas recombinant hamster N-ethylmaleimide-sensitive fusion protein and bovine alpha-SNAP augmented release. Guanosine 5'-3-O-(thio)triphosphate, but not cAMP, augmented secretion in the presence but not in the absence of ATP. The T. gondii NSF/SNAP/SNARE/Rab machinery participates in dense granule release using parasite protein components that can interact functionally with their mammalian homologues.
Dendritic cells (DCs) express several receptors for the Fc portion of immunoglobulin (Ig)G (FcgammaR), which mediate internalization of antigen-IgG complexes (immune complexes, ICs) and promote efficient major histocompatibility complex (MHC) class II-restricted antigen presentation. We now show that FcgammaRs have two additional specific attributes in murine DCs: the induction of DC maturation and the promotion of efficient MHC class I-restricted presentation of peptides from exogenous, IgG-complexed antigens. Both FcgammaR functions require the FcgammaR-associated gamma chain. FcgammaR-mediated MHC class I-restricted antigen presentation is extremely sensitive and specific to immature DCs. It requires proteasomal degradation and is dependent on functional peptide transporter associated with antigen processing, TAP1-TAP2. By promoting DC maturation and presentation on both MHC class I and II molecules, ICs should efficiently sensitize DCs for priming of both CD4(+) helper and CD8(+) cytotoxic T lymphocytes in vivo.
BACKGROUND: Few studies have dealt with the behavior of the corrected (QTc) and uncorrected QT intervals during exercise and recovery. HYPOTHESIS: Based on previously published dynamics of the QT interval during treadmill testing, this study attempted to reevaluate the computer-proposed underlying mechanisms of these dynamics and to determine whether the so-called memory phenomenon could be operative in some subjects without evidence of structural heart disease. METHODS: This study included 42 unmedicated healthcare volunteers, 23 men and 19 women aged between 20 and 42 (mean 31.7) years. All had normal physical examinations, x-rays, and transthoracic echocardiograms. The electrocardiograms were also normal with 12-lead QT interval dispersions of < 90 ms. RESULTS: During exercise and recovery, the behavior of the QT intervals permitted the categorization into two groups. In Group 1 (31/42; 73.8% of subjects) the uncorrected QT interval showed a biphasic pattern consisting of a gradual decrease during incremental exercise followed by a gradual increase during recovery. In contrast, the QTc interval had a triphasic pattern resulting from a slight increase during the early phase of exercise, a gradual decrease at the highest rates, and a final increase during recovery as the rate slowed to control values. In Group 2 (11/42; 36.2% of subjects) the behavior was considered as paradoxical since the uncorrected QT interval displayed in a triphasic pattern whereas the QTc interval yielded a tetraphasic pattern due to the fact that both showed a second decrease during recovery while the rate was decreasing. CONCLUSIONS: Analysis of dynamics behavior of the QTc and the uncorrected QT intervals during exercise showed that some normal subjects had a paradoxical behavior which, because of its temporal relation to the phases of exercise, could be an expression of the so-called memory phenomenon.
The effect of N-ethyl-maleimide (NEM) on delta5- and delta6-desaturase activities and the incorporation of substrates and products into different microsomal lipid classes and phospholipid (PL) subclasses were studied in human fetal liver microsomes, obtained after legally approved therapeutic abortion. Desaturase activities were measured by a radiochemical method using reversed-phase high-performance liquid chromatography (HPLC). After nonphospholipid (NPL) and PL separation on silica cartridges, the radioactivity in different lipids of the NPL group was assessed by two-dimensional thin-layer chromatography, and their fatty acid (FA) composition by gas-liquid chromatography. The PL subclasses were separated, and the distribution of radioactivity between products and substrates was determined in PL subclasses. NEM inhibited the delta5- and delta6-desaturase activities in the n-6 series of FA but not the delta6-desaturase activity in the n-3 series, which suggests the existence of two distinct delta6-desaturases, one for the n-6 series and another for the n-3 series. Whether NEM was present or absent, most of the radioactivity was recovered in the free FA form (about 80%). The desaturation products, obtained in the presence or absence of NEM, were preferentially incorporated into PL, suggesting a channeling of the newly synthesized FA toward microsomal PL. The comparison of the distribution of substrates and products incorporated into the different PL classes showed that most of the labeled FA were incorporated into phosphatidylcholine and to a lesser degree into phosphatidylethanolamine.
BACKGROUND: Traumatic aortic rupture is highly lethal, and its surgical treatment is complicated by a high rate of paraplegia. METHODS: The charts of 263 patients with traumatic aortic rupture from vehicular accidents treated between 1971 and 1998 were reviewed. Patients were grouped according to four periods: group 1, 1971 to 1975, (n = 31); group 2, 1976 to 1985, (n = 83); group 3, 1986 to 1994, (n = 82); and group 4, 1994 to 1998 (n = 67). Seventy-one patients died of exsanguination before definitive care. One hundred-ninety two patients had surgical repair with the following techniques: clamp and sew, 6 in group 1, 22 in group 2, 54 in group 3, none in group 4; shunt, 23 in group 1, 39 in group 2, 2 in group 3; cardiopulmonary bypass, 2 in group 1, 1 in group 3. Forty-three patients had partial bypass with the centrifugal pump and heparin-coated circuits in group 4. RESULTS: Operative mortality was 6 of 31 (19%) in group 1, 22 of 61 (36%) in group 2, 15 of 57 (26%) in group 3, and 7 of 43 (16%) in group 4. There was one case of paraplegia in group 1 (4%), ten in group 2 (18%), 11 in group 3 (26%), and none in group 4. This difference of paraplegia between the groups was significant (p<0.002). Significant factors for paraplegia were intraoperative hypotension (p<0.000002), cross-clamp time longer than 30 minutes (p<0.008), pump versus no pump (p<0.008), and younger age group (28+/-11 versus 39+/-17 years) (p<0.03). CONCLUSIONS: There were no statistically significant improvements in mortality rate over the four periods, although, the mortality rate was lowest in the last period when partial bypass with the centrifugal pump was used exclusively. Further, the use of the centrifugal pump with heparin-coated circuits, with femoral vein cannulation into the right atrium and distal aortic perfusion, reduced paraplegia significantly.
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OBJECTIVE: To compare the degree of absorption and the effect on uterine contractility of the prostaglandin E1 analogue misoprostol after vaginal and oral administration. METHODS: Thirty women with a normal intrauterine pregnancy between 8 and 11 weeks' gestation who requested termination of pregnancy were given either 0.2 mg (orally n = 5; vaginally n = 6) or 0.4 mg (orally n = 10; vaginally n = 9) of misoprostol. Intrauterine pressure was recorded using a Grass polygraph connected to a pressure transducer 30 minutes before misoprostol was given and for 4 hours thereafter. At the end of the recording, suction curettage was performed. Blood samples were obtained at 0, 0.5, 1, 2, 4, and 6 hours for measurement of misoprostol, which was assayed by high-pressure liquid chromatography-mass spectrometry. RESULTS: In all patients, the first effect was an increase in uterine tonus. After 0.4 mg of misoprostol administered orally, uterine tonus started to increase after a mean (+/- standard deviation) time of 7.8+/-3.0 minutes and reached its maximum after 25.5+/-5.0 minutes. The corresponding times after vaginal administration were 20.9+/-5.3 minutes and 46.3+/-20.7 minutes, respectively. The initial increase in tonus was also more pronounced after oral than after vaginal administration. After vaginal administration, all patients developed uterine contractions; the activity, measured in Montevideo units, increased continuously during the observation period. This was not the case after oral administration. Plasma levels of misoprostol were measured in 18 patients. The highest levels were found 30 minutes after oral treatment and 1-2 hours after vaginal administration. CONCLUSION: The long-lasting and continuously increasing uterine contractility after vaginal administration can be explained only in part by a direct effect of misoprostol. The longer period of elevated plasma levels of misoprostol may also have initiated the prolonged events leading to increased uterine contractility.
Despite its proven clinical application for protection-preservation of the brain and heart during cardiac surgery, hypothermia research has fallen in and out of favor many times since its inception. Since the 1980s, there has been renewed research and clinical interest in therapeutic hypothermia for resuscitation of the brain after cardiac arrest or TBI and for preservation-resuscitation of extracerebral organs, particularly the abdominal viscera in low-flow states such as HS. Although some of the fears regarding the side effects of hypothermia are warranted, others are not. Without further laboratory and clinical studies, the significance of these effects cannot be determined and ways to overcome these problems cannot be developed. Currently, at the turn of the century, there are significant data demonstrating the benefit of mild-to-moderate hypothermia in animals and humans after cardiac arrest or TBI and in animals during and after HS. The clinical implications of uncontrolled versus controlled hypothermia in trauma patients and the best way to assure poikilothermia for cooling without shivering are still unclear. It is time to consider a prospective trial of therapeutic, controlled hypothermia for patients during traumatic HS and resuscitation. The authors believe that the new millennium will witness remarkable advantages of the use of controlled hypothermia in trauma. Starting in the prehospital phase, mild hypothermia will be induced in hypovolemic patients, which will not only decrease the immediate mortality rate but perhaps also will protect cells and reduce the likelihood of secondary inflammatory response syndrome, multiple organ failure, and late deaths. The most futuristic applications will be hypothermic strategies to achieve prolonged suspended animation for delayed resuscitation in traumatic exsanguination cardiac arrest.
In all its forms and applications, sonography plays a significant role in the management of injured patients, from the emergency department to beyond hospital discharge. The use of new and existing sonographic technology will increase because sonographic imaging and measurements are generally less invasive; are inexpensive; use no ionizing radiation; and are portable, repeatable, and, in many instances, as accurate as the so-called "contemporary gold standards." The training and credentialing of physicians in sonography is in evolution and will be an increasingly important issue with more widespread use and broader applications. The future of sonography in trauma care in the next millennium is bright, and surgeons and surgical residents are encouraged to gain proficiency and learn about this new surgical frontier as it evolves.
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