Search PubMed⌕ Search

Biomedical subjects

A Sala

Publications and source records attributed to A Sala.

At least 91 records · Page 5Linked to original sources

14,15-Dehydroleukotriene A4: a specific substrate for leukotriene C4 synthase.

We studied the metabolism of 14,15-dehydro-leukotriene A4 (14, 15-dehydro-LTA4) by human platelet leukotriene C4 (LTC4) synthase and polymorphonuclear leucocyte (PMNL) leukotriene A4 (LTA4) hydrolase. Metabolites were separated and identified using reversed-phase HPLC coupled to diode-array UV detection. Human platelets metabolize 14,15-dehydro-LTA4 to 14,15-dehydro-LTC4 with apparent kinetics identical with authentic LTA4. Metabolism to 14, 15-dehydro-LTC4 is inhibited by MK-886, a reported LTC4 synthase inhibitor in human platelets, with a potency comparable with that shown by LTA4. In contrast, neither human red-blood-cell lysates nor human PMNL enzymically convert 14,15-dehydro-LTA4 into 14, 15-dehydro-leukotriene B4. Minor amounts of 14,15-dehydro-LTC4, observed in some PMNL preparations, result from variable eosinophil contamination, as confirmed using highly purified neutrophil and eosinophil-enriched preparations. In addition, 14,15-dehydro-LTA4 irreversibly inhibits PMNL LTA4 hydrolase with an IC50 of 0.73 microM. The geometry of the methyl terminus of LTA4 does not influence the metabolism by human platelet LTC4 synthase. The double bond at C-14,15 is essential for the catalytic activity of LTA4 hydrolase but not for binding to this enzyme.

Binding Sites↗

Comparative analysis of isolated human bronchi contraction and biosynthesis of cysteinyl leukotrienes using a direct 5-lipoxygenase inhibitor.

Quantitation of cysteinyl leukotriene production and smooth muscle contraction upon immunological challenge of isolated human bronchi was evaluated. Analysis of picomole amounts of leukotriene C4, D4, and E4 was achieved using HPLC separation and enzyme immunoassay quantitative determination. The aim of the study was to correlate the contraction of airway smooth muscle and cysteinyl leukotriene production with and without 5-lipoxygenase inhibition. In human isolated bronchial tissue treated with indomethacin and pyrilamine to make their contractile responses leukotriene dependent only, the novel 5-lipoxygenase inhibitor 5,6-Dihydroxy-2-(N,N-Dimethylhydrazino)-1,2,3,4-tetrahydro-naph talene bromide (CHF 1909) caused a concentration-dependent inhibition of the immunologically induced contraction, showing an IC50 value of 13 +/- 2.2 microM (mean +/- CV). At the concentration of 30 microM, this compound caused more than 90% inhibition of the maximal bronchoconstriction in vitro, and inhibited cysteinyl leukotriene production by 90% as well. Contemporary measurement of immunologically induced contraction and production of cysteinyl leukotrienes in isolated human bronchi provided a direct correlation between smooth muscle contraction and synthesis of leukotriene C4, D4, and E4.

Bronchi↗

Inhibition of leukocyte leukotriene B4 production by an olive oil-derived phenol identified by mass-spectrometry.

We have evaluated the effects of hydroxytyrosol (HT), a potent antioxidant present in olive oil, on the formation of arachidonic acid 5-lipoxygenase metabolites by leukocytes in vitro. HT, a simple phenolic compound, extracted from first-pressure oil, was isolated by HPLC and characterized by gas chromatography/mass spectrometry. HT inhibited in a dose-related manner the production of leukotriene B4 (LTB4) by calcium ionophore-stimulated leukocytes. As expected, similar inhibition was observed for omega-oxidized metabolites of LTB4, namely 20-hydroxy and 20-carboxy-LTB4. The results disclose a new biological activity of olive oil-derived phenols on leukocyte eicosanoid production.

Antioxidants↗

Pharmacological modulation of human platelet leukotriene C4-synthase.

The aim of this study was to test if human platelet leukotriene C4-synthase (LTC4-S) is pharmacologically different from cloned and expressed LTC4-S and, in light of the significant homologies between 5-lipoxygenase activating protein (FLAP) and LTC4-S, if different potencies of leukotriene synthesis inhibitors acting through binding with FLAP (FLAP inhibitors) reflect in different potencies as LTC4-S inhibitors. Leukotriene C4 (LTC4) synthesis by washed human platelets supplemented with synthetic leukotriene A4 (LTA4) was studied in the absence and presence of two different, structurally unrelated FLAP inhibitors (MK-886 and BAY-X1005) as well as a direct 5-lipoxygenase inhibitor (zileuton). LTC4 production was analyzed by RP-HPLC coupled to diode array detection. We report that human platelet LTC4-S was inhibited by MK-886 and BAY-X1005 (IC50 of 4.7 microM and 91.2 microM, respectively), but not by zileuton (inactive up to 300 microM); all 3 compounds were able to inhibit 5-lipoxygenase metabolite biosynthesis in intact human polymorphonuclear leukocytes (IC50 of 0.044 microM, 0.85 microM, and 1.5 microM, respectively). Platelet LTC4-S does not appear pharmacologically different from expression cloned LTC4-S. LTC4-S inhibition by FLAP inhibitors is in agreement with the significant homology reported for expression-cloned LTC4-S with FLAP, Furthermore, functional homology of the binding sites for inhibitors on LTC4-S and FLAP is suggested by the conservation of the relative potencies of MK-886 and BAY-X1005 vs FLAP-dependent 5-lipoxygenase activity and LTC4-S inhibition: MK-886 was 19.3-fold more potent than BAY-X1005 as FLAP inhibitor and 19.6-fold more potent than BAY-X1005 as LTC4-S inhibitor.

Blood Platelets↗

Activation of human B-MYB by cyclins.

B-MYB expression is associated with cell proliferation and recent studies have suggested that it promotes the S phase of mammalian cells. Based on its homology to the transcription factors c-MYB and A-MYB, B-MYB is thought to be involved in transcriptional regulation; however, its activity is not detectable in several cell lines. It was postulated that B-MYB function may depend on the presence of a cofactor, and recent studies suggested that B-MYB is phosphorylated specifically during S phase in murine fibroblasts. In this report we provide evidence that the product of the human B-myb gene can be activated in vivo by coexpression with cyclin A or cyclin E. Transfection studies showed that B-MYB was a weak transcriptional activator in SAOS-2 cells and was unable to promote their proliferation. In contrast, overexpression of both B-MYB and cyclin A or cyclin E caused a drastic increase in the number of SAOS-2 cells in S phase. Also, overexpression of cyclin A and cyclin E in SAOS-2 cells enhanced the ability of B-MYB, but not c-MYB, to transactivate various promoters, including the cdc2 promoter, the HIV-1-LTR, and the simian virus 40 minimal promoter. A direct role for cyclin-dependent activation of B-MYB was demonstrated using an in vitro transcription assay. These observations suggest that one mechanism by which cyclin A and E may promote the S phase is through modification and activation of B-MYB.

Cell Cycle↗

Endothelial cells as target for antiphospholipid antibodies. Human polyclonal and monoclonal anti-beta 2-glycoprotein I antibodies react in vitro with endothelial cells through adherent beta 2-glycoprotein I and induce endothelial activation.

OBJECTIVE: To investigate the ability of human anti-beta 2-glycoprotein I (anti-beta 2 GPI) antibodies to recognize the cofactor adherent on endothelial cells (EC) and to modulate endothelial functions. METHODS: Six human affinity-purified polyclonal anti-beta 2 GPI IgG and 2 IgM monoclonal antibodies (MAb) were obtained from patients with the antiphospholipid syndrome. The antibodies were tested for their ability to 1) bind to endothelial monolayers through the adherent beta 2 GPI and 2) modulate endothelial adhesion molecule expression and interleukin-6 (IL-6) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) secretion. RESULTS: The affinity-purified IgG and the MAb with anti-beta 2 GPI activity, but not the respective controls, displayed EC binding, which declined on cells incubated in serum-free medium and was restored in a dose-dependent manner by exogenous human beta 2 GPI. After EC binding, both polyclonal and monoclonal antibodies up-regulated adhesion molecule expression. Anti-beta 2 GPI MAb also significantly increased IL-6 and 6-keto-PGF1 alpha secretion. CONCLUSION: These findings support the hypothesis that anti-beta 2 GPI antibodies bind and activate EC through the adherent cofactor beta 2 GPI, likely leading to a procoagulant state.

6-Ketoprostaglandin F1 alpha↗

Additional approaches to DNA typing of skeletal remains: the search for "missing" persons killed during the last dictatorship in Argentina.

DNA typing techniques are among the most advanced tools for human identification and can contribute to the identification of poorly preserved skeletal remains. Ten thousand people are thought to have been killed during the last dictatorship in Argentina (1976-1983) and there are few official records on the identity of the victims or the location of burials. A mass grave containing 340 skeletons was excavated using archeological methods. A small number of individuals was identified by traditional forensic methods and one family group by mitochondrial DNA (mtDNA) analysis. Due to the lack of antemortem physical information on many of the victims, the application of molecular methods is imperative to speed up the identification process. We have tested two molecular screening methods, Y chromosome-specific short tandem repeats (DYS19, DYS385, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS393) and amplification of autosomal microsatellites using nested primers. These methods can complement solely matrilineal mtDNA sequence data in the identification of "missing" persons.

Argentina↗

Effects of warm ischemia on valve endothelium.

BACKGROUND: This study investigates the time-dependent resistance of the endothelium of porcine aortic and pulmonary valves to different periods of warm ischemia (WIT). METHODS: Twenty-five 9-month-old swine were divided after death into five groups of WIT (0, 6, 12, 24, and 36 hours). Aortic and pulmonary valves were removed and a total of 15 aortic and 15 pulmonary valve specimens were obtained for each WIT interval. Valves were then examined for (1) their viability rate by the trypan blue dye exclusion method at light microscopy (percent of viability compared with 0 hours of WIT); (2) ultrastructural signs of irreversible or reversible ischemic damage by transmission electron microscopy (cell disruption, dilation of endoplasmic reticulum, cytoplasmic edema, nuclear and mitochondrial changes); (3) endothelial function by pharmacologic evaluation of both the endothelial-releasing capacity of prostacyclin and the endothelial-dependent dynamic responses to relaxing (acetylcholine from 1 x 10(-10) mol/L to 1 x 10(-4) mol/L) in aortic and pulmonary valve segments precontracted with norepinephrine (1 x 10(-6) mol/L) and contracting (NG-monomethyl-L-arginine, 1 x 10(-4) mol/L) drugs. RESULTS: Our results showed an endothelial progressive time-dependent ischemic injury, which reached significance after 12 hours of exposure. Viability and functional data indicated that 6 hours of WIT only provoked slight endothelial damage (p > 0.05 respect to time 0 hours), with signs at transmission electron microscopy consistent with a reversible injury. At 12 hours of exposure, we observed a significant reduction (p < 0.05) with respect to time 0 of the viability rate of prostacyclin production and of the endothelium-dependent dynamic responses to acetylcholine and NG-monomethyl-L-arginine. These functional impairments, although significant, were not consistent, however, with a complete loss of viability. Transmission electron microscopic observations confirmed the appearance of signs of irreversible injury; nevertheless, some elements were found to be well preserved or presented reversible damage. After 24 hours of WIT, ultrastructural and functional data were consistent with a dramatic decrease compared with controls in endothelial viability and functions (p < 0.01). Finally, after 36 hours of WIT, there was a subtotal loss of viability, of functions (p < 0.001) and, at transmission electron microscopic observations, of the endothelial layer of the valves. CONCLUSIONS: Our data show that the endothelial cells are resistant to short periods of WIT (up to 6 hours), and suggest that these cells can endure longer exposures, up to 12 hours of warm ischemia. Periods of 24 and 36 hours of WIT provoke progressive irreversible damage.

Acetylcholine↗

Surgery of descending thoracic aortic aneurysms with centrifugal pump support.

Fifty-five patients with descending thoracic aortic aneurysms were operated upon between October 1987 and October 1994. All patients were supported by a centrifugal pump during operation. The mean(s.d.) duration of cross-clamping was 39(13) min. In order to evaluate the efficacy of the centrifugal pump, haemodynamic and metabolic measurements were made on four occasions (before cross-clamping, immediately after cross-clamping and before cross-clamp removal) and again after cross-clamp removal. The haemodynamic data remained stable throughout the procedure: central venous pressure (15(4.6) versus 16(4.8) versus 16(4.6) versus 15(4.6) mmHg; P = n.s.), pulmonary artery pressure (25(6.2) versus 24(5.1) versus 22(5.3) versus 23(4.4) mmHg; P = n.s.), radial systolic pressure (119(19.9) versus 116(25.2) versus 111(25.9) versus 111(20.7) mmHg; P = n.s.) and heart rate (75(12.6) versus 77(14) versus 76(15.6) versus 78(16) beats/min; P = n.s.). The acid-base status deteriorated slowly during surgery. Values before and after cross-clamping were: pH (7.42 (0.04) versus 7.37(0.06); P < 0.05), base excess (-0.67(2.20) versus -3.70(2.50); P < 0.05) and bicarbonates (24(8.9) versus 20(1.9); P < 0.05). The cerebrospinal fluid pressure remained constant: 20(5.7) versus 19(5.9) versus 18(5) versus 19(5) mmHg; P = n.s. Renal function, measured before, and at 1, 3 and 7 days after the operation also remained stable (creatinine: 1.1(0.4) versus 1.2(0.4) versus 1.2(0.4) versus 1.2(0.4); P = n.s.; blood urea nitrogen: 46(18.7) versus 46(18.6) versus 51(24.9) versus 55(27.9); P = n.s.). Step-wise multiple linear regression comparing cerebrospinal fluid pressure against haemodynamic and metabolic data showed that during aortic cross-clamping there was a significant relationship between central venous pressure (P < 0.0013) and arterial pH (P < 0.0148), while before and after cross-clamping multivariate analysis showed a relationship only between central venous pressure and cerebrospinal fluid pressure (P < 0.0035). The results confirm that centrifugal pump support is effective in stabilizing haemodynamics and protecting the kidney during thoracoabdominal aneurysm repair.

Acid-Base Equilibrium↗

Treatment of childhood Hodgkin's disease with COPP or COPP-ABV (hybrid) without radiotherapy in Nicaragua.

BACKGROUND: Childhood Hodgkin's disease (HD) in low-income countries has been reported to have distinct presenting features, including a high prevalence of the mixed cellularity subtype, which also seems to be associated with poorer prognosis. Further investigations are needed to evaluate these issues. Another controversial aspect of childhood HD is the use of radiotherapy (RT) in its treatment and the growing concern about its serious adverse side effects. In this paper, data on the diagnosis and outcome of children treated without RT in a low-income country (Nicaragua) are reported. PATIENTS AND METHODS: Forty-eight consecutive children aged 0-15 years, diagnosed at 'La Mascota' Hospital of Managua (Nicaragua) from January 1990 to October 1995. entered this study. Follow-up was updated in May 1996. Clinical and histopathological staging was performed according to Ann Arbor and Rye criteria, respectively. Treatment consisted of COPP (six cycles) for stages I or IIA, or COPP-ABV hybrid): eight cycles for stages IIB or III, and ID cycles for stage IV. Total cumulative doses of adriamycin and bleomycin in this protocol are, respectively, 200 and 80 mg:sqm for stages II B or III and 250 and 100 mg/sqm for stage IV. RESULTS: The median age of the 48 patients at diagnosis was seven years, and the mean age was 7.9 years (range 3-15 years). Clinical stages were IA in 5, IIA in 9, IIB in 6, IIIA in 5, IIIB in 14, and IVB in 9. Histopathologically, 25 cases presented with mixed cellularity, 15 with nodular sclerosis, 5 with lymphocytic predominance and 3 with lymphocytic depletion. Four patients did not proceed with treatment and were lost to follow-up. Two patients (stages IIIB and IVB), who never achieved complete remission (CR) during treatment, presented progressive disease at the end of the scheduled chemotherapy. The remaining 42 patients were in complete remission at the end of chemotherapy. Following discontinuation of therapy, one patient (stage IA) was lost to follow-up and two patients with stage IIIB, who were in CR after the second chemotherapy cycle, relapsed 20 and 9 months following the diagnosis. EFS at three years is 100% for the 25 patients with stages I, II, IIIA and 74.9% for the 23 patients with stages IIIB or IV. CONCLUSION: The presenting features found in these patients are similar to those reported from other low-income countries. In our experience, however, the high prevalence of the mixed cellularity subtype was not associated with poorer prognosis. Satisfactory results have been achieved in patients with stages I, II or IIIA HD using COPP or COPP-ABV (hybrid) regimens without RT. The treatment was also well tolerated and can thus be recommended for these patients in low-income countries, where RT facilities may be scarce or unavailable. The use of more aggressive treatment schedules and/or RT on involved fields in front-line treatment may, however, be needed for the more advanced stages IIIB or IV. Large studies with adequate follow-up are needed to evaluate whether, if RT is omitted, higher cumulative doses of more toxic drugs are required and thus compare the long-term toxic effects of different treatment modalities.

Adolescent↗

Nitric oxide modulation of transcellular biosynthesis of cys-leukotrienes in rabbit leukocyte-perfused heart.

1. We have studied the role of nitric oxide (NO) in the regulation of the transcellular biosynthesis of sulphidopeptide leukotrienes (cys-LT) generated upon neutrophil-vascular wall interactions and their functional consequences, in the spontaneously beating, cell-perfused, heart of the rabbit. 2. Hearts were perfused under recirculating conditions (50 ml) with 5 x 10(6) purified human neutrophils (PMNL), and challenged with 0.5 microM A-23187 for 30 min. Coronary perfusion pressure (CPP) and left-ventricular end-diastolic pressure (LVEDP) were monitored. Cys-LT formation was measured by reversed phase high performance liquid chromatography (h.p.l.c.) and u.v. spectral analysis. Myeloperoxidase (MPO) enzyme activity, assayed in aliquots of the recirculating buffer, was used as a marker of PMNL, adhesion to the coronary endothelium. 3. Basal CPP and LVEDP values averaged 45 +/- 1.4 mmHg and 5 +/- 0.1 mmHg, respectively; A-23187 triggered an increase in CPP (134 +/- 9 mmHg, at 30 min) which was significantly attenuated by pretreatment with L-arginine, 100 microM (90 +/- 3 mmHg, at 30 min). Pretreatment with NG-monomethyl-L-arginine, 10 microM (L-NMMA), induced a marked increase in CPP (290 +/- 40 mmHg, at 20 min) and in LVEDP (47 +/- 16 mmHg), so pronounced that it caused cardiac arrest in systole in 5 out of 6 hearts and these were prevented by L-arginine, 100 microM, (CPP 115 +/- 10 mmHg, LVEDP 6 +/- 1.1 mmHg, at 30 min). 4. The increase in CPP was accompanied by the release of cys-LT in the circulating buffer, which was reduced significantly by L-arginine. Pretreatment with L-NMMA, caused a marked rise in cys-LT concentrations which was prevented by L-arginine. 5. Neither L-arginine nor L-NMMA affected directly the A-23187-induced arachidonic acid (AA) metabolism in isolated PMNL alone. 6. Pretreatment with L-NMMA caused a prompt drop in myeloperoxidase (MPO), activity, suggesting rapid adhesion of PMNL to the coronary wall; this effect was significantly blunted by L-arginine. 7. This study suggests that NO provides cardioprotection in an organ model of transcellular metabolism of cys-LT by preventing PMNL adhesion to the coronary intima.

Animals↗

Cyclooxygenase-2 and synthesis of PGE2 in human bronchial smooth-muscle cells.

The purpose of this study was to determine the mechanism of enhanced prostaglandin synthesis in cultured human bronchial smooth-muscle cells challenged with interleukin-1 beta (IL-1 beta). Cells were incubated with IL-1 beta (10 to 50 U/ml) for 0 to 24 h. Prostaglandin E2 (PGE2) production was evaluated through the conversion of exogenous (14C)-arachidonic acid and specific enzyme immunoassay of endogenous products. IL-1 beta enhanced PGE2 formation in a concentration- and time-dependent manner, reaching its peak at 6 to 8 h and fading at 18 to 24 h. Immunoblot analysis showed that the inducible cyclooxygenase enzyme (COX-2) was expressed only in IL-1 beta treated cells, whereas the constitutive isoform of cyclooxygenase (COX-1) remained unaltered. COX-2 expression and PGE2 formation were inhibited by dexamethasone (2 microM), cycloheximide (10 microM), and IL-1-receptor antagonist (IL-1 ra) (250 ng/ml), independently. PGE2 synthesis was significantly reduced by compound SC-58125, a specific COX-2 inhibitor. The close parallelism between the kinetics of COX-2 protein expression and PGE2 accumulation, as well as the constitutive nature of COX-1 isoform, indicate that IL-1 beta-driven PGE2 formation in human bronchial smooth-muscle cells is mediated by de novo expression of COX-2 enzyme.

Arachidonic Acid↗

Transcellular metabolism of leukotriene A4 by rabbit blood cells: lack of relevant LTC4-synthase activity in rabbit platelets.

The objective of this study was to determine the transcellular metabolism of leukotriene A4 by rabbit blood cells. mixed peripheral blood leukocyte preparations with and without platelets in a ratio of 1:40 were challenged with the Ca(2+)-ionophore A23187. 5-Lipoxygenase metabolites production was assessed by RP-HPLC coupled with diode-array UV detection. In light of the observation that leukotriene C4 production in leukocyte-platelet coincubation was the same as with leukocytes alone, mixed coincubation of human and rabbit blood cells was tested. Rabbit leukocytes with human platelets resulted in a significant increase of leukotriene C4 production, while no changes were observed in human leukocytes with or without rabbit platelets. In agreement with these results, intact rabbit platelets or rabbit platelet lysates, unlike human platelets, were not able to convert synthetic leukotriene A4 free acid to leukotriene C4. These data provide evidence that rabbit leukocytes are able to make a significant amount of leukotriene A4 available for transcellular metabolism, while rabbit platelets, unlike human platelets, lack leukotriene C4-synthase activity.

Animals↗

HPLC determination of ITF 188 and its metabolite ITF 1078 in urine after intranasal administration of new heparin salt ITF 1300 to dogs.

Heparin salt ITF 1300 in which low molecular weight heparin is salified with a new counterion, di-[3-(N,N-dibutylamino)]propyl carbonate (ITF 188), was selected for the pharmacological development. A specific, sensitive and reproducible HPLC method for the determination of ITF 188 and its alcoholic metabolite ITF 1078 in urine was developed. The method was employed for the study of urinary excretion of the counterion after intranasal administration of ITF 1300 to dogs. The total amount of ITF 188 and ITF 1078 found in urine within 72 hours after the nasal instillation of ITF 1300 accounts for about 5% of the administered dose.

Administration, Intranasal↗

[Cellular viability and immune response in homologous cryopreserved cardiac valves].

BACKGROUND: Long-term durability of homograft valves is related to cellular viability and immunological compatibility. In this paper, we carry out a critical update of the relationship between cellular viability and immunology of cryopreserved homograft valves. BIOLOGICAL ISSUE: In the first section, we review the theoretical background of the superior durability of cryopreserved valves with high cellular viability, with respect to the "fresh" antibiotic-stored valves, with lower viability rates. Afterwards, a brief review of the principal factors influencing the homograft valve viability rate during harvesting, preparation, sterilization and cryopreservation phases, is performed. CELLULAR VIABILITY: In the second paragraph, we analyze the problem of cellular viability in cryopreserved valves, both for matrix cells--fibroblast--and for endothelium. In particular, we report the current methods of quantifications of viability and more recent biological researches. Correlations between biological and clinical data are also discussed. IMMUNOLOGICAL ISSUE: Finally, the immunological issue and its relationships with cellular viability is analyzed, with particular regard to experimental evidence and clinical implications. Moreover, we have described the recent hypothesis on the influence of cryopreservation on the antigenicity of tissues, and the laboratory researches on the long-term antibody response in humans after homograft valve implantation.

Cell Survival↗

Simultaneous determination of ITF 296 and two metabolites in plasma by a validated HPLC method.

A simple and sensitive HPLC validated method was developed for the simultaneous determination of ITF 296 (a new orally active nitrate) and its metabolites ITF 1124 and ITF 1577 in biological samples. Quantitation was performed by peak height ratios between the three compounds and the internal standard (ITF 1721). Detection limit of the method was 0.005 microgram/ml using 1 ml of plasma. The results indicated that the method is reproducible, accurate, precise, sensitive and specific for the measurement of the three compounds in plasma samples. Those characteristics allowed us to conclude that the method is suitable for analysing samples obtained after administration of ITF 296 to humans at therapeutic dosage and to animals for formulation studies.

Animals↗