Biomedical subjects

F Sobrino

Publications and source records attributed to F Sobrino.

At least 19 recordsLinked to original sources

PubMed1997

Inhibitory effect of albumin-derived advanced glycosylation products on PMA-induced superoxide anion production by rat macrophages.

Advanced glycosylation end products (AGE) are implicated in many of the complications of diabetes. In the same way, infectious diseases are frequently associated with this disease. An impaired respiratory burst in macrophages may be a cause of infectious complications in diabetic patients. To establish a possible mechanism of this altered cell function, we have analyzed the effect of AGE-modified proteins on PMA-dependent superoxide anion production (O2.-) from normal rat peritoneal macrophages. We have used AGE-modified bovine serum albumin (AGE-BSA) prepared by incubation with glucose. AGE-BSA partially inhibits the phorbol ester-dependent superoxide production by macrophages in vitro. The specificity of this inhibitory effect is demonstrated by the fact that aminoguanidine, an inhibitor of the formation of AGE products, fully prevents the effect of AGE-BSA in vitro. Macrophages from diabetic rats shown an inhibition on PMA dependent-O2.- production. However, the treatment in vivo with aminoguanidine produced a cancelation of the inhibitory effect observed in the diabetic state. These data suggest that AGE-modified proteins could be implicated in the impairment of macrophage respiratory burst in diabetes.

Animals↗

PubMed1997

Nicotinamide inhibits inducible nitric oxide synthase enzyme activity in macrophages by allowing nitric oxide to inhibit its own formation.

Nitric oxide (NO) production by macrophages is mainly regulated by induction of nitric oxide synthase (iNOS) by cytokines and microbial products. Nicotinamide (NIC) inhibits NO production by activated macrophages in a dose dependent manner. NIC also inhibits NOS enzyme activity in extracts from activated macrophages. The inhibition was noncompetitive with L-arginine (Ki 13.37 +/- 4.40 mM, n=3), uncompetitive versus NADPH (Ki 3.06 +/- 0.17 mM, n=3) and tetrahydrobiopterin. Finally, the inhibition by nicotinamide was fully reversed by scavenging NO with hemoglobin. We suggest that NIC acts by allowing NO to inhibit its own formation.

Animals↗

PubMed1997

A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants.

A large-scale vaccination experiment involving a total of 138 cattle was carried out to evaluate the potential of synthetic peptides as vaccines against foot-and-mouth disease. Four types of peptides representing sequences of foot-and-mouth disease virus (FMDV) C3 Argentina 85 were tested: A, which includes the G-H loop of capsid protein VP1 (site A); AT, in which a T-cell epitope has been added to site A; AC, composed of site A and the carboxy-terminal region of VP1 (site C); and ACT, in which the three previous capsid motifs are colinearly represented. Induction of neutralizing antibodies, lymphoproliferation in response to viral antigens, and protection against challenge with homologous infectious virus were examined. None of the tested peptides, at several doses and vaccination schedules, afforded protection above 40%. Protection showed limited correlation with serum neutralization activity and lymphoproliferation in response to whole virus. In 12 of 29 lesions from vaccinated cattle that were challenged with homologous virus, mutant FMDVs with amino acid substitutions at antigenic site A were identified. This finding suggests the rapid generation and selection of FMDV antigenic variants in vivo. In contrast with previous studies, this large-scale vaccination experiment with an important FMDV host reveals considerable difficulties for vaccines based on synthetic peptides to achieve the required levels of efficacy. Possible modifications of the vaccine formulations to increase protective activity are discussed.

Amino Acid Sequence↗

PubMed1996

Enhancement of Antigen-specific functional responses by neutrophils from allergic patients.

It has been demonstrated that neutrophils from healthy donors or from patients with inflammatory disorders can bind immunoglobulin (Ig) E proteins through binding to Mac-2/epsilon bp. Functional responses to allergens were assessed by measuring the respiratory burst and intracellular Ca2+ levels, and binding of allergens to neutrophils was assessed by flow cytometry analysis and fluorescence microscopy. In this article, we demonstrate that neutrophils sensitized to specific allergens (from allergic patients), but not from healthy donors, are sensitive to allergens of the same type as those that produce clinical allergic symptoms. The activation of neutrophils was analyzed by the induction of a respiratory burst that was detected with luminol-dependent chemiluminescence. Intracellular Ca2+ levels increased parallel to those of the inducing allergens. In addition, the specific binding of allergens on the cell surface was revealed by flow cytometry and allergen-FITC-labeled staining analyses. The present data suggest a restricted recognition of allergen by sensitive neutrophils, probably associated with the specific binding of the allergen to its corresponding IgE molecule, which is bound to the Mac-2/epsilon bp structure. These findings demonstrate a functional role of allergen-associated neutrophils during the allergic state.

Allergens↗

PubMed1996

Nitric oxide and superoxide anion production decrease with age in resident and activated rat peritoneal macrophages.

Superoxide anion and nitric oxide production have been studied in resident and activated peritoneal macrophages of 3-, 12-, and 24-month-old rats. Some key enzymes involved in the metabolism of glucose were also studied in relation to aging. Production of O2 and NO was reduced in all cases in middle-aged (12 months) and old (24 months) animals. Malic enzyme and citrate synthase activity shows a progressive reduction with age. Hexoquinase, pyruvate quinase, and lactate dehydrogenase activities decrease sharply from 3 to 12 months with no significant change between 12 and 24 months. Taken as a whole, the results of enzyme activity suggest that aging may reduce the capacity for glucose utilization in macrophages.

Aging↗

PubMed1996

Anti-CD69 antibodies enhance phorbol-dependent glucose metabolism and Ca2+ levels in human thymocytes. Antagonist effect of cyclosporin A.

The human activation antigen CD69 is an early inducible surface glycoprotein acquired by T cells in the thymus at the stage of positive selection and during activation of mature lymphoid cells both in vivo and in vitro. We have studied the regulatory influence of CD69 activation pathway on the glycolytic process and transduction signals of thymocytes. Treatment of human thymocytes with different anti-CD69 monoclonal antibodies (mAbs), in the presence of submitogenic doses of phorbol ester, produced an enhanced release of lactate without significant alterations in Fru 2,6-P2 levels or phosphofructokinase-2 (PFK-2) and pyruvate kinase activities. A small increase in phosphofructokinase-1 (PFK-1) activity was also detected. Furthermore, anti-CD69 mAb increased the glucose detritiation from [2-3H] and [3-3H]glucose, thus indicating an enhanced flux through hexokinase and PFK-1 steps. In addition, de novo synthesis of diacylglycerol and intracellular Ca2+ levels increased after anti-CD69 mAb treatment. The stimulatory effects of anti-CD69 mAb on both glycolysis and Ca2+ levels were inhibited by cyclosporin A. Because CD69 molecules are present in certain subset populations of immature thymocytes, the ability of anti-CD69 mAb to stimulate the glycolysis, the synthesis of diacylglycerol and the intracellular Ca2+ levels suggest that the activation signals delivered through CD69 molecules could play a role in the thymus cells maturation.

Antibodies, Monoclonal↗

PubMed1996

Streptozotocin-induced diabetes increases fructose 2,6-biphosphate levels and glucose metabolism in thymus lymphocytes.

Acute effect of streptozotocin-induced diabetes on several parameters of glucose metabolism was investigated in thymus lymphocytes (thymocytes). The cells from diabetics rats accumulated in vitro about 2-fold more fructose 2,6-bisphosphate (Fru-2, 6-P2) in the presence of increasing glucose concentration than cells from normal rats. An increased production of lactate was also observed. Phosphofructokinase-1 (PFK-1) and phosphofructokinase-2 (PFK-2) activities were enhanced in cells from diabetic rats compared with those from normal rats. [U-14C]glucose incorporation into glycogen was also increased in cells from diabetic rats and the 14CO2 liberation was lesser than in cells from normal animals. From these data it may be concluded that the response of thymocytes to streptozotocin-induced diabetes is similar to that observed in other extrahepatic tissues.

Animals↗

PubMed1996

A porcine CD8+ T cell clone with heterotypic specificity for foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV)-specific T cell lines and clones have been obtained from a swine lymphocyte antigen (SLA) inbred miniature pig vaccinated with chemically inactivated virus. One of the clones obtained, CE3, showed a specific and heterotypic proliferation against infectious but not inactivated FMDV in the presence of syngeneic peripheral blood mononuclear cells (PBMC). Adherent cells from PBMC were sufficient to support specific activation of the clone and the proliferation was abolished when the contact between CE3 and adherent cells was prevented. Phenotypic characterization of CE3 cells revealed expression of CD2, CD25 (interleukin-2 receptor), SLA class I and SLA class II. Furthermore, the cells were highly CD8 positive but showed low expression of CD4. The expression of T cell receptor (TCR) alpha and beta genes confirmed their T cell nature. Consistent with the CD8 phenotype, the proliferative response of CE3 was inhibited with MAbs to SLA class I and CD8. Altogether, these results indicate that CE3 is a porcine SLA class I-restricted CD8+ T cell clone, that recognizes a heterotypic FMDV antigen.

Animals↗

PubMed1996

Protection from nicotinamide inhibition of interleukin-1 beta-induced RIN cell nitric oxide formation is associated with induction of MnSOD enzyme activity.

We have studied the long-term effects of nicotinamide (NIC) on the synthesis of NO by insulin producing cells. NIC delays the formation of nitrite by interleukin (IL)-1 beta-(IL-1, 25 U/ml)-stimulated RINm5F cells, and previous exposure of cells to IL-1 for 15 h prevents this effect. The delay is associated with a lack of cytokine-induced inducible nitric oxide synthase (iNOS) enzyme activity in cell extracts. NIC (20 mM) inhibits NO synthase (NOS) activity in extracts from cells incubated with IL-1 for 6 h and 24 h, and oxyhemoglobin counteracts this inhibition. Hence, NIC could scavenge O2- and allow NO to inhibit the enzyme. The NO donor SIN-1 inhibits in a concentration-dependent manner iNOS activity, and the effect is potentiated by NIC. In intact cells, protection from NIC is associated with IL-1-induced expression of MnSOD activity, and reversible blockade of iNOS expression with pyrrolidine dithiocarbamate counteracts the NIC effect. We conclude that O2- plays a role in preventing NO inhibition of iNOS. The loss of this action coincides with the induction of MnSOD enzyme activity. In addition, the stimulation by NIC of IL-1-induced nitrite production in pyrrolidine dithiocarbamate-treated cells is a novel action that should be considered when the drug is proposed as potential agent for the prevention of insulin-dependent diabetes mellitus.

Animals↗

PubMed1996

Respiratory burst is decreased by human hyperlipemic serum in rat peritoneal macrophages.

The effect of hyperlipemic human serum on superoxide anion (O2-) production by rat peritoneal macrophages was investigated. Phorbol myristate acetate (PMA)-stimulated O2- production was inhibited when cells were preincubated with hyperlipemic human serum. This inhibition was specifically carried out by a lipid fraction and was dependent on both cholesterol and triglyceride serum levels. This inhibitory effect was not exerted by a direct effect on NADPH-oxidase activity, nor by a putative superoxide dismutase activity present in the serum. With human neutrophils, we observed a decreased mobility of the cytosolic factor p47-phox to the membrane during the activation process, caused by hyperlipemic serum. We did not find any effect of hyperlipemic serum on NO2- production by cultured rat macrophages. These results suggest that a pathological increase of circulating plasma lipids may be associated with an impaired inflamatory capacity of macrophages.

Animals↗

PubMed1995

Decrease in free-radical production with age in rat peritoneal macrophages.

The respiratory-burst reaction has been studied in rat peritoneal macrophages of different ages (3, 12 and 24 months) using phorbol 12-myristate 13-acetate (PMA) to stimulate NADPH oxidase. Production of O2-. and H2O2 decreased with age (about 50 and 75% respectively); however, no difference in NADPH oxidase activity was found. NO. production was also reduced with age (40%). Furthermore, a progressive and significant decrease in the pentose phosphate flux was detected as a function of age in control and PMA-stimulated macrophages. The NADPH/NADP+ ratio decreased with age in control and PMA-stimulated macrophages. Glucose uptake was lower in middle-aged (12 months) and old (24 months) animals but no differences were found between these groups.

Aging↗

PubMed1995

Effect of thimerosal and other sulfhydryl reagents on calcium permeability in thymus lymphocytes.

We have studied the effects of thimerosal, a mercurial compound extensively used as a preservative, as well as other sulfhydryl reagents (e.g. p-hydroxymercurybenzoate, hydrogen peroxide, bromophenacyl bromide, and mercuric chloride) on Ca2+ homeostasis and the redox status of sulfhydryl groups in thymus lymphocytes. They all induced an increase in [Ca2+]i which was blocked with dithiothreitol, suggesting that they act via the oxidation or blockade of sulfhydryl groups. [Ca2+]i increase could be directly related to the effect of the different reagents on cellular protein sulfhydryl content. Experiments with ethidium bromide indicate that the observed rise in [Ca2+]i was not due to a non-specific increase in membrane permeability. Thimerosal differs from the other agents studied in its oxidative properties, which is probably linked to the production of a potent reductor molecule, thiosalicylic acid, which may modulate its oxidative capacity.

Animals↗

PubMed1995

A method for determining the position and size of optimal sequence regions for phylogenetic analysis.

The availability of fast and accurate sequencing procedures along with the use of PCR has led to a proliferation of studies of variability at the molecular level in populations. Nevertheless, it is often impractical to examine long genomic stretches and a large number of individuals at the same time. In order to optimize this kind of study, we suggest a heuristic procedure for detection of the shortest region whose informational content can be considered sufficient for significant phylogenetic reconstruction. The method is based on the comparison of the pairwise genetic distances obtained from a set of sequences of reference to those obtained for different windows of variable size and position by means of a simple index. We also present an approach for testing whether the informative content in the stretches selected in this way is significantly different from the corresponding content shown by the larger genomic regions used as reference. Application of this test to the analysis of the VP1 protein gene of foot-and-mouth-disease type C virus allowed us to define optimal stretches whose informative content is not significantly different from that displayed by the complete VP1 sequence. We showed that the predictions made for type C sequences are valid for type O sequences, indicating that the results of the procedure are consistent.

Animals↗

PubMed1995

In vitro synthesis of foot-and-mouth disease virus specific antibodies by porcine leukocytes.

We have characterized the in vitro secondary antibody response to FMDV of peripheral blood mononuclear cells (PBMC) from immunized pigs. The results obtained indicated that primed swine leukocytes can support an in vitro T-B cell cooperation which is functional and leads to the production of viral specific antibodies. The response was shown to be independent of viral replication, being induced by both infective and inactivated virus as well as by recombinant polypeptides VP1 and VP3. In all cases, concentration of PBMC supernatants allowed the detection of viral-specific IgG antibodies by ELISA. Significant titers of foot-and-mouth disease virus (FMDV) specific neutralizing antibodies were detected in concentrated supernatants after stimulation with either infective or inactivated whole virus, whereas no neutralizing activity was found in supernatants from PBMC responding to individual capsid polypeptides. The titers of IgG1 and IgG2 were similar for PBMC incubated with viruses, while IgG2 predominated when VP1 or VP3 were used as stimulators. In addition, significant titers of IFN-gamma were detected in supernatants of PBMC stimulated with infectious or chemically inactivated FMDV.

Animals↗

PubMed1995

Modulation of phorbol ester-induced respiratory burst by vanadate, genistein, and phenylarsine oxide in mouse macrophages.

The effect of the inhibitors of tyrosine phosphatase (vanadate and phenylarsine oxide) and of an inhibitor of tyrosine kinase (genistein) on O2.- production in mouse peritoneal macrophages was examined. Vanadate and phenylarsine oxide produced a dose-dependent inhibition of phorbol myristate acetate (PMA)-induced O2.- production, whereas genistein potentiated O2.- production triggered by phorbol ester. Vanadate had no effect on the respiratory burst in human neutrophils challenged with fMLP, in agreement with previously published data on human intact neutrophils. It did not alter reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity in membrane preparations of mouse peritoneal macrophages. These data suggest that the phosphorylation of protein(s) in tyrosine residues blocked the PMA-dependent respiratory burst in mouse macrophages.

Animals↗

PubMed1995

Inhibitory effect of cyclosporin A and FK506 on nitric oxide production by cultured macrophages. Evidence of a direct effect on nitric oxide synthase activity.

Casein-elicited peritoneal macrophages from mice were cultured either alone or with interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS), and the effect of cyclosporin A (CsA) and FK506 on NO2- production (due technical difficulties NO2- was taken as the index for NO) was analysed. We observed an inhibitory effect of CsA and FK506 on NO2- production. The IC50 for NO2- production by casein-elicited macrophages was 0.1 microgram/ml for CsA and 0.3 microgram/ml FK506. The effect of both drugs was dose-dependent and was more clear in non-stimulated macrophages. The presence of IFN-gamma and LPS in the culture increased NO2- production by casein-elicited macrophages and partially eliminated the inhibition exerted by CsA and FK506. Both drugs acted directly on the nitric oxide synthase (NOS), since CsA and FK506 reduced by 35% and by 17%, respectively, NOS activity in the crude cytosolic fraction. However, CsA and FK506 did not alter 14CO2 production from [1-14C]glucose, suggesting that the pentose monophosphate pathway activity was not modified. These data add new insight into the interpretation of the immunosuppressive properties of both drugs.

Amino Acid Oxidoreductases↗

PubMed1994

Antigenic specificity of porcine T cell response against foot-and-mouth disease virus structural proteins: identification of T helper epitopes in VP1.

The contribution of each of the viral capsid proteins of foot-and-mouth disease virus (FMDV) in the T cell response of vaccinated pigs has been studied. Viral polypeptides, VP1 to VP4, were expressed as fusion proteins in Escherichia coli, and were used to stimulate peripheral blood mononuclear cells of vaccinated animals. Significant, dose-dependent responses to whole virion were detected in the seven animals analyzed and, in five of them, responses to recombinant polypeptides VP1, VP2, and VP3 were noticed, VP4 was recognized only by one of the pigs. Among the responder animals, VP1 and VP3 induced the higher proliferative responses. The patterns of recognition of a nested set of VP3 fragments expressed as fusions in E. coli were different among the animals studied and were consistent with the presence of different T cell epitopes on the protein. Likewise, three of the four VP1 fragments induced significant responses and were differentially recognized by each of the animals tested. Partially overlapping synthetic peptides spanning VP1 amino acids 41 to 209 were used to identify T cell epitopes in this protein. The significant responses obtained in three of seven additional FMDV vaccinated outbred pigs analyzed revealed the existence of at least 11 different T cell epitopes distributed throughout the sequence studied, which were distinctly recognized by each of the responder animals. A peptide corresponding to a relevant B cell antigenic site, around amino acids 140-160, was shown to stimulate lymphocytes from two of the responder animals. Thus, the results obtained indicate that different T cell epitopes of capsid proteins VP1 and VP3 are recognized by pig populations. The different patterns of recognition of recombinant polypeptides and synthetic peptides observed among outbred animals support an important contribution of genetic restriction, probably mediated by MHC genes, to the individual T cell response in swine.

Animals↗

PubMed1994

Cyclosporin A antagonizes phenylephrine, oxytocin and angiotensin effects on glucose metabolism in rat thymus lymphocytes.

Effects of phenylephrine, oxytocin and angiotensin on fructose 2,6-bisphosphate (Fru 2,6-P2) content and glycolytic parameters were studied in incubated thymus lymphocytes. These hormones modified Fru 2,6-P2 content dependent upon the energetic status of the cells. In non-preincubated thymus lymphocytes (with relatively high levels of glycogen and ATP), phenylephrine, oxytocin and angiotensin depressed Fru 2,6-P2 content in a dose-dependent manner. The opposite was found when the cells were preincubated for 2 h without substrates (low levels of ATP and glycogen). Changes in lactate release were less evident, but significant. Phenylephrine did not modify the maximal activities of phosphofructokinase (PFK)-1 or PFK-2. However, both submaximal PFK-1 and PFK-2 activities were inhibited by phenylephrine, and the response to exogenous Fru 2,6-P2 on PFK-1 was also altered. The activities of Fru 1,6-P2 and pyruvate kinase were not modified by phenylephrine or A23187 treatment. Simultaneous presence of Cyclosporin A (CsA), an immunosuppressive drug, antagonizes the alpha-adrenergic effect on Fru 2,6-P2 content. CsA alone did not alter basal levels of ATP, hexose phosphate or Fru 2,6-P2, and its opposing effect to alpha-agonist was dose-dependent. CsA cannot change the positive action of PMA or the negative action of A23187 on Fru 2,6-P2 content. The present data suggest that CsA acts prior to calcium liberation and protein kinase C activation. Different possible molecular models are discussed.

Adenosine Triphosphate↗