Biomedical subjects

C M Riera

Publications and source records attributed to C M Riera.

At least 19 recordsLinked to original sources

PubMed2001

Modulation of arachidonic acid turnover in macrophages by cadmium.

The effects of cadmium (Cd) induced redox changes on arachidonic acid (AA) turnover in mouse resident peritoneal macrophages (pM) were studied. The pre-incubation of pM in a medium containing glutathione (GSH, 0.1 or 1 mM) for 6 h protects pM from loss of viability and AA uptake diminution induced by Cd with regard to non pre-incubated cultures. The exposure of macrophages to Cd 10 microM decreases AA uptake within 2 h and increases AA release in relation to non-exposed macrophages. It also enhances AA mobilization and reactive oxygen species (ROS) release induced by okadaic acid and opsonized zimosan and decreases those induced by lipopolysaccharide, but does not modify either AA mobilization or ROS release induced by phorbol ester. These results might suggest that redox changes induced by Cd produce an important impact on AA turnover in macrophages; information that is relevant in the understanding of the cellular toxicity of this metal.

Animals↗

PubMed2001

Identification of a CD8alpha(+) dendritic cell subpopulation in rat spleen and evaluation of its OX-62 expression.

Rat spleen DC and bone marrow-derived DC were isolated and characterized by morphology and flow cytometry. We found a CD8alpha(+) DC subpopulation representing 19-48% (27.4 +/- 12.0) of total spleen DC. The OX-62 expression on total spleen DC was 41-59% (51.8 +/- 7.5). Myeloid bone marrow-derived DC were negative for CD8alpha and OX-62. We demonstrated the coexpression of CD8alpha and OX-62 molecules, at least in a portion CD8alpha(+) spleen DC. Both CD8alpha(+) and CD8alpha(-) spleen DC subpopulations separated by MACS were able to induce an in vivo primary immune response to OVA. The immune response induced by the CD8alpha(-) DC subpopulation was higher (P < 0.05). We identified a CD8alpha(+) DC subpopulation in rat spleen less effective in inducing an immune response than CD8alpha(-) DC. Moreover, our results suggest the presence of DC subpopulations with different lineages in DC preparations based on OX-62 expression.

Animals↗

PubMed2001

Identification of rat prostatic steroid binding protein (PSBP) as an immunosuppressive factor.

Prostatic steroid binding protein (PSBP) is the major protein produced ( approximately 20% of the total cytosolic protein) and secreted into the seminal fluid by the rat ventral prostate but its physiological function has not been elucidated yet. Since PSBP is secreted into the seminal fluid (which is itself a potent immunosuppressor) and has strong homology with uteroglobin (which possess an important anti-inflammatory function) our aim was to determine what effect, if any, PSBP would have on the immune system. With that purpose in mind we performed mononuclear cell cultures in the presence or absence of purified PSBP and analysed the effect of this protein on different functional parameters. PSBP inhibits the mitogen-induced proliferation of normal rat spleen mononuclear cells (MNC) specifically and in a dose-dependent manner. It reduces the production of IL-2 and the expression of its receptor (analysed by flow cytometry) which are important events for lymphocyte proliferation. Also, PSBP was able to inhibit OVA-specific proliferation of lymph node cells from previously primed animals. The immunosuppressive effect of PSBP is not due to an inherent toxic effect to the cells, since the cell viability was kept intact at the different times of culture studied. We also analysed the effect of rat PSBP on mitogen-induced proliferation of mouse spleen and human blood MNC. The proliferation was strongly abolished in a dose-dependent and non-species specific fashion. Moreover, PSBP strongly inhibits the human mixed lymphocyte reaction. Taken together, the present data support evidence for a new type of function for PSBP. We report that PSBP is a potent immunosuppressor factor and we describe its effect on the immune function in vitro. Here, we discuss the possible implications of these findings in the protection of sperm from immunologic damage in the feminine reproductive tract.

Androgen-Binding Protein↗

PubMed2000

Evidence of a role for galectin-1 in acute inflammation.

Galectin-1 (Gal-1), a member of a family of beta-galactoside-binding proteins, has been suggested to play key roles in immunological and inflammatory processes. The present study deals with the concept of an in vivo role for Gal-1 in acute inflammation by using the rat hind paw edema test. Local administration of Gal-1 (0.5, 2, 4 and 8 microg/ml) inhibited acute inflammation induced by bee venom phospholipase A(2) (PLA(2)) when it was injected 30 min before the enzyme or co-injected together with PLA(2). The anti-inflammatory effect was prevented by a specific antibody, but independent of its carbohydrate-binding properties. In contrast, Gal-1 failed to inhibit histamine-induced edema. Histopathological studies showed a clear reduction of the inflammatory process when Gal-1 was injected before PLA(2), evidenced by a diminished number of infiltrated polymorphonuclear neutrophils and scarce degranulated mast cells. The anti-inflammatory effect was also assessed in vitro, showing that Gal-1 treatment reduced prostaglandin E(2) secretion and arachidonic acid release from stimulated peritoneal macrophages. Results presented here provide the first evidence for a role of Gal-1 in acute inflammation and suggest that the anti-inflammatory effect involves the inhibition of both soluble and cellular mediators of the inflammatory response.

Animals↗

PubMed2000

Molecular mechanisms implicated in galectin-1-induced apoptosis: activation of the AP-1 transcription factor and downregulation of Bcl-2.

Galectins are emerging as a new class of bioactive molecules with specific immunomodulatory properties. Galectin-1 (Gal-1), a member of this family, has been shown to induce apoptosis of mature T cells and immature thymocytes. To gain insight into the intracellular signals transduced by Gal-1 upon binding to mature T cells, we investigated whether this protein triggered activation of the dimeric AP-1 transcription factor. A marked increase in the binding of nuclear extracts to synthetic oligonucleotides containing the AP-1 consensus sequence, could be detected by an electrophoretic mobility shift assay, when T cells were cultured for 30 min in the presence of Gal-1. This DNA-binding activity was preceded by a rapid increase in the levels of c-Jun mRNA, as determined by Northern blot analysis. Requirement of AP-1 for Gal-1-induced apoptosis was confirmed by the dose-dependent reduction on the level of DNA fragmentation observed when cells were pre-treated with curcumin (an inhibitor of AP-1 activation) before exposure to Gal-1. Finally, evidence is also provided by Western blot analysis, showing that Gal-1 inhibits Concanavalin A (Con A) induction of Bcl-2 protein. Results presented in this study provide the first experimental evidence regarding AP-1 and Bcl-2 as targets of the signal transduction pathway triggered by Gal-1 and set the basis for a more in depth understanding of the molecular mechanisms of T-cell death regulation.

Animals↗

PubMed2000

[Induction of primary immune response by rat dendritic cells].

In this work we generated dendritic cells (DC) from rat bone marrow cultures stimulated with GM-CSF and IL-4. After 10 days of culture, we obtained numerous mature DC showing morphological characteristics of DC and high levels of MHC class II molecules. Also, we isolated DC from rat spleen on the bases of their differential adherence and low-density properties. The purity of these cells was > 90% according to morphology and MHC class II expression. To evaluate the capacity of bone marrow DC, immature spleen DC or spleen DC cultured 24h with GM-CSF (mature spleen DC), to elicit an immune response to ovalbumin (OVA), DC were loaded with this antigen and transferred to normal rats. Both bone marrow and spleen DC induced delayed type hypersensitivity responses (DTH). However, mature DC from spleen induced a stronger immune response against OVA with the highest DTH values (p < 0.05). These differences in the induction of the immune response correlated with higher expression of MHC class II molecules on mature DC.

Animals↗

PubMed1999

Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T cell apoptosis.

Galectin-1 (GAL-1), a member of a family of conserved beta-galactoside-binding proteins, has been shown to induce in vitro apoptosis of activated T cells and immature thymocytes. We assessed the therapeutic effects and mechanisms of action of delivery of GAL-1 in a collagen-induced arthritis model. A single injection of syngeneic DBA/1 fibroblasts engineered to secrete GAL-1 at the day of disease onset was able to abrogate clinical and histopathological manifestations of arthritis. This effect was reproduced by daily administration of recombinant GAL-1. GAL-1 treatment resulted in reduction in anticollagen immunoglobulin (Ig)G levels. The cytokine profile in draining lymph node cells and the anticollagen IgG isotypes in mice sera at the end of the treatment clearly showed inhibition of the proinflammatory response and skewing towards a type 2-polarized immune reaction. Lymph node cells from mice engaged in the gene therapy protocol increased their susceptibility to antigen-induced apoptosis. Moreover, GAL-1-expressing fibroblasts and recombinant GAL-1 revealed a specific dose-dependent inhibitory effect in vitro in antigen-dependent interleukin 2 production to an A(q)-restricted, collagen type 2-specific T cell hybridoma clone. Thus, a correlation between the apoptotic properties of GAL-1 in vitro and its immunomodulatory properties in vivo supports its therapeutic potential in the treatment of T helper cell type 1-mediated autoimmune disorders.

Animals↗

PubMed1999

Peritoneal antigen-presenting cells pulsed in vivo with myelin basic protein induce the suppression of experimental autoimmune encephalomyelitis (EAE) in Wistar rats.

Suppression of Experimental Autoimmune Encephalomyelitis (EAE) can be achieved by i.p. administration of soluble myelin basic protein (MBP) in adult Wistar rats before the immunization. In the present work, we analyze the role of peritoneal antigen-presenting cells (APC) in the induction of tolerance to EAE. Peritoneal cells (PC) pulsed in vivo with MBP were obtained from rats that had been intraperitoneally injected 2 h previously with soluble MBP (MBP-PC) and then inoculated in recipient rats before the induction of EAE. Our findings show that the i.p. treatment of the animals with MBP-PC before the immunization was able to diminish the incidence and severity of the disease, reduce the histological alterations, abrogate the proliferative response against MBP and change the pattern of the humoral response to MBP. Moreover, when spleen mononuclear cells (MNC) from tolerant animals were cultured together with spleen MNC from sick animals, a dose-dependent inhibition of the proliferative response was observed, arguing for the presence of a regulatory cell population in the tolerant animals. It is also demonstrated that the MBP-PC are activated and their capability of inducing suppression of EAE is highly associated with the enhanced expression of MHC class II IA molecule. Our results show that peritoneal cells pulsed in vivo with MBP are able to induce tolerance and suggest that the up-regulation of MHC class II on MBP-PC is a necessary event for tolerance induction in our model.

Animals↗

PubMed1999

Granulocyte-macrophage colony-stimulating factor protects dendritic cells from liposome-encapsulated dichloromethylene diphosphonate-induced apoptosis through a Bcl-2-mediated pathway.

Liposome-encapsulated dichloromethylene diphosphonate (L-MDP) has been used for depleting cells of the monocyte-macrophage lineage. We have undertaken this study to investigate whether dendritic cells are susceptible to this liposome-encapsulated compound. Dendritic cells were cultured in the presence of L-MDP and further processed for apoptosis detection. The highly characteristic DNA cleavage into oligonucleosome-sized fragments, incorporation of biotinylated dUTP into DNA strand breaks and the typical ultrastructural features of apoptosis were evident in dendritic cells exposed to the drug. More importantly, we demonstrated that granulocyte-macrophage colony-stimulating factor protects dendritic cells not only from apoptosis induced by the exogenous compound but also from spontaneous apoptosis. Western blot analysis revealed that this protection was tightly correlated with the activation of a Bcl-2-mediated pathway. Regulation of the apoptotic threshold of dendritic cells will be advantageous for the generation of new insights in immunotherapy.

Analgesics, Non-Narcotic↗

PubMed1999

Galectins: a key intersection between glycobiology and immunology.

Galectins are a family of evolutionarily conserved animal lectins, widely distributed from lower invertebrates to mammals. They share sequence and structure similarities in the carbohydrate recognition domain and specificity for polylactosamine-enriched glycoconjugates. In the last few years significant experimental data have been accumulated concerning their participation in different biological processes requiring carbohydrate recognition such as cell adhesion, cell growth regulation, inflammation, immunomodulation, apoptosis and metastasis. In the present review we will discuss some exciting questions and advances in galectin research, highlighting the significance of these proteins in immunological processes and their implications in biomedical research, disease diagnosis and clinical intervention. Designing novel therapeutic strategies based on carbohydrate recognition will provide answers for the treatment of autoimmune disorders, inflammatory processes, allergic reactions and tumor spreading.

Apoptosis↗

PubMed1999

Galectin-1, an alternative signal for T cell death, is increased in activated macrophages.

Galectin-1 belongs to an evolutionarily conserved family of animal beta-galactoside-binding proteins, which exert their functions by crosslinking the oligosaccharides of specific glycoconjugate ligands. During the past decade, attempts to identify the functional role of galectin-1 suggested participation in the regulation of the immune response. Only in the last few years has the molecular mechanism involved in these properties been clearly elucidated, revealing a critical role for galectin-1 as an alternative signal in the generation of T cell death. In the present study we will discuss the latest advances in galectin research in the context of the regulation of the immune response, not only at the central level but also at the periphery. Moreover, we will review the purification, biochemical properties and functional significance of a novel galectin-1-like protein from activated rat macrophages, whose expression is differentially regulated according to the activation state of the cells. The novel role of a carbohydrate-binding protein in the regulation of apoptosis is providing a breakthrough in galectin research and extending the interface between immunology, glycobiology and clinical medicine.

Animals↗

PubMed1999

Humoral response against myelin antigens in two strains of rats with different susceptibility to experimental allergic encephalomyelitis (EAE).

Lewis (Lw) rats are susceptible and Wistar (Wr) rats are usually resistant to the induction of experimental allergic encephalomyelitis (EAE). In this study we analyze the humoral response to myelin antigens, providing evidence for different B cell response to myelin basic protein (MBP) and other myelin proteins between these two strains of rats with different susceptibility to EAE. In fact, IgG anti-MBP titers in Wr rats were markedly higher than in Lw ones. Moreover, an inverse relationship between the amount of antigen injected to induced EAE and the level of anti-MBP antibodies was observed in Wr rats, while IgG anti-MBP varied in a positive dose-depending manner in sera from Lw rats. Also, sera from Wr rats analyzed by immunoblotting showed a strong reactivity with MBP and other myelin proteins, but sera from Lw rats reacted only with MBP. Evaluation of IgA and IgM against MBP in Wr rats showed again higher titers of these isotypes when compared with the titers observed in Lw rats. The distribution of IgG subclasses in sera from both strains indicated that Wr produced low titers of specific IgG1, while Lw rats did not produce specific IgG1. However, Wr rats showed high levels of IgG2a, IgG2b and IgG2c subclasses while lesser titers of these isotypes were observed in Lw animals. These findings indicate that both strains have the capacity to develop antibodies against portions of the MBP molecule, but antibody production is greater in the resistant Wistar rats suggesting a B cell activation in these animals, that could be related to their lower susceptibility.

Animals↗

PubMed1998

Activated rat macrophages produce a galectin-1-like protein that induces apoptosis of T cells: biochemical and functional characterization.

Galectins, a family of closely related beta-galactoside-binding proteins, show specific immunomodulatory properties. We have recently identified the presence of a galectin-like protein in rat peritoneal macrophages by means of a cross-reactivity with a polyclonal Ab raised against a galectin purified from adult chicken liver. Galectin expression was up-regulated in inflammatory and activated macrophages, revealing a significant increase in phorbol ester- and formylmethionine oligopeptide-treated cells. In an attempt to further explore its functional significance, rat macrophage galectin was purified from activated macrophages by a single-step affinity chromatography on a lactosyl-Sepharose matrix. The eluted fraction was resolved as a single protein band of approximately 15,000 Da by SDS-PAGE that immunoreacted strongly with the anti-chicken galectin serum. Gel filtration studies revealed that the protein behaved like a dimer under native conditions, and saccharides bearing a beta-D-galactoside configuration were able to inhibit the hemagglutinating activity displayed by the purified galectin. In agreement with its isoelectric point of approximately 4.8, the amino acid analysis showed a definitive acidic pattern. Internal amino acid sequencing of selected peptides obtained by proteolytic cleavage revealed that this carbohydrate-binding protein shares all the absolutely preserved and critical residues found in other members of the mammalian galectin-1 subfamily. Finally, biochemical and ultrastructural evidence, obtained by genomic DNA fragmentation and transmission electron microscopy, are also provided to show its potential implications in the apoptotic program of T cells. This effect was quantified by using the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling assay and was found to be associated to the specific carbohydrate-binding properties of galectin.

Amino Acid Sequence↗

PubMed1998

Chronic varied stress modulates experimental autoimmune encephalomyelitis in Wistar rats.

Stress disturbs homeostasis by altering the equilibrium of various hormones which have a significant impact on immune responses. Few studies have examined the influence of stressors on autoimmune disease in animal models. In our work, we studied the effects of long-term exposure (14 days) to chronic varied stress (CVS) in a model of experimental autoimmune encephalomyelitis (EAE) in Wistar rats. We studied whether the exposure to CVS before or after the immune challenge would correlate with differences in the clinical course of the disease. We also examined whether the CVS would modulate the magnitude of the cellular or the humoral immune response. We observed opposite effects on the clinical signs in animals stressed before or after the immune challenge. The clinical signs of the disease were attenuated in animals stressed before but not after the immune challenge. Relationships were found in the modulation of the clinical severity related to the time of exposure to the CVS, the histological alterations and the proliferative results. Stressed animals with milder clinical signs presented an exacerbated humoral response against myelin antigens while stressed animals with more severe clinical symptoms exhibited a significantly diminished one. Besides, we detected the presence of specific IgG1 associated with the exposure to CVS before the induction of EAE. Our results show that, depending on the timing of the exposure of Wistar rats to the CVS, the neuroendocrine disbalance favors a more pronounced humoral or cellular profile of the response.

Animals↗

PubMed1998

Non-obese diabetic (NOD) mice are genetically susceptible to experimental autoimmune prostatitis (EAP).

Rodents develop inflammatory, non-infectious, prostatitis upon autoimmuniz-ation with male accessory gland (MAG) extracts in complete Freund's adjuvant (CFA). Although there appears to be differences among strains, with respect to susceptibility to induction, specific details are not known about the genetic bases of such differences. Because NOD mice have inherited a genetic predisposition to autoimmune lesions affecting, apart from the islets of Langerhans, a large array of secretory glands such as salivary glands, thyroid, parathyroids and adrenal cortex, we selected this strain to assess the influence of inherited genes upon experimentally-induced autoimmune prostatitis (EAP). Indeed, MAG extracts injected into young NOD males in association with CFA cause a severe inflammatory reaction in the prostate, accompanied by a humoral and T cell-mediated response. NOD mice develop a more aggressive form of EAP than Wistar rats, the strain of reference used to establish the model. In NOD mice, disease begins earlier, affects 100% of the animals, does not require boosting and leads to florid infiltrates circumscribed to lateral and dorsal prostatic lobes. Immune mice develop a T cell-mediated response to MAG assessed by in vitro proliferation and accompanied by the release of IFN-gamma, whereas IL-4 is not detectable in the same culture super-natants. To assess the influence of the NOD background genes upon EAP susceptibility, we tested C57BL/6.H2(g7) mice in parallel. NOD mice are considerably more susceptible to EAP induction than congenic C57BL/6.H2(g7) mice. Both strains demonstrate a detectable humoral and cell-mediated response against MAG, but the histopathological manifestations are considerably more dramatic in NOD than in the C57BL/6.H2(g7) strain. Our results thus support the notion that NOD mice have background genes which favour severe autoimmune manifestations, irrespective of the target tissue.

Animals↗

PubMed1998

Prostatein (or rat prostatic steroid binding protein) is a major autoantigen in experimental autoimmune prostatitis.

Experimental autoimmune prostatitis (EAP) is a disease that could be considered an experimental model of human non-bacterial prostatitis. In this experimental model, male rats are intradermally immunized with a saline extract of male sex accessory glands (RAG) in an adequate adjuvant. The prostatitis observed in the immunized animals develops as a consequence of the immune response against RAG antigens, and the histological lesion is strikingly similar to the pattern of prostatic inflammation observed in the human disease. In this study, we purified one of the prostatic autoantigens recognized by the autoantibodies in our model. Amino acid sequence analysis identified the purified protein as prostatein or rat prostatic steroid binding protein, a member of the uteroglobin superfamily. Prostatein was recognized not only by the humoral autoimmune response, but also by the cellular autoimmune response. Certainly, the DTH response and lymph node cell proliferative assays against prostatein in immunized animals yielded positive results. Prostatein is not only the target of the autoimmune response in animals immunized with the whole extract, but also an inducing antigen of the disease. Purified prostatein, when incorporated to an adequate adjuvant, elicited cellular and humoral autoimmune response and lesion in the prostate gland. The identification of one of the target antigens in autoimmune prostatitis has provided a further refinement and characterization of our model, which could serve for a better understanding of the aetiology, pathogenesis and pathophysiology of non-bacterial prostatitis.

Androgen-Binding Protein↗

PubMed1997

Modulation of the inflammatory response by corticotropin-releasing factor.

Peptides of the corticotropin-releasing factor (CRF) family have been shown to have either pro- or anti-inflammatory activities. CRF (10-30 micrograms/kg) administered subcutaneously or intravenously could inhibit edema and dye leakage in the rat paw produced by several injuries. These findings are opposed to some results suggesting a predominantly pro-inflammatory effect of CRF mainly in arthritic processes. The purpose of this work was to identify in vivo and in vitro the conditions for the pro- or anti-inflammatory actions of CRF in order to clarify its physiological and pharmacological function. Using the rat paw edema test we observed that only the highest doses of CRF employed (5 micrograms) induced a moderate and sustained swelling. Pre-treatment with low doses of CRF (0.5-5 ng) was able to inhibit the edema induced by Naja naja phospholipase A2, carrageenin or histamine. Higher doses (50 ng-5 micrograms) had no anti-inflammatory activity. When co-inhibited with Naja naja phospholipase A2 or histamine the peptide did not modify the swelling at doses up to 500 ng, showing at 5 micrograms an additive edema with Naja naja phospholipase A2. In vitro, CRF did not modify the release of histamine but slightly increased the release of arachidonic acid to the medium. Our findings show a clear dose dependence on the local effects of CRF in inflammatory responses. These results suggest that the mechanisms of the two dose-related phenomena may be distinct.

Animals↗

PubMed1997

Involvement of peritoneal dendritic cells in the induction of autoimmune prostatitis.

We have been working within a model of autoimmune prostatitis induced by the intraperitoneal administration of saline extract of rat male accessory glands (RAG) associated to liposomes. The intraperitoneal administration of RAG-liposomes elicits both primary and secondary cellular autoimmune responses to RAG as well as organ-specific lesions. To evaluate the participation of dendritic cells (DC) in the induction of the autoimmune response, we purified peritoneal DC (PDC) after a single injection of RAG-liposomes and we characterized this population by morphology and phenotype. Based on adherence and morphologic criteria, we determined that PDC comprised approximately 1% of the total peritoneal cells. The ultrastructure of the dendritic cell enriched fraction was assessed by electron microscopy. By FACS analysis, PDC showed a two to three-fold increase in expression of the IA molecule compared to macrophages. They expressed low but positive levels of the CD14 marker, and intermediate levels of both CD11b (Mac-1) and CD54 (ICAM-1) adhesion molecules. In addition, PDC transferred either intravenously or intraperitoneally efficiently elicited the autoimmune response to RAG in normal receptors. These results support the involvement of peritoneal dendritic cells in the induction of autoimmune prostatitis, modifying the idea of macrophages as the single antigen presenting cell in the peritoneal cavity.

Animals↗