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Biomedical subjects

N Fernandez

Publications and source records attributed to N Fernandez.

At least 37 records · Page 2Linked to original sources
PubMed2000

Rapid desensitization and slow recovery of the cyclic AMP response mediated by histamine H(2) receptors in the U937 cell line.

The present study focused on the desensitization process of the H(2) receptor in U937 cells and the recovery of the cyclic AMP (cAMP) response. Treatment of U937 leukemic cells with the H(2) histamine receptor agonists (+/-)-N(1)-[3-(3, 4-difluorophenyl)-3-(pyridin-2-yl)propyl]-N(2)-[3-(1H-imidazol-4-yl)p ropyl]guanidine (BU-E-75) and amthamine produced a rapid desensitization characterized by decreased cAMP production (T(1/2) = 20 min). Pretreatment with 10 microM BU-E-75 did not induce modifications in the responses to prostaglandin E(2), isoproterenol, or forskolin. H(2) receptor desensitization was not affected by protein kinase A and C inhibitors, but was reduced drastically by Zn(2+) and heparin, known to act as inhibitors of G protein-coupled receptor kinases. Recovery studies of the cAMP response showed that cAMP levels reached 50% of the initial values within 5 hr. Furthermore, desensitization produced an important decrease in the basal level of this cyclic nucleotide. The minimal value was observed 12 hr later, and corresponded to approximately 1.3% of the initial basal level (7.5 vs 0.1 pmol/10(6) cells). This result could be explained by an increase in phosphodiesterase activity following 10 microM BU-E-75 treatment. When cells were exposed for 2 hr to an H(2) agonist, binding assays showed no modification in the number of H(2) receptors; internalization began just after 8 hr. Although the initial desensitization seems to involve G protein-coupled receptor kinases, results indicate that additional mechanisms of regulation were triggered by the H(2) agonists.

Cimetidine↗
PubMed2000

Effects of sea lice (Lepeophtheirus salmonis Kröyer, 1837) infestation on macrophage functions in Atlantic salmon (Salmo salar L.).

Experiments were conducted to determine the effects of sea lice, Lepeophtheirus salmonis, on non-specific defence mechanisms in Atlantic salmon, Salmo salar, by experimentally infesting hatchery-reared 1 and 2 year old post-smolts, S1 and S2, with laboratory grown infective copepodids at moderate to high infection intensities ranging from 15-285 lice per fish. The effects of sea lice-induced stress were investigated by measuring the blood levels of cortisol and glucose as indicators of primary and secondary stress responses, and by changes in macrophage respiratory burst activity and phagocytosis as indicators of tertiary stress responses as well as non-specific defence mechanisms. Fish were sampled prior to sea lice infestation at day 0 and at days 3, 7, 14 and 21 post-infestation. Sea lice were at copepodid stage at day 3, at chalimus stages at days 7 and 14, and at pre-adult stage at day 21. Blood levels of cortisol and glucose were found to be significantly increased at day 21 in fish-infested with the highest levels. Macrophage respiratory burst and phagocytic activities were found to be significantly decreased only at day 21. These results indicate that sea lice do not suppress host defence mechanisms during the earlier stages of infestation. They do have effects on the development of chronic stress and on the host non-specific defence mechanisms soon after the lice reach the pre-adult stage.

Age Factors↗
PubMed2000

High affinity interactions of Coxsackievirus A9 with integrin alphavbeta3 (CD51/61) require the CYDMKTTC sequence of beta3, but do not require the RGD sequence of the CAV-9 VP1 protein.

Integrins are transmembrane molecules involved in numerous cell matrix, cell-cell adhesion phenomena and also utilised as viral receptors. These interactions with integrins are mediated by brief oligopeptide recognition sequences. The Arg-Gly-Asp sequence (RGD), is recognized by many integrins, including integrin alphavbeta3 (CD51/61). Coxsackievirus A9 (CAV-9), a human pathogen that has an Arg-Gly-Asp sequence in the VP1 capsid protein, has been known to be one of the many viruses that utilise integrin alphavbeta3 as a receptor. In order to determine important binding sites of CAV-9 on integrin alphavbeta3, we performed binding studies of CAV-9 on CHO-alphavbeta3, CHO-alphavbeta1 and CHO-alphavbeta1-3-1 mutant cell line, in the presence of function blocking mAb specific for integrin alphavbeta3 and natural ligand vitronectin. Our experiments show that the CYDMKTTC sequence (187-193 residue) of integrin beta3, which has been shown to be involved in ligand specificity, is an important binding site for CAV-9. We also report that an RGD-less Coxsackievirus A9 mutant can bind efficiently on the ligand binding site of integrin alphavbeta3. Thus documenting the capability of this RNA virus to interact with integrin alphavbeta3, without the presence of an Arg-Gly-Asp sequence.

Amino Acid Sequence↗
PubMed2000

Human major histocompatibility molecules have the intrinsic ability to form homotypic associations.

We have investigated the homotypic associations of major histocompatibilty, class II and class I molecules using immunoprecipitation from detergent solubilised cell extracts. A 120-kDa structure corresponding to an HLA-DR dimer of dimers was immunoprecipitated by the HLA-DR specific mAb L243 from both biotinylated cell-surface and metabolically labeled B cells and transfectant fibroblasts. The thermostability of this structure in SDS was examined. It was detected at 4 degrees C, 22 degrees C, and 37 degrees C, but not at 50 degrees C or 100 degrees C. Experiments performed with L243 Fab fragments and with purified HLA-DR molecules, indicated the presence of HLA-DR dimers of dimers and single heterodimers on B cells. HLA-DQ was also found to form SDS-stable dimers of dimers and single heterodimers on the cell surface of B cells, demonstrating that HLA class II isotypes, other than HLA-DR, also form homotypic associations. Similar experiments performed with HLA class I specific mAb, W632, revealed the existence of a 90 kDa and a 135-kDa structure corresponding to a MHC class I multimers. Under the same conditions, non-MHC molecules such as CD14 were found not to self-associate. These findings indicate that major histocompatibility molecules have the intrinsic ability to form homotypic associations at the cell surface of antigen presenting cells.

Antigens, Differentiation, B-Lymphocyte↗
PubMed2000

Rough and smooth forms of fluorescein-labelled bacterial endotoxin exhibit CD14/LBP dependent and independent binding that is influencedby endotoxin concentration.

Lipopolysaccharide (LPS, or endotoxin), is a major constituent of the outer membrane of Gram-negative bacteria. Bacteria express either smooth LPS, which is composed of O-antigen (O-Ag), complete core oligosaccharides, and the lipid A, or rough LPS which lack O-Ag but possess lipid A and progressively shorter core oligosaccharides. CD14 has been described as the receptor for complexes of LPS with LPS-binding protein (LBP). Using flow cytometry we have compared the binding of Salmonella minnesota rough LPS (ReLPS) and Escherichia coli smooth LPS labelled with fluorescein isothiocyanate (FITC-LPS) to Chinese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14-CHO), to MonoMac 6 cells and to endothelial cells. Our results showed that both forms of LPS display the same binding characteristics, and that the binding of FITC-LPS to cells was both CD14- and LBP-dependent for LPS concentrations up to 100 ng.mL-1. At LPS concentrations higher than 100 ng.mL-1 we observed CD14/LBP-independent binding. CD14/LBP-dependent binding was dose dependent, saturable, and enhanced in the presence of human pooled serum (HPS), and the monoclonal anti-CD14 antibody (MY4) or unlabelled LPS could outcompete it.

Animals↗
PubMed2000

Human parechovirus 1 utilizes integrins alphavbeta3 and alphavbeta1 as receptors.

Human parechovirus 1 (HPEV1) displays an arginine-glycine-aspartic acid (RGD) motif in the VP1 capsid protein, suggesting integrins as candidate receptors for HPEV1. A panel of monoclonal antibodies (MAbs) specific for integrins alphavbeta3, alphavbeta1, and alphavbeta5, which have the ability to recognize the RGD motif, and also a MAb specific for integrin alpha2beta1, an integrin that does not recognize the RGD motif, were tested on A549 cells. Our results showed that integrin alphav-specific MAb reduced infectivity by 85%. To specify which alphav integrins the virus utilizes, we tested MAbs specific to integrins alphavbeta3 and alphavbeta1 which reduced infectivity significantly, while a MAb specific for integrin alphavbeta5, as well as the MAb specific for alpha2beta1, showed no reduction. When a combination of MAbs specific for integrins alphavbeta3 and alphavbeta1 were used, virus infectivity was almost completely inhibited; this shows that integrins alphavbeta3 and alphavbeta1 are utilized by the virus. We therefore proceeded to test whether alphav integrins' natural ligands fibronectin and vitronectin had an effect on HPEV1 infectivity. We found that vitronectin reduced significantly HPEV1 infectivity, whereas a combination of vitronectin and fibronectin abolished infection. To verify the use of integrins alphavbeta3 and alphavbeta1 as HPEV1 receptors, CHO cells transfected and expressing either integrin alphavbeta3 or integrin alphavbeta1 were used. It was shown that the virus could successfully infect these cells. However, in immunoprecipitation experiments using HPEV1 virions and allowing the virus to bind to solubilized A549 cell extract, we isolated and confirmed by Western blotting the alphavbeta3 heterodimer. In conclusion, we found that HPEV1 utilises both integrin alphavbeta3 and alphavbeta1 as receptors; however, in cells that express both integrins, HPEV1 may preferentially bind integrin alphavbeta3.

Animals↗
PubMed2000

The Th2 cytokine environment of the placenta.

It is now accepted that local changes to the balance of Th1/Th2-type cytokines occur during pregnancy within the maternal uterus and fetoplacental unit. These changes in cytokine profiles contribute to implantation of the embryo, development of the placenta, and survival of the fetus to term. Overall within the placenta there is a bias in the ratio of Th1:Th2 cytokines towards the Th2-type cytokines. However, there are specific fluctuations in this balance at implantation and during the initiation of parturition. The predominant cytokines at each stage of gestation function both to limit maternal immune rejection of the semi-allogeneic embryo/fetus, especially at the maternofetal interface; and to facilitate the on-going physiological processes within the maternal reproductive tract. These two, at times conflicting, roles are discussed in this review, with key evidence concerning cytokine expression and function from mouse and humans.

Allergens↗
PubMed2000

Atypical protein kinase C-zeta stimulates thyrotropin-independent proliferation in rat thyroid cells.

Several reports have indicated that protein kinase C (PKC) is an important regulator of proliferation in thyroid cells. Unlike TSH, the mitogenic effects of phorbol esters are accompanied by de-differentiation. The role of individual PKC isoforms in thyroid cell proliferation and differentiation has not been examined. Recent studies have implicated the atypical PKCzeta, a phorbol ester-unresponsive isozyme, in cell proliferation, death, and survival. We overexpressed PKCzeta in Wistar rat thyroid (WRT) cells and determined that PKCzeta conferred TSH-independent DNA synthesis and cell proliferation. Cells overexpressing PKCzeta show higher levels of phosphorylated p42/p44 MAPK compared with vector-transfected cells. Experiments using a luciferase reporter for Elk-1 revealed that PKCzeta overexpressing cells exhibit higher basal Elk-1 transcriptional activity than vector-transfected control cells. Interestingly, stimulation of Elk-1 transcriptional activity by MEK1, a p42/p44 MAPK kinase, was significantly enhanced in cells overexpressing PKCzeta. Strikingly, TSH retained the ability to stimulate Tg expression in cells expressing PKCzeta. These results suggest that PKCzeta stimulates TSH-independent mitogenesis through a p42/p44 MAPK-dependent pathway. Unlike overexpression of Ras or phorbol ester treatment, PKC overexpression does not impair thyroglobulin (Tg) expression.

Animals↗
PubMed2000

Molecular associations and microdomains in antigen-presenting cell-T-cell interactions.

Protein-protein interactions are utilized by the cell for almost all biological processes. Homotypic associations-physical associations between related proteins-are commonly employed as regulatory mechanisms with diverse physical and biological outcomes. X-ray crystallography has helped to reveal such protein-protein interactions. The field of antigen presentation has been especially helped by X-ray crystallography. For example, HLA-DR, CD4, and the TCR have all been crystallized as individual homodimers. One of the surprises from the early crystallographic work on MHC class II molecules was the observation that individual class II molecules existed as dimers of dimers or "superdimers," as they were called. Their existence was later confirmed by biochemical and single-cell fluorescent imaging methods. A "noninvasive" fluorescent-based technique confirmed the existence of MHC class II dimers of dimers on human living antigen-presenting cells (APCs) and ruled out potential artifacts that could have been introduced during the preparation of material for either crystallographic or biochemical studies. Little functional information has emerged so far as to the physiological significance of the MHC class II dimers of dimers. In this article, we review all the past and the latest advances in the field, taking into account the discovery of the immunological synapse and supramolecular activation clusters, and discuss the possibility of different molecular associations and microdomains involved in APC-T-cell recognition.

Animals↗
PubMed1999

Intracellular and cell surface heterotypic associations of human leukocyte antigen-DR and human invariant chain.

The intracellular and cell-surface heterotypic associations of HLA-DR in the presence and absence of the invariant chain were investigated. Simultaneous confocal microscopy imaging of the Golgi apparatus and HLA-DR molecules revealed that cells transfected only with HLA-DR and not the invariant chain or HLA-DM, accumulate class II molecules mostly in the Golgi apparatus, proximal to the cell nucleus. In contrast, in cells transfected with both HLA-DR and the invariant chain, or HLA-DR, the invariant chain and HLA-DM, the class II molecules are more evenly distributed in intracellular compartments. Confocal microscopy and flow cytometry revealed that in the absence of the invariant chain, a greater number of HLA-DR molecules are transported to the cell surface. Biochemical experiments and nonequilibrium pH gradient electrophoresis revealed that HLA-DR associates with surface invariant chain in the presence of HLA-DM. In cells that lack HLA-DM, no cell-surface association of HLA-DR and Ii was observed. Taken together, these results reveal two separate and distinct functions for surface and intracellular invariant chain subsets. The intracellular invariant chain "arrests" the class II molecules in the endocytic pathway. In contrast, cell-surface invariant chain associates with class II molecules at the cell surface, possibly playing a role in recycling empty class II molecules or as an accessory molecule.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1998

Mobility of cell surface receptors: a re-evaluation.

It has long been known from fluorescence recovery after photobleaching experiments that the mobility of most cell surface receptors is much smaller than expected for free diffusion of proteins in a fluid lipid bilayer. Single-particle tracking experiments are currently revealing the complexity of the constraints to free diffusion. Evidence has been obtained for several different processes: domain-limited diffusion, temporary confinement and anomalous diffusion. The type of motion exhibited by a given receptor will profoundly influence the rate of any functional process which requires movement in the plane of the membrane. In particular, anomalous diffusion greatly reduces the distance travelled by a receptor on a time scale of minutes.

Diffusion↗
PubMed1998

Gamma-ray irradiation induces B7.1 costimulatory molecule neoexpression in various murine tumor cells.

The use of gene-modified tumor cells as a strategy for active immunotherapy is currently undergoing intensive fundamental and clinical research. Most clinical trials use gamma-ray-irradiated tumor cells as vaccine, although little is known about the effects of irradiation on the immunogenicity of tumor cells. In particular, no data have been reported so far concerning the effects of gamma-ray irradiation on the expression of B7 molecules in tumor cells. In this paper, we show a neoexpression of the B7.1 molecule after gamma-ray irradiation in tumor cell lines from different tissues, while the B7.2 molecule remains unexpressed in all the cell lines tested. Furthermore, the induction of B7.1 molecule membrane expression after irradiation is shown to result from the neoexpression of B7.1 mRNA, and to be reproduced with H2O2 oxidative stress. These data could explain the enhanced immunogenicity of many tumor cells after irradiation, and could lead to new immunotherapy protocols.

Animals↗
PubMed1998

Regulation of major histocompatibility complex and TAP gene products in preimplantation mouse stage embryos.

PROBLEM: To determine the ontogeny of major histocompatibility complex (MHC) expression and TAP products in mouse embryos. METHOD OF STUDY: mRNAs encoding MHC and associated molecules were identified by reverse transcriptase-polymerase chain reaction, and the protein products were localized by confocal microscopy. RESULTS: mRNAs encoding class Ia (H-2Db) and class Ib (Q7/9) were present in one-cell embryos, whereas beta 2-microglobulin (beta 2-m) transcripts were not detected until the two-cell stage. Transporter TAP1, but not TAP2, transcripts were detected only in blastocysts. H-2 class Ia (classical) protein was detected on the surface of two-cell embryos, H-2 class Ib (nonclassical) protein was detected on one-cell embryos, and beta 2-m transcripts were detected on eight-cell embryos; TAP1 protein was present at low levels in the cytoplasm from the one-cell stage onward, increasing in expression in blastocysts. CONCLUSIONS: In mice, MHC class I mRNAs encoding the heavy chain of H-2- and Q7/9-encoding Qa2 molecules are synthesized soon after conception prior to implantation. Similarly, the nonpolymorphic MHC class I-associated molecule beta 2-m also is expressed before implantation. TAP1, but not TAP2, is first detected at the blastocyst stage, thus preceding the onset of TAP2 in embryonic development.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Unique biochemical properties of human leukocyte antigen-E allow for a highly specific function in immune recognition.

PROBLEM: Does a correlation exist between the expression of human leukocyte antigen (HLA) class Ia and HLA-E and what is its biological significance? METHOD OF STUDY: HLA-E transcripts were detected by reverse transcriptase-polymerase chain reaction. Metabolically labeled HLA-E heavy chains were immunoprecipited and analyzed by one-dimensional isoelectric focusing. Mouse RMA-S cells defective with regard to transporter associated with antigen processing (TAP) function were transfected with HLA-E and human beta 2-microglobulin to investigate TAP dependence of the cell-surface expression of HLA-E. RESULTS: HLA-E is transcribed regardless of the down-regulation of polymorphic HLA class Ia expression. HLA-E is transported to the cell surface in the absence of TAP-controlled peptide loading. In human cells, the amount of HLA-E protein is very low regardless of the presence of correct peptide ligands. CONCLUSIONS: HLA-E regulates immune functions in cells that have down-regulated the expression of polymorphic HLA-class Ia molecules, either by preventing harmful natural killer cells from attacking targets that have physiologically decreased HLA-class Ia expression or by activating effector cells against virus-infected and tumor cells with impaired HLA-class Ia expression.

Animals↗
PubMed1998

Active specific T-cell-based immunotherapy for cancer: nucleic acids, peptides, whole native proteins, recombinant viruses, with dendritic cell adjuvants or whole tumor cell-based vaccines. Principles and future prospects.

Whereas tumor cells are poor immunogens, recombinant tumor cells or dendritic cells as well as engineered viruses have been demonstrated to elicit specific antitumor immune responses leading to tumor growth suppression and long-lasting immunity in mouse tumor models. Single cytotoxic T lymphocyte-defined epitope-based strategies have proved useful for immunization in tumor-bearing mice. This strategy is under investigation in human melanoma, along with adjuvants such as cytokines or dendritic cells. Flt3L is an in vivo dendritic-cell growth factor that offers new prospects in the field of active specific immunotherapy. These immunotherapeutic approaches are being tested in clinical trials, and may open up novel avenues for disease-free patients with poor prognostic factors.

Adoptive Transfer↗
PubMed1997

Beta2-chimaerin is a high affinity receptor for the phorbol ester tumor promoters.

Beta2-chimaerin, a member of the GTPase-activating proteins for the small GTP-binding protein p21Rac, possesses a single cysteine-rich domain with high homology to those implicated in phorbol ester and diacylglycerol binding in protein kinase C (PKC) isozymes. We have expressed beta2-chimaerin in Sf9 insect cells using the baculovirus expression system and determined that, like PKCs, beta2-chimaerin binds phorbol esters with high affinity in the presence of phosphatidylserine as a cofactor. Scatchard plot analysis using the radioligand [3H]phorbol 12,13-dibutyrate revealed a dissociation constant of 1.9 +/- 0.2 nM for beta2-chimaerin. Likewise, beta2-chimaerin is a high affinity receptor for the bryostatins, a class of atypical PKC activators. A detailed comparison of structure-activity relations using several phorbol ester analogs revealed striking differences in binding recognition between beta2-chimaerin and PKCalpha. Although the diacylglycerol 1-oleoyl-2-acetylglycerol binds with similar potency to both beta2-chimaerin and PKCalpha, the mezerein analog thymeleatoxin has 56-fold less affinity for binding to beta2-chimaerin. To establish whether beta2-chimaerin responds to phorbol esters in cellular systems, we overexpressed beta2-chimaerin in COS-7 cells and monitored its subcellular distribution after phorbol ester treatment. Interestingly, as described previously for PKC isozymes, beta2-chimaerin translocates from cytosolic to particulate fractions as a consequence of phorbol ester treatment. Our results demonstrate that beta2-chimaerin is a novel target for the phorbol ester tumor promoters. The expansion of the family of phorbol ester receptors strongly suggests a potential for the "non-kinase" receptors as cellular mediators of the phorbol ester responses.

Animals↗
PubMed1997

Analysis of HLA class Ib gene expression in male gametogenic cells.

We have investigated mRNA expression for nonclassical MHC class I genes (HLA-E,-F,-G) in human gametogenic cells. Testicular tissue was treated by collagenase and the resulting cell suspension was further purified by fractionation on Percoll gradients in a two-step procedure. Three gametogenic cell fractions were analyzed: purified heterogenous suspension of gametogenic cells, fraction of round spermatids and fraction of elongated spermatids. Total RNA isolated from each cell population was subjected to both reverse transcriptase/polymerase chain reaction and Northern blot analysis using oligonucleotides specific for HLA-E, -F and -G. Both method gave similar results. We have found a considerable level of HLA-E mRNA, very low amounts of reamplified cDNA for HLA-F and both a complete lack of mRNA and reamplified cDNA for the HLA-G gene in the analyzed gametogenic cell fractions. Additionally, we have localized HLA-E molecules on the cells of the adluminal compartment within seminiferous tubules using immunostaining with monoclonal antibodies specific for HLA-E heavy chain followed by confocal microscopy analysis. The unique expression pattern of HLA class I antigens in the male gonad could play an important role in an efficient protection against an autoimmunological attack toward germ cells.

Blotting, Northern↗