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N Fernandez

Publications and source records attributed to N Fernandez.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cell-surface expression of human histocompatibility leucocyte antigen (HLA) class II-associated invariant chain (CD74) does not always correlate with cell-surface expression of HLA class II molecules.

The human histocompatibility leucocyte antigen (HLA) class II-associated invariant chain comprises at least four polypeptides. One of these is expressed as a membrane-bound subunit and has been designated as CD74. We investigated the expression of CD74 on B- and T-cell types utilizing monoclonal antibodies (mAb) specific for the c-terminus of CD74. All B cells tested expressed CD74 regardless of their HLA phenotypes. High and low CD74 expressors were provisionally assigned based on quantitative variations found in some haplotypes. A mutant cell with selective deletions of class II loci DR and DQ but with retention of DP and additional class II heterodimers synthesize normal amounts of CD74. In contrast, however, a mutant with a total class II deletion had diminished expression of CD74, suggesting that at least one of the class II isotypes is essential for full constitutive expression of CD74. Major histocompatibility complex (MHC) class II negative T-cell lines and CD3+ T cell isolated from peripheral blood did not express CD74. However, allostimulated T cells which express de novo induced class II determinants also did not express CD74. Taken together, these results show that CD74 is expressed on B cells and unexpectedly is not synthesized on de novo-induced class II positive T-cell clones.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1992

The cytokines effect on EBV-immortalized human B cells producing antisperm antibody.

We have analyzed the antisperm antibody production of autoimmunized male subjects using Epstein-Barr virus (EBV) immortalization of B lymphocytes. We evaluated the influence of several in vitro culture variants applied prior to EBV infection on the frequence of antibody-producing cells and affinity of secreted antibodies. The following variants were applied: a) polyclonal antigenic stimulation of lymphocytes with PWM, b) PWM (pokeweed mitogen) + IL-2 + interferon gamma and c) PWM + IL-2 + interferon gamma + sperm antigenic extract. The variants where the cytokines were added did not increase the frequency of EBV-infected antibody-producing cells as comparing to EBV infection previously amplified by the use of polyclonal activator. Furthermore the cytokine activation either in combination with mitogen or in vitro secondary antigenic sensitization (prior to EBV transformation) did not seem to have beneficial effect on affinity of antibodies produced by EBV-infected cells in comparison to straight EBV infection. On the other hand, the attempt to promote an immunoglobulin secretion (IgM) by previously obtained human-human antisperm hybridomas by adding of IL-2 was quite successful.

Antibody Affinity↗
PubMed1992

A ligand-epitope in vitro analysis of major histocompatibility determinants expressed on B and T lymphocytes.

Human histocompatibility leucocyte antigen (HLA)-specific monoclonal antibody probes were used to determine the affinity constant, and cell-surface density of HLA class I and class II determinants. The measurements were estimated for single-cell units of B-lymphoblastoid cell line (B-LCL) and cloned activated T cells in different functional states. Each HLA subset showed unimodal affinity constant values for the interaction with the corresponding HLA-specific antibodies. Such values ranged between 2.2 x 10(7) M-1 (class I) and 4.0 x 10(7) M-1 (class II) for different histocompatibility epitopes. In both B and T cells there was a rank order of epitope expression, class I being highly expressed (5 x 10(6) epitopes/cell) followed by DR, DQ and DP, (1.1-3.0 x 10(6) epitopes/cell). Suppressive clones carrying functionally defined stimulating determinants previously designated 'DY' carried similar numbers of DR, DQ and DP binding sites to DY- non-suppressive clones, but showed selective increases of class II determinants reactive with broad class II-specific antibodies. The results are discussed in the context of the functional consequences of different patterns of HLA epitope expression in immune responses.

Antibodies, Monoclonal↗
PubMed1991

Variation in somatic cell count, California mastitis test, and electrical conductivity among various fractions of ewe's milk.

Variation of three estimates of udder inflammation (SCC, California mastitis test, and electrical conductivity) among the foremilk, machine milk, and hand stripping samples were studied. Foremilk and hand stripping milks were taken from each individual teat; machine milk was taken from the entire udder. For this experiment a total of 30 Manchega ewes were subjected to machine milking during the 9th wk of lactation. Samples were taken in duplicate during two milkings on consecutive mornings. Significant differences were observed in the SCC and electrical conductivity, but not in California mastitis test, between foremilk and stripping milk. The stripping fraction had higher SCC (70 +/- 12%) and lower electrical conductivity (difference = .52 +/- .03 mS/cm) than did the foremilk fraction in both healthy and mastitic udders. Machine milk, foremilk, and stripping milk fractions in udders where both halves were healthy were compared. The electrical conductivity values for the machine milk were intermediate and significantly different from conductivity of foremilk and stripping fractions. The log SCC of the machine milk did not differ from that of the foremilk fraction but was significantly less than the stripping milk.

Animals↗
PubMed1990

Major histocompatibility complex (MHC) protein analysis by optimised two-dimensional electrophoretic methods.

Human histocompatibility molecules HLA-Class I and Class II (DR, DQ, DP) were analysed using three two-dimensional protocols: nonequilibrium pH gradient electrophoresis (NEPHGE), isoelectric focusing-acidic gradient (IEF-AG) and isoelectric focusing-basic gradient (IEF-BG). The three methods differ in their carrier ampholyte combinations and electrophoretic conditions. They provide different pH gradients and therefore different electrofocusing profiles. The NEPHGE protocol was adequate for separating proteins across a broad range of pI mobilities, i.e. 4.4 pH units between the acidic and the basic end. In contrast, the IEF-AG and the IEF-BG protocols gave a separation power across a narrow pH range, 1.9 and 1.7 pH units respectively. Thus, whereas the NEPHGE protocol provides a tool for a global major histocompatibility complex (MHC) antigen profile analysis, the IEF-AG and -BG allows one to investigate subcomponents of the individual MHC chains. For example, NEPHGE analysis of the HLA Class I heavy chain revealed a single spot. However, IEF-BG revealed the presence of six equidistantly spaced spots spanning a short pH gradient with identical molecular weight. Similar improved resolution was seen for the HLA-DR, DQ, and DP molecules. The IEF acidic gradient was adequate for separating the alpha chain; the IEF basic gradient gave better resolution of the beta chains. This data provides a baseline set of conditions for both analytical and preparative MHC protein studies prior to amino acid sequencing.

Alleles↗
PubMed1990

Fluid-phase endocytosis in yeasts other than Saccharomyces cerevisiae.

A FITC-dextran internalization assay with Saccharomyces cerevisiae as positive control was used to determine whether fluid-phase endocytosis is a general characteristic of yeasts. Schizosaccharomyces pombe, Pichia polymorpha, Kluyveromyces phaseolosporus, Yarrowia lipolytica and Candida albicans were clearly positive, whereas results obtained with Debaryomyces marama were inconclusive. In all cases internalized FITC-dextran was found to be localized in the vacuoles and the process was always time- and temperature-dependent. Lower eucaryotes, particularly yeasts, appear to have the ability to incorporate substances from the extracellular medium through fluid-phase endocytosis.

Dextrans↗
PubMed1989

Separation of the different classes of intrahepatic lipoproteins from various animal species. Their binding with 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(a)pyrene.

Using several analytical methods, including sucrose density gradient and potassium bromide density gradient ultracentrifugations, we have demonstrated that liver cells contain a range of lipoproteins somewhat distinct from those found in plasma. In addition to very low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL), many heavier entities have been found in the cytosol of various animal species. These heavier entities might represent either anabolic or catabolic intermediates of lipoproteins. Labelled hydrophobic xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo(a)pyrene strongly bind to the various classes of lipoproteins and may be used as radioactive tracers in the analysis and possibly in the metabolic studies of intracellular lipoproteins. Moreover, this binding may be a prerequisite for a storage or/and a carrier--roles of lipoproteins in the intracellular distribution of lipophilic xenobiotics within the cells.

Animals↗
PubMed1989

Intracellular lipoproteins as carriers for 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(a)pyrene in rat and mouse liver.

The possible role of hepatic lipoproteins as intracellular carriers in the transport of 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(a)pyrene was assessed by in vitro and in vivo studies. Following administration of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin or unlabelled 2,3,7,8-tetrachlorodibenzofuran to C57 BL/6 mice or Sprague-Dawley rats these compounds were bound to lipoproteins which subsequently underwent rapid and pronounced degradative processing, possibly catalysed by lipoprotein lipase, to heavier entities. At the highest doses of xenobiotics administered, an almost complete disappearance of lipoprotein particles was observed. The in vitro incubation of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin-lipoprotein and [3H]benzo(a)pyrene-lipoprotein complexes with separated Ah receptor and 4S protein, respectively, demonstrated that a passive transfer occurred; the latter was likely dependent on both the relative affinities of the ligands towards the different cellular binding components as well as on their quantitative binding capacity. Taken together, these findings support the idea of a carrier-role for lipoproteins in the intracellular transport of hydrophobic xenobiotics and it may be asked whether the widespread modulators of lipoprotein level such as fibrates or others affect drug transfer or action.

Animals↗
PubMed1989

Biochemical analysis of a novel H-2 class I-like glycoprotein expressed in five AKR-(Gross virus) derived spontaneous T cell leukemias.

The H-2 class I Ag profiles of five spontaneous AKR (H-2K) Gross virus leukemic cell lines were analyzed. A novel H-2 class I, "alloantigen"-like glycoprotein was immunoprecipitated and isolated from all the tumor cell lines using an H-2Dd-specific mAb 35-5-8. The novel Ag was also recognized in vitro by anti-H-2Dd-specific CTL. In addition, DNA from all the thymomas, but not the DNA from normal adult AKR thymic cells showed a transcribed gene detectable with an H-2Dd-specific oligonucleotide probe. The molecular profile of the novel antigen was further studied by two-dimensional gel electrophoresis and analyzed by a computer based image analyzer system and reverse-phase HPLC tryptic peptide mapping. Its molecular pattern was different from the syngeneic H-2Kk, H-2Dk, and the allogeneic H-2Dd gene products. The two-dimensional gel pattern of the novel H-2 class I molecule had a different overall structure reflected in isoelectric point, number, and distribution of polypeptide spots. The tryptic peptide map analysis showed six peaks exclusively identified with the novel Ag. The calculated degree of homology with the corresponding H-2Dd, H-Dk, and H-Kk peptides was 41, 56, and 51%, respectively. In addition, an unusual cell surface distribution of the novel Ag was observed in most of the leukemic lines. The removal of sialic acid residues by neuraminidase treatment facilitated the detection of the allodeterminants by anti-H-2Dd-specific mAb and CTL. Furthermore, we showed that in one AKR tumor line, 424, there is a close association of the novel Ag with the syngeneic class I molecules. Prior preclearance of the syngeneic class I molecules revealed the presence of the H-2Dd-like allospecificity. The genetic and molecular relationship between the expression of this novel class I-like glycoprotein and the recently sequenced Q5 gene is under current investigation.

AKR murine leukemia virus↗
PubMed1989

[Extracorporeal lithotripsy of gallbladder calculi. Tolerability, complications and early results].

The purpose of this prospective study, conducted on 88 patients, was to assess the tolerance, efficiency and early complications due to a piezo-electric lithotriptor in the destruction of gallbladder stones. One hundred and sixty one sessions were performed in 82 patients. All patients had symptomatic, uncomplicated lithiasis, the diameter of which was less than, or equal to, 30 mm. All patients had less than 7 stones in a functional gallbladder. In 22 patients, the stones were calcified. Lithotripsies were carried out without anesthesia or premedication, except in an 8-year-old child who had to be anesthetized. In 3 cases it was impossible to visualize the gallstones and in 3 other patients, the procedure was discontinued because of abdominal pain. Following the procedure, biliary pain occurred in 20 per cent of the patients. One patient only had biliary colic with transient anicteric cholestasis. Clinical examination, sonography, biological tests were found to be normal in all other patients. Endoscopic sphincterotomy or emergency surgery was never required. Stones were found to be unaltered in 6 patients following 2 lithotripsy sessions. The 76 other patients had fragmentation of their stones. The free gallbladder rate was 15.8 per cent between 0 and 2 months, 24.6 per cent between 2 and 4 months and 51.3 per cent between 4 and 8 months. These results tend to show that the destruction of biliary stones by piezo-electric lithotriptor is efficient and well tolerated. As repeated routine examinations were always negative, the three-day hospitalisation period no longer seems necessary.

Adolescent↗
PubMed1988

Solubilisation effect of Nonidet P-40, triton X-100 and CHAPS in the detection of MHC-like glycoproteins.

We have analysed the differential solubilisation effect of three detergents on cell-membrane histocompatibility glycoproteins. Two nonionic detergents (Nonidet P-40 and Triton X-100) which are extensively used in the extraction of MHC proteins and a zwitterionic detergent (CHAPS) which is sulphobetaine derivative of cholic acid were used. An AKR (H-2k) derived spontaneous leukaemic cell line--424--was used as the experimental model. In this tumour cell line a class I-like antigen is expressed but not directly detected by cell-binding radioimmunoassay or immunoprecipitation from NP-40 or Triton X-100 solubilised glycoproteins. However, 46 kDa and 12 kDa bands consistent with the classical H-2 class I pattern were seen by SDS-PAGE after immunoprecipitation with the 34.5.8 anti-H-2Dd MoAb using CHAPS solubilised 424 glycoproteins. The H-2Dd-reactive molecule appears to be associated with at least one of the syngeneic class I specificities (H-2Kk, H-2Dk) and not accessible to react with the specific anti H-2Dd MoAb. The detergents NP-40 and Triton X-100 appear to be less efficient than CHAPS in breaking protein-protein interactions. This property of CHAPS permitted the adequate solubilisation of the novel antigen and its direct detection. The results of this study suggest that the alternative use of a non-denaturing zwitterionic detergent may contribute to the detection and characterisation of MHC-related, membrane-bound proteins of tumours and normal cells.

Animals↗
PubMed1988

Binding characteristics of Ah receptors from rats and mice before and after separation from hepatic cytosols. 7-Hydroxyellipticine as a competitive antagonist of cytochrome P-450 induction.

The Ah receptor, a soluble protein implicated in the mechanism of action of the toxic halogenated aryl hydrocarbons has been examined in rodent livers. Due to the difficulty of making reliable quantitative determinations on binding parameters for hydrophobic compounds in cytosols that contain several components, Ah receptors from livers of Sprague-Dawley rats and C57BL/6 mice have been separated, in a preparative manner, using sucrose density gradient centrifugation in a vertical rotor. The binding characteristics of Ah receptors, before and after separation, were assessed by competition of various chemicals as 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, 3-methylcholanthrene, benzo[a]pyrene, beta-naphthoflavone and ellipticines with [3H]3-methylcholanthrene and 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin as radioligands. The rationale of this approach is supported by the results obtained and the major conclusions are as follows. 1. The intrinsic binding characteristics of Ah receptors were dependent on the presence or absence of other cytosolic binding components (light-density component and 4-S carcinogen-binding protein). 2. In contrast with many previous unsuccessful attempts, the separation of the C57BL/6 Ah receptor allowed the unambiguous detection of a 9-S binding peak with [3H]benzo[a]pyrene as a radioligand. 3. The intrinsic binding characteristics of the separated Ah receptors of Sprague-Dawley rats and C57BL/6 mice were similar if not identical. 4. A good correlation exists between the competitive potency (IC50) of chemicals and their ability to induce aryl hydrocarbon hydroxylase activity, except for 7-hydroxyellipticine which binds to the Ah receptors of rat and mouse liver (IC50 approximately 5-10 microM) without inducing aryl hydrocarbon hydroxylase. 5. When coadministered with various inducers, 7-hydroxyellipticine antagonizes (from about 20% to 65%) the inducing ability of chemicals displaying similar (ellipticines) or weaker (chlorpromazine, phenothiazine) binding affinities for the Ah receptor.

Alkaloids↗
PubMed1988

Binding characteristics of 4-S proteins from rat and mouse liver. High affinity of ellipticines.

The binding characteristics of 4-S components (carcinogen-binding protein) from livers of Sprague-Dawley rats, C57BL/6 and DBA/2 mice have been examined before and after separation from other binding components presents in the cytosol. Competitive potency of 3-methylcholanthrene, benzo[alpha]pyrene, beta-naphthoflavone and 20 ellipticines, a series of compounds differently substituted on the dimethyl-pyrido-carbazole nucleus and deprived of carcinogenic activity, has been determined with [3H]3-methylcholanthrene and/or [3H]benzo[a]pyrene as radioligands. The inducing ability of the same compounds for aryl hydrocarbon hydroxylase and for ethoxyresorufin-O-deethylase has been compared to their affinity for the 4-S protein and the Ah receptor respectively. The main results of this study are as follows. 1. The intrinsic binding characteristics of 4-S proteins were dependent on both the nature of the radioligand used and the presence or absence of other cytosolic binding components. 2. The heterocyclic ellipticines were revealed as strong ligands for the carcinogen-binding protein (stronger than benzo[alpha]pyrene for five derivatives substituted in the A ring of ellipticine), with IC50 values ranging from 0.047 microM (8-hydroxyellipticine) to 5.8 microM (N2-ethyl-9-hydroxyellipticinium). 3. When the affinity of ellipticines was plotted versus their inducing ability of aryl hydrocarbon hydroxylase and ethoxyresorufin-O-deethylase, it appears that a good correlation exists for the Ah receptor but not for the 4-S protein. It is concluded that these data, as well as the lack of enzymatic induction after benzo[alpha]pyrene treatment of DBA/2 mice, which display a high level of 4-S protein, do not support the implication of this binding component in the positive control of cytochrome P-450 induction.

Alkaloids↗
PubMed1988

DY determinants, possibly associated with novel class II molecules, stimulate autoreactive CD4+ T cells with suppressive activity.

A set of T cell clones (TCC) isolated from HLA-DR-, Dw-, DQ-matched allogeneic MLCs was found to proliferate autonomously when stimulated with cells carrying a wide range of class I or II specificities. This apparently unrestricted proliferation was relatively weak, and only low levels of IL-2 were present in the supernatants of stimulated cells. Autologous as well as allogeneic PBMC and B lymphoblastoid cell lines (B-LCL) were capable of stimulating such clones, which were also restimulated by suppressive, but not by helper, TCC. Moreover, such clones displayed the unusual property of autostimulation. mAb inhibition experiments suggested that class II- or class II-restricted antigens were involved in stimulation. Thus, certain "broad" mAbs (TU39, SG520) reacting with multiple locus products inhibited activation of these reagents, but none of those reacting more specifically with DR (TU34, TU37, L243, Q2/70, SG157), DQ (TU22, SPV-L3, Leu 10), or DP (B7/21), or mixtures of these mAbs, were able to do so. Evidence from sequential immunoprecipitation experiments suggested that mAb TU39 bound class II-like molecules other than DR, DQ, and DP on TCC and B-LCL, and it is therefore proposed that such putative novel class II-like molecules may carry the stimulating determinants for these autoreactive clones. DY-reactive clones lacked helper activity for B cells but mediated potent suppressive activity on T cell proliferative responses that was not restricted by the HLA type of the responding cells. Suppressive activity was induced in normal PBMC by such clones, as well as by independent suppressive clones, which was also inhibited only by mAb TU39. These findings lead to the proposal that DY-reactive autostimulatory cells may constitute a self-maintaining suppressive circuit, the level of activity of which would be regulated primarily by the availability of IL-2 in the microenvironment.

Antibodies, Monoclonal↗
PubMed1988

A novel HLA class II molecule.

Molecular evidence has been obtained for a novel monomorphic HLA class II molecule distinct from HLA-DP/DQ/DR using a panel of lymphoblastoid cells which include HLA-loss mutants. The expression of this molecule was investigated using monomorphic affinity-purified mouse monoclonal antibodies (mAbs), including one of the IgG2a subclass designated EDU-1. This antibody reacts strongly in a cell-binding radioimmunoassay with HLA-DR and -DQ loss mutants derived from a lymphoblastoid parental cell. The EDU-1 mAb also reacted with a local panel of homozygous Epstein-Barr virus-transformed cell lines. The reactive molecules were further detected on allostimulated T-cell clones and various leukemic cells including those of myeloid origin which lack surface expression of HLA-DQ molecules. Thus the class II molecule described in this study corresponds to a monomorphic HLA class II determinant expressed on a variety of cells of different origin and HLA phenotypes. Moreover, this antigen structure is distinct from that of HLA-DP/DQ/DR as shown by direct immunoprecipitation, serial immunodepletion experiments, and two-dimensional gel electrophoresis. The molecule could be specified by new class II genes between DP and DQ. An alternative explanation for the genetic basis of the novel molecule is the existence of isotypic associations between alpha and beta chains of various class II molecules (DP, DX, DZ, and DO) but not DR and DQ as the mutant cells tested lack the latter genes.

Antibodies, Monoclonal↗