Procedure for radiolabeling gizzerosine and basis for a radioimmunoassay.

A method for the labeling of gizzerosine (GZ), a biogenic amine found in fish meal, is described. The labeling procedure with (125)I using a water-soluble Bolton-Hunter reagent and a mild water-insoluble oxidant (Iodogen) reagent is rapid and reproducible. The (125)I-GZ hapten was demonstrated to be immunologically active in a radioimmunoassay developed with polyclonal antibodies to GZ absorbed with a histamine-Sepharose column. The curves were linear in the range of 0.0001 and 0.1 microgram/mL. Samples of fish meal previously extracted of histamine with methanol and submitted to acid hydrolysis were contaminated with known amounts of GZ and submitted to the assay. The fish meal samples contaminated with GZ show a dose-response effect similar to the standard curve, and apparently the other component present in the sample did not interfere with the binding of the antibodies to (125)I-GZ. These data indicate the suitability of the radioimmunoassay to determine specifically GZ in fish meal.

Apoptosis induction in nonirradiated human HL-60 and murine NSO/2 tumor cells by photoproducts of indole-3-acetic acid and riboflavin.

The effect of the photoproducts of indole-3-acetic acid sensitized by riboflavin on nonirradiated human HL-60 and murine NSO/2 tumor cells was studied. Severe damage with a dose-response effect was observed on both cell types. The effect was greater than that previously described for the tryptophan riboflavin photoproducts. Electron microscopy studies and flow cytometry analysis of DNA fragmentation allowed us to conclude that the photoproducts studied in this work induce cell death by an apoptotic mechanism.

CD8+ T cells are the effectors of the contact dermatitis induced by urushiol in mice and are regulated by CD4+ T cells.

BACKGROUND: The exposure of human skin to leaves and branches of litre (Lithraea caustica), a Chilean endemic tree, induces a severe contact dermatitis characterized by swelling and pruritus in susceptible individuals. The allergenic priniciple of litre is 3-pentadecyl (10-enyl) catechol (litreol), which is structurally similar to the allergens isolated from poison oak and poison ivy. All of them belong to a family of compounds named urushiols. As a proelectrophilic allergen, litreol must be intracellularly activated before modifying proteins of individuals exposed to it. As a result, self-peptides derived from litreol-modified intracellular proteins would be presented in the context of class I MHC molecules. We hypothesized that CD8+ T lymphocytes would play a major role during the effector phase of the immune response induced by those modified peptides. In order to test this hypothesis, we investigated the cellular immune response to litreol in Balb/cJ mice. The role of the different lymphocyte subpopulations in this response was assessed by immunodepleting mice of CD4+ or CD8+ T lymphocytes using specific monoclonal antibodies (mAbs). We report the observation that the contact dermatitis induced by litreol has two components: a primary response which does not require TCRalpha beta+ T cells, and a secondary response mediated mainly by CD8+ T cells and regulated by CD4+ T cells. Our results show that CD8+ lymphocytes play a central role as effectors of the secondary response to litreol. Furthermore, our data suggest that two functionally different CD4+ T subpopulations serve as regulators of the CD8+ T cell function: a CD4+ T helper population sensitive to a low dose of the depleting mAb, and CD4+ T suppressor population which is eliminated only with a high dose of depleting mAb.

Development of monoclonal antibodies to gizzerosine, a toxic component present in fish meal.

This study is the first report of the development of monoclonal antibodies (MAbs) against gizzerosine (GZ), one of the causative agents of black vomit, a serious poultry disease. Balb/c mice were immunized with different GZ conjugates; the most immunogenic conjugate in experimental animals was determined by enzyme-linked immunoadsorbent assays (ELISA). Somatic fusions were carried out using splenic lymphocytes from GZ-immune mice and the NSO/2 myeloid cell line. Primary selection of hybridomas secreting antibodies to GZ was done using a direct ELISA, with GZ bound to bovine serum albumin (BSA), GZ directly bound to maleinimide preactivated plates and histamine bound to BSA, a GZ related biogenic amine present in fish meal. Four MAbs--3H4, 3H10, and 5B1 of the IgG1 isotype, and 8G7 of the Ig2a isotype-were specific to GZ and did not cross-react with histamine. Only monoclonals 3H4 and 8G7 bound GZ in solution by means of a competitive ELISA. Finally, to determine the performance of the competitive ELISA developed with the MAbs, experiments were conducted with GZ in solution (0 to 10 microg/ml) and with GZ labeled with horseradish peroxidase (HRP) as the tracer; the antibody complex was captured by using rabbit anti-mouse IgG preactivated ELISA plates. These experiments showed that monoclonal anti-GZ-3H4 generates a more sensitive assay close to linearity in the range about of 0.1 to 10 microg/ml of GZ. No cross-reaction was observed with histamine, histidine, or lysine at all concentrations tested.

Modulation of fatty acid oxidation alters contact hypersensitivity to urushiols: role of aliphatic chain beta-oxidation in processing and activation of urushiols.

Lithraea caustica, or litre, a tree of the Anacardiaceae family that is endemic to the central region of Chile, induces a severe contact dermatitis in susceptible human beings. The allergen was previously isolated and characterized as a 3-(pentadecyl-10-enyl) catechol, a molecule belonging to the urushiol group of allergens isolated from poison ivy and poison oak plants. Because urushiols are pro-electrophilic haptens, it is believed that the reactive species are generated intracellularly by skin keratinocytes and Langerhans cells. The active species are presumed to modify self proteins which, after proteolytic processing, would generate immunogenic peptides carrying the hapten. The presence of a 15-carbon-length hydrophobic chain should impair antigen presentation of self-modified peptides by class I MHC molecules, either by steric hindrance or by limiting their sorting to the ER lumen. We have proposed that the shortening of the aliphatic chain by beta-oxidation within peroxisomes and/or mitochondria should be a requirement for the antigen presentation process. To test this hypothesis we investigated the effect of drugs that modify the fatty acid metabolism on urushiol-induced contact dermatitis in mice. Clofibrate, a peroxisomal proliferator in mice, increased the immune response to the urushiols from litre by 50%. Conversely, tetradecyl glycidic acid, an inhibitor of the uptake of fatty acids by mitochondria, decreased the hypersensitivity to the hapten. An increase in the level in glutathione by treatment of the animals with 2-oxotiazolidin-4-carboxilic acid lowered the response. Those findings strongly support a role for the fatty acid oxidative metabolism in the processing and activation of urushiols in vivo.

Development of monoclonal antibodies against a riboflavin-tryptophan photoinduced adduct: reactivity to eye lens proteins.

We describe here the development of monoclonal antibodies to the hapten tryptophan-riboflavin, generated by irradiation of a solution of bovine serum albumin in the presence of riboflavin. The specificity of the three obtained monoclonal antibodies, named 1E6, 5H5, 5A8 all belonging to the IgG1 isotype, was assessed by a competitive enzyme-linked immunosorbent assay in the presence of an increasing concentration of the tryptophan-riboflavin adduct, obtained from an irradiated riboflavin-sensitized tryptophan solution. It was demonstrated that the tryptophan-riboflavin antibodies react with the soluble proteins of the eye lens; this reaction was more intense in the old rat lenses as compared to the young ones, and a maximum binding of the antibodies was obtained with the soluble protein fraction from the human cataractous lens. By indirect immunofluorescence, a reactivity associated with the protein matrix, localized in the lens central zone, was observed. In the peripheral zone of the lens, where the younger cells are found, a marked immunofluorescent emission was observed on structures preferentially localized in the nuclei.

An alternative ELISA for T4 determination based on idiotype anti-idiotype interaction and a latex method for anti-idiotype monoclonal antibody selection.

This paper is the first report on the use of an idiotype-anti-idiotype monoclonal antibody reaction to develop an enzyme immunoassay for thyroxine (T4). We have developed a monoclonal antibody against T4, named 1F10 of IgG1 subclass and KA 5.21 x 10(8) M-1 which was used to obtain anti-idiotypic monoclonal antibodies. Anti-idiotypic antibodies were selected by a novel method, a passive agglutination assay with the idiotype monoclonal 1F10 absorbed on latex particles and subsequently characterized by RIA. One of these anti-idiotype antibodies, named 5B3--type beta antibody--of IgG1 subclass, was used to develop an enzyme-linked T4 idiotype-anti-idiotype immunosorbent assay. The T4 calibration curve, using the 1F10 idiotypic antibody adsorbed to solid phase and the 5B3 anti-idiotypic antibody conjugated to alkaline phosphatase (ALP), shows adequate performance in the range between 0.7-25 micrograms% of the analyte. The reliability of the proposed method is demonstrated by the correlation coefficient r = 0.74, found between T4 measured by RIA and our assay, with a panel of sera from euthyroid, hypothyroid and hyperthyroid individuals. The correlation coefficient was r = 0.93 within assays and r = 0.88 between assays. These results provide the basis for a new non isotopic assay for the study and diagnosis of T4-related human disease and provides a model to develop immunoassays for other haptens and small molecules of clinical interest.

Visible light anaerobic photoconversion of tyrosine sensitized by riboflavin. Cytotoxicity on mouse tumoral cells.

The anaerobic phototransformation of tyrosine under visible light sensitized by riboflavin is reported. The cytotoxicity of the anaerobic photoproducts on in vitro-cultured myeloid mouse tumoral cells was demonstrated. A radical mechanism is proposed. Dityrosine was identified as one of the main anaerobic photoproducts by using absorption, emission and 1H-NMR spectra.

Visible light effects on tumoral cells in a culture medium enriched with tryptophan and riboflavin.

When NSO/2 myeloid cell line and teratocarcinoma F9 cells were irradiated in Dulbecco's modified Eagle medium enriched with tryptophan and riboflavin, toxic photoproducts for these tumoral cells were generated. The active participation of 1O2 and .OH was established using specific scavengers and quenchers. A cytotoxic effect was also observed when unirradiated tumoral cells were incubated in a previously irradiated culture medium enriched with tryptophan and riboflavin. When irradiated medium was used alone, enriched only with tryptophan or only with riboflavin, no toxic effect was observed. The relevance of charge transfer processes between triplet riboflavin and tryptophan in the generation of cytotoxic photoproducts is discussed.

Development of anti-human B blood group monoclonal antibodies suitable as a blood typing reagent.

An improved procedure for the generation of high-avidity anti-human B blood group monoclonal antibodies (MAbs) was developed. One of them, termed 7A1-2, showed excellent qualities of titer, avidity, and intensity required for use as human B blood typing reagent. Hemagglutination inhibition studies with monosaccharides and oligosaccharides were carried out to determine the specificity of the MAb 7A1-2. These studies indicate that the antibody reacts with the immunodominant region of the antigen which is known to confer the serologic specificity of this blood group.

Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space.

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization--by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin--of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the antiacrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa.

Mucin allows survival of Salmonella typhi within mouse peritoneal macrophages.

Salmonella typhi is a facultative intracellular human specific pathogen. Both immunocompetent and immunodeficient mice are resistant to S. typhi. However, when they are infected with S. typhi suspended in mucin, the bacteria become pathogenic and infect peritoneal phagocytic cells. The LD50 for mice was 10(5) bacteria suspended in 5% mucin; mouse survival was approximately 48 hours after injection. A high number of bacteria was recovered from peritoneal cells; transmission electron microscopy disclosed a large number of vesicles filled with S. typhi cells in peritoneal cells from infected animals. The addition of mucin to cultures of the reticuloendothelial cell line J774.3 also allowed invasion of the mammalian cells with S. typhi. These data indicate that mucin allows intracellular survival of S. typhi.

A light-induced tryptophan-riboflavin binding: biological implications.

We review here the covalent photo-binding induced by visible light between the essential amino acid tryptophan and the vitamin riboflavin. We discuss the biological implications of this photoadduct in relation to the hepatotoxic and cytotoxic effect associated to parenteral nutrients and to culture media exposed to the action of light, respectively. We also analyze the formation of a photo-binding between riboflavin and the residues of tryptophan present in the proteins of the eye lens, a tissue which is permanently exposed to visible light.

Round-headed spermatozoa: a model to study the role of the acrosome in early events of gamete interaction.

Gamete interactions in mouse involves at least two steps: the first is the interaction of a spermatozoa receptor located in the plasma membrane and ZP3, a zona pellucida (ZP) glycoprotein. ZP3 also can induce the acrosome reaction, making possible the second step: a closer interaction between ZP2 and an inner acrosomal membrane receptor. Our aim was to study gamete interaction in round-headed spermatozoa to determine at which functional level fertility is impaired. These spermatozoa are predominant in some infertile male and are characterized by the absence of acrosome; they also present an abnormal pattern of chromatin condensation. Human ZP and zona free hamster oocytes were used to study gamete interaction. No binding to ZP was observed either with light or electron microscopy. Our findings suggest that the presence of the acrosome could be necessary for the sorting and right organization of plasma membrane proteins. Round-headed spermatozoa could also present a general alteration of membrane protein synthesis. The lack of fusion with zona-free hamster oocytes may be explained by an altered reorganization of plasma membrane proteins in the post acrosomal region as a result of the absence of the acrosome reaction in round headed spermatozoa.

Amino terminal sequence of heavy and light chains from ratfish immunoglobulin.

The ratfish, Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (Mr 960,000), composed of heavy (Mr 71,000), light (Mr 22,500), and J (Mr 16,000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark, Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.

[Synthesis and secretion of the surface antigen from hepatitis B virus in animal cell cultures]. / Síntesis y secreción del antígeno de superficie del virus de Hepatitis B en cultivo de células animales.

Stable mammalian cell lines synthesizing and secreting Hepatitis B surface particles have been obtained through genetic engineering techniques. These particles show by electron microscopy a size of 22 nm, they are structurally and immunochemically similar to the particles present in the plasma from chronic hepatitis B patients. Therefore these particles are an excellent source for the preparation of a vaccine against the virus.