Antiparasite activity of sea-anemone cytolysins on Giardia duodenalis and specific targeting with anti-Giardia antibodies.

The killing activity of sea-anemone cytolysins on Giardia duodenalis was investigated. Three different toxins, sticholysin I and II from Stichodactyla helianthus (St I and St II) and equinatoxin II from Actinia equina (EqtII) were all found to be active in an acute test, with a C50 in the nanomolar range (St I, 0.5 nM; St II, 1.6 nM; and EqtII, 0.8 nM). A method to target the cytolysin activity more specifically towards the parasite cells by using anti-Giardia antibodies was then investigated. Parasite cells were sensitised with a primary murine monoclonal or polyclonal antibody followed by a biotinylated secondary anti-mouse-IgG monoclonal antibody. Subsequently, avidin and a biotinylated EqtII mutant were added, either in two separate steps or as a pre-formed conjugate. When the monoclonal antibody was used, the C50 of biotinylated EqtII was 1.3 nM with sensitised cells and 5 nM with non-sensitised cells, indicating a four-fold enhancement of activity with the cell treatment. Treatment with the polyclonal antibody was somehow more effective than with the monoclonal antibody in an acute test. This indicates that sea-anemone cytolysins can efficiently kill Giardia cells, and that it is possible to improve, to a certain extent, the anti-parasite specificity of these toxins with anti-Giardia antibodies. However, the feasibility of this approach "in vivo" remains to be demonstrated.

Partial characterization of the epitope on excretory-secretory products of Fasciola hepatica recognized by monoclonal antibody ES78.

This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens in serum and stool samples of cattle and humans with fasciolosis, using a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA). The epitope was characterized by periodate oxidation, alkaline borohydride reduction, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, and enzymatic proteolysis. These results, together with those of the 2-site ELISA, lectin immunoassays, and beta-galactosidase digestion, showed that MAb ES78 reacts with a partly protein/partly carbohydrate antigenic determinant that is found on several ES molecules of adult specimens of F. hepatica and contains at least 1 disulfide bond and beta-galactose probably as galactose-beta(1-3)-N-acetylgalactosamine disaccharide.

A 10 microliter indirect ultramicro ELISA for detection of monoclonal antibodies against human alphafetoprotein.

An ultramicro ELISA, which uses 10 microliter of reaction volume developed for quantitation of human alphafetoprotein (AFP), has been adapted to use in screening hybridoma products in order to take advantage of the efficiency and sensitivity of this system. Positive samples were detected with a low background level.

Generation of murine triomas secreting bi-specific monoclonal antibodies that recognize HBsAG ad and ay subtypes.

We report the generation of murine triomas by fusing splenocytes from mice previously immunized with HBsAg ay-subtype and a hybridoma, secreting anti-HBsAg ad-subtype monoclonal antibody, which was rendered HGPRT- by induced mutagenesis with N-methyl-N'nitro-N-nitrosoguanidine. The fusion yielded a 83.8% of hybrids showing the antigen specificity of the parental hybridoma and a 16.1% of bi-specific monoclonal antibodies. One of them, coded as 1C8A5, showing a heavy chain isotype (IgG1/IgG2b) was used as capture reagent in an ultramicro-ELISA. As little as 0.78 I.U. of both HBsAg ad- and ay-subtypes could be realiably detected.

Lysosomal enzyme release from macrophages: a model of food yeast toxicity evaluation.

The role of lysosomal enzyme released by macrophages was examined in relation to the toxic effect caused by food yeast. Mouse peritoneal macrophages exposed to yeast in culture showed marked release of N-acetyl glucosaminidase, beta-galactosaminidase and beta-glucuronidase below the median lethal dose (LD50). LD50 was measured from the dose response curves of the cytoplasmic lactate dehydrogenase enzyme. Saccharomyces cerevisiae showed the highest LD50 followed by Kluyveromyces fragilis and Candida utilis yeast. LD50 values obtained as well as the in vitro lysosomal release by mouse peritoneal macrophages may be relevant to assess the toxic capacity of food yeast intended for human consumption.

Comparative study of epitopes recognized by two monoclonal antibodies that protects mice against Trichomonas vaginalis challenge.

Trichomonas vaginalis is a flagellated protozoan which infects the urogenital tract of humans. Previous studies have demonstrated that monoclonal antibodies (MAbs) against a 62 kDa proteinase (4D8 and 1A8) decreased cytoadherence of the parasite to epithelial cells in vitro and passive inoculation of mice with two MAbs 24 h before the intraperitoneal challenge resulted in different grade of protections to T. vaginalis infection. In the present paper we describe the characterization of the epitopes recognized by MAbs 4D8 and 1A8. The epitopes were characterized by heat treatment, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, enzymes proteolysis and periodate oxidation. The results showed that the two MAbs 4D8 and 1A8 each react with a different protein epitope of repetitive nature found on the same excretory-secretory molecules of T. vaginalis and it could explain the variation in the protection grade obtained in the challenge experiments.

In vivo effect of clofazimine in the lysosomal enzyme level and immune complex phagocytosis of mouse peritoneal macrophages.

The effects of clofazimine on macrophages obtained from mice fed by gavage with various drug concentrations were studied. The results obtained demonstrated an increase in the activity of various lysosomal enzymes and in the amount of labeled immune complexes phagocytosed at drug concentrations of 1 mg/kg and 10 mg/kg body weight. This confirms and extends the effects reported by us of clofazimine's action on the lysosomal apparatus.

The action of Clofazimine on the level of lysosomal enzymes of cultured macrophages.

Mouse peritoneal and calf alveolar macrophage cultures were exposed to various concentrations of Clofazimine, 3 (p-chloroanilino)-10-p-Chlorophenyl 2, 10-dihydro-2-isopropylimino, for 120 hr and an increase of four lysosomal enzymes were found with 0 . 3 micrograms/ml of the drug. In mouse peritoneal macrophage cultures, higher concentrations were toxic. Cycloheximide inhibited the lysosomal enzyme activity increase found. No change in enzymatic activity was observed when a lysosomal enriched granular fraction was incubated with various drug concentrations. Our results strongly suggest that Clofazimine at concentrations close to therapeutic serum levels induces de novo synthesis of lysosomal enzymes in macrophage cultures.

Phagocytosis and intracellular degradation of 125I-labelled immune complexes by Clofazimine treated macrophage cultures.

Mouse peritoneal and calf alveolar macrophage cultures were exposed to various Clofazimine concentrations for 5 days. Cultures exposed to drug concentrations near the blood therapeutic level phagocytize and digest more immune complexes than control cultures. Our results allow us to suggest that the beneficial action of Clofazimine in lepromatous leprosy could be at least partially mediated by the removal and degradation of circulating immune complexes by macrophages.

Effects of antiallergic drugs on cyclic nucleotides in guinea pig ileum and trachea.

In former papers we have provided evidence on the protective effect of disodium cromoglycate (DSCG) and ketotifen (Ke) against the contractions induced by various agonists (histamine, serotonin, acetylcholine, prostaglandins) in ileal and tracheal smooth muscle of guinea pigs. To offer an insight into the mode of action of these antiallergic drugs we quantified concentrations of cyclic AMP and GMP in these tissues. We observed that DSCG (10(-3)-10(-2) M) significantly diminished the concentration of cyclic AMP in ileum and trachea of guinea pigs, but it increased the level of cyclic GMP in ileum. Ketotifen (10(-6)-10(-4) M) does not modify cAMP and cGMP in ileum, however, in trachea it significantly decreases the concentrations of cGMP. We also studied the effects of oxatomide, ICI 74.917 and BRL 10.833 on cyclic nucleotides concentrations in guinea pig ileum BRL 10.833 increases cAMP at a relatively high dose (10(-4) M). ICI 74.917 at the same dose decreases cGMP. Oxatomide (10(-6)-10(-4) M) does not modify concentrations of both nucleotides. Our results demonstrate that these antiallergic drugs do not have a unique effect on cyclic nucleotides. Our findings do not support a relevant role of these nucleotides on the mechanism of action of these antiallergic drugs.

Monoclonal antibodies against a 62 kDa proteinase of Trichomonas vaginalis decrease parasite cytoadherence to epithelial cells and confer protection in mice.

Trichomonas vaginalis infects the epithelium of the genital tract. The mechanism by which it invades the tissue leading to the disease is not thoroughly understood. However, results of several studies seem to agree that parasite adhesion to epithelium cells is the initial step leading to infection in women. T. vaginalis is associated with high levels of proteolytic activity. The role of some of these proteinases in the development of infection has been demonstrated. The current study establishes the role of a 62 kDa excretion-secretion proteinase in parasite cytoadherence. Monoclonal antibodies (MAbs) against this enzyme were tested for their ability to inhibit this process. Three stable hybrid producers of IgG(1)class MAbs (4D8, 1A8, 3C11) against the 62 kDa proteinase were obtained. Two of them (4D8 and 1A8) showed parasite recognition by immunofluorescence. Parasite cytoadherence to a monolayer of HeLa cells was inhibited by the 4D8, 1A8 and 3C11 antibodies. MAb 4D8 administered 24 h before a challenge with T. vaginalis by the intraperitoneal route was able to protect the majority of mice. Nitric oxide levels in the serum of animals inoculated with MAb 4D8 and challenged with the parasite were significantly different from those recorded in mice treated with an unrelated MAb. These studies show that an appropriate antibody against 62 kDa proteinase can help the host resist a challenge by the intraperitoneal route with T. vaginalis.

Actividad biologica e inmunologica de la prolactina en varones infectados con el virus de la inmunodeficiencia humana / Biological and immunological activity of prolactin in men with human immunodeficiency virus infection

Antecedentes. Aunque existen múltiples evidencias sobre los efectos reguladores de la prolactina (Prl) sobre la respuesta inmune tanto humoral como mediada por células, no se conoce cuál es la función de esta hormona en la infección por el virus de la inmunodeficiencia humana (VIH). El objetivo de la presente investigación fue determinar si existen cambios en las concentraciones de Prl inmunorreactiva (determinadas por radioinmunoensayo [RIA]), bioactiva (bioensayo con células Nb2) o en las isoformas de pesos moleculares en las muestras plasmáticas o en los medios de cultivo de las células mononucleares de sangre periférica (CMSP) de varones infectados con el virus del sida. Métodos. Se estudió la producción de Prl in vivo e in vitro en 20 varones no tratados infectados con el VIH y un grupo de 14 varones sanos. Los estudios se realizaron en condiciones basales de ayuno o bajo estimulación de la síntesis de Prl por administración de metoclopramida endovenosa y toma de muestras a intervalos de 30 min durante 5 h. Resultados. En los varones infectados con el virus del sida las concentraciones de Prl bioactiva ­aunque no las de Prl inmunorreactiva­ en ayunas fueron superiores (p = 0,03), pero durante la prueba de estimulación las concentraciones de Prl bioactiva e inmunorreactiva fueron inferiores en los varones infectados (p 0,01). En los sujetos controles se encontró una correlación positiva entre las concentraciones de Prl bioactiva e inmunorreactiva (r = 0,728), mientras que en los infectados el coeficiente de correlación fue de 0,041. La forma molecular mayoritaria encontrada en las muestras plasmáticas de los sujetos controles y varones infectados por el virus del sida fue la de 23,5 kDa. La síntesis de Prl bioactiva por los cultivos de las CMSP de los pacientes infectados con el virus del sida (grupo II) no se estimuló por la adición de concanavalina A (Con A), a diferencia con las células de los sujetos sanos (grupo I). Sin embargo, las concentraciones en condiciones basales de cultivo siempre fueron mayores para las células mononucleares de los varones infectados con el virus del VIH. Conclusiones. Los resultados demuestran que los pacientes infectados con el virus del sida tienen un tono dopaminérgico disminuido, que sus células linfocitarias no son capaces de sintetizar Prl como respuesta a la estimulación mitogénica y que se pierde la correlación entre las concentraciones de Prl bioactiva e inmunorreactiva debido a mecanismos asociados a la presencia de la enfermedad. Las diferencias entre las concentraciones de Prl bioactiva e inmunorreactiva encontradas entre los dos grupos no están relacionadas con la presencia de distintas formas polimórficas de la hormona (AU)

Cultivos primarios de queratinocitos epidérmicos humanos utilizando una capa alimentadora combinada / Primary cultures of human epidermal keratinocytes using a compound feeder layer

Una de las aplicaciones prácticas más importantes del cultivo de queratinocitos epidérmicos humanos es la obtención de sábanas de epidermis, para utilizarlas en el tratamiento de pacientes que han recibido grandes quemaduras y que con la utilizacuón de los métodos convencionales tienen pocas posibilidades de recuperación. Estos cultivos, comúnmente desarrollados sobre un soporte de células SWISS 3T3 letalmente irradiadas, se hicieron crecer sobre un nuevo soporte combinado, primero de fibroblastos primarios de embrión de ratón y posteriormente en presencia de fibroblastos diploides humanos de la propia muestra de piel. Se logró obtener un epitelio escamoso estratificado, comprobado con la tinción de azul de rodanilo, el cual fue desprendido posteriormente de la placa de cultivo. Ensayando la línea celular NIH 3T3 como capa alimentadora, no se obtuvieron resultados positivos