A specific immunoradiometric assay of cationic trypsin(ogen) that does not recognize trypsin-alpha-1-proteinase inhibitor complex.

An immunoradiometric assay using two monoclonal antibodies directed to human trypsin 1 was developed for measuring trypsin(ogen) in biological fluids. The assay is different from other assays in that it is specific for cationic trypsinogen and does not recognize the alpha-1-proteinase inhibitor-trypsin complex. It can be used as a complement to classical immunoassays to characterize trypsinogen activation in pathological cases. The evaluation and the specificity of the assay are presented.

Monoclonal antibodies to human pancreatic trypsin 1 inhibit the activation of human trypsinogens 1 and 2.

Two monoclonal antibodies (Mab) raised against human pancreatic trypsin 1, Mab G6 and A8, were previously isolated and characterized. The two Mab which recognize trypsinogen 1 are found to inhibit the activation of trypsinogen 1 by enterokinase. The inhibition of activation by the two Mab is concentration-dependent, rapid and virtually complete with Mab G6. Activation of trypsinogen 2 is totally inhibited by Mab G6, while Mab A8 has no effect on the activation of trypsinogen 2. The two monoclonal antibodies have opposite effects on the proteolytic activity of trypsin 1; Mab G6 increases proteolytic activity while Mab A8 inhibits trypsin activity by as much as 40%. This inhibition is concentration dependent but cannot account for the complete inhibition of activation of trypsinogen 1. Neither monoclonal antibody significantly inhibits the esterolytic activity of either form of human trypsin. Western-blot analysis of the reactivity of the two monoclonal antibodies with trypsinogens of various species shows that only Mab G6 cross-reacts with dog trypsinogen.

Contamination of dairy products by fungal metabolites.

The contamination of dairy products with various mycotoxins or other undesirable fungal metabolites can be attributed a.o. to ingestion of contaminated feed or the accidental development of molds and by consequence the excretion of fungal metabolites into the intermediate product. Different dairy products of commercial origin were examined: milk powder, reconstituted infant milk powder, and cheese. In addition to that, environmental factors contributing to the formation of the undesired fungal metabolites were studied. It was found that the presence of mycotoxins in dairy products is more related to the environmental factors causing mold growth on dairy products than to the ingestion of moldy feed by the cow.

Production and characterization of two monoclonal antibodies to human pancreatic trypsin 1.

Two monoclonal antibodies (MAb) G6 and A8 directed against human trypsin 1 have been produced by hybridization of myeloma cells with spleen cells of OF1 immunized mice. Antibodies were screened by radioimmunoassay. Monoclonal antibodies were purified by affinity chromatography on Protein A-Sepharose, and we found that MAb G6 was of the IgG2b class and MAb A8 of the IgG2a class. Both MAbs had a high affinity for trypsin 1 with the respective affinity constants equal to 1.3 x 10(8) l/mol for G6 and 3.2 x 10(7) l/mol for A8. Epitope specificity was studied by western blotting, using human trypsinogens 1 and 2. Both MAbs gave a positive reaction with native trypsinogen 1 and no reaction with the same protein after reduction. Only MAb G6 reacted with trypsinogen 2 in the native form. Its affinity for trypsin 2 was found similar to that for trypsin 1 with a constant equal to 2.7 x 10(7) l/mol. Both antibodies appeared directed against conformational and not sequential epitopes.

Transplacental transfer of T2-toxin: pathological effect.

Radioactivity was recovered in fetuses of pregnant rats following administration of radioactive T2-toxin. The transplacental effects of T2-toxin were studied by ip administration or oral feeding of pregnant rats at doses equivalent to natural contaminations and compatible with the maintenance of pregnancy. Body weights of the newborn rats from treated females were similar to the body weights of the control animals but their thymuses were atrophic. This atrophy was reversible in one week. Since the measurement of antibody production for fetuses and newborns is not feasible, the lymphoblastic response to mitogen of the splenic and thymic cells of baby rats from treated and control females was tested. At 4 and 6 days after birth, a good response to PHA for the thymic cells of the mother treated young rats was observed. Histological examination of the thymus showed that one day after birth the cortex was atrophic while the medulla was proliferative; on day six the situation was reversed. For the spleen, both B and T cells were impaired and their responsiveness to PHA and LPS decreased. On days 1 and 6, the periarteriolar sheats, as well as the follicles, appeared atrophic. These results show that T2-toxin easily passes the placental barrier and that T2-toxin injection or feedings at levels (2 ppm) similar to those in naturally contaminated foods, produced an impairment of the immune system of the newborn.

[Modification of the koch bacillus by a material extracted from tuberculosis lesions]. / Modification du bacille de Koch par un matériel extrait de lésions de tuberculose.

Lymph node lesions of tuberculous cattle and swine gave, after disruption, centrifugation through 2.2 M sucrose, and ultrafiltration, a material that brings about, in vitro, modification of the tubercle bacilli or chromogenic mycobacteria into bacterial elements that are not acid fast, rapidly growing on nutritive agar supplemented with glycerol. The phenomenon is similar to that which the authors have previously described, using an inducing agent extracted from cultures of mycobacteria, called "endometallaxic conversion."

[Effect of mycotoxins on the rate of fermentation in Saccharomyces cerevisiae]. / Effets de mycotoxines sur l'activité fermentaire de Saccharomyces cerevisiae.

The rate of fermentation of Saccharomyces cerevisiae is partially inhibited by different mycotoxins. This effect is remarkable with T2-toxin and diacetoxyscirpenol, slight with aflatoxin-B1, penicillic acid and patulin. On the contrary, the butenolide appears as a stimulator of the alcoholic fermentation.

[Photodensitometric determination of "PR-toxin", metabolite of Penicillium roqueforti (author's transl)]. / Dosage photodensitométrique de la "PR-toxine" métabolite de Penicillium roqueforti.

Two methods are proposed for the determination of PR-toxin. One allows a direct quantitative estimation of the mycotoxin, the other utilizes an imine formation after reaction with ammonium hydroxide. In both cases a fluorodensitometric assay on thin-layer chromatographic plates is carried out after spraying with p-dimethylaminobenzaldehyde reagent. The two procedures can be applied to estimate the toxin in foodstuffs at levels as low as 10 and 1 microgram/kg, respectively.

[Contamination of cow's milk during ingestion of small quantities of aflatoxin]. / Etude de la contamination du lait de vache lors de l'ingestion de faibles quantités d'aflatoxine.

Sixteen cows, some in the early and the rest in the late lactation period, were given daily, during a eight day period a ration of peanut meal naturally contaminated by aflatoxin. The analysis for aflatoxin M1 of the milk obtained from these cows indicates the following points: --the level of aflatoxin M1 is not influenced by the volume of the daily milk secretion; --the quantity of this hydroxy-derivative expressed as a percentage of the parent toxin varies from 0.14 to 0.34 in the animals in the late lactation period, and from 0.66 to 0.95 in the cows producing some twenty litres of milk per day; --in order to achieve a tolerance limit of 20 ng/kg of aflatoxin M1, in the milk, the daily ingestion of the mycotoxin by cows should not exceed 90--100 micrograms per animal.

[Formation of nitrosamines in cheese products]. / Formation de nitrosamines dans les produits fromagers.

Several strains of micromycetes used as fermentation agents in the cheese industry or having led to accidents during cheese making are able to favor the formation of nitrosamines in 60% of the cases. The concentrations observed are similar to those found by other authors with other microorganisms. The results obtained in a semi-synthetic medium are checked during the ripening of experimental camembert type cheese made from milk containing nitrates and cultured with a strain of Penicillium camemberti, which favors very much the synthesis of nitrosamines. The amount of nitrosodimethylamine formed in this cheese increases from 5 to 20 ppb during ripening. A tentative explanation of the mechanism of formation is outlined.

[Effect of sterigmatocystin on the toxinogenesis of the Aspergillus flavus group]. / Effet de la sterigmatocystine sur la toxinogenese d'Aspergillus du groupe flavus.

The aflatoxinogenesis of Aspergillus parasiticus is significantly enhanced by the presence, in the medium, of sterigmatocystin at a high level (35--50 microgram/ml); low concentrations, in the order of 175 microgram/ml, have no effect on the production of aflatoxins. During the period where the aflatoxinogenesis of the culture is high, no variation of the sterigmatocystin level is noted, Experiments with 14C-sterigmatocystin indicate that the mold does not utilize the metabolite itself as a precursor of aflatoxins.

Determination of mycophenolic acid, penicillic acid, patulin, sterigmatocystin, and aflatoxins in cheese.

A method has been developed for detection of aflatoxins, mycophenolic acid, patulin, penicillic acid, and sterigmatocystin in cheese. It is based on selective extraction with a mixture of equal volumes of 5% sodium chloride, methanol, and aceton, precipitation of caseins at -25 C, defatting with hexane, and removal of extraneous matter by transfer of mycotoxins to chloroform and ethyl acetate. The extract is purified further by column chromatography. Mycotoxins are quantitated on thin layer chromatograms by fluorescence comparisons. Mycophenolic acid, patulin, and penicillic acid are visualized with diethylamine. The limits of detection in cheese are about 20 micrograms/kg for mycophenolic acid, patulin, and sterigmatocystin, 30 microgram/kg for pencillic acid, and 1 microgram/kg for aflatoxins B1 and M1.

[Transmissible modification induced in Mycobacterium tuberculosis and Mycobacterium bovis in vitro]. / Modification transmissible induite in vitro chez Mycobacterium tuberculosis et Mycobacterium bovis.

A biologically active material (fraction "S") is isolated from cultures of scotochromogenic mycobacteria. Mycobacterium tuberculosis, or Mycobacterium bovis by disrupting the cells, sedimentation through 2.2 M sucrose, and ultrafiltration. The fraction "S" induces the modification of tubercle bacilli into non acid-fast bacteria forming smooth colonies on nutritive glycerol agar within 24-36 h of incubation. Three new phenotypes are thus obtained; two proved to be stable upon subculturing. Frequently the phenomenon occurs with a very large part of the Koch's bacillus population exposed to the inducing agent effect. It can be reproduced with crude preparations of DNA obtained from the fraction "S." It is inhibited by concanavalin A. The observed modification does not correspond to a transfer of characteristics of the inducing agent from the donor mycobacteria; furthermore it can be manifested even in the strain used for the preparation of the fraction "S."