An immunological approach to prion diseases.

Ovine scrapie and bovine spongiform encephalopathy are genetic diseases, presenting probably autoimmunity transmissible by the oral route. The absence of immune response in prion diseases indicates a tolerant state for PrP(C) and PrP(SC). The tolerant state against these diseases should be overcome before immunizing animals. We suggest that an early diagnosis may be possible using polyclonal and monoclonal antibodies specific for either ovine or bovine PrP(SC). Such reagents could be obtained by immunizing corresponding animals with peptides from beta sheet molecules bound to a linker or with the complete molecule (27-30 kDa).

Mice ascites as a source of polyclonal and monoclonal antibodies.

A large quantity of polyclonal anti-ovalbumin antibodies was obtained from mice by a simple modification of the method described by Kurpisz et al. (1988). In addition, the cells from ascitic fluid were used to produce monoclonal antibodies. Egg ovalbumin hyperimmunized BALB/c mice were injected successively with pristane, antigen and a non-antibody secreting myeloma cell line: the production of ascitic fluid containing antiovalbumin antibody activity was observed after 10-25 days. Cells from ascitic fluid were harvested, washed and fused together with polyethylene glycol to produce monoclonal antibodies. Two fusions were performed and a large number of monoclonal anti-ovalbumin antibodies was obtained. This method is simple, reproducible, allows many fusions to be obtained from one mouse, and allows the use of ascitic B cells rather than the more, frequently used splenic B cells.

Common antigenic properties of a g-type (goose) and a c-type (duck) egg white lysozyme: antibody responses in rabbits and mice.

Embden goose (GEWL) and Barbary duck (DEWL) egg white lysozymes possess different amino acid sequences corresponding to the g-type and c-type, respectively. GEWL was shown to be a better immunogen than DEWL in both rabbits and mice. The antigenicity of the two lysozymes was tested using different techniques (i.e. indirect ELISA, inhibition tests and immunoabsorption experiments). Injection of either GEWL or DEWL into rabbits and mice induced both specific antibodies and cross-reacting antibodies. Moreover, anti-GEWL antibodies, in contrast to anti-DEWL antibodies, did not cross-react with hen egg white lysozyme (HEWL), a c-type lysozyme. While the structure of GEWL was not modified after binding to plastic, DEWL was denaturated, but it did keep some native epitopes. It was concluded that g-type and c-type lysozymes, which have different amino acid sequences, exhibit strong common antigenic properties.

Ultrasound treatment for harvesting an aminopeptidase from lactic Acid bacteria and quantitation of the enzyme by enzyme-linked immunosorbent assays.

Ultrasound treatment of Lactococcus lactis subsp. cremoris AM2 was optimized to release a maximum amount of intracellular aminopeptidase without modifying the antigenicity of the enzyme. The cells were sonicated three times for 30 s at 23 W. Antibodies produced against the aminopeptidase purified from L. lactis subsp. cremoris AM2 enabled us to use immunoblotting to detect the enzyme in the lysates of all of the lactococci tested but not in the lysates of Leuconostoc strains, lactobacilli, and Streptococcus salivarus subsp. thermophilus. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the purified aminopeptidase; the detection limit was 4 ng/ml. The aminopeptidase in the supernatant obtained after the ultrasound treatment of strain AM2 cells was detected with the ELISA starting with a total protein concentration of 200 ng/ml. The proportion of equivalent purified aminopeptidase in the supernatant of L. lactis subsp. cremoris AM2 was about 2% of the total protein. Similarly, the aminopeptidase was quantified in different lactococci; the percentages varied between 0.16 and 2%, depending on the strain. The aminopeptidase content in a mixture of several lactic bacteria was also determined with the sandwich ELISA.

Immunochemical probes for food proteins after heat processing.

While better hygiene controls and vaccinations have diminished the occurrence of infectious diseases in humans, food-borne diseases have increased. Thus sterilization of food products is of prime importance. The introduction of new technologies applied to food has necessitated new methods for the control of food safety and food quality. This review aims to point out the importance of immunochemistry in the identification of structural changes induced in food proteins during food processing. New technologies have introduced the use of additives in food products, therefore it is important to identify and quantify such additives, even after complete hear denaturation. Toxic chemicals, toxins and pesticides which can contaminate food products before or during processing should also be identified. Finally, the use of immunochemical tests as a control of sterilization procedures in heterogeneous foodstuffs is discussed.

[Immunochemical analysis of structural changes in ovalbumin caused by heat: possible application to the quick control of food sterilization]. / Analyse immunochimique des modifications structurales de l'ovalbumine par la chaleur: application possible au contrôle rapide de la stérilisation des aliments.

Bacteriological methods, to determine proper hygiene control for food for human consumption suffers from inordinate delay (1-4 days) in the methodology before an accurate answer can be given regarding the food safety. In contrast by immunochemistry results can be obtained in 15 to 30 minutes. As it is known that during heating processing protein structure is modified, a careful identification of epitopic changes by monoclonal antibodies could be of help. It was found that after heat treatment, ovalbumin structure modified depending upon the temperature of heating. Monoclonal antibodies were raised against either native or heat denatured ovalbumin. Such monoclonal antibodies bound to plastic can be used for the immunocapture of ovalbumin in solution which is then revealed by polyclonal antibodies. With five different monoclonal antibodies it was possible to identify either native ovalbumin or heat denatured ovalbumin between 50 degrees C and 65 degrees C or heat denatured ovalbumin between 85 degrees C and 100 degrees C. Work is in progress to recognize epitopes specific of temperature and time of heating.

[Are industrial toxic substances a risk factor in gallbladder cancer?]. / Les toxiques industriels sont-ils un facteur de risque du cancer de la vésicule biliare?

Increased prevalence of gallbladder cancer has been related to specific occupations and suggests a possible professional exposure to carcinogens. The authors report the case of a 64-year-old woman who had a squamous cell carcinoma of the gallbladder with hepatic metastases, and professional exposure to trichloroethylene in a degreasing metal laboratory. Although trichlorothylene could not be assumed to be a true carcinogen for gallbladder cancer, this case underscores that epidemiologic studies taking into account professional exposure, in addition to the usual risk factors of gallbladder cancer, are requested, in order to detect a new patient group at risk for gallbladder cancer.

Natural killer (NK) activity and interferon (IFN) production by a fraction of spleen and blood lymphocytes in swine.

After carbonyl iron treatment and gradient isolation, spleen and blood pig lymphocytes exhibited NK activity and produced IFN after viral induction. Removal of plastic-adherent cells, including the majority of B cells, did not change these activities. The plastic-non-adherent cells were further separated into two subsets of roughly similar size by panning using a monoclonal, anti-T, and anti-null cell antibodies (81 + cells). NK activity and IFN production were found in the 81 - cell fraction. A significantly higher proportion of null lymphocytes from blood and of splenic Fc-gamma receptor-bearing lymphocytes was also found among the 81 - cell fraction as compared to the 81 + fraction, without any change among other subsets. Similar proportions of helper (PT4+), cytotoxic (PT8+) and total T cells (MSA4+) were found among lymphocytes bound to target K562 cells and among the whole lymphocyte population. In contrast, lymphocytes that bound K562 cells demonstrated a striking increase in the proportion of Fc-gamma receptor-positive cells of high affinity. These results show that NK cells and IFN-producing cells are mainly included in the same blood and spleen fraction, and suggest that among 81 - cells only those expressing an Fc-gamma receptor of high affinity are active.

[Rapid collection of swine lymphocyte subpopulations in large quantity]. / Obtention rapide de sous-populations lymphocytaires de porc en grande quantité.

Lymphocyte subpopulations derived from different pig organs (blood, spleen, mesenteric lymph nodes) were separated by a simple, rapid and cheap panning technique, using either normal or ozone treated Petri dishes with bovine serum albumin. Slg+ lymphocytes could thus be obtained with a purity of up to 95% by adhesion onto Corning plastic dishes. The purified cells retained their proliferative activity with regard to lectins. The subpopulation including PT4 and PT8 was then separated by another panning on ozone-treated plastic dishes with a purity of 80-90%.

A cytotoxic murine monoclonal antibody that recognizes a porcine T cell subset involved in lectin-induced proliferation.

A panel of cytotoxic monoclonal antibodies (Moab) derived from mice immunized with porcine thymocytes has been developed which reacts with monomorphic determinants on porcine peripheral blood lymphocytes (PBL). These Moab recognized from 25% to 100% of PBL as tested by flow microfluorimetry One of the Moab (PT 81) that bound 39% of PBL, 30% of splenocytes and 65% of thymocytes as determined by flow microfluorimetry was selected for initial characterization. PT 81 specifically lysed 37% of Ficoll/Hypaque-prepared PBL depleted of monocytes by carbonyl iron ingestion. This antibody lysed 47% of T cells (PBL that formed dextran-enhanced, sheep erythrocyte rosettes; P less than 0.001) and 64% of a T cell subset (PBL that formed dextran-enhanced, porcine erythrocyte rosettes; P less than 0.001) when compared to cells treated with complement alone. Lysis of PBL with PT 81 plus complement caused a 100% enrichment in the number of surface immunoglobulin positive cells (P less than 0.01). The conclusion that PT 81 does not recognize B cells was further supported by double labelling experiments. Removal of PT 81 positive cells did not significantly affect the number of cells with C3b (zymosan-complement rosettes) or Fc gamma (erythrocyte-antibody rosettes) receptors. PBL that were treated with PT 81 plus complement were passed over Ficoll/Hypaque (density 1.09) to remove dead cells. The PT 81-depleted lymphocyte population displayed only 20%, 20%, 26% and 29% of the proliferative responses of control cells treated with complement alone (P less than 0.01) to several concentrations of the mitogens phytohemagglutinin, concanavalin A, soybean agglutinin and pokeweed mitogen, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid outside-inside translocation in lymphocyte plasma membranes is a protein-mediated phenomenon.

We have measured the transbilayer diffusion of spin-labeled analogs of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in pig lymphocyte plasma membrane. At 4 degrees C and 37 degrees C the aminophospholipids are rapidly transported from the outer to the inner leaflet of the membrane, whereas the choline-containing phospholipids experience a slower diffusion. This selectivity is abolished after cell treatment by SH-group reagents indicating that the aminophospholipid translocation is protein-dependent and must be driven by a system analogous to the one existing in the human red cell membrane. The fact that the selectivity exists at low temperature, that it does not depend on cytoskeleton integrity and that there is a competition between the two aminophospholipids show that this translocation is not purely an endocytic process.

Purification of three porcine immunoglobulin classes from the same biological source.

Porcine IgG, IgA and IgM were isolated from sow milk. They were systematically purified by a combination of gel filtration and ion exchange chromatography. The yields were 85%, 64% and 32%, respectively. These immunoglobulins were very pure, as checked by SDS-PAGE and Ouchterlony immunodiffusion. SDS-PAGE showed moreover the evidence of secretory component S in the IgA molecule and of joining piece J both in IgA and IgM molecules. The procedure can be used to purify immunoglobulin classes from other biological sources.

A method of purifying sheep sIg+ lymphocytes as a tool for class II MHC antigen analysis.

A method is described for the purification of sheep lymphocytes carrying class II MHC antigens. After incubation of purified blood lymphocytes on anti-IgM-coated petri dishes, the adherent fraction contained 95% sIg-positive cells determined by immunofluorescence. When tested with cross-reacting anti-class II (bovine and human) monoclonal antibodies, more than 95% of these cells were positive either by immunofluorescence or cytotoxicity. This technique will permit studies of the polymorphism of sheep class II antigens.