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1.
Immunology ; 76(2): 299-304, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1634252

ABSTRACT

In order to understand better how cytokines modulate porcine lymphocyte-mediated natural cytotoxicity and to develop a rapid and reliable colorimetric assay to study that activity in young pigs, we studied inherent and cytokine induced in vitro natural killer (NK) activity. The cytokines we studied were human recombinant interleukin-1 alpha (IL-1 alpha), IL-2, IL-4 and interferon-gamma (IFN-gamma). Natural killer activity by peripheral blood mononuclear cells (PBMC), reported as per cent specific lysis (%SL), was determined by the colorimetric measurement of lactate dehydrogenase released from tumour cell targets, YAC-1 and K562. Inherent NK activity was low and remained relatively unchanged by alterations of assay length or effector cell concentration. Low NK activity was also observed in response to IL-4 and IFN-gamma. IL-2 and, to a lesser extent, IL-1 alpha induced significant NK activity with trends towards increasing %SL with increasing cytokine dose. Optimal IL-1 alpha- and IL-2-induced NK activity could be observed at 18 hr, with significant activity stimulated by IL-2 as early as 4 hr. IL-2-induced NK activity was sensitive to effector cell concentration; %SL decreased as the effector to target ratio decreased. IL-1 alpha- and IL-2-induced NK activities were decreased in the presence of IL-4. These results indicate porcine PBMC are sensitive to in vitro modulation by human recombinant IL-1 alpha, IL-2 and IL-4. The ability of IL-1 alpha and IL-2 to induce swine NK activity and the ability of IL-4 to inhibit that activity are similar to the actions of those cytokines in human NK systems.


Subject(s)
Cytotoxicity, Immunologic/immunology , Interleukins/immunology , Killer Cells, Natural/immunology , Swine/immunology , Animals , Cells, Cultured , Dose-Response Relationship, Immunologic , Interferon-gamma/immunology , Interleukin-1/immunology , Interleukin-2/immunology , Interleukin-4/immunology , Recombinant Proteins/immunology
2.
Infect Immun ; 58(11): 3627-32, 1990 Nov.
Article in English | MEDLINE | ID: mdl-2228233

ABSTRACT

Geographically distinct lines of Tritrichomonas foetus were assayed for their ability to cause cytotoxicity in nucleated mammalian cells and lysis of bovine erythrocytes. T. foetus was highly cytotoxic toward a human cervical cell line (HeLa) and early bovine lymphosarcoma (BL-3) but displayed low levels of cytotoxicity against African green monkey kidney (Vero) cells. In addition to variation in the extent of cytotoxicity toward different targets, differences in the levels of cytotoxicity in the same nucleated target occurred with different parasite lines. Whole T. foetus, unfractionated whole-cell extracts, and parasite-conditioned medium (RPMI 1640 without serum) all caused lysis of bovine erythrocytes. Lytic activity in the conditioned medium was substantially reduced by repeated freezing and thawing or heating to 90 degrees C for 30 min. Damage of mammalian target cells by live T. foetus could be reduced by the presence of protease inhibitors; however, such inhibitors did not diminish the lytic effects of conditioned medium. These results suggested that proteolytic enzymes were necessary for the lytic mechanism of the live parasites but were not required once lytic factors were released into the parasite-conditioned medium. They further suggested that the lytic molecules were either proteins or had proteinaceous components.


Subject(s)
Cells, Cultured/parasitology , Hemolysis , Protozoan Infections/pathology , Tritrichomonas/physiology , Animals , Endopeptidases/pharmacology , Freezing , Hot Temperature , Humans
3.
J Parasitol ; 75(6): 977-80, 1989 Dec.
Article in English | MEDLINE | ID: mdl-2693679

ABSTRACT

Sections of bovine placenta from cases of bovine trichomoniasis were examined for the presence of Tritrichomonas foetus by standard histological methods using phase-contrast microscopy and by indirect immunofluorescence assay (IFA) employing monoclonal antibodies (mAbs) specific for T. foetus. Parasites were identified readily in deparaffinized tissue up to 4 yr old by IFA with 2 mAbs previously shown to bind to the surface of living T. foetus. These results indicated that the IFA provided a rapid and specific method of identifying T. foetus in tissue sections as compared to standard histological methods.


Subject(s)
Abortion, Veterinary/parasitology , Cattle Diseases/parasitology , Placenta/parasitology , Protozoan Infections, Animal , Tritrichomonas/isolation & purification , Animals , Antibodies, Monoclonal/immunology , Cattle , Cross Reactions , Female , Fluorescent Antibody Technique , Microscopy, Phase-Contrast , Pregnancy , Protozoan Infections/parasitology , Tritrichomonas/immunology
4.
Dev Biol Stand ; 60: 63-72, 1985.
Article in English | MEDLINE | ID: mdl-3876250

ABSTRACT

KC 2000 TM is a serum-free medium designed and developed for use in all phases of hybridoma technology providing maximum growth and antibody production. This unique medium provides consistent results as its performance is not affected by variation found in serum. In addition, the use of KC 2000 TM reduces the risk of contamination of cultures by mycoplasma and viruses which may be present in low concentrations in serum. Antibody production and assays are performed in KC 2000 TM without high concentrations of interfering proteins. Only those proteins necessary to produce maximum performance in all hybridoma applications are present (250 micrograms protein/ml). Extensive testing has shown that this serum-free medium yields maximum results in all aspects of hybridoma technology, including the generation of antibody secreting hybridomas, as well as their subcloning. With a single exception, all cell lines tested could be transferred directly from medium containing serum into KC 2000 TM. Consistent growth and antibody production was maintained during subsequent passaging of these cells. These results show that KC 2000 TM is unique among serum-free media as it is the only serum-free medium that can be successfully utilized in the selection of HAT-resistant hybrid cell clones after use of a single type of medium throughout the entire process of development and maintenance of hybridoma cell lines.


Subject(s)
Culture Media , Hybridomas/cytology , Animals , Antibodies, Monoclonal/biosynthesis , B-Lymphocytes/immunology , Cell Division , Cell Line , Humans , Hybridomas/immunology , In Vitro Techniques , Lymphocyte Activation , Lymphokines/biosynthesis , Mice , T-Lymphocytes/immunology
5.
Exp Parasitol ; 55(3): 320-30, 1983 Jun.
Article in English | MEDLINE | ID: mdl-6602063

ABSTRACT

Using a sensitive, economical, and reproducible microassay, the relationship of toxoplasma inhibiting factor to interleukin 2 has been examined. The assay developed took advantage of the observation that (1) Toxoplasma gondii tachyzoites replicated efficiently in the murine monocytic cell line, RAW 264; (2) treatment of RAW 264 cells with toxoplasma inhibiting factor prevented intracellular replication of the parasite to an extent similar to that observed with identical treatment of freshly isolated murine peritoneal exudate cells; and (3) [3H]uracil incorporation was an efficacious means to quantify replication (or inhibition of replication) of tachyzoites within the cell line. Although toxoplasma inhibiting factor and interleukin 2 were both present in the same lectin- and antigen-stimulated splenocyte supernatant fluids, results from microassays strongly suggested that the molecules were two distinct entities.


Subject(s)
Interleukin-2/analysis , Lymphokines/analysis , Toxoplasma/immunology , Animals , Cell Line , Cells, Cultured , Immunization , Interleukin-2/pharmacology , Lymphokines/pharmacology , Mice , Mice, Inbred BALB C , Rats , Rats, Inbred F344 , Spleen/cytology , Toxoplasma/drug effects , Toxoplasma/growth & development
6.
Vet Immunol Immunopathol ; 3(4): 365-79, 1982 Jul.
Article in English | MEDLINE | ID: mdl-6981877

ABSTRACT

Bovine peripheral blood leukocytes were examined for blast transformation in response to T-cell lectins in serum-containing RPMI 1640 medium and serum-free Iscove's medium. Phytohemagglutinin-induced blastogenesis was significantly greater in Iscove's medium than in RPMI containing ten percent fetal calf serum. Concanavalin A-induced blast transformation was equivalent in both media. However, the kinetics of lectin response and the quantity of lectin required for optimum blastogenesis was considerably different in the two culture media. Concanavalin A-induced blast transformation of bovine thymocytes in Iscove's medium revealed that a concentration of 10(6) cells/ml, inconsequential blastogenesis ensued; but at 10(7) cells/ml blast transformation was significant and dose-dependent. Therefore, conditioned media from concanavalin A-stimulated bovine peripheral blood leukocytes, prepared in serum-free Iscove's medium, were assayed for costimulator activity using bovine thymocytes at 10(6) cells/ml in Iscove's medium as indicator cells. Both optimum lectin requirements and cell concentrations for production of costimulator activity were found. Conditioned medium, operated with the total exclusion of serum and with optimal costimulator activity, was fractionated via gel exclusion chromatography. A quantitative assay was described, and results indicated that bovine costimulator had an approximate molecular weight of 20,000 daltons.


Subject(s)
Interleukin-1/biosynthesis , Interleukin-2/biosynthesis , Lymphocyte Activation , T-Lymphocytes/immunology , Animals , Cattle , Culture Media , Interleukin-1/isolation & purification , Lectins/pharmacology
7.
Vet Immunol Immunopathol ; 3(4): 381-97, 1982 Jul.
Article in English | MEDLINE | ID: mdl-6181609

ABSTRACT

Bovine peripheral blood leukocytes, activated with concanavalin A, were cultured in bovine costimulator-containing conditioned medium prepared in a totally defined, serum-free medium. A population of leukocytes subsequently grew exponentially. These bovine cells had the morphology of lymphoblasts, were negative for chloroacetate esterase, slightly positive for conspecific esterases, and highly peanut agglutinin-positive. These data suggested that the bovine leukocytes were of the T-cell lineage and that the active factor in the costimulator-containing conditioned medium might be the bovine equivalent of interleukin 2. A quantitative microassay, subsequently developed, revealed that the lymphoblastoid cell line was costimulator-dependent and lectin-independent. Further utilization of the microassay supported this contention and strengthened the concept of a bovine interleukin 2-dependent bovine T-cell line: Phytohemagglutin-M, phytohemagglutinin-P, and concanavalin A induced active factor from peripheral blood leukocytes, while lipopolysaccharide, a potent inducer of Interleukin 1 in other systems, failed to induce activity; and both T-cells and macrophages were required for optimal factor activity. Finally, a means by which to optimize production of the active moiety, utilizing lymph node cells, as opposed to peripheral blood leukocytes, was examined.


Subject(s)
Interleukin-2 , Animals , Cattle , Cell Line , Growth , Histocytochemistry , Interleukin-2/analysis , Interleukin-2/metabolism , Interleukin-2/pharmacology , Lymphocytes/physiology , Lymphoid Tissue/metabolism , Mice , Staining and Labeling
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