Bovine Peripheral Lymphocytes Research Articles

Effect of N-metylcarbamate pesticide bendiocarb on cattle lymphocytes afterin vitroexposure

Bendiocarb is a carbamate broad-spectrum insecticide used to control disease vectors such as mosquitoes and flies, as well as household and agricultural pests. Nowadays, only few papers reporting cytogenetic or possible genotoxic effect of this insecticide on mammalian cells are available. In the present study 24-hour exposure to bendiocarbamate at concentrations ranging from 20 to 160 μg/ml was used for investigation of unstable chromosomal aberrations (CA), sister chromatid exchanges (SCE) and stable chromosomal aberration induction in cultured bovine peripheral lymphocytes. The slight but no significant increase of chromatide breaks frequency was observed after the exposure of lymphocytes to 80 μg/ml of bendiocarb. At the highest concentration added to the cell cultures (160 μg/ml) mitotic index decrease was shown in both donors (p < 0.05; p < 0.01). Both statistically significant elevation of SCEs (p < 0.05) and a reduction of proliferative indices (PI) (p < 0.01) were shown at a dose of 80 μg/ml. By means of two fluorescent-labelled whole chromosome-painting probes, stable aberrations such as bovine chromosome 1 and 5 translocation as well as numerical aberrations (polyploidies, heteroploidies) were visualised under fluorescent microscope in some examined metaphases.

Escherichia coli Shiga toxin 1 enhances il-4 transcripts in bovine ileal intraepithelial lymphocytes

Shiga toxin 1 (Stx1) blocks the activation of bovine peripheral and intraepithelial lymphocytes (IEL), implying that the toxin has the potential to retard the host's immune response during intestinal colonization of cattle with human pathogenic Stx-producing Escherichia coli (STEC). Since Stx1 does not eliminate affected lymphocytes by causing cellular death, we assumed that Stx1 disturbs the integrity of the immune regulatory network. We therefore assessed the impact of Stx1 on the expression of selected chemokine and cytokine genes in vitro by real-time RT-PCR and by quantitation of intracellular cytokine proteins. While Stx1 did not alter the amount of mRNA specific for interleukin (IL)-2, IL-10, gamma interferon (IFN-γ), transforming growth factor beta (TGF-β), IL-8, 10 kDa interferon inducible protein (IP-10), and monocyte chemoattractant protein 1 (MCP-1) in cultured ileal IEL (iIEL), minute concentrations of Stx1 led to an up to 40-fold increase of il-4 transcripts within 6–8 h of incubation. Comparative experiments with peripheral lymphocytes revealed that the effect was specific for iIEL. The enhancement of il-4 transcripts in iIEL was not accompanied by apoptosis but required the enzymatic activity of the holotoxin. Nevertheless, iIEL retained their ability to synthesize proteins in the presence of Stx1: 40% of iIEL could be stimulated to synthesize IFN-γ while less than 10% expressed IL-4 or TGF-β. Furthermore, iIEL were found to produce granulocyte chemoattractants, but the release of these substances was not different in iIEL cultures incubated with or without Stx1. Although Stx1 did not affect the numbers of iIEL producing either cytokine, these findings point to an altered responsiveness of IEL during bovine STEC infections and shed light on the initial effects Stx1 exerts on the local adaptive immune system.

Cytogenetic effect of technical glyphosate on cultivated bovine peripheral lymphocytes

A technical herbicide containing isopropyl amine salt of glyphosate was tested for induction of chromosome aberrations (CA) and sister chromatid exchanges (SCE) in cultured bovine peripheral lymphocytes. Cultures were exposed to a glyphosate formulation at concentrations ranging from 28 to 1120 μmol/l without and with metabolic activation. No clastogenic effect of the herbicide was found. Its genotoxic effect was confirmed in the SCE assay after 24 h of incubation. A statistically significant elevation in SCE induction was observed in each of the donors after application of the product at doses ranging from 56 to 1120 μmol/l. The highest concentrations (560 and 1120 μmol/l) also caused reduction of mitotic and proliferation indices. In the 2 h-assay with metabolic activation a statistically significant frequency of SCE was observed only in cultures treated with the agent at a concentration of 140 μmol/l.

The effect of glyphosate on the frequency of micronuclei in bovine lymphocytes in vitro

Glyphosate is a widely used broad-spectrum herbicide that has expanded its applications on plant varieties that are genetically modified to tolerate glyphosate treatment. The aim of this study was to determine the frequency of micronuclei (MNi) in bovine peripheral lymphocytes after exposure to glyphosate in vitro. The cytokinesis block micronucleus assay (CBMN) for estimation of genotoxic activity was used. The obtained results indicate that glyphosate weakly induced micronuclei in bovine peripheral lymphocytes. Significant elevations of MNi (p <0.05) were observed at concentrations of glyphosate of 280 μW and 560 μW, respectively. Treatment of bovine lymphocytes did not result in the induction of micronuclei in a dose-dependent manner. From cytotoxicity data it is evident that CBPI does not reflect the reduction of cell proliferation. The influence of metabolic activation on the genotoxic activity of glyphosate was investigated, too. When lymphocyte cultures were treated with glyphosate together with a liver membrane fraction (S9) from Aroclor 1245-induced rat liver, the number of micronuclei in binucleated cells did not increase significantly.

Bovine ileal intraepithelial lymphocytes represent target cells for Shiga toxin 1 from Escherichia coli.

The discovery that bovine peripheral lymphocytes are sensitive to Stx1 identified a possible mechanism for the persistence of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) in the bovine reservoir host. If intraepithelial lymphocytes (IEL) are also sensitive to Stx1, the idea that Stx1 affects inflammation in the bovine intestine is highly attractive. To prove this hypothesis, ileal IEL (iIEL) were prepared from adult cattle, characterized by flow cytometry, and subjected to functional assays in the presence and absence of purified Stx1. We found that 14.9% of all iIEL expressed Gb(3)/CD77, the Stx1 receptor on bovine lymphocytes, and 7.9% were able to bind the recombinant B subunit of Stx1. The majority of Gb(3)/CD77(+) cells were activated CD3(+) CD6(+) CD8 alpha(+) T cells, whereas only some CD4(+) T cells and B cells expressed Gb(3)/CD77. However, Stx1 blocked the mitogen-induced transformation to enlarged blast cells within all subpopulations to a similar extent and significantly reduced the percentage of Gb(3)/CD77(+) cells. Although Stx1 did not affect the natural killer cell activity of iIEL, the toxin accelerated the synthesis of interleukin-4 (IL-4) mRNA and reduced the amount of IL-8 mRNA in bovine iIEL cultures. Because the intestinal system comprises a rich network of interactions between different types of cells and any dysfunction may influence the course of intestinal infections, this demonstration that Stx1 can target bovine IEL may be highly relevant for our understanding of the interplay between STEC and its reservoir host.

Mitotic Index and Cell Proliferation Kinetics as Additional Variables for Assessment of Genotoxic Effect of The Herbicide Modown

·ivikova K. , J . Dianovsk : Mitotic Index and Cell Proliferation Kinetics as Additional Variables for Assessment of Genotoxic Effect of The Herbicide Modown. Acta Vet. Brno 2000, 69: 45–50. The in vitro effect of the herbicide Modown (with active component bifenox) was tested for the ability to influence cell proliferation of PHA-stimulated bovine peripheral lymphocytes. Mitotic (MI) and proliferation (PI) indices were determined as an alternative for the screening of the cytostatic activity. The herbicide Modown exerted a clear effect on the inhibition of MI and PI over a concentration-tested range of 25 μg/ml to 1000 μg/ml. An expressive proliferation delays was found after the treatment with herbicide at a dose of 250 μg/ml (P < 0.001), while the higher doses of 500 and 1000 μg/ml had caused nearly complete mitotic inhibition in each donor (P < 0.05 and P < 0.001, respectively). A correlation between the PI and MI inhibition refers rather to cytostatic than cytotoxic effects of the herbicide. The results support the possibility of immunosuppression by herbicide exposure. Herbicide Modown, bovine peripheral lymphocytes, cell proliferation, cytostatic effect Nitrodiphenylether herbicides are persistent and lipophilic environmental contaminants may contribute to the impairment of animal health and production. The presence of pesticide residues has been demonstrated in raw bovine milk and in different domestic animal tissues (Lioi et al. 1998). All nitrodiphenylether herbicides exert their phytotoxic activity in chlorophyll of plants by the reduction itself to radical species, which initiate destructive reactions in membrane lipids, leading to the cell leakage (Corbet t et al. 1984). Members, as acifluorfen and oxyfluorfen, e.g. are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane–bound enzyme involved in the heme and chlorophyll biosynthesis pathways (Camadro et al. 1995). The later was studied for the induction of haematological diseases in human erythroid progenitor cells but a cytotoxic effect had been seen only at very high concentrations (Rio et al. 1997). Bifenox is a selective herbicide used in control of annual broad-leaved weeds and some grasses, e. g. in cereals, maize, soya beans, rice and other crops. J inno et al. (1999) described the cytotoxic and porphyrogenic effects of bifenox in rat hepatocytes. They found the maximum porphyrin accumulation at 0.25 mM of the herbicide and an inhibion of protoporphyrogen oxidase, resulting in the accumulation of protoporphyrin IX. Francis et al. (1999) evaluated maternal and developmental toxicity of ten diphenylether herbicides (including bifenox) in mice. They found a correlation among the position of chlorine substituents and the potential for inducing prenatal and postnatal syndroms. But no prenatal and postnatal embryotoxicity was shown. ACTA VET. BRNO 2000, 69: 45–50 Address for correspondence: RNDr. Katarina ·ivikova, CSc. Department of Veterinary Genetics, University of Veterinary Medicine Komenskeho 73, 041 81 Ko‰ice, Slovak Republic Phone: +421 95 63 321 11, (ext. 482) Fax: +421 95 63 236 66 E-mail: sivikova@uvm.sk http://www.vfu.cz/acta-vet/actavet.htm

Genotoxic activity of the commercial herbicide containing bifenox in bovine peripheral lymphocytes

The commercial herbicide with active element bifenox (principal tradename Modown) was tested for the evaluation of genotoxicity in cultured cow peripheral lymphocytes in vitro. Several cytogenetic endpoints as chromosome aberrations (CA), sister chromatid exchanges (SCE), mitotic (MI) and proliferation (PI) indices were investigated in different sampling times. To detect possible metabolic modifications in herbicide genotoxicity, the cultures for SCE determination were also treated with S9 fraction. Cultures of lymphocytes were exposed to the herbicide at concentrations of 25, 50, 250, 500 and 1000 μg/ml. A slight increase of CAs was found after exposure of this agent to doses ranging from 25 to 250 μg/ml for 24 h. In the CA assay no statistical significance was seen. Both higher doses (500 and 1000 μg/ml) caused a decrease of chromosome damage in comparison to the last active dose or control values correlated to induced cytotoxicity. Four concentrations (all except the highest one) of the herbicide were applied into cultures in SCE assays both with and without metabolic activation. Significant elevations of SCE were observed after applications of herbicide tested at doses of 250 and 500 μg/ml in each donor (P<0.001 and P<0.05, respectively) for 24 h. These concentrations also caused a statistically significant decrease in the MI and PI. Treatment for 48 h provided inadequate evidence for the genotoxic activity of the herbicide.

Chromosome Damage in Cultured Bovine Peripheral Lymphocytes Induced by Herbicide Chloridazon

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Proto-oncogene expression in bovine peripheral blood leukemic lymphocytes during their spontaneous proliferation, differentiation and apoptosis in vitro

The expression of various proto-oncogenes in primary culture of lymphocytes from peripheral blood of bovine with chronic lymphocytic leukemia (CLL) was studied. Cellular proto-oncogenes encode proteins that propagate growth, differentiation or apoptosis signals from cell membrane to nucleus. The proliferation and differentiation of normal eukaryotic cells are precisely controlled. Tumor cells usually are characterized both by the continuous growth signal and by the block of cell differentiation. We have previously reported that along with spontaneous proliferation, bovine CLL lymphocytes continuously differentiate and enter apoptosis in vitro. CLL cells with an autocrine growth mechanism and at the same time undergoing spontaneous differentiation and apoptosis in vitro provide a new model system to investigate the possible involvement of various proto-oncogenes in the regulation of cellular proliferation, differentiation and apoptosis. Northern blot analysis revealed simultaneous expression of a number of proto-oncogenes in CLL cells. Transcripts of c-fos, c-myc, c-myb, Araf, c-raf1, hck, IL-2 receptor α-chain ( IL-2 Rα) were found in lymphocytes at the peak of their proliferative activity in culture. Kinetics studies demonstrated that CLL cells constitutively express transcripts of so-called immediate response nuclear proto-oncogenes c-myc, c-fos as well as cytoplasmic proto-oncogenes hck and c-raf1, i.e., genes coding for tyrosine and serine-threonine protein kinases, respectively. Expression level did not change significantly during all stages of CLL cells in culture. The results show that continuous expression of c-myc mRNA does not prevent CLL cell differentiation and may be associated with apoptotic cell death.

Dairy cattle as a bioindicator of exposure to genotoxic substances in a heavily polluted area in Northern Bohemia

The exposure of dairy cattle to genotoxic agents in two districts with different levels of environmental pollution was estimated using cytogenetic analysis of bovine peripheral lymphocytes. The Teplice district represented an industrialized area where the air pollution rate is extremely high mainly in the winter, and the Prachatice district – an agricultural area with a relatively low level of pollution. The Ames test was used to examine feed samples for the content of mutagenic substances. Cows in the Teplice district showed a significantly higher count of aberrant cells (4.83±2.36) than cows in the Prachatice district (3.63±2.12). The sum of revertants induced by rinsings or extracts of feeds in both of the two test strains ( Salmonella typhimurium TA 98 and TA 100) was significantly higher in the district of Teplice than in the district of Prachatice. The percentages of findings with mutagenic responses were 56.3 and 34.8% for the districts of Teplice and Prachatice, respectively. No mutagenic activity was found in milk samples collected in any of the districts. Apparently, the cows kept in the Teplice district were more exposed to genotoxic substances than the cows in the Prachatice district. The major source of this exposure was probably fresh fodder contaminated by industrial emissions.

T cell-mediated destruction of bovine leukemia virus-infected peripheral lymphocytes by bovine leukemia virus env-vaccinia recombinant vaccine.

T cell-mediated destruction of bovine leukemia virus-infected peripheral lymphocytes by bovine leukemia virus env-vaccinia recombinant vaccine.

Immunomodulatory effects of fusarochromanones TDP-1 and TDP-2.

An in vitro peripheral lymphocyte blastogenesis system was used to investigate the biological activities of the fungal toxin fusarochromanone (TDP-1) and its monoacetyl derivative TDP-2. Briefly, cultures of human or bovine peripheral lymphocytes were exposed to TDP-1 or TDP-2 and a mitogen (PHA, Con A or PWM). After a standard incubation time, cell proliferation was quantified using the MTT bioassay. Human and bovine lymphocyte proliferation was inhibited by high concentrations of TDP-1; however, bovine lymphocyte proliferation was significantly increased at low concentrations of TDP-1. TDP-2 has similar but less pronounced effects on lymphocyte proliferation.

Co‐mitogenic assay for assessing the effects of aflatoxin B1 on interleukin‐1 production in bovine macrophages

AbstractPrevious research has shown that aflatoxin B1 (AFB1) markedly decreases the uptake of tritiated thymidine in bovine peripheral lymphocytes. Currently, the immunosuppressive activities of aflatoxin B1 are being investigated further using bovine macrophages as a model cell type. Polystyrene‐adhered macrophages demonstrated a 50% reduction in up‐take of tritiated thymidine at 10 μg/mL aflatoxin B1. The distribution of 3H‐AFB1 in bovine macrophages was investigated to study mechanisms by which AFB1 may be exerting immunosuppressive effects. Only 12.96% of the incorporated 3H‐AFB1 was taken into nuclear DNA, whereas 80.13% remained in the cytosolic fraction. A co‐mitogenic assay was developed to ascertain the correlation between reduced tritiated thymidine uptake, binding of 3H‐AFB1 to nuclear DNA, and interleukin‐1 (IL‐1) production and/or activity. Conditioned media containing interleukin‐1 activity was prepared by culturing macrophages for 24 hr with 2.5 μg/mL lipopolysaccharide, 45 ng/mL phorbol myristate acetate, with or without 10 μg/mL aflatoxin B1. The conditioned media was used in a co‐mitogenic assay with calf thymocytes as target cells. In this assay, the response of calf thymocytes to conditioned media from AFB1‐treated cells did not differ from the response to control conditioned media. In addition, AFB1 added to thymocyte cultures co‐stimulated with conditioned media and concanavalin A (ConA) greatly diminished the thymocyte response to co‐stimulation. The data support the conclusions that the immunosuppressive effects of AFB1 on the bovine macrophage and T lymphocyte may not be directly related via impaired IL‐1 production. The blinding of AFB1 to macrophage DNA was not sufficient to produce any detectable effect on interleukin‐1 production by bovine macrophages.

Bovine monoclonal antibody specific for Brucella abortus lipopolysaccharide

The development of a bovine monoclonal antibody against Brucella abortus smooth lipopolysaccharide (BM-8) by interspecies fusion of bovine peripheral lymphocytes from an immunized cow and a murine plasmacytoma cell line is described. The twice cloned cell line secreted bovine IgG 1 subclass antibody. Ascites fluid was prepared in pristane treated nu/nu mice by intraperitoneal injection. The pooled ascites fluid was purified by affinity chromatography and the functions of the antibody assessed in various serological tests. The BM-8 antibody did not agglutinate well at a neutral pH, however, under acid conditions it was efficient at agglutinating B. abortus cells. The antibody did not precipitate B. abortus LPS in double agar gel immunodiffusion but was very active in the direct complement fixation test and the indirect enzyme immunoassay, although it was unable to compete with a murine monoclonal antibody in a competitive enzyme immunoassay.

In vitro reactivity to concanavalin A of bovine lymphocytes after cryopreservation

Cryopreservation of bovine peripheral lymphocytes and its effect on the in vitro response to concanavalin A tested in a microculture system is described. Using DMSO as cryoprotectant in the medium, the cells were cooled to −30°C at 1.3°C/minute and further to −80°C at 6°C/minute and then rapidly to −196°C by dropping in liquid nitrogen. The cells were recovered by rapid thawing in water at 30–35°C and washed twice before use in the stimulation test. Ten percent DMSO had a much better protective effect than 5%; addition of 25% fetal bovine serum to the freezing had no favourable effect. In most of the 16 animals used in the experiments the frozen lymphocytes gave the same or a higher response to Con A than those kept in the DMSO containing medium at 4°C for two hours. The responses of the frozen cells were comparable to those of fresh lymphocytes (kept at 4°C for two hours in medium without DMSO).