concanavalin-a and deoxynivalenol

The

Topics: Animals; CD28 Antigens; CD8 Antigens; Cell Proliferation; Cell Survival; Concanavalin A; Dose-Response Relationship, Drug; Gene Expression; Lymphocyte Activation; Swine; T-Lymphocyte Subsets; Trichothecenes; Tumor Necrosis Factor Receptor Superfamily, Member 7

Deoxynivalenol (DON) is one of the most prevalent mycotoxins, contaminating cereals and cereal-derived products. Its derivative deepoxy-deoxynivalenol (DOM-1) is produced by certain bacteria, which either occur naturally or are supplemented in feed additive. DON-induced impairments in protein synthesis are particularly problematic for highly proliferating immune cells. This study provides the first comparison of the effects of DON and DOM-1 on the concanavalin A-induced proliferation of porcine, chicken, and bovine peripheral blood mononuclear cells (PBMCs). Therefore, isolated PBMCs were treated with DON (0.01-3.37 µM) and DOM-1 (1.39-357 µM) separately, and proliferation was measured using a bromodeoxyuridine (BrdU) assay. Although pigs are considered highly sensitive to DON, the present study revealed a substantially higher sensitivity of bovine (IC50 = 0.314 µM) PBMCs compared to chicken (IC50 = 0.691 µM) and porcine (IC50 = 0.693 µM) PBMCs. Analyses on the proliferation of bovine T-cell subsets showed that all major subsets, namely, CD4⁺, CD8β⁺, and γδ T cells, were affected to a similar extent. In contrast, DOM-1 did not affect bovine PBMCs, but reduced the proliferation of chicken and porcine PBMCs at the highest tested concentration (357 µM). Results confirm the necessity of feed additives containing DON-to-DOM-1-transforming bacteria and highlights species-specific differences in the DON sensitivity of immune cells.

Topics: Animals; Cattle; Cell Proliferation; Cells, Cultured; Concanavalin A; Leukocytes, Mononuclear; Poultry; Swine; Trichothecenes

In this study, the in vitro effects of the treatment of concanavalin A (Con A)--stimulated splenic lymphocytes with DON were examined. Splenic lymphocytes isolated from chickens were stimulated with 12.5 μg/mL Con A and exposed to deoxynivalenol (DON) (0-50 μg/mL) for 48 h. The intracellular calcium concentration ([Ca(2+)]i), pH, calmodulin (CaM) mRNA levels, and Na(+),K(+)-ATPase and Ca(2+)-ATPase activities were detected. With the DON exposure concentrations increased, the [Ca(2+)]i and CaM mRNA levels gradually increased in a dose-dependent manner, and all the evaluated conconcentrations affected ATPase activity to the same extent. There were significant differences (P<0.05 or P<0.01) between the treatment groups and the control group. These results indicate that an imbalance in calcium homeostasis and intracellular acidification are components of DON cytotoxicity in chicken lymphocytes.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Calmodulin; Cell Culture Techniques; Cells, Cultured; Chickens; Concanavalin A; Dose-Response Relationship, Drug; Environmental Pollutants; Homeostasis; Lymphocyte Activation; Lymphocytes; Real-Time Polymerase Chain Reaction; Sodium-Potassium-Exchanging ATPase; Spleen; Trichothecenes

Deoxynivalenol (DON) immunotoxicity and its induction of cytokines and related genes in the splenic lymphocytes of chickens have not been completely elucidated. In the present study, we aimed to evaluate the effects of 48 h of different DON treatments (0 μg/mL, 0.2 μg/mL, 0.8 μg/mL, 3.2 μg/mL, 12.5 μg/mL, and 50 μg/mL) on the secretion and the mRNA expressions of some cytokine genes, such as interleukin-1beta (IL-1β), IL-1RI, IL-2, IL-4, IL-6, IL-10, IL-12β, and IFN-γ in chicken splenic lymphocytes. The concentrations of IL-1RI, IL-6, IL-10, and IFN-γ were increased with the DON concentrations increasing (P<0.05 or P<0.01). However, the concentrations of IL-1β, IL-2, IL-4, and IL-12β were decreased with the DON concentrations increasing (P<0.05 or P<0.01). Except IL-1β, the mRNA expressions of the other cytokines were up-regulated by DON. The highest mRNA expressions values of IL-1RI, IL-4, IL-10, IL-12β, and IFN-γ were at 50 μg/mL DON treatment groups (P<0.05 or P<0.01), while the highest mRNA expressions values of IL-2 and IL-6 were at 12.5 μg/mL DON treatment groups (P<0.05 or P<0.01). Our data revealed that the potent effects of DON in affecting the secretion and the mRNA expression of the related cytokines in chicken splenic lymphocytes in vitro.

Topics: Animals; Chickens; Concanavalin A; Cytokines; Lymphocytes; Male; Primary Cell Culture; Spleen; Trichothecenes

The effects of four mycotoxins, T-2 toxin, deoxynivalenol (DON), ochratoxin A (OTA) and fumonisin B1 (FB1) on the response of bovine peripheral blood mononuclear cells (PBM) in vitro to the mitogens concanavalin A (Con A), phytohaemagglutinin A (PHA) and pokeweed mitogen (PWM) were assayed after 4 days' incubation using 3H-thymidine uptake and the MTT bioassay. The concentrations of mycotoxin required to reduce the proliferative response of PBM by 50% for Con A, PHA and PWM as measured by 3H-thymidine incorporation was for T-2 toxin 0.30, 0.40 and 0.18 ng ml-1; for DON 0.07, 0.09 and 0.04 microgram ml-1; for OTA 0.10, 0.20 and 0.15 microgram ml-1, and for FB1 35, 18 and 11 micrograms ml-1 respectively. The concentrations of mycotoxin required to reduce the proliferative response of PBM by 50% for Con A, PHA and PWM as measured by the MTT bioassay were for T-2 toxin 2.0, 2.0 and 1.0 mg ml-1; for DON 0.70, 0.50 and 0.50 microgram ml-1; OTA 1.5, 1.5 and 1.5 micrograms ml-1; and FB1 > 50, > 50 and 20 micrograms ml-1 respectively. Further cytotoxicity assays including the LDH bioassay and Trypan blue exclusion were performed only on Con A-stimulated PBM cells after 72 h incubation. With the LDH-bioassay the 50% inhibition levels were T-2 toxin 0.3 ng ml-1, DON 0.4 microgram ml-1, OTA 1.4 micrograms ml-1 and FB1 3.5 micrograms ml-1; for Trypan blue uptake the 50% inhibition levels were T-2 toxin 5 ng ml-1, DON 2.3 micrograms ml-1 and OTA 4 micrograms ml-1 respectively.

Topics: Animals; Carboxylic Acids; Cattle; Cell Division; Concanavalin A; Female; Fumonisins; Immunity, Cellular; Leukocytes, Mononuclear; Mitogens; Mycotoxins; Ochratoxins; Phytohemagglutinins; Pokeweed Mitogens; T-2 Toxin; Time Factors; Trichothecenes

The effects of continuous in vitro exposure to the trichothecene, vomitoxin (VT) or another protein synthesis inhibitor, cycloheximide (CHX), on interleukin (IL) secretion and mRNA levels were evaluated in murine splenic CD4+ cells. Significant increases were seen in supernatant IL-2, IL-4 and IL-5 obtained from 7 day Concanavalin A (Con A)-stimulated CD4+ cultures containing VT concentrations of 250, 100 and 100 ng/ml, respectively, compared with controls run in the absence of VT. The effect of VT on CD4+ cell proliferation was also assessed after culturing for 3, 5 and 7 days with Con A. Although total cell numbers were not affected at day 3, cultures at day 5 with 50 or more ng VT/ml and at day 7 with 100 or more ng VT/ml had significantly lower cell numbers than controls. In addition, viable cell number was unaffected at day 3, but was significantly decreased at day 5 by VT concentrations of 12.5 ng or more ml and at day 7 by 100 or more ng VT/ml. Elevations in IL-2, IL-4 and IL-5 were also observed in 7-day Con A-stimulated CD4+ cell cultures containing CHX at 50-100, 50 and 10 ng/ml, respectively. When CD4+ cells were stimulated with Con A in the absence of inhibitors and then subjected to reverse transcriptase-polymerase chain reaction coupled with Southern analysis, maximal IL-2, IL-4 and IL-6 mRNA levels were induced at 48 hr whereas peak IL-5 mRNA was observed at 72 hr. Superinduction of IL-2 mRNAs was observed in the presence of VT at 50-100 ng/ml and CHX at 50-250 ng/ml. IL-4 and IL-5 mRNAs were superinduced by VT at 100 ng/ml and CHX at 50 ng/ml. The results suggest that VT and CHX could superinduce both interleukin secretion and mRNA transcript levels in CD4+ cell cultures and that, for VT, these effects occurred concurrently with inhibition of cell proliferation.

Topics: Analysis of Variance; Animals; Blotting, Southern; CD4-Positive T-Lymphocytes; Cell Division; Cells, Cultured; Concanavalin A; Cycloheximide; Enzyme-Linked Immunosorbent Assay; Female; Interleukin-2; Interleukin-4; Interleukin-5; Interleukin-6; Interleukins; Mice; RNA, Messenger; Trichothecenes

The effects of trichothecene structure on cytokine secretion and gene expression were assessed in primary CD4+ T-cells from murine spleen. CD4+ T-cells were stimulated with concanavalin A (Con A) for 2 or 7 days in the presence of various concentrations of the trichothecenes, vomitoxin (VT or deoxynivalenol), nivalenol (NIV), 15-acetyl deoxynivalenol (15-ADON), 3-acetyl deoxynivalenol (3-ADON), T-2 toxin (T-2) and verrucarin A (Ver A). Culture supernatants were subsequently analyzed for interleukin (IL)-2, IL-4 and IL-5 by ELISA. At day 2, all trichothecenes were found to have inhibited production of IL-2, IL-4, and IL-5. However, at day 7, supernatant IL-2 was significantly increased (2-5.5-fold) in cultures containing VT, NIV, 3-ADON, and 15-ADON at 250, 250, 2500, and 1000 ng/ml doses, respectively, when compared to control Con A-stimulated cultures; significant increases in IL-2 were not observed with T-2 and Ver-A. Similarly, at day 7, IL-4 and IL-5 were significantly increased in the presence of VT (100 ng/ml), NIV (100 ng/ml), 3-ADON (1000 ng/ml), 15-ADON (500 ng/ml), T-2 (1 ng/ml), and Ver A (50 pg/ml, only IL-5) when compared to control cultures. IL production was inhibited at trichothecene concentrations exceeding the aforementioned optima. When total RNA of 2-day cultures was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) in conjunction with Southern analysis, IL-2 mRNA was also found to be superinduced by VT (50 and 100 ng/ml), NIV (50, 100 and 250 ng/ml), 3-ADON (1500 ng/ml), 15-ADON (100 ng/ml), T-2 (0.5 ng/ml) and Ver A (25, 50 and 100 pg/ml); IL-4 mRNA by VT (50 ng/ml), NIV (50 ng/ml), and Ver A (25, 50 and 100 pg/ml); IL-5 mRNA by VT (50 ng/ml); and IL-6 mRNA by 15-ADON (100 ng/ml) and Ver A (50 pg/ml). As the trichothecene concentration increased from these levels, inhibition of mRNA transcript levels was also observed for many of the interleukins. Taken together, the results suggest that trichothecenes as a group can either inhibit or superinduce both IL secretion and mRNA levels in CD4+ T-cells. Superinduction exhibited a rank order of macrocyclic > type A > type B trichothecenes and was dependent on acylation of the trichothecene nucleus.

Topics: Acylation; Animals; Antibiotics, Antineoplastic; Blotting, Southern; CD4-Positive T-Lymphocytes; Cells, Cultured; Concanavalin A; Cytokines; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Interleukin-2; Interleukin-4; Interleukin-5; Mice; RNA, Messenger; Structure-Activity Relationship; T-2 Toxin; Transcription, Genetic; Trichothecenes

The effects of 8 weeks of dietary exposure to the fungal toxin vomitoxin (25 ppm) on the kinetics of in vitro immunoglobulin (Ig) production and appearance of IgA-secreting cells in lymphocyte culture were assessed in the B6C3F1 mouse. The feeding regimen resulted in an IgA:IgG serum ratio of 2.4 compared to 0.4 in controls indicating that there was dysregulation of IgA production in the systemic compartment. Prior toxin feeding had no effect on viability of Peyer's patch (PP) or splenic lymphocyte cultures. IgA production, as determined by enzyme-linked immunosorbent assay, was significantly greater in treatment PP and splenic lymphocytes cultured for 2-11 days than in corresponding controls. Similar trends were found for IgG production in PP cultures although levels were much lower. There were 1.7 and 2.0 times more IgA-producing cells, as measured by the ELISPOT assay, in freshly prepared PP and splenic lymphocytes from treatment mice compared to control mice, respectively. In contrast, after 2 days there were 10.9, 3.2, and 12.4 times more IgA-secreting cells in concanavalin A (Con A), LPS, and unstimulated treatment PP cultures, respectively, and 4.0, 2.0, and 3.5 times times more IgA-secreting cells in 2-day treatment spleen cultures, respectively. Both IgA and IgG secretion in Con A-stimulated cultures were significantly greater when treatment T cells and control B cells were combined than when control T cells and control B cells were combined. Increased Ig secretion attributable to T cell effects was not observed in LPS-stimulated or unstimulated PP reconstitution cultures or in spleen reconstituted cultures with and without mitogen. The results provide evidence that dietary vomitoxin enhances terminal differentiation of IgA secreting cells in PP. This and resultant migration of IgA secreting cells into the systemic compartment favor a shift from IgG to IgA as the primary serum isotype.

Topics: Animals; B-Lymphocytes; Cells, Cultured; Concanavalin A; Diet; Female; Immunoglobulin A, Secretory; Mice; Peyer's Patches; Spleen; T-Lymphocytes; Trichothecenes

Prolonged dietary exposure of mice to the trichothecene vomitoxin induces abnormally high levels of serum IgA and kidney mesangial IgA accumulation in a manner that is highly analogous to the human glomerulonephritis IgA nephropathy. In this study, the capacity of Peyer's patch and splenic lymphocytes to produce IgA and IgG were compared in B6C3F1 mice that were fed diets with and without 25 ppm vomitoxin for up to 12 wk. Serum IgA increased 2-, 4- and 8-fold after 4, 8 and 12 wk, respectively, of vomitoxin exposure and it became the primary serum isotype, whereas serum IgG was unaffected. On termination of the experiment there were increased numbers of IgA-secreting cells in Peyer's patches after 8 wk of toxin exposure and in the spleen after 4, 8 and 12 wk of toxin exposure. There were also increased numbers of IgG-secreting cells in Peyer's patches on termination of the experiment at 4, 8 and 12 wk but no effects was observed in the spleen. Supernatant IgA and IgA-secreting cell numbers were also markedly elevated in lymphocyte cultures obtained from Peyer's patches and, to a lesser extent, from spleens of treated mice compared with controls. Based on output of treated mice relative to corresponding controls, IgA secretion was greatest in concanavalin-A-stimulated and unstimulated Peyer's patch cultures. Enhanced IgG secretion and IgG-secreting cells were also observed in mitogen-stimulated and unstimulated Peyer's patch lymphocyte cultures of treated relative to control mice, but differences in splenocyte cultures were negligible. Based on total Ig output, IgA production was 8- to 20-fold greater than IgG production in both control and treatment Peyer's patch cultures. In contrast, vomitoxin treatment caused a shift from primarily IgG production in lipopolysaccharide-stimulated spleen cultures to equivalent IgA production. These data provide in vitro evidence that ingestion of vomitoxin promotes terminal differentiation of IgA-secreting progenitors in the Peyer's patch and, to a lesser extent, in the spleen. These functional changes are consistent with the shift from IgG to IgA as the primary serum isotype.

Topics: Animals; Cells, Cultured; Concanavalin A; Female; Immunoglobulin A; Immunoglobulin G; Lipopolysaccharides; Lymphocytes; Mice; Peyer's Patches; Random Allocation; Spleen; Trichothecenes

Topics: Animals; Concanavalin A; Cycloheximide; In Vitro Techniques; Interleukin-1; Lymphocyte Activation; Macrophages; Rats; Sesquiterpenes; Trichothecenes