Juan I. Azcona-Olivera

Effects of trichothecene structure on cytokine secretion and gene expression in murine CD4+ T-cells.

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.

Comparative assessment of fumonisin in grain-based foods by ELISA, GC-MS, and HPLC

Seventy-one (71) food samples were analyzed for the mycotoxin fumonisin by a monoclonal antibody based competitive enzyme-linked immunosorbent assay (ELISA). Fumonisins were detected primarily in corn-based products with 7/12, 2/2 and 1/3 and 1/7 yellow cornmeal, blue cornmeal, corn muffin mix, and mixed grain cereal samples yielding positive results, respectively. When the positive samples and randomly selected negative samples were assessed by other methods, correlations (r values) between ELISA and gas chromatography-mass spectrometry (GC-MS), ELISA and high-pressure liquid chromatography (HPLC) and GC-MS and HPLC were 0.478 (p < 0.05), 0.512 (p < 0.05), and 0.946 (p < 0.01), respectively. The results suggested that although the immunoassay could be used for screening of fumonisin in food samples, higher estimates were attained by ELISA than by the other two methods particularly in the more contaminated samples. These observations may result from differences in sample preparation among the methods or because of the presence of structurally related compounds in extracts that are detectable by ELISA but not the other two methods.

Effects of vomitoxin (Deoxynivalenol) and cycloheximide on IL-2, 4, 5 and 6 secretion and mRNA levels in murine CD4+ cells

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.

Vomitoxin (deoxynivalenol)-induced IgA nephropathy in the B6C3F1 mouse: dose response and male predilection.

Oral exposure to the trichothecene vomitoxin (VT or deoxynivalenol) in mice induces marked elevation of total and autoreactive IgA, IgA immune complexes, and mesangial IgA deposition in a manner that is highly analogous to human IgA nephropathy. In this study, immunopathologic markers indicative of IgA nephropathy were compared in male and female B6C3F1 mice fed semipurified AIN-76A diet containing 0, 2, 10 or 25 ppm VT for 12 weeks. Males fed 10 and 25 ppm VT and females fed 25 ppm VT had increased serum IgA at 4 weeks. At week 8, male mice fed the minimal dose of 2 ppm VT and female mice fed 10 ppm also exhibited elevated serum IgA. IgA levels were consistently higher in treatment males than females with significant differences being observed in the 10-ppm dose group at 4 and 12 weeks. IgA coproantibodies were marginally increased (maximum of 2-fold) in mice of both genders fed 10 and 25 VT. At 8 and 12 weeks, serum IgM was depressed in male and female mice eating 10 and 25 ppm VT, whereas consistent effects on serum IgG or IgE were not observed. In similar fashion, male mice in the 2, 10 and 25 ppm VT groups exhibited microscopic hematuria as early as 4 weeks, whereas this occurred in females fed 10 and 25 ppm VT only at week 10 with urinary erythrocyte counts being lower than male counterparts. Mesangial deposition of IgA and C3 was significantly increased in males exposed to 2, 10 and 25 ppm VT and in females exposed to 10 and 25 ppm VT, with males exhibiting a greater deposition than corresponding females. Based on these immunological parameters, males appeared more susceptible than female mice to VT-induced IgA dysregulation and IgA nephropathy in terms of latency, threshold dose, and severity.

Role of gender and strain in vomitoxin‐induced dysregulation of IgA production and IgA nephropathy in the mouse

Prolonged dietary exposure of female B6C3F1 mice to the trichothecene vomitoxin results in hyperproduction of immunoglobulin A (IgA) with a concurrent immunopathology that mimics human IgA nephropathy. To assess the role of gender and strain in the mouse model, semipurified AIN-76A diet containing 25 ppm vomitoxin was fed to B6C3F1 male mice and to B6C3F1, BALB/c, C3H/HeN, C3H/HeJ, and C57BL/6 female mice for 8 wk, and immunopathologic indicators of IgA nephropathy were compared to mice fed clean diet. At the cessation of the experiment, all treatment groups weighed less than respective controls. Serum IgA was increased in male and female B6C3F1 mice as well as in C3H/HeJ, C57BL/6, and BALB/c female mice compared to corresponding controls. Serum IgA levels were two- to sixfold higher in B6C3F1 male treatment animals compared to female treatment groups from all strains. In contrast, at wk 8 serum IgG levels were unaffected or decreased, and serum IgM was decreased in all groups at wk 8. There was a trend toward increased IgA production by Peyers patch (PP) lymphocytes isolated from treatment mice as compared to controls in all groups except the C3H/HeJ mice. Notably, IgA levels were 18-fold higher in B6C3F1 male treatment PP cultures than in B6C3F1 female treatment cultures. Hematuria was significantly greater in treatment mice than respective controls at both wk 4 and 8. Increased mesangial IgA deposition was also detectable in all treatment groups except the C57BL/6 mouse. The results suggested that the male B6C3F1 mouse and the five strains of female mice exhibited many of the immunopathologic effects found in IgA nephropathy and that IgA elevation was more marked in male B6C3F1 than female B6C3F1 mice.

Effects of intermittent vomitoxin exposure on body weight, immunoglobulin levels and haematuria in the B6C3F1 mouse

Continuous dietary exposure of female B6C3F1 mice to the trichothecene vomitoxin (VT) results in reduced body weight gain, elevated production of serum immunoglobulin A (IgA), kidney mesangial IgA deposition and glomerulonephritis. To assess whether intermittent consumption of dietary VT, as might occur during natural animal and human exposures, has similar effects to those for continuous consumption, a comparison was made between two schedules of dietary exposure. Female B6C3F1 mice were fed for 13 weeks with either a semipurified AIN-76A diet containing 20 ppm VT continuously or with 20 ppm VT intermittently (every other week). The effect these diets had on body weight gain, serum immunoglobulin (Ig) profile, mesangial Ig deposition and haematuria were assessed and compared with each other as well as with mice fed a control diet. Reduced body weight gains in the treatment groups were seen as early as 2 weeks. After week 4, the mean body weight of the intermittent group appeared higher than the continuous group during the weeks when it was fed a control diet, but dropped to continuous group levels during the weeks they were fed VT. Serum IgA levels in the intermittent group remained at control levels and were significantly lower than the continuous group during the course of the study. In contrast, serum IgG and serum immunoglobulin M (IgM) levels for the intermittent and continuous groups were significantly decreased compared with control. Mesangial IgA deposition was significantly lower in the intermittent group compared with the continuous group, and had levels comparable to mice on the control diet. Haematuria was significantly greater in both treatment groups compared with control at weeks 5 and 13 when the intermittent group was fed VT containing diet, but haematuria in the intermittent group dissipated at week 10 when it was fed control diet. The results presented here suggest that the type of dietary exposure regimen is critical in determining the extent of toxic effects induced by VT. Thus, when animal models are used for assessing the toxic effects of mycotoxins, it may be useful to consider the effects of intermittent and sporadic exposure.

Detection of fumonisins in Fusarium cultures, corn, and corn products by polyclonal antibody-based ELISA : Relation to Fumonisin B1 detection by liquid chromatography

A specific sheep polyclonal antiserum was applied to the competitive direct enzyme-linked immunosorbent assay (CD-ELISA) of fumonisins in Fusarium cultures, fresh corn, animal feed, and human foods, and the results were related to fumonisin B1 (FB1) levels determined by liquid chromatography (LC). The limit of detection of FB1 for the CD-ELISA was 0.1 ng/ml. Cross reactivity (defined as [ng/ml FB1 required for 50% inhibition]/[ng/ml of analogue required for 50% inhibition] × 100) for the antiserum in the ELISA was 100, 24, and 30%, for FB1 FB2, and FB3, respectively. FB1 was detected in F. moniliforme cultures grown on corn (≤760 μg/g) but not in those for F. graminearum . Fumonisin estimates were 2.8-fold higher by CD-ELISA than FB1 estimates by LC. Upon using the antiserum in a commercial CD-ELISA format (Veratox), mean recovery of FB1 from spiked corn (0.1 to 3 μg/g)was 85.2 ± 25.3%, whereas the mean recovery by LC was 74.1 ± 6.2%. Analyses of 43 fresh corn, 28 animal feed, and 14 human food samples revealed that estimates of total fumonisins by the commercial ELISA were 2.9-, 2.2-, and 1.3-fold higher than FB1 values determined by LC. For each sample type, the ELISA significantly correlated with LC (r > 0.9; P < 0.05). Taken together, the results suggest that the polyclonal antibody-based CD-ELISA could be a reliable first-tier screening procedure for fumonisins in corn, animal feed, and human food when used in conjunction with LC confirmation. The differences between the two methods suggests the further presence of FB2 and FB3 or structurally related compounds.

Effects of vomitoxin ingestion on murine models for systemic lupus erythematosus.

Dietary exposure to the trichothecene vomitoxin (VT) results in reduced body weight gain, elevated serum IgA, terminal differentiation of Peyers patch B cells to IgA secreting plasma cells haematuria, and increased kidney mesangial IgA accumulation in B6C3F1 mice and other inbred strains. These effects closely mimic a human autoimmune-like kidney disease known as IgA nephropathy. Using NZBW/F1, MRL/lpr, and BXSB mouse strains as models of systemic lupus erythematosus, we assessed whether consumption of diet containing 5 ppm or 10 ppm VT will similarly affect mice genetically prone to autoimmunity. Reduced weight gains were seen in NZBW/F1 and MRL/lpr mice fed both doses of VT within 2-3 weeks. In contrast, VT had little effect on weight gain by BXSB mice. Serum Ig levels in all three strains generally did not differ from control mice. Haematuria was significantly increased when all three strains were fed VT. In NZBW/F1 Peyers patch cultures stimulated with lipopolysaccharide (LPS), prior VT exposure significantly increased the IgG and IgM secretion but had no effect on IgA. In MRL/lpr Peyers patch cultures stimulated with LPS, VT exposure increased IgA secretion but not IgM or IgG. BXSB Peyers patch cultures prepared from VT treatment groups produced significantly more IgA than controls when cultured with LPS or Concanavalin A. Whereas mesangial deposition of IgA and IgG was significantly lower in the treatment groups of NZBW/F1 and MRL/lpr mice compared with control, BXSB mice had significantly higher IgA, IgG, and complement (C3) deposition when fed VT. The results suggest that although dietary VT differentially affected mice with aberrant immune systems, these strains did not appear to be any more sensitive to the mycotoxin than were more immunologically robust inbred strains.