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 Peyer's 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 Peyer's 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 Peyer's patch cultures stimulated with LPS, VT exposure increased IgA secretion but not IgM or IgG. BXSB Peyer's 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.

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.

Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development.

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.

Vomitoxin-mediated IL-2, IL-4, and IL-5 superinduction in murine CD4+ T cells stimulated with phorbol ester calcium ionophore: relation to kinetics of proliferation.

The effects of the trichothecene vomitoxin (VT) on the kinetics of cell proliferation and cytokine production were evaluated in murine CD4+ T cells. The CD4+ cultures were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to activate protein kinase C and increase cytoplasmic free calcium, respectively, in a range of VT concentrations. Total and viable cell counts at 3, 5, 7, 9, and 11 days revealed delayed or impaired cell proliferation in cultures containing between 50 and 100 ng/ml VT, with complete inhibition being observed at 250 and 500 ng/ml of VT. The VT concentration required to inhibit protein synthesis in a 3-day culture by 50% in this model was 40 ng/ml. When enzyme-linked immunosorbent assay (ELISA) was used to quantitate cytokines, IL-2 levels in control cultures were highest at Day 1 and declined rapidly thereafter, whereas, in VT groups, IL-2 levels were highest at Day 3 and remained elevated up to 11 days. IL-2 levels were elevated by continuous exposure to 100-500 ng/ml of VT with more than 100-fold differences being observed between control and 250 ng/ml VT from Days 5 to 11. When IL-2 levels were expressed on a per viable cell basis, increases were even more marked with as much as 6 log differences being observed between the treatments at 250-500 ng/ml VT and control cultures at Day 7. Supernatant IL-4 and IL-5 levels were also elevated by 100 and 250 ng/ml VT in a dose- and time-dependent fashion compared to control cultures, whereas 500 ng/ml VT was inhibitory. When relative IL mRNA abundance was analyzed during the first 3 days of culture by reverse transcriptase-polymerase chain reaction (RT-PCR) in conjunction with Southern hybridization analysis, IL-2 mRNA levels in Days 1, 2 and 3 in cultures containing 100 and 250 ng/ml VT were greater than corresponding controls. IL-2 mRNA abundance in both control and VT-treated cultures was maximal at Day 1 and decreased rapidly thereafter in controls, whereas much slower rates of IL-2 disappearance were noted in 100 and 250 ng/ml of VT. IL-4 and IL-5 mRNA levels at VT doses of 50 and 100 ng/ml were also elevated compared to controls. Pulsed VT (8 to 48 hr) or cycloheximide (4 to 48 hr) exposure of CD4+ cells enhanced supernatant levels of IL-2 but not IL-4 upon incubation for 24 hr in fresh medium. This effect was not persistent. Taken together, VT enhanced and/or delayed peak IL-2, IL-4, and IL-5 gene expression and secretion in CD4+ T cells stimulated with PMA and ionomycin. Remarkably, cytokine superinduction occurred simultaneously with partial or maximal inhibition of cell proliferation.

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.

Induction of cytokine mRNAs in mice after oral exposure to the trichothecene vomitoxin (deoxynivalenol): relationship to toxin distribution and protein synthesis inhibition.

The effects of oral exposure to 0, 5, and 25 mg/kg body wt vomitoxin (VT) on cytokine mRNA levels in spleen, Peyer's patches (PP), liver, kidney, and small intestine were evaluated in B6C3F1 mice at 2 and 4 hr postexposure using RT-PCR in conjunction with Southern hybridization analysis. The abundance of mRNAs for several cytokines was increased in VT-exposed mice with maximal effects occurring in the 25 mg/kg group at 2 hr. Specifically, IL-1 beta and IL-6 mRNA levels increased in spleen and PP following exposure to VT. TNF-alpha mRNA levels were markedly elevated in spleen and liver of VT-exposed mice. TGF-beta mRNA was increased in treatment kidneys and to a lesser extent in liver and small intestine. IFN-gamma mRNAs were elevated according to the rank order: spleen > PP > small intestine > liver > kidney, whereas IL-2 mRNAs were increased primarily in spleen and PP. VT had little effect on abundance of mRNAs for the TH2 cytokines, IL-4 and IL-5, or the housekeeping gene, hypoxanthine guanine ribosyl transferase. In order to relate cytokine mRNA abundance to toxin distribution, mice were administered 5 and 25 mg/kg VT body wt containing [3H]VT and tissue levels were monitored over time. Maximum VT molar equivalents for both doses were found at 30 min or 1 hr in all tissues with a rapid clearance following two-compartment kinetics over 24 hr. When effects of oral VT exposure on in vivo protein synthesis at 3 hr postexposure was measured using [14C]leucine uptake, it was found to be inhibited by > or = 20 and > or = 50% in tissues of mice receiving 5 and 25 mg/kg VT, respectively. While recovery was observed in tissues of the 5 mg/kg group at 6 hr, protein synthesis was still significantly inhibited (> or = 70%) at 9 hr for all tissues in the 25 mg/kg group. The results suggest that acute oral VT exposure resulted in the transient elevation of mRNAs for proinflammatory and TH1 cytokines. These effects occurred immediately after peak VT accumulation and concurrently with marked in vivo protein synthesis inhibition.

Effects of dihydrotestosterone and estradiol on experimental IgA nephropathy induced by vomitoxin.

Ingestion of the trichothecene vomitoxin (VT) by mice induces effects that mimic the common human glomerulonephritis, IgA nephropathy (IgAN). These include elevation of serum IgA, IgA immune complexes, and mesangial IgA deposition. Based on previous observations that male mice are more prone to VT-induced IgAN, the effects of castration of male and female B6C3F1 mice and sex hormone supplementation on several immunopathologic indicators of the disease were compared. In the first study, castrated and intact male and female mice were fed control AIN-76A diet or the same diet containing 10 ppm VT for 12 weeks. At Week 12, all but the intact female group fed VT exhibited increased serum IgA, with castrated female mice having greater levels than intact females. When microscopic hematuria was used as an indicator of disease severity in intact VT-fed mice, erythrocyte counts for males exceeded those for females at weeks 4 and 12. VT-fed, castrated females exhibited greater hematuria than intact counterparts, whereas VT-fed, castrated males had lower urinary erythrocyte counts than intact counterparts. In a second study, castrated male and female mice were implanted with controlled release pellets of placebo, 5 alpha-dihydrotestosterone (DHT), or 17 beta-estradiol (E2) and then were fed either control diet or a 10 ppm VT diet for 8 weeks. Castrated male and female mice treated with VT and DHT pellet exhibited more severe hematuria, higher IgA levels, and greater mesangial IgA deposition than mice exposed to the same diet with placebo or E2 pellet at Week 8. While VT-fed animals with an E2 pellet exhibited greater hematuria and mesangial IgA deposition at Week 8 than the placebo groups, their IgA levels were not significantly elevated over those for VT-fed mice with a placebo pellet. Relative to two other pathologic markers for IgAN, the aforementioned effects in both studies were generally consistent with mesangial deposition of complement component C3 but not IgG. The results suggest that (1) enhanced male susceptibility to VT-induced IgAN may be related to modulation by the biologically active androgen DHT and (2) while castration of females increased severity of VT-induced IgAN, supplementation of castrated male or female mice with E2 did not reverse this effect but rather increased disease severity.

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 Peyer's 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.

Elevated gene expression and production of interleukins 2, 4, 5, and 6 during exposure to vomitoxin (deoxynivalenol) and cycloheximide in the EL-4 thymoma.

Aberrant elevation of serum IgA and induction of murine IgA nephropathy following dietary exposure to the naturally occurring trichothecene vomitoxin (VT or deoxynivalenol) may involve dysregulation of cytokine production at the T cell level. EL4.IL-2 (EL-4), a cloned thymoma that produces interleukins (IL)-2, 4, 5, and 6, was used as a T cell model to investigate the in vitro effects of VT on interleukin production and gene expression. When supernatants of cells stimulated with phorbol 12-myristate 13-acetate (PMA) were assessed by enzyme-linked immunosorbent assay, IL-2, 4, and 5 were increased in the presence of 50 and/or 100 ng/ml VT for 2 and/or 8 days of culture. IL-2, 5, and 6 were also significantly elevated in the presence of 10-100 ng/ml of cycloheximide (CHX), another protein synthesis inhibitor, after 8 days of culture. As demonstrated by Northern analysis, VT at the levels between 50 and 100 ng/ml superinduced IL-2, 4, 5, and 6 mRNAs in PMA-stimulated EL-4 cells during a 24 hr culture period. Similar effects in PMA-treated samples were observed for CHX at 50, 100, 250, 1000, and 10000 ng/ml. mRNA levels for both IL-4 and IL-5, but not IL-2 and IL-6, were increased in unstimulated EL-4 cultures exposed to 50 and 100 ng/ml VT for 48 hr when analyzed by reverse transcriptase-polymerase chain reaction. Using [3H]leucine incorporation as a measurement of protein synthesis, IC50s for VT and CHX were estimated to be 280 and 55 ng/ml, respectively. This study indicates that VT as well as CHX could increase production of several interleukins in the EL-4 model even when present at concentrations that partially inhibited protein synthesis, whereas IL mRNA superinduction occurred across a broader range of concentrations that included maximal protein synthesis inhibition.

Production of polyclonal antibodies to the trichothecene mycotoxin 4,15-diacetylnivalenol with the carrier-adjuvant cholera toxin.

The trichothecene mycotoxin 4,15-diacetylnivalenol (DNIV) was conjugated to cholera toxin (DNIV-CT) for use as an immunogen and as an adjuvant for specific antibody production. Repeated intravenous injection of 7.5 micrograms of the conjugate was effective at generating specific antibodies to DNIV in rabbits as determined by enzyme-linked immunosorbent assay (ELISA). When small amounts (1 to 10 micrograms per animal) of DNIV-CT were used to immunize mice, polyclonal antibodies were observed as early as 4 weeks of immunization. The relative affinity of the antibodies to DNIV increased with the immunogen dose in mice. Antibodies were not detectable in either rabbits or mice that were injected with DNIV conjugated to the carrier protein bovine serum albumin or when DNIV-CT was blocked with glutaraldehyde. Competitive ELISA of mouse and rabbit serum revealed that the antibodies were most specific for DNIV but reacted to a small extent with fusarenone-X, deoxynivalenol, and nivalenol. No reactivity was observed with 3- or 15-acetyldeoxynivalenol. The results suggest that specific polyclonal antibodies can be prepared against a trichothecene when CT is used as an adjuvant and carrier protein. DNIV antibodies will be useful for monitoring the compound in food in conjunction with other trichothecene antibodies, detection of DNIV-producing cultures, and investigation of 8-ketotrichothecene biosynthesis.

Generation of antibodies reactive with fumonisins B1, B2, and B3 by using cholera toxin as the carrier-adjuvant.

Murine polyclonal antibodies reactive with fumonisins B1, B2, and B3 were produced after a novel immunization procedure with cholera toxin as both a hapten carrier protein and adjuvant. Immunization of mice with two 7.5-micrograms doses of fumonisin B1-cholera toxin conjugate without adjuvant resulted in the production of fumonisin B1-specific antibodies in all mice within 15 days when intraperitoneal, subcutaneous, and intravenous routes were used. In contrast, conventional immunization procedures with fumonisin B1-bovine serum albumin conjugates with and without Freund's adjuvant were largely ineffective. Fumonisin antibodies could be readily mass-produced in ascites fluid by using cholera toxin as a carrier-adjuvant. A competitive indirect enzyme-linked immunosorbent assay (ELISA) was devised whereby immobilized fumonisin B1-ovalbumin and free fumonisin B1 competed for antibody binding. The detection limit for fumonisin B1 in the ELISA was 100 ng/ml. The antiserum cross-reacted with fumonisins B2 and B3 but not with the hydrolyzed backbone of fumonisin B1 and tricarballylic acid. Concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 260, 300, and 650 ng/ml, respectively. These polyclonal antibodies should find wide usage in the ELISA for fumonisins in foods, feeds, and tissues.