A joint AOAC/IUPAC (International Union of Pure and Applied Chemistry) interlaboratory study of an enzyme-linked immunosorbent screening assay (ELISA) for aflatoxins was conducted in laboratories in Canada, France, Japan, South Africa, Switzerland, The Netherlands, Tunisia, and the United States. Twenty-eight samples of raw and roasted peanuts, corn, whole cottonseed, cottonseed meal, ammoniated cottonseed meal, and poultry feed containing various quantities of natural aflatoxins and supplemented when appropriate with aflatoxin B1 were distributed to participating laboratories for testing. The assay is based on conjugation of pure aflatoxin B1 to an enzyme and the competition between this conjugate and (free) aflatoxins in the product for aflatoxin-specific antibodies coated onto microtiter well walls. After a wash step to remove all unbound aflatoxins, a substrate, added to each well, is catalyzed from a colorless to a green solution by any bound enzyme-conjugated aflatoxin Bf present. The intensity of the color decreases as the amount of free aflatoxin B1 in the product increases. Overall correlation was good between ELISA and thin-layer chromatographic (TLC) results for cottonseed products and mixed feed. Variable results were reported for corn and peanut product samples. Although some positive samples (> 15 ng/g) of cottonseed products and mixed feed were reported to contain < 15 ng/g by visual determination, a review of data for absorbance measurements showed that the contamination level was close to the ≥ 15 ng/g standard and would not have been reported as negative under routine screening. Variation in ELISA results may have been due to several factors such as: lack of homogeneity of the aflatoxin contamination in the samples (prestudy TLC analysis samples were collected randomly from a pool of subsamples), interferences that resulted from incomplete removal of hexane during the filtration step, and antibody strips at or past their expiration date. The ELISA method has been adopted official first action as a screening method to determine the presence or absence of aflatoxin B1 at a concentration of ≥ 15 ng/g in cottonseed products and mixed feed.
Alveolar macrophages (AMs) are a critical element of the innate immune response to inhaled agents, yet functional and genetic studies of this unique macrophage population are lacking. Current strategies to obtain large quantities of AMs are cumbersome and inefficient. This is due largely to both the high cost of time and resources involved in the extraction of AMs and the inability to effectively culture AMs ex vivo for extended periods of time. While bone marrow derived macrophages (BMDMs) are modeled in numerous immortalized cell lines, AMs currently lack an acceptable model that can be used in vitro. Recently, self-replicating cells derived from the fetal mouse liver, termed “MPI” cells, have been shown to possess AM-like characteristics. Here, we show that early after isolation, these cells are SiglecFhi, Cd11chi, and Cd14low, while also expressing high levels of Pparg, Marco, Itgax, and Car4, akin to AMs. Additionally, like AMs, MPI cells effectively efferocytose dead cell debris and phagocytose silica particles. While these cells lose their “AM-likeness” over time, addition of the cytokine TGF-β dramatically delays this shift away from the AM-like phenotype. Gene expression analysis shows that in contrast to cells treated with TGF-β, untreated MPI cells cease expressing Tgfbr1, the receptor for TGF-β, concurrent with the shift away from the AM-phenotype. Further, these cells are amenable to viral transduction, and we have successfully employed CRISPR/Cas9 targeted genetic editing in MPI cells. These findings further our understanding of MPI cells as an accessible and genetically tractable model for AMs that allow for long-term and large-scale studies that are not possible with AMs isolated ex vivo.
Nuclear erythroid 2 related factor 2 (Nrf2) is a transcription factor activated by cell stress, including oxidative and electrophilic stimuli, resulting in the upregulation of cytoprotective genes. Nrf2 can be activated experimentally by a number of different pharmacological agents, including tert-butylhydroquinone (tBHQ), which is also used commercially as a food additive. We recently showed that activation of Nrf2 by tBHQ promotes Th2 differentiation and inhibits Th1 differentiation in isolated CD4+ T cells. Given that tBHQ is a food additive and promotes Th2 differentiation, the purpose of the present studies was to determine the effect of tBHQ in a mouse model of food allergy. Mice were administered a diet either with or without tBHQ (0.001%) for two weeks prior to sensitization with ovalbumin. Notably, this dose of tBHQ is lower than that normally present in standard rodent chow (0.0016%). The mice were sensitized weekly transdermally for 4 weeks. Sensitization to ovalbumin caused an increase in ovalbumin-specific IgE in plasma, which was greater in animals exposed to tBHQ. After sensitization, the mice were orally challenged with ovalbumin, causing mild/moderate immediate hypersensitivity, which was exacerbated by tBHQ. Overall, these studies suggest that low doses of the food additive, tBHQ, increase IgE response to food allergen and exacerbate clinical signs of immediate hypersensitivity. (This work was funded by NIH grant: ES018885.)
Deoxynivalenol (DON) is a Fusarium mycotoxin that has been long known to suppress weight gain of monogastric animals. We tested the hypothesis that DON consumption will reduce weight and adiposity in diet‐induced obese (DIO) mice. Female B6C3F1 mice were fed 60% kcal high fat diet (HFD) for 8 wk to induce obesity. They were then regrouped and fed HFD containing either 0, 2, 5,10 or ppm DON for an additional 8 wk. Animals fed HFD containing 5 ppm and 10 ppm DON exhibited reductions in body weights of 16% and 23%, respectively, while periuterine fat pad weights of the DON 10 ppm HFD group were reduced by 40%. Insulin like growth factor acid labile subunit (IGFALS) was reduced by 20 and 42% in the 5 and 10 ppm DON‐fed mice, respectively. In subsequent experiments, we determined that DIO mice fed DON exhibited reduced food intake and that these animals regained weight upon removal of DON from the diet. Taken together, our data indicate that DON attenuated weight and adiposity in DIO mice in a reversible manner and that this corresponded to both decreased food intake and deregulation of the growth hormone axis. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59‐0790‐4‐119 and by PHS Grant ES03358
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