ToxSci Advance Access originally published online on November 7, 2006
Toxicological Sciences 2007 95(2):412-426; doi:10.1093/toxsci/kfl153
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Deoxynivalenol Exacerbates Viral Bronchopneumonia Induced by Respiratory Reovirus Infection
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* Department of Microbiology and Molecular Genetics
Food Science and Human Nutrition
Center for Integrative Toxicology
Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824
¶ Department of Microbiology, Immunology, and Cell Biology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia 26506
1 To whom correspondence should be addressed at 234 G. M. Trout Building, Michigan State University, East Lansing, MI 48824-1224. Fax: (517) 353-8963. E-mail: pestka{at}msu.edu.
Received August 28, 2006; accepted November 1, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The trichothecene mycotoxin deoxynivalenol (DON), a frequent contaminant of cereal grains, is known to dysregulate mucosal and systemic immunity. In this study, we tested the hypothesis that DON interferes with the murine immune response to viral respiratory infection. Female Balb/c mice (5 weeks old) were orally gavaged with DON (10 mg/kg body weight [bw]) or saline vehicle and then intranasally instilled with 107 plaque-forming units of reovirus serotype 1, strain Lang (T1/L). At 10-day postinstillation (PI), both viral titers and reovirus L2 gene expression were 10-fold higher in lungs of DON-treated mice than in saline controls. The lowest observed effective DON dose that impaired viral clearance was 2 mg/kg bw. Although DON amplified reovirus-induced interferon (IFN)-ß and IFN-
mRNA responses in lung, the toxin suppressed mRNA expression for IFN-
, IFN-ß receptor (IFNAR), and IFN- receptor (IFNGR). DON also impaired induction of two type 1 IFN-dependent antiviral genes, double-stranded RNA activated protein kinase R (PKR) and oligoadenylate synthase 2 (OAS2). Respiratory reovirus infection caused a mild bronchopneumonia in mice which was markedly exacerbated by DON as evidenced by severe inflammatory cell infiltration, marked alveolar damage, and a higher volume density of intraepithelial mucosubstances in pulmonary airways. At 3- and 7-day PI, elevations in total protein, MCP-1, TNF-, total cells, macrophages, neutrophils, and lymphocytes were observed in bronchoalveolar lavage fluid (BALF) of control mice infected with reovirus. DON markedly enhanced viral-induced elevations of protein, MCP-1, TNF-, and inflammatory cells in the BALF at 3-day PI. DON exposure also upregulated induction of reovirus-specific immunoglobulin A (IgA) in BALF, fecal pellets, and serum. DON's effect on BALF IgA was preceded by elevated IL-6 expression and secretion in the lung. Taken together, the results suggest that DON compromised resistance to respiratory viral infection. Reduced expression of IFNAR and type 1 IFN-mediated genes in the lung might contribute to DON impairment of pulmonary reovirus clearance, whereas exacerbation of bronchopneumonia and IgA responses corresponded to increased MCP-1, TNF-, and IL-6 expression. Key Words: deoxynivalenol; reovirus; respiratory infection; histopathology; cytokines; flow cytometry bead array; host resistance.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Deoxynivalenol (DON or "vomitoxin") is a type B trichothecene mycotoxin that commonly occurs in wheat, barley, and corn following infection in the field by the fungus Fusarium graminearum (Bai and Shaner, 2004
). DON and other trichothecenes target leukocytes and are further capable of dysregulating immune function (Pestka and Smolinski, 2005). At the molecular level, DON induces phosphorylation of mitogen-activated protein kinases (MAPKs) via a mechanism known as the ribotoxic stress response (Pestka et al., 2004). MAPK activation modulates expression of genes associated with immune response, chemotaxis, inflammation, and apoptosis (Chung et al., 2003a,b; Moon and Pestka 2002, 2003; Zhou et al., 2003a,b,c, 2005a,b).
DON and other trichothecenes can both enhance and suppress systemic responses to experimental infections by Salmonella (Bottex et al., 1990; Kubena et al., 2001; Tai and Pestka, 1990; Ziprin and Elissalde, 1990), Listeria (Corrier et al., 1987; Ziprin and McMurray, 1988), Staphylococcus (Cooray and Jonsson, 1990), and herpes simplex virus (Friend et al., 1983a,b).
Trichothecenes have also been shown to suppress mucosal immune function. Reovirus, a double-stranded RNA virus isolated from respiratory and gastrointestinal (GI) systems of human and animals induces marked cellular and humoral responses (Tyler et al., 2001), making it useful to study the mucosal effects and mechanisms of immunotoxicants (Cuff et al., 1998). When oral exposure to reovirus was used to investigate the suppressive effects of DON and the type A trichothecene, T-2 toxin, on GI mucosal immunity in the mouse, doses as low as 2 and 0.2 mg/kg body weight (bw) of these toxins, respectively, depressed interferon (IFN)- expression and increased intestinal reovirus burden (Li et al., 2005, 2006a).
Reoviruses might also be useful for studying whether trichothecene exposure interferes with immune responses at another critical mucosal site—the respiratory tract. Airway exposure to reovirus causes murine lung infection in the mouse which generates a spectrum of age- and strain-dependent immunopathologic and inflammatory sequelae (Bellum et al., 1996; London et al., 2002a,b; Majeski et al., 2003a,b; Morin et al., 1996). Notably, reovirus infection evokes only mild pulmonary inflammation in C3H mice (London et al., 2002a; Periwal and Cebra, 1999) but causes a more severe inflammation in Balb/c or CD-1 mice (Thompson et al., 1996, 1999). Reovirus-infected CBA/J mice exhibit the most severe pathologic effects which closely mimic bronchiolitis obliterans organizing pneumonia and acute respiratory distress syndrome.
Recently, we determined that T-2 toxin suppresses murine host resistance to respiratory infection by reovirus in Balb/c mice (Li et al., 2006b). Given the frequent occurrence of DON in foods (Abouzied et al., 1991; Pestka and Smolinski, 2005), it is important from the perspective of safety assessment to understand how this mycotoxin might affect viral respiratory infection. This is particularly critical since studies with the enteric reovirus model suggest that inherent differences exist between T-2 and DON relative to minimum effective doses and virus persistence as well as immunoglobulin A (IgA) and cytokine responses (Li et al., 2005, 2006a). The objective of this research was to test the hypothesis that DON exposure impairs the murine immune response to respiratory reovirus infection. Specifically, we assessed the effects of a single oral exposure to DON on reovirus clearance, lung injury, and IgA responses. Acute DON exposure not only impaired reovirus clearance but markedly exacerbated reovirus-induced inflammation and pulmonary damage. These effects corresponded to suppression of type 1 IFN-mediated responses, upregulation of proinflammatory cytokine and chemokine expression, and elevated IgA responses in the lung.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals and animals.
All chemicals including DON and tissue culture components were purchased from Sigma Chemical Co (St Louis, MO) unless otherwise noted. Female Balb/c mice were purchased from Charles River Labs (Portage, MI). Mice were housed in microisolator cages under negative pressure laminar flow with humidity and temperature control at the MSU University Research Containment Facility. All animal studies were overseen by the Michigan State University Committee on Animal Use and Care and followed National Institutes of Health (NIH) guidelines.
Virus.
Reovirus serotype 1, strain Lang (T1/L), was grown in L929 fibroblast cells at 34°C in DMEM medium with 5% (vol/vol) fetal bovine serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 0.25 µg/ml of amphotericin B (Invitrogen, Carlsbad, CA). Virions were purified as described previously (Furlong et al., 1988; Silvey et al., 2001). Virus titers were determined by a plaque-forming assay (Major and Cuff, 1997).
Experimental design.
DON was dissolved in endotoxin-free water. For most experiments, 5-week-old BALB/c female mice were orally gavaged with 10 mg/kg bw DON or vehicle in a 100-µl volume using a 22 G intubation needle (Popper and Sons, New Hyde Park, NY). For the dose-response study, mice were gavaged with DON 0, 2, 5, 10, or 25 mg/kg bw or vehicle in a 100-µl volume. After 2 h, mice were lightly anesthetized with isoflurane (Abbott Labs, North Chicago, IL) and intranasally instilled with 1 x 107 plaque-forming units (PFU) of reovirus 1/l in a total volume of 30 µl (15 µl per nostril) of sterile injectable grade saline. Control animals were instilled with 30 µl (15 µl per nostril) of the saline vehicle.
Fecal pellets were collected at 0-, 2-, 4-, 6-, 8-, and 10-day PI. Pellets were weighed and suspended in phosphate-buffered saline (PBS, 10% [wt/vol]), held on ice for 2 h, sonicated for 15 s, and cleared by high-speed centrifugation for 10 min at 4°C. Supernatants were used directly for specific antibody detection by ELISA.
Mice were killed 10 days after intranasal instillation. At the time of necropsy, each animal was anesthetized with an ip injection 100 µl of sodium pentobarbital 12% (wt/vol). After induction of anesthesia, a midline laparotomy was performed, and the mouse was exsanguinated via the abdominal aorta. Immediately after death, the trachea was exposed and cannulated, and the heart and lung were excised en bloc. The right extrapulmonary bronchus was ligated, and the right cranial and middle lung lobes were excised and stored at 20°C for later processing for virus quantitation by plaque-forming assay. Right caudal and accessory lobes were removed and then immersed and stored in RNAlater solution (Ambion, Inc., Austin, TX) until further processing to extract RNA from the lung tissue.
Following removal of the right lung lobes, the left lung lobe was intratracheally perfused with 10% (wt/vol) neutral buffered formalin at a constant pressure of 30 cm of fixative. After 1 h, the trachea was ligated, and the inflated left lung lobe was immersed in a large volume of the same fixative for at least 24 h. After fixation, the left lobe was microdissected along the main axial airway, and two transverse tissue blocks were excised at the level of the fifth (proximal) and eleventh (distal) airway generation (G5 and G11, respectively) for further processing, as has been previously reported (Farraj et al., 2003). Tissue blocks were embedded in Immuno-Bed plastic embedding media (Polysciences, Inc., Warrington, PA), and the anterior surface of each block was cut at a thickness of 2–3 microns using a glass knife. Lung sections were stained with hematoxylin and eosin for routine light microscopic examination or alcian blue (pH 2.5)/periodic acid Schiff sequence (AB/PAS) for detection of stored intraepithelial mucosubstances in the conducting airways.
To further investigate inflammatory cell and cytokine responses to reovirus and DON, additional mice were treated with 0 or 10 mg/kg bw DON and reovirus as described above, but bronchoalveolar lavage fluid (BALF) was collected postmortem at 3- and 7-day PI in situ by intratracheal instilling and withdrawing 1 ml of PBS twice with an intubation cannula (22 G). Samples were pooled from each animal.
Quantitation of reovirus L2 gene.
Total RNA was extracted from lung using TRIZOL reagent (Invitrogen). Reverse transcriptase reaction and real-time PCR were performed as described previously (Li et al., 2005) using PCR primers selected from published sequence of 
2 core spike (L2 gene) of reovirus T1/L (Breun et al., 2001) (Table 1).
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Real-time PCR quantitation of antiviral and inflammation-related genes.
Real-time PCR for antiviral and inflammation-related genes was performed as previously described (Kinser et al., 2004
) using primer sequences that were designed from GenBank sequences (Table 1).
Semiquantitative scoring of lung histopathology.
A board-certified, veterinary pathologist, without previous knowledge of exposure history of the individual mice, conducted the light microscopic examinations and ranked the severity of treatment-induced pulmonary histopathology in the lung sections of each mouse using a criteria-based numeric scoring system. A score of 0 was given when no exposure-related lung lesions were observed in the sections. If an exposure-related morphologic alteration affected less than one fourth of the lung section, it was given a histopathologic severity score of 1 (minimal). When exposure-related alterations involved greater than one fourth, but less than on half of the lung section, it was scored as 2 (mild). A severity score of 3 (moderate) was given when the exposure-induced lesions involved more than one half but less than three fourths of the lung section. If induced morphologic alterations affected greater than three fourths of the lung section, it was given a severity score of 4 (marked).
Morphometry.
Quantitative analyses for the volume densities of mucosubstances stored in surface epithelium lining the axial airway of the left lung lobe were made using G5 lung sections stained with AB/PAS to identify acidic (AB positive; blue stain) and neutral (PAS positive; red stain) mucosubstances within mucous goblet cells. A semiautomatic computerized image analysis (Harkema et al., 1997) was used to make these measurements. The area of AB/PAS–positive intraepithelial mucosubstances was calculated by the image analysis software program from the manually or automatically circumscribed perimeter of the stained material. The method used to estimate the amount of stored mucosubstance per unit length of surface area of epithelial basal lamina (volume density per surface area) has been previously described in detail (Harkema et al., 1987).
Analysis of BALF.
Total leukocytes in BALF were counted with a hemocytometer, and fractions of neutrophils, macrophages, and lymphocytes were determined in cytospin samples with Diff-Quik reagent (Baxter, Miami, FL). IL-6, IL-10, MCP-1, IFN-, TNF-, and IL-12p70 concentration in BALF were determined with a Cytometric Bead Array Mouse Inflammation Kit (BD, Biosciences, San Diego, CA) according to manufacturer's instructions using a FACScalibur and BD CBA Analysis Software (BD Bioscience, San Jose, CA). Assay sensitivities for these analytes were 5, 17.5, 52.7, 2.5, 7.3, and 10.7 pg/ml, respectively.
Detection of reovirus-specific IgA.
BALF, fecal supernatant, and serum were assayed for virus-specific IgA by ELISA using a modification of the procedure of Major and Cuff (1997). Absorbance at 450 nm was used as end point for BALF and fecal suspensions. For sera, titers were designated as the highest serum dilution that yielded absorbances of 0.2 or higher; the geometric mean antibody titer was then calculated.
Statistics.
All data were analyzed with SigmaStat v 3.1 (Jandel Scientific; San Rafael, CA) with the criterion for significance set at p < 0.05. Morphometric and real-time PCR data were statistically analyzed using one-way ANOVA with Student-Newman-Keuls posttest. Data from histopathological severity of treatment-induced lesions were analyzed using the Mann Whitney Rank Sum Test (nonparametric test).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reovirus Clearance from Lung
To test the capacity of DON to modulate reovirus respiratory infection, 5-week-old female BALB mice were intubated with 10 mg/kg bw DON or water vehicle and then intranasally instilled 2 h later with reovirus or saline vehicle. After 10 days, viral titers and virus RNA expression of lung in infected mice pretreated with water vehicle were approximately 105 PFU/lung (Fig. 1A) and 200 copies L2 gene/µg total lung RNA (Fig. 1B), respectively. However, in DON-treated mice, both PFU and L2 gene copy number were 10 times higher. When the dose-response effects of DON on L2 RNA in lung were assessed 3 days PI, the mycotoxin was found to significantly elevate L2 RNA expression in lung at 2, 5, 10, and 25 mg/kg bw (Fig. 2). Thus, as little as 2 mg/kg DON increased the reovirus burden in lung early during the infection.
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Type 1 IFN Antiviral Response
Since IFN-
, IFN-ß, and other type I IFNs play a critical role in the early innate antiviral response (Garcia-Sastre and Biron, 2006), expression of genes that mediated his response were monitored in the lung after treatment with DON and/or reovirus (Fig. 3). IFN- expression was suppressed by DON at 3- and 7-day PI and by reovirus at 3 days, and the suppression was magnified at 3 days following coexposure (Figs. 3A and 3B). In contrast, reovirus-induced mRNA expression of IFN-ß in lungs was enhanced by DON at 3-day PI but not at 7-day PI (Figs. 3C and 3D). DON alone markedly suppressed mRNA expression of IFN-ß receptor (IFNAR), a type 1 IFN receptor, in the lungs of both uninfected mice after 3 days and reovirus-infected mice after 3 and 7 days (Figs. 3E and 3F).
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Respiratory reovirus infection also induced expression of mRNAs for several type 1 IFN-driven antiviral genes including 2'-5' oligoadenylate synthase 2 (OAS2), double-stranded RNA activated protein kinase R (PKR), the myxovirus resistance gene Mx1, and ubiquitin-like protein ISG15 in lungs at 3- and 7-day PI (Fig. 4). In virus-infected mice pretreated with DON, OAS2 mRNA expression was impaired at 7-day PI (Figs. 4A and 4B) and PKR mRNA expression was suppressed at 3- and 7-day PI (Figs. 4C and 4D). Induction of Mx1 and ISG15 mRNAs were unaffected by DON (Figs. 4E–4H). Thus, DON-induced suppression of IFNAR expression corresponded to interference with type 1 IFN-mediated OAS2 and PKR antiviral pathways.
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IFN-
and IFN- receptor ExpressionIFN-
is also very important to the antiviral response and is produced as part of innate and acquired immune responses (Biron and Sen, 2001). Reovirus did not induce detectable IFN- protein secretion or IFN- mRNA expression in the lung at 3-day PI (data not shown). However, both parameters were upregulated 100- and 30-fold, respectively, at 7-day PI (Figs. 5A and 5B), suggesting that IFN- might be produced as part of the acquired response in this model. DON exposure markedly enhanced both IFN- protein and mRNA responses at 7-day PI. In contrast, DON suppressed mRNA expression of IFN- receptor (IFNGR) expression in reovirus-infected mice at 3-day PI (Fig. 5C). By 7-day PI, IFNGR expression was markedly suppressed in reovirus-infected mice, but DON did not further modulate this suppressive effect (Fig. 5D).
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Reovirus-Induced Lung Pathology
No pulmonary histopathology was evident in mice exposed to DON alone or control mice exposed only to the saline vehicle (Figs. 6A–6D). Incidence and severity of lung lesions were greatest in the mice exposed to both reovirus and DON. At 10-day PI, all the mice in this group had either a mild or moderate, subacute bronchopneumonia. This airway orientated lung lesion was characterized by a peribronchial mononuclear inflammatory cell infiltrate, composed mainly of large and small lymphocytes and monocytes. The airway inflammation was centered around the large-diameter, main axial conducting airway but extended also into the more distal, small-diameter, preterminal and terminal bronchioles, proximal alveolar ducts, and alveolar septa and airspaces in the adjacent alveolar parenchyma (Fig. 6D). Perivascular cuffing of inflammatory cells was also present in these areas as well as increased numbers of alveolar macrophages/monocytes in the affected alveolar airspaces. The inflammatory response, however, rarely extended to the outer pulmonary pleura. There was a noticeable proximal to distal decrease in the severity of the bronchopneumonia in the lung lobe of each animal with more inflammatory lesions in the proximal lung section (G5 axial airway level; closest to the hilus) compared to the more distal section (G11 axial airway level).
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Interestingly, only two of the five mice exposed to reovirus alone had histopathologic evidence of a viral-induced mononuclear cell bronchopneumonia at 10-day PI. The extent and severity of the lung lesions in these mice were less than that in the mice that were exposed to both reovirus and DON (Figs. 6E and 6F).
Reovirus-Induced Mucous Cell Metaplasia
Associated with the inflammatory response, there was mild hypertrophy and mucous cell metaplasia in the respiratory epithelium lining the main axial airway and minimal to mild alveolar type two cell hyperplasia lining some of the affected alveolar septa. The amount of AB/PAS–stored mucosubstances in this airway epithelium of reovirus-infected mice was markedly less than that measured in the mice coexposed to reovirus and DON (Figs. 7A–7D). There was approximately three times the level AB/PAS–stained mucosubstances in the airway epithelium lining the G5 axial airway of these coexposed mice compared to that morphometrically measured in similar airways of saline-instilled control mice (Fig. 7E).
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Reovirus-Induced Leukocyte Migration to the Alveolar Space
To further understand the mechanisms by which DON contributes to lung inflammation in reovirus-infected mice, BALF cellularity was evaluated (Table 2). Exposure to reovirus alone caused marked increases in total cell, macrophage, neutrophil, and lymphocyte numbers at 3- and 7-day PI. Both macrophage and total inflammatory cell number in the BALF from mice exposed to DON alone were increased markedly at 3-day PI but recovered by 7-day PI. DON potentiated migration of lymphocytes and total cells into the BALF of reovirus-treated mice after 3 days, with a similar trend being evident for neutrophils. By 7 days, total cell numbers in BALF of cotreated mice were greatly elevated compared to the reovirus group with macrophages and neutrophils primarily contributing to this increase. These increases were accompanied by a decrease in the lymphocyte population. Thus, prior DON exposure selectively altered cell migration into the lung of reovirus-infected mice.
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Protein and Cytokine Secretion into Alveolar Space Following Reovirus Infection
BALF protein concentrations in saline- and DON-treated control mice did not differ after 3 and 7 days (Figs. 8A and 8B). Reovirus infection induced protein secretion at 3- and 7-day PI, whereas protein concentrations were 4- and 1.5-fold higher at 3- and 7-day PI, respectively, in DON-treated mice infected with reovirus. Respiratory reovirus infection also induced secretion of the chemokine MCP-1 (Figs. 8C and 8D) and the proinflammatory cytokine TNF-
at 3- and 7-day PI (Figs. 8E and 8F) into alveolar airspaces but did not induce IL-10 or IL-12 (data not shown). DON pretreatment in reovirus-infected mice, potentiated production of MCP-1 at 3-day PI and TNF- at 3- and 7-day PI as compared to saline-treated infected mice. Thus, prior DON exposure enhanced protein, MCP-1, and TNF- concentrations in the BALF of reovirus-infected mice.
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Mucosal and Systemic IgA Response to Respiratory Reovirus Infection
Reovirus-specific IgA responses in BALF and feces were measured as an indicator of lung and gut mucosal responses, respectively. The virus induced robust IgA responses in both tissues which were amplified by DON in BALF at 7-day PI (Fig. 9A) and in fecal supernatants from 6- to 10-day PI (Fig. 9B). Similarly, DON enhanced reovirus-specific IgA titers in serum (Fig. 9C). DON is known to potently upregulate IgA secretion in the mouse, and this upregulation is driven by increased IL-6 expression (reviewed by Pestka, 2003
). Reovirus induced IL-6 mRNA expression in lung tissue and IL-6 secretion into BALF at 3- and 7-day PI (Figs. 10A–10D). Exposure to DON amplified IL-6 mRNA expression and IL-6 secretion at 3 days (Figs. 10A and 10C) but not 7 days (Figs. 10B and 10D). The results suggested that DON potentiated reovirus-induced IgA responses to respiratory reovirus infection and, in the lung, these effects were preceded by elevated IL-6 expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunotoxic chemicals have the potential to increase susceptibility to the viral respiratory infections by modulating mucosal and systemic immune responses. DON has a unique capacity to up- and down-regulate immune function by disrupting intracellular signaling within leukocytes (Pestka et al., 2004
). In this study, reovirus was chosen to investigate effects of DON on respiratory immune response because it (1) causes a range of pathological changes in the lungs of mice depending on the age, species, and viral dose (Thompson et al., 1996, 1999), (2) induces a robust mucosal immune responses (Cuff et al. 1998), (3) has been widely used a model for viral pathogenesis (Tyler et al., 2001, (4) has recently been shown to cause bronchiolitis and aveolitis in mice exposed to T-2 toxin (Li et al., 2006b), and (5) presents a minimal risk to laboratory personnel or laboratory animal facilities. The results of the present study demonstrate that intranasal exposure to reovirus initiated mild bronchopneumonia in 5-week-old Balb/c mice. However, infection of DON-treated mice increased lung reovirus burden and suppressed virus clearance from the infected lung. In addition, more severe histopathological changes were evident in lung with marked exacerbation of inflammatory cell infiltration and mucous metaplasia in conducting airways. The finding that as little as 2 mg/kg bw DON suppressed host resistance to reovirus in the lung is consistent with our previous observations in the intestinal model (Li et al., 2005).
While most reovirus infections are asymptomatic in humans, this virus can cause mild respiratory or GI symptoms that are virtually indistinguishable from upper respiratory infections and diarrheal illnesses often encountered during infancy and early childhood (Tyler, 2001). The capacity of DON to exacerbate bronchopneumonia in young reovirus-infected mice is very important because the toxin might similarly exacerbate asymptomatic or mild human infections by reovirus or other viruses.
Immunity to reovirus and other viruses includes both innate and adaptive components. These typically involve cell-mediated and antibody effector responses that are finely regulated by cytokines. IFNs play a particularly critical role in immunity by binding to surface receptors on effector cells and inducing transcription of genes that mediate antiviral responses (Goodbourn et al., 2000). IFN- and IFN-ß induce several antiviral genes that include PKR, OAS 2, and Mx proteins (Garcia-Sastre and Biron, 2006). Both PKR and OAS pathways can be activated by viral double-stranded RNA. Upon activation, PKR will shut down viral and protein translation, while OAS can activate RNase L which digests viral RNA and mRNA. The observations that DON exposure reduced mRNA levels of PKR and OAS2 in DON-treated mice following infection were consistent with depressed IFN- and IFNAR expression as well as with the elevated viral titers observed in lung tissue. Several questions arise from these observations. Typically, these innate mechanisms play a very important role within the first 72–96 h following infection. Since the effects observed here occurred later (7 days), it is not readily apparent whether DON directly impairs these important antiviral pathways or if they are downstream consequences of an earlier event. It should be further noted that the effects of DON on IFNAR, PKR, and OAS2 were modest, as were the effects on IFNGR. Also, DON did not appear to affect induction of Mx1 and ISG15, even though elevated levels of these genes were observed in lung tissues of reovirus-treated mice. Thus, further exploration into the kinetics of mRNA and protein expression of antiviral genes as well as responsible cell phenotypes is warranted in this model.
Severe lung damage, consistent with inflammatory cell infiltration and protein secretion, in DON-treated mice following infection were likely to be mediated, in part, by increased TNF- and MCP-1 concentrations. TNF-, a cytokine that plays an important role in many inflammatory diseases, is produced by macrophages, monocytes, dendritic cells, B cells, neutrophils, mast cells, and CD4+ cells (Aggarwal et al., 2002). Once released in the airway, TNF- acts by inducing a general inflammatory response through the enhanced release of proinflammatory/chemotactic mediators. In addition, TNF- upregulates adhesion molecules, such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1), matrix melloproteinase 9 (MMP9), and cyclooxygenase 2 (COX2) (Aggarwal et al., 2002; Russo and Polosa, 2005), all of which can facilitate the migration of inflammatory cells and pulmonary cell damage. MCP-1 is produced by a wide variety of cell types including monocytes and epithelial cells (Shyy et al., 1993; Taub et al., 1995). This chemokine promotes migration of monocytes, T cells, neutrophils, and NK cells during inflammatory response (Carr et al., 1994; Loetscher et al., 1994; Taub et al., 1995).
Enhanced mucosal and systemic reovirus-specific IgA responses were consistent with several previous investigations demonstrating that chronic DON exposure elevates total serum IgA, fecal IgA, and ex vivo IgA production as well as specific IgA to microbial and self-antigens (reviewed by Pestka, 2003). IL-6 was assessed here because DON is an highly effective inducer of IL-6 (Zhou et al., 2003b) and, furthermore, elevated IL-6 plays a key role in aberrantly increased IgA responses in mice fed DON for extended time periods (Pestka and Zhou, 2000). Taken together, the current and previous findings suggest that elevated IL-6 in reovirus-infected mice might contribute to increased mucosal and systemic IgA responses. While an enhanced reovirus-specific IgA seems counterintuitive to the observed increases in reovirus, this was an acquired immune response that might impact viral clearance beyond the 10-day window employed here.
Virus-induced mucous cell metaplasia is complex and involves a novel immune axis for growth factor and cytokine production and downstream events that modulate ciliated epithelial cell survival and transdifferentiation (Holtzman et al., 2006). The results presented here suggest that reovirus evoked prominent mucous cell metaplasia but that this was markedly exacerbated by DON. Both TNF- and IL-6 can stimulate airway mucin gene expression (Voynow et al., 2006). It is thus tempting to speculate that DON-mediated increases in these two proinflammatory cytokines contributed to the enhanced mucous cell metaplasia observed in mice cotreated with reovirus and DON.
The mechanisms by which DON upregulates cytokine and chemokine production might be direct or indirect. Regarding direct effects, DON is known to increase gene expression in T cells and macrophages by enhancing both activation of key transcription factors (Li et al., 2000; Ouyang et al., 1996; Wong et al., 2002; Zhou et al., 2003b) and cytokine mRNA stability (Chung et al., 2003a; Li et al., 1997; Wong et al., 2002). Both enhanced transactivation and mRNA stabilization of immune and inflammatory genes by DON have been linked to activation of p38 and ERK 1/2 MAPKs via the ribotoxic stress response (Chung et al., 2003b; Moon and Pestka, 2002, 2003). Double-stranded RNA activated protein kinase R (Zhou et al., 2003c) and the Src-family tyrosine kinases (Zhou et al., 2005b) are two signaling transducers upstream of the MAPKs that mediate DON's action. The possibility for interaction with reovirus in inducing these intracellular signaling cascades is a potential target for further study. An alternative explanation is that cytokine and chemokine upregulation by DON of might occur indirectly as an outcome of increased reovirus burden. Both direct and indirect mechanisms are not mutually exclusive.
An alternative explanation for DON suppression of host response to reovirus might be increased death of lymphocytes or lung epithelial cells. Since the sublethal DON dose (10 mg/k bw) used for most experiments does not induce apoptosis in thymus, peyer's patches, spleen, or bone marrow (Zhou et al., 1999, 2000), altered reovirus clearance might not be readily explainable by lymphocyte death in the mucosal or systemic immune compartments. However, lymphocyte recruitment was significantly reduced in mice treated with virus and DON compared to mice treated with virus alone. It is possible that DON could synergize with virus or a virus-induced mediator to cause apoptotic death in a critical lymphocyte population. In support of this latter contention, Uzarski et al. (2003) demonstrated that DON-induced lymphocyte apoptosis is amplified by TNF-, which was upregulated in the model described here.
Since respiratory epithelium is a primary barrier against pathogens and toxins, epithelial damage could facilitate viral infection and exacerbate pathologic sequelae (Becker and Soukup, 1999; Chauhan et al., 2003; Li and Holian, 1998; Miyamoto, 1997; Romieu et al., 2002; Spannhake et al., 2002; Wanner, 1993). However, histopathological changes were neither evident here nor are there reports that DON induces specific damage or cell apoptosis in lung tissues at the concentrations employed in this study.
In summary, the results presented here suggest that DON compromised resistance to respiratory reovirus infection. Multiple mechanisms might be involved in DON-induced suppression of the antiviral response and resultant downstream sequelae (Fig. 11). The pathologic effects described herein would likely increase morbidity and discomfort to a person during the course of respiratory infection and thus are relevant from the perspective of human safety assessment. The finding that as little as 2 mg/kg bw of DON potentiated reovirus L2 RNA levels in lung is consistent with the threshold for immunosuppression observed previously for this toxin in the reovirus-GI model (Li et al., 2005). Thus, both studies suggest that a relatively low dose of DON was sufficient to increase the viral burden in the mouse. This dose would be equivalent to consumption of 13 ppm of DON in mouse diet over the course of 1 day. DON concentrations exceeding these values have been found in contaminated human food samples (Abouzied et al., 1991). From a safety assessment perspective, additional studies are needed to determine how chronic low-dose exposure to DON might impact host resistance to reovirus. Another critical question that remains to be answered relates to how the toxin might modulate host resistance to other respiratory viruses such as influenza and enhance their pathologic effects.
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ACKNOWLEDGMENTS |
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This study was supported by a Strategic Research Grant from the Michigan State University Foundation and by Public Health Service grants AI034544 (CC), ES03553 (JP), and DK058833 (JP) from the NIH. We thank Annette Thelen, Zahidul Islam, Lori Bramble, and Chidozie J Amuzie for technical assistance.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abouzied MM, Azcona JI, Braselton WE, Pestka JJ. (1991) Immunochemical assessment of mycotoxins in 1989 grain foods: Evidence for deoxynivalenol (vomitoxin) contamination. Appl. Environ. Microbiol. 57:3672–677.
Aggarwal BB, Shishodia S, Ashikawa K, Bharti AC. (2002) The role of TNF and its family members in inflammation and cancer: Lessons from gene deletion. Curr. Drug Targets. Inflamm. Allergy 1:4327–341.[CrossRef][Medline]
Bai G and Shaner G. (2004) Management and resistance in wheat and barley to fusarium head blight. Annu. Rev. Phytopathol. 42:135–161.[CrossRef][Web of Science][Medline]
Becker S and Soukup JM. (1999) Effect of nitrogen dioxide on respiratory viral infection in airway epithelial cells. Environ. Res. 81:2159–166.[Medline]
Bellum SC, Hamamdzic D, Thompson AH, Harley RA, London SD, London L. (1996) Experimental reovirus serotype 1/strain Lang infection of the lung: A model for the study of the lung in the context of mucosal immunity. Lab. Invest. 74:1221–231.[Web of Science][Medline]
Biron CA and Sen GC. (2001) Interferons and Other Cytokines. In Knipe DM and Howley PM (Eds.). Fields Virology 4th ed. (Lippincott Williams & Wilkins, Philadelphia, PA) pp. 321–351.
Bottex C, Martin A, Fontanges R. (1990) Action of a mycotoxin (diacetoxyscirpenol) on the immune response of the mouse—interaction with an immunomodulator (OM-89). Immunopharmacol. Immunotoxicol. 12:2311–325.[Web of Science][Medline]
Breun LA, Broering TJ, McCutcheon AM, Harrison SJ, Luongo CL, Nibert ML. (2001) Mammalian reovirus L2 gene and lambda2 core spike protein sequences and whole-genome comparisons of reoviruses type 1 Lang, type 2 Jones, and type 3 Dearing. Virology 287:2333–348.[CrossRef][Web of Science][Medline]
Carr MW, Roth SJ, Luther E, Rose SS, Springer TA. (1994) Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc. Natl. Acad. Sci. U.S.A. 91:93652–3656.
Chauhan AJ, Inskip HM, Linaker CH, Smith S, Schreiber J, Johnston SL, Holgate ST. (2003) Personal exposure to nitrogen dioxide (NO2) and the severity of virus-induced asthma in children. Lancet 361:93731939–1944.[CrossRef][Web of Science][Medline]
Chung YJ, Yang GH, Islam Z, Pestka JJ. (2003a) Up-regulation of macrophage inflammatory protein-2 and complement 3A receptor by the trichothecenes deoxynivalenol and satratoxin G. Toxicology 186:1–251–65.[CrossRef][Web of Science][Medline]
Chung YJ, Zhou HR, Pestka JJ. (2003b) Transcriptional and posttranscriptional roles for p38 mitogen-activated protein kinase in upregulation of TNF-alpha expression by deoxynivalenol (vomitoxin). Toxicol. Appl. Pharmacol. 193:2188–201.[CrossRef][Web of Science][Medline]
Cooray R and Jonsson P. (1990) Modulation of resistance to mastitis pathogens by pretreatment of mice with T-2 toxin. Food Chem. Toxicol. 28:10687–692.[CrossRef][Web of Science][Medline]
Corrier DE, Ziprin RL, Mollenhauer HH. (1987) Modulation of cell-mediated resistance to listeriosis in mice given T-2 toxin. Toxicol. Appl. Pharmacol. 89:3323–331.[CrossRef][Web of Science][Medline]
Cuff CF, Fulton JR, Barnett JB, Boyce CS. (1998) Enteric reovirus infection as a probe to study immunotoxicity of the gastrointestinal tract. Toxicol. Sci. 42:299–108.
Farraj AK, Harkema JR, Jan TR, Kaminski NE. (2003) Immune responses in the lung and local lymph node of A/J mice to intranasal sensitization and challenge with adjuvant-free ovalbumin. Toxicol. Pathol. 31:4432–447.[CrossRef][Web of Science][Medline]
Friend SC, Babiuk LA, Schiefer HB. (1983a) The effects of dietary T-2 toxin on the immunological function and herpes simplex reactivation in Swiss mice. Toxicol. Appl. Pharmacol. 69:2234–244.[CrossRef][Web of Science][Medline]
Friend SC, Schiefer HB, Babiuk LA. (1983b) The effects of dietary T-2 toxin on acute herpes simplex virus type 1 infection in mice. Vet. Pathol. 20:6737–760.[Abstract]
Furlong DB, Nibert ML, Fields BN. (1988) Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles. J. Virol. 62:1246–256.
Garcia-Sastre A and Biron CA. (2006) Type 1 interferons and the virus-host relationship: A lesson in detente. Science 312:5775879–882.
Goodbourn S, Didcock L, Randall RE. (2000) Interferons: Cell signalling, immune modulation, antiviral response and virus countermeasures. J. Gen. Virol. 81:pt 102341–2364.
Harkema JR, Hotchkiss JA, Griffith WC. (1997) Mucous cell metaplasia in rat nasal epithelium after a 20-month exposure to ozone: A morphometric study of epithelial differentiation. Am. J Respir. Cell Mol. Biol. 16:5521–530.[Abstract]
Harkema JR, Plopper CG, Hyde DM, St George JA, Dungworth DL. (1987) Effects of an ambient level of ozone on primate nasal epithelial mucosubstances. Quantitative histochemistry. Am. J Pathol. 127:190–96.[Abstract]
Holtzman MJ, Battaile JT, Patel AC. (2006) Immunogenetic programs for viral induction of mucous cell metaplasia. Am. J Respir. Cell Mol. Biol. 35:129–39.
Kinser S, Sneed R, Roth R, Ganey P. (2004) Neutrophils contribute to endotoxin enhancement of allyl alcohol hepatotoxicity. J Toxicol. Environ Health A 67:12911–928.[CrossRef][Web of Science][Medline]
Kubena LF, Bailey RH, Byrd JA, Young CR, Corrier DE, Stanker LH, Rottinghaust GE. (2001) Cecal volatile fatty acids and broiler chick susceptibility to Salmonella typhimurium colonization as affected by aflatoxins and T-2 toxin. Poult. Sci. 80:4411–417.
Li L and Holian A. (1998) Acrolein: A respiratory toxin that suppresses pulmonary host defense. Rev. Environ. Health 13:1–299–108.[Medline]
Li M, Cuff CF, Pestka J. (2005) Modulation of murine host response to enteric reovirus infection by the trichothecene deoxynivalenol. Toxicol. Sci. 87:1134–145.
Li M, Cuff CF, Pestka JJ. (2006a) T-2 toxin impairment of enteric reovirus clearance in the mouse associated with suppressed immunoglobulin and IFN-gamma responses. Toxicol. Appl. Pharmacol. 214:3318–325.[CrossRef][Web of Science][Medline]
Li M, Harkema JR, Islam Z, Cuff CF, Pestka JJ. (2006b) T-2 toxin impairs murine immune response to respiratory reovirus and exacerbates viral bronchiolitis. Toxicol. Appl. Pharmacol. (in press).
Li S, Ouyang Y, Yang GH, Pestka JJ. (2000) Modulation of transcription factor AP-1 activity in murine EL-4 thymoma cells by vomitoxin (deoxynivalenol). Toxicol. Appl. Pharmacol. 163:117–25.[CrossRef][Web of Science][Medline]
Li S, Ouyang YL, Dong W, Pestka JJ. (1997) Superinduction of IL-2 gene expression by vomitoxin (deoxynivalenol) involves increased mRNA stability. Toxicol. Appl. Pharmacol. 147:2331–342.[CrossRef][Web of Science][Medline]
Loetscher P, Seitz M, Clark-Lewis I, Baggiolini M, Moser B. (1994) Monocyte chemotactic proteins MCP-1, MCP-2, and MCP-3 are major attractants for human CD4+ and CD8+ T lymphocytes. FASEB J. 8:131055–1060.[Abstract]
London L, Majeski EI, Paintlia MK, Harley RA, London SD. (2002a) Respiratory reovirus 1/L induction of diffuse alveolar damage: A model of acute respiratory distress syndrome. Exp. Mol. Pathol. 72:124–36.[CrossRef][Web of Science][Medline]
London L, Majeski EI, tman-Hamamdzic S, Enockson C, Paintlia MK, Harley RA, London SD. (2002b) Respiratory reovirus 1/L induction of diffuse alveolar damage: Pulmonary fibrosis is not modulated by corticosteroids in acute respiratory distress syndrome in mice. Clin. Immunol. 103:3 pt 1284–295.[CrossRef][Web of Science][Medline]
Majeski EI, Harley RA, Bellum SC, London SD, London L. (2003a) Differential role for T cells in the development of fibrotic lesions associated with reovirus 1/L-induced bronchiolitis obliterans organizing pneumonia versus acute respiratory distress syndrome. Am. J. Respir. Cell Mol. Biol. 28:2208–217.
Majeski EI, Paintlia MK, Lopez AD, Harley RA, London SD, London L. (2003b) Respiratory reovirus 1/L induction of intraluminal fibrosis, a model of bronchiolitis obliterans organizing pneumonia, is dependent on T lymphocytes. Am. J. Pathol. 163:41467–1479.
Major AS and Cuff CF. (1997) Enhanced mucosal and systemic immune responses to intestinal reovirus infection in beta2-microglobulin-deficient mice. J. Virol. 71:85782–5789.[Abstract]
Miyamoto T. (1997) Epidemiology of pollution-induced airway disease in Japan. Allergy 52:Suppl. 38, 30–34.[Web of Science][Medline]
Moon Y and Pestka JJ. (2002) Vomitoxin-induced cyclooxygenase-2 gene expression in macrophages mediated by activation of ERK and p38 but not JNK mitogen-activated protein kinases. Toxicol. Sci. 69:2373–382.
Moon Y and Pestka JJ. (2003) Cyclooxygenase-2 mediates interleukin-6 upregulation by vomitoxin (deoxynivalenol) in vitro and in vivo. Toxicol. Appl. Pharmacol. 187:280–88.[CrossRef][Web of Science][Medline]
Morin MJ, Warner A, Fields BN. (1996) Reovirus infection in rat lungs as a model to study the pathogenesis of viral pneumonia. J. Virol. 70:1541–548.[Abstract]
Ouyang YL, Li S, Pestka JJ. (1996) Effects of vomitoxin (deoxynivalenol) on transcription factor NF-kappa B/Rel binding activity in murine EL-4 thymoma and primary CD4+ T cells. Toxicol. Appl. Pharmacol. 140:2328–336.[CrossRef][Web of Science][Medline]
Periwal SB and Cebra JJ. (1999) Respiratory mucosal immunization with reovirus serotype 1/L stimulates virus-specific humoral and cellular immune responses, including double-positive (CD4(+)/CD8(+)) T cells. J. Virol. 73:97633–7640.
Pestka JJ. (2003) Deoxynivalenol-induced IgA production and IgA nephropathy-aberrant mucosal immune response with systemic repercussions. Toxicol. Lett. 140–141:287–295.[CrossRef]
Pestka JJ and Smolinski AT. (2005) Deoxynivalenol: Toxicology and potential effects on humans. J. Toxicol. Environ. Health B Crit Rev. 8:139–69.[Web of Science][Medline]
Pestka JJ and Zhou HR. (2000) Interleukin-6-deficient mice refractory to IgA dysregulation but not anorexia induction by vomitoxin (deoxynivalenol) ingestion. Food Chem. Toxicol. 38:7565–575.[CrossRef][Web of Science][Medline]
Pestka JJ, Zhou HR, Moon Y, Chung YJ. (2004) Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: Unraveling a paradox. Toxicol. Lett. 153:161–73.[CrossRef][Web of Science][Medline]
Romieu I, Samet JM, Smith KR, Bruce N. (2002) Outdoor air pollution and acute respiratory infections among children in developing countries. J. Occup. Environ. Med. 44:7640–649.[CrossRef][Web of Science][Medline]
Russo C and Polosa R. (2005) TNF-alpha as a promising therapeutic target in chronic asthma: A lesson from rheumatoid arthritis. Clin. Sci. (Lond) 109:2135–142.[Medline]
Shyy YJ, Li YS, Kolattukudy PE. (1993) Activation of MCP-1 gene expression is mediated through multiple signaling pathways. Biochem. Biophys. Res. Commun. 192:2693–699.[CrossRef][Web of Science][Medline]
Silvey KJ, Hutchings AB, Vajdy M, Petzke MM, Neutra MR. (2001) Role of immunoglobulin A in protection against reovirus entry into Murine Peyer's patches. J. Virol. 75:2210870–10879.
Spannhake EW, Reddy SP, Jacoby DB, Yu XY, Saatian B, Tian J. (2002) Synergism between rhinovirus infection and oxidant pollutant exposure enhances airway epithelial cell cytokine production. Environ. Health Perspect. 110:7665–670.[Web of Science][Medline]
Tai JH and Pestka JJ. (1990) T-2 toxin impairment of murine response to Salmonella typhimurium: A histopathologic assessment. Mycopathologia 109:3149–155.[CrossRef][Web of Science][Medline]
Taub DD, Proost P, Murphy WJ, Anver M, Longo DL, van DJ, Oppenheim JJ. (1995) Monocyte chemotactic protein-1 (MCP-1), -2, and -3 are chemotactic for human T lymphocytes. J. Clin. Invest. 95:31370–1376.[Web of Science][Medline]
Thompson AH, London L, Bellum SC, Hamamdzic D, Harley RA, London SD. (1996) Respiratory-mucosal lymphocyte populations induced by reovirus serotype 1 infection. Cell Immunol. 169:2278–287.[CrossRef][Web of Science][Medline]
Thompson AH, McRoberts JG, Crowe SR, London L, London SD. (1999) Optimal induction of upper respiratory tract immunity to reovirus 1/L by combined upper and lower respiratory tract inoculation. Vaccine 17:11–121404–1415.[CrossRef][Web of Science][Medline]
Tyler KL, Clarke P, DeBiasi RL, Kominsky D, Poggioli GJ. (2001) Reoviruses and the host cell. Trends Microbiol. 9:11560–564.[CrossRef][Web of Science][Medline]
Uzarski RL, Islam Z, Pestka JJ. (2003) Potentiation of trichothecene-induced leukocyte cytoxicity and apoptosos by TNF-alpha and fas activation. Chem Biol. Interact. 146:2105–109.[CrossRef][Web of Science][Medline]
Voynow JA, Gendler SJ, Rose MC. (2006) Regulation of mucin genes in chronic inflammatory airway diseases. Am. J Respir. Cell Mol. Biol. 34:6661–665.
Wanner HU. (1993) Effects of atmospheric pollution on human health. Experientia 49:9754–758.[CrossRef][Web of Science][Medline]
Wong SS, Zhou HR, Pestka JJ. (2002) Effects of vomitoxin (deoxynivalenol) on the binding of transcription factors AP-1, NF-kappaB, and NF-IL6 in raw 264.7 macrophage cells. J. Toxicol. Environ. Health A 65:161161–1180.[CrossRef][Web of Science][Medline]
Zhou HR, Harkema JR, Hotchkiss JA, Yan D, Roth RA, Pestka JJ. (2000) Lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol) synergistically induce apoptosis in murine lymphoid organs. Toxicol. Sci. 53:2253–263.
Zhou HR, Harkema JR, Yan D, Pestka JJ. (1999) Amplified proinflammatory cytokine expression and toxicity in mice coexposed to lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol). J. Toxicol. Environ. Health 57:2115–136.
Zhou HR, Islam Z, Pestka JJ. (2003a) Kinetics of lipopolysaccharide-induced transcription factor activation/inactivation and relation to proinflammatory gene expression in the murine spleen. Toxicol. Appl. Pharmacol. 187:3147–161.[CrossRef][Web of Science][Medline]
Zhou HR, Islam Z, Pestka JJ. (2003b) Rapid, sequential activation of mitogen-activated protein kinases and transcription factors precedes proinflammatory cytokine mRNA expression in spleens of mice exposed to the trichothecene vomitoxin. Toxicol. Sci. 72:1130–142.
Zhou HR, Islam Z, Pestka JJ. (2005a) Induction of competing apoptotic and survival signaling pathways in the macrophage by the ribotoxic trichothecene deoxynivalenol. Toxicol. Sci. 87:1113–122.
Zhou HR, Jia Q, Pestka JJ. (2005b) Ribotoxic stress response to the trichothecene deoxynivalenol in the macrophage involves the SRC family kinase Hck. Toxicol. Sci. 85:2916–926.
Zhou HR, Lau AS, Pestka JJ. (2003c) Role of double-stranded RNA-activated protein kinase R (PKR) in deoxynivalenol-induced ribotoxic stress response. Toxicol. Sci. 74:2335–344.
Ziprin RL and Elissalde MH. (1990) Effect of T-2 toxin on resistance to systemic Salmonella typhimurium infection of newly hatched chickens. Am. J. Vet. Res. 51:111869–1872.[Web of Science][Medline]
Ziprin RL and McMurray DN. (1988) Differential effect of T-2 toxin on murine host resistance to three facultative intracellular bacterial pathogens: Listeria monocytogenes, Salmonella typhimurium, and Mycobacterium bovis. Am. J. Vet. Res. 49:71188–1192.[Web of Science][Medline]
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