[Investigation on population dynamics and Echinococcus infections in small rodents around human settlement in Yushu City, Qinghai Province].

OBJECTIVE: To investigate the population dynamics and Echinococcus infections in small rodents around human settlement in Yushu City, Qinghai Province. METHODS: Rodents were captured using the mouse trap method in pastures from Batang Township and Longbao Township of Yushu City, Qinghai Province on May, August and October, 2018. The body weight and snout-vent length of all captured rodents were measured, and the species was identified according to the rodent morphology. Genomic DNA was extracted from rodent liver specimens and lesion specimens, and the mitochondrial cox1 gene of Echinococcus was amplified using PCR assay for identification of parasite species. In addition, the tissue specimens positive for PCR assay were sampled for pathological examinations. The prevalence of Echinococcus infections was estimated in rodents, and a phylogenetic tree was created based on Echinococcus cox1 gene sequences. RESULTS: A total of 285 small rodents were captured, including 143 Ochotona curzoniae (50.2%), 141 Lasiopodomys fuscus (49.5%), and 1 Neodon irene (0.3%), and there was a remarkable variation in habitat selection among these three rodent species. The number of L. fuscus correlated positively with vegetation coverage (r = 0.350, P = 0.264), with the greatest number seen in August, and the number of O. curzoniae negatively with vegetation coverage (r = -0.371, P = 0.235), with the highest number seen in August and the lowest number in May. The female/male ratios of O. curzoniae and voles were 1:0.96 and 0.82:1, respectively. The body weight (r = 0.519, P < 0.01) and snout-vent length (r = 0.578, P < 0.01) of O. curzoniae showed a tendency towards a rise with month, while the body weight (r = -0.401, P < 0.01) and snout-vent length (r = -0.570, P < 0.01) of voles presented a tendency towards a reduction with month. No Echinococcus infection was detected in voles, while 2.1% prevalence of E. shiquicus infection was seen in O. curzoniae. Phylogenetic analysis revealed consistent sequences of cox1 gene from E. shiquicus in Yushu City of Qinghai Province and Shiqu County, Ganzi Tibetan Autonomous Prefecture of Sichuan Province. CONCLUSIONS: The small rodents around the human settlement in Yushu City of Qinghai Province mainly include O. curzoniae and L. fuscus, with the greatest numbers seen in May and August, respectively. Following the concerted efforts for echinococcosis control, the prevalence of Echinococcus infections is low in small rodents around the human settlement in Yushu City; however, there is still a risk of echinococcosis transmission.

[Establishment of a recombinase-aided isothermal amplification assay for nucleic acid detection of Echinococcus multilocularis and its preliminary application].

OBJECTIVE: To establish a rapid nucleic acid detection technique for identification of Echinococcus multilocularis based on the recombinase aided isothermal amplification assay (RAA) and assess its diagnostic efficiency. METHODS: The mitochondrial gene sequence of E. multilocularis (GenBank accession number: AB018440) was used as a target sequence. The primers were designed according to the RAA reaction principle and synthesized, and RAA was performed using the generated primers. E. multilocularis genomic DNA at various concentrations and the pMD19-T (Simple) vector containing various copies of the target gene fragment were amplified using RAA to evaluate its sensitivity for detection of E. multilocularis, and RAA was em- ployed to detect the genomic DNA of E. granulosus G1 genotype, Taenia saginata, T. asiatica, T. multiceps, Dipylidium caninum, Toxocara canis, Trichuris trichiura, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Fasciola gigantica and Clonorchis sinensis to evaluate its specificity. In addition, the optimized RAA was employed to detect nine tissue specimens of E. granulosus-infected animals, 3 fecal samples from E. granulosus-infected dogs and 2 fecal samples from field infected dogs to examine its reliability and feasibility. RESULTS: The established RAA was able to detect the specific target gene fragment of E. multilocularis within 40 min. The lowest detect limit of RAA was 10 pg if E. multilocularis genomic DNA served as a template. If the re- combinant plasmid was used as a template, the minimally detectable copy number of RAA was 104. In addition, RAA was nega- tive for the genomic DNA of E. granulosus G1 genotype, T. saginata, T. asiatica, T. multiceps, D. caninum, T. canis, T. trichiura, G. lamblia, F. hepatica, P. westermani, F. gigantica and C. sinensis. The established RAA was positive for detection of the tissue specimens of infected animals, and simulated and field dog stool samples. CONCLUSIONS: A rapid, sensitive and specific RAA is established, which shows promising values in identification of E. multilocularis and gene diagnosis of alveolar echinococcosis.

[Analysis of colorectal cancer screening results in Kunming from 2014 to 2017].

This study analyzed the distribution of high-risk population, the compliance and detected lesions of colorectal cancer screening from the Cancer Screening Program in urban areas of Kunming,Yunnan Province from 2014 to 2017. A total of 127 960 residents were included,of which 14 791 (11.70%) cases were diagnosed with high risk of colorectal cancer by the National Cancer Center High Risk Population Assessment System. A total of 3 484 cases completed colonoscopy clinical screening and the rate of participation was 23.55%. The screening results showed that 592 positive cases were detected, and the positive rate was 17.17%. The detection rates of polyps,adenomas,advanced adenomas,precancerous lesions and colorectal cancer were 16.27%,13.12%,7.18%,7.63% and 0.26%, with 567, 457, 250, 266 and 9 cases, respectively.

[Clinical significance of hypermethylation of DLC-1 gene in myelodysplastic syndrome patients and effects of decitabine on DLC-1 gene expression].

Objective: To detect the methylation status of DLC-1 gene in the patients with myelodysplastic syndrome(MDS), the effect of abnormal methylation of DLC-1 gene on the expression of DLC-1 gene, the clinical significance of methylation of DLC-1 gene in MDS patients, and the effect of decitabine on DLC-1 gene expression. Methods: A total of 43 MDS patients were treated in Fujian Medical University Union Hospital from 2013 to 2015. Methylation status of DLC-1 gene in MDS patients were detected by the methylation specific PCR(MSP). The expression of DLC-1 gene mRNA was determined with real-time fluorescence quantitative PCR(RTFQ-PCR). MDS patients were divided into 5 groups (very low-risk, low-risk, intermediate-risk, high-risk and very high-risk, n=0, 8, 7, 18, 10) according to WPSS classification. And the clinical significance of methylation of DLC-1 gene in patients with MDS were investigated. In order to investigate the change in gene methylation and expression of DLC-1 gene after treatment with decitabine, methylation statuses of DLC-1 gene in MDS patients before and after be treated with decitabine were detected by the bisulfite sequencing PCR(BSP). The expressions of DLC-1 gene mRNA of these patients were determined with RTFQ-PCR. Results: Hypermethylation of CpG island of DLC-1 gene was observed in 55.16%(22/43)MDS patients. The expressions of DLC-1 gene mRNA in methylation positive patients were significantly lower than that in methylation negative patients (0.32±0.06 vs 0.91±0.11)(P=0.008). For MDS patients, the DLC-1 methylation rate of intermediate-and high-risk patient was 21/35, which was significantly higher than that of low-risk patient(1/8, P=0.006). The methylation status of DLC-1 gene were monitored in 8 patients before and after treatment with the decitabine (decitabine 20 mg/m(2,) d1-d5/d28, more than 4 courses) , the methylation rate of DLC-1 gene dropped from 57.50%±5.11% to 14.13%±2.07% after treatment(P=0.010). The expression of DLC-1 gene increased after treatment with decitabine(0.67±0.08 vs 0.28±0.06, P=0.015). Conclusions: Methylation of DLC-1 gene is common in MDS patients and may be associated with poor prognosis. Decitabine may activate the expression of DLC-1 gene by demethylation, which may be one of the mechanisms for the treatment of patients with MDS.

Differential expression of calcium-regulating genes in heat-stressed turkey breast muscle is associated with meat quality.

Aberrant postmortem Ca(2+)-regulation in the early postmortem period is associated with the occurrence of inferior meat quality in turkeys, described as pale, soft, and exudative (PSE). The objective of the current study was to quantify expression of 4 candidate genes responsible for maintaining Ca(2+) homeostasis in turkey skeletal muscle as a function of heat stress: α and ß ryanodine receptors (RYR; Ca(2+)-release channels), the sarco/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1), and the sarcoplasmic reticulum, Ca(2+)-storage protein calsequestrin (CASQ1). Two genetic lines of turkeys were used: a growth-selected commercial line and a randombred control line. Market-age birds were subjected to one of 5 heat stress treatments: no heat, 1 d, 3 d, 5 d, or 7 d of heat followed by 7 d of ambient temperature. Breast muscle samples were harvested and classified as normal or PSE using the meat quality parameters percentage of marinade uptake and percentage of cook loss. These parameters differed significantly by line, heat stress treatment, and meat quality status. Expression of candidate genes was measured using TaqMan quantitative PCR. Heat treatment was associated with significantly enhanced expression of αRYR, ßRYR, and CASQ1 in normal muscle from both lines. Conversely, mRNA abundance of these genes was reduced in PSE muscle from both lines and recovered or increased by 7 d + 7 d of rest. Genetic line differences were observed at several time points. Expression of SERCA1 in both normal and PSE samples from both lines was unchanged or trended downward with heat stress. Taken together, genetic line and heat-stress treatment affected the expression of important Ca(2+)-regulating genes in association with meat quality status. The data suggest that birds whose meat leads to PSE may fail to respond to heat stress appropriately due to a delay in the upregulation of the important calcium-regulating genes: αRYR, ßRYR, and CASQ1.

Effects of tumor necrosis factor type 1 and 2 receptor deficiencies on anorexia, growth and IgA dysregulation in mice exposed to the trichothecene vomitoxin.

Dietary exposure of mice to vomitoxin (VT), a trichothecene mycotoxin, causes anorexia and impaired growth as well as inducing elevated serum IgA and kidney mesangial IgA deposition in a manner analogous to human IgA nephropathy. Based on the observations that TNF-alpha is induced by in vitro and in vivo VT exposure, it was hypothesized that this cytokine plays a role in the nutritional and immunological effects of this toxin. To test this hypothesis, the effects of dietary VT on feed intake, weight gain, serum IgA levels and kidney mesangial IgA deposition in mice homozygous for targeted disruption of the two known TNF-alpha cell surface receptors, TNFR1(p55) or TNFR2(p75), were compared to effects in corresponding C57BL/6J wild-type (WT) mice with normal receptor function. The capacity of VT to cause feed refusal or impair weight gain over a 12-week feeding period was not impaired in TNFR1 knockout (KO) or TNFR2-KO as compared to WT mice. Both WT and TNFR-KO mice fed VT exhibited reduced (P<0.05) feed conversion efficiency, but surprisingly, feed conversion efficiency was significantly higher (P<0.05) in TNFR1-KO and TNFR2-KO fed either control or VT diets than in corresponding WT mice. By week 12, serum IgA concentrations in all three mouse groups fed VT were significantly higher than those for corresponding mice fed control diets (P<0.05). Serum IgA levels in the VT-fed TNFR1-KO group were significantly less (P<0.05) than those for the VT-fed WT mice at 4, 8 and 12 weeks, whereas no differences in this parameter were found between the TNFR2-KO and WT groups. Serum IgA immune complex concentrations were measured at wk 12 and found to follow an identical pattern to IgA. Kidneys taken from VT-fed TNFR2-KO and WT mice after 12 weeks had significantly increased mesangial IgA deposition as compared to controls. While slight increases in mesangial IgA were also observed in VT-fed TNFR1-KO mice, these levels were significantly less (P<0.05) than that found in VT-fed TNFR2-KO and WT mice. Taken together, the data suggest that while VT-mediated anorexic and growth effects were largely independent of TNF-alpha, VT-induced dysregulation of IgA production was dependent, in part, on the interaction of TNF-alpha with TNFR1.

Interleukin-6-deficient mice refractory to IgA dysregulation but not anorexia induction by vomitoxin (deoxynivalenol) ingestion.

Dietary exposure to the trichothecene vomitoxin (VT) causes feed refusal and elevates IgA production in the mouse. Based on the observations that IL-6 can cause anorexia and promote IgA production and that gene expression of this cytokine is increased in vivo and ex vivo on VT exposure, we hypothesized that IL-6 is an essential cytokine in VT-induced feed refusal and IgA dysregulation. To test this hypothesis, the effects of dietary VT on feed intake, weight gain, serum IgA levels and kidney mesangial IgA deposition in an IL-6-"knockout" mouse (B6129-IL6(tmi Kopf)) were compared to those in both a corresponding "wildtype" (B6129F2) and a previously characterized "sentinel" strain (B6C3F1) that possess the intact gene for this cytokine. IL-6 deficiency did not alter the capacity of VT to cause feed refusal or impair weight gain. VT-fed B6129F2 and B6C3F1 mice had significantly higher serum IgA concentrations than did their corresponding controls fed clean diet, whereas significant differences were not observed between IL-6 KO mice fed VT or control diets. Kidneys taken from VT-fed wild-type and sentinel mice had significantly increased mesangial IgA deposition as compared to controls. While slight increases in mesangial IgA were observed in VT-fed IL-6 KO mice, mean fluorescence intensities were significantly less than that found in the corresponding wild-type and sentinel strains. IL-6 KO mice appeared to be less prone to the development of microscopic haematuria following VT exposure than were the corresponding wild-type and sentinel strains. In total, the results suggested that IL-6-deficient mice were refractory to VT-induced dysregulation of IgA production and development of IgA nephropathy, whereas chronic VT-mediated nutritional effects related to feed intake and weight gain were unaffected.

Lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol) synergistically induce apoptosis in murine lymphoid organs.

Human exposure to Gram-negative bacterial lipopolysaccharide (LPS) is common and may have an important influence on chemical toxicity. LPS has been shown previously to enhance synergistically the toxicity of trichothecene mycotoxins. Because either of these toxin groups alone characteristically target lymphoid organs at high doses, we evaluated the effects of coexposure to subthreshold doses of Salmonella typhimurium LPS and vomitoxin (VT) administered by intraperitoneal injection and oral gavage of B6C3F1 mice, respectively, on apoptosis in lymphoid tissues after 12-h exposure. The capacity of LPS (0.5 mg/kg body weight) and VT (25 mg/kg body weight) to act synergistically in causing apoptosis in thymus, spleen, and Peyer's patches was suggested by increased internucleosomal DNA fragmentation in whole cell lysates as determined by gel electrophoresis. Following terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end-labeling (TUNEL) of tissue sections, a dramatic enhancement of fluorescence intensity indicative of apoptosis was observed in thymus, spleen, Peyer's patches, and bone marrow from coexposed animals as compared to those given the agents alone. Evaluation of hematoxylin and eosin-stained tissue sections of treatment mice revealed the characteristic features of lymphocyte apoptosis, including marked condensation of nuclear chromatin, fragmentation of nuclei, and formation of apoptotic bodies in tissues from mice. Combined treatment with VT (25 mg/kg body weight) and LPS (0.5 mg/kg body weight) significantly increased (p<0.05) the amount of apoptotic thymic and splenic tissue as compared to the expected additive responses of mice receiving either toxin alone. When apoptosis was examined in cell suspensions of thymus, spleen, Peyer's patches, and bone marrow by flow cytometry in conjunction with propidium iodide staining, the percentage of apoptotic cells was significantly increased (p<0.05) in cotreatment groups as compared to the additive responses to LPS and VT given alone. The results provide qualitative and quantitative evidence for the hypothesis that LPS exposure markedly amplifies the toxicity of trichothecenes and that the immune system is a primary target for these interactive effects.

Amplified proinflammatory cytokine expression and toxicity in mice coexposed to lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol).

A single oral exposure to the trichothecene vomitoxin (VT) has been previously shown in the mouse to increase splenic mRNA levels for several cytokines in as little as 2 h. Since one underlying mechanism for these effects likely involves superinduction of transiently expressed cytokine genes, VT may also potentially amplify cytokine responses to inflammatory stimuli. To test this possibility, the effects of oral VT exposure on tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1beta expression were measured in mice that were intraperitoneally injected with lipopolysaccharide (LPS), a prototypic inflammatory agent. As anticipated, VT alone at 1, 5, and 25 mg/kg body weight increased splenic mRNA expression of all three cytokines after 3 h in a dose-response fashion. LPS injection at 1 and 5 mg/kg body weight also induced proinflammatory cytokine mRNA expression. There was a synergistic increase in TNF-alpha splenic mRNA levels in mice treated with both VT and LPS as compared to mice treated with either toxin alone, whereas the effects were additive for IL-6 and IL-1beta mRNA expression. When relative mRNA levels were examined over a 12-h period in mice given LPS (1 mg/kg) and/or VT (5 mg/kg), significant enhancement was observed up to 6, 12, and 3 h for TNF-alpha, IL-6, and IL-1beta, respectively. When plasma cytokine concentrations were measured, TNF-alpha was found to peak at 1 h and was significantly increased at 1, 3, and 6 h if mice were given LPS and VT, whereas LPS or VT alone caused much smaller increases in plasma TNF-alpha Plasma IL-6 peaked at 3 h in LPS, VT, and LPS/VT groups, with the combined toxin group exhibiting additive effects. Plasma IL-1beta was not detectable. The potential for VT and LPS to enhance toxicity was examined in a subsequent study. Mortality was not observed up to 72 h in mice exposed to a single oral dose of VT at 25 mg/kg body weight or to an intraperitoneal dose of LPS at 1 or 5 mg/kg body weight; however, all mice receiving VT and either LPS dose became moribund in less than 40 h. The principal histologic lesions in the moribund mice treated with VT and LPS were marked cell death and loss in thymus, Peyer's patches, spleen, and bone marrow. In all of these lymphoid tissues, treatment-induced cell death had characteristic histologic features of apoptosis causing lymphoid atrophy. These results suggest that LPS exposure may markedly increase the toxicity of trichothecenes and that the immune system was a primary target of these interactive effects.

Effects of yogurt ingestion on mucosal and systemic cytokine gene expression in the mouse.

To assess the potential for ingestion of yogurt to modulate immunity, its effects on basal gene expression of cytokines in systemic and mucosal sites were determined in mice. Yogurts were manufactured from pasteurized nonfat dry milk using five commercial starter cultures with or without Bifidobacterium sp. and Lactobacillus acidophilus. Treatment mice were fed the AIN-93G diet mixed 1:1 with unheated yogurt or heat-treated yogurt (wt/wt) for 2 and 4 weeks, and control mice were fed the AIN-93G diet mixed 1:1 (wt/wt) with nonfat dry milk. The viability of the various bacterial groups in unheated yogurts was maintained above 10(6) CFU/g throughout the feeding period. The yogurt-feeding regimens did not significantly affect weight gain. Relative mRNA levels in spleen, mesenteric lymph nodes, or Peyer's patches for the cytokines interferon-gamma, tumor necrosis factor-alpha, interleukin-2, -4, and -6, and the "housekeeping gene" beta2-microglobulin were determined by reverse transcriptase-polymerase chain reaction in conjunction with hybridization analysis. Prolonged feeding of some yogurts decreased expression of several cytokine mRNAs, the depression of tumor necrosis factor-alpha mRNA in the spleen being the most prominent effect. Heat-treated yogurts were more effective in altering cytokine mRNA expression than were unheated yogurts containing viable organisms. Generally, yogurts either had no effect or decreased specific cytokine mRNA in the test organs, regardless of whether they contained Bifidobacterium sp. and L. acidophilus. These results suggest that, in contrast with previous studies in vitro, some yogurt formulations may reduce rather than stimulate basal cytokine expression and that these effects are most prominent in the systemic compartment.

Induction of cytokine gene expression in mice after repeated and subchronic oral exposure to vomitoxin (Deoxynivalenol): differential toxin-induced hyporesponsiveness and recovery.

A single oral exposure to vomitoxin (VT) in mice has been previously shown to induce in lymphoid tissues the rapid expression of cytokine mRNAs that are produced by both macrophages and T cells. To determine whether prior VT exposures positively or negatively modulate the cytokine response to the toxin in this model, we evaluated the effects of short-term oral (two to seven consecutive daily doses) and subchronic dietary (4 weeks) exposure to VT on expression of a panel of cytokine mRNAs. Effects of a single oral exposure to 0, 5, and 25 mg/kg body wt of VT or of two such daily consecutive doses on splenic cytokine mRNA abundance were compared 2 h after the last toxin administration using RT-PCR in combination with hybridization analysis. While robust cytokine mRNA responses occurred after a single VT exposure, attenuated but significant induction of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-12p40 mRNA was observed after a second VT dose. Similar but insignificant trends occurred with interferon (IFN)-gamma, IL-2, IL-4, and IL-10 mRNAs. Serum TNF-alpha and IL-6 proteins mimicked cytokine mRNA responses although attenuation responses were less marked. Mice were also dosed with VT at 0, 0.5, 2, or 5 mg/kg body wt consecutively for 2, 4, or 7 days and cytokine mRNAs were assessed 2 h after the last treatment in spleen and Peyer's patches. Upon exposure to 2 and 5 mg/kg body wt VT, the relative abundance of IL-1beta, IL-6, TNF-alpha, IL-12 p35, IL-12p40, IL-2, and IL-10 mRNAs increased with dose frequency whereas IFN-gamma and IL-4 mRNAs were unaffected. When mice were fed 0, 10, and 25 ppm VT for 4 weeks, increased expression of mRNAs for TNF-alpha, IL-2, IFN-gamma, and IL-10 was most prominent. However, when VT-fed mice were also challenged with an oral dose of VT equivalent to daily intake at 2 h prior to RNA isolation, vigorous mRNA responses were observed for IL-1beta, IL-6, TNF-alpha, IL-12p40, IL-12p35, IL-2, IFN-gamma, IL-4, and IL-10. In general, spleens were more responsive to the above effects than Peyer's patches. The results indicate that, following a single prior VT exposure, a significant but attenuated cytokine mRNA response occurred upon a second VT treatment. This hyporesponsiveness was overcome upon repeated exposures to the toxin. These data further support the contention that elevated cytokine expression may play a contributory role in the pathophysiologic and immunologic effects of VT and other trichothecene mycotoxins.

Modulation of IL-1beta, IL-6 and TNF-alpha secretion and mRNA expression by the trichothecene vomitoxin in the RAW 264.7 murine macrophage cell line.

Oral exposure of mice to vomitoxin (VT) has been previously shown to enhance gene expression of several cytokines associated with macrophage activation. Here, the effects of exposure to VT in vitro on cytokine secretion and mRNA expression were determined in the murine macrophage cell line RAW 264.7. Enzyme-linked immunosorbent assay (ELISA) of supernatants revealed that significant increases in secreted tumour necrosis factor alpha (TNF-alpha) were observed 2 days after exposure to VT at 100 ng/ml and 250 ng/ml, both with and without lipopolysaccharide (LPS) activation. While VT did not affect IL-6 secretion in the absence of LPS, significantly increased IL-6 production was observed in culture supernatants after 1, 2 and 5 days of exposure to VT at 250 ng/ml in the presence of LPS. Soluble IL-1beta was not detected in control or VT-treated cell cultures with or without LPS activation. Immunochemical staining of intracellular cytokines in conjunction with flow cytometric analysis was used to detect the effects of VT on the percentage of positive cells and output per cell. The percentage of cells that produced intracellular TNF-alpha were significantly increased at 100 and 250 ng/ml VT with and without LPS whereas increased IL-6 output per cell was observed at 100 and 250 ng/ml VT with LPS. To assess the effects of VT on cytokine mRNA expression, RAW 264.7 cells were analysed semi-quantitatively using reverse transcription-polymerase chain reaction(RT-PCR) in conjunction with Southern hybridization analysis. Elevated TNF-alpha mRNA was observed at 100 and 250 ng VT/ml at 6 and 24 hr in the absence of LPS. With the addition of LPS, superinduction of TNF-alpha was not observed in the presence of VT. Increased IL-1beta and IL-6 mRNAs were observed at 100 and 250 ng VT/ml at 24 hr in the presence of LPS. These results demonstrated that VT could superinduce both cytokine secretion and mRNA levels in macrophage cultures.

Role of macrophages in elevated IgA and IL-6 production by Peyer's patch cultures following acute oral vomitoxin exposure.

Oral vomitoxin (VT) exposure in mice results in elevated cytokine gene expression, increased production of IgA, and IgA nephropathy. To determine the potential role of macrophages (Mphi) in these effects, an ex vivo model was devised whereby Peyer's patch (PP) and spleen cells were prepared from mice 2 h after oral exposure to 0 or 25 mg/kg body wt VT, cultured, and then evaluated for IgA and cytokine IL-6 production. Both PP and, to a lesser extent, spleen cells from treatment mice produced more IgA over a 7-day period than did corresponding control cells when cultured without a costimulus or in the presence of either phorbol myristate acetate plus ionomycin (PMA + ION) or lipopolysaccharide (LPS); IgA elevation was most marked in LPS-treated cultures. The VT effect was completely ablated in PP cultures that were depleted of Mphi but not in Mphi-depleted spleen cultures. VT exposure similarly increased production of IL-6, an important helper factor for IgA secretion, in LPS-stimulated PP and spleen cell cultures. IL-6 production was also ablated by Mphi depletion. A potential costimulatory role for Mphi was further suggested because both IgA and IL-6 production increased when Mphi-depleted PP cells from VT-treated animals were cocultured with peritoneal Mphi from VT-treated animals. Similar effects were observed when an analogous ex vivo approach was used with purified PP B cells and peritoneal Mphi. PP B cells from control animals also secreted elevated levels of IgA when cocultured with splenic CD4(+) cells from VT-treated animals, thus confirming previous studies showing that T cell help also contributes to increased IgA production. Potential roles for soluble mediators and cell contact in this process were suggested when IgA production was measured in cultures of PP cells separated from VT-treated Mphi by a semipermeable membrane. Taken together, these and previous results suggest that Mphi may play a key mechanistic role in elevated IgA production and IgA nephropathy in VT-exposed mice.

Potential role for IL-5 and IL-6 in enhanced IgA secretion by Peyer's patch cells isolated from mice acutely exposed to vomitoxin.

Dietary exposure to vomitoxin (VT) results in hyperelevated serum IgA and IgA nephropathy in mice. To assess the possible role of cytokines in this IgA dysregulation, the effects of a single oral exposure in B6C3F1 male mice to 0, 5 or 25 mg/kg BW VT on production of IgA and cytokines in Peyer's patch (PP) and spleen cell cultures were evaluated. IgA levels were increased significantly in PP cell cultures prepared from mice at 2 or 24 h after oral exposure to VT and subsequently stimulated with phorbol myristate acetate (PMA) and ionomycin (ION) or with lipopolysaccharide (LPS). Significant effects on IgA production were not observed in spleen cell cultures. Since cytokines such as IL-2, IL-4, IL-5 and IL-6 have been shown to promote IgA production, the effect of the same VT exposure regimen on secretion of these mediators was determined in PP and spleen cultures. Supernatant IL-2 and IL-4 levels were unaffected by the prior treatment of animals with VT. In contrast, IL-5 levels were increased significantly in 7-day PP cell cultures obtained 2 h after VT exposure both with and without PMA + ION exposure but not in other cultures. IL-6 levels were increased significantly in LPS-treated cultures prepared from PP at 2 and 24 h following exposure to VT. IL-6 levels were also elevated significantly in both PMA + ION or LPS treated cultures from spleen isolated at 2 h but not 24 h post VT exposure. To determine whether IL-5 or IL-6 play a role in IgA hyperelevation in vitro, PP and spleen cells from mice obtained 2 h after exposure to 25 mg/kg VT were cultured in the presence of neutralizing cytokine antibodies (Abs) and IgA production was monitored. Consistent with IL-5's previously documented role in IgA production, anti-IL-5 decreased IgA levels to background in cultures of both control and VT-exposed PP or spleen cells in the presence of either PMA + ION or LPS. Similar results were seen with addition of anti-IL-6. IgA levels were decreased to a lesser extent in PP cells cultured with LPS and in spleen cells cultured with PMA + ION from VT-exposed mice to which anti-IL-2 Ab was added. Thus, the potential for enhanced IgA production exists in lymphocytes as early as 2 h and as late as 24 h after a single oral exposure to VT and this may be related to the increased capacity to secrete helper cytokines of T cell and macrophage origin. Taken together, the results suggest that the superinduction of cytokine expression may, in part, be responsible for upregulation of IgA secretion in mice exposed orally to VT.

Differential cytokine mRNA expression in mice after oral exposure to the trichothecene vomitoxin (deoxynivalenol): dose response and time course.

Acute oral exposure of B6C3F1 mice to vomitoxin (VT) has been previously shown to induce expression of mRNAs for cytokines that are characteristically produced in lymphoid tissues by macrophage and T cells. The purpose of this study was to evaluate the effects of VT dose on the expression of mRNAs for a cytokine profile consisting of TNF-alpha, IL-1beta, IL-6, IL-12, IFN-gamma, IL-2, IL-4, IL-5, IL-10, IL-12, and TGF-beta and to measure the kinetics of these responses. The effects of a single oral exposure to 0, 0.1, 0.5, 1, 5, and 25 mg/kg BW of VT on cytokine mRNA abundance in spleen and Peyer's patches (indicators of systemic and mucosal immune compartments, respectively) were assessed at 2 hr postexposure using reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with hybridization analysis. Both 5 and 25 mg/kg VT significantly induced the mRNAs for the proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha; the T helper 1 cytokines IFN-gamma and IL-2; and the T helper 2 cytokines IL-4 and IL-10, whereas lower doses had no effect. IL-12p40 mRNA was also induced but not IL-12p35 mRNA. The effects were more pronounced in spleen than in the Peyer's patches. IL-5 and TGF-beta mRNAs were expressed constitutively in spleen and Peyer's patches but were not affected by VT. When cytokine mRNA levels were measured at 1, 2, 4, 8, and 24 hr after oral exposure to 25 mg/kg BW of VT, mRNA expression kinetics varied among cytokines or between spleen and Peyer's patches. The duration of elevated mRNA expression in spleen was 1-8 hr for TNF-alpha, IL-6, IFN-gamma, and IL-10 and was 1-4 hr for IL-1beta, IL-12p40, IL-2, and IL-4. In Peyer's patches, duration periods were 1-8 hr for IL-6, IL-2, and IL-10; 1-4 hr for IL-1beta, IL-12p40, and TNF-alpha; and 1-2 hr for IFN-gamma. Serum levels of TNF-alpha, IL-6, and IFN-gamma were elevated 3 hr after exposure to 25 mg/kg VT, thus suggesting that elevation of splenic and Peyer's patch mRNA abundance correlated with increases in systemic production of these cytokines. Taken together, the results indicate that a single VT exposure rapidly induces gene expression in vivo for a wide range of cytokines with apparently complete recovery occurring after 24 hr. Elevated cytokine expression may play an important role in the pathophysiologic effects of VT and other trichothecenes.

Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone.

The heavy-chain and kappa light-chain variable region genes of an antizearalenone hybridoma cell line (2G3-6E3-2E2) were isolated by PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a single-chain Fv (scFv) DNA fragment. The scFv DNA fragment was cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with E tag and phage M13 p3 in Escherichia coli TG1. In the presence of helper phage M13K07, the scFv fusion protein was displayed on the surfaces of recombinant phages. High-affinity scFv phages were enriched through affinity selection in microtiter wells coated with zearalenone-ovalbumin conjugate. The selected recombinant phages were used to infect E. coli HB2151 for the production of soluble scFv antibodies. One selected clone (pQY1.5) in HB2151 secreted a soluble scFv antibody (QY1.5) with a high zearalenone-binding affinity (concentration required for 50% inhibition of binding, 14 ng/ml), similar to that of parent monoclonal antibody in a competitive indirect enzyme-linked immunosorbent assay. However, scFv QY1.5 exhibited higher cross-reactivity with zearalenone analogs and had greater sensitivity to methanol destabilization than the parent monoclonal antibody did. Nucleotide sequence analyses revealed that the light-chain portion of scFv QY1.5 had a nucleotide sequence identity of 97% to a mouse germ line gene VK23.32 in mouse kappa light-chain variable region subgroup V, whereas the heavy-chain nucleotide sequence was classified as mouse heavy-chain subgroup III (D) but without any closely related members having highly homologous complementarity-determining region sequences. The potential of soluble scFv QY1.5 for routine screening of zearalenone and its analogs was demonstrated with zearalenone-spiked corn extracts.

Polychlorinated biphenyls release insulin from RINm5F cells.

Polychlorinated biphenyls (PCBs) possess a variety of biological effects, including alterations in growth, development and metabolism, that may be dependent on insulin. However, no reports on the action of PCBs on cells which produce and secrete insulin are available. The current study examined the ability of a commercial mixture of PCBs (Aroclor 1254) and three specific PCB congeners, to alter the release of insulin using the hormone producing cell line RINm5F. Exposure of cells to Aroclor 1254 (A-1254) produced a concentration-dependent increase in media insulin reaching a peak, when expressed as percent of control, at 30 min. In spite of continued exposure, media insulin relative to control declined and no treatment-related difference was observed at 48 hrs. Cellular levels of the hormone declined as much as 50% by that time. The insulin releasing action of A-1254 was mimicked by each of the non-coplanar congeners 2,2',4,4'-tetrachlorobiphenyl (TCB) and 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) but the coplanar congener 3,3',4,4'-TCB showed no significant activity. These results indicate that PCBs are capable of producing a release of insulin from RINm5F cells, an effect that is unlikely to be associated with coplanar congeners that initiate their action by binding to the Ah-receptor.

Production of a hybridoma cell line secreting retinoic acid-specific monoclonal antibody.

A stabilized hybridoma cell line secreting anti-retinoic acid monoclonal antibodies of subclass IgG1 with kappa chains was produced by fusing NS-1 myeloma cells with the spleen cells from BALB/c female mice immunized with all-trans-4-oxoretinoic acid-oxime-chicken IgG conjugate. The antibody titer of mice ascitic fluid ranged from 1/12,800 to 1/25,600, as determined by competitive indirect enzyme-linked immunosorbent assay (ELISA). 50% inhibition dosage of all-trans-retinoic acid at a 1/20,000 dilution of mice ascitic fluid was 6.6 ng/ml, as determined by ELISA. The anti-retinoic acid monoclonal antibody was generated in mice ascitic fluid and purified by protein G affinity chromatography. Cross-reactivity of the monoclonal antibody was determined at 0.1 microgram/ml concentration of retinoids and indicated high specificity to both all-trans-retinoic acid (86% inhibition) and 13-cis-retinoic acid (87% inhibition), and strong cross-reactivity with 4-oxoretinoic acid (77%) and 4-oxoretinoic acid oxime (109%). Specificity was confirmed by the horseradish peroxidase-linked immunostaining method and immunoradioassay. The affinity constant of the monoclonal antibody, K, was determined to be 3.6 X 10(9) l/mol. A calibration curve for retinoic acid using the monoclonal antibody to retinoic acid was developed; the detection limit for all-trans-retinoic acid is 1 ng/ml in the competitive indirect ELISA. The antibody counteracts the effect of retinoic acid on growth inhibition and differentiation in HL-60 cells.