Assessment of deoxynivalenol metabolite profiles in UK adults

Deoxynivalenol (DON) requires no activation for toxicity, though susceptibility may reflect individual variations in detoxification. This study reports the measurement of un-metabolised urinary DON (free DON) and DOM-1 in samples previously analysed for the combined measure of free DON + DON-glucuronide (fD + DG), with a concentration >5 ng/ml, for 34 UK adults. Four consecutive daily urine samples were analysed from twenty-two individuals, whilst from 12 individuals only a single sample was analysed. The mean (median) concentration of urinary fD + DG in this sub-set was 17.8 ng/ml (13.8 ng/ml), range 5.0-78.2 ng/ml. In 23/34 (68%) individuals, free DON was detected, mean 2.4 ng/ml; range 0.5-9.3 ng/ml. Urinary DOM-1 was detected in 1/34 (3%) of individuals; present at ∼1% of urinary fD + DG concentration for that individual. The concentration of fD + DG combined was significantly correlated with urinary free DON (p < 0.001, R² = 0.65), but not with the percentage of free DON to fD + DG (p = 0.615, R² = 0.01), suggesting that the level of DON exposure did not affect the metabolism to DG within the range observed. In this survey most individuals had no detectable urinary DOM-1 and 68% did not detoxify all of the ingested DON to DON-glucuronide. This study needs to be extended to understand whether the fD / DG ratio provides a phenotypic measure of DON susceptibility.     

Effects of pretransfusion warming of platelets to 35°C on posttransfusion platelet viability

BACKGROUND: Three of four prior studies suggested that warming platelets (PLTs) to 37°C before transfusion into patients with thrombocytopenia gave improved corrected PLT count increments.
STUDY DESIGN AND METHODS: Eighteen normal subjects had apheresis PLTs collected that were stored at 22°C for 5 days in two storage bags. One bag of PLTs was warmed to 35°C before infusion, and one remained at 22°C. Three different methods of warming the donor's autologous PLTs before reinfusion were evaluated: warming PLTs to 35°C for 10 or 60 minutes followed by radiolabeling or radiolabeling the PLTs followed by warming to 35°C for 60 minutes. In the first two methods, the warmed PLTs would have returned to 22°C before infusion, and in the third, the PLTs would still be warm when injected. The paired test and control PLTs were radiolabeled with either ¹¹¹In or ⁵¹Cr to determine posttransfusion PLT recoveries and survivals. PLT morphology score, pH, hypotonic shock response, extent of shape change, and annexin V binding were determined just before transfusion.
RESULTS: There were no differences in posttransfusion autologous radiolabeled PLT recoveries and survivals or in the in vitro measurements for the PLTs maintained at 22°C versus those warmed to 35°C using any of the three methods of PLT warming before infusion.
CONCLUSION: Based on these 5-day-stored autologous radiolabeled PLT recovery and survival measurements, there is no evidence that warming PLTs to 35°C before infusion improves postinfusion PLT viability.     

Prevention of Dystrophic Pathology in Severely Affected Dystrophin/Utrophin-deficient Mice by Morpholino-oligomer-mediated Exon-skipping

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon-skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore dystrophin expression, which has been demonstrated in vitro and in vivo. In particular, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been shown to induce widespread high levels of dystrophin expression in the mdx mouse model. Here, we report the efficiency of the PPMO-mediated exon-skipping approach in the utrophin/dystrophin double-knockout mouse (dKO) mouse, which is a much more severe and progressive mouse model of DMD. Repeated intraperitoneal (i.p.) injections of a PPMO targeted to exon 23 of dystrophin pre-mRNA in dKO mice induce a near-normal level of dystrophin expression in all muscles examined, except for the cardiac muscle, resulting in a considerable improvement of their muscle function and dystrophic pathology. These findings suggest great potential for PPMOs in systemic treatment of the DMD phenotype.     

Hormonal influences of early postnatal indomethacin and ibuprofen in neonatal rats

ObjectiveIndomethacin and ibuprofen are administered to preterm neonates for symptomatic patent ductus arteriosus. The drugs suppress prostaglandins (PGs) which modulate growth and secretion of various hormones. We examined the hypothesis that early postnatal indomethacin and ibuprofen influence growth and GH-IGF-I-insulin and HPA axes in neonatal rats.DesignRat pups received IP injections of saline (Sal) on P1, P2, and P3; 10 mg/kg ibuprofen on P1 followed by 5 mg/kg on P2 and P3; or 0.2 mg/kg indomethacin on P1 followed by 0.1 mg/kg on P2 and P3. Serum and hepatic GH, GHBP and IGF-I; and serum corticosterone and insulin levels were determined.ResultsIbuprofen suppressed somatic growth in the sucking rats, but the effect was transient, resolving by P14. Indomethacin had an opposite, latent effect on body weight and liver to body weight ratios in weanling rats. Both indomethacin and ibuprofen had profound hormonal effects that differed in magnitude and timing. Indomethacin resulted in a sustained elevation in corticosterone levels at P21, while ibuprofen increased serum and hepatic GH levels. Both drugs suppressed GHBP in serum at P7 and P14; and liver at P4 and P7, but a rebound increase in serum GHBP was noted at P21 with Ibuprofen only. Both drugs increased serum IGF-I at P7. The effect remained sustained with indomethacin.ConclusionsThese results provide evidence for an involvement of PGs in the regulation of growth as well as the GH-IGF and HPA axes. Therefore, early postnatal exposure to PG inhibitors may further exacerbate postnatal growth restriction and ability to cope with stress.     

Autophagy: assays and artifacts

Autophagy is a fundamental and phylogenetically conserved self‐degradation process that is characterized by the formation of double‐layered vesicles (autophagosomes) around intracellular cargo for delivery to lysosomes and proteolytic degradation. The increasing significance attached to autophagy in development and disease in higher eukaryotes has placed greater importance on the validation of reliable, meaningful and quantitative assays to monitor autophagy in live cells and in vivo in the animal. To date, the detection of processed LC3B‐II by western blot or fluorescence studies, together with electron microscopy for autophagosome formation, have been the mainstays for autophagy detection. However, LC3 expression levels can vary markedly between different cell types and in response to different stresses, and there is also concern that over‐expression of tagged versions of LC3 to facilitate imaging and detection of autophagy interferes with the process itself. In addition, the realization that it is not sufficient to monitor static levels of autophagy but to measure ‘autophagic flux’ has driven the development of new or modified approaches to detecting autophagy. Here, we present a critical overview of current methodologies to measure autophagy in cells and in animals. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.