Measurement of canine IgE using the alpha chain of the human high affinity IgE receptor

In vitro assays for allergen specific immunoglobulin E (IgE) are a convenient and reproducible alternative to intradermal skin testing in dogs. Such tests may be used to support a diagnosis of atopic dermatitis and to define appropriate allergens for immunotherapy. Current in vitro assays rely upon monoclonal or polyclonal antibodies as IgE detection reagents. However, in sera where allergen-specific IgG occurs in great excess, any IgE:IgG cross-reactivity of the detection reagent may result in lowered assay specificity. Therefore, we have developed an assay for canine IgE which uses a recombinant form of the extracellular part of the alpha chain of the human high affinity IgE receptor (FcvarepsilonRIalpha). Biotinylated FcvarepsilonRIalpha shows no significant binding to purified canine IgG, and recognizes a heat labile antibody in serum, with a detection limit of 73-146pg/ml. Comparison of assay signals using the labeled FcvarepsilonRIalpha and a highly specific anti-canine IgE monoclonal antibody (MAb) shows good agreement. The FcvarepsilonRIalpha is therefore a sensitive and specific alternative to polyclonal or monoclonal antibodies for canine serum IgE measurement.     

Viability of OPS Vitrified Sheep Embryos After Direct Transfer

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.     

Immune Modulating Brevetoxins: Monocyte Cytotoxicity,Apoptosis, and Activation of M1/M2 Response Elements Is Dependent on Reactive Groups

Brevetoxins are a suite of marine neurotoxins that activate voltage-gated sodium channels (VGSCs) in cell membranes, with toxicity occurring from persistent activation of the channel at high doses. Lower doses, in contrast, have been shown to elicit neuroregeneration. Brevetoxins have thus been proposed as a novel treatment for patients after stroke, when neuron regrowth and repair is critical to recovery. However, findings from environmental exposures indicate that brevetoxins may cause inflammation, thus, there is concern for brevetoxins as a stroke therapy given the potential for neuroinflammation. In this study, we examined the inflammatory properties of several brevetoxin analogs, including those that do and do not bind strongly to VGSCs, as binding has classically indicated toxicity. We found that several analogs are toxic to monocytes, while others are not, and the degree of toxicity is not directly related to VGSC binding. Rather, results indicate that brevetoxins containing aldehyde groups were more likely to cause immunotoxicity, regardless of binding affinity to the VGSC. Our results demonstrate that different brevetoxin family members can elicit a spectrum of apoptosis and necrosis by multiple possible mechanisms of action in monocytes. As such, care should be taken in treating “brevetoxins” as a uniform group, particularly in stroke therapy research.     

Comparative Study on the Performance of Three Detection Methods for the Quantification of Pacific Ciguatoxins in French Polynesian Strains of Gambierdiscus polynesiensis

Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis. fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient (r² = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus. These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management.