In the Schwann cells and neuronal plasma membranes the gangliosides are organized in clusters forming complexes of gangliosides in the microdomains termed lipid rafts. We investigated frequency, clinical correlates, fine specificity and pro-inflammatory properties of antibodies to ganglioside complexes (GSCs) in a Guillain Barre syndrome (GBS) population. In 63 patients with different GBS variants we performed an ELISA for antibodies to Campylobacter Jejuni (C. jejuni), gangliosides and GSCs. We studied the fine specificity of antibodies to GSCs by immunoabsorption study and performed a complement activation assay. Twenty-seven percent of patients had antibodies to GSCs and 71 percent had antibodies either to single gangliosides or to GSCs. Patients with antibodies to GSCs had more frequent involvement of cranial nerves but did not present more frequent antecedent respiratory, gastrointestinal or C. jejuni infection, did not have a preferential demyelinating or primary axonal GBS variant and did not develop greater disability at six months. The absorption study showed in 2 sera that antibodies to the complex GD1a/GD1b did not react with the gangliosides forming the complex or other single gangliosides, suggesting that antibodies to GSCs are targeted to new conformational glycoepitopes different from the ones displayed by the single gangliosides. Antibody anti-GSCs activated the complement more frequently than antibodies to single gangliosides. Complement activation indicates that antibodies to GSCs have high avidity, pro-inflammatory properties and may exert a pathogenic role in GBS.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.A c c e p t e d M a n u s c r i p t Abstract 16Because no suitable products are at the moment available to safely control the spread of 17 BTV-16 in Europe, an inactivated vaccine was produced from the reference field isolate 18 of bluetongue virus serotype 16. One group of six sheep was vaccinated subcutaneously 19 with the inactivated vaccine twice, on days 0 and 28, whereas a second group of 8 20 sheep was inoculated with saline solution and used as mock-vaccinated control 21 animals. Seventy eight days after the first vaccination, all sheep were inoculated 22 subcutaneously with a suspension containing 10 6.3 TCID 50 of a virulent reference BTV-23 16 isolate. Apart from a transient inflammatory reaction at the injection site, no adverse 24 effects were reported following vaccination. All vaccinated animals developed high titres 25 (7.3-9.3 Log 2 (ED50%/50µl)) of virus-specific neutralizing antibodies and were resistant 26 * Manuscript

Four hybridoma cell lines (C4B, 10C2G5, 6C5F4C7, 2D10G11) were adapted to grow in serum-free conditions. Cell proliferation, viability, and antibody production in Nutridoma SP and Ex-Cell HSF 610 media were evaluated and results compared with those obtained using DMEM containing 10% fetal bovine serum (control medium). Clone C4B showed the best IgG productivity in control medium, but viability and number of cells per milliliter were similar for the three media. For clone 10C2G5, the highest values of cell viability were obtained with both control medium and Nutridoma SP; no significant differences in IgG yields and number of cells/mL were observed among the three media. Clone 6C5F4C7 provided no significant differences when grown in the two serum-free media and in control medium. Clone 2D10G11 showed the best IgG productivity in control medium and in Ex-Cell HSF 610, even if Ex-Cell HSF 610 provided the lowest number of cells/mL; no significant differences among the three media were obtained for viability. Purified antibodies produced from each hybridoma cell line grown in serum-free media showed a higher degree of purity than those produced by the same cell lines grown in control medium. Purified MAbs were also titrated by ELISA to test MAb-antigen affinity.

Yessotoxins (YTXs) are polycyclic ether compounds produced by phytoplanktonic dinoflagellates and accumulated in filter-feeding shellfish. Mouse bioassay is still the official method to detect these toxins, even if it is lacking of specificity and sensitivity. Moreover, there is growing resistance against the use of animal experiments. Many efforts have been made to determine YTXs with other methods. The detection of YTX using a functional assay allows its quantification with an automated and repetitive technique at concentrations in the range of the 1 mg of YTX equivalent/kg European regulatory limit. In this study, an in vitro functional assay based on YTX treatment of MCF-7 cells and resulting in the accumulation of a 100-kDa fragment of E-cadherin was developed on samples of Mytilus galloprovincialis collected from the Adriatic Sea, Italy, along the coasts of Abruzzo, Molise, and Emilia Romagna regions. The YTX concentrations ranged from 0.2 to 1.8 mg of YTX equivalent/kg. The occurrence of levels exceeding the above mentioned limit was observed only in samples of Emilia Romagna region. This last result could represent a risk for human health, but these shellfish were not intended to consumers, because they belonged to a preventive monitoring program.

Monoclonal antibodies (MAbs) against horse IgG were produced by immunizing Balb/c mice with purified horse IgG and were characterized in indirect ELISA versus purified immunoglobulins from donkey, cow, buffalo, sheep, pig, and chicken. Three MAbs (1B10B6C9, 1B10B6C10, 1B10B6E9) reacted only with horse and donkey IgG and IgM and, in western blotting, were specific for the Fc fragment of equine IgG. MAb 1B10B6E9 was used in chemiluminescent immunoblotting assay for the diagnosis of dourine and in indirect immunofluorescence assay (IFA) for the diagnosis of African horse sickness and dourine.