Passive transfer of IgG anti-GM1 antibodies impairs peripheral nerve repair.

Anti-GM1 antibodies are present in some patients with autoimmune neurological disorders. These antibodies are most frequently associated with acute immune neuropathy called Guillain-Barré syndrome (GBS). Some clinical studies associate the presence of these antibodies with poor recovery in GBS. The patients with incomplete recovery have failure of nerve repair, particularly axon regeneration. Our previous work indicates that monoclonal antibodies can inhibit axon regeneration by engaging cell surface gangliosides (Lehmann et al., 2007). We asked whether passive transfer of human anti-GM1 antibodies from patients with GBS modulate axon regeneration in an animal model. Human anti-GM1 antibodies were compared with other GM1 ligands, cholera toxin B subunit and a monoclonal anti-GM1 antibody. Our results show that patient derived anti-GM1 antibodies and cholera toxin beta subunit impair axon regeneration/repair after PNS injury in mice. Comparative studies indicated that the antibody/ligand-mediated inhibition of axon regeneration is dependent on antibody/ligand characteristics such as affinity-avidity and fine specificity. These data indicate that circulating immune effectors such as human autoantibodies, which are exogenous to the nervous system, can modulate axon regeneration/nerve repair in autoimmune neurological disorders such as GBS.

An update on pathobiologic roles of anti-glycan antibodies in Guillain-Barré syndrome.

Anti-glycan antibodies directed against gangliosides are now considered the major immune effectors that induce damage to intact nerve fibers in some variants of the monophasic neuropathic disorders that comprise Guillain-Barré syndrome. Recent experimental studies elucidating the complexity of anti-glycan antibody-mediated pathobiologic effects on intact and injured nerves undergoing repair are discussed.

Molecular structure of antibodies against small ligands

Antibodies which bind bioactive ligands can serve as a template for the generation of a second antibody which may react with the physiological receptor. This phenomenon of molecular mimicry by antibodies has been described in a variety of systems. In order to understand the chemical and molecular mechanisms involved in these interactions, monoclonal antibodies directed against two pharmacologically active alkaloids, morphine and nicotine, were carefully studied using experimental and theoretical molecular modeling techniques. The molecular characterization of these antibodies involved binding studies with ligand analogs and determination of the variable region amino acid sequence. A three-dimensional model of the anti-morphine binding site was constructed using computational and graphics display techniques. The antibody response in BALB/c mice to morphine appears relatively restricted, in that all of the antibodies examined in this study contained a $\lambda$ light chain, which is normally found in only 5% of mouse immunoglobulins. This study represents the first use of theoretical and experimental modeling techniques to describe the antigen binding site of a mouse Fv region containing a $\lambda$ light chain. The binding site model indicates that a charged glutamic acid residue and aromatic side chains are key features in ionic and hydrophobic interactions with the ligand morphine. A glutamic acid residue is found in the identical position in the anti-nicotine antibody and may play a role in binding nicotine. ^

Production of antibodies to canine IL-1beta and canine TNF to assess the role of proinflammatory cytokines

IL-1 and TNF are important proinflammatory cytokines implicated in both antimicrobial host defense and pathogenesis of diseases with an immune-mediated and/or inflammatory component. Respective studies in the dog have been hampered by the unavailability of reagents allowing the specific measurement of canine cytokine proteins and the effect of canine cytokine neutralization by Ab. Starting with recombinant canine (rcan) IL-1beta and rcanTNF, four polyclonal antisera and 22 mAb specific for rcanIL-1beta and rcanTNF were generated. Their usefulness in neutralization assays was determined. Using cytokine-containing supernatants of canine cells in bioassays, polyclonal antisera neutralized either canine IL-1beta or TNF. TNF was also neutralized by three antibodies developed in this study and one commercial mAb. The usefulness of monoclonal and polyclonal Ab in canine cytokine-specific Ab capture ELISA's was assessed. This resulted in the identification of a commercial mAb combination and one pair developed in this study allowing low levels of TNF to be detected by antibody capture ELISA. The detection limit was 141 pg/ml rcanTNF for both combinations. Using rcanIL-1beta as an antigen allowed the detection of lower concentrations of rcanIL-1beta (20 pg/ml, on the average) by a pair of polyclonal antisera than when monoclonals were used. By using such IL-1beta-specific and TNF-specific ELISA's, the respective cytokines were detected in supernatants of canine PBMC stimulated with LPS or heat-killed Listeria monocytogenes and interferon-gamma combined. Thus, monoclonal and polyclonal reagents were identified allowing the quantitation of canine IL-1beta and TNF production in vitro, and the neutralization of these cytokines.

Prevalence of antibodies against bluetongue virus serotype 8 in bulk-tank milk samples from dairy cattle herds located in risk areas for bluetongue virus transmission after a vaccination programme in Switzerland.

Switzerland had been affected by the bluetongue virus serotype 8 (BTV-8) epidemic in Europe in the years 2007 to 2009. After three years of mandatory vaccination and comprehensive surveillance, Switzerland showed to be free of BTV-8 in 2012. In the future Elisa testing of bulk-tank milk (BTM) samples as a very sensitive and cost-effective method should be used for the surveillance of all serotypes of BTV. To determine the prevalence of seropositive herds, BTM from 240 cattle herds was sampled in July 2012. The results showed an apparent seroprevalence of 98.7% in the investigated dairy herds. Most plausible, the high prevalence was caused by the vaccination campaigns rather than by infections with BTV-8. In the outbreak the cumulative number of BTV-8 cases in Switzerland had been 75.Thus it is very likely that the used inactivated vaccines induced long-term antibody titres. Due to the high seroprevalence, investigating for BT-antibodies cannot be used for early recognition of a new introduction of BTV at the moment. Nonetheless, testing of BTM samples is appropriate for an annual evaluation of the seroprevalence and especially as an instrument for early recognition for incursions as soon as the antibody prevalence declines.To determine this decline the BTM testing scheme should be conducted each year as described in this work.

Immunohistochemical panel for the diagnosis of Hirschsprung's disease using antibodies to MAP2, calretinin, GLUT1 and S100.

AIMS The diagnosis of Hirschsprung's disease is currently based on the identification of aganglionosis and the presence of an increase in acetylcholinesterase-positive hypertrophic nerve fibres in the large bowel submucosa. However, acetylcholinesterase staining is laborious and requires a skilled technician. The aim of this study was to identify a method for diagnosing Hirschsprung's disease reliably using an immunohistochemical panel of recently proposed markers. METHODS AND RESULTS Sixty-nine specimens from 37 patients were evaluated. MAP2 and calretinin antibodies were shown to stain ganglia reliably in the submucosal and myenteric plexuses of normal tissue. By contrast, reduced staining of ganglia was observed in patients with Hirschsprung's disease. Staining for GLUT1 and S100 was used to evaluate the number and thickness of nerve fibres. Gain of GLUT1 and S100 expression was in contrast to the loss of calretinin and MAP2. Hypertrophic submucosal nerve fibres in Hirschsprung's disease develop a perineurium with a ring-like GLUT1 staining pattern similar in size and intensity to that observed in deeper subserosal tissue. CONCLUSIONS The diagnosis of Hirschsprung's disease using immunohistochemical panels could be as accurate as with conventional frozen section techniques. In particular, the use of a combination of markers for ganglia and hypertrophic nerve fibres highlighting a prominent perineurium in Hirschsprung's disease could be an alternative method.

Prevalence of antileptospiral serum antibodies in dogs in Ireland.

A total of 474 serum samples from client owned Irish dogs were tested for the presence of antibodies to serovars Canicola, Icterohaemorrhagiae, Bratislava, Autumnalis, Pomona, Altodouro, Grippotyphosa, Mozdok, Hardjobovis and Ballum. Six per cent of dogs presented to veterinary practitioners for problems unrelated to leptospirosis showed evidence of prior exposure to leptospiral serovars belonging to the serogropus Ballum, Australis, Pomona and Sejroe. One unvaccinated dog suspected to have leptospirosis showed seroconversion to serogroup Icterohaemorrhagiae. Based on these results the authors conclude that canine exposure to serogroup Ballum should be monitored because dogs may serve as sentinels for this serovar in the environment. Vaccination with multivalent vaccines containing serovar Bratislava in addition to serogroups Icterohaemorrhagiae and Canicola is advisable.

Evaluation of a Commercial ELISA for the Detection of Antibodies to Sarcoptes scabiei in Wild Boar (Sus scrofa).

Sarcoptic mange occurs in free-ranging wild boar (Sus scrofa) but has been poorly described in this species. We evaluated the performance of a commercial indirect enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of sarcoptic mange in domestic swine when applied to wild boar sera. We tested 96 sera from wild boar in populations without mange history ("truly noninfected") collected in Switzerland between December 2012 and February 2014, and 141 sera from free-ranging wild boar presenting mange-like lesions, including 50 live animals captured and sampled multiple times in France between May and August 2006 and three cases submitted to necropsy in Switzerland between April 2010 and February 2014. Mite infestation was confirmed by skin scraping in 20 of them ("truly infected"). We defined sensitivity of the test as the proportion of truly infected that were found ELISA-positive, and specificity as the proportion of truly noninfected that were found negative. Sensitivity and specificity were 75% and 80%, respectively. Success of antibody detection increased with the chronicity of lesions, and seroconversion was documented in 19 of 27 wild boar sampled multiple times that were initially negative or doubtful. In conclusion, the evaluated ELISA has been successfully applied to wild boar sera. It appears to be unreliable for early detection in individual animals but may represent a useful tool for population surveys.