Rest in poultry

Resting behaviour as well as other maintenance behaviours were studied in a group of Jungle Fowl (10 ♀♀, 1 ♂) and in a group of a medium hybrid commercial laying strain (9 ♀♀, 1 ♂). Observations were carried out under spacious and varied housing conditions. Both groups were observed for seven 24-h periods. Sleeping and dozing are defined and described and 24-h patterns of rest and other maintenance behaviours are shown. Results and differences between groups are discussed.     

Lack of an association between candidate gene loci and idiopathic generalized epilepsy in Kuwaiti Arab children

Summary Idiopathic generalized epilepsies (IGEs) are the most common types of epilepsy in childhood and adolescence. A variety of data suggest that IGEs have a predominant genetic etiology. Recently, a number of gene mutations have been found to be associated with various types of epilepsy in mainly the Caucasian populations. The objective of this study was to investigate the association of three different candidate genes with IGE in Kuwaiti Arab children. This study includes 123 Kuwaiti patients with a confirmed diagnosis of epilepsy. Most of the patients have had a diagnostic EEG with generalized spike-wave discharges (GSWs). All patients were evaluated by using a validated seizure questionnaire. The clinical type of epilepsy was determined by a trained neurologist/pediatrician. The study also include 100 controls, the control subjects were children which did not have any history of neurological disorders. Blood samples were collected from all patients and control subjects after taking informed consent. DNA was isolated and analyzed by molecular methods. A FokI polymorphism in neuronal nicotinic acetylcholine receptor alpha-4 subunit (CHRNA4) gene was detected by PCR-RFLP method. A missense mutation (Ser248Phe) in CHRNA4 gene was analyzed by PCR-RFLP using HpaII. A C121W mutation in sodium-channel beta-1 subunit (SCN1B) gene was screened by a PCR-RFLP method using HinPI. A 2-bp deletion in Cystatin B gene was detected by PCR-RFLP using XcmI. The incidence of three FokI polymorphism genotypes in Kuwaiti IGE patients was 1,1 (85%), 1,2 (14%) and 2,2 (1%) respectively. The missense mutation Ser248Phe of CHRNA4 gene was not detected at all in Kuwaiti IGE patients. The C387G transversion resulting in C121W change in third exon of the SCN1B gene was detected in 3/123 patients (2%). The patients carrying this mutation also exhibited febrile seizures. The incidence of 2 bp deletion in the cystatin B gene was found to be 4% (5/123 IGE patients). The data obtained from molecular analysis show a lack of association between three candidate genes and clinical expression of IGE in Kuwaiti Arab children. This is completely different from the findings reported from Caucasian populations of France, Australia and USA in which case a strong association has been reported between IGE and these genes. To whom corresspondence should be addressed. Tel: +965-5319486; Fax: +965-5338940; E-mail: haider@hsc.edu.kw     

A human IgE bispecific antibody shows potent cytotoxic capacity mediated by monocytes

The generation of bispecific antibodies (bsAbs) targeting two different antigens opens a new level of specificity and, compared to mAbs, improved clinical efficacy in cancer therapy. Currently, the different strategies for development of bsAbs primarily focus on IgG isotypes. Nevertheless, in comparison to IgG isotypes, IgE has been shown to offer superior tumor control in preclinical models. Therefore, in order to combine the promising potential of IgE molecules with increased target selectivity of bsAbs, we developed dual tumor-associated antigen-targeting bispecific human IgE antibodies. As proof of principle, we used two different pairing approaches - knobs-into-holes and leucine zipper–mediated pairing. Our data show that both strategies were highly efficient in driving bispecific IgE formation, with no undesired pairings observed. Bispecific IgE antibodies also showed a dose-dependent binding to their target antigens, and cell bridging experiments demonstrated simultaneous binding of two different antigens. As antibodies mediate a major part of their effector functions through interaction with Fc receptors (FcRs) expressed on immune cells, we confirmed FcεR binding by inducing in vitro mast cell degranulation and demonstrating in vitro and in vivo monocyte-mediated cytotoxicity against target antigen-expressing Chinese hamster ovary cells. Moreover, we demonstrated that the IgE bsAb construct was significantly more efficient in mediating antibody-dependent cell toxicity than its IgG1 counterpart. In conclusion, we describe the successful development of first bispecific IgE antibodies with superior antibody-dependent cell toxicity–mediated cell killing in comparison to IgG bispecific antibodies. These findings highlight the relevance of IgE-based bispecific antibodies for clinical application.     

Behavioural and physiological consequences of acute social defeat in growing gilts: effects of the social environment

Endocrine, behavioural and immunologic processes, together with body growth, were evaluated in gilts that were defeated at 10 weeks of age in resident-intruder tests. Immediately after defeat, gilts were either separated from or reunited with a familiar conspecific (litter-mate; always a barrow). Gilts were assigned to one of four treatments: (a) DI: defeat, followed by isolation (separation from original litter-mate; n=8); (b) I: no defeat, isolation (control group; n=9); (c) DP; defeat, followed by pair-housing (reunion with original litter-mate; n=8); and (d) P: no defeat, pair-housing (control group; n=8). The following general conclusions were derived: (1) social defeat caused pronounced short-term elevations in hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal medullary activities, and of prolactin levels. Moreover, as soon as 1h after defeat, percentages of blood lymphocytes and neutrophilic granulocytes were, respectively, decreased and increased; (2) social defeat had some long-lasting influence on behaviour and physiology, but isolation predominantly determined responses in the longer term. Defeat, as well as isolation, resulted in increased cardiovascular activities compared to P controls, as observed in a novel object test (NOT: +7 days) and an aversion test (AVT: +14 days). Moreover, defeated as well as isolated gilts did not habituate to a repeated novel environment test (NET: -7, +2 and +7 days) in terms of frequencies of vocalising, whereas P controls did. Isolation, through the separation from any other pig, was responsible for the other observed long-term characteristics, which developed progressively. Isolated gilts showed high mobilities and high cortisol responses in the repeated NET (+7 days), not being habituated. This contrasted the reactions of pair-housed gilts, which were much reduced. In addition to their high cardiovascular activities in the NOT and the AVT, isolated gilts also displayed higher heart rates in the repeated NET and during human presence following the NOT, compared to pair-housed gilts. Finally, isolated gilts were more inhibited to approach a novel object (in the NOT) than pair-housed pigs; and (3) stress responses of defeated gilts were modulated by the subsequent social environment. Stimulation of the HPA-axis (plasma- and salivary cortisol) was prolonged in those defeated gilts which were isolated (observed in the first hour). Changes in leucocyte subsets were still observed after 3 days in DI, but were 'normalised' within 1 day in DP gilts. Two days after defeat, habituation to the repeated NET in terms of mobility and salivary cortisol responses occurred in control and DP gilts, but not in DI gilts. We argue that these effects of the social environment shortly after defeat were related to a stress-reducing effect of a stable social relationship, i.e. social support.     

Progranulin directly binds to the CRD2 and CRD3 of TNFR extracellular domains

We previously reported that PGRN directly bound to TNF receptors (TNFR) in vitro and in chondrocytes (Tang, et al., Science, 2011). Here we report that PGRN also associated with TNFR in splenocytes, and inhibited the binding of TNFα to immune cells. Proper folding of PGRN is essential for its binding to TNFR, as DTT treatment abolished its binding to TNFR. In contrast, the binding of PGRN to Sortilin was enhanced by DTT. Protein interaction assays with mutants of the TNFR extracellular domain demonstrated that CRD2 and CRD3 of TNFR are important for the interaction with PGRN, similar to the binding to TNFα. Taken together, these findings provide the molecular basis underlying PGRN/TNFR interaction and PGRN-mediated anti-inflammatory activity in various autoimmune diseases and conditions.     

Antigen Binding Characteristics of Immunoglobulin Free Light Chains: Crosslinking by Antigen is Essential to Induce Allergic Inflammation

Beside the production of complete immunoglobulins IgG, IgE, IgA, IgM and IgD, consisting of tetrameric heterodimers of immunoglobulin heavy and light chains, B cells also secrete immunoglobulin free light chains (Ig-fLC). Previous studies showed that Ig-fLCs are able to induce immediate hypersensitivity reactions. It is apparent that recognition and binding of antigen are crucial steps in the onset of these inflammatory responses. In this study, the binding characteristics of Ig-fLC to antigen were further investigated using various biochemical approaches. In addition, we investigated whether antigen-mediated crosslinking of Ig-fLC is required to initiate allergic skin inflammation in vivo. Our study shows that binding of Ig-fLCs to antigen can be measured with different experimental setups. Surface plasmon resonance analysis showed real-time antigen binding characteristics. Specific antigen binding by Ig-fLCs was further detected using immunoblotting and ELISA. Using the ELISA-based assay, a binding affinity of 76.9±3.8 nM was determined for TNP-specific Ig-fLC. Antigen-induced ear swelling in mice passively sensitized with trinitrophenol-specific Ig-fLC was inhibited when multivalent antigen was combined with excess of monovalent antigen during challenge. We conclude that Ig-fLCs are able to interact with antigen, a prerequisite for antigen-specific cellular activation. In analogy to antigen-specific Fc receptor-induced mast cell activation, crosslinking of Ig-fLCs is necessary to initiate a local allergic response.