Ameliorative effect of dietary supplementation of copper oxide nanoparticles on inflammatory and immune reponses in commercial broiler under normal and heat-stress housing conditions.

Heat stress (HS) is one of the most serious adverse conditions that affect poultry causing immunosuppression and decreasing production. In a novel approach, we investigated effects of supplementing copper oxide nanoparticles (CuO-NPs) on the immune response in two commercial broiler strains (Ross 308 and Cobb 500). At one day old, birds were divided into 3 groups with 3 replicates for each. The first group received diet supplemented with 100% of their recommended copper requirements as CuO while, in the second and third groups, birds were given diets supplemented with 100% and 50% of the recommended Cu requirements in the form of CuO-NPs, respectively. At age of 21 day, each group was subdivided randomly into normal (24 ±â€¯2 °C) and heat stressed (33 ±â€¯2 °C for 5 h per day for two successive weeks) groups. Under normal housing temperature, CuO-NPs, significantly enhanced the immune response in these birds, compared to CuO shown by the increased levels of phagocytic activity (PA), lysozyme serum activity, and by upregulating immune-modulator genes including NF-κß, PGES, IL-1ß, TGF-1ß, IFN-γ, BAX and CASP8. The responses were different between the two studied strains especially at the level of gene expression. In HS birds, supplementation of CuO-NPs reduced HS induced inflammatory conditions, as shown by lower gene expression levels, lower degenerative changes in the spleen, and altered heterophils/lymphocytes (H/L) ratio. We suggest CuO-NPs supplementation, especially in those chickens that received diet supplemented with 50% of their recommended Cu requirements, could be used under normal housing temperature to enhance the birds' immune response, and during HS to lower heat stress-induced degenerative changes depending on the magnitude of the HS.

Expression and Regulation of Cholecystokinin Receptor in the Chicken's Immune Organs and Cells.

Cholecystokinin (CCK) is a neuropeptide that affects growth rate in chickens by regulating appetite. CCK peptides exert their function by binding to two identified receptors, CCKAR and CCKBR in the GI tract and the brain, respectively, as well as in other organs. In mammals, CCK/CCKAR interactions affect a number of immunological parameters, including regulation of lymphocytes and functioning of monocytes. Thus, food intake and growth can potentially be altered by infection and the resulting inflammatory immune response. It is uncertain, however, whether chicken express CCKAR in immune organs and cells, and, if so, whether CCKAR expression is regulated by pathogen derived inflammatory stimuli. Herein, we identify expression of CCKAR protein in chicken peripheral blood mononuclear cells (PBMC) including monocytes, and expression of the CCKAR gene in PBMC, thymus, bursa, and spleen, in selected commercial and pure chicken breeds. Further, stimulation with various types of E. coli heat-labile enterotoxins or lipopolysaccharide significantly regulated expression of CCKAR on monocytes in the different breeds. Ligation of CCKAR with antibodies in PBMC induced mobilization of Ca2+, indicating that CCKAR is signal competent. Injection with polyinosinic: polycytidylic acid (poly I:C), a synthetic analogue of double stranded viral RNA that binds Toll-Like Receptor-3 (TLR3), also regulated gene expressions of CCKAR and proinflammatory cytokines, in the different breeds. Interestingly, variations in the expression levels of proinflammatory cytokines in the different breeds were highly correlated with CCKAR expression levels. Taken together, these findings indicate that the physiological function of CCKAR in the chicken is tightly regulated in immune organs and cells by external inflammatory stimuli, which in turn regulate growth. This is the first report CCKAR expression in immune organs and cells, in any species, and the initial observation that CCKAR is regulated by inflammatory stimuli associated with bacterial and viral infection.

The Quest for an HIV-1 Vaccine Adjuvant: Bacterial Toxins as New Potential Platforms.

While tremendous efforts are undergoing towards finding an effective HIV-1 vaccine, the search for an HIV-1 vaccine adjuvant lags behind and is understudied. More recently, however, efforts have focused on testing adjuvant formulations that can boost the immune response and generate broadly neutralizing antibodies to HIV-1 ENV (gp160). Despite this, there remain a number of challenges towards achieving this goal. These include safety of adjuvant formulations; stability of the incorporated antigens; maintenance of ENV immunogenicity; optimal inoculation sites; the effective combination of adjuvants; stability of ENV neutralizing epitopes in some adjuvant formulations; mucosal immunity; and long-term maintenance of the immune response. A new class of adjuvants for HIV-1 proteins is suggested to overcome many of the limitations of some other adjuvants. Type 1 (LT-I) and type 2 (LT-II) human E. coli enterotoxins (HLTs) and their non-toxic B-subunits derivatives are strong systemic and mucosal adjuvants and effective carriers for other proteins and epitopes. Their stable molecular structure in the presence of fused proteins and epitopes, and their ability to target surface receptors on antigen presenting cells make them ideal for the delivery of HIV-1 ENV or HIV other proteins. Importantly, unlike some other adjuvants, HLTs and derivatives have well-defined modes of immune system activation. The challenges in finding optimal HIV-1 vaccine adjuvant formulation and the important properties of HLTs are discussed.

E. coli Heat-labile Enterotoxin B Subunit as a Platform for the Delivery of HIV Gag p24 Antigen.

Multiple vaccination strategies have been devised against HIV-1 including delivery of HIV moieties in attenuated or replication defective recombinant microbial agents alone or in combination with priming agents in form of soluble proteins or naked DNA. For the priming agents to be effective, adjuvants might be essential in directing the immune response to a desired outcome. E. coli enterotoxin B subunit (LTB) is an effective adjuvant and carrier for other proteins and epitopes. Here we show that conjugation of HIV gag p24 to LTB enhances the T cell response to gag p24 by increasing rate of T cell division compared to other treatments. Because HIV vaccines are likely to be multivalent, we further investigated whether gag p24 inhibits antigen presentation of an unrelated antigen, OVA. Addition of gag p24 to OVA-responsive DO.11.10 cell culture did not have adverse effects on antigen presentation. Interestingly, the presence of LTB in these cultures significantly increased proliferation of DO.11.10 cells. In all, the results suggest the use of LTB to boost immune responses against HIV gag p24 in systemic priming regimens with oral recombinant HIV vaccines.

Functional and structural requirements for the internalization of distinct BCR-ligand complexes.

Antigen (Ag) binding to the BCR rapidly initiates two important events: a phosphorylation cascade that results in the production of secondary signaling intermediaries and the internalization of Ag-BCR complexes. Previous studies using anti-BCR antibodies (Ab) have suggested that BCR signaling is an essential requirement for BCR endocytosis and have further implicated lipid rafts as essential platforms for both BCR functions. However, published data from our laboratory indicate that lipid rafts and consequently raft-mediated signaling are dispensable for BCR-mediated internalization of Ag-specific BCR. Therefore, we investigated the relationship between BCR signaling and endocytosis by defining the role of early kinase signaling in the BCR-mediated internalization of a model Ag (haptenated protein). The results demonstrate that Src kinases and Syk-mediated BCR signaling are not essential for BCR-mediated Ag internalization. Moreover, by comparing Ag and Ab, it was determined that while both localize to clathrin-coated pits, the internalization of Ab-BCR complexes is more susceptible to inhibition of signaling and highly sensitive to disruption of lipid rafts and the actin cytoskeleton compared to Ag-BCR complexes. Thus, these results demonstrate that the nature of the ligand ultimately determines the functional requirements and relative contribution of lipid rafts and other membrane structures to the internalization of BCR-ligand complexes.

Dynamics of MHC class II-activating signals in murine resting B cells.

MHC class II (MHC II) proteins are competent signaling molecules on APC. However, little is known about the mechanisms that control generation of their activating signals. Previous reports highlighted a number of factors that could affect the nature and outcome of MHC II signals, including the inability of MHC II ligation on resting vs activated murine B cells to induce mobilization of Ca2+. In the present study, we report that ligation of MHC II on resting murine B cells reproducibly induces mobilization of intracellular Ca2+ using both mAbs and cognate T cells as ligands. Mobilization of Ca2+ was independent of MHC II haplotype, isotype, or mouse genetic background. MHC II-mediated mobilization of Ca2+ is completely inhibited by inhibitors of src-like kinases and syk, and MHC II ligation increases overall tyrosine phosphorylation level. Moreover, MHC II ligation results in specific up-regulation of CD86. However, induction of these responses is dependent on the type of anti-MHC II Ab used, suggesting that epitope specificity and/or the nature of ligation is important. Moreover, we demonstrate that MHC II-derived signals are strictly regulated by the order and timing of BCR and CD40 signals, suggesting coordination of these signals preserves the integrity of early B cell priming events. Thus, the mode and the context of MHC II ligation influence generation of MHC II-derived activating signals in resting B cells. Based on these results, a new model that highlights the role of MHC II-activating signals in regulation of Ag presentation by B cells is proposed.

The pathway of antigen uptake and processing dictates MHC class II-mediated B cell survival and activation.

The influence of the pathway of Ag uptake and processing on MHC class II (CII)-mediated B cell function is unknown. In this study, we investigate in resting and activated (via the BCR or CD40) B cells the biological properties of CII-peptide complexes (CII-peptide) generated by either the BCR-mediated Ag processing (type I complex) or fluid phase Ag processing (type II complex). Compared with type I complex, ligation of type II complex by either specific Ab or the TCR in Ag-presenting assay results in significant decreases in B cell survival rate (50-100%) and expression levels of CII, CD86, and CD54. Loss of B cells following ligation of type II complex occurs in the presence of a comparatively good level of specific CD4(+) T cell division, indicating that B cell loss is a late event following T cell stimulation. Comparative analysis of T and B cell conjugates after Ab ligation of type I or II complex reveals decreased efficiency of the latter in forming conjugates. Neither initial differential levels of CII and other studied surface markers, B cell type inherent differences, BCR signaling, T cell proliferation, nor initial density of CII-peptide complexes could explain the T cell-induced B cell loss. We propose that the context in which CII-peptide complexes are present in the membrane following BCR uptake and processing leads to B cell survival. Thus, appropriate targeting of Ag ensures generation of relevant immune responses.

Antigen binding to GM1 ganglioside results in delayed presentation: minimal effects of GM1 on presentation of antigens internalized via other pathways.

Plasma membrane rafts are sphingolipid- and cholesterol-rich patches that function as membrane trafficking and surface signalling regions. Ganglioside GM1 is an integral component of these microdomains, and Escherichia coli enterotoxin B subunit (EtxB) is a pentamer that binds with high affinity to GM1 resulting in GM1 cross-linking. We previously demonstrated that antigen coupled directly to EtxB resulted in enhanced presentation relative to antigen taken up by fluid-phase endocytosis. Here we demonstrate a new role for GM1 in antigen presentation by examining the effects of cross-linking GM1 on the kinetics of presentation and processing of antigen by the B-cell receptor (BCR), fluid-phase endocytosis and GM1-targeted antigen. EtxB bound to B cells does not augment the subsequent kinetics or magnitude of presentation of either BCR-internalized antigen or soluble antigen. Moreover, presentation of GM1-bound antigen is significantly slower than antigen presentation following BCR-mediated uptake. In contrast to the rapid internalization of BCR-bound antigen (which has a half life of 60 min), the majority of EtxB-bound antigen forms a plasma membrane depot detectable for many hours after initial incubation (and with a half life of 12 hr). We conclude that cross-linking of GM1 by EtxB minimally affects the processing and presentation of antigens internalized via other pathways. Nevertheless, binding of antigens to GM1 results in delayed presentation that has important implications for in vivo immunization using GM1-targeted adjuvants.