This research determined the in vitro, intraperitoneal injection and dietary immunostimulatory effects on the grouper Epinephelus malabaricus of chitosan and its derivatives with different molecular weight, chitosan, polyglucosamine and N-acetyl-chitooligosaccharides. Respiratory burst activity of head-kidney phagocytes isolated from the grouper incubated in vitro with the chitosans at a range of concentrations was studied. Respiratory burst activity generally decreased with increasing dosage of chitosan products. N-acetyl-chitooligosaccharides were significantly more potent in enhancing respirtatory burst activity than the other two chitosans. Respiratory burst activity of head-kidney phagocytes of the grouper injected with three kinds of chitosans at 4 dosages was assayed. N-acetyl-chitooligosaccharides caused significantly higher respiratory burst activity than the other two chitosans. N-acetyl-chitooligosaccharides at the dosage of 10 µg/g was found to enhance the highest respiratory burst activity among treatments. In the time series assay with intraperitoneal injection by N-acetyl-chitooligosaccharides at a dosage of 10 µg/g, it was found that enhancement of NBT reduction occurred early in the time course of the study and is similar to the time series response of the glucan treatment. When the groupers (120g) were fed with diets containing 5 concentrations of N-acetyl-chitooligosaccharides including 0, 0.5, 1, 1.5, and 2 g/100g and stocked in indoor closed recirculation systems for 7 weeks, weight gain of the fish was not significantly affected by the dietary treatments. The immune status measured by respiratory burst activity, alternative complement pathway, agglutination titer, lysozyme activity and superoxide dismutase activity was not significantly affected by N-acetyl-chitooligosaccharides supplement. But feeding the grouper with N-acetyl-chitooligosaccharides at 1 g/100g diet seems to lower the immunity of the fish, although the effects were statistically insignificant.

Consumption of fish oils (FO) enriched with the n-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), is beneficial to a variety of inflammatory disorders due, in part, to the alteration of membrane composition of T-lymphocytes and other immune cells. We previously observed that down-regulation of proliferation and cytokine synthesis by CD4+ T-cells in mice fed diets rich in n-3 PUFA was dependent on the involvement of CD28, a co-stimulatory molecule necessary for T-cell activation. Since the co-receptor homologues, CD28 and CTLA-4, have opposing effects on T-cell activation, we hypothesized that the balance of costimulatory and downregulatory properties of CD28 and CTLA-4, respectively, would be altered by diet. A significant increase (p<0.05) in CD28 and CTLA-4 surface expression was observed in CD4+ T-cells post-stimulation with phorbol ester and calcium ionophore (PMA/Iono) or anti-CD3 and anti-CD28 (&#945;CD3/CD28) antibodies in all diet groups. A significant increase (p<0.01; 20%) in the number of CD28 molecules was observed in n-3 PUFA vs. CO-fed mice after 48 h of in vitro CD4+ T-cell activation, and both CTLA-4 mRNA transcript and protein levels were upregulated by 50% at 72 h post-activation (p<0.01). Treatment with anti-CTLA-4 mAb in vivo in Mycobacterium bovis (BCG)-vaccinated mice did not alter the suppressive effects of dietary n-3 PUFA on antigen (PPD)-induced lymphocyte proliferation or delayed hypersensitivity reactions. T-cells from both the C57BL/6 and IL-10mice fed dietary n-3 PUFA after 72 h of in vitro stimulation with &#945;CD3/CD28. CD4T-cells from C57BL/6 mice fed DHA produced significantly less IFN&#947; and IL-10, while CD4T-cells from IL-10Ligation of CD28 upregulates IL-10 receptor (IL-10R) expression on CD4+ T-cells. Therefore, we hypothesized that dietary n-3 PUFA would suppress T-cell function through the effects of IL-10. Surprisingly, the proliferation of purified splenic CD4+ T-cells activated in vitro with &#945;CD3/CD28 was suppressed by dietary n-3 PUFA in both conventional mice (C57BL/6) and IL-10 gene knockout (IL-10(-/-)) mice. Furthermore, IL-10R cell surface expression was significantly down-regulated on CD4+ T-cells from both the C67BL/6 and IL-10(-/-) mice fed dietary n-3 PUFA produced significantly more IFN&#947; compared to the CO-fed group.

The aim of this study was to identify common messenger molecules used in both the immune and the neuroendocrine systems in birds, and to shed light on a cell type within the bursa of Fabricius that has historically been postulated as a potential neuroendocrine-immune link, the bursal secretory dendritic-like cells (BSDC). An immunocytochemical approach was used to identify neuroendocrine cell populations in the thymus, pituitary and bursa of Fabricius in the chicken. Molecular confirmation of the neuroendocrine cell marker, chromogranin A (CgA) in the thymus tissue of the chicken was reported. Previously the serine protease inhibitor, ovoinhibitor, was localized in bursal follicles, specifically the cortico-medullary border region. The presence of ovoinhibitor was identified and confirmed in the chicken pituitary by this study. Continued focus on the neuroendocrine-immune interactions in chicken immune tissue narrowed the study around the BSDC population. The BSDC are a component of the stromal, non-lymphoid cellular environment of the bursa of Fabricius and are thought to play a role in B-cell maturation and differentiation. They are located mainly along the cortico-medullary border of the bursal follicles in the same area as the majority of the ovoinhibitor-positive cell population. During attempts to isolate the BSDC population by flow cytometry and laser capture microdissection, a cell culture method was developed that enriched the BSDC population by 10-fold. This enriched population was used to evaluate protein product secretion following lipopolysaccharide (LPS) challenge and compared to in vivo challenge with live Salmonella. For the first time, up-regulation of the pro-inflammatory cytokine IL-12 was documented in the chicken following in vivo challenge. In addition, the gene expression of serine protease inhibitors was markedly decreased in the adherent cell population following LPS stimulation. As a result of this research a novel method for the enrichment of an adherent population, including the BSDC, was developed, providing a valuable tool for the analysis of this population during immune stimulation.

Drosophila TGFbeta-Activating-Kinase 1 (dTAK1) is an essential component of both the Immune Deficiency (IMD) innate immune and TNF/Eiger apoptotic cascades. The IMD and JNK pathways bifurcate at the level of dTAK1. Hence, elucidating the regulatory mechanism of dTAK1 is pertinent to understanding the regulation of both innate immunity and apoptosis. In this study, Trabid was identified as a novel negative regulator of the Drosophila IMD pathway. Trabid interacted with dTAK1 and decreased K63-linked ubiquitination, thereby reducing immune signalling. Three tandem Npl4 Zinc Fingers (NZF) and C518 were required for Trabid activity. Lysines 142 &amp; 156 were identified as the K63 Ub acceptor sites of dTAK1, required for K63-linked ubiquitination and signalling. Also, results show Lys 156 functioned as the K48 Ub acceptor site. Further, the ZF domain of TAK1-associated Binding Protein 2 (dTAB2) was important in modulating dTAK1 K63-linked ubiquitination and thereby the immune signal. These results indicate an elaborate and multi-tiered mechanism for regulating dTAK1 activity and modulating the immune signal. Further, Ariadne-2 (Ari-2) was identified as a novel component of the Drosophila TNF/Eiger pathway which functioned at the level of dTAK1. Results indicate that Ari-2 is essential for normal development and longevity. It enhances the apoptotic signal when concomitantly over-expressed with Eiger. Further, Ari-2 interacts with dTAK1, dTAB2 and dTRAF2 which are all implicated in TNF/Eiger signalling. Thus, evidence supports the hypothesis that Ari-2 functions as an adaptor, involved in assembling a distinct signalling complex which transduces the apoptotic signal without activating immunity.

Previous studies employing the generation of MHC-incompatible embryonic chicken chimaeras by injecting MHC-incompatible stem cells resulted in an unexpected finding. Chimaeras made late in gestation developed as adults a severe autoimmune syndrome resembling the human syndrome of Systemic Lupus Erythematosus. Work in our laboratory aims to understand the role of CD8 T cells in immunity and/or autoimmunity. We have tested a three-cell model of CD4 T cell activation and differentiation during the development of the immune response specific for MHC transplantation antigens in one way mixed lymphocyte reactions. Our model proposes that whether Th1 or Th2 immunity is generated depends on both the ratio of CD4:CD8 T cells specific for antigen at the initiation of the immune response and on the ability of antigens to coordinately induce both CD4 and CD8 T cells. Previous studies employing parent into F1 models of graft-versus-host disease in mice have shown that the injection of parental cells results in two distinct outcomes. Parental cells which do not have a sufficient number of CD8 T cells present produce an autoimmune syndrome characteristic of systemic lupus erythematosus and a chronic graft-versus-host disease mediated by a Th2 response. Conversely, the presence of an adequate number of CD8 T cells results in a Th1 immune response and acute graft-versus-host disease resulting in the death of the F1 host. Our findings indicate that the ratio of the number of CD4 T cells to the number of CD8 T cells present in the spleen is crucial in whether naive CD4 T cells differentiate into Th1 or Th2 cells. We refer to this ratio as the CD4:CD8 T cell ratio or CD4:CD8 ratio. Thus, the differentiation of naive CD4+ T cells towards a differentiated Th1 phenotype is critically dependent on the concomitant induction of CD8 T cells by the same antigen, driven by a low CD4:CD8 ratio. In contrast, inefficient induction of CD8 T cells during the initial priming of lymphocytes greatly facilitates the differentiation of CD4 T cells towards the Th2-type lineage, and occurs when the CD4:CD8 ratio is high. Given our findings on the significance of the ratio of CD4:CD8 T cells in the decision making process of CD4 differentiation stimulated by antigen, we hypothesized that different CD4: CD8 ratios at different stages of development might contribute to the immune response generated at these stages. We tested this hypothesis in mice by comparing the CD4:CD8 ratio in adults and neonates and the Th1/Th2 responses generated in vitro. This CD4:CD8 T cell ratio is significantly higher in neonates than adults resulting in predominant Th1 responses by adult spleen cells and Th1/Th2 responses by neonatal spleen cells as demonstrated by the ELISPOT assay. We have compared the CD4:CD8 T cell ratio of a large number of adult and neonatal spleens in several mouse strains and have studied it systematically in BALB/c and C57BL/6 mice by flow-cytometry. We have consistently found a 3-5 fold higher CD4:CD8 T cell ratio in neonates as compared to adults in the strains tested. Furthermore, we found that neonatal spleen cells generate a predominant Th2 response whereas adult spleen cells generate CD4 and CD8 Th1 immunity when activated under the same conditions. We have further studied the role of CD8 T cells in CD4 T cell differentiation by reconstructing the adult CD4:CD8 T cell ratio in neonatal spleen cells with age-matched, isolated CD8 T cells. We found that in these “CD4:CD8 ratio-reconstructed cultures”, the Th2/IL-4 immunity is suppressed with concomitant generation of Th1/IFN-γ immunity upon activation by allo-antigen. Additionally, we have characterized the phenotype of the T cell mediating Th1/IFNγ immunity in the “CD4:CD8 ratio reconstructed cultures” and we found that while CD8 T cells produce exclusively IFN-γ, CD4 T cells now produce IFN-γ rather than IL-4. We suggest that physiologically distinct CD4:CD8 ratios at different stages of life should be considered in designing protocols of neonatal vaccination against pathogens that are contained by Th1-type immunity upon infection as adults. Moreover, as elaborated in the discussion, our studies might be pertinent in understanding by which mechanism autoimmunity arises in some cases.

The in vitro and in situ immune responses of carp Cyprinus carpio to Ichthyophthirius multifiliis were investigated in order to characterise the immune mechanisms involved in protection. 'O' group and adult carp were immunised against I.multifiliis by controlled infection procedures. Sterile immunity was not achieved; theronts were observed to penetrate the skin of immunised fish, although in the majority of cases this did not lead to successful trophozoite establishment. It was concluded that most parasites prematurely exited the epidermis of immunised fish within two hours of penetration as an active survival strategy. Trophozoites remaining in immunised fish beyond two hours post infection were able to complete normal development. Serum from carp immunised against I.multifiliis displayed specific in vitro theront immobilising activity, and antibody was detected against parasite ciliary membranes and mucocyst organellae; similar activity was not detected in cutaneous mucus. Significant amounts of antibody could not be located at the immediate host/parasite interface of trophozoites in situ in immune skin; prevention of antibody binding may be achieved by means of a mucocyst-derived &quot;sheath&quot; around the parasite and the formation of a layer of necrotic host tissue debris. Parasite development in immunised fish initiated a localised cellular infiltration, predominated by type III granulocytes (&quot;basophils'') and mast cell-like cells, the activity of which may augment further cellular and humoral infiltration. Sites of premature parasite exit from the epidermis of immunised fish were infiltrated by actively phagocytic cells, predominantly macrophages, probably in response to localised antibody/antigen complex deposition. Pronephric leucocytes of immunised fish displayed greater in vitro non-specific phagocytic activity than cells of carp naive to I.multifiliis; the relevance of this to enhanced antigen uptake in immunised fish is discussed. Based on results of the present study, a model for the Mode of protection in fish against I.multifiliis is proposed.

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No description available.

No description available.

Plants have evolved sophisticated defence systems against pathogen infection. Initiation of induced defence signalling often involves specific recognition of invading pathogens by the products of specialized host Resistance (R) genes. Consequently, the pathogen is stopped at the site of infection. A unique dominant mutant in Arabidopsis thaliana, snc1, constitutively expresses pathogensis-related (PR) genes and exhibits enhanced resistance to bacterial and oomycete pathogens. SNC1 encodes an R-gene – a single amino acid change renders this protein constitutively active without interaction with pathogens. snc1 displays a stunted phenotype that may be caused by both the accumulation of toxic compounds and energy squandered on unnecessary defence instead of normal growth. The distinctive morphological phenotype of snc1 is intimately associated with the other resistance phenotypes, and provides a robust genetic tool for dissecting the signalling events downstream of snc1. To identify genes important for defence signalling, we carried out a suppressor screen to identify modifier of snc1 (mos) mutants that restore the wild type size and morphology in the snc1 background. Furthermore, in most cases, a loss of sneakiness in mos mutants correlated with a reduced or abolished constitutive PR gene expression, SA accumulation and pathogen resistance in snc1 plants. These loss of function mutants represent defects in positive regulators of the snc1 pathway. I cloned and characterized two mos mutants, and showed that they both have roles in Arabidopsis innate immunity as well. mos6 partially suppresses snc1 and exhibits enhanced disease susceptibility (EDS) to an oomycete pathogen. MOS6, identified by map-based cloning, encodes an alpha-importin subunit, one of 8 found in Arabidopsis, and has a demonstrated role in nucleocytoplasmic partitioning (protein import). Two other genes cloned by others from this screen, MOS3 and MOS7, encode components of the nuclear pore complex, implicating nuclear trafficking as a key regulator in plant innate immunity. mos4 exhibits EDS to virulent and avirulent bacterial and oomycete pathogens. There is evidence that MOS4-mediated resistance is independent of the signalling protein NPR1. MOS4 encodes a protein with homology to human Breast Cancer Amplified Sequence 2 and with predicted protein-protein interaction domains. Subcellular localization of MOS4-GFP shows that MOS4 is localized to the nucleus. To illuminate the biochemical function of MOS4, a yeast-2-hybrid screen was conducted. One MOS4-interactor was a putative myb transcription factor, MOS4-Associated Complex Protein 1 (MAC1), also known at AtCDC5. MAC1 interacts directly with MOS4 in vitro and in planta. mac1 insertional mutants exhibit defects in immune responses to pathogens similar to that of mos4. In addition, mac1 also partially suppressed snc1 morphology and enhanced resistance. Both MOS4 and MAC1 have homologs in humans and fission yeast that are members of a discrete protein complex that has been implicated in several different biological processes including RNA splicing, apoptosis and protein degradation. Using proteomics data from yeast and human, we found genes with homology to additional components of the orthologous complex in Arabidopsis, and isolated insertion mutants in these. Mutations in PRL1, which encodes a WD protein, display similar disease phenotypes to that of mos4 and mac1. AtCDC5 has DNA binding activity, suggesting that this complex may regulate defence responses through transcriptional control. Since the complex components along with their interactions are highly conserved from fission yeast to Arabidopsis and human, they may also have a yet-to-be identified function in mammalian innate immunity.