Although granzymes (Gzms) A-and B-induced cell death pathways have been defined, little is known about how other orphan Gzms function in CTL-mediated cytotoxicity. GzmK and A are tryptases among all the Gzms of humans and they are closely linked on the same chromosome. In this study, we showed that GzmK can be efficiently delivered into target cells with a cationic lipid protein transfection reagent Pro-Ject. We found human GzmK triggers rapid cell death independently of caspase activation. The features of death are characterized by rapid externalization of phosphatidylserine, nuclear morphological changes and single-stranded DNA nicks. GzmK hydrolyzes the nucleosome assembly protein SET in its recombinant and native forms or in intact cells. Cleavage of SET by GzmK abrogates its nucleosome assembly activity. After GzmK loading, SET and DNase NM23H1 rapidly translocate into the nucleus and SET is cleaved, where the nuclease activity of NM23H1 is activated to nick chromosomal DNA.
The object of this trial was to investigate the effect of dietary β-1,3/1,6-glucan supplementation on the performance and immunological response of broiler chickens. Two hundred and forty 1-day old male broilers (39±1 g) were separated into six treatments which were given six different feeds containing 0 (control), 25, 50, 75, 100 and 125 mg/kg dietary β-1,3/1,6-glucan supplementation. On days 21 and 42, body weight gain, feed consumption and feed conversation rate were recorded as measures of growth performance. The levels of key cytokines in the immuno-regulating pathway: interleukin-1 (IL-1), interleukin-2 (IL-2), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and the concentrations of signal molecules: peripheral blood plasma globulin, serum Immunoglobulin G (IgG) and intestinal secretary Immunoglobulin A (sIgA), were measured as indices of the immune response to determine suitable levels of dietary β-1,3/1,6-glucan supplementation. The results indicated that performance was elevated quadratically with dietary β-1,3/1,6-glucan supplementation. Maximal growth performance and an enhanced immunological response were obtained at a supplemented level of 50 mg/kg.
Beta-1,3/1,6-glucan (beta-glucan) was tested as a possible immunomodulator. Chicken macrophages from a macrophage cell line MQ-NCSU and from abdominal exudate of broiler chickens were exposed to various concentrations of beta-glucan in vitro. In addition, day-old broiler chicks were fed a diet containing 0, 20, and 40 mg/kg beta-glucan in the starter and 0, 20, and 20 mg/kg in the grower diet. Several baseline immune parameters were examined following such exposures. The results showed that beta-glucan exposure increased nitrite and interleukin-1 (IL-1) production as well as induced macrophage to proliferate in culture. However, IL-6 production was not affected. Dietary beta-glucan supplementation increased the macrophage phagocytic activity, anti-sheep red blood cells antibody response post-boost, as well as the PHAP-mediated lymphoproliferative response measured as a toe-web swelling. The percentage of CD4+, CD8+, and CD4+/CD8+ double positive lymphocytes in the intestinal intraepithelial leukocytes was increased in beta-glucan supplemented chicks. Furthermore, the primary and secondary lymphoid organs such as bursa of Fabricius, thymus and spleen were larger in beta-glucan-supplemented chicks as compared to the chicks on basal diet. The findings of these studies which showed that beta-glucan improves several baseline immune responses in the chicken imply that beta-glucan can be used as a possible immunomodulator in food animals such as the chicken.
Granule-mediated cytolysis is the major pathway for killer lymphocytes to kill pathogens and tumor cells. Little is known about how granzyme K functions in killer lymphocyte-mediated cytolysis. We previously showed that human GzmK triggers rapid cell death independently of caspase activation with singlestranded DNA nicks, similar to GzmA. In this study we found that GzmK can induce rapid reactive oxygen species generation and collapse of mitochondrial inner membrane potential (⌬⌿m). Blockade of reactive oxygen species production by antioxidant N-acetylcysteine or superoxide scavenger Tiron inhibits GzmK-induced cell death. Moreover GzmK targets mitochondria by cleaving Bid to generate its active form tBid, which disrupts the outer mitochondrial membrane leading to the release of cytochrome c and endonuclease G. Thus, we showed herein that GzmK-induced caspase-independent death occurs through Bid-dependent mitochondrial damage that is different from GzmA.
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