水飞蓟对动物机体免疫功能的影响研究

将小鼠分为四组，分别饲喂１０％水飞蓟、２０％水飞蓟、环磷酰胺（０．０１ｇ／ｋｇ）和空白对照（常规饲料），研究对小鼠免疫功能的影响。实验结果证明，水飞蓟各剂量组能增高小鼠外周血白细胞数目（Ｐ＜０．０１、０．００１）；提高小鼠腹腔巨噬细胞的吞噬率（Ｐ＜０．００１，０．００１）及吞噬指数（Ｐ＜０．０５、０．００１）并随剂量加大有增强的趋势；能促进小鼠淋巴细胞转化，提高淋巴细胞转化百分率（Ｐ＜０．０１、０     

Effects of autophagy inhibitor bafilomycin A1 on macrophages polarization

AIM:To investigate the effect of bafilomycin A1 (Baf A1) on polarization in mouse macrophages RAW264.7. METHODS:The macrophages RAW264.7 were treated with Baf A1, the concentration of M1/M2 polarization related cytokines were determined by ELISA. The markers of M1/M2 polarization in the macrophages were determined by flow cytometry. The formation of autophagicbody was observed by immunofluorescence. Western blot was used to detect the expression of autophagy related protein levels. RESULTS:The concentration of M1 related proinflammatory cytokine tumor necrosis factor-α (TNF-α) was increased significantly after Baf A1 intervention (P<0.01). However, the concentration of M2 related anti-inflammatory cytokines IL-10 and IL-13 showed no significant difference. The double positive rate of CD197 and HLA-DR (M1 marker) positive cells in Baf A1 treated group were significant higher than that in control group (P<0.05), indicated that Baf A1 induced polarization of macrophage to M1. The results of immunofluoresence showed that the autophagosomes formation was notable increased in Baf A1 group (P<0.05), meanwhile Western blot results showed the expression of autophay related protein LC3-Ⅱ but not P62 was marked up-regulated (P<0.05). For autophagy activator rapamycin (Rapa) treated group, autophagosome formation was also significant increased (P<0.05), but the expression of P62 was notable down-regulated (P<0.05). CONCLUSION:Baf A1 induces polarization of mouse macrophage RAW264.7 to M1, which may be related to the inhibitory effect on the formation of autolysosome.     

奶牛腹腔巨噬细胞的分离与鉴定

以无血清的RPMI-1640培养液灌洗奶牛腹腔,分离获取奶牛腹腔巨噬细胞,在含10%小牛血清的RPMI-1640培养液中培养。采用倒置显微镜观察细胞形态,并通过瑞氏染色计算其纯度。结果表明,采用无血清的RPMI-1640培养液灌洗奶牛腹腔,能够获得较高纯度的巨噬细胞,能为奶牛胞内寄生菌存活机制的研究提供很好的试验材料。     

Local pulmonary immune responses in domestic cats naturally infected with Cytauxzoon felis

Cytauxzoonosis is a hemoprotozoal disease of cats and wild felids in the South and Southeastern United States caused by Cytauxzoon felis. Although the causative agent has been recognized since the seventies, no study has examined the local immune response in affected organs, such as the lung, and compared them to the lungs of uninfected domestic cats. Previous studies have suggested that the histopathologic findings in the lungs of C. felis-infected cats are caused by the release of pro-inflammatory mediators, such as cytokines and increased production of inducible nitric oxide synthase (iNOS), by the infected macrophages. Our laboratory had previously found an upregulation of the adhesion molecule CD18, which can stimulate the release of these pro-inflammatory mediators. The objective of this study was to characterize local pulmonary immune responses in cats naturally infected with C. felis. Immunohistochemistry was performed to detect tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, iNOS, and major histocompatibility complex (MHC) II in 19 lungs from affected cats that died between 2005 and 2013. Results showed increased expression of all of these molecules when compared to lungs from uninfected, healthy cats. Furthermore, MHC II is expressed in the endothelium of C. felis naturally infected cats. These results support that there is a marked, local, pro-inflammatory immune response that can contribute to the pathogenesis of cytauxzoonosis in the lungs.     

Screening of proteins interacting with activation domain of macrophage migration-inhibitory factor

AIM: To screen the proteins that interact with the catalytic thiol-protein oxidoreductase (TPOR) domain of macrophage migration-inhibitory factor (MIF). METHODS: Yeast two-hybrid system was used to study the proteins that interacted with MIF. The bait vector pBTM116-MIF was constructed and transfected into L40 yeast strain. L40 competent cells expressing the TPOR domain were prepared and used to screen the proteins interacting with the TPOR domain of MIF in a human osteosarcoma cDNA library. The proteins interacting with TPOR domain were identified by HIS3 reporter gene and β-galactosidase assay. The techniques of co-immunoprecipitation and immunofluorescence were also used to verify the proteins interacting with TPOR domain. RESULTS: Wild-type pBTM116-MIF and pACT2-TXNL2 (thioredoxin-like 2 protein) were constructed and co-transfected into L40 yeast strain. The activation of HIS3 reporter gene and the positive result of β-galactosidase assay indicated that TXNL2 was the candidate protein that interacted with the catalytic TPOR domain of MIF. The vector of pcDNA3.1-Myc-TXNL2 was constructed and transfected into MCF-7 cell line. The result of co-immunoprecipitation assay showed that the catalytic TPOR domain of MIF co-immunoprecipitated with Myc-TXNL2 protein. The result of immunofluorescence assay demonstrated that the catalytic TPOR domain of MIF and TXNL2 co-localized in the cytoplasm of the cells. CONCLUSION: TXNL2 is the protein that interacts with the catalyzed TPOR domain of MIF.     

Killing of Brucella antigen-sensitized macrophages by T lymphocytes in bovine brucellosis

The present study was an investigation into the role of T lymphocytes in the killing of antigen-sensitized macrophages (MΦ) in bovine brucellosis. Following confirmation of bovine T lymphocyte cell lines derived from Brucella abortus Strain 19 vaccinated steers as antigen-specific in proliferation studies using various antigens, we adapted an apoptosis assay for evaluation of cytotoxicity by these bovine T cells against autologous monocyte-derived macrophages (MDMΦ) as target cells. Various B. abortus antigen preparations were tested including whole γ-irradiated B. abortus bacteria (γBA), a soluble cytosolic protein fraction and a membrane-associated protein fraction. Both polyclonal and cloned T lymphocyte cell lines exhibited cytotoxicity against MDMΦ targets in an antigen-specific fashion. Polyclonal and cloned T lymphocyte cell lines demonstrated cytotoxic responses to varying degrees against B. abortus antigens regardless of whether the antigen used was whole nonviable bacteria, a soluble protein extract or a membrane-associated fraction of extracted bacteria. To further develop correlation of these responses to an in vivo host defense mechanism, cytotoxicity was evaluated using target cells that had been infected with live B. abortus S19 or B. abortus Strain 2308. Cytotoxic responses were also demonstrated consistently against infected targets with either strain of B. abortus although in most cases, cytotoxicity was higher against target cells sensitized with γBA compared to those infected with live bacteria. Cloned T lymphocyte cell lines were all CD4⁺, CD8⁻ cells indicating that the observed cytotoxic responses were most likely due to an inflammatory T_h1 response and may represent an important host defense mechanism induced by vaccination with live attenuated strains of B. abortus in cattle.     

Bacillus subtilis-based direct-fed microbials augment macrophage function in broiler chickens

The present study was conducted to evaluate the function of Bacillus subtilis-based direct-fed microbials (DFMs) on macrophage functions, i.e., nitric oxide (NO) production and phagocytosis in broiler chickens. DFMs used in this study were eight single strains designated as Bs2084, LSSAO1, 3AP4, Bs18, 15AP4, 22CP1, Bs27, and Bs278, and one multiple strain DFM product (Avicorr™) containing equal amount of Bs2084, LSSAO1 and 15AP4. NO concentrations were monitored in plasma and in the supernatants from the peripheral blood-derived monocytic cells (PBMC)-derived macrophages stimulated by either chicken recombinant interferon gamma (IFNγ) or lipopolysaccharide (LPS) from Escherichia coli or Salmonella typhi. In addition, phagocytosis of fluorescent beads and green fluorescent protein (GFP)-labeled Salmonella by PBMC-derived macrophage was assayed. Plasma NO levels were significantly higher in groups given 3AP4 or Bs27 diets compared with the control group at days 7 and 14. NO production by PBMC-derived macrophages stimulated with IFNγ or LPS was apparent, although the effect was strain-dependent. Phagocytosis of fluorescent beads or GFP-labeled Salmonella by macrophages was augmented in groups on DFM-supplemented diets compared with those fed the control diet. This study describes the immunomodulatory effects of Bacillus-based DFMs on innate immunity in broiler chickens.     

小茴香对小鼠免疫功能的影响

[目的]研究小茴香对环磷酰胺模型小鼠免疫功能的影响。[方法]以环磷酰胺制备小鼠免疫低下模型并结合体外药理试验,测定灌胃给药后小鼠体内巨噬细胞吞噬率、血清溶血素抗体水平,并观察植物血凝素诱导T淋巴细胞的增殖反应。采用含药血清体外培养小鼠腹腔巨噬细胞,MTT法考察巨噬细胞的活性,并观察体外培养的巨噬细胞吞噬红细胞的吞噬百分率和吞噬指数。[结果]小茴香提高免疫抑制小鼠碳粒廓清率,促进血清溶血素形成以及促进T淋巴细胞增殖。[结论]小茴香有提高小鼠免疫功能的作用。