内毒素、TNF-α及IL-6在诱导生长激素不敏感中的作用

目的：在受体和受体后水平探讨内毒素（LPS）、TNF－α及IL－6在诱导生长激素不敏感中的作用。方法：采用雄性SD大鼠，分别静注LPS、TNF－α及IL－6，不同时相处死大鼠。应用RT－PCR法测定肝组织IGF－1、GHR和SOCS－3mRNA的表达；应用放射免疫法测定血清GH水平；应用酶联免疫吸附法测定血清TNF－α和IL－6水平。结果：静注LPS后，各时间点血清GH水平与对照组无明显差异，血清TNF－α和IL－6迅速升高，但TNF－α很快即恢复至正常水平。肝组织IGF－1和GHR mRNA的表达分别在注射LPS后12h和8h下调最多。正常大鼠肝组织SOCS－3mRNA微弱表达，但在注射LPS后立即上调，在1h时即上升了7．84倍，线性回归分析显示IL－6水平与SOCS－3mRNA的表达呈高度正相关。静注TNF－α后，肝组织SOCS－3 mRNA的表达无明显变化，但可明显下调GHR mRNA的表达。静注IL－6后，肝组织GHR mRNA的表达无明显变化，但SOCS－3 mRNA的表达却明显上调。结论：静注LPS可以诱导生长激素的不敏感，在受体水平与GHR mRNA的表达下调有关，在受体后水平与SOCS－3mRNA的表达上调有关。体内LPS的生物活性主要是由TNF－α和IL－6介导的，TNF－α和IL－6分别在受体和受体后水平发挥作用，但TNF－α的作用具有滞后性。     

绿原酸诱导肺癌细胞凋亡及其机制研究

目的研究绿原酸对人肺癌A549细胞凋亡的作用及其机制,为临床应用提供依据。方法四氮唑盐(MTT)法检测不同浓度的绿原酸对A549细胞增殖的影响;流式细胞术检测细胞凋亡率;采用间接荧光标记法测定细胞内活性氧水平的变化;分光光度法检测绿原酸对表皮生长因子受体酪氨酸激酶(epidermal growth factor receptor tyrosine kinase,EGFR-TPK)、基质金属蛋白酶(matrix metalloproteinase 2,MMP-2)和氨肽酶N(aminopeptidase,APN)活性的变化。结果MTT法检测显示,绿原酸在24 h、48 h和72 h时均可抑制A549细胞增殖,且呈浓度和时间依赖性关系趋势;绿原酸作用48 h后,细胞凋亡率呈现明显增高的趋势,与对照组比较差异有显著性(P0.01);在100μg/ml质量浓度的绿原酸作用下使细胞内活性氧(ROS)百分率达到了(42.32±3.3)%,与对照组相比比较差异有显著性(P0.01);绿原酸对APN、EGFR-TPK和MMP-2半数抑制浓度(IC_(50))分别为(8.12±0.78)、(29.43±2.76)和(1.54±0.13)μg/ml,显示出了较好的抑制效果。结论绿原酸可以抑制A549的增殖和转移,促进细胞凋亡,其凋亡机制可能与细胞内活性氧组分的增多及APN、EGFR-TPK和MMP-2活性降低有关。     

Differential activation of host cell signalling pathways through infection with two variants of influenza A/Puerto Rico/8/34 (H1N1) in MDCK cells

In cell culture-based influenza vaccine production, few efforts have been undertaken to characterise virus–host cell interactions in detail. Two influenza virus strains that grew to different virus titres, and differed in virus dynamics, apoptosis induction and proteome changes were observed.     

An inhibitory effect of resveratrol in the mitotic clonal expansion and insulin signaling pathway in the early phase of adipogenesis

Resveratrol is known as a potent antiobesity compound that acts partly through inhibition of adipogenesis. However, the direct targets responsible for its antiadipogenic action are unclear. Our hypothesis is that resveratrol inhibits adipogenesis through modulation of mitotic clonal expansion (MCE) and cell signaling pathways in the early phase of differentiation. To test this, we examined the effects of resveratrol on MCE and insulin signaling pathway in the early phase of adipogenesis in murine preadipocytes. We observed that the antiadipogenic action of resveratrol is largely limited to the early phase of adipogenesis. Specifically, the presence of resveratrol in the first 24 hours of adipogenesis was required for its antiadipogenic effect. During the first 24 hours of adipogenesis, resveratrol impaired the progression of MCE by suppressing the cell cycle entry of preadipocytes to G2/M phase, and expression of cell cycle regulators cyclin A and cyclin-dependent kinase 2. Concomitantly, resveratrol inhibited insulin signaling pathway in the early phase of adipogenesis. Furthermore, we revealed an inhibitory effect of resveratrol on insulin receptor (IR) activity, and this is likely through a direct physical interaction between resveratrol and IR. The antiadipogenic effect of resveratrol is through inhibition of the MCE and IR-dependent insulin signaling pathway in the early phase of adipogenesis.     

Non-alcoholic fatty liver disease and its treatment with n-3 polyunsaturated fatty acids

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Capsaicin attenuates palmitate-induced expression of macrophage inflammatory protein 1 and interleukin 8 by increasing palmitate oxidation and reducing c-Jun activation in THP-1 (human acute monocytic leukemia cell) cells

Capsaicin, a spicy component of hot peppers, has been shown to improve inflammatory disease and obesity. In this study, we tested the hypothesis that the anti-inflammatory activity of capsaicin can be used to improve free fatty acid (FFA)-induced inflammation by reducing gene expression of macrophage inflammatory protein 1 (MIP-1) and interleukin 8 (IL-8) in THP-1 (human acute monocytic leukemia cell) macrophages. To investigate whether capsaicin ameliorates palmitate-induced MIP-1 and IL-8 gene expressions, we treated THP-1 cells with palmitate in the presence or absence of capsaicin and measured MIP-1 and IL-8 by real-time polymerase chain reaction. To elucidate the mechanism by which capsaicin effects on palmitate-induced MIP-1 and IL-8 gene expressions, we performed immunoblotting with stress kinase-related antibodies and measured palmitate oxidation and palmitate oxidation-related gene expression. Palmitate and stearate but not the unsaturated FFA oleate significantly increased MIP-1 and IL-8 expressions in THP-1 macrophages. Treatment with capsaicin or FFA oxidation stimulators inhibited palmitate-induced MIP-1 and IL-8 expressions in THP-1 macrophages. Capsaicin increased the gene expression of carnitine palmitoyltransferase 1 and the β-oxidation of palmitate. Furthermore, capsaicin significantly reduced palmitate-stimulated activation of c-Jun N-terminal kinase, c-Jun, and p38. Our data suggest that the attenuation of palmitate-induced MIP-1 and IL-8 gene expressions by capsaicin is associated with reduced activation of c-Jun N-terminal kinase, c-Jun, and p38 and preserved β-oxidation activity.     

Acute exposure to high-fat diets increases hepatic expression of genes related to cell repair and remodeling in female rats

High-fat diets (HFD) promote the development of both obesity and fatty liver disease through the up-regulation of hepatic lipogenesis. Insulin resistance, a hallmark of both conditions, causes dysfunctional fuel partitioning and increases in lipogenesis. Recent work has demonstrated that systemic insulin resistance occurs in as little as the first 72 hours of an HFD, suggesting the potential for hepatic disruption with HFD at this time point. The current study sought to determine differences in expression of lipogenic genes between sexes in 3-month-old male and female Long-Evans rats after 72 hours of a 40% HFD or a 17% fat (chow) diet. Owing to the response of estrogen on hepatic signaling, we hypothesized that a sexual dimorphic response would occur in the expression of lipogenic enzymes, inflammatory cytokines, apoptotic, and cell repair and remodeling genes. Both sexes consumed more energy when fed an HFD compared with their low fat–fed controls. However, only the males fed the HFD had a significant increase in body fat. Regardless of sex, HFD caused down-regulation of lipogenic and inflammatory genes. Interestingly, females fed an HFD had up-regulated expression of apoptotic and cell repair–related genes compared with the males. This may suggest that females are more responsive to the acute hepatic injury effects caused by HFDs. In summary, neither male nor female rats displayed disrupted hepatic metabolic pathways after 72 hours of the HFD treatment. In addition, female rats appear to have protection from increases in fat deposition, possibly due to increased caloric expenditure; male rats fed an HFD were less active, as demonstrated by distance traveled in their home cage.     

Design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens

Liposomes (phospholipid bilayer vesicles) are versatile and robust delivery systems for induction of antibody and T lymphocyte responses to associated subunit antigens. In the last 15 years, liposome vaccine technology has matured and now several vaccines containing liposome-based adjuvants have been approved for human use or have reached late stages of clinical evaluation. Given the intensifying interest in liposome-based vaccines, it is important to understand precisely how liposomes interact with the immune system and stimulate immunity. It has become clear that the physicochemical properties of liposomal vaccines - method of antigen attachment, lipid composition, bilayer fluidity, particle charge, and other properties - exert dramatic effects on the resulting immune response. Here, we present a comprehensive review of the physicochemical properties of liposomal vaccines and how they influence immune responses. A discussion of novel and emerging immunomodulators that are suitable for inclusion in liposomal vaccines is also presented. Through a comprehensive analysis of the body of liposomal vaccine literature, we enumerate a series of principles that can guide the rational design of liposomal vaccines to elicit immune responses of a desired magnitude and quality. We also identify major unanswered questions in the field, pointing the direction for future study.     

A novel nanoemulsion vaccine induces mucosal Interleukin-17 responses and confers protection upon Mycobacterium tuberculosis challenge in mice

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is contracted via aerosol infection, typically affecting the lungs. Mycobacterium bovis bacillus Calmette-Guerin (BCG) is the only licensed vaccine and has variable efficacy in protecting against pulmonary TB. Additionally, chemotherapy is associated with low compliance contributing to development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb. Thus, there is an urgent need for the design of more effective vaccines against TB. Experimental vaccines delivered through the mucosal route induce robust T helper type 17 (Th17)/ Interleukin (IL) -17 responses and provide superior protection against Mtb infection. Thus, the development of safe mucosal adjuvants for human use is critical. In this study, we demonstrate that nanoemulsion (NE)-based adjuvants when delivered intranasally along with Mtb specific immunodominant antigens (NE-TB vaccine) induce potent mucosal IL-17 T-cell responses. Additionally, the NE-TB vaccine confers significant protection against Mtb infection, and when delivered along with BCG, is associated with decreased disease severity. These findings strongly support the development of a NE-TB vaccine as a novel, safe and effective, first-of-kind IL-17 inducing mucosal vaccine for potential use in humans.     

Use of protective antigen of Bacillus anthracis as a model recombinant antigen to evaluate toll-like receptors 2, 3, 4, 7 and 9 agonists in mice using established functional antibody assays,antigen-specific antibody assays and cellular assays

Toll-like receptor (TLR) agonists can act as immune stimulants alone or as part of alum or oil formulations. Humoral and cellular immune responses were utilized to assess quantitative and qualitative immune response enhancement by TLR agonists using recombinant protective antigen (rPA) of B. anthracis as a model antigen. To rPA, combined with aluminum hydroxide (Alhydrogel; Al(OH)3) or squalene (AddaVax?), was added one of 7 TLR agonists: TLR2 agonist Pam3CysSK4 (PamS), TLR3 agonist double stranded polyinosinic:polycytidylic acid (PolyIC), TLR4 agonists Monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA), TLR7-8 agonists 3M?052 or Resiquimod (Resiq), or TLR9 agonist CPG 7909 (CPG). CD-1 or BALB/c mice received two intraperitoneal or intramuscular immunizations 14 days apart, followed by serum or spleen sampling 14 days later. All TLR agonists except PamS induced high levels of B. anthracis lethal toxin-neutralizing antibodies and immunoglobulin G (IgG) anti-PA. Some responses were >100-fold higher than those without a TLR agonist, and IP delivery (0.5 mL) induced higher TLR-mediated antibody response increases compared to IM delivery (0.05 mL). TLR7-8 and TLR9 agonists induced profound shifts of IgG anti-PA response to IgG2a or IgG2b. Compared to the 14-day immunization schedule, use of a shortened immunization schedule of only 7 days between prime and boost found that TLR9 agonist CPG in a squalene formulation maintained higher interferon-γ-positive cells than TLR4 agonist GLA. Variability in antibody responses was lower in BALB/c mice than CD-1 mice but antibody responses were higher in CD-1 mice. Lower serum 50% effective concentration (EC50) values were found for rPA-agonist formulations and squalene formulations compared to Al(OH)3 formulations. Lower EC50 values also were associated with low frequency detection of linear peptide epitopes. In summary, TLR agonists elicited cellular immune responses and markedly boosted humoral responses.