双歧杆菌总DNA对小鼠IL—2,NK活性影响的研究

目的 从双歧杆菌、大肠杆菌提取ＤＮＡ,用ＤＮＡ免疫小鼠,观察免疫功能的变化,探讨双歧杆菌ＤＮＡ对小鼠免疫功能的影响,并作对比研究。方法 肌肉注射提取的双歧杆菌ＤＮＡ、大肠杆菌ＤＮＡ,颈椎处死后,检测脾细胞的免疫功能,同时提取ＩＥＬ细胞与ＤＮＡ共孵育,检测它对ＩＥＬ细胞的激活情况及细胞因子产生情况,以自然杀伤细胞（ＮＫ）活性,白细胞介素２（ＩＬ－２）产生能力为指标,测定小鼠上述各项指标变化。结果 双歧杆菌ＤＮＡ、大肠杆菌ＤＮＡ肌肉注射后,小鼠以上两项指标与相应对照组相比较均明显提高（Ｐ〈０．０５）。双歧杆菌ＤＮＡ提高小鼠ＮＫ活性与ＩＬ－２水平程度大于大肠杆菌ＤＮＡ的作用（Ｐ〈０．０１）。结论 双歧杆菌ＤＮＡ可快速激活ＮＫ活性,提高体内ＩＬ－２水平。其效能优于大肠杆菌ＤＮＡ。     

青春双歧杆菌DM8504菌株对小鼠体内HCa—F25／16A3—F肿瘤细胞 …

本实验应用加热处死的青春双歧杆菌ＤＭ８５０４菌株皮下注射荷瘤ＨＣａ－Ｆ２５／１６Ａ３－Ｆ肿瘤的ＢＡＬＢ／ｃ小鼠,酶联法测定小鼠体内ＴＮＦ－α、ＩＬ－６的含量,ＴＵＮＥＬ法及电镜观察肿瘤组织中是否有凋亡细胞的存在。实验结果指出：双歧杆菌能提高荷瘤小鼠体内ＴＮＦ－２的含量,较对照组明显升高,但ＩＬ－６的含量处理组与对照组之间无显著性差异。ＴＵＮＥＬ法除观察到不同程度的坏死组织外,未见到散在凋亡的肿瘤细     

青春双歧杆菌DM8504菌株对小鼠体内HCa-F_(25)/16A_3-F肿瘤细胞抑制作用的初步研究

本实验应用加热处死的青春双歧杆菌ＤＭ８５０４菌株皮下注射荷瘤ＨＣａ－Ｆ２５／１６Ａ３－Ｆ肿瘤的ＢＡＬＢ／ｃ小鼠,酶联法测定小鼠体内ＴＮＦ－α、ＩＬ－６的含量,ＴＵＮＥＬ法及电镜观察肿瘤组织中是否有凋亡细胞的存在。实验结果指出：双歧杆菌能提高荷瘤小鼠体内ＴＮＦ－２的含量,较对照组明显升高,但ＩＬ－６的含量处理组与对照组之间无显著性差异。ＴＵＮＥＬ法除观察到不同程度的坏死组织外,未见到散在的凋亡的肿瘤细胞,电镜观察与ＴＵＮＥＬ结果相一致,肿瘤细胞呈现坏死细胞的超微结构,未见凋亡细胞所具有的典型的形态特征。结果提示：双歧杆菌通过调节机体的免疫系统发挥抗肿瘤的作用,其对ＨＣａ－Ｆ２５／１６Ａ３－Ｆ肿瘤细胞的杀伤是通过坏死的方式实现的。     

双歧杆菌脂磷壁酸与5-氟尿嘧啶联用的抗肿瘤研究

目的探讨双歧杆菌脂磷壁酸与5-氟尿嘧啶（5-Fu）联用对H22荷瘤小鼠的抗肿瘤作用及免疫功能的影响。方法双歧杆菌脂磷壁酸单独或联合5-Fu处理H22荷瘤Balb／c小鼠,定期测量肿瘤大小,观察小鼠一般状况;计算抑瘤率、血红细胞数和白细胞数,取脾和胸腺计算脏器指数;HE染色分析肿瘤组织变化;MTT法检测小鼠脾T淋巴细胞增殖转化功能以及ELISA法检测小鼠脾淋巴细胞分泌IFN-γ含量。结果双歧杆菌脂磷壁酸及5-Fu单独应用均可抑制肿瘤生长,但单独5-Fu处理组小鼠一般状况差,毒性反应重;双歧杆菌脂磷壁酸与5-Fu联合应用,与单独5-Fu处理组比较,不仅抑瘤率明显提高（P〈0．01）,且荷瘤小鼠一般状况改善,白细胞数升高,脏器指数增加,小鼠脾T淋巴细胞增殖能力强,脾淋巴细胞分泌IFN-γ,水平提高;光镜观察HE染色瘤体组织,双歧杆菌脂磷壁酸处理组可见大量炎症细胞浸润。结论双歧杆菌脂磷壁酸联合5-FU能增强化疗的抑瘤作用,并能扭转化疗引起的免疫低下现象,起到增效减毒作用。     

IL—12抗病毒作用的研究

白细胞介素－１２（ＩＬ－２）是一种异源二聚体细胞因子。ＩＬ－１２通过调节Ｔｈ１／Ｔｈ２细胞分化,诱导ＩＦＮ－γ等细胞因子的生成,与ＩＬ－１２雷同作用增强ＮＫ细胞、ＬＡＫ细胞及ＣＴＬ细胞活性发挥抗病毒作用。本文介绍ＩＬ－１２的分子生物学,生物学活性及抗病毒作用的研究进展。     

双歧杆菌及其表面分子对小鼠肠内致癌相关性细菌酶活性的影响

本文研究了分叉双歧杆菌及其脂磷壁酸、细胞壁肽聚糖对小鼠粪便中肠内细菌酶－β－葡萄糖醛酸酶、偶氮还原酶和硝酸盐还原酶活性的影响。结果表明,小鼠摄入双歧杆菌活菌、死菌、脂磷壁酸、细胞壁肽聚糖３周期间,肠内细菌偶氮还原酶、β－葡萄糖醛酸酶、硝酸盐还原酶活性降低,以活菌、死菌作用更为显著,二者无显著差异。     

双歧杆菌和乳杆菌在诱发抗肿瘤免疫中的作用

双歧杆菌和乳杆菌给封闭群昆明小鼠腹腔注射,在体内激活后,胸腺细胞和脾细胞对ＣｏｎＡ刺激的增殖反应,脾贴附性细胞对ＹＡＣ－１,Ｌ９２９的细胞毒作用,以及脾贴附性细胞产生对上述二株瘤细胞的肿瘤坏死因子（ＴＮＦ）的活性都比对照动物明显增强。结果提示短双歧杆菌和嗜酸性乳杆菌给小鼠腹腔注射后,通过激活脾脏淋巴细胞和贴附性细胞（巨噬细胞）所介导的免疫功能而明显地增强宿主的抗肿瘤活性。     

双歧杆菌对RAW264.7细胞的免疫刺激作用

为了检测双歧杆菌是否能增强巨噬细胞免疫因子的产生,用ＲＡＷ２６４．７细胞作为巨噬细胞模型,将８种不同的经热处理杀死的双歧杆菌分别与培养的ＲＡＷ２６４．７细胞温育。所有的双歧杆菌均能诱导ＴＮＦ－α及ＩＬ－６的产生,且产生ＴＮＦ－α及ＩＬ－６的量与双歧杆菌的剂量及诱导时间有关,在２４ｈ之内,与对照组相比,８种双歧杆菌均能增强ＴＮＦ－α和ＩＬ－６的产生,但是如果双歧杆菌与ＲＡＷ２６４．７细胞温育时间短（只有１４ｈ）,低剂量的双歧杆菌（１０μｇ／ｍＬ和５０μｇ／ｍＬ）就不能诱导ＴＮＦ－α和ＩＬ－６的产生。双歧杆菌     

短小双歧杆菌对机体几种重要细胞因子诱生作用研究

本实验观察了热杀的短小双歧杆菌对小鼠ＴＮＦ—α、ＩＬ—１、ＩＬ—２等细胞因子的诱生活性的影响。结果表明,短小双歧杆菌腹腔注射后,小鼠ＴＮＦ—α、ＩＬ—１、ＩＬ—２诱生活性显著增强,本文结果提示,短小双歧杆菌可能通过其细胞壁免疫活性成分而发挥免疫调节作用。     

双歧杆菌,大肠杆菌菌体多糖iIFL激活作用的对比研究

采用ＰｅｒｃｏⅡ非连续密度梯度离心法从小鼠小肠提取小肠上皮间细胞（ｉｎｔｅａｌｉｎａｌ ｉｎｔｒａｅ」ｐｉｔｈｅｌｉａｌ ｌｙｍｐｈｏｃｙｔｅ,ｉＩＥＬ）,应用双抗原标记间接免疫荧光法对其抗原表型进行测定,同时,应用有机溶剂提取的双歧杆菌、大肠杆菌菌体多糖与ｉＩＥＬ细胞共同孵育徨,检测ｉＩＥＬ细胞的自然系伤活性、ＩＦＮγ和ＩＬ－２产生能力。结果表明：ｉＩＥＬ细胞的表型８５．１％ＣＤ３＾＋７３．８Ｃ     

Synergistic proliferation and activation of natural killer cells by interleukin 12 and interleukin 18.

We investigated the effects of IL-12 and IL-18 on unstimulated murine splenocytes and observed that the two cytokines strongly synergized for their proliferation, whereas IL-12 and IL-18 alone were essentially inactive in this respect. Phenotypical and functional analyses of cells proliferating in response to IL-12 and IL-18 revealed that large granular Ly-49C(+)DX5(+)CD3(-)NK blasts were expanded in these cultures and that they displayed cytotoxic activity against Yac-1 cells, a murine NK cell target. Further analyses indicated three major differences between NK cells appearing in response to IL-12 and IL-18 and those derived in the presence of other NK cell growth factors, such as IL-2 or IL-15. First, a population of T-NK cells, i.e. expressing T cell (TCRalphabeta, CD3) and NK cell (Ly-49) markers, was detected amongst cells growing in IL-2 or IL-15 but not in cultures supplemented with IL-12 and IL-18. Second, most NK cells derived with IL-2 or IL-15 expressed the NK1.1 antigen, while those derived with IL-12 and IL-18 did not. Finally, striking differences were observed regarding cytokine production. Cells stimulated with IL-12 and IL-18 in combination, but not with IL-2 or IL-15, produced IFN-gamma, IL-3, IL-6 and TNF. IFN-gamma was not involved in the response of NK cells to IL-12 and IL-18, as indicated by experiments demonstrating that the combination of the two cytokines displayed similar effects on spleen cells from IFN-gammaR-knock-out mice. Receptor (IL-12Rbeta1, IL-12Rbeta2 and IL-18R) gene expression studies did not indicate that the mechanism underlying the synergy between IL-12 and IL-18 involved reciprocal induction of their receptors. Taken together, our results demonstrate that IL-12 and IL-18 exert striking synergistic activities for NK cell proliferation and activation, distinct from those induced by IL-2 or IL-15.     

IL-15 and macrophage secretory factors facilitate immune activation of neonatal natural killer cells by lipoteichoic acid

Neonates possess a relatively “naive”, yet inducible immune system. Our hypothesis is that upon strategic antigen exposure, cytokine priming and sensitization by accessory cells, natural killer (NK) cells could be activated to become a functional phenotype. We investigated the in vitro stimulation of cord blood (CB) and adult NK cells upon challenge with lipoteichoic acid (LTA), interleukin (IL)-15 and LTA-primed autologous macrophage-conditioned medium, using CD107a and CD69 phenotypes as indicators of activation. We also examined response of CB macrophages to LTA, in terms of P44/42 extracellular signal-regulated kinases (ERK1/2) activation and cytokine secretion. LTA significantly induced secretion of inflammatory cytokines tumor necrotic factor (TNF)-α, IL-6, IL-12 and activated the upstream signal of ERK1/2 phosphorylation in neonatal macrophages. The magnitude of responses to stimulation differed between neonatal and adult NK cells. Co-stimulation with IL-15 was critical for expansion of the CD69 and CD107a NK subpopulations in both neonatal and adult cells, upon a LTA challenge. NK cell activation could be enhanced by LTA-primed autologous macrophages through secretory factors. Our results indicated that neonatal macrophages and NK cells can evoke immunologic responses to a Gram-positive bacterial antigen. The combinatory priming strategy is relevant for development of novel protocols, such as IL-15 treatment, to compensate for the immaturity of the innate immune system in newborns against bacterial infections.     

IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.     

Enhancement of IL-2-induced T cell proliferation by a novel factor(s) present in murine spleen dendritic cell-T cell culture supernatants

The mechanism(s) underlying the potent accessory cell function of dendritic cells (DC) remains unclear. The possibility was considered that a soluble factor(s) released during the interaction of DC and T cells might contribute to the potent T cell activating function of DC. Culture supernatants were generated from mixtures of murine spleen DC and periodate-treated spleen T cells and were examined for the presence of known cytokine activities and factors capable of enhancing T cell responsiveness to IL-2. Serum-free supernatants from 24 h DC-T cell co-cultures exhibited high levels of IL-2, detectable levels of IL-3, and negligible levels of IL-1, -4, -5, -6, and TNF. A factor(s) was also identified with an apparent Mr of 12.5 to 17.0 kDa, henceforth designated IL-2 enhancing factor (IL-2EF), which enhanced the IL-2-induced proliferation of murine thymocytes, CTLL, and HT-2 cells by approximately three- to fourfold. This enhancement was also observed in the presence of neutralizing antibodies to murine IL-1 alpha, -1 beta, -3, -4, -5, -6, granulocyte-macrophage (GM)-CSF, TNF, and IFN-gamma. However, IL-2EF failed to enhance: 1) the activity of IL-1, -3, -4, -5, or -6 on cells responsive to these cytokines; 2) IL-2-augmented, IL-5-induced BCL1 proliferation; and 3) either PHA- or Con A-stimulated thymocyte proliferation. Moreover, neither IFN-gamma nor GM-CSF exhibited IL-2EF activity. When DC and T cells were cultured separately (after an initial 12 h co-culture period), IL-2EF activity resided predominantly in the T cell-derived supernatants. These and other data indicate that IL-2EF, a heat-labile T cell-derived 12.5 to 17.0 kDa protein, is distinct from IL-1 alpha, -1 beta, -2, -3, -4, -5, -6, TNF, IFN-gamma, GM-CSF, and previously described factors that co-stimulate thymocyte proliferation in the presence of Con A or PHA. It is suggested that IL-2EF functions to specifically enhance IL-2-driven T cell proliferation and contributes to the potent activation of T cells induced by DC.     

In vivo and in vitro induction of natural killer cells by cloned human tumor necrosis factor

Summary The natural killer (NK) cell activity of mice in the peritoneal cavity is very low or undetectable and testing peritoneal NK cells is a useful model for studying the influence of activating substances upon local injection. Injection of tumor necrosis factor (TNF) at doses of 10–200 ng caused a marked activation of NK cell activity which was maximal after 24 h and declined rapidly on day 2. A similar effect was observed when interferons alpha and beta were injected, and there were additive results when interferon was injected together with TNF. The NK cell nature of the effector cells activated by TNF was substantiated by the finding that previous injection with anti-asialo GM 1 antibody prevented activation. Interferon could not be detected in the peritoneal wash fluid after injection of TNF suggesting interferon-independent activation. In further experiments after i.p. injection of TNF peritoneal exudate cells (PECs) only killed YAC-1 targets in a 4-h assay. There was no additional killing in an 18-h assay towards neither YAC-1 cells or P815 cells, suggesting that macrophages were not involved. Furthermore TNF was also active in vitro by activating NK cells in isolated human peripheral blood cells. However in the PECs stimulated in vitro no significant induction of cytotoxic capacities by TNF was measured. Our data suggest that the action of TNF is not restricted to the lysis of tumor cells but can also induce immunological properties in the host defense against virus infections and neoplasms.     

NK cell tolerance to TLR agonists mediated by regulatory T cells after polymicrobial sepsis

As sensors of infection, innate immune cells are able to recognize pathogen-associated molecular patterns by receptors such as TLRs. NK cells present in many tissues contribute to inflammatory processes, particularly through the production of IFN-γ. They may display a protective role during infection but also a detrimental role during sterile or infectious systemic inflammatory response syndrome. Nevertheless, the exact status of NK cells during bacterial sepsis and their capacity directly to respond to TLR agonists remain unclear. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors. The aim of this study was to gain insight into TLR2/TLR4/TLR9 expression on/in murine NK cells at the protein level and determine if their agonists were able to induce cytokine production. We show, by flow cytometry, a strong intracellular expression of TLR2 and a low of TLR4 in freshly isolated murine spleen NK cells, similar to that of TLR9. In vitro, purified NK cells respond to TLR2, TLR4, and TLR9 agonists, in synergy with activating cytokines (IL-2, IL-15, and/or IL-18), and produce proinflammatory cytokines (IFN-γ and GM-CSF). Finally, we explored the possible tolerance of NK cells to TLR agonists after a polymicrobial sepsis (experimental peritonitis). For the first time, to our knowledge, NK cells are shown to become tolerant in terms of proinflammatory cytokines production after sepsis. We show that this tolerance is associated with a reduction of the CD27(+)CD11b(-) subset in the spleen related to the presence of regulatory T cells and mainly mediated by TGF-β.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

Murine NK cell heterogeneity: subpopulations of C57BL/6 splenic NK cells detected by NK-1.1 and NK-2.1 antisera

NK-1.1 antiserum - (BALB/c X C3H)F1 anti-CE - and NK-2.1 antiserum - NZB anti-BALB/c - detect genetically distinct alloantigens on C57BL/6 natural killer (NK) cells. We have analyzed whether these two alloantigens are associated with functional subsets of NK cells. For this study, nylon wool nonadherent C57BL/6 spleen cells (SC) were treated with complement (C) and NK-1.1 or NK-2.1 antisera and then tested for NK activity against a panel of tumor targets in 6- and 19-hour 51Cr release assays. The NK activity against the prototype NK target YAC-1 was reduced equally by both antisera. Similar reductions by both antisera were also observed when SC were tested against another murine lymphoma target, L5178c127v, against the C57BL/6 melanoma B16, and against the human liver cell line Chang. In contrast, NK activity to the lymphoma FBL-3 and the human erythroleukemia K562 was significantly reduced in SC treated with NK-2.1 antiserum and C, whereas SC treated with NK-1.1 antiserum and C showed either less reduction or no reduction in activity against these two cell lines. With two other targets, E male G2 and RBL-5, neither serum produced significant depletion of activity, Analysis of SC indirectly labeled with either NK-1.1 or NK-2.1 antiserum and fluorescein-labeled goat anti-mouse Ig, however, did not detect significant differences between NK-1+ and NK-2+ cell populations.     

Lack of optimal activation of natural killer levels by interleukin-2 in rat spleen cells: evidence for suppression

The effect of human recombinant IL-2 on the levels of natural killer (NK) activity in spleen cells derived from BALB/c mice and different strains of rats was studied. Enhancement of murine NK activity in response to IL-2 was readily demonstrable. Levels of NK activity in control as well as IL-2-treated spleen cells from Wistar, Sprague-Dawley, Fischer, and Long-Evans rats increased initially and peaked on Day 1 or 2 of culture. No significant differences between the control and the IL-2-treated cultures was found in this duration. The subsequent fall in NK activity was slower in IL-2-treated cultures of Fischer and Long-Evans rat spleen cells resulting in a significant difference between the NK levels in control and IL-2-treated cells on Day 5 and Day 7 of the culture. In the case of Wistar and Sprague-Dawley rats, the boosting effect of IL-2 on NK levels was either poor or nonexistent. Even though NK activation was poor, spleen cells of all strains of rats did proliferate in response to IL-2, indicating that the IL-2 preparation used was biologically active on rat spleen cells. Rat spleen cells cultured alone or with IL-2 released a factor(s) in the culture supernatant which could suppress the IL-2-induced NK activation in mouse spleen cells. Indomethacin could block the release of suppressor factor by cultured rat spleen cells. Moreover the NK levels in rat spleen cells could be augmented by IL-2 in the presence of indomethacin. These results indicate that the subdued IL-2-induced NK activation in bulk cultures of rat spleen cells could be due to spontaneous release of prostaglandins by the cultured cells.     

T and B cells are not required for clearing Staphylococcus aureus in systemic infection despite a strong TLR2-MyD88-dependent T cell activation

Staphylococcus aureus infection elicits through its mature lipoproteins an innate immune response by TLR2-MyD88 signaling, which improves bacterial clearing and disease outcome. The role of dendritic cells (DCs) and T cells in this immune activation and the function of T and B cells in defense against S. aureus infection remain unclear. Therefore, we first evaluated DC and T cell activation after infection with S. aureus wild type (WT) and its isogenic mutant, which is deficient in lipoprotein maturation, in vitro. Lipoproteins in viable S. aureus contributed via TLR2-MyD88 to activation of DCs, which promoted the release of IFN-γ and IL-17 in CD4(+) T cells. This strong effect was independent of superantigens and MHC class II. We next evaluated the function of T cells and their cytokines IFN-γ and IL-17 in infection in vivo. Six days after systemic murine infection IFN-γ, IL-17, and IL-10 production in total spleen cells were MyD88-dependent and their levels increased until day 21. The comparison of CD3(-/-), Rag2(-/-), and C57BL/6 mice after infection revealed that IFN-γ and IL-17 originated from T cells and IL-10 originated from innate immune cells. Furthermore, vaccination of mice to activate T and B cells did not improve eradication of S. aureus from organs. In conclusion, S. aureus enhances DC activation via TLR2-MyD88 and thereby promotes T(H)1 and T(H)17 cell differentiation. However, neither T cells and their MyD88-regulated products, IFN-γ and IL-17, nor B cells affected bacterial clearing from organs and disease outcome.