Manganese superoxide dismutase expressed in silkworm larvae,Bombyx mori L enhances the NK activity and splenocyte proliferation against Sarcoma 180 tumor cells in vivo

Natural killer cell (NK) is known as a major immune system in body through mediating cell death via several possible pathways, and one of three subpopulations of lymphocytes functioning as scavenger of tumor, virus infected cells etc. Our present results found that the SOD-contained silkworm larvae powder caused an enhancement of the effect on NK cell cytotoxicity, which implied this material modulated the immune system in mice in vivo. The NK cell activities of S180 tumor modeled mice treated with silkworm powder including SOD were enhanced significantly ranging from 30% to 48%, respectively, compare to a distilled water feeding control and silkworm powder without SOD. Meanwhile, the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control both in T cells or B cells. The average tumor weight of S180 modeled mice treated with doses of SOD-contained silkworm powder was lighter than that of water control showing the tumor inhibition rates (IR) reached to 22.51% to 37%, respectively. In conclusion, these findings demonstrate that administration of silkworm larvae powder containing SOD results in activation of NK cells and immune T-cell and B-cell, suggesting the silkworm larvae powder containing SOD play a positive role in tumor inhibition.     

Anti-oxidation and immune responses in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L

With manganese superoxide dismutase expressed in silkworm larvae, Bomby mori L, we investigate the effects of silkworm larvae powder containing SOD on the antioxidation and the immune system of mouse. The contents of MDA both in mice plasma or liver organ treated with silkworm larvae powder containing manganese superoxide dismutase were reduced compare to control. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities both in plasma or liver organ of the treated mice were significantly higher than that of both control and bromobenzene treated mice (group-BM), suggesting the silkworm larvae powder containing SOD play a positive role in anti-oxidation in mice. This experiment was also designed to investigate the effects of silkworm larvae powder containing SOD on the immune system of mouse, focused on hemolysin response, hemagglutination against SRBC and the activity of natural killer (NK) cells. All treated mice showed significant increase in hemolysin response to SRBC and demonstrated an activation of NK cell function by the SOD-contained silkworm larvae powder, which suggest a promotion in humoral immunity. The results suggested the SOD expressed in silkworm maybe have potential application in medicine.     

Immunity promotion and proteomic identification in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae

With manganese superoxide dismutase (SOD) expressed in silkworm larvae, Bomby mori L, we investigated the effects of silkworm larvae powder containing SOD on the immune system of mouse and employed a proteomics approach to examine this phenomenon. Our data on the effects of continuous treatment with SOD-containing silkworm larvae powder showed that the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control. The results of PFC assay also revealed that antibody production was higher in all three treated groups than controlled mice. We investigated the phagocytosis of mouse macrophages. The SOD treatment led to a dose-dependent increase of phagocytic activity. We identified six proteins that related to immunity of mice. The data showed all these six matched proteins related immunity presented the increase of expression level in plasma of mouse administrated with silkworm powder including SOD compared to that of control. These findings demonstrate that administration of silkworm larvae powder containing SOD results in enhancement of immunity activities in the mouse. The results also suggested that the SOD expressed in silkworm maybe have potential application in medicine.     

灰树花孔菌超微粉体内抗肿瘤作用研究

通过研究肿瘤抑制率、脏器指数、肿瘤切片的观察以及血清中影响因子干扰素-γ（IFN-γ）、白细胞介素-2（IL-2）、血管内皮细胞生长因子（VEGF）、过氧化氢酶（CAT）、谷胱甘肽-过氧化物酶（GSH-PX）和超氧化物歧化酶（SOD）等指标的变化对比分析灰树花孔菌 Grifola frondosa子实体粗粉及灰树花孔菌子实体超微粉对Hep-A-22荷瘤小鼠体内抗肿瘤和免疫调节作用。结果表明,各剂量组分均具有抑制肿瘤生长作用,其中灰树花孔菌超微粉高剂量组（1 500mg/kg）抑瘤效果最佳,与模型组比较有极显著性差异（ P<0.01）。通过HE染色后的肿瘤细胞可观察到坏死面积增大,同时该组血清中IFN-γ、IL-2、VEGF、GSH-PX和SOD含量显著增加（ P<0.01或 P<0.05）,并与抑制肿瘤具有一定相关性。     

十全育真汤对H22荷瘤小鼠免疫功能的影响

目的：研究十全育真汤对荷瘤小鼠免疫功能的影响,探讨十全育真汤抗肿瘤的作用机制。方法：SPF级雄性昆明种小鼠30只,制成荷H₂₂小鼠肝癌细胞移植瘤模型,随机分为模型组、阳性对照组及十全育真汤组（n=10）;另选择未接种小鼠10只为正常对照组。正常对照组、模型组每天按10 ml/kg灌胃生理盐水及蒸馏水,阳性对照组、十全育真汤组每天按8 g/kg、18 g/kg灌胃参一药液（80 mg/ml）与十全育真汤剂。连续给药14 d后处死小鼠,测量胸腺、脾脏指数及抑瘤率,检测外周血中白细胞、淋巴细胞含量及T细胞亚群CD3、CD4、CD8细胞百分比,血清中白细胞介素2（IL-2）、肿瘤坏死因子α（TNF-α）及干扰素-β（IFN-β）的含量,淋巴细胞增殖能力及NK细胞杀伤功能。结果：较正常对照组,十全育真汤组小鼠体重明显增加,脾脏指数显著增大（P<0.05）;白细胞、淋巴细胞CD4、CD8、CD3及TNF-α含量明显升高（P<0.05）;IL-2、IFN-β含量显著下降（P<0.05）;淋巴细胞增殖能力、NK细胞杀伤功能明显升高（P<0.05）。与模型组相比,十全育真汤组小鼠胸腺、脾脏指数显著增大（P<0.05）;白细胞、淋巴细胞CD3、CD4、IL-2、IFN-β及TNF-α含量明显升高（P<0.05）,CD8含量则显著降低（P<0.05）;NK细胞杀伤功能及淋巴细胞增殖能力显著增加（P<0.05）。结论：十全育真汤可促进H₂₂荷瘤小鼠免疫器官的生长,增强机体免疫功能,有利于肿瘤机体的恢复。     

IL-28 elicits antitumor responses against murine fibrosarcoma

IL-28 is a recently described antiviral cytokine. In this study, we investigated the biological effects of IL-28 on tumor growth to evaluate its antitumor activity. IL-28 or retroviral transduction of the IL-28 gene into MCA205 cells did not affect in vitro growth, whereas in vivo growth of MCA205IL-28 was markedly suppressed along with survival advantages when compared with that of controls. When the metastatic ability of IL-28-secreting MCA205 cells was compared with that of controls, the expression of IL-28 resulted in a potent inhibition of metastases formation in the lungs. IL-28-mediated suppression of tumor growth was mostly abolished in irradiated mice, indicating that irradiation-sensitive cells, presumably immune cells, are primarily involved in the IL-28-induced suppression of tumor growth. In vivo cell depletion experiments displayed that polymorphonuclear neutrophils, NK cells, and CD8 T cells, but not CD4 T cells, play an equal role in the IL-28-mediated inhibition of in vivo tumor growth. Consistent with these findings, inoculation of MCA205IL-28 into mice evoked enhanced IFN-gamma production and cytotoxic T cell activity in spleen cells. Antitumor action of IL-28 is partially dependent on IFN-gamma and is independent of IL-12, IL-17, and IL-23. IL-28 increased the total number of splenic NK cells in SCID mice and enhanced IL-12-induced IFN-gamma production in vivo and expanded spleen cells in C57BL/6 mice. Moreover, IL-12 augmented IL-28-mediated antitumor activity in the presence or absence of IFN-gamma. These findings indicate that IL-28 has bioactivities that induce innate and adaptive immune responses against tumors.     

Effects of macrophage-colony-stimulating factor on cyclophosphamide-injected mouse NK1.1+ cell activity

 We injected cyclophosphamide into mice and examined their natural killer (NK) activity both in vitro and in vivo. Cyclophosphamide injection temporarily abrogated the lung clearance activity of Yac-1 lymphoma cells, which is considered to be an index of NK activity in vivo. However, administration of recombinant human macrophage-colony-stimulating-factor (rhM-CSF) to cyclophosphamide-injected mice restored the lung clearance activity. To clarify whether the administration of rhM-CSF activated NK cells, we purified NK1.1⁺ cells from mice treated with cyclophosphamide and/or rhM-CSF and examined their functions (cytotoxicity, proliferation, and interferon γ production) in vitro. Cyclophosphamide injection decreased the number, but did not suppress the functions of NK1.1⁺ cells. The numbers of NK1.1⁺ cells in cyclophosphamide-injected mice restored by rhM-CSF administration. And the functions of NK1.1⁺ cells from both saline-injected and cyclophosphamide-injected mice were accelerated by rhM-CSF administration. These results suggested that the temporary abrogation of NK activity in vivo caused by cyclophosphamide injection was due to a decrease in the number and not to suppression of the functions of NK1.1⁺ cells. The injection of cyclophosphamide into mice increased the number of tumor (B16 melanoma) nodules formed in the lungs and liver. However, treatment with rhM-CSF recovered the anti-metastatic activity in the lungs of cyclophosphamide-injected mice. These results show that administration of rhM-CSF restores NK activity suppressed by cyclophosphamide injection in vivo. Received: 28 September 1999 / Accepted: 23 December 1999     

白花蛇舌草豆甾醇对肝癌细胞的体内外抑制作用及对其增殖周期、凋亡的影响

目的:研究白花蛇舌草豆甾醇(stigmasterol from Hedyotis diffusa willd.,SHD)对人肝癌细胞SMMC-7721、BEL-7402的体外抑制作用,对肝癌H22的体内抑制作用及对其增殖周期、凋亡的影响。方法:MTT法评价SHD对人肝癌细胞SMMC-7721、BEL-7402的抑制率变化规律。昆明雄性小鼠60只,随机取10只为正常对照组,余接种H22瘤株,随机分为模型对照组、5-FU阳性对照组(30mg/kg)和高中低剂量SHD给药组(剂量分别为15、30、60mg/kg),腹腔给药10 d后,比较各组瘤重抑制率、H22细胞周期分布、凋亡率。结果:SHD对SMMC-7721、BEL-7402细胞具有体外抑制作用;SHD显著抑制H22肿瘤,增加G0-G1期细胞比例,降低G2/M期细胞比例,促进肿瘤细胞凋亡。结论:SHD在体外、体内均具有抑制肝癌细胞的作用,此作用与阻滞肿瘤细胞增殖周期,促进肿瘤细胞凋亡有关。     

Elevated natural killer cell-mediated cytotoxicity, plasma interferon, and tumor cell rejection in mice persistently infected with lymphocytic choriomeningitis virus

To assess the effects of chronic virus infection on NK cells, the related phenomena of interferon (IFN) production, NK cell activation, and resistance to tumor implants were studied in mice persistently infected with lymphocytic choriomeningitis virus (LCMV). NK cells from these LCMV-carrier mice displayed augmented killing of the NK-sensitive YAC-1 target cell. They did not lyse the more resistant targets L-929 and P815, whereas NK cells from acutely infected mice efficiently lysed all three cell types. The plasma from LCMV-carrier mice contained an antiviral substance identified as IFN type I, based on species specificity, virus nonspecificity, resistance to pH 2, and sensitivity to antibody to type I IFN. IFN titers in plasma from LCMV-carrier mice were 32 to 64 U/ml, about 20-fold less than those in acutely infected mice. Both the IFN and NK cell levels continuously remained elevated in the LCMV carrier mice up to at least 6 months of age. IFN is known to activate NK cells and to induce their blastogenesis in vivo. As determined by centrifugal elutriation, large NK blast-size cells were isolated from the spleens of acutely infected mice, but not from either normal or LCMV-carrier mice, suggesting augmented NK cell-mediated lysis in the absence of enhanced proliferation. Poly inosinic-cytidylic acid induced high levels of NK cell-mediated cytotoxicity and blastogenesis in both control and LCMV-carrier mice, but IFN was induced to lower levels in carriers as compared with controls. Coincidental with augmented NK cell activity, the LCMV-carrier mice rejected intravenously injected 125IUdR-labeled tumor cells more efficiently than did normal mice. Thus, LCMV carrier mice have low levels of type I IFN, moderately augmented NK cell activity lasting for at least 6 months, and increased resistance to tumor cell implants. This indicates that augmented NK cell-mediated cytotoxicity can be maintained in vivo over prolonged periods of time in the presence of chronic low-level IFN stimulation.     

NK cell activation and tumor infiltration are involved in the antitumor mechanism of Virulizin

Previous studies have demonstrated antitumor efficacy of Virulizin in several human tumor xenograft models and a critical role for macrophages in the antitumor mechanism of Virulizin. Although there is growing support for an immune stimulatory mechanism of action for Virulizin, the details remain to be elucidated. The aim of this study was to determine whether infiltration of natural killer (NK) cells into xenografted tumors is altered by Virulizin treatment, and whether such alterations contribute to the antitumor activity of Virulizin. Immunohistochemical analysis demonstrated that xenografted tumors from Virulizin-treated mice had an increase in infiltration of F4/80⁺ (macrophages) and NK1.1⁺ (NK) cells. The increase in NK1.1⁺ cell infiltration occurred at an early stage of Virulizin treatment, which correlated with an early sign of apoptosis. In addition, Virulizin resulted in an increase in the number of NK cells in the spleens, and NK cells isolated from the spleen exhibited increased cytotoxicity to tumor cells in vitro. In NK cell–deficient SCID-beige mice, the antitumor activity of Virulizin was compromised, providing additional support to the hypothesis that NK cells are necessary for inhibition of tumor growth by Virulizin. Finally, depletion of macrophages resulted in the loss of Virulizin-induced increase in NK1.1⁺ cell infiltration into xenografted tumors, suggesting the involvement of macrophages in NK cell infiltration into tumors. Taken together, these results strongly support a mechanism in which Virulizin stimulates a sustained expansion and infiltration of NK cells and macrophages into tumors with subsequent activation of NK cells that is responsible for the observed antitumor activity.     

香菇C91-3菌丝发酵液蛋白LFP91-3C小鼠体内抗肿瘤免疫机制研究

目的探讨香菇C91-3菌丝发酵液蛋白LFP91-3C的体内抗肿瘤免疫机制。方法H22瘤细胞荷瘤纯系BALB／C小鼠建立动物模型,随机分为生理盐水（NS）对照组、环磷酰胺（CTX）治疗组和LFP91-3C治疗组,观察LFP91-3C对荷瘤小鼠生存期、实体瘤块生长抑制及病理、荷瘤小鼠免疫细胞（NK细胞活性、淋巴细胞增殖率）和免疫因子（血清IL-2、IFN-γ含量）的影响。结果LFP91-3C能显著延长荷瘤小鼠的生存期,抑制实体肿瘤的生长,可在病理切片看到大量的炎细胞浸润,以及NK细胞活性、淋巴细胞增殖率、血清IL-2和IFN-γ含量也显著提高。与NS对照组和CTX治疗组比较差异有显著性。结论LFP91-3C能通过激活机体免疫系统来实现其抗肿瘤的作用。     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

山野木层孔菌子实体中抑制H22荷瘤小鼠肿瘤的活性成分研究

采用梯度提取法对山野木层孔菌子实体进行提取,得到石油醚层、甲醇层及水层3 种提取物及石油醚层中获得化合物4,6,8(14),22(23)-四烯-3-酮-麦角甾烷,并采用H22 荷瘤小鼠进行体内抗肿瘤活性研究,以抑瘤率、免疫器官指数、免疫因子为指标检测抗肿瘤活性。结果表明,石油醚高剂量组（100mg/kg）、单体化合物中剂量组（7.5mg/kg）抑制率分别为62.21%、57.67%;脾指数、胸腺指数均高于对照组和环磷酰胺（CTX）组,白介素-2（IL-2）的含量明显高于对照组和环磷酰胺（CTX）组（P＜0.01）;肿瘤坏死因子-α（TNF-α）含量明显低于对照组（P＜0.01）。因此认为上述石油醚提取物和单体化合物对H22荷瘤小鼠肿瘤有抑制作用,并且均能改善小鼠的免疫功能。     

Effect of tumor immunity on the distribution of labeled lymphoid cells in tumor-challenged mice

The distribution of ⁵¹Cr-labeled lymphoid cells from normal mice and mice immunized against a tumor were compared after intravenous inoculation of the labeled cells into normal syngeneic recipients. Spleen cell preparations from immune donors contained increased percentages of spleen and bone marrow-seeking cells, thus suggesting expansion of these cell populations when immunity to a tumor exists. Homing of labeled normal cells in tumor cell-injected normal animals was somewhat different from that seen in tumor cell-inoculated mice that were immunized against the tumor. In the latter case, accumulations of lymph node and spleen cells in recipient lymph nodes and bone marrow were consistently lower. In contrast, lymphoid cells from animals immunized against the tumor were found to accumulate in virtually the same percentages in lymphoid organs of normal and immune recipients. The behavior of lymphoid cell populations from thymus or bone marrow that consist mainly of precursor cells was unaffected by presence of malignancy and/or tumor immunity.     

Role for TLR2 in NK cell-mediated control of murine cytomegalovirus in vivo

Natural killer (NK) cells are essential for the early control of murine cytomegalovirus (MCMV) infection. Here, we demonstrate that toll-like receptor 2 (TLR2) plays a role in the NK cell-mediated control of MCMV. TLR2 knockout (KO) mice had elevated levels of MCMV in the spleen and liver on day 4 postinfection compared to C57BL/6 mice. In vivo depletion of NK cells with anti-NK1.1 antibodies, however, eliminated the differences in viral titers between the two groups, suggesting that the effect of TLR2 on MCMV clearance on day 4 was NK cell mediated. The defect in early antiviral control was associated with a decreased NK cell population in the spleen and liver and reduced amounts of interleukin-18 and alpha/beta interferon secreted in the TLR2 KO mice. Our studies suggest that in addition to the reported involvement of TLR9 and TLR3, TLR2 is also involved in innate immune responses to MCMV infection.     

CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-beta-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.     

大豆皂苷对荷瘤小鼠免疫功能的影响

目的：观察大豆皂苷（soyasaponins,SS）对荷瘤小鼠免疫功能的影响。方法：建立S180荷瘤小鼠模型,随机分为模型对照组、环磷酰胺组（CTX）组（20mg／kg）、SS低、中、高剂量组（10、20、30mg／kg）,每组12只。连续给药15天后处死小鼠,测量小鼠免疫器官指数、淋巴细胞转化率（采用MTT法）、溶血空斑数（采用Jeme改良玻片法）、巨噬细胞吞噬功能（采用半体内法）以及NK细胞活性（采用乳酸脱氢酶法）。结果：SS中、高剂量组提高小鼠脾脏指数、脾淋巴细胞转化率、溶血空斑数、巨噬细胞吞噬率及吞噬指数、NK细胞活性,与模型对照组比较有显著性差异（P〈0．05）。与模型对照组比较,SS各剂量组胸腺指数无明显差异（P〉0．05）。结论：一定剂量的SS具有增强荷瘤小鼠免疫功能的作用,从而发挥抗肿瘤作用。     

樟芝液体发酵粉对肝癌H22的体内生长抑制作用(英文)

本文采用肝癌H22荷瘤小鼠的肿瘤模型,对樟芝液体发酵粉的体内抗肿瘤活性进行评价。结果表明,樟芝液体发酵粉(500或1000 mg/kg b.w.)能够体内显著抑制H22肿瘤的生长,抑制率分别为42.0%和46.4%。同时,与模型组相比,樟芝发酵粉能够延长荷瘤小鼠的生存周期,延长率为29.4%。组织病理学研究结果表明,模型组小鼠的肿瘤细胞生长旺盛,而樟芝发酵粉处理组的小鼠肿瘤细胞具有明显的皱缩和坏死症状。樟芝发酵产物具有较好的体内抗肿瘤活性。