Biological activities and antitumor mechanism of an immunopotentiating organogermanium compound, Ge-132 (review)

The biological activities and antitumor mechanism of an immunopotentiator, Ge-132, is reviewed herein. Ge-132 exhibited antitumor activity against certain syngeneic and allogeneic experimental tumors. It was shown that T-cells and macrophages were involved when tumor-bearing mice were protected by the compound. This protective effect could be transferred to tumor-bearing mice, not treated with the compound, by a macrophage fraction and serum specimens obtained from Ge-132-treated mice. Interferon gamma (IFN gamma) was detected in the circulation of Ge-132-treated mice and when sera obtained from Ge-132-treated mice were treated with anti-IFN gamma antiserum in vitro, the antitumor activity was abolished. On the other hand, in mice treated with anti-IFN gamma antiserum, Ge-132 did not induce serum IFN and failed to protect against death due to ascites tumor progression. The in vivo administration of monoclonal anti-Thy 1.2 antibody prevented the expression of the antitumor activity of Ge-132. However, serum specimens obtained from Ge-132-treated mice effectively inhibited the tumor growth of T-cell-depleted mice bearing ascites tumors. Since it has been reported that T-lymphocytes produce IFN gamma, this suggested that Ge-132 may first stimulate T-cells to produce IFN gamma in the expression of the observed antitumor efficacy. In addition, sera obtained from Ge-132-treated mice did not show any antitumor activity in mice depleted of macrophage functions. Additionally, passive transfer of macrophages from mice treated with these serum specimens to tumor-bearing mice also resulted in the inhibition of tumor growth. Pretreatment of these serum specimens with anti-IFN gamma antiserum effectively prevented the generation of cytotoxic macrophages. Also, tumor-bearing mice treated exogenously with this antiserum did not differ significantly in survival as compared to controls, despite the administration of Ge-132. Furthermore, the antitumor activity of Ge-132 was detected in NK cell-depleted mice. Therefore, the antitumor mechanism of Ge-132 in the murine ascites tumor system may be expressed as follows: (a) Ge-132 stimulates T-cells to induce IFN gamma when mice are treated orally with the compound, (b) IFN gamma activates macrophages to become cytotoxic, and (c) the cytotoxic macrophages eliminate tumor cells.     

Protective effects of hochu-ekki-to, a Chinese traditional herbal medicine against murine cytomegalovirus infection.

The innate immunity against murine cytomegalovirus (MCMV) at the early phase of infection is mediated by NK cells and macrophages. We studied the effects of hochu-ekki-to (HET), a traditional Chinese herbal medicine, on the regulation of innate immunity mediated by NK cells and macrophages. We found the oral administration of HET to increase both the number of leukocytes in the spleen and liver and the splenic NK cell cytotoxicity associated with the increased induction of serum IFN-alpha/beta after an MCMV infection but it had no effect on liver NK cells. However, no differences were found in the serum IL-12, IFN-gamma, TNF-alpha and nitric oxide (NO) production in the culture of macrophages between the HET- and PBS-treated mice on day 2 after MCMV infection. In addition, HET-treated splenic and peritoneal macrophages were found to show a higher intrinsic resistance against in vitro MCMV infection than that of PBS-treated mice. Therefore, the HET-induced effects on NK cells and macrophages selectively reduced the viral load in the spleen but not in the liver at an early phase of MCMV infection. HET may thus be useful in the treatment of human cytomegalovirus infection which commonly occurs in HIV-infected AIDS patients.     

Immunogenicity of an interferon-beta1a product

In order to determine whether Blastoferon?, a biosimilar interferon (IFN)- beta 1a formulation, shares epitopes with other known IFN-beta products, a series of neutralization bioassays were performed with a set of well-characterized anti-IFN- beta monoclonal antibodies and human sera (World Health Organization Reference Reagents). The bioassay was the interferon-induced inhibition of virus cytopathic effect on human cells in culture (EMC virus and A-549 cells). Computer-calculated results were reported as Tenfold Reduction Units (TRU)/ml. To further assess Blastoferon? immunogenicity, in vivo production of anti-IFN beta antibodies was determined in sera of patients included in the pharmacovigilance plan of Blastoferon? by the level of IFN- beta 1a binding antibodies (by enzyme immunoassay -EIA) and neutralizing antibodies (in the Wish-VSV system). The highly characterized neutralizing monoclonal antibodies A1 and A5 that bind to specific regions of the IFN- beta molecule reacted positively with the three beta 1a IFNs: Blastoferon?, Rebif?, and the IFN- beta WHO Second International Standard 00/572. As expected, the non-neutralizing monoclonal antibodies B4 and B7 did not neutralize any of the IFN- beta preparations. The commercially available monoclonal antibody B-02 reacted essentially equally with Rebif? and Blastoferon?. The WHO Reference Reagent human serum anti-IFN- beta polyclonal antibody neutralized all the IFN- beta products, whereas the WHO Reference Reagent human serum anti-IFN-alpha polyclonal antibody G037-501-572 appropriately failed to react with any of the IFN- beta products. On the basis of in vitro reactivity with known, well-characterized monoclonal and polyclonal antibody preparations, Blastoferon? shares immunological determinants with other human interferon- beta products, especially IFN- beta 1a. In vivo antibodies were detected by EIA in 72.9% of 37 chronically treated multiple sclerosis patients, whereas neutralizing antibodies were found in 8.1% of them. Blastoferon? appears to have immunological characteristics comparable to other IFN- beta 1a products.     

Ganoderma tsugae supplementation alleviates bronchoalveolar inflammation in an airway sensitization and challenge mouse model

Ganoderma tsugae (a Chinese mushroom Songshan lingzhi) cultivated in Taiwan is extensively used in Chinese traditional medicine to treat different diseases. To determine whether G. tsugae has anti-inflammatory effects on bronchoalveolar inflammation in vivo, we investigated the anti-inflammatory effects of G. tsugae products, YK01 and YK07, on bronchoalveolar inflammation using an airway sensitization and challenge mouse model. Female BALB/c mice were weekly sensitized by intraperitoneal injection of ovalbumin (OVA) three times and challenged with aerosolized OVA twice. Differential cell counts of infiltrating leukocytes, inflammatory mediators, cytokines in bronchoalvelor lavage fluid (BALF) of OVA-challenged mice were examined after continuously consuming G. tsugae diets for 5 weeks. We found that supplementation of G. tsugae significantly decreased total infiltrating leukocytes and lymphocyte percentage in BALF in the experimental groups. Supplementation of G. tsugae also significantly reduced inflammatory mediators in BALF including histamine, prostaglandin E2, eotaxin, and protein levels, however the levels of pro-inflammatory cytokines, interleukin (IL)-1beta and IL-6, in BALF did not significantly change. These results suggest that both G. tsugae supplementation diets YK01 and YK07 might alleviate bronchoalveolar inflammation via decreasing the infiltration of inflammatory cells and the secretion of inflammatory mediators into the local tissues of lungs and airways. Further, these results indicate that the relief of bronchoalveolar inflammation in an airway sensitization murine model provides a possible therapeutic application for G. tsugae in allergic asthma.     

Induction of cytolytic activity and interferon-gamma production in murine natural killer cells by polymyxins B and E

Natural killer (NK) cells are the primary effector cells of the innate immune system and have well-established roles in tumor rejection and resistance to viruses, bacteria and certain parasites. There is a need for more specific immune modulators of NK cell activity that lack the wide-ranging side effects of NK cell-stimulatory interleukins. The polycationic antibiotic polymyxin B (PMB) has been shown to have a unique ability to enhance activities of some immune cells, independent of its antibiotic properties. Here we report that both PMB and its analog polymyxin E (PME) markedly enhanced the activity of NK cells enriched from the murine spleen. Maximal activation of NK cell activity was obtained after 24 h of incubation with PMB at a dose of 300 mug/ml. PMB nonapeptide, one of the two PMB domains, and PME methanesulfonate, the negatively charged derivative of PME, had little effect on NK cell activity. PMB induced interferon (IFN)-gamma and tumor necrosis factor-alpha production in NK cells. Proliferation of NK cells in vitro was significantly stimulated by being incubated with PMB. Administration of PMB to mice for 7 consecutive days stimulated splenic NK cell activity and increased NK cell populations in the spleen. These results suggest that the polycationic antibiotics PMB and PME may up-regulate innate and adaptive immune responses by induction of NK cell activity and IFN-gamma production.     

Immunomodulatory activity of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a potent anti-HIV nucleotide analogue, on in vivo murine models.

In order to evaluate the influence of antiviral nucleoside analogues upon the natural immune system, we investigated the immunomodulatory activity of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a nucleotide analogue with potent anti-HIV and anti-herpes activity, in a murine system. C57BL/6 mice were inoculated intraperitoneally with 10, 25 and 50 mg PMEA/kg. Mononuclear cells were isolated from their spleens, and some natural immune functions were evaluated. The results show that PMEA significantly increases the levels of natural killer (NK)-cell cytotoxicity. We also found that alpha/beta IFN production was substantially increased in PMEA-treated mice, while both IL-1 and IL-2 production was decreased. Thus, PMEA can increase some natural immunity functions, such as NK activity and IFN production. These results suggest that PMEA might be active in vivo against HIV and herpes viruses both as an immunomodulator and as an antiviral compound.     

口服环孢素对鼠巨细胞病毒慢性感染的影响及其机理

长期口服环孢素(cyclosporine,CsA)使感染鼠巨细胞病毒(murine cytomegalovirus,MCMV)的小鼠脾和唾液腺中病毒滴度显著增高,而且维持脾中病毒的慢性感染。CsA显著降低血清干扰素(interferon,IFN)水平,但对脾天然杀伤细胞(na-tural killer cell,NK cell)活性无明显影响。被动输入免疫鼠脾细胞可保护小鼠免受MCMV感染,而且该保护依赖T细胞,具有病毒特异性和受H-2组织相容抗原(H-2 histocompatibility antigen)限制。长期使用CsA取消了上述保护作用。本实验结果表明CsA可能通过抑制细胞毒性T淋巴细胞及干扰素诱生使MCMV在脏器中滴度升高,并且维持了病毒的慢性感染。     

Varying role of alpha/beta interferon in the antiviral efficacy of synthetic immunomodulators against Semliki Forest virus infection.

The question of whether interferon alpha/beta is the common mechanism of antiviral action of synthetic immunomodulators was investigated in B6C3F1 mice infected with Semliki Forest virus. Mice were treated with various concentrations of normal sheep serum or potent anti-alpha/beta interferon antiserum, inoculated with the immunomodulators, and infected 24 hours later with virus. Three patterns emerged. The antiviral action of the pyrimidinone (ABMP) and the oral interferon inducer (CL246,738) appeared to be mediated primarily by interferon alpha/beta; their protective ability was almost completely abrogated by treatment with low levels of anti-alpha/beta interferon antiserum. The antiviral action of two other immunomodulators, a mismatched polyribonucleotide (Ampligen) and a polyanionic copolymer (MVE-2) at least partially involved interferon. Activity of these compounds was reduced, but not consistently eliminated by treatments with high doses of antiserum. The antiviral activity of another polyribonucleotide, polyriboinosinic-cytidylic acid complexed with lysine carboxymethylcellulose (poly ICLC), was not affected by treatment with even the highest amount of antiserum (two injections of 100,000 neutralizing units each). Almost complete protection by poly ICLC was observed despite the fact that this high concentration of antiserum, when given alone, caused a decrease in natural resistance to Semliki Forest virus infection. Taken together, these results indicate that induction of interferon alpha/beta does not appear to be the major common mechanism of antiviral activity among these diverse synthetic immunomodulators.     

毛蚶多肽提取物的体外免疫活性

目的考察毛蚶多肽提取物(PEAS)的体外免疫活性。方法 MTT法测定PEAS对小鼠脾淋巴细胞增殖的影响及对小鼠NK细胞杀伤活性的影响;中性红吞噬法测定PEAS对小鼠腹腔巨噬细胞吞噬功能的影响;流式细胞术测定PEAS所致小鼠脾淋巴细胞周期的变化;双抗体夹心ELISA法测定PEAS对主要细胞因子IFN-γ和IL-4分泌水平的影响。结果 PEAS能够显著促进小鼠脾淋巴细胞增殖(P<0.05或0.01),能够增强小鼠腹腔巨噬细胞吞噬中性红活性和小鼠NK细胞杀伤活性(P<0.05或0.01);PEAS能够促进脾淋巴细胞G0/G1期向DNA合成期(S期)转化;PEAS协同Con A作用,能够增加IFN-γ的分泌(P<0.05或0.01),并且能够抑制IL-4的分泌(P<0.05或0.01)。结论 PEAS体外可促进小鼠脾淋巴细胞、腹腔巨噬细与NK细胞的免疫功能,其机制可能与促进脾淋巴细胞DNA合成,增加IFN-γ的分泌量有关。     

Roles of interferon and natural killer cells in the antiviral activity of 7-thia-8-oxoguanosine against Semliki Forest virus infections in mice.

7-Thia-8-oxoguanosine is a novel biological response modifier with broad-spectrum antiviral activity against many DNA and RNA viruses in vivo. Since two of its properties are to induce interferon and to activate natural killer (NK) cells, we investigated the roles of the lymphokine and NK cells in the antiviral activity of the compound against Semliki Forest virus. Antibody to interferon alpha/beta could completely abolish the protective activity of the nucleoside against virus infection in mice, whereas antibodies to interferons beta and gamma could not, indicating that interferon alpha was of major importance to confer protection to the animals. Reduced activation of NK cells was also observed in mice treated with 7-thia-8-oxoguanosine and antibody to interferon alpha/beta. The role of NK cells in the protective activity of the compound was directly assessed in beige mice or in Swiss Webster mice treated with asialo GM1 antibody. In both experiments, the animals were protected from lethal virus infection by treatment with nucleoside. Spleen cells primed by 7-thia-8-oxoguanosine and adoptively transferred to untreated mice could not save them from virus-induced mortality. These three results provide evidence that natural killer cells activated by 7-thia-8-oxoguanosine play a minimal role in protection from acute Semliki Forest virus infections in mice.     

Effects of proxigermanium on interferon production and 2',5'-oligoadenylate synthetase activity in the lung of influenza virus-infected mice and in virus-infected human peripheral blood mononuclear cell cultures.

Proxigermanium (SK-818) is a synthesized organic germanium compound having various biological activities. The effects of proxigermanium on interferon (IFN) production in mice infected with influenza virus and virus-infected human peripheral blood mononuclear cells (hPBMC) were investigated. Proxigermanium alone did not induce IFN production in normal mice or in hPBMC without viral infection. On the other hand, proxigermanium enhanced alpha/beta IFN production in viral-infected mice and hPBMC. Since proxigermanium is known to have antiviral activity in vivo but not in vitro, it is likely that the IFN production augumenting activity of proxigermanium is involved in its antiviral activities.     

The role of natural killer cells in protection of mice against death and corneal scarring following ocular HSV-1 infection

C57BL/6 mice depleted of NK (natural killer) cells with anti-asialo-GM1 antibody were more susceptible to lethal HSV-1 ocular challenge (12% survival) than control C57BL/6 mice (100% survival), CD4+ depleted mice (100% survival), CD8+ depleted mice (80% survival), or macrophage depleted mice (85% survival). NK depletion also resulted in significantly higher levels of HSV-1 induced corneal scarring than was seen with any of the other groups. C57BL/6 mice depleted of NK cells with PK136 (anti-NK1.1 antibody which is more specific for NK cells than is anti-asialo-GM1 antibody) were also more susceptible to HSV-1 ocular challenge than T cell or macrophage depleted mice. Vaccination completely protected NK depleted mice against death and corneal scarring. In contrast to C57BL/6 mice, in BALB/c mice, NK depletion had no effect on survival or corneal scarring following ocular HSV-1 challenge. Experiments with IFN-gamma knockout mice (IFN-gamma(o/o) mice) suggested that IFN-gamma played a minor role in protection of na?ve mice against death following HSV-1 challenge. However, IFN-gamma did not appear to be an important factor in protection against HSV-1 induced eye disease. Thus, protection against HSV-1 induced corneal scarring in naive mice appeared to be due to a non-INF-gamma NK function. Our results therefore suggest that NK cells were very important in protecting naive C57BL/6 mice but not vaccinated C57BL/6 mice against corneal scarring and death following ocular HSV-1 challenge.     

Chemotherapeutic agents and modulation of natural killer cell activity in vitro

This study investigated the effect of various clinically used chemotherapeutic agents on non-modulated and human alpha Interferon (IFN) modulated natural killer cell (NK) activity. The inhibitors of DNA synthesis, Adriamycin, Cis-Platinum, 5-Fluorouracil and Methotrexate, did not alter NK cell activity at comparable clinically used doses. Similarly, Vincristine and Vinblastine, which are antimitotic agents, did not affect the NK activity. Only inhibitors of RNA synthesis L-Asparaginase and Actinomycin D reduced non-modulated and IFN modulated NK cell activity.     

松杉灵芝菌丝体多糖(FI_0-c)对人组织瘤细胞和人外周血白细胞产生炎症性细胞因子的影响(Ⅱ)(英文)

目的:比较研究水溶性多糖(FI_0-c)及其氯磺酸修饰产物(FI_0-c-S)对人炎症性细胞因子产生的影响.方法:应用氯磺酸修饰法对多糖进行化学修饰.用放射免疫分析法(RIA)及逆转录聚合酶链反应(RT-PCR)测FI_0-c和FI_0-c-S对人组织瘤细胞(THP-1)和人外周血单核细胞(PBMC)分泌各种与炎症有关的细胞因子,白介素-1(IL-1α)和肿瘤坏死因子α(TNFα)的影响及对mRNA表达的影响.结果:FI_0-c和FI_0-c-S(浓度分别为4,40,400 mg/L)显著提高 低剂量组LPS 10 mg/L协同PMA 200 nmol/L诱导的THP-1细胞产生TNFα的量,然而,这些多糖明显地抑制高剂量组LPS 100 mg/L协同PMA诱导的THP-1细胞产生TNFα.在无刺激的条件下FI_0-c能够诱导比较多量的IL-1α产生,但是FI_0-c或FI_0-c-S却都明显抑制高剂量或低剂量LPS和PMA诱导的THP-1细胞产生IL-1α.低浓度FI_0-c 4 mg/L显著抑制高剂量组LPS 100 mg/L协同PMA诱导的THP-1细胞产生IL-1或TNFα mRNA及蛋白质的量.结论:松杉灵芝菌丝体水溶性多糖在不同的刺激条件下具有双向免疫调节作用.化学修饰的多糖可改变原多糖对细胞因子产生的调节方向.     

中脑导水管周围灰质微量注射纳洛酮对鞘内吗啡诱导的免疫抑制的调节

AIM: To study the involvement of opioid receptor of periaqueductal gray (PAG) and hypothalamic-pituitary-adrenal (HPA) axis in the effect of intrathecal morphine on immune function. METHODS: Rat splenic natural killer (NK) cell activity was determined by a europium release assay; the concanavalin A-induced splenic IL-2 production, TNF-beta activity, and serum TNF-alpha level were determined by colorimetric thiazolyl blue tetrazolium bromide (MTT) and gentian violet assay, and serum corticotrophin (ACTH) level by radio-immunological method after intrathecal injection of morphine and PAG microinjection of naloxone. RESULTS: Intrathecal morphine inhibited splenic NK cell activity, IL-2 production, TNF-beta activity, and increased in serum ACTH level. Microinjection of naloxone 1 microgram into PAG partially antagonized the inhibition of NK cell activity and the elevation of serum ACTH level by morphine. CONCLUSION: The opioid receptor of PAG involved in the suppression of NK cell activity by intrathecal morphine, which was accompanied by an activation of HPA axis.     

Activation of natural killer T cells by NK-4, a criptocyanine dye

We previously reported that oral administration of NK-4, a criptocyanine dye, enhances interleukin (IL)-12-depend- ent interferon (IFN)-γ production by lipopolysaccharide (LPS)-stimulated mouse splenocytes. These findings raised a possibility that NK-4 potentiated IFN-γ production by T cells, natural killer (NK) cells or natural killer T (NKT) cells in response to IL-12 produced by macrophage and dendritic cells. To explore this possibility, we first analyzed percentages of T, NK or NKT cells in splenocytes of mice that were administered NK-4 orally for three days. The percentage of NKT cells in splenocytes from NK-4-treated mice was significantly (p<0.05) increased compared to vehicle-treated mice. When splenocytes were stimulated with α-galactosylceramide (α-GalCer), an NKT cell ligand, IFN-γ production by splenocytes from NK-4-treated mice tended to increase, while no difference in the IL-4 production and proliferation were observed between the vehicle- and NK-4-treated mice. When IFN-γ/IL-4 ratios were calculated in individual mice, the ratios were significantly (p<0.05) elevated in NK-4-treated mice. Furthermore, IL-12 production by α-GalCer-stimulated splenocytes from NK-4-treated mice was also significantly (p<0.05) increased. These results suggest that oral administration of NK-4 increases the population of type I NKT cells with potent IFN-γ-producing activities. Since IL-12 and IFN-γ have been shown to play important roles in anti-tumor immunity as well as in the defence against bacterial infection, our results further imply that NK-4 may provide a potential therapeutic tool in cancer immunotherapy.     

Oral administration of heat-killed Lactobacillus plantarum L-137 enhances protection against influenza virus infection by stimulation of type I interferon production in mice

We have previously reported that heat-killed Lactobacillus plantarum L-137 (HK-LP) stimulates macrophage/dendritic cells to produce T helper (Th) 1-related cytokines in vitro and in vivo in mice. We here examined the effect of oral administration of HK-LP on protection against influenza virus infection in mice. C57BL/6 mice were orally given HK-LP from day − 7 to 7 and intranasally infected with influenza virus A/FM/1/47 (H1N1, a mouse-adapted strain) at 100 pfu on day 0. The survival time was significantly prolonged in mice treated with HK-LP than that in mice treated with PBS as controls. The viral titers in the lung were significantly lower in mice treated with HK-LP than controls at the early stage after influenza virus infection. An appreciable level of interferon (IFN)-β was detected in the serum of mice treated with HK-LP, while no IFN-β was detected in controls after influenza infection. Our results suggest that HK-LP, a potent IFN-β inducer, is useful for prevention against influenza infection.     

鲨肝活性肽的免疫调节和抗细胞凋亡作用

目的　为了选择鲨肝活性肽 (SHAP)临床应用的适应症并了解其作用机制。方法　分离培养人外周血单核细胞 (PBMC) ,检测SHAP对其分泌IFN α和IFN γ的影响 ;用环磷酰胺 (Cy)建立免疫低下模型 ,检测SHAP对免疫低下小鼠血清溶血素的生成及血清IL 2水平的影响 ;用流式细胞仪分析SHAP对对乙酰氨基酚 (AAP)诱导小鼠肝损伤模型中细胞凋亡的影响 :用Fas单克隆抗体诱导小鼠暴发性肝炎和抑制肝癌细胞株SMMC772 1的增殖 ,用SHAP处理后 ,分析小鼠血清谷丙转氨酶 (GPT)的水平及SMMC772 1增殖的变化情况。结果　SHAP能够有效诱导PBMC分泌IFN α和IFN γ ,促进免疫低下小鼠血清溶血素的生成和提高血清IL 2的水平 ,显著降低AAP诱导的肝细胞凋亡。 5 0mg·L- 1的SHAP可消除 5mg·L- 1Fas单克隆抗体对SMMC772 1增殖的抑制作用。 3mg·kg- 1的SHAP能显著降低Fas单克隆抗体诱导的暴发性肝炎小鼠的血清GPT水平。结论　SHAP具有免疫调节作用和抗细胞凋亡的作用 ,可能是SHAP防治肝炎的作用机制之一。     

绞股蓝总皂甙对Lewis肺癌荷瘤小鼠肿瘤生长及免疫功能的影响

观察绞股蓝总皂甙对Ｌｅｗｉｓ肺癌荷瘤小鼠肿瘤生长,脾淋巴细胞数及ＮＫ活性的影响。方法整体动物的抑瘤试验,脾淋巴细胞计数及ＮＫ活性测定。结果：ＧＰｓ对荷瘤小鼠Ｌｅｗｉｓ肺癌细胞具有明显的抑制作用,在剂量１０、２０、４０ｍｇ／ｋｇ腹腔注射给药条件下,其抑瘤率分别为（３０．７±１．２）％,（５１．５±２．５）％（Ｐ〈０．０１）。同时,ＧＰｓｉｐ给药后荷瘤小鼠脾淋巴细胞总数明显增加,外周血淋巴细胞ＮＫ活性