In vitro augmentation of cytotoxicity by N-CWS in peritoneal polymorphonuclear leukocytes (PMNs) induced by PSK,OK-432 or N-CWS

Peritoneal polymorponuclear leukocytes (PMNs) were collected from the peritoneal cavity of C3H/He mice 6 hrs after intraperitoneal (i.p.) injection of 2.5 mg/head of PSK, 1 KE (100 µg)/head of OK-432 or 200 µg/head ofNocardia rubra cell wall skeleton (N-CWS). Withoutin vitro stimulation, these PMNs did not show cytotoxicity to syngeneic MM46 mammary carcinoma cells in⁵¹Cr release assay. Cytotoxicity of these PMNs was augmented by the addition of 25 µg/ml of N-CWS but not of PSK or OK-432 to cultures for the assay at the beginning of the culture. H₂O₂ production of PSK-induced PMNs was increased by thein vitro addition of 25 µg/ml of N-CWS but not of PSK. These results suggest that PSK as well as OK-432 and N-CWS can induce PMNs capable of responding further to N-CWS as the second stimulant.     

Morphological study of cytotoxicity produced by PSK-induced polymorphonuclear leukocytes (PMNs) andNocardia rubra cell wall skeleton

The morphologic changes in PMNs induced by an i.p. injection of PSK, a polysaccharide from the mycelia ofCoriolus versicolor, and tumor cells undergoing cell death, were evaluated by immunohistochemical staining and electron microscopy. Male C3H/He mice, 8–10-weeks old, received an i.p. injection of 125 mg/kg of PSK. Their PMNs were obtained 6 h after the PSK injection by peritoneal lavage. N-CWS (Nocardia rubra cell wall skeleton) was added at the start of the chromium release assay using the MM46 mammary carcinoma cell line, which is syngeneic to C3H/He mice, as target cells. During the cytotoxic assay, the cells were fixed at various time points. The MM46 cells expressed ICAM-1 while the PMNs expressed both ICAM-1 and LFA-1 as determined by immunohistochemical staining and immunoelectron microscopy using anti-ICAM-1 and anti-LFA-1 antibodies. PMNs with ruffle-like microvilli adhered to the MM46 tumor cells 30 min after the addition of N-CWS. Immunoelectron microscopic findings suggested that the adhesion molecules were LFA-1 on the PMNs and ICAM-1 on the MM46 tumor cells, but cell fusion between the PMNs and tumor cells was not observed. The MM46 tumor cells gradually lost their microvilli, which showed cell damage, and died 6–7 h after the addition of the N-CWS. This time course of tumor cell death is compatible with the results of the cytotoxic assay. Pretreatment of PMNs by anti-LFA-1 antibody suppressed % lysis of MM46 tumor cells from 90 % to 10 %(p<0.01). These data suggest that adhesion molecule on the surface of PMNs such as LFA-1 might play an important role on signal transduction of these PMNs cytotoxic function in this experimental system.     

Induction of tumoricidal macrophages from bone marrow cells of normal mice or mice bearing a colony-stimulating-factor-producing tumor

Summary Nonadherent cells of the bone marrow of C3H/HeN mice were incubated for 3 days with the culture supernatant of an L-929 cell line containing macrophage-colony-stimulating factor. Approximately, 70% of the cells became phagocytic, adherent to plastic dishes and positive for nonspecific esterase staining. The adherent cells exhibited a weak tumoricidal activity against MM48 syngeneic mammary carcinoma cells, and the cytotoxicity was strongly augmented by the addition of bacterial lipopolysaccharide to the cytotoxicity assay. The cytotoxicity induced by lipopolysaccharide was also shown to be mediated by Thy1.2^– and asialo-GM1⁺ cells, and was abrogated by the addition of carrageenan. Macrophage-colony-stimulating-factor-producing (D66) and nonproducing (A23) variants were separated from the MM48 tumor line in in vitro culture following limiting dilution. There was no difference between these two variants in either the in vitro growth rate or the susceptibility to macrophage-mediated cytotoxicity. C3H/HeN mice inoculated i.p. with D66 survived longer than did those inoculated i.p. with A23. C3H/HeN mice bearing D66 or A23 as an ascitic form were given i.p. injections of Nocardia rubra cell wall skeleton (N-CWS). N-CWS significantly prolonged the survival period of mice bearing D66, whereas it exhibited no apparent antitumor effect on mice bearing A23. The increase in the cell number of D66 in the peritoneal cavity was significantly retarded, compared with that of A23. In contrast, the number of peritoneal macrophages increased more in D66-bearing mice than in A23-bearing mice. The increase in the peritoneal macrophage number was further augmented by an i.p. injection of N-CWS. Peritoneal macrophages of D66-bearing mice exhibited apparent tumoricidal activity against MM48 tumor cells in the presence of lipopolysaccharide, and the cytotoxicity was significantly augmented by i.p. injection of N-CWS. On the other hand, the responsiveness of peritoneal macrophages to lipopolysaccharide was found to be poor in A23-bearing mice and the tumoricidal activity was only weakly augmented by N-CWS. These results strongly suggest that M-CSF plays an important role not only in the maturation of macrophage progenitors but also in the induction and the accumulation of activated macrophages. Abbreviations used: M-CSF, macrophage-colony-stimulating factor; NABMC, nonadherent bone marrow cells; CM, conditioned medium; NK, natural killer; N-CWS, Nocardia rubra cell-wall skeleton     

Effect ofNocardia rubra cell-wall skeleton on cancer prevention in humans

Summary The retired workers at the chemical weapons plant in Japan are regarded as a high-risk group for cancers. Under the Cancer Preventive Program,Nocardia rubra cell-wall skeleton (N-CWS) was administered to 80 workers directly involved in the production of sulfur mustard and 66 workers engaged in work related to sulfur mustard production. Untreated workers whose age, sex, duties and duration of work at this factory were individually matched to the N-CWS-treated workers were used as controls. During a 4.5-year observation period, development of cancers was found in 7 treated workers and 17 untreated controls. After elimination of the influence of the difference in smoking level, the incidence of subjects who developed cancers was compared statistically between the N-CWS-treated workers and the untreated controls and a significant suppression of development of cancers was noted in the N-CWS-treated workers. Thus, it was concluded that the administration of N-CWS could prevent cancer development in humans.     

In situ activation of tumoricidal properties in rat alveolar macrophages and rejection of experimental lung metastases by intravenous injections of Nocardia rubra cell wall skeleton

Summary The IV injection of squalene-treated cell wall skeleton of Nocardia rubra (N-CWS) into F344 rats rendered their alveolar macrophages (AM) tumoricidal. Maximum tumoricidal activity developed in AM by 24 h after the IV, but not IP or SC, injection of 300 g N-CWS. Tumoricidal activity of AM was maintained for 48–72 h after one IV injection of N-CWS. Experimental lung metastases were produced in female F344 rats by the IV injection of viable syngeneic mammary adenocarcinoma cells. Treatments twice weekly with Hank's balanced salt solution, N-CWS placebo or N-CWS began 3, 7, or 10 days later and were continued for 3 or 4 weeks for a total of six or eight treatments. Practically all the rats (>90%) treated with N-CWS beginning on either day 3 or day 7 after tumor cell challenge survived until day 210, when the experiment was terminated. In contrast, 90% of the rats treated with balanced salt solution or N-CWS placebo died by day 70 of the experiment. Therapy with N-CWS preparation was not successful when the first injection was administered 10 days after tumor cell challenge, suggesting that this therapeutic regimen is effective only against minimal tumor burden. We conclude that in this animal tumor model, the IV injection of N-CWS preparations can render AM tumoricidal and aid in the eradication of pulmonary micrometastases.     

Antitumor activity of Nocardia cell wall skeleton preparations in transplantable tumors in syngeneic mice and patients with malignant pleurisy

Summary The antitumor activity of the cell wall skeleton preparations of four species of Nocardia, N. brasiliensis strain 146, N. coeliaca strain 122, N. polychromogenes strain 6, and N. rubra, which showed potent adjuvant activity on the induction of cell-mediated cytotoxicity in allogeneic mice, was examined with the aid of EL-4 leukemia, melanoma B16, and MH-134 hepatoma in syngeneic mice. Preliminary clinical trials were performed and the results suggest that the cell wall skeleton of N. rubra, upon intrapleural injection, may be useful as an immunotherapeutic agent for patients with malignant pleurisy. The chemical properties of these cell wall skeleton preparations are described.     

Effect of Nocardia rubra cell wall skeleton on augmentation of cytotoxicity function in human pleural macrophages

Summary The ability of Nocardia rubra cell wall skeleton (N-CWS) to augment macrophage cytotoxicity function was examined using human pleural macrophages prepared from 32 malignant pleural effusions and 53 pleural washings. The cytostatic activity of pleural macrophages for human lung cancer cells (PC-9) was augmented following incubation of pleural mononuclear cells with 10 g/ml N-CWS for 24 h. Macrophage activity was increased by direct interaction of macrophages with N-CWS or by incubation of macrophages with supernatant culture fluids from pleural lymphocytes with N-CWS. The cytotoxic potential of the pleural macrophages obtained from patients treated with 500 g of N-CWS intrapleurally was also increased. The heat and acid stability studies revealed that the culture fluids from pleural lymphocytes treated with N-CWS contained macrophage activation factor in addition to interferon-. These results suggest that direct and indirect macrophage activation is part of the mechanism in which N-CWS has a clinical effect on malignant pleural effusions.     

Augmentative effect of Nocardia rubra cell-wall skeleton (N-CWS) on lymphokine-activated killer (LAK) cell induction

Summary The present study elucidated that N-CWS augments the cytolytic activity against 3LL tumor cells of LAK cells from N-CWS-immunized mice administered i.p. with rIL-2. This augmentative effect of N-CWS was not seen when the LAK cells were prepared from normal mice. The cytolytic activity was predominantly expressed in the NAPC prepared from the site of injection of rIL-2, and repeated administrations of rIL-2 were required to induce and maintain this potent cytolytic activity in vivo. Serological analysis revealed that the LAK cells were positive for Thy 1.2 and asialo GM1 antigens and that they were not classical CTL or NK cells. The administration of rIL-2 statistically prolonged the MST of mice bearing LAK-sensitive 3LL cells but not the MST of mice bearing LAK-resistant EL-4 leukemia. Furthermore, combination therapy with N-CWS and rIL-2 prolonged the MST of the mice more than the therapy with rIL-2 alone. These results suggest that LAK cells potentiated with N-CWS would be useful for immunotherapy of malignant neoplasms. Abbreviations used: N-CWS, Nocardia rubra cell-wall skeleton; rIL-2, recombinant interleukin 2; LAK, lymphokine-activated killer; RPMI 1640, Roswell Park Memorial Institute 1640; FCS, fetal calf serum; TCM, tumor culture medium; PC, peritoneal cells; NAPC, nonadherent PC; APC adherent PC; MST, mean survival time; NK, natural killer; E:T ratios, effector to target ratios; Poly I:C, polyinosinic-polycytidylic acid; CTL, cytotoxic T lymphocytes; RLNC, regional lymphnode cells     

N-CWS灌胃对小鼠移植性肿瘤及免疫功能的影响

为了考察红色诺卡氏菌细胞壁骨架(Nocardia rubra cell wall skeleton,N-CWS)灌胃给药对小鼠的体内抑瘤效应及其免疫调节作用,采用小鼠肉瘤S_(180)的移植性肿瘤模型,检测N-CWS灌胃给药的抑瘤活性;同时观察N-CWS体内细胞毒作用和对正常小鼠的毒性、免疫器官重量、巨噬细胞(MΦ)吞噬功能及脾淋巴细胞转化的影响.结果显示,小鼠灌服N-CWS的LD_(50)＞1.2 g/kg;N-CWS 200、400、800 mg/kg剂量灌胃小鼠对S_(180)有明显的抑制作用,其抑瘤率分别为63.33%、71.11%、64.88%;与对照组比较,N-CWS可增加免疫器官重量和提升外周血白细胞数量、能明显提高小鼠MΦ的吞噬活性及显著提高淋巴细胞转化率(P＜0.05),同时N-CWS激活了的MΦ对肿瘤细胞的细胞毒效应亦明显增强(P＜0.05).因此N-CWS适用于口服给药,对小鼠移植性肿瘤有明显的抑制作用,其抗肿瘤作用可能与增强机体免疫功能有关.     

Analysis of therapeutic effect in experimental chemoimmunotherapy for rat ascites tumor

Summary The lyophilized, squalene-treated Nocardia rubra cell wall skeleton (N-CWS) was confirmed to produce tumoricidal peritoneal macrophages resulting in inhibition of tumor growth when injected locally into the syngeneic ascites fibrosarcoma, AMC 60 in ACI/N rats. Furthermore, N-CWS was found to augment therapeutic effect when administered repeatedly after a single local injection of mitomycin-C (MMC). To analyze the effects, various in vitro cytolysis assays were performed using N-CWS-activated peritoneal macrophages. When tumor target cells were exposed in vitro to MMC, the resulting cytolysis in the presence of N-CWS-activated macrophages was similar to cytolysis of intact target cells. On the other hand, when N-CWS-activated macrophages were exposed to MMC, the tumoricidal activity was lost significantly, depending on exposure to MMC. When tumor target cells and N-CWS-activated macrophages were simultaneously exposed to MMC, tumor-cell cytolysis was strikingly depressed. In the final experiment, combined injection of MMC and N-CWS into the ascites tumor resulted in remarkable increases not only in peritoneal exudate cell number, but also in in vitro tumoricidal activity of peritoneal macrophages as compared to those induced by either agent alone. In addition, the production of tumoricidal macrophages by IP injection of MMC alone was also noticeable, as described previously. These results possibly indicate the involvement of macrophage activation in induction of therapeutic effect in chemoimmunotherapy.This work was supported in part by grants from the Ministry of Health and Welfare, and the Ministry of Education, Science and Culture     

Effects of periodic administration ofNocardia rubra cell-wall skeleton on immunoglobulin production and B-cell-stimulatory factor activity in vitro in workers at a poison gas factory

Summary The former workers at the Okunojima poison gas factory (poison gas workers) are a high-risk group for malignant neoplasms and show abnormalities in cellular immunity. At the same time, poison gas workers often have chronic respiratory diseases, such as chronic bronchitis, and are highly susceptible to respiratory infections. To explore the possibility of immunological cancer prevention, we have periodically administered 200 µgNocardia rubra cell-wall skeleton (N-CWS) to poison gas workers once every 3 months since December 1978. During this period, we noted a significantly lower incidence of influenza among poison gas workers receiving N-CWS than in those not receiving the drug during the influenza epidemic. This finding suggested that the administration of N-CWS enhanced the resistance of these workers to infections. Therefore, periodical administration of N-CWS to poison gas workers was considered to enhance the reduced T-cell function of normalizing antibody production by stimulating the production of B-cell-stimulatory factor (BSF). In the present study, to clarify the mechanism of immunosuppression in the poison gas workers and to examine the effects of continual administration of N-CWS on this condition, we compared the immunoglobulin production and the proliferative and differentiative activities of B-cell-stimulatory factor (BSF) of peripheral blood mononuclear cells (PBMC), in poison gas workers treated or not treated with N-CWS. Comparisons were also made with age-matched healthy controls. In the untreated poison gas workers, immunoglobulin and BSF production of PBMC were reduced as compared with the control group. On the other hand, in the poison gas workers receiving N-CWS, immunoglobulin and BSF production of PBMC were restored nearly to the control level. These results show that in vitro antibody production in the poison gas workers was reduced and that a reduction in BSF production of T cells was one of its causes.     

Localization of human sarcoma with radiolabeled monoclonal antibody

Summary We performed a randomized controlled study of postoperative adjuvant immunochemotherapy with Nocardia rubra cell wall skeleton (N-CWS) and Tegafur for gastric carcinoma between September 1979 and March 1983. A total of 309 patients were entered into this trial. Of the 309 patients, there were 98 evaluable patients in the chemotherapy group and 115 evaluable patients in the immunochemotherapy group. In both groups, Tegafur was given as chemotherapy at a daily dose of 400 to 800 mg, starting at 24–29 days after gastrectomy. In the immunochemotherapy group, 400 g of N-CWS was injected i. d. within the 2nd postoperative week. It was given weekly during the first month and subsequently monthly for as long as practicable. The patients were surveyed for length of survival in March 1985. The postoperative survival rate was analyzed for all cases, and for patients with various histopathological stages of carcinoma for comparison between the two treatment groups. No statistical difference was detected between the two groups in terms of age, sex, surgical curabilities, or stage of carcinoma. The overall survival rate for all patients was significantly higher in the immunochemotherapy group than in the chemotherapy group (p<0.05). With stage III plus IV disease, 53 patients from the chemotherapy group and 61 patients from the immunochemotherapy group were included for the analysis. As a consequence, a highly significant survival rate was observed in patients with stage III plus IV carcinoma in the immunochemotherapy group (p<0.005) as compared to the chemotherapy group. The overall 5-year (1800 days) survival rate after surgical treatment was 60.2% for the chemotherapy group and 73.2% for the immunochemotherapy group. In patients with stage III plus IV disease, the 5-year survival rates of the two treatment groups were 28.8% and 52.4%, respectively. Accordingly, the 50% survival period of patients with stage III plus IV cancer was 1800 days or more in the immunochemotherapy group, whereas it was only 722 days in the chemotherapy group. These results emphasize the effectiveness of N-CWS as an adjuvant immunotherapeutic agent in postoperative gastric cancer patients.The main side effects of N-CWS were skin lesions in the injected sites and fever, but these were temporary and not serious.     

Human blood monocyte activation byNocardia rubra cell wall skeleton for productions of interleukin 1 and tumor necrosis factor-α

Human blood monocytes were obtained from peripheral blood of healthy donors by counter-flow centrifugal elutriation. Functional integrity of monocytes for production of interleukin 1 (IL-1) and tumor necrosis factor (TNF-) in response toNocardia rubra cell wall skeleton (N-CWS) was examined by bioassay and enzyme immunoassay. Monocytes treated with N-CWS at more than 0.5 g/ml produced IL-1 and TNF- extracellularly. Extracellular TNF activity appeared within 4 h, and maximally, 16 h after N-CWS stimulation, whereas longer time was needed for IL-1 activity to appear, the peak production being at 24 h. The neutralizing experiment also showed that anti TNF- antibody did not affect IL-1 production by the monocytes treated with N-CWS, suggesting independen cy of IL-1 production of TNF-.These results suggest that the therapeutic antitumor effect of N-CWS is due, in part at least, to the augmented production of these monokines.     

Direct activation of tumoricidal properties in rat alveolar macrophages by Nocardia rubra cell wall skeleton

Summary Alveolar macrophages (AM) lavaged from lungs of normal F344 rats were rendered tumoricidal following their direct interaction with squalene-treated Nocardia rubra cell wall skeleton (N-CWS) present in the culture medium. Maximum tumoricidal activity was obtained by incubating AM with 1 g N-CWS/ml for a 24-h period. These AM were cytolytic to syngeneic, allogeneic, and xenogeneic tumorigenic cells. Tumoricidal activity following interaction with N-CWS decreased gradually and was lost completely by 96 h. A second in vitro exposure to N-CWS reactivated AM to their full tumoricidal potential. The present studies suggest that N-CWS can directly activate AM to render them tumoricidal.     

Augmentative effect ofNocardia rubra cell-wall skeleton on the induction of human lymphokine-activated killer (LAK) cells by the production of LAK cell helper factor(s)

Summary Nocardia rubra cell wall skeleton (N-CWS) was found to synergistically augment lymphokine-activated killer (LAK) cell generation from human peripheral blood mononuclear cells (PBMC) in the presence of a suboptimal dose of recombinant interleukin-2 (rIL-2). N-CWS increased the number of PBMC expressing IL-2 receptor on their surfaces, and the presence of N-CWS at the early stage of the culture period was essential for the exertion of its augmentative activity on the LAK induction. The predominant phenotype of LAK precursor cells responding to N-CWS and rIL-2 was CD3^– CD16⁺. Culture supernatant from N-CWS-stimulated PBMC was found to act as a substitute for N-CWS in the induction of LAK generation in the presence of rIL-2, suggesting that these cells produced a factor capable of augmenting LAK cell induction (LAK helper factor, LHF). LHF was found to have a molecular mass of 29 kDa by gel filtration, and could also function as a killer helper factor to augment allo-antigen-specific cytotoxic T lymphocyte generation from human peripheral blood T cells as well as murine thymocytes. LHF showed no species specificity, indicating that it is different from IL-4. The enhancing activity of LHF was not neutralized with anti-TNF, anti-IL-1, or anti-IL-1 antibodies. Furthermore, no tumor necrosis factor- (TNF), TNF, IL-1, , IL-2, IL-5, IL-6 or interferon activity was detected in semi-purified LHF during enzyme-linked immunosorbant assay and biological assays. The present findings indicate that LHF produced from N-CWS-stimulated PBMC is a molecule distinct from TNF, TNF, interferon, IL-1, -2, -4, -5, and -6, and suggest that LHF might be a novel lymphokine involved in LAK generation.This work was supported by a Grant-in-aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan     

The biological macromolecule Nocardia rubra cell-wall skeleton as an avenue for cell-based immunotherapy

Promoting the antitumor effects of cell-based immunotherapy for clinical application remains a difficult challenge. Nocardia rubra cell-wall skeleton (N-CWS) is an immunotherapeutic agent for cancers that have been proven to possess the ability to activate immune response without showing toxicity. However, its effects on immune cells that are derived from tumor patients and cultured in vitro remain unclear. As expected, N-CWS can enhance the proliferation and viability of cytokine-induced killer (CIK) cells, dendritic cells (DCs), and natural killer (NK) cells. The maturation of DCs and specific cytotoxicity against NK cells and CIK cells were consistently promoted. The TUNEL-staining and the Annexin V/propidium iodide assay revealed that after treatment with N-CWS, the stimulated CIK/NK cells could induce DNA breaks in tumor cells. Furthermore, quantitative real-time polymerase chain reaction and western blot analysis showed upregulation of proapoptotic biomarkers (caspase-3 and caspase-9) and a downregulation of the antiapoptotic biomarker Bcl-2 in the tumor cells of the N-CWS-treated group, indicating that N-CWS could induce hepatocellular carcinoma cell apoptosis via CIK/NK cells. Finally, CIK/NK cells could notably suppress the invasion and migration of tumor cells in the presence of N-CWS. Our study provides evidence that N-CWS could significantly increase the growth of CIK cells, DCs, and NK cells, particularly due to its robust antitumor activities by inducing apoptosis, and attenuate the invasion and migration of tumor cells.     

Effect of Nocardia rubra cell-wall skeleton treatment on tumor formation in two-stage chemical carcinogenesis of mouse skin

Summary In two-stage chemical carcinogenesis of mouse skin, Nocardia rubra cell-wall skeleton (N-CWS), a potent immunopotentiator, was injected SC at various times. The dorsal skin of C57BL/6 male mice (about 10 cm²) was painted with 20 g 7,12-dimethylbenz(a)anthracene (DMBA) in 0.1 ml acetone when the animals were 11 weeks old (initiation). Seven weeks later, they were painted with 2.5 g 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in 0.1 ml acetone twice weekly for 30 weeks (promotion). The timing of N-CWS treatment was important. N-CWS treatment before initiation reduced the incidence of skin tumor and the mean number of skin tumors per mouse most effectively. It is speculated that the antitumor activity of N-CWS may be composed of at least two mechanisms, being achieved through the enhancement of immunological surveillance and through changes in the metabolism of chemical carcinogens.     

红色诺卡氏菌Nr-8206株培养形态与其有效物质及杂质关系分析

筛选红色诺卡氏菌（Nocardia rubra）Nr-8206株适宜投产的最佳培养形态。将红色诺卡氏菌Nr-8206株复壮,通过涂布、形态学考察筛选典型菌落形态。通过发酵技术获得各种菌落形态菌株的生物量,进一步通过细胞破碎、化学提纯等方法获得细胞壁多糖产物,紫外可见光分光光度法进行有效物质含量测定及杂质的检测。结果表明,红色诺卡氏菌Nr-8206株的最佳菌落形态为菌落直径1.68 mm、橘色、有突起、有褶皱、菌落边缘丝状,编号RY2。进行菌株RY2发酵,其菌体量最多,经破碎、提纯后,其有效物质糖含量及胞壁酸含量均高于其他形态的菌落,并高于出发菌株Nr-8206,且其杂质蛋白质残余量更低,杂质更容易去除。该研究可供生产企业以该菌株作为工作菌株时提供形态选择参考。     

The effect of combination therapy of radiation and Z-100, an arabinomannan on tumor growth in mice

The effect of radiation combined with intraperitoneal administration of Z-100, an immunomodulatory arabinomannan extracted fromMycobacterium tuberculosis, was studied using Meth A fibrosarcoma in BALB/c mice and metastasis of Lewis lung carcinoma, 3LL, in C57BL/6 mice.In mice bearing Meth A fibrosarcoma, a moderate degree of growth inhibition was observed in the group of single therapy with Z-100 or radiation (10 Gy). When radiation was combined with Z-100, the tumor growth was significantly inhibited. In mice bearing 3LL, slight inhibition of pulmonary metastasis was observed in the group of single therapy, while significant degrees of inhibition of primary tumor growth and pulmonary metastasis were observed in the combination group. This suggests the usefulness of combined use of Z-100 in radiation therapy.Abbreviations 3LL Lewis lung carcinoma - IL-3 Interleukin-3 - BRM(s) Biological response modifier(s) - N-CWS Nocardia-Cell wall skeleton - CSF Colony stimulating factor     

红色诺卡菌固体培养基配方的优化

应用响应面优化设计法优化固体培养基配方,增大红色诺卡菌的固体培养细胞生物量。首先用Plackett-Burman法从现有培养基组分中找到影响红色诺卡菌细胞生物量的关键因素,再通过最陡爬坡法确定细胞生物量最大的配方,用作中心组合设计(Central Composite Design, CCD)实验的基础起始值,拟合数学模型方程,最后找到最优组分的组合。优化的配方转移至企业实施放大实验,对结果进行验证和比较。试验结果表明,培养基各组分中影响红色诺卡菌细胞生物量的关键因素为蛋白胨、NaCl、牛肉膏;最优固体培养基配方:蛋白胨42 g/L、牛肉膏8 g/L、NaCl 1.2 g/L、甘油10 mL/L、Na_2HPO_4·12H_2O 0.3 g/L、琼脂20 g/L。在细胞生物量方面最优固体培养基配方比原配方高104%。响应面优化设计可用于提高红色诺卡菌细胞生物量固体培养基的优化,也为红色诺卡菌培养条件、液体发酵的优化研究提供参考。