Nocardia Rubra Cell Wall Skeleton Research Articles

Antitumor activity of squalene-treated cell-wall skeleton of Nocardia rubra in mice.

Antitumor activity of squalene-treated cell-wall skeleton of Nocardia rubra in mice.

Cytostatic activity of peripheral blood monocytes against bronchogenic carcinoma cells in patients with lung cancer.

Cytostatic acitivity of peripheral blood monocytes against cultured cell lines of bronchogenic carcinoma was examined in patients with lung cancer. Cytostatic activity in lung cancer patients with neither augmented nor suppressed as compared with that of controls such as normal healthy persons, patients with malignancies other than lung cancer, and patients with benign respiratory diseases. There was no correlation between the cytostatic activity of monocytes and the advance of clinical stages of the disease. Conventional modalities of anticancer treatments such as surgery, radiotherapy, chemotherapy, and their combination therapy had no effect on cytostatic activity of peripheral blood monocytes. However, adequate immunotherapy with nocardia rubra cell-wall skeleton augmented the cytostatic activity of peripheral blood monocytes, although adequate immunotherapy with Mycobacterium bovis BCG cell-wall skeleton had no effect.

Observation of the curative effect between Nocardia rubra cell wall skeleton-injection combined with the intrapleural chemotherapy and following the intrapleural chemotherapy

To observe and compare the curative effect between the intrapleural-chemotherapy combined Nocardia rubra cell wall skeleton-injection and the Nocardia rubra cell wall skeleton-injection after control of the malignant pleural effusion by chemotherapy. Every time after elimination of pleural effusion, we injected DDP and Nocardia rubra cell wall skeleton at the same time into pleural cavity; or after we emplaced an improved central vena catheter into pleural cavity then took out closed-drainage or eliminated the pleural effusion every day, then injected 5-FU 0.5 qd intrapleural-space. After the malignant pleural effusion had being controlled, 800 μg Nocardia rubra cell wall skeleton was injected intrapleural-space. (We call it as “improved following-therapy”). Using the improved following-therapy, the control rate of malignant pleural effusion increased: CR + PR, 79.07% > 65.79% (P < 0.05), and the incidence rate of the encapsulated pleural effusion that would impact on patient’s respiratory faction decreased. Conclusion: Giving chemotherapy after closed-drainage or immediately after taking out pleural effusion everyday, then giving biotherapy after pleural effusion had been controlled, the curative effect would be enhanced and the side-reaction would be reduced.

Immune therapy for lung cancer: are we getting closer?

It is well known that lung cancer patients have abnormalities in their immune system and that the extent of these abnormalities may have prognostic relevance (1, 2). The promise of immune therapy for lung cancer has been suggested for many years, but real improvements in survival have remained elusive. An early report suggested that resected lung cancer patients who developed empyema had a superior outcome (3), perhaps on the basis of immune stimulation. This led to a number of trials of nonspecific immune stimulants, such as Calmette-Guerin bacillus (BCG) and nocardia rubra cell wall skeleton (4, 5). Although several small, uncontrolled trials showed promise to these approaches, larger randomized trials failed to show any survival benefit (6–9). Prior studies also evaluated the use of thymosin factor V, interferon, and other nonspecific immune stimulants. Once again, promising early study results could not be confirmed for thymosin factor V (10, 11), interferon (12–15), IL2 with or without adopted cells (16), or other means. However, immune approaches remain appealing because initial therapy with surgery, chemotherapy, and radiotherapy alone and in combination produce high response rates in all histologic types of lung cancer, but relapse is nearly inevitable. Over the past decade, a number of tumor antigens, which are expressed on the majority of lung cancer cells, were described. Some of these are expressed in nearly all small-cell lung cancers (SCLC) and are overexpressed on these tumor cells in comparison with normal lung tissues. These tumor antigens provide a new specific means for development of successful vaccine approaches. Tumor antigens that are being explored for potential vaccines include mutant oncogene proteins such as p53, ras, or erb B2, and also cell surface proteins such as CEA, MUC1, GD2, or HuD. At this point, the ideal antigen for immune therapy is unknown. There are also many ways in which these antigens may be developed as vaccines. One approach is to isolate the tumor protein, such as mutant p53, and administer the protein alone or with nonspecific immune stimulants. A group from the National Cancer Institute (NCI) evaluated the clinical relevance of human antitumor immune responses to tumor antigens, including p53 and HuD (17). They found serum antibodies against autologous tumor proteins in 50% of SCLC and 75% of non–small-cell lung cancer (NSCLC) patients, including two patients who had anti-p53 antibodies and two patients who had anti-HuD antibodies. The presence of antibodies was found to correlate with improved survival and limited stage. In subsequent studies, the NCI showed that missense p53 mutations give rise to new tumor-specific peptide sequences, which are efficiently processed and presented by human lung cancer cells. They then showed that these mutant p53 peptides were effectively targeted by cytotoxic T cells specific for the endogenous mutant epitopes (18). They are currently conducting a clinical trial using the patient-specific p53 peptides as the immunogen. However, this approach is very labor intensive because the mutant p53 protein must be identified and isolated from each patient and then mass produced. Another approach to using oncogene mutations evaluated activating mutations of the p21 ras proto-oncogene because these activating mutations are limited in number and occur in many common malignancies and in otherwise completely conserved regions. Triozzi and colleagues (19) engineered a chimeric ras immunogen incorporating a promiscuous T-cell epitope to enhance the immunogenicity of an oligopeptide corresponding to a weakly immunogenic substitution, and they are evaluating this immunogene as a vaccine. Many lung cancers overexpress erb B1 and its protein, epidermal growth factor receptor (EGFR), and/or erb B2 and its protein, Her2/neu. These oncoproteins have also been used in vaccine development. Gonzalez and associates (20) linked the human EGF protein to either tetanus toxoid or Neisseria meningitidis recombinant protein and immunized patients with intradermal injection using aluminum hydroxyde as an adjuvant. Anti-EGF antibodies developed in 60% of the immunized patients without major toxicities. Finally, Amici and coworkers developed a plasmid DNA encoding the rat neu NT oncogene (21). In a transgenic mouse model, intramuscular injections of neu NT plasmids drastically reduced the outgrowth of new and mammory neoplasms. Common tumor antigens such as CEA, MUC1, GD2, and HuD have been used as the basis for vaccine strategies. Human MUC1 is overexpressed in many human car( Received in original form May 6, 1999 )

Local expression of cytokines in rat bladder carcinoma tissue after intravesical treatment with Nocardia rubra cell wall skeleton and bacille-Calmette-Guerin.

This study aimed to investigate local immunotherapy with Nocardia rubra cell wall skeleton (N-CWS) and bacille-Calmette-Guerin (BCG) in chemically induced rat bladder carcinoma. After being fed with 0.025% N-butyl-N-(4-hydroxybutyl) nitrosamine for 26 weeks, female Wistar rats were treated once a week by intravesical instillation of 100 microg N-CWS or 5 x 10(6) Colony-Forming Units (CFU) BCG. Tissue specimens were obtained 4 h after the ninth instillation and analyzed by reverse transcription polymerase chain reaction (RT-PCR) for mRNA expression of rat cytokines. The analysis showed high expression of interleukin (IL)-1 alpha, tumor necrosis factor (TNF)-alpha, IL-2, and interferon (IFN)-tau in BCG (7/7, 7/7, 7/7, 5/7) and N-CWS (9/9, 9/9, 8/9, 8/9) treated tumors in comparison to low expression in controls (3/9, 0/9, 3/9, 3/9). Interestingly, intravesical instillation of N-CWS tended to be less effective at preventing local invasion of the tumors. It is speculated that this difference may result from a more strongly induced expression of T-helper cell-derived lymphokines (IL-2, IFN-tau) by BCG.

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

The morphologic changes in PMNs induced by an i.p. injection of PSK, a polysaccharide from the mycelia of Coriolus 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 1% 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.

Postoperative immunostimulation after complete resection improves survival of patients with stage I nonsmall cell lung carcinoma

approximately 40% of primary lung carcinoma patients who die within 1 month after a complete resection have residual tumor in regional or distant organs, emphasizing the importance of postoperative adjuvant therapy. In this study, the effectiveness of transfer factor (TF) and nocardia rubra-cell wall skeleton (N-CWS) as adjuvant therapy for patients with primary, completely resected nonsmall cell carcinoma of the lung was evaluated in a randomized controlled trial A total of 82 patients with Stage I disease who had a complete resection were allocated randomly into 2 groups: TF + N-CWS (n = 41) or control (surgery only) (n = 41). The distributions of age, sex, histology, differentiation, T classification, tumor size, visceral pleural invasion, and the site of origin, were similar in the two groups. The 5- and 10-year disease specific survival rates in the TF + N-CWS group were 85% and 85%, respectively, and those in the control group were 72% and 64%, respectively. There was a statistically significant difference between the two groups (P = 0.041). When the survival was analyzed according to clinical characteristics, significant differences were observed in patients with no visceral pleural invasion or with T1 disease. The frequency of distant metastasis was significantly less in the TF + N-CWS group than in the control group. These results indicate that TF + N-CWS is beneficial as adjuvant therapy after surgical treatment of Stage I nonsmall cell carcinoma of the lung.

Former poison gas workers and cancer: incidence and inhibition of tumor formation by treatment with biological response modifier N-CWS.

Mustard gas is known to have mutagenic and carcinogenic effects on animal and human cells. In this report, 1,632 male Japanese who worked in poison gas factories at some time between the years 1927 and 1945 were studied to determine comparative risk for development of cancer, the reference population being data on Japanese males overall. The standardized mortality ratio (SMR) for lung cancer in workers directly and indirectly involved in the production of mustard gas was significantly elevated. In addition, SMR for lung cancer in worker who had worked for more than 5 years was also significantly elevated. Thus, poison gas workers who had engaged in the production of mustard gas or related work for more than 5 years are a high-risk group for lung cancer. Under the cancer preventive program, Nocardia rubra cell-wall skeleton (N-CWS) was administered to 146 former poison gas workers. 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 smoking level, a significant suppression of development of cancers was noted in the N-CWS-treated workers as compared to the untreated controls. Although the molecular mechanisms of carcinogenesis in former poison gas workers remains unclear, our study proposes the possible effect of biological response modifiers in the prevention of cancer development in high-risk human subjects.

Adoptive immunotherapy of poorly immunogenic tumors with in vitro sensitized cells generated by intratumoral administration of biological response modifiers

We investigated the efficacy of intratumoral administration of biological response modifiers (BRM) in induction of in vitro sensitized (IVS) cells for adoptive immunotherapy of the poorly immunogenic MCA 102 sarcoma and B16-BL6 (BL6) melanoma. We used the bacterial immunoadjuvant Nocardia rubra cell wall skeleton (N-CWS), and a streptococcal preparation, OK-432, for MCA 102 and BL6, respectively. After C57BL/6 (B6) mice were inoculated subcutaneously (s.c.) with viable MCA 102 or BL6 tumor cells in the foot-pad, mice were injected intratumorally (i.t.) with N-CWS ranging from 10 to 400 gg or OK-432 ranging from 1 to 100 μg. Draining popliteal lymph nodes (LN) were harvested 7 days after i.t. administration of BRM, and LN cells were cultured with irradiated tumor cells in the presence of IL-2 for 11 days. These IVS cells (7.5 × 10 6 or 2 × 10 6) were transferred intravenously (i.v.) to B6 mice with 4 day pulmonary metastases established by i.v. injection of viable MCA 102 cells (1 × 10 6) or viable BL6 cells (3 × 10 5). The mice were also received intraperitoneally 4 × 10 4 IU/day of IL-2 for 4 days after adoptive transfer. The transfer of IVS cells from mice immunized by i.t. injection of 100 μg of N-CWS 1 week after inoculation of tumor cells significantly reduced MCA 102 pulmonary metastases, compared with control IVS cells without administration of N-CWS. Moreover, the transfer of IVS cells from mice immunized by i.t. injection of 10 gg of OK-432 3 days after inoculation of tumor cells significantly reduced BL6 pulmonary metastases compared with control IVS cells without administration of OK-432. The administration of N-CWS resulted in no enhancement of in vitro cytotoxicity. Although the administration of 10 gg of OK-432 augmented in vitro cytotoxicity of 1VS cells against BL6, cytotoxic activity was lower than that of IVS cells immunized with N-CWS. The major phenotype was CD8 + cells in IVS cells immunized with N-CWS or OK-432. These results suggest that i.t. administration of N-CWS and OK-432 facilitates the production of sensitized T-cells, and this administration route of BRM may be useful in the adoptive immunotherapy of human cancer.

Recombinant human granulocyte colony-stimulating factor augments cytotoxicity of OK-432-induced polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMNs) recovered from the peritoneal cavity of mice treated with the streptococcal preparation OK-432, exhibited strong cytotoxicity after the in vitro addition of Nocardia rubra cell wall skeleton (N-CWS). In this study, we investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) could augment the cytotoxicity of OK-432-induced PMNs after the addition of N-CWS in vitro. PMNs recovered from the peritoneal cavity of 8- to 10-week-old, male C3H/He mice induced by intraperitoneal (i.p.) injection of 50 KE/kg (1 KE = 0.1 mg) of OK-432 were used in a 51Cr release assay against MM46 mammary carcinoma cells. While addition of rhG-CSF in vitro did not augment the cytotoxicity of OK-432-induced PMNs, marked augmentation of the cytotoxicity of OK-432-induced PMNs was observed following a single subcutaneous (s.c.) or i.p. injection of 125 μg/kg of rhG-CSF. The effect of in vivo administered rhG-CSF was dependent on the timing of the injection with respect to OK-432 administration and differed from s.c. or i.p. injections. Interestingly, the cytotoxicity of OK-432-induced PMNs was rather weak following consecutive s.c. or i.p. administration of rhG-CSF for 7 – 14 days. H 2O 2 is likely involved in mediating the cytotoxicity of OK-432-induced PMNs since activity was significantly reduced by the in vitro addition of low concentration of catalase Generation of H 2O 2 by the PMNs correlated with cytotoxicity. These results suggest that in vivo administration of rhG-CSF augments the cytotoxicity of OK-432-induced PMNs in a time dependent fashion and that H 2O 2 plays an important role in mediating their cytotoxicity.

Clinical usefulness of continuous administration of Nocardia rubra cell wall skeleton (N-CWS) in diffuse panbronchiolitis (DPB).

Eight patients with diffuse panbronchiolitis (DPB) who had repeated intractable airway infections were continuously treated with Nocardia rubra cell wall skeleton (N-CWS), a biological response modifier. As a result, subjective symptoms were reduced in 6 patients. Antibiotics therapy could be discontinued completely in two patients and the dose of antibiotics could be reduced considerably in two other patients. No adverse reactions in relation to N-CWS were observed. These results suggest that N-CWS is effective in treating erythromycin-resistant DPB.

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on cytotoxicity of PSK-induced peritoneal polymorphonuclear leukocytes (PMNs).

We investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced the cytotoxicity of PSK-induced polymorphonuclear leukocytes (PMNs) in the peritoneal cavity. Male C3H/He mice, 8- to 10-week-old, received single subcutaneous (s.c.) or intraperitoneal (i.p.) injection of 2.5 micrograms/animal of rhG-CSF at different time points before or after an i.p. administration of PSK. In other experiments, mice were s.c. or i.p. treated with the same dosage of rhG-CSF every day for 7 or 14 consecutive days and i.p. injected with 2.5 mg/animal of PSK on the last day. Peritoneal PMNs were harvested 6 hrs after the administration of PSK and purified to more than 95% by Ficoll-Paque for in vitro cytotoxic assay. In vitro cytotoxic assays with 51Cr labeled MM46 mammary carcinoma cells were added with 5-20 micrograms/ml of Nocardia rubra cell wall skeleton (N-CWS) at the beginning of the assay to augment the cytotoxic activity of PMNs. In vitro addition of rhG-CSF to the assay did not enhance the cytotoxicity of PSK-induced PMNs. However, the cytotoxicity was significantly increased when rhG-CSF was s.c. administered 12 hrs before a PSK injection or 2 or 5 hrs after that. On the other hand, the cytotoxicity was rather weak when mice s.c. or i.p. received consecutive injections of rhG-CSF. This cytotoxicity may be mediated by H2O2, since H2O2 production of PMNs during the cytotoxic assay appears to correlate with the levels of cytotoxicity under suppressed H2O2 generation by catalase or enhanced generation by rhG-CSF. These results suggest that rhG-CSF augments the cytotoxicity of PSK-induced PMNs when administered in vivo timely.

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 alpha (TNF-alpha) in response to Nocardia rubra cell wall skeleton (N-CWS) was examined by bioassay and enzyme immunoassay. Monocytes treated with N-CWS at more than 0.5 microgram/ml produced IL-1 and TNF-alpha 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-alpha antibody did not affect IL-1 production by the monocytes treated with N-CWS, suggesting independency of IL-1 production of TNF-alpha. These results suggest that the therapeutic antitumor effect of N-CWS is due, in part at least, to the augmented production of these monokines.

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

Peritoneal polymorphonuclear 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 micrograms)/head of OK-432 or 200 micrograms/head of Nocardia rubra cell wall skeleton (N-CWS). Without in vitro stimulation, these PMNs did not show cytotoxicity to syngeneic MM46 mammary carcinoma cells in 51Cr release assay. Cytotoxicity of these PMNs was augmented by the addition of 25 micrograms/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 the in vitro addition of 25 micrograms/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.

In vitro Activation of Bovine Macrophage and Peripheral Blood Mononuclear Cells by Nocardia rubra Cell Wall Skeleton (N‐CWS)

Nocardia rubra cell wall skeleton (N-CWS) was used for immunotherapy to bovine leukemia virus (BLV)-positive cattle with enlarged subcutaneous lymphatic nodules. Electron microscopical observations showed the infiltration of macrophage and T cells around N-CWS treated lesions. Antitumor effect induced by N-CWS was examined in vitro. The maximum cytolytic activity of macrophage was observed, when the cells were incubated with 10 micrograms/ml of N-CWS. Chemiluminescence response of peripheral blood mononuclear cells (PBMC) using N-CWS as stimulant was observed with a high level of activity for a long period, 5 hr. Mitogenic effect of N-CWS to PBMC was observed in the presence of macrophages but not without macrophages. Furthermore, interleukin 2 activity was recognized in supernatant of PBMC cultured with N-CWS. Maximum cytotoxic T lymphocyte response was induced when PBMC were cultured with 0.1 micrograms/ml of N-CWS.

Properties of tumor infiltrated cells induced by N-CWS.

Analysis of surface marker of cells after intratumor injection with Nocardia rubra cell wall skeleton (N-CWS) resulted in gradually increasing percentage of macrophage, Pan T and BoCD4+ cells. Proportion of BoCD8+ cells gradually increased from 4th day and then decreased from 8th day after the injection. Fresh tumor infiltrated cells obtained from lymphatic nodule at 8 days after injection of N-CWS showed cytotoxic activity against bovine leukemia cell line, but this activity decreased with the time of cultivation and no activity could be detected after 14 days cultivation. These cultured cells were injected twice to lymphatic nodule at one week interval for adoptive immunotherapy and found to induce complete regression of nodule after 5 weeks from first injection.

Synergy of Nocardia rubra Cell Wall Skeleton and Interleukin 2 in the in vivo Induction of Murine Lymphokine‐activated Killer Cell Activity

Combination of an ip injection of Nocardia rubra cell wall skeleton (N‐CWS) and 3 daily sc injections of human recombinant interleukin 2 (rIL 2) into C3H/HeN mice resulted not only in a significant increase in the number of peritoneal cells (PC) but also in a potent induction of their lymphokine‐activated killer (LAK) activity, compared with results obtained with N‐CWS or rIL 2 alone. The augmented LAK activity of PC was mediated by nonadherent, nonphagocytic, Thy‐1.2+∼− and asialo GM1+ cells. Nonadherent PC induced by an ip injection of N‐CWS bound more 125I‐labeled rIL 2 than did normal, nonadherent PC, and generated high LAK activity when cultured overnight with rIL 2. In contrast, normal, nonadherent PC responded only weakly to the overnight stimulation with rIL 2. The phenotype of N‐CWS‐induced PC with an elevated IL 2 responsiveness was Thy‐1.2+∼−, Lyt‐1.1−, Lyt‐2.1− and asialo GM1+, suggesting that the N‐CWS‐stimnlated LAK precursors were derived mainly from the NK cell lineage. However, mature T cells may also be involved in this mechanism, because N‐CWS failed to augment the IL 2 responsiveness of nonadherent PC in BALB/c nu/nu mice. Treatment of CS7BL/6N mice bearing solid Lewis lung carcinoma (3LL) tumors with an intratumoral injection of N‐CWS followed by 6 daily sc injections of rIL 2 resulted in the apparent suppression of tumor growth, while N‐CWS or rIL 2 alone produced no such suppression. These results suggest that N‐CWS augments the antitumor effect of rIL 2 by accumulating LAK precursors and elevating their responsiveness to rIL 2 at the injection site.

Augmentation of Murine Lymphokine‐activated Killer Cell Induction by a Factor Produced by Nocardia rubra Cell Wall Skeleton‐stimulated T Cells

Four‐hour exposure of C3H/HeN mouse spleen cells to Nocardia rubra cell wall skeleton (N‐CWS) before 4‐day culture with a suboptimal dose of human recombinant interleukin 2 (rIL 2) augmented the induction of lymphokine‐activated killer (LAK) cell activity, whereas the treatment with N‐CWS alone induced no cytotoxicity. In accordance with this, the IL 2 binding activity of spleen cells was augmented by combined stimulation with N‐CWS and rIL 2. The augmented cytotoxicity was mediated by Thy‐1.2+, Lyt‐1.1−, Lyt‐2.1− and asialo GM1+ cells. Cell cultures in diffusion chambers revealed that N‐CWS‐treated spleen cells produced a LAK cell induction‐helper factor (LAK‐helper factor, LHF) when cultured with rIL 2. The LHF production required Thy‐1.2+, Lyt‐1.1+, Lyt‐2.1+ and asialo GM1− cells, and the coexistence of unstimulated accessory cells was also essential for the LHF production. Cells responding to both LHF and rIL 2 to generate LAK activity were Thy‐1.2−, Lyt‐1.1−, Lyt‐2.1− and asialo GM1+. The culture fluid of spleen cells stimulated with both N‐CWS and rIL 2 contained no tumor necrosis factor (TNF) activity, and the additional stimulation with N‐CWS caused no production of either IL 2 or interferon (IFN). Murine recombinant interleukin la (Mu‐rIL 1α) could not replace the augmentative effect of N‐CWS on LAK cell induction. These results suggest that in the presence of rIL 2, N‐CWS stimulates murine T cells to produce LHF that is probably distinct from IL 1, IL 2, TNF and IFN.

Fast atom bombardment combined with tandem mass spectrometry for analysis of a complex mixture of lipo-oligopeptides fromNocardia rubra cell wallskeleton

Experimental variables in fast atom bombardment combined with tandem mass spectrometry based on low-energy collision-induced dissociation have been systematically invetigated in order to optimize parameters for analysis of synthetic lipo-oligopeptides. The parameters studied were matrix, ion polarity, collision gas, collision gas pressure and collision energy. The optimal parameters recommended in this study have been applied to the N-terminal amino acid seuquence determination of lipopeptide obtained from Nocardia rubra cell wall skeleton. Daughter and parent ion experiments with negative ion fast atom bombardment combined with tandem mass spectrometry have proven to be very useful for structure elucidation of a complex mixture of lipo-oligopeptides.

Therapeutic efficacy of sequential therapy with OK-432, cyclophosphamide, IL2-cultured lymphocytes and in vivo IL2 against advanced murine plasmacytoma.

BALB/c mice inoculated IP with a syngeneic plasmacytoma MOPC104E were treated with a combination of a streptococcal preparation, OK-432 (1 KE, 0.1 mg/mouse), low-dose of cyclophosphamide (CPA, 1 mg/kg) and adoptive transfer of tumor-bearer-spleen cells (2 x 10(7) cells) cultured with IL2 and sonicated tumor extract (adoptive immunotherapy; AIT). The consecutive protocol of OK-432 (day 8, 9 post inoculation) - CPA (day 10) - AIT (day 11) was the most effective. Rate of complete remission was highest when recombinant (r-) IL2 was injected to the mice after AIT. Moreover, another bacterial preparation, Nocardia rubra cell wall skeleton and another low-dose chemotherapy, Mitomycin C could be used successfully instead of OK-432 or CPA. Transfer test of intraperitoneal cells (tumor cells plus host cells) of mice on day 11 post inoculation (on the day of AIT) revealed that OK-432 augmented the susceptibility of peritoneal cells to cultured lymphocytes in inhibition of transplantability, and that CPA after OK-432 augmented the anti-tumor effect of tumor-bearer-spleen cells which act synergistically with cultured lymphocytes. This therapy schedule seems to be the best model to augment the effect of AIT with minimal side effect.