Beneficial effects of LK-4, an analog of dextromethorphan on lipopolysaccharide-induced sepsis in rats

Summary Dextromethorphan (DM), an anti-tussive agent, has been claimed to have anti-inflammatory and immunomodulatory effects in vitro. In our preliminary screening test, LK-4, an analog of DM, can afford more protection against circulatory failure induced by LPS than that of DM. Thus, the aim of this study was to evaluate the effects of LK-4 on sepsis induced by intravenous (i.v.) administration of lipopolysaccharide (LPS; 10 mg/kg) in anesthetized Wistar rats and survival rate by intraperitoneal administration of LPS (70 mg/kg) in conscious ICR mice. Results demonstrated that posttreatment with LK-4 (3 and 5 mg/kg, i.v.) significantly attenuated the deleterious hemodynamic changes (e.g., hypotension and tachycardia) in rats treated with LPS. Meanwhile, LK-4 (3 mg/kg) significantly inhibited the elevation of plasma tumor necrosis factor-α, as well as values of GOT and GPT, and BUN and creatinine caused by LPS. The induction of inducible NO synthase and the overproduction of NO and superoxide anions by LPS were also reduced by LK-4. Moreover, infiltration of neutrophils into the lungs and liver of rats 8 h after treatment with LPS was also reduced by LK-4. Furthermore, LK-4 increased the survival rate of mice insulted by toxic dose of LPS. In conclusion, the beneficial effects of LK-4 on LPS-induced sepsis result from its anti-inflammatory and anti-oxidant effects. Thus, LK-4 can be potentially used as a therapeutic agent for sepsis in the future.     

Effect of carboxymethyl-chitin-glucan on cyclophosphamide induced mutagenicity

The effect of high molecular carboxymethyl-chitin-glucan (CMCG), administered either intraperitoneally, intravenously or orally prior to cyclophosphamide injection, on the frequency of micronucleated reticulocytes was evaluated in peripheral blood of female ICR mice. Both intraperitoneal and intravenous administration of CMCG decreased the clastogenic effect of cyclophosphamide. The protective effect of CMCG was concentration dependent, with a higher decrease achieved by 100 mg/kg than by 50 mg/kg body weight. On the other hand, not even five peroral pretreatments with CMCG in the dose of 200 mg/kg body weight during the week prior to simultaneous administration of CMCG and cyclophosphamide induced a decrease of micronucleated reticulocytes in peripheral blood. It is therefore conceivable that CMCG failed to pass through the gastrointestinal tract, probably due to its high molecular weight. The antimutagenic effect of CMCG against cyclophosphamide was manifested by its intraperitoneal and intravenous administration to female ICR mice.     

Protective effect of an acidic glycoprotein obtained from culture of Chlorella vulgaris against myelosuppression by 5-fluorouracil

 An acidic glycoprotein prepared from a culture of Chlorella vulgaris (CVS) was examined for its protective effect on 5-fluorouracil(5FU)-induced myelosuppression and indigenous infection in mice. Subcutaneous administration of CVS greatly reduced the mortality of non-tumor-bearing mice given a high dose of 5FU, and could increase the LD₅₀ value of 5FU for these mice. After 5FU treatment, indigenous infection developed probably as a result of the impairment of the host defense system. CVS reduced the incidence of indigenous infections and this effect was attributable to the acceleration of recovery from 5FU-induced myelosuppression. Early recovery of hematopoietic stem cells, or cells responding to interleukin-3 or granulocyte/macrophage-colony-stimulating factor, was especially observed in the bone marrow of CVS-treated mice on days 4 – 9 after the injection of 5FU. When tumor-bearing mice were given CVS during treatment with 5FU, CVS prolonged the survival of mice without affecting the antitumor activity of 5FU. In addition, CVS was itself shown to exert an antitumor effect. These results suggested that CVS may be beneficial for the alleviation of side-effects in cancer chemotherapy without affecting the antitumor activity of the chemotherapeutic agent. Received: 15 August 1995 / Accepted: 23 April 1996     

Interleukin-1α synergistic in vivo enhancement of cyclophosphamide- and carboplatin-mediated antitumor activity

 Interleukin-1α (IL-1α) has potent acute antitumor activity in vivo and can enhance the efficacy of chemotherapeutic drug-mediated antitumor responses. Studies were undertaken to examine the ability of IL-1α to enhance the activity of cyclophosphamide (CTX) administered in combination with carboplatin. To determine the in vivo effect of IL-1α, CTX and/or carboplatin, mice bearing 14-day RIF-1 tumors were treated on day 0 with a concurrent i.p. injection of varying doses of CTX (5–150 mg/kg), human IL-1α (125 μg/kg), and carboplatin (50 mg/kg) and examined 24 h later for the surviving fraction by the in vivo excision clonogenic-tumor-cell assay. Even at the lowest doses of CTX, IL-1α significantly enhanced the clonogenic tumor cell kill when compared to treatment with CTX alone. When carboplatin was added to the treatment schema, significantly greater clonogenic cell killing and tumor regrowth delay were observed as compared to any agent alone or a two-drug combination (CTX/IL-1α or CTX/carboplatin). Significant enhancement was observed even at low doses of CTX in combination with carboplatin and IL-1α. The interaction between the three-drug combination was found to be synergistic as determined by the median dose effect with significant dose reduction apparent for IL-1α and CTX when used in this combination. These results demonstrate that IL-1α can synergistically enhance the antitumor efficacy of CTX and the combination of CTX and carboplatin. Received: 11 September 1996 / Accepted: 20 May 1997     

Inhibition of morphine dependence by a lipopolysaccharide from Pantoea agglomerans.

A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified and examined for relief of morphine dependence by observing its inhibition of the jumping of mice on naloxone-precipitate withdrawal. Administration of LPSp either intravenously or intradermally showed marked inhibition of the jumping. Beta-endorphin in mouse serum and brain tissue were recognized to be in synchrony with the time course of the relief. Administration of TNF-alpha gave similar effect, suggesting that LPSp induces a cytokine cascade to produce endogenous TNF followed by ACTH/beta-LPH gene products and beta-endorphin. The effect of LPSp was better than that of LPS from E. coli or Bordetella pertussis, and thus is considered to be applicable for clinical use.     

Experimental antimetastatic activity of aclarubicin

Marked antimetastatic activity of aclarubicin, an anthracycline antibiotic, was demonstrated on models of spontaneous and artificial metastases of murine tumors such as Lewis lung carcinoma and melanoma B16. The activity depended on the antibiotic dose and administration regimen. The highest antitumor effect of aclarubicin was observed when the antibiotic was used at the earliest periods after intravenous injection of the tumor cells (the model of artificial metastases) or after amputation of the limb with the tumor (spontaneous metastases). Aclarubicin was active after administration by any of the routes used: intravenous, intraperitoneal and oral, the latter by its efficiency being not inferior to the parenteral administration. When used intravenously aclarubicin showed activity similar to that of adriamycin. However, after oral administration only aclarubicin had antimetastatic action.     

In vivo effects of recombinant human lymphotoxin on human medulloblastoma xenograft: Enhancement of antitumor activity of etoposide

The authors investigated the antitumor activities of rHuLT alone and in combination with etoposide on human medulloblastoma xenografts growing subcutaneously in nude mice. Intravenous administration of rHuLT (1.0×10⁵U/kg, 5.0×10⁵U/kg, 2.5×10⁶U/kg, three times a week for three weeks) suppressed medulloblastoma growth depending on the dose. However, the highest dosage caused serious side effects. Combining rHuLT (intravenously, 5.0×10⁵U/kg, three times a week for three weeks) with etoposide (intraperitoneally, 20mg/kg, once a week for three weeks) increased the antitumor activity without causing serious toxicity. Microscopically, tumor specimen showed thrombosed tumor vessels and massive necrosis 3 weeks after rHuLT treatment. Ultrastructural examination revealed that 120 minutes after the administration of rHuLT alone, disruption of interendothelial junctions was evident, and that the endothelial cells were destroyed at 240 minutes.Concentration of etoposide in tumor tissue peaked 30 minutes after intraperitoneal administration, and then decreased with time. When etoposide was administered in combination with rHuLT, the concentration of etoposide in tumor tissue after 60 to 240 minutes was significantly higher than when etoposide was given alone, and the area under the concentrationversus time curve was also greater for the tumors of mice with combination treatment.The findings suggest that the proper combination of rHuLT and etoposide may have synergistic antitumor activities. Histological changes suggest that increased concentrations of etoposide within the tumor after combination therapy may occur due to increased vascular permeability and/or decreased etoposide clearance which is the result of blood stasis in the tumor vasculature.Abbreviations AUC area under the concentration versus time curve - LT lymphotoxin - rHuLT recombinant human lymphotoxin - rHuTNF recombinant human tumor necrosis factor - TNF tumor necrosis factor     

Single administration of low dose cyclophosphamide augments the antitumor effect of dendritic cell vaccine

Single administration of low dose cyclophosphamide (CTX) was previously reported to enhance the antitumor efficacy of immunotherapies. To investigate the possible mechanisms for this effect, we examined whether a single administration of low dose CTX could augment the immunogenicity of dendritic cell (DC) vaccines. Fifty milligrams per kilogram body weight dose of CTX was administrated intraperitoneally to mice after B16 melanoma or C26 colon carcinoma tumor models were established, DC vaccine generated from mouse bone marrow and pulsed with B16 or C26 tumor cells lysates were vaccinated 4 days later. CTX treatment potentiated the antitumor effects of the DC vaccine, and increased the proportion of IFN-γ secreting lymphocytes in spleens. Furthermore, a significantly reduced proportion of CD4+CD25+FoxP3+ regulatory T (T_reg) cells was detected by flow cytometry in spleen lymphocytes from tumor-bearing mice treated with CTX. Thus, a single administration of low dose CTX could augment antitumor immune responses of DC vaccine by reducing the proportion of CD4+CD25+FoxP3+ T_reg cells in tumor-bearing mice. Our results suggested a possible mechanism of CTX-induced immunopotentiation and provided a strategy of immunotherapy combining a low dose CTX with DC vaccine. J.-Y. Liu and Y. Wu contributed equally to this work.     

Evaluation of therapy with methanol extraction residue of BCG (MER)

Summary The current status of therapy with the methanol extraction residue of BCG (MER) is reviewed. We have identified 41 evaluable clinical trials of MER therapy, involving approximately 3,000 patients with malignant disease. The diagnoses have included lung, colon, and breast cancer, malignant melanoma, acute leukemia, and a small number of other malignancies. MER has been used as an adjunct to therapy for advanced disease and as prophylaxis against recurrence after surgery. Most studies have used the intradermal route but subcutaneous, intralesional, and intravenous routes have also been explored. The major local toxicity is pain and sterile abscess formation. The major systemic toxicity with administration by the intravenous route includes fever, malaise, and the development of pulmonary infiltrates. With the intradermal route little activity has been observed and there is no confirmed example of an increased remission rate, remission duration, or survival induced by MER therapy. When given by the intralesional route MER can cause regression of metastatic malignant melanoma nodules, and when given by the intravenous route MER is a potent immunoadjuvant. Antibody-dependent cellular cytotoxicity and natural killer cell activity were both boosted after one dose of intravenous MER. In rare patients receiving either intradermal or intravenous MER alone, without other therapy, tumor regression has been noted. These cases have included gastrointestinal cancer, lymphoma, and leukemia. Overall, the data indicate that the future of therapy with mycobacterial fractions awaits the development of more potent, less toxic fractions that can be administered systemically.     

Systemic availability and abortion-inducing effects of nocloprost after intravenous, subcutaneous and intragastric administration to pregnant guinea pigs

Nocloprost was administered to 3 groups of 4 pregnant guinea pigs intravenously and subcutaneously in a dose of 30 micrograms/kg and intragastrically in a dose of 100 micrograms/kg. Plasma nocloprost levels were measured at defined times up to 24 h p. adm. with a specific radioimmunoassay and induction of abortion monitored simultaneously. In one animal per group uterus pressure was recorded continuously up to 8 hours p.adm. Animals were sacrificed 7 days p.adm. and the number and state of fetuses in utero evaluated. Systemic availability of unchanged drug was 100% after intravenous (AUCi.v. = 8.6 +/- 2.0 ng h/ml) and subcutaneous (AUCs.c. = 11.5 +/- 1.2 ng h/ml) administration and approximately 30% after intragastric administration (AUCi.g. = 8.9 +/- 2.0 ng h/ml). The incidence of abortion after intragastric administration corresponded to that after subcutaneous administration. After intravenous injection the abortion rate was somewhat less, indicating that equal AUC-values do not necessarily indicate identical pharmacological effects.     

Tolerance to low-dose endotoxin in awake sheep

Dose response and tolerance to a small intravenous dose of Serratia marcescens lipopolysaccharide (LPS) were studied in awake sheep. Core temperature significantly increased after a dose of 0.002 micrograms/kg; changes in pulmonary arterial pressure, pulmonary vascular resistance, plasma thromboxane B2, and circulating leukocyte concentration occurred after 0.02 micrograms/kg; plasma 6-keto-prostaglandin F1 alpha increased after 0.2 micrograms/kg. Development of acute tolerance was studied by injection of S. marcescens LPS (0.02 micrograms/kg iv) on 3 consecutive days: pulmonary arterial pressure and thromboxane B2 levels were significantly lower than controls after the second dose, whereas fever and the degree of leukopenia were not diminished until the third dose. After intravenous administration of LPS given in increasing doses from 0.1 to 3.2 micrograms/kg three times weekly over 7 wk, there were no measurable changes in any of the above parameters after challenge with S. marcescens LPS (0.02 micrograms/kg) after a 1-wk rest period. In awake sheep, small intravenous doses of LPS can cause physiologically important changes of the pulmonary circulation and can alter the hemodynamic and eicosanoid mediator responses to subsequent challenges with LPS. Large intravenous doses of LPS can ablate the physiological responses to subsequent small doses of LPS.     

In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4⁺ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4⁺ cells. Received: 17 September 1996 / Accepted: 8 November 1996     

Autoantibody to p185 erbB2/neu oncoprotein by vaccination with xenogenic DNA

 The passive transfer of antibodies and vaccination procedures against p185, the erbB2/neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4⁺ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year. Received: 29 August 1996 / Accepted: 31 October 1996     

Efficacy against lung metastasis with a tumor-targeting mutant of Salmonella typhimurium in immunocompetent mice

Salmonella typhimurium double leu-arg auxotrophs have been shown to be highly effective as antitumor agents in nude mouse models of human metastatic cancer. In order to proceed to clinical development of the S. typhimurium double auxotroph, termed A1-R, it is necessary to evaluate antitumor efficacy in immunocompetent mice. In the present study, we have observed the efficacy of A1-R on the Lewis lung (LLC) carcinoma in vitro as well as in C57BL/6 (C57) immunocompetent mice. In vitro, A1-R treatment of LLC began to induce cell death within one hour. Various doses and schedules of A1-R were administered to C57 mice implanted with LLC, including bolus single intravenous injection; medium dose with weekly intravenous administration and metronomic treatment with small intravenous doses twice a week. Bolus treatment was toxic to the immunocompetent host, in contrast to nude mice. Lower-dose weekly doses and metronomic doses were well tolerated by the immunocompetent host. Weekly intravenous injection with 2 x 10⁷ bacteria and twice a week intravenous injection with 10⁷ bacteria significantly inhibited metastasis formation, while bolus injection was toxic. Intra-thoracic administration was carried out with 10⁸ bacteria A1-R injected into Lewis lung-bearing C57 mice weekly for three weeks. Lung metastasis was significantly inhibited by intrathoracic bacterial administration, without toxicity. The results in this report, demonstrating the anti-metastatic efficacy of S. typhimurium A1-R in immunocompetent mice, indicate the clinical potential of bacterial therapy of cancer.     

A comparison of benflurone effects after intravenous infusion or oral administration. A study on laboratory rats

The effects of oral administration and intravenous infusion of benflurone were compared using the single dose of 100 mg/kg b.w. Oral administration of benflurone induced more moderate changes in both white and red blood picture than the equal dose of orally administered cyclophosphamide. In contrast to oral benflurone administration, intravenous benflurone induced deep anaemia, deep reticulocytopenia, transient neutrophilia and slight decrease in small lymphocyte counts. The recovery from intravenous benfluorone effects was rapid.     

Gene-modified tumor cells as cellular vaccine

 The identification and characterization of many tumor antigens and the parallel explosion of knowledge of the cellular and molecular mechanisms of antigen recognition by the immune system have given renewed hopes that immunogenetherapy could be a promising modality to treat certain tumors. Many different novel strategies have been developed to derive genetically modified tumor cells and use them as cellular vaccines to induce useful antitumor immunity in a variety of animal tumor models. This review discusses induction of tumor immunity by injecting tumor cells that are genetically engineered to secrete various cytokines and to express major histocompatibility complex molecules and/or costimulatory molecules. While there has been a great success in inducing excellent antitumor immunity in a variety of tumor models, there are some difficulties and limitations in the application of these gene-modified tumor cells for the treatment of preexisting tumors. A number of improvements and modifications are already underway to overcome some of these problems. Received: 6 August 1996 / Accepted: 20 September 1996     

Hemorphins Act as Homeostatic Agents in Response to Endotoxin-Induced Stress

The effect of synthetic LVV-hemorphin-7 and hemorphin-7 on hypothalamo-pituitary-adrenocortical axis activity in response to endotoxin-induced stress was studied. The intraperitoneal (ip) endotoxin (lipopolysaccaride, LPS) (0.5 mg/kg) administration in combination with hemorphin (1 mg/kg) induce significant decrease in plasma corticosterone and modest decrease in plasma levels of tumor necrosis factor-alpha (TNFα) in compare with elevated levels of both corticosterone and TNFα in plasma of rats received LPS administration alone. Increased activity of calcineurin in both plasma and brain of rats received ip administration of LPS, was recovered under LPS + hemorphin treatment. In two independent proteome analysis, using 2-dimensional fluorescence difference gel electrophoresis and the isotope coded protein label technology, peptidyl-prolyl cis-trans-isomerase A (cyclophilin A) was identified as regulated by hemorphins protein in mouse brain. A therapeutic potential of hemorphins and mechanisms of their homeostatic action in response to endotoxin-induced stress are discussed.     

Antitumor effects of the molecule-downsized immunoconjugate composed of lidamycin and Fab’ fragment of monoclonal antibody directed against type IV collagenase

Type IV collagenase plays an important role in tumor invasion and metastasis through cleaving type IV collagen in the basement membrane and extracellular matrix. In this study a molecule-downsized immunoconjugate (Fab’-LDM) was constructed by linking lidamycin (LDM), a highly potent antitumor antibiotic, to the Fab’ fragment of a monoclonal antibody directed against type IV collagenase and its antitumor effect was investigated. As assayed in 10% SDS-PAGE gel, the molecular weight of Fab’-LDM conjugate was 65 kD with a 1: 1 molecular ratio of Fab’ and LDM. The Fab’-LDM conjugate maintained most part of the immunoreactivity of Fab’ fragment to both type IV collagense and mouse hepatoma 22 cells by ELISA. By MTT assay, Fab’-LDM conjugate showed more potent cytotoxicity to hepatoma 22 cells than that of LDM. Administered intravenously, Fab’-LDM conjugate proved to be more effective against the growth of subcutaneously transplanted hepatoma 22 in mice than free LDM in two experiment settings. In Experiment I, the drugs were given intravenously on day 1 and day 8. Fab’-LDM at the doses of 0.025 mg/kg, 0.05 mg/kg and 0.1 mg/kg inhibited tumor growth by 76.7%, 93.3% and 94.8%, while free LDM at 0.05 mg/kg inhibited tumor growth by 76.1%, respectively. In experiment II, the drugs were given intravenously on day 4 and day 11, Fab’-LDM at the doses of 0.025 mg/kg and 0.05 mg/kg inhibited tumor growth by 74.2%, 80.9%, while free LDM at 0.05 mg/kg inhibited tumor growth by 60.5%, respectively. In terms of survival time, Fab’-LDM was more effective than free LDM. The results suggest that the molecule-downsized immunoconjugate directed against type IV collagenase is of high efficacy in experimental cancer therapy.     

The ubiquity of natural adsorbable organic halogen production among basidiomycetes

 Recently, several species of basidiomycetes were shown to produce de novo high concentrations of chloroaromatic metabolites. Since these lignocellulose-degrading fungi play a major role in the ecosphere, the purpose of this study was to determine the ubiquity of organohalogen production among basidiomycetes. A total of 191 fungal strains were monitored for adsorbable organic halogen (AOX) production when grown on defined liquid media. Approximately 50% of the strains tested and 55% of the genera tested produced AOX. A low production of 0.1–0.5 mg AOX/l was observed among 25% of the strains, a moderate production of 0.5–5.0 mg AOX/l was observed among 16% of the strains and 9% of the strains produced high levels (5–67 mg AOX/l). The latter group was dominated by species belonging to the genera Hypholoma, Mycena and Bjerkandera, showing specific AOX productions in the range 1074–30893 mg AOX/kg dry weight of mycelial biomass. Many highly ecologically significant fungal species were identified among the moderate to high producers. These species were also able to produce AOX when cultivated on natural lignocellulosic substrates. Hypholoma fasciculare and Mycena metata respectively produced up to 132 mg and 193 mg AOX/kg dry weight of forest litter substrate in 6 weeks. Received: 5 October 1995/Received revision: 28 December 1995/Accepted: 12 February 1996     

The concurrent administration of OK432 augments the antitumor vaccination effect with tumor cells by sustaining locally infiltrating natural killer cells

 The effect of a local injection with a streptococcal preparation OK432 on the antitumor vaccination with tumor cells was investigated. Natural killer (NK) cells, which were detected by anti-NK1.1 monoclonal antibody (mAb), increased in the peritoneal exudate cells after an intraperitoneal (i.p.) injection with syngeneic B16 melanoma cells. Furthermore, a concurrent i.p. injection with OK432 efficiently sustained the locally infiltrating NK cells. The OK432 treatment also sustained the augmented NK and lymphokine-activated killer activities in the peritoneal exudate cells. This treatment also increased the ability of the locally infiltrating NK cells to produce interferon γ in response to the tumor cells. In addition, the concurrent i.p. injection with OK432 in combination with the tumor cells enhanced the capacity of the spleen cells to turn into anti-(B16 melanoma) cytotoxic T lymphocytes after in vitro restimulation. This augmenting effect of OK432 was dependent on NK cells. Moreover, the concurrent injection with OK432 at the time of antitumor vaccination significantly enhanced the protective immunity against B16 melanoma at the rechallenge. Taken together, these findings indicate that a concurrent local injection with OK432 in combination with tumor cells efficiently augments the antitumor vaccination effect, in part, by sustaining the locally infiltrating activated NK cells. Received: 6 March 1996 / Accepted: 30 May 1996