In vivo antitumor activity of various forms of delayed-type hypersensitivity in mice.

Relationships among various forms of delayed-type hypersensitivity (DTH) and nonspecific resistance to Lewis lung tumor were studied in syngeneic and semisyngeneic mice. Only the tuberculin type of DTH obviated a virulent inoculum of 10(6) tumor cells. The Jones-Mote type of DTH, even modified by cyclophosphamide pretreatment, produced a significant local inflammatory reaction which was unable to destroy tumor cells. The antitumor effect of the tuberculin type was observed in BCG-or in Corynbacterium parvum-immune mice and also in sheep red blood cell-immunized mice, but only after the modulating effect of BCG. The antitumor activity of the DTH reaction was anatomically restricted and time related, and it required local persistence of specific antigen. A minimal number of bacteria, 1 times 10(6) living or heat-killed BCG organisms, were equally able to eradicate 10(5) tumor cells in BCG-immune mice. Biphasic effects on tumor growth were observed when systemic specific inflammatory reactions were elicited in BCG-immune mice. However, tumor-specific immunity was never observed, inasmuch as BCG-immune mice surviving injection of a mixture of BCG and tumor cells did not resist a second tumor cell challenge.     

Synergistic antitumor effects of BCG and monoclonal antibodies capable of inducing antibody-dependent cell-mediated cytotoxicity

Three monoclonal antibodies (MAbs) of different immunoglobulin subclasses against MH134 murine ascitic hepatoma cells, detecting the same antigenic determinant of tumor-associated antigen of the tumor cells, were tested for their ability to produce a synergistic antitumor effect with Mycobacterium bovis BCG in C3H/HeN mice. 12A2 (IgG2a) induced both antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against the tumor cells. C3H/HeN mice were inoculated ip with MH134 tumor cells (day 0), and received an ip injection of BCG (day 1) and/or 12A2 (day 5). The combined therapy with BCG and 12A2 brought about a significant prolongation of the survival period, whereas either BCG or the MAb alone exhibited poor therapeutic effectiveness. 11G2 (IgG1), inducing ADCC but not CDC against MH134 tumor cells, was shown to exhibit antitumor effects as potent as those of 12A2, when used in combination with BCG. However, 7C2 (IgM), capable of inducing CDC but not ADCC to the tumor cells, produced no apparent synergistic effect with BCG. ADCC of BCG-induced peritoneal cells was mediated by the adherent cell population of the cells and abolished by the addition of carrageenan, suggesting that the effector cells of the cytotoxicity were macrophages. Moreover, carrageenan abolished the combined antitumor effect of BCG and these MAbs in the serological Winn assay. These results suggest that activated macrophages play a major role in the synergistic antitumor effect of BCG and MAbs capable of inducing ADCC against MH134 tumor.     

Inhibition and stimulation of the growth of Krebs-2 carcinoma by BCG vaccine.

The effect of BCG vaccine on the growth of imtransplants of Krebs-2 carcinoma in mice was studied. The simultaneous injection of BCG and tumor cells either inhibited tumor growth (BCG given in admixture with tumor cells) or stimulated it (BCG injected contralateral to the tumor transplantation site). The BCG dose was directly related to the effect. Tumor growth was also stimulated by the ip injection of starch or liquid paraffin. In these experiments, the BCG effect was attributed to the redistribution of cells involved in nonspecific and specific tumor resistance. Shortly after BCG prevaccination, particularly when BCG doses were high and mice were susceptible to vaccine infection, BCG was either without effect or stimulated tumor growth; later, however, tumor growth was inhibited regardless of the BCG dose and the injection site of the BCG. The effect of BCG prevaccination was suggested to be due to: 1)the distraction of macrophages and T-lymphocytes to defend the host against the multiplying mycobacteria, and 2)the activation of the pool of these cells that become capable to participate in antitumor resistance after mycobacteria elimination.     

S－180瘤苗联用BCG治疗效应的研究

本文观察荷瘤小鼠（接种１×１０５Ｓ－１８０瘤细胞）用异体同基因Ｓ－１８０瘤苗联用ＢＣＧ治疗的主动免疫试验的效应．结果表明，９０％（１８／２０）实验鼠产生迟发型皮肤赵敏反应，与对照组或单一治疗组（ＢＣＧ组、瘤苗组）相比，其生存期明显延长（Ｐ＜０．０５）和转移瘤肺结节数显著下降（Ｐ＜０．０１）．ＤＣＨ反应持久的四只小鼠显示了强的抗癌免疫反应．结果证实，采用Ｈａｎｎａ免疫方案可以提高接种１×１０５瘤细胞的荷瘤鼠抗病免疫反应，并可借ＤＣＨ皮试反应监测其ＡＳＩ疗效。     

Enhanced host resistance to transplantable murine lymphosarcoma in Swiss mice by combined immunostimulation with BCG and polyinosinic-polycytidylic acid

Combined immunostimulation with BCG and double-stranded polyinosinic-polycytidylic acid (poly I . poly C) was more effective than single-modality immunostimulation in suppressing tumor growth in inbred Swiss mice. BCG sensitization followed by administration of poly I . poly C on the day of tumor cell injection significantly prolonged the survival period against parental lymphosarcoma (LS) and its ascites variant (LS-A). BCG and poly I . poly C given together on the day of tumor cell injection suppressed only LS-A and not LS. BCG or poly I . poly C given alone did not result in tumor cures. Silica injection given 2 days before poly I . poly C injection completely abrogated the antitumor effect of sequential treatment with BCG and poly I . poly C. Silica treatment given on and beyond the day of poly I . poly C injection did not abrogate the antitumor effect. This observation indicated that intact macrophage effector function was essential at the time of tumor cell inoculation to obtain an effective antitumor action.     

Antitumor effect of actinomycin D entrapped in liposomes bearing subunits of tumor-specific monoclonal immunoglobulin M antibody

Sonicated liposomes containing actinomycin D in the membranes were chemically coated with the subunits of monoclonal immunoglobulin M (IgM) antibody against a mouse mammary tumor-associated antigen (MM antigen) and examined for their in vitro and in vivo antitumor effects against MM46 (MM+) and MM48 (MM-) tumors of C3H/He mouse origin. The antibody-bearing, actinomycin D-containing liposomes (chemoimmunoliposomes) were selectively bound to MM+ tumor cells and showed much more in vitro cytotoxicity against the tumor cells than that shown by free actinomycin D. The in vivo antitumor effect of the chemoimmunoliposomes was tested on the mammary tumor cells (5 X 10(4) to 5 X 10(6) transplanted i.p. into syngeneic mice. A single i.p. injection of the chemoimmunoliposomes containing 0.3, 0.5, or 1 microgram of actinomycin D into MM46 tumor-bearing mice resulted in the cure of some mice and a prolonged survival time in the rest of the mice as compared to results in controls. In this test, free actinomycin D, anti-MM IgM antibody, and bovine serum albumin-coated liposomes containing actinomycin D were marginally effective or ineffective. To examine a systemic antitumor effect of chemoimmunoliposomes, mice were inoculated with MM46 tumor cells and then treated with a single i.v. injection of liposomes 4 days later. If the mice were pretreated with an i.v. injection of unmodified multilamellar liposomes, an injection of the chemoimmunoliposomes containing 1 microgram of actinomycin D resulted in a significant inhibition of tumor growth. Both free actinomycin D and bovine serum albumin-coated liposomes containing actinomycin D were ineffective against the s.c. tumor. These results indicate that an antitumor drug entrapped in the membranes of small sonicated liposomes bearing antitumor monoclonal antibodies can be delivered to antigenic tumor cells and exert more efficient antitumor activity than does the free drug.     

Responses of tumors induced in inbred guinea pig strain JY=1 and strain Hartley/F to BCG.

A transplantable fibrosarcoma induced in inbred JY-1 guinea pig strain by 3-methylcholanthrene (MCA) and designated J4, an allotransplantable subline of J4 (JH4) which was obtained by the transplantation of J4 into the inbred Hartley/F guinea pig strain and maintained by passages in this strain, and a syngeneic liposarcoma H10 induced in a Hartley/F guinea pig by MCA were tested for their immunotherapeutic response with BCG. The growth of J4 and H10 tumors was suppressed in most of the animals when tumor cells were mixed with BCG before being injected sc into BCG-immune or BCG-nonimmune recipients. The growth of the JH4 tumor was suppressed at the sites of injection with a mixture of tumor cells and BCG in BCG-immune recipients but not in nonimmune animals. All guinea pigs surviving the injection of a tumor cell-BCG mixture resisted a second tumor cell challenge. When subcutaneous sarcomas grew to about 8-15 mm in diameter, BCG was injected into the tumors. The growth of JH4 tumor was not influenced by the injection in either BCG-immune or BCG-nonimmune animals, while the regression of the established J4 transplants was produced in 2 of 3 nonimmune recipients. The growth of the H10 tumor was not inhibited with an intratumor injection into nonimmune guinea pigs, while the H10 tumor regressed in BCG-immune animals for 4-5 weeks after intratumor injection and thereafter grew progressively. Skin reactions in animals that received repeated intradermal injections of the tumor cells and BCG were tested with 10(6) viable tumor cells as eliciting antigens. Typical delayed-type hypersensitivity reactions that were specific to the homologous antigens were observed. The possible reasons for the different responses to BCG among the guinea pig tumors, including line-10 hepatocarcinoma in strain-2 guinea pigs, were discussed.     

In vivo induction of antitumor immunity and protection against tumor growth by injection of CD154-expressing tumor cells

As they should enhance tumor-specific antigen presentation by dendritic cells, tumor cell lines genetically modified to express CD154 molecules have been used in an attempt to induce protective antitumor immunity. Two murine models were used: the major histocompatibility complex (MHC) class I negative melanoma B16F10 and the MHC class I positive mammary adenocarcinoma TS/A. CD154 or mock-transfected B16F10 or TS/A cells were injected subcutaneously into H-2-compatible B6D2 mice. CD154 expression by tumor cells induced a complete rejection (in the TS/A model) or a striking reduction (in the B16F10 model) of modified tumors growth, but also a significant protection against the growth of mock tumor cells injected simultaneously, either mixed with the CD154-expressing tumor cells, or in the other flank of mice. Thirty days after CD154-expressing tumor rejection, splenic lymphocytes from surviving tumor-free mice were able to inhibit tumor proliferation in vitro and significant amounts of IFN-gamma were detected in the sera of these mice. Growth kinetics of mock and CD154-expressing tumors in immunocompetent versus nude mice suggest that T lymphocytes and natural killer cells responses are implicated in this antitumor immunity. The injection of CD154-expressing tumor cell induced an antitumor protective response, both locally and distant from the injection site. The effect was most pronounced in MHC class I expressing TS/A tumor model.     

Passive local anaphylaxis: demonstration of antitumor activity and complementation of intratumor BCG.

When an extracellular dye, Lissamine green, or 51Cr-labeled spleen cells were injected iv into C3H mice bearing small, partially necrotic 3-methylcholanthrene-induced transplantable fibrosarcomas (McC3), the tumor content of these circulating elements per unit weight was substantially lower than that of other selected organs. The level of these blood-borne materials was, however, significantly augmented by the intratumor induction of passive local anaphylaxis (PLA). The PLA-induced augmentation was inhibited by administration of the histamine and serotonin antagonist cyproheptadine; comparable increases were also induced by the intratumor injection of a histamine and serotonin mixture or BCG. The weekly intratumor induction of PLA in McC3 tumors resulted in the complete regression of a significant number of the tumors, and this therapeutic effect was eliminated by cyproheptadine treatment. The intratumor injection of BCG induced the regression of approximately 50% of injected tumors, and the combination of this immunostimulant treatment with the generation of PLA was more therapeutically effective than either treatment alone. PLA in the vicinity of solid tumors may, by increasing vascular permeability, potentiate antitumor effector mechanisms, particularly when these are BCG-stimulated. Despite this demonstration of a possible role of anaphylactic reactions in tumor immunity, no definitive evidence was found that active reagin-mediated local anaphylaxis occurred in C3H mice bearing the McC3 tumor, whether or not they were treated with immunostimulants.     

Toxicity of intrapleural Bacillus Calmette-Guérin treatment in animals.

The toxicity of intrapleural Tice strain Bacillus Calmette-Guérin (BCG) infection was tested in hamsters. Doses above 10(6) colony-forming units caused significant systemic infection, which could be controlled with conventional antituberculosis therapy. Living BCG in the pleural space did not prevent the healing of bronchial or vascular closures after pulmonary resection. Prophylactic intrapleural BCG (10(6) colony-forming units) significantly reduced tumor growth in the lungs of mice following i.v. injection of 5 x 10(5) syngeneic sarcoma cells. These animal experiments suggest that intrapleural BCG may be administered in the pleural space after lung resection in limited doses if followed by a complementary course of antimicrobial therapy.     

Inhibition of experimental and spontaneous pulmonary metastasis of murine RCT (+) sarcoma by beta-cyclodextrin-benzaldehyde

The effect of beta-cyclodextrin-benzaldehyde (CDBA) on pulmonary metastasis in C3H/He mice was examined. In experimental metastasis that was induced by iv injection of 1 X 10(6) RCT (+) cells, the highest inhibition was observed in the mice that were treated daily with CDBA (5 mg/day) for 1 week before tumor cell inoculation and further treated for 3 weeks after inoculation, when compared with those in other experimental groups that were given only pretreatment or posttreatment. The inhibitory effect was dose-dependent. In spontaneous metastasis that was induced by sc injection of 3 X 10(6) RCT (+) cells, the inhibition of metastasis was also observed in the mice treated with CDBA (5 mg/day) in the same manner as described above. However, the development of the primary tumor was not inhibited. CDBA-treated tumor-bearing mice showed almost as much NK activity as normal mice. Furthermore, although injection of 5-fluorouracil suppressed this activity to about 50% of that in normal mice, the combined treatment with CDBA could maintain the NK cell activity at the normal level. The results suggested that the inhibition of pulmonary metastasis might be induced by a combined effect of CDBA; that is, the direct inhibition of tumors and the maintenance of NK cell activity.     

Combination of direct intratumoral administration of dendritic cells and irradiation induces strong systemic antitumor effect mediated by GRP94/gp96 against squamous cell carcinoma in mice

We tested a new therapeutic modality for head and neck and esophageal cancers, a combination of direct intratumoral (i.t.) administration of dendritic cells (DCs) and radiation therapy (RT) in mouse squamous cell carcinoma (SCC). We also evaluated the functions of gp96, which can enhance systemic antitumor activity, and the mechanism of the abscopal effect. Mouse SCC cells (1 x 10(5)), SCCVII, were inoculated into the left femur of C3H/He mice subcutaneously, and also similarly inoculated into chest subcutaneous tissue. Only the left femur tumor was exposed to 4 or 10 Gy of ionizing radiation, and then 1 x 10(6) DCs i.t. was injected only into the femur tumor. Following this procedure, tumor volumes of the femur and chest were measured. We evaluated whether gp96 could enhance the antitumor effect. With DCs i.t. and RT, tumor growth was markedly suppressed. Tumor growth of non-treated tumors were also suppressed, indicating that the combination therapy of DCs and RT evoked systemic antitumor activity. In vitro, the enhancement of gp96 expression was strongly detected by immunostaining after irradiation, DCs with gp96 induced strong cytotoxic activity in vitro, and tumor growth inhibition was observed by direct i.t. injection of gp96. A combination of DCs i.t. and RT can induce a strong antitumor effect not only against treated local tumor but also against non-treated distant tumor, indicating that this treatment can evoke a strong systemic antitumor effect. Gp96 is thought to be one of the target molecules to explain the abscopal effect.     

Effects of Photodynamic Therapy Using Mono-l-aspartyl Chlorin e6 on Vessels and Its Contribution to the Antitumor Effect

The effect of photodynamic therapy (PDT) on the vascular system has a significant role in tumor tissue destruction. We investigated the contribution of vascular damage to the antitumor effects of PDT and analyzed the quantitative vascular changes after PDT. Fibrosarcoma-bearing BALB/c male mice were injected with mono-L-aspartyl chlorin e6 (NPe6) at a dose of 0.25, 5 or 15 mg/kg, and photoradiation was performed with a diode laser 10 min, 2 h or 24 h after injection, respectively. Ten minutes after injection of 0.25 mg/kg, NPe6 was found to be present only in plasma, while at 2 h after injection of 5 mg/kg it was present in both plasma and tumor, and 24 h after injection of 15 mg/kg it was present only in the tumor. The antitumor effects observed in the 5 mg/ kg-2 h and 0.25 mg/kg-10 min groups were virtually the same, whereas the effect in the 15 mg/kg- 24 h group was weaker. The damage to the tumor vasculature and tumor cells in the 15 mg/kg-24 h group occurred later than under the other conditions, and vascular damage in the tumor-surrounding tissue was also less marked even 24 h after PDT. These results suggested that the plasma NPe6 concentration during laser irradiation contributed more than the tumor NPe6 concentration to the antitumor effect, and that the minimal damage to blood vessels around the tumor at the low plasma NPe6 concentration may be one reason for the failure to obtain a marked antitumor effect.     

Effects of photodynamic therapy using mono-L-aspartyl chlorin e6 on vessels and its contribution to the antitumor effect.

The effect of photodynamic therapy (PDT) on the vascular system has a significant role in tumor tissue destruction. We investigated the contribution of vascular damage to the antitumor effects of PDT and analyzed the quantitative vascular changes after PDT. Fibrosarcoma-bearing BALB / c male mice were injected with mono-L-aspartyl chlorin e6 (NPe6) at a dose of 0.25, 5 or 15 mg / kg, and photoradiation was performed with a diode laser 10 min, 2 h or 24 h after injection, respectively. Ten minutes after injection of 0. 25 mg / kg, NPe6 was found to be present only in plasma, while at 2 h after injection of 5 mg / kg it was present in both plasma and tumor, and 24 h after injection of 15 mg / kg it was present only in the tumor. The antitumor effects observed in the 5 mg / kg-2 h and 0. 25 mg / kg-10 min groups were virtually the same, whereas the effect in the 15 mg / kg-24 h group was weaker. The damage to the tumor vasculature and tumor cells in the 15 mg / kg-24 h group occurred later than under the other conditions, and vascular damage in the tumor-surrounding tissue was also less marked even 24 h after PDT. These results suggested that the plasma NPe6 concentration during laser irradiation contributed more than the tumor NPe6 concentration to the antitumor effect, and that the minimal damage to blood vessels around the tumor at the low plasma NPe6 concentration may be one reason for the failure to obtain a marked antitumor effect.     

Antitumor activity of bacterial infection. II. effect of Listeria monocytogenes on growth of a guinea pig hepatoma.

Growth of a guinea pig hepatoma was suppressed when tumor cells were mixed with viable Listeria monocytogenes (LM) before intradermal (id) injection into syngeneic recipients. Heat-killed LM were less effective than viable organisms in suppressing tumor growth. A vaccine containing oil droplets and LM cell walls lacked antitumor activity. Intratumor injection of viable LM on the 7th day after id injection of tumor cells prolonged survival of guinea pigs that did not succumb to LM infection. After intratumor injection of 0.6 times 10-8-1.0 times 10-8 LM, 5 of 22 guinea pigs died from acute infection (23 percent). In the 17 survivors, 3 tumors regressed completely (18 percent). Animals surviving injections of LM and tumor cells were immune to a second challenge with tumor cells. Immunization ofguinea pigs with an intravenous injection of LM decreased the mortality from intratumor injection of LM, but the intratumor injection of LM failed to cure a significant fraction of LM-immune animals bearing 7-day hepatoma transplants. BCG was more effective than LM in producing tumor regression. Synergism between LM and BCG was not observed, and simultaneous intratumor injection of BCG and LM was no more effective than intratumor injection of BCG alone in the treatment of 12-day tumor transplants.     

Specific simian virus 40 (SV40)-induced immunity against transplantable SV40 tumor in Syrian hamsters: abrogation by BCG vaccination.

Syrian hamsters were immunized with simian virus 40 (SV40) and/or BCG (in different time and sequence combinations) and were subsequently challenged with SV40-induced transplantable tumor cells. Immunization of hamsters with some but not all BCG preparations alone induced high levels of resistance to SV40 transplantable tumor cells. The immunization with SV40 of hamsters preliminarily inoculated with BCG induced antitumor immunity, the level of which was equal to resistance induced by immunization of normal animals with SV40. No cumulative effects of BCG and SV40 immunizations were noticed in any of our experiments even when BCG prepartions alone induced considerable increase of antitumor resistance. Inoculation of animals with the mixture of SV40 and BCG was generally less effective than that with SV40 alone. Inoculation of BCG preparations into hamsters preimmunized with SV40 resulted in the complete or partial abrogation of the resistance induced by SV40. All preparations of BCG, independent from their antitumor activity per se, decreased the resistance induced by SV40. The effect of this BCG-induced abrogation of resistance was observed in animals immunized with SV40 7-427 days before inoculation of BCG. The effect was not short-term, as it was still observed 3 months after BCG inoculation.     

Ethylchlorformate polymerized tumor protein immunotherapy of the murine bladder tumor

Using murine transplantable transitional cell carcinoma (MBT2), the effect of ethylchlorformate (ECF) polymerized tumor protein was compared with that of bacillus Calmette-Guerin (BCG). Seventy-five C3H/He mice were challenged with an intradermal inoculation of 5 X 10(5) viable MBT2 tumor cells and divided into five groups. Each group was intradermally administered with 0.01 mg of ECF (low ECF), 0.25 mg of ECF (high ECF), 0.1 mg of ECF and 10(6) CFU BCG (ECF/BCG), 10(6) CFU of BCG alone or normal saline (control) weekly for 10 weeks. The mean survival rate for the treatment groups was 64 to 73 days and significantly longer than that for the control group (P less than 0.001, Savage). The incidence of biologically active tumor progression was significantly less for the treatment groups (low ECF, 53%; high ECF, 33%; ECF/BCG, 7%; BCG, 27%) compared with the control group (87%; P less than 0.5, chi-square. The mean rate of tumor growth was significantly lower for all treatment groups than for the control group (P less than 0.001, ANOVA and SNK), and the ECF/BCG group had the lowest growth rate despite a higher incidence of local granulomatous reaction. In this study, immunotherapy significantly prolonged the survival rate, decreased the incidence of biologically active tumor progression, and slowed the rate of tumor growth. The combination of ECF polymerized tumor protein and BCG had the greatest effect, suggesting that the effect of the vaccine was increased with BCG.     

Antitumor activity of the DNA fraction from Mycobacterium bovis BCG. II. Effects on various syngeneic mouse tumors

MY-1, a fraction extracted from BCG and composed of 70.0% DNA and 28.0% RNA, was examined for its antitumor activity against 9 different syngeneic mouse tumors. Tumor regression was induced in almost all of the mice bearing any of five kinds of solid tumors by repeated intralesional injections of 100 micrograms MY-1. When cells of some tumors were inoculated intradermally together with MY-1, tumor growth was suppressed, lung metastases were inhibited, and the survival times of mice bearing 1 of 3 leukemic tumors were prolonged. Repeated sc injections with MY-1 in sites remote from tumor cell inoculation or repeated iv injections were more or less effective against three kinds of solid tumors. Mice inoculated with Lewis lung carcinoma cells in a hind footpad and whose legs were amputated 9 days later were given iv or sc injections of MY-1 every other day (8 times in total), resulting in substantial prolongation of survival. No direct cytotoxicity of MY-1 for these tumors could be shown in three kinds of experiments, which indicates that the antitumor mechanism of MY-1 is host mediated. MY-1 was equally effective in mice with or without presensitization with BCG, whereas BCG was much more effective in BCG-sensitized mice. This finding suggests that a delayed-type hypersensitivity reaction elicited by BCG protein is not required for the antitumor activity of MY-1.     

Antitumor Effect of Intratumoral Administration of Biological Response Modifiers: Induction of Immunosuppressive Acidic Protein, a Type of α1-Acid Glycoprotein, in Mice

The antitumor effects of biological response modifiers (BRMs) in an experimental mouse model, the "double grafted tumor system" were analyzed. Male BALB/c mice received simultaneous inoculations of Metn-A fibrosarcoma cells on the right flank (10⁶ cells) and left flank (2 × 10⁵ cells) on day 0, and BRMs were injected intratumorally into the right tumor on days 3, 4 and 5. PSK (a protein-bound polysaccharide preparation), interleuldn-1 (IL-1) and cepharanthin (R) cured not only the right, but also the left, non-treated tumor in a double grafted tumor system. OK-432 (a Streptococcus preparation) and BCG and tumor necrosis factor (TNF) cured the right tumor and inhibited the growth of the left tumor. Lentinan (a polysaccharide preparation) and IL-6 inhibited neither the right nor the left tumor. Immunosuppressive acidic protein (IAP) in serum was increased transiently soon after intradermal injection of PSK, CR, OK-432 and TNF in BALB/c mice. Lentinan, however, did not induce IAP. IAP in serum was gradually increased after intradermal inoculation of Meth-A tumor in BALB/c mice. The biochemical difference between PSK-induced IAP (early, inflammatory IAP) and Meth-A-induced IAP (late, tumor-induced IAP) was investigated by crossed affinity immunoelectrophoresis with concanavalin A. IAP of murine serum was separated into 4 peaks. IAP in normal mouse was rich in high-mannose type sugar chain (Peak 3) and contained no hybrid-type sugar chain (Peak 4), which was present in inflammatory and tumor-induced IAP. Inflammatory IAP was rich in biantennary sugar chain (Peak 2) and tumor-induced IAP was rich in tri-tetraantennary sugar chain (Peak 1).     

Mechanistic analysis of high antitumor effect of intradermal administration of lipopolysaccharide fromPantoea Agglomerons

Lipopolysaccharide fromPantoea Agglomerans (LPSp) has a remarkably high antitumor activity even against poorly immunogeneic tumors when given by intradermal injection combined with cyclophosphamide (CY). We have extended this study to gain an insight into the mechanism of this antitumor effect, and especially into the induction of cell mediated immunity. In immuno-histological studies, extensive necrosis and marked infiltration of the inflammatory cells at the tumor were observed after intradermal injection of LPSp combined with CY, but not after CY alone or after no treatment. The cells around the tumors were mostly neutrophils and macrophages (Mac 1 + ); T cells (CD4 +, CD8 + ) were also present. The serum levels of cytokines, induced after intradermal injection of LPSp, were determined and compared with intravenous administration of LPSp or recombinant TNF-Sam2. TNF-α, IL-1, IL-6 and GM-CSF were measured by ELISA as a marker of cytokine induction. The peak level of TNF-α induced by intradermal injection of LPSp was about 5000 pg ml^-1, which was considered relatively small since this level was observed even in clinical trial. There seems to be a longer period of release of TNF-α after an intraderrnal injection than after an intravenous injection. This may produce the remarkably high antitumor effect of the intradermal injection. The antitumor effect of intradermal administration combined with CY was evaluated in nude mice to clarify the role of T cells in high antitumor activity. In this experiment, antitumor activity was found to be much less in BALB/c nu/nu mice without regression, while complete regression was frequently observed in syngeneic mice, show-ing the crucial role of T cells in this treatment. These observations suggest that intradermal administration of LPSp in combination with CY continuously releases and induces not only extensive necrosis of the tumor but also cell mediated antitumor immunity, which may be indispensable for complete regression of the tumor. Clinical application of this treatment for advanced cancer patients is in progress.