Effects on antitumor activity and cytokine production in the thoracic cavity by intrapleural administration of Lactobacillus casei in tumor-bearing mice

The effects Lactobacillus casei YIT9108 (LC 9018) on antitumor activity and cytokine production in Meth A fibrosarcoma (Meth A)-bearing BALB/c mice were examined. Intrapleural (i.pl.) administration of LC 9018 was effective in prolonging the survival of Meth A-bearing mice, and frequently cured mice of the tumor. However, the results also indicated that the effect of LC 9018 was in part inhibited in mice treated with anti-CD3 or anti-CD8 antibody, but not affected in anti-CD4 antibody-treated mice. In contrast, LC 9018 had little effect on Meth A-bearing SCID or nude mice. These results demonstrated that CD8⁺ T cells participated in prolonging the survival of Meth A-bearing mice. Moreover, the examination of the production of several cytokines revealed that the production of interferon-γ and interleukin-6 was, in particular, augmented in the exudated fluid of the thoracic cavity in BALB/c mice injected with LC 9018 i.pl. These results suggested that i.pl. administration of LC 9018 induced those cytokines which had the potential to activate the thoracic macrophages or proliferate the thoracic lymphocytes to the cytotoxic T cells. Taken together, these findings demonstrated that the prolonging effects on survival by i.pl. administration of LC 9018 depended on CD8⁺ T cells, and the i.pl. administration of LC 9018 into i.pl. Meth A-bearing mice induced several cytokines which participated in the subsequent immunoresponses. Received: 24 June 1996     

Induction of tumoricidal peritoneal exudate cells by administration of Lactobacillus casei

The lifespan of mice into which Meth A fibrosarcoma was transplanted intraperitoneally (i.p.) was prolonged by i.p. treatment with Lactobacillus casei YIT 9018 (LC 9018). Treatment with LC9018 before the tumor inoculation (pre-treatment), was more effective than after the tumor inoculation (post-treatment). When tumor cells were inoculated subcutaneously along with peritoneal exudate cells (PEC) harvested from LC 9018-treated mice in the Winn type test, the tumor growth was significantly inhibited, but it was not inhibited by spleen cells. Adherent and nonadherent cells of LC 9018-induced PEC suppressed tumor growth in the Winn type test. PEC induced with LC 9018 showed significant cytolytic activity for 51Cr-labeled Meth A from 3 days to at least 2 weeks after a single injection of LC 9018. The adherent cells lysed tumor cells in the cytolytic test, though nonadherent cells did not show cytolytic activity. These results show that LC 9018 can induce two cell populations possessing the ability to kill tumor cells in vivo. One may be activated macrophages which directly kill tumor cells and the other may be T-lymphocytes which can induce cytotoxic cells.     

The role of tumor necrosis factor (TNF)-α in the antitumor effect of intrapleural injection of Lactobacillus casei strain Shirota in mice

The involvement of several cytokines in the antitumor effect induced by intrapleural (i.pl.) injection of heat-killed cells of Lactobacillus casei strain Shirota (LC 9018) in mice was investigated. Injection of LC 9018 i.pl. into Meth A fibrosarcoma (Meth A)-bearing mice not only significantly prolonged the survival of the mice, but also effectively inhibited the accumulation of malignant pleural fluid in the thoracic cavity. In the thoracic cavity of tumor-bearing mice treated with LC 9018, we observed large amounts of several cytokines including interleukin (IL)-1β, interferon (IFN)-γ, IL-12 and tumor necrosis factor (TNF)-α. Both anti-IFN-γ and anti-IL-12 monoclonal antibody (mAb) treatments partially diminished the antitumor activity of LC 9018 in vivo, while the treatment of anti-IL-1β mAb did not influence the survival of the mice. However, anti-TNF-α mAb treatment completely abolished the antitumor effect of LC 9018 in vivo, suggesting that in this model LC 9018 has a survival-prolonging effect involving certain cytokines. Moreover, i.pl. injection of mouse recombinant TNF-α into Meth A-bearing mice pretreated with anti-TNF-α mAb partially restored the survival-enhancing effect of LC 9018. These results led us to conclude that TNF-α induced by i.pl. injection of LC 9018 plays an important role in the antitumor effect of LC 9018 in vivo. Received: 22 February 1999     

Increased production of cytotoxic macrophage progenitors by Lactobacillus casei in mice

Heat-killed Lactobacillus casei YIT9018 (LC9018), when administered intravenously to normal mice, induced increase in Mac-1+ cells and Mac-2+ cells but not in Mac-3+ cells in spleen. The number of both populations changed in the same time course and was maximal 14 d after the administration. To know the effect of LC9018 on hematopoietic progenitor level, we examined the number of macrophage colony-forming cells (M-CFC), granulocyte-macrophage CFC (GM-CFC), and colony-forming units in spleen (CFU-S) in bone marrow 3 d after the administration. LC9018 stimulated the proliferation of M-CFC but not that of GM-CFC and CFU-S. LC9018-induced M-CFC were similar to normal M-CFC in dependence on macrophage colony-stimulating factor (M-CSF) and buoyant density. M-CFC-derived macrophages cultured in the presence of M-CSF expressed Mac-1 and Mac-2 but not Mac-3. They showed cytotoxic activity against syngenic tumor cells, Meth A, via direct contact, when assayed by using an in vitro colony inhibition assay or an in vivo Winn test. These results indicate that LC9018 stimulates the proliferation of cytotoxic macrophage progenitors in bone marrow and induces their differentiation in spleen. These effects may be one of the ways in which LC9018 suppresses tumor growth.     

The role of lymph node cells in the inhibition of metastasis by subcutaneous injection of Lactobacillus casei in mice

The role of lymph node cells in the inhibition of metastasis of B16-BL6, a highly metastatic variant of B16 melanoma, by Lactobacillus casei YIT9018 (LC 9018) in C57BL/6 mice was determined. Subcutaneous (s.c.) injection of LC 9018 into the left inguinal or the left axillary region inhibited both axillary lymph node and lung metastasis of B16-BL6 after surgical excision of the tumors resulting from inoculation into the left front footpad of the mice. There was, however, little inhibition of the metastasis when LC 9018 was given in the inguinal or axillary region opposite the tumor inoculation. The s.c. injection of LC 9018 into the left inguinal region also augmented not only the cytolytic activity of the left inguinal lymph node cells but also the left axillary lymph node cells against B16-BL6 in vitro. However, the antimetastatic effect of LC 9018 was decreased when LC 9018 was injected s.c. into mice in which the left inguinal lymph nodes had been resected. Furthermore, natural killer cell activity of the left inguinal lymph node cells from mice injected s.c. with LC 9018 was augmented. These results suggest that the lymph node cells activated by the s.c. injection of LC 9018 played an important role in the inhibition of the metastasis and that the treatment with LC 9018 augmented the host immune response.     

Augmentation of antimetastatic effect on Lewis lung carcinoma (3LL) in C57BL/6 mice by priming with Lactobacillus casei

The augmentation of the antimetastatic effect of heat-killed cells of Lactobacillus casei YIT9018 (LC 9018) on Lewis lung carcinoma (3LL) in C57BL/6 mice by presensitization (priming) with LC 9018 was examined. Intralesional injection of LC 9018 into 3LL-bearing mice inhibited both the growth of the primary tumors and the formation of lung metastases, and this effect was significantly augmented by subcutaneous injection of LC 9018 before the tumor inoculation. In the LC 9018-primed mice, intraperitoneal administration of LC 9018 into syngeneic hosts after priming induced a high level of interleukin-2 (IL-2) and interferon- (IFN-) in the peritoneal cavity. At this time, T cells of the spleen cells from the LC 9018-primed mice proliferated and produced IL-2 when co-cultured with LC 9018 as antigen in vitro. Also, the phenotype of these T cells was found to be L3T4⁺ and Ly-2.2^– T cells by analysis by flow cytometry. These results suggest that LC 9018-reactive helper T (T_h) cells were induced by the priming and subsequent challenge with LC 9018, and that IL-2 or IFN-, which was produced by the activated LC 9018-reactive T_h cells, augmented a host immune response resulting the antitumor activity.     

Role of colony-stimulating activity in antitumor activity of Lactobacillus casei in mice

Wide-ranging differences were observed between the antitumor activities of 23 lactobacilli (13 species; 23 strains) and their capacities to elevate the level of serum colony-stimulating activity (CSA) by intraperitoneal administration in mice, and a good correlation existed between the two activities. The mechanism of enhanced production of CSA by administration of Lactobacillus casei YIT 9018 (LC 9018), one of the bacteria that had the strongest activities, and the role of CSA in antitumor activity of LC 9018 were studied. Colony-stimulating activity in the washing fluid from the peritoneal cavity of mice that had been administered LC 9018 intraperitoneally was elevated at 3 to 24 h after the injection, and CSA was also detected at elevated levels in the serum of the mice 6 to 12 h after injection. The cells responsible for the production of CSA after stimulation with LC 9018 seem to be the resident macrophages at the site of administration, because the resident macrophages of mice lavaged 1 h after an intraperitoneal administration of LC 9018 released CSA when they were cultured in vitro. Moreover, resident peritoneal macrophages of normal mice cultured with LC 9018 in vitro also produced CSA. Similar results were obtained with athymic nude mice, and the CSA-inducing activity of LC 9018 was diminished in the mice pretreated with carrageenan, which is selectively toxic to mature macrophages. Bone marrow cells matured to macrophages and polymorphonuclear cells by culture with the CSA induced by LC 9018 for 7 days. These matured macrophages showed strong antitumor activity both in vivo and in vitro. These results suggest that CSA plays important roles in the antitumor activity of LC 9018: it enhances not only the multiplication of committed precursor cells for macrophages and polymorphonuclear cells, but also the functional maturation of the precursor cells for macrophages which serve as potent effectors for tumor cells.     

Oxygen radical production by peritoneal macrophages and Kupffer cells elicited with Lactobacillus casei.

BALB/c mice were injected intraperitoneally (i.p.) or intravenously (i.v.) with Lactobacillus casei YIT9018 (LC 9018). The i.p. injected LC 9018 augmented oxygen radical (OR) production by peritoneal macrophages (PM) and suppressed the production of prostaglandin E2 by PM. The growth of i.p. inoculated Meth A fibrosarcoma was also inhibited by an i.p. injection of LC 9018. i.v. injection of LC 9018 stimulated OR production by fixed macrophages and inhibited the growth of Listeria monocytogenes in the liver. Furthermore, glucose-6-phosphate dehydrogenase activity in the liver was significantly increased (10 to 20 times) by LC 9018 i.v. injection. A significant correlation was observed between the augmentation of OR production by PM or fixed macrophages in the liver and inhibition of growth of Meth A or L. monocytogenes. The augmentation of OR production by LC 9018 was more marked and was maintained for a longer period of time than that by other bacterial immunostimulants.     

Role of macrophages in serum colony-stimulating factor induction by Lactobacillus casei in mice.

Heat-killed Lactobacillus casei YIT9018 (LC9018), when injected intravenously into mice at a dose of 4 to 40 mg/kg, induced the production of serum colony-stimulating factor (CSF). Since this induction was observed in both C3H/HeJ and C3H/HeN mice, LC9018 was considered to act differently from lipopolysaccharide. The amount of serum CSF induced by LC9018 in nude mice and whole-body-X-ray-irradiated mice was similar to that in control mice, but the induction of serum CSF was suppressed by the previous administration of carrageenan, indicating that macrophages, but not T cells, were responsible for serum CSF induction by LC9018. To determine whether macrophages themselves produce CSF or help other cells produce CSF in response to LC9018, we prepared adherent cells from the peritoneal cavity of normal mice and examined CSF activity in their conditioned media. Peritoneal adherent cells did not produce CSF without LC9018, but when cultivated with 1 mg of LC9018 per ml, they produced CSF at the same time that serum CSF was induced after the intravenous administration of LC9018. Additionally, in vitro-induced CSF formed macrophage, granulocyte, and mixed colonies, as serum CSF did. CSF production by peritoneal adherent cells was completely inhibited by cycloheximide (50 micrograms/ml), and neither the elimination of T cells from the peritoneal adherent cells by treating them with anti-Thy-1.2 antibody plus complement nor the addition of T cells affected CSF production. These results suggest that heat-killed LC9018 induces serum CSF in mice via direct stimulation of macrophages to produce CSF de novo.     

Enhancement of hematopoietic response of mice by subcutaneous administration of Lactobacillus casei.

Mice that had received heat-killed Lactobacillus casei (LC 9018) subcutaneously (s.c.) showed enhanced resistance to systemic (i.e., intravenous) infection with Listeria monocytogenes, but the antilisterial resistance of mice was less augmented by s.c. administration of Propionibacterium acnes ("Corynebacterium parvum"). Though there was little change in the total number of splenic leukocytes after s.c. administration of LC 9018, the monocyte-macrophage ratio increased after treatment, reaching its peak on day 5 to 7 after injection. The number of progenitor cells that form macrophage colonies under the stimulus of L-cell-conditioned medium in a semisolid agar culture system increased in the spleens of mice pretreated s.c. with LC 9018, showing a peak response on day 5 after injection. The increase corresponded to the increase in the dose administered, and increased numbers were detected even 10 days after treatment. The number of macrophage colonies in the femurs of mice pretreated s.c. with LC 9018 showed a temporary increase on day 3 after injection but then a decrease until day 10. Colony-stimulating activity was detected in the sera of mice administered LC 9018 s.c. 18 h previously, and the colonies produced were of three types: granulocyte (8%), macrophage (56%), and granulocyte-macrophage (36%). Administration of C. parvum s.c. had little effect on these hematopoietic responses of mice.     

Concomitant Immunity against Tumor Development is Enhanced by the Oral Administration of a Kampo Medicine, Hochu-Ekki-To (TJ-41: BU-ZHONG-YI-QI-TANG)

The oral administration of a kampo herbal medicine, Hochu-ekki-to (TJ-41: Bu-Zhong-Yi-Qi-Tang) using a water-supplying bottle resulted in a slight but significant inhibition of Meth A growth. The oral administration of TJ-41 with gastric gavage significantly enhanced the specific antitumor activity against Meth A at rechallenge on day 9. In a tumor-neutralizing assay, the tumor draining LN cells of the TJ-41 administered mice showed an antitumor activity against Meth A. In a cytolytic assay, the anti-Meth A specific cytolytic T lymphocyte activity was not detected in the spleen cells of the Meth A bearing and TJ-41 administered mice. The oral administration of TJ-41 enhanced the natural killer (NK) activity of the spleen cells in naive mice but could not improve the decreased NK activity of spleen cells from the tumor bearing mice. In a cytostatic assay, the peritoneal exudate cells from the Meth A bearing and TJ-41 administered mice showed a significantly higher amount of cytostatic activity against Meth A than that from either Meth A bearing or TJ-41 administered mice. These results indicate that the oral administration of TJ-41 into the tumor bearing mice may thus be able to enhance concomitant antitumor immunity through the augmentation of the cytostatic activity.     

Protective effect of Lactobacillus casei on Pseudomonas aeruginosa infection in mice.

The protective effect of heat-killed Lactobacillus casei YIT9018 (LC 9018) against Pseudomonas aeruginosa infection in mice was compared with that of Corynebacterium parvum. Survival of mice after intraperitoneal (i.p.) infection with P. aeruginosa was augmented in mice that had been pretreated i.p. with LC 9018 5 days previously. Similar treatment of mice with C. parvum, however, was not effective at all. Moreover, mice became more susceptible to infection with P. aeruginosa after such treatment. Growth of P. aeruginosa in the peritoneal cavity and spleen was markedly inhibited in LC 9018-pretreated mice, whereas such inhibition of bacterial growth was not observed in C. parvum-treated mice. The protective effect of LC 9018 was observed in mice subjected to 800 rads of whole body irradiation but was abrogated when mice were treated with carrageenan. These results suggest that augmentation of the resistance of mice to P. aeruginosa was caused by the induction of activated macrophages. The number of macrophages detectable in the peritoneal cavity was almost the same in LC 9018- and C. parvum-treated mice. Growth of Listeria monocytogenes was inhibited by pretreatment with LC 9018. Inhibition of L. monocytogenes was also observed after the same pretreatment with C. parvum. It was suggested that macrophages activated with LC 9018 were involved in the protective immunity to P. aeruginosa.     

Amelioration of Skewed Th1/Th2 Balance in Tumor-Bearing and Asthma-Induced Mice by Oral Administration of Agaricus blazei Extracts

We showed in a previous study that hot-water extracts of Agaricus blazei (Agaricus extracts) had anti-tumor activity to Meth A fibrosarcoma, but it remains unclear whether the Agaricus extracts ameliorate the skewed balance of type-1 T helper (Th1) and type-2 T helper (Th2) cells. We examined whether Agaricus extracts effect the skewed Th1/Th2 balance in tumor-bearing and asthma-induced mice. When Meth A-bearing mice were given orally either Agaricus extracts or water once a day starting 5 days after tumor implantation, spleen T cells, prepared from tumor-bearing mice treated with Agaricus extracts, in response to anti-CD3 monoclonal antibody produced significantly higher levels of interferon γ (IFN-γ) than that of controls. The mRNA expression of IFN-γ-inducing protein 10 and the frequency of CD69⁺ or CD49d⁺ cells, among activated T cells infiltrated into tumors, significantly increased in Agaricus-treated mice, compared with those of tumor-controls. In asthma-induced mice, treatment with the Agaricus extracts caused significant downregulation of OVA-specific antibody responses of IgG1 and IgE but not of IgG2a, and significantly decreased total cell numbers, levels of interleukin 5, and eosinophil numbers in bronchial alveolar lavage fluids. IFN-γ production by anti-CD3-stimulated spleen cells, obtained from Agaricus-treated mice, significantly increased. Our results strongly suggest that oral administration of Agaricus extracts ameliorates the Th1/Th2 balance from the Th2-skewed conditions.     

Mitogenic effect of 12-0 tetradecanoyl phorbol 13-acetate on non-human primate mononuclear cells andin vitro interaction with Epstein-Barr virus transformation

Summary 12-0 tetradecanoyl phorbol 13-acetate (TPA), known to promote tumors in mice and also to enhance viral transformation as well as induction of viral antigens, was demonstrated to be mitogenic to peripheral blood mononuclear cells from rhesus monkeys and three species of marmosets. Even though mitogenic response varied between species and within species, the mitogenic dose response due to TPA was comparable to the response of phytohemagglutinin (PHA-P). A significant synergistic effect of PHA-P and TPA on mononuclear cells from marmosets was evident when they were used together at optimal doses. TPA also increased the efficiency ofin vitro transformation of marmoset lymphocytes by Epstein-Barr virus.With 5 FiguresThis study was supported partly by National Cancer Institute, Division of Cancer Cause and Prevention, NIH, Program Resource Contracts No. 1-CP-8-1023, 1-CP-VO-81039-66 and Grant No. R-01-CA-21665 to M.N.     

Regression and prevention of autochthonous tumors induced by 3-methylcholanthrene after injection of a T-cell receptor alpha /beta positive and CD4/CD8 double negative T-cells.

Both the therapeutic and preventative effects of a murine T-cell line, tMK-2, with T-cell receptor (TCR) alpha/beta positive and CD4-/8- double negative (DN) phenotype against autochthonously tumors induced by subcutaneous (s.c.) injection of 3-methylcholanthrene (MC) were examined. Complete regression of the tumor was observed when administration of tMK-2 cells was begun on tumors 5 mm in diameter. The tumor mass in five out of five mice was reduced in size after the administration of tMK-2 cells regardless of the routes of administration: s.c. injection of tMK-2 cells (5 x 10(7) cells) once a week around tumors, intraperitoneal (i.p.) injection (5 x 10(7) cells), or intravenous (i.v.) injection (1 x 10(7) cells). The tumors regressed to the status of a scar within 1 month of initial injection, and this status was maintained throughout the remainder of the 3 months period of tMK-2 cell injection. One month after discontinuation of tMK-2 cell administration, the diameter of the tumors had not increased regardless of the route of injection. The control groups consisted of either untreated mice, mice with i.v. injection of 1 microg of recombinant murine interleukin (IL)-12 once a week, or mice with s.c. injection of autologous splenocytes (5 x 10(7)) from BALB/c mice once a week. Continuous growth of tumors was observed in each group and all control mice died due to bleeding ulcerations of the tumors. Tumor development was effectively prevented when tMK-2 cells were administrated 1 week after the s.c. injection of MC. In the groups receiving s.c., i.p., and i.v. injection of tMK-2 cells, no MC-induced tumors developed, whereas four out of five of the control mice developed autochthonous tumors. The tMK-2 cells also exerted in vitro NK-like cytotoxic activity, and their killing activity was strongly increased in the presence of both IL-2 and IL-12. These results suggest that the injected T-cells with TCR alpha/beta positive and CD4- /8- DN phenotype and NK-like activity are important in the therapy as well as the prevention of tumor development.     

Comparison of the humoral and cellular immune responses to two preparations ofCryptosporidium parvumCP15/60 recombinant protein

This study compares the immune responses produced by immunising mice and rabbits with two preparations of the recombinant 15/60 kDa protein ofCryptosporidium parvum. GenomicC. parvumDNA was amplified and the recombinant protein was synthesized as a fusion protein with glutathione-S-transferase inEscherichia coliand in the eukaryotic system of baculovirus/insect cells. Both recombinant proteins induced similar levels of serum antibodies against the fusion recombinant protein, but the eukaryotic recombinant protein triggered a stronger humoral response toC. parvum. Similarly, increased lymphoproliferation occurred only after stimulation of spleen cells from mice immunised with the eukaryotic recombinant protein. This suggests that the eukaryotic protein is a better candidate for immunological studies on cryptosporidiosis.     

Immunity to transplantable carcinogen-induced fibrosarcomas in B2/B2 chickens. III. Tumor growth inhibition by local delayed hypersensitivity reactions to unrelated antigens

Delayed hypersensitivity (DH) reaction to human Ig, corynebacterium parvum or allogeneic cells at the site of tumor cell injection, suppressed the fibrosarcoma (SCFS) growth in chickens. Spleen cells of SC chickens sensitized to human Ig or C. parvum suppressed SCFS growth when adoptively transferred with tumor cells, but only when the sensitizing antigen was present locally. This suppression did not occur in irradiated recipients. SCFS I cells injected into wattles of chickens immune to the tumor, provoked a local DH reaction. Spleen cells from donors sensitized to SCFS I adoptively transferred immunity to both SCFS I and SCFS II when cells of the two tumors were mixed together before injection, but not when SCFS II cells were injected alone. Tumor-specific and nontumor-specific DH may be essential for local suppression of fibrosarcoma growth in chickens.     

The Role of Superoxide Anion and Lysosomal Enzymes in Anti-Listerial Activity of Elicited Peritoneal Macrophages

The in vitro effect of superoxide anion and lysosomal enzyme activity on the killing of Listeria monocytogenes EGD (listeria) by peritoneal macrophages (PM) was investigated. Generation of superoxide anion by PM stimulated with phorbol myristate acetate (PMA) was significantly increased by intraperitoneal injection of Lactobacillus casei YIT 9018 (LC9018) or Corynebacterium parvum (CP), but not by injection of peptone. However, superoxide anion generation by LC9018-elicited PM stimulated with listeria was not increased any more than that by peptone-elicited PM, and generation of superoxide anion by the PM was affected by the difference in stimuli. The killing of listeria by LC9018- or CP-elicited PM in vitro was significantly less than that by peptone-elicited PM or resident PM. Significant correlation was observed between the anti-listerial activity of PM and the intracellular killing of listeria by PM. On the other hand, beta-glucuronidase and beta-N-acetyl-D-glucosaminidase activities of LC9018-elicited, CP-elicited, or resident PM were significantly weaker than those of peptone-elicited PM, and no significant correlation was observed between the increase in beta-glucuronidase and beta-N-acetyl-D-glucosaminidase activities and the increase in anti-listerial activity. These results suggest that increase in the activity of lysosomal enzymes such as beta-glucuronidase and beta-N-acetyl-D-glucosaminidase is not correlated with the anti-listerial activity of PM, and that superoxide anion has very little effect on the anti-listerial activity of PM in vitro.     

Inhibition of bacillus Calmette-Guérin-induced nitric oxide in bladder tumor cells may improve BCG treatment

Bacillus Calmette-Guérin (BCG) is considered to be one of the most effective treatments for superficial and in situ bladder cancer. However, either failure to respond initially or relapse within the first 5 years of treatment has been observed in some patients. As nitric oxide (NO) has been detected in the bladder of BCG-treated patients, we analyzed the role of endogenous NO generated after BCG treatments on human (T24) and murine (MB49 and MBT2) bladder tumor cells in the viability of tumor and immune cells, both in vitro and in vivo. In vitro inhibition of cancer cells after BCG treatment was evaluated by cell titer assay. NO production was determined as nitrite by Griess reagent. The death of immunocytes was evaluated by 51Cr release. Tumor histology with hematoxylin and eosin and Masson's trichrome staining was performed. BCG induced a direct inhibition of tumor cell growth in vitro, independently of NO levels. Besides, BCG-mediated NO production by tumor cells induced the death of spleen and peritoneal cells in syngeneic mice. The in vivo inhibition of NO synthase (NOS) activity by NG-nitro-L-arginine methyl ester in combination with BCG, improved tumor regression by generating a healing tissue. The increase of NO generated after BCG administration may induce the death of immunocytes. The in vivo inhibition of NO ameliorated immunotherapy with BCG by additional tumor growth inhibition. Our results suggested the possibility that the final outcome of patients with bladder tumors may improve by modulating NOS activity concomitantly with BCG therapy.     

Antibody treatment of human tumor xenografts elicits active anti-tumor immunity in nude mice

Athymic nude mice bearing subcutaneous tumor xenografts of the human anti-colorectal cancer cell line SW480 were used as a preclinical model to explore anti-tumor immunotherapies. Intratumor or systemic treatment of the mice with murine anti-SW480 serum, recombinant anti-SW480 polyclonal antibodies, or the anti-colorectal cancer monoclonal antibody CO17-1A, caused retardation or regression of SW480 tumor xenografts. Interestingly, when mice that had regressed their tumors were re-challenged with SW480 cells, these mice regressed the new tumors without further antibody treatment. Adoptive transfer of spleen cells from mice that had regressed their tumors conferred anti-tumor immunity to na?ve nude mice. Pilot experiments suggest that the transferred anti-tumor immunity is mediated by T cells of both gammadelta and alphabeta lineages. These results demonstrate that passive anti-tumor immunotherapy can elicit active immunity and support a role for extra-thymic gammadelta and alphabeta T cells in tumor rejection. Implications for potential immunotherapies include injection of tumor nodules in cancer patients with anti-tumor antibodies to induce anti-tumor T cell immunity.