Endogenous tumor necrosis factor induction with Bordetella pertussis vaccine as a triggering agent and its therapeutic effect on MM46 carcinoma-bearing mice

Induction of endogenous tumor necrosis factor (TNF) by administration of Bordetella pertussis vaccine (BPV) as a triggering agent and its therapeutic effect against MM46 carcinoma were investigated in C3H/He mice. Test triggering agents were injected intravenously into mice after intravenous injection of 4-fold dilution of macrophage activating factor (MAF) or 10(4) units of murine interferon-gamma (Mu-IFN-gamma). Then sera were obtained from the mice, and their TNF activities were assayed on L-929 cells by the method of Ruff and Gifford. The triggering activity of BPV was the highest among those of conventional triggers, such as lipopolysaccharide (LPS) of Escherichia coli, and OK-432. The levels of serum TNF activity triggered by BPV (4 X 10(9) cells), LPS of E. coli (3 micrograms) and OK-432 (3 KE) were 5350, 85 and 102 units/ml, respectively. Growth of MM46, a spontaneous mammary carcinoma cell line of C3H/He was observed for 35 days after tumor inoculation and was suppressed significantly by intravenous injection of MAF and BPV (4 X 10(9) cells). On local injection of BPV (2 X 10(9) cells) into murine tumors, complete regression was observed in 67% of the mice tested with or without MAF priming on day 25 after tumor inoculation, and intratumoral TNF activity was observed even in the case of the single injection of BPV.     

The Changing Pattern of the Splenic Lymphocyte Subsets in Tumor-bearing Mice after Oral Treatment with OK-432

The aim of this study was to investigate the influence of oral administration of OK-432 on the tumor growth of tumor-bearing mice. In addition, the changing pattern of the splenic lymphocyte subsets of tumor-bearing mice was evaluated by flow cytometry. OK-432 at a dose of 0.1, 1 or 10 KE was administered orally every 3 days or every other day for 30 days to subcutaneously Meth A tumor-inoculated mice. The tumor growth was significantly inhibited in the 1 KE every 3 days group, in the 1 KE every other day group and in the 10 KE every 3 days group. In the 10 KE every other day group, OK-432 inhibited the tumor growth on days 10 and 20, while the agent did not show a marked inhibitory effect on day 30. The percentages of splenic L₃T₄-positive cells and splenic asialo GM₁-positive cells were significantly increased in the 1 KE every other day group, while the Lyt2⁺/ Thyl.2⁺ratio was decreased. On the other hand, in the 10 KE every other day group, OK-432 showed no effect on the percentages of splenic L₃T₄-positive cells and Lyt2⁺/Thyl.2⁺ratio on days 20 and 30. Our results suggest that the antitumor effect of oral administration of OK-432 may be correlated with the changing pattern of L₃T₄-positive cells and Lyt2⁺/Thyl.2⁺ratio.     

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).     

Enhancement of Blood Stasis and Vascular Permeability in Meth-A Tumors by Administration of Hyperthermia in Combination with Tumor Necrosis Factor

Blood stasis and vascular permeability induced by tumor necrosis factor (TNF) in Meth-A tumors transplanted in BALB/c mice were significantly enhanced by hyperthermia at 40°C for 30 min immediately following TNF administration. A dose-dependent, sustained decline in the intratumoral blood flow rate occurred following the administration of TNF alone (i.v.; 5 × 10³, 5 × 10⁴, or 5 × 10⁵ JRU/kg) and was enhanced by the administration of hyperthermia in combination with the TNF, even though no decline occurred with hyperthermia alone. The combination of TNF at 5 × 10⁵ JRU/kg and hyperthermia resulted in a blood flow ratio (ratio of blood flow after administration to that before) of 0.47 at 1 h compared with a ratio of 0.65 at 1 h after TNF alone. The blood flow in normal skin sites did not decrease in any case. The permeability of the intratumoral vasculature similarly increased in a dose-dependent manner after the administration of TNF alone and was further increased by combination with hyperthermia, even though no increase occurred with hyperthermia alone. The mean permeability in mice receiving TNF alone at 5 × 10⁵ JRU/kg was 1.35 times that in untreated mice. In mice receiving TNF at the same dose together with hyperthermia, the ratio was increased to 1.65. The results suggest that TNF selectively suppresses intratumoral blood flow, that this effect is enhanced by mild hyperthermia, and that the mechanism of the suppression by TNF with or without hyperthermia partly involves an increase in blood vessel permeability.     

Analysis of CD8 T-cell response by IFNgamma ELISPOT and H-2L(d)/pRL1a tetramer assays in pRL1a multiple antigen peptide-immunized and RL male 1-bearing BALB/c and (BALB/c x C57BL/6) F(1) mice

We previously identified an H-2L^d-binding peptide pRL1a (IPGLPLSL) on RL male 1 that is predominantly recognized by cytotoxic T-lymphocytes (CTLs). MAP is a multibranched lysine core with antigenic peptides. Immunization of BALB/c mice with pRL1a MAP effectively induced pRL1a CTLs. Here, we demonstrate the presence of pRL1a-recognizing CD8⁺ T-cells in pRL1a MAP-immunized and RL male 1-bearing BALB/c and (BALB/ cxC57BL/6)F₁ mice by using IFNγ ELISPOT and H-2L^d/pRL1a tetramer assays. A few IFNγ ELISPOTs and no tetramer-positive cells were detected ex vivo in spleen cells from BALB/c mice immunized with pRL1a MAP. After a single in vitro stimulation with RL male 1, 432 and 741 IFNγ ELISPOTs/10⁵ cells were detected and tetramer-positive CD8⁺ T-cells occurred at relative frequencies of 5.7% and 30.8% in splenic CD8⁺ T-cells from mice that had been doubly and triply immunized, respectively, against pRL1a MAP. Tetramer-positive cells displayed two distinct cell populations, CD62L^low and CD62L^high. Secondary in vitro stimulation expanded CD62L^high cells more efficiently than CD62L^low cells. Furthermore, a higher frequency of IFNγ-producing and tetramer-positive CD8⁺ T-cells was detected ex vivo in RL male 1-bearing semi-allogeneic (BALB/cxC57BL/6)F₁ than in BALB/c mice on day 14 after tumor inoculation.     

Intratumoral induction of tumour necrosis factor by systemic administration of Bordetella pertussis vaccine

Intratumoral induction of tumour necrosis factor (TNF) by administration of Bordetella pertussis vaccine (BPV) as compared with that by the agent OK-432 was investigated in mice. Two hours after such administration tumour tissues tested were resected from the mice, homogenised, and the TNF activities in the homogenate were assayed using a L-929 fibroblast assay. Intravenous injection of BPV into mice bearing the MM46 carcinoma resulted in a greater concentration of TNF in the tumour homogenate than in the serum. With OK-432, however, there was a greater concentration of TNF in the serum than in the tumour homogenates. A high level of intratumoral TNF induction by BPV was also observed in mice bearing Meth A fibrosarcoma or Lewis lung carcinoma. The therapeutic effect against the Meth A fibrosarcoma was in parallel with the intratumoral TNF activity. Intratumoral TNF activity is therefore believed to be a good index of therapeutic effect.     

Dose of Adenoviral Vectors Expressing Interleukin-2 Plays an Important Role in Combined Gene Therapy with Cytosine Deaminase/5–Fluorocytosine: Preclinical Consideration

Using a syngeneic murine model, we investigated the therapeutic efficacy of combined gene therapy using adenoviral vectors expressing murine interleukin-2 (AdmIL-2) and Escherichia coli cytosine deaminase (AdCD). In a subcutaneous tumor model, tumor-bearing mice were treated with an intratumoral injection of adenoviral vectors and received an intraperitoneal administration of 5–fluorocytosine (5–FC). Only the mice treated with AdCD (2×10⁸ pfu) and an intermediate dose of AdmIL-2 (1×10⁶ pfu) survived significantly longer than mice treated with AdCD alone ( P <0.01). Moreover, 40% of these treated mice obtained complete remission from tumor-bearing status. The cytotoxicity of splenocytes obtained from the treated mice was related to the survival period. Tumor-specific cytotoxic T lymphocyte assay showed that the cell-mediated cytotoxic response was specific for parental tumor cells. In a hepatic metastasis model, mice treated with an intravenous administration of both AdCD (2×l0⁸ pfu) and an intermediate dose of AdmIL-2 (1×10⁶ pfu) demonstrated the most significant reduction of metastatic foci and the longest survival following a 5–FC administration. These results suggest that gene therapy combined with AdmIL-2 and AdCD may be a promising strategy for clinical application and, in addition, that translation of combined gene therapy from murine models into the clinical setting will require careful attention to the variables of cytokine expression levels in the design of clinical trials and in the evaluation of treatment efficacy.     

Epidermal Growth Factor Prolongs Survival Time of Tumor-bearing Mice

We observed that human epidermal growth factor (hEGF) alone prolonged the survival time of mice bearing various murine syngeneic tumors as well as athymic nude mice bearing human xenografts. No changes in the subcutaneous solid tumor mass volume were observed. Prolongation of survival time by hEGF was observed in mice bearing murine epidermoid carcinoma (BSC) and human gastric carcinoma (KATO III), but not in murine epidermoid carcinoma (KLN205) or human epidermoid carcinoma (A431). Human tumor cells such as A431, KATO III, and murine tumor cells, KLN205, BSC had roughly 2 × 10⁶, 3 × 10⁴, 1.3 × 10³ and 1 × 10³ EGF receptors/cell, respectively. Although KLN205 and BSC tumor cells maintained nearly the same number of EGF receptors, the effects of hEGF were very different. Although A431 tumor cells had nearly 100 times more receptors than KATO III cells, the prolongation of survival time of mice bearing A431 by hEGF was no better than that of mice bearing KATO III. Accordingly, it appears that this prolongation of survival time by hEGF is independent of the number of EGF receptors on tumor cells. In addition, hEGF was shown to inhibit experimental pulmonary metastasis of murine BSC tumor, but was ineffective with murine KLN205 tumor. These results suggest that prolongation of survival time by hEGF may result from the inhibition of tumor cell metastasis and EGF may play a role in preventing the metastasis of certain malignant neoplasms unrelated to its effects through the EGF receptor on tumor cells.     

Chemosensitivity of peritoneal micrometastases as evaluated using a green fluorescence protein (GFP)-tagged human gastric cancer cell line

The Chemosensitivity of micrometastases in the peritoneal cavity to a 5-fluorouracil derivative (TS-1) was examined with a micrometastasis model featuring a human gastric cancer cell line tagged with the green fluorescence protein ( GFP ) gene in nude mice. Peritoneal metastases on the omentum and mesentery could be specifically visualized even when minute or dormant and also externally monitored noninvasively under illumination with blue light from 1 day after intraperitoneal (i.p.) injection of tumor cells. Metastatic deposits formed after i.p. injection of 2×10⁶ tumor cells were significantly reduced by TS-1 in a dose-dependent manner (15–20 mg/kg), when it was orally administered from day 1 post-injection for 4 weeks (early administration). No such inhibition was evident after injection of 1×10⁷ tumor cells. When 2×10⁶ tumor cells given injection, the ascites-free period in TS-1-treated mice was significantly longer than in their untreated counterparts. Survival of TS-1-treated mice (5/15) was also significantly higher than the zero rate in controls (0/15), with 4 out of 5 surviving mice being free from peritoneal metastasis and the exception having only a few dormant metastases. In contrast, when TS-1 was administered starting from day 7 post-injection for 4 weeks (late administration), the survival and ascites-free period of the TS-1-treated mice were not significantly influenced. The results indicate that the Chemosensitivity of peritoneal metastases to TS-1 is dependent on the number of i.p. tumor cells and the timing of drug administration. Peritoneal micrometastases at an early stage are most susceptible and can be effectively eliminated by oral administration of an anti-cancer agent, which leads to the longer survival and better quality of life (QOL) of the mice. (Cancer Sci 2003; 94: 112–118)     

Enhanced Antitumor Effect of Recombinant Human Tumor Necrosis Factor in Combination with Recombinant Human Granulocyte Colony-stimulating Factor in BALB/c Mice

The synergistic antitumor effect of tumor necrosis factor (TNF) and granulocyte colony-stimulating factor (G-CSF) was investigated. G-CSF was administered subcutaneously to BALB/c mice inoculated with Meth-A cells at a dose of 2.5 μ g/day for 5 consecutive days. When TNF (1 × 10³ U) was administered intravenously to mice which had been pretreated with G-CSF, tumor growth showed a 74.1% inhibition 17 days after the tumor cell inoculation, compared to that of untreated mice. In this experiment, G-CSF significantly ( P <0.025) enhanced the antitumor effect of TNF. The in vitro cytotoxicity of TNF (10 U/ml) towards Meth-A cells was increased about 5.2-fold in the presence of neutrophils (E/T=50) as compared to the cytotoxicity obtained with TNF alone. A combination of TNF and G-CSF (50 ng/ml) in the presence of neutrophils, resulted in a 2.1 times greater cytotoxicity against Meth-A cells as compared to that obtained without G-CSF. Significant augmenting effects of G-CSF on superoxide (O₂⁻) production by TNF-stimulated neutrophils were observed. These observation suggest that the neutrophil plays an important role in the antitumor action of TNF on Meth-A cells, and that the antitumor effect of TNF is enhanced by combination with G-CSF.     

Changes in Lymphocyte Subsets Following Multiple Administration of Recombinant Interleukin-2 plus Recombinant Interferon-beta or -gamma in Tumor-bearing Mice

Treatment with a combination of recombinant human interleukin-2 (rHIL-2) and recombinant mouse interferon-beta (rIFN-β) or -gamma (rIFN-γ) showed a significant antitumor effect against sc adenocarcinoma 755 in mice, although treatment with either one alone had almost no effect. The combination of rHIL-2 and rIFN-β caused regression of the tumor but the combination of rHIL-2 and rIFN-γ did not. Injection of tumor-bearing mice with the combinations of rHIL-2 and rIFN resulted in marked increases in the total number of peritoneal lymphocytes, and the frequency of Lyt-2⁺ cells was more markedly increased by the combination of rHIL-2 and rIFN-β than by the combination of rHIL-2 and rIFN-γ. In Winn assay, elimination of the Lyt-2⁺ population abolished the protective capacity of the peritoneal cells. The subsets of thymocytes were drastically changed when mice were bearing a tumor or were treated with cytokines. In particular, Lyt-2⁺/L3T4⁺ cells were decreased in tumor-bearing mice, but many Lyt-2⁺/L3T4⁺ cells were maintained in the thymus by treatment with a cytokine alone. When treated with rHIL-2 and rIFN-β the Lyt-2⁺/L3T4⁺ cells were markedly decreased, while Lyt-2^-/L3T4^- T-cells were increased, but these subsets were little changed by treatment with rHIL-2 plus rIFN-γ. Thus, injections of rHIL-2 and rIFN-β into tumor-bearing mice resulted in a high frequency of Lyt-2⁺/L3T4^- cells in the peritoneal cavity, together with changes in the T-cell subsets in the thymus. These results suggest that maturation of T-cells in the thymus may be an important step in the pathway by which cytokine treatment brings about regression of tumors.     

Radioimmunotherapy of human synovial sarcoma using a monoclonal antibody against FZD10

We previously reported Frizzled homolog 10 ( FZD10 ), a member of the Frizzled family, to be a promising therapeutic target for synovial sarcomas. In this report, we established a murine monoclonal antibody (MAb), namely, MAb 92-13 that had specific binding activity against native FZD10 product expressed in synovial sarcoma cell lines. Subsequent immunohistochemical analyses with the MAb 92-13 confirmed an absence or hardly detectable level of FZD10 protein in any normal human organs. We confirmed the specific binding activity of this MAb in vivo after injection of fluorescent-labeled MAb i.p. or i.v. into the mice carrying synovial sarcoma xenografts by the use of the in vivo fluorescent imaging system as well as radioisotopes. Moreover, MAb 92-13 was effectively internalized into the synovial sarcoma cells after its binding to FZD10 on the cell surface. A single i.v. injection of the Yttrium-90 (⁹⁰Y)-MAb 92-13 drastically suppressed tumor growth of synovial sarcoma in mice without any severe toxicity. Median time to tumor progression was 58 days for mice treated with ⁹⁰Y-MAb 92-13 and 9 days for mice treated with non-labeled antibody control or untreated mice (difference = 49 days; P  = 7 × 10⁻⁵). This result indicates that MAb 92-13 could be utilized as the novel treatment modality for synovial sarcoma and other FZD10-positive tumors. ( Cancer Sci 2008; 99: 432–440)     

Modulation of Immune Response against Tumor Cells by the in vivo Administration of an Autoreactive Th Clone

The immunization of C3H mice with irradiated syngeneic MM48 tumor cells induced specific tumor-neutralizing cells (TNC). The TNC activity was mediated by the L3T4⁺, Ly-2⁻ T cell population, and the generation of TNC coincided with the appearance of delayed-type hypersensitivity response against MM48 antigen. The administration of an auto-I-A^k reactive T helper cell clone MS202 to normal C3H mice resulted in the facilitation of growth of MM48 tumor due to the induction of T suppressor (Ts) cells. Splenic T cells from animals given this T cell clone inhibited the TNC activity of immunized mice resulting in the escape of MM48 from the TNC effect. The surface phenotype of the Ts cells was L3T4⁺, Ly-2⁻. The Ts cells induced by the clone MS202 were totally antigen-nonspecific, and were able to suppress both the effector and inductive processes of TNC. The results suggest the presence of a physiological MHC-restricted T cell circuit that regulates immune responses against the growing tumors.     

A SYNTHETIC SINGLE-STRANDED DNA, POLY(dG, dC), INDUCES TNTERFERON-α/β AND -γ, AUGMENTS NATURAL KILLER ACTIVITY, AND SUPPRESSES TUMOR GROWTH

When various synthetic double- or single-stranded DNAs were incubated with spleen cells from mice (BALB/c or CDF¹) at 37° for 20 hr, it was found that some of the DNAs augmented NK activity and produced factors in the culture supernatants which showed antiviral activity and activity to render mouse macrophages cytotoxic toward tumor cells. Poly(dG, dC) showed the strongest activities, when incubated with spleen cells from lipopolysaccharide-nonresponsive mice, C3H/HeJ. The activity of the culture supernatant to activate macrophages was completely abolished by a small amount of anti-IFNγ antibody. On the other hand, the virus-inhibitory activity of the supernatant was mostly neutralized by anti-IFNα/β. When IMC tumor cells (5 × 10⁵ cells) were mixed with poly(dG, dC) (100 μg) and then inoculated intradermally into CDF¹ mice, the tumor did not take, while tumors grew progressively and killed the mice in a control group inoculated with tumor cells alone. Direct cytotoxicity of poly(dG, dC) at a concentration of 1,000 μg/ml against IMC cells was not observed in vitro .     

Effect of Human Macrophage Colony-stimulating Factor on Granulopoiesis and Survival in Bone-marrow-transplanted Mice

Human macrophage colony-stimulating factor (hM-CSF) has been isolated from normal human urine and purified to a homogenous protein. The effect of hM-CSF on granulopoiesis was investigated in BALB/c mice transplanted with a suboptimal number of bone marrow cells. Lethally irradiated (7.8 Gy) mice were transplanted with 1 × 10⁶ syngeneic mouse bone marrow cells and treated with a daily intraperitoneal dose of 64 μg/kg of hM-CSF for 5 days following the transplant. The hM-CSF injection resulted in stimulation of the recovery of blood neutrophils as well as an increase in the number of granulocyte-macrophage progenitor cells (CFU-GM) in the femur and spleen. The survival of lethally irradiated mice was dependent on the cell number transplanted; most mice transplanted with 2 × 10⁴ cells died within 2 weeks. The recovery of hematopoiesis in mice transplanted with 2 × 10⁴ cells was modestly but significantly stimulated by hM-CSF administration initiated from 5 days before or 1 day after transplantation for a 5-day period. Furthermore, the hM-CSF administrations markedly reduced the mortality in these mice during the early period after the transplantation. Since anaerobic bacteria were frequently detected in arterial blood immediately before the deaths but were not found in the surviving mice, it is speculated that early deaths occurring within 2 weeks after the transplant may be caused by opportunistic infections, and hM-CSF injection may prevent these mortal infections through its stimulating effect on monocyte-macrophage functions that are responsible for the production of hematopoietic regulators.     

Hyperthermic Enhancement of the Antitumor Effect of Natural Human Tumor Necrosis Factor-α and -β: an in vitro and in vivo Study

A synergistic antitumor effect of natural human tumor necrosis factor-β (TNF-β) in combination with hyperthermia was found, in comparison with that of TNF-α, using an in vitro antiproliferative assay on a human colon cancer cell line (RPMI4788) and an in vivo tumor growth inhibition assay on Meth A sarcoma cells. In vitro combined treatment with TNF-β (10,000 U/ml) and hyperthermia (at 43° for 60 min) synergistically inhibited the proliferation of the cells. Combined effects of TNF-α or natural human interferon-α or -γ (IFN-α, -γ) and hyperthermia were also examined, and furthermore, the combinations of TNFs and IFNs were examined in combination with hyperthermia at 42°; their antiproliferative effects were further augmented by hyperthermia. In vivo growth of Meth A sarcoma cells (5 × 10⁵), transplanted subcutaneously into BALB/c mice, was inhibited significantly ( P <0.05) with the combination of TNF-α or -β (2 × 10⁵ U/mouse) and hyperthermia (at 43° for 60 min) as compared to either a single intravenous injection of TNF-α or -β alone or the hyperthermia alone. The influence of TNF-β and hyperthermia on the cell cycle was examined. Flow cytometric analysis showed that RPMI4788 cells treated with TNF-α or -β accumulated in the S phase of the cell cycle, and that hyperthermia (at 42° for 60 min) alone had no influence on the cell cycle and did not augment the S phase accumulation of the cells treated with TNF-α or -β.     

Insulin and Insulin-like Growth Factor 1 Stimulate Proliferation of Metastatic Variants of Colon Carcinoma 26

The proliferation rate of malignant cells in vivo is one of the important factors which affect the formation of tumor metastasis. A highly metastatic variant of mouse colon adenocarcinoma 26 (NL- 17) grew more rapidly than a low-metastatic variant (NL-44) both in vitro and in vivo. The effect of growth factors on the proliferation of NL-17 and NL-44 cells was examined in serum-free medium. Among growth factors examined, human insulin and insulin-like growth factor 1 (IGF-1), which were produced by gene engineering techniques, stimulated the growth of metastatic NL-17 and NL-44 cells as determined by thymidine incorporation and cell counts. DNA synthesis and cell proliferation of the high-metastatic NL-17 was stimulated to a greater extent by insulin and IGF-1 than those of the low-metastatic NL-44. These findings suggest that circulating growth factors could enhance the formation of tumor metastasis. Scatchard analysis of [¹²⁵I]IGF-1 binding to NL-17 and NL-44 showed that each cell line had an almost equal number of IGF-1 receptors (1.37 × 10⁵/cell and 1.26 × 10⁵/cell, respectively), which had similar dissociation constants (8.94×10⁻¹⁰ M and 9.54×10⁻¹⁰ M , respectively). Since the number and affinity of IGF-1 receptors are equivalent between low- and high-metastatic cells, the intracellular events which result in the cell growth after binding of IGF-1 may differ between NL-17 and NL-44 cells.     

Murine Asialo GM1+CD8+ T Cells as Novel Interleukin-12-responsive Killer T Cell Precursors

Freshly isolated CD8⁺ T cells, but not CD4⁺ T cells, contained 20–30% of asialo GM1⁺ (ASGM1⁺) T cells which were distinct from ASGM1⁺NK1.1⁺ natural killer cells. This novel ASGM1⁺CD8⁺ T cell subpopulation showed a strong proliferative response to interlenkin-12 (IL-12) in the presence of IL-2. Culture of ASGM1⁺CD8⁺ T cells with IL-12 plus IL-2 allowed the generation of anomalous killer T cells concomitantly with the accumulation of cytolytic molecules. Moreover, ASGM1⁺CD8⁺ T cells produced high levels of interferon-γ (IFN-γ), but not IL-4, upon stimulation with IL-12 plus IL-2. Such immune responses were not observed in ASGM1⁻ CD8⁺ T cell snbpopulations constituting the majority of CD8⁺ T cells. These results demonstrated that ASGM1⁺CD8⁺ T cells are a novel subpopulation of IL-12-responsive and IFN-γ-producing killer T cell precursors.     

Activation of Intestinal Mucosal Immunity in Tumor-bearing Mice by Lactoferrin

We have previously demonstrated that oral administration of bovine lactoferrin (bLF) markedly increases CD4⁺ and CD8⁺ T cells and NK (asialoGM1⁺) cells in the blood of tumor-bearing mice and enhances anti-metastatic activity. In this paper, we document that oral administration of bLF and bLF-hydrolysate (bLFH) is associated with strong increases in CD4⁺ and CD8⁺ T, as well as asialoGM1⁺ cells in lymphoid tissues and lamina propria of the small intestine in mice, especially in tumor-bearing animals in which Co26Lu cells were implanted subcutaneously. Moreover, IgM⁺ and IgA⁺ B cells in lamina propria of the small intestine were also significantly increased by bLF and bLFH. Bovine apo-transferrin (bTF) did not exhibit such activity. In the colon, only CD8⁺ cells were significantly increased by treatment with bLF, while asialoGM1⁺ cells were significantly decreased. bLF and bLFH induced cytokines to activate T, B and asialoGM1⁺ cells. Administration of bLF and bLFH, but not bTF, increased production of interleukin-18 (IL-18), interferon-gamma (IFN-γ) and caspase-1 in the mucosa of the small intestine. Particularly high levels of IL-18 were found in the epithelial cells of the small intestine. Moreover, administration of bLF and bLFH, but not bTF, induced IFN-γ presenting cells in the small intestine. Caspase-1, which processes proIL-18 to mature IL-18, was also induced in the epithelial cells of the small intestine following treatment with bLF and bLFH, but not with bTF. These results suggest that enhanced production of IL-18 and IFN-γ and caspase-1 induction by treatment with bLF may be important for elevation of intestinal mucosal immunity.     

Priming effect of interferons and interleukin 2 on endogenous production of tumor necrosis factor in mice

The effects of interferons (IFNs) and interleukin 2 (IL 2) on endogenous production of tumor necrosis factor (TNF) were investigated in mice. Production of serum TNF was triggered by iv injection of OK-432 and tested by in vitro cytotoxicity assay. Injection of recombinant IFN-gamma with OK-432 and tested by in vitro cytotoxicity assay. Injection of recombinant IFN-gamma with OK-432 or of IFN-alpha/beta, recombinant IFN-beta, recombinant IFN-alpha A/D or recombinant IL 2 six hours before OK-432 enhanced TNF production about 10-fold, which indicated priming actions of these compounds in TNF production. These findings suggest that these compounds could also be used as priming agents for endogenous production of TNF in cancer patients.