The contribution of mesangial cell proliferation to progressive glomerular injury

Toll-like receptor signaling in anti-cancer immunity

Masato Okamoto and Mitsunobu Sato

Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, Tokushima, Japan

Abstract: It is important to augment the anti-cancer host response in cancer treatment. Recent studies suggested that the signaling via Toll-like receptors (TLRs) which are newly identified receptor molecules recognizing many pathogens, are involved in the induction of anti-cancer immunity. Seya et al. demonstrated that maturation of dendritic cells (DCs) and cytokine induction by the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guerin (BCG-CWS) are induced via both TLR2 and TLR4. Akira et al. discovered a new molecule of TLR family, TLR9, recognizing unmethylated bacterial CpG-DNA, whose clinical use is expected for cancer therapy as a potent inducer of a helper T cell1 (Th1)-type T-cell response. TLR9-deficient mice did not show any responses to CpG-DNA, including Th1cytokine production and maturation of DCs. We have obtained two molecules, a lipoteichoic acid-related molecule isolated from streptococcal agent OK-432, and a plant-derived 55-kDa protein that can induce Th1 response and elicit a strong anti-cancer effect in vivo and in vitro. Our basic experiments demonstrate that TLR4 signaling is intimately involved in anti-cancer immunity induced by these immunopotentiators. Our clinical examination in oral cancer patients also suggests the requirement of both TLR4 and MD-2 in the OK-432-induced anti-cancer host response. Establishment and clinical use of the methodology for human cancer therapy by utilizing TLR signaling is greatly expected. J. Med. Invest. 50:9-24, 2003

Keywords:anti-cancer immunity, Toll-like receptor (TLR), Bacterial CpG-DNA, OK-432, plant-derived protein

INTRODUCTION
Toll-like receptors (TLRs) that are expressed mainly on macrophages and dendritic cells (DCs), are recently identified receptor molecules that recognize many types of pathogens in addition to host-derived proteins. Macrophages and DCs are not only primarily involved in innate immunity, but are also essential for establishment of adaptive immunity as antigen-presenting cells (APCs). Thus, TLR signaling promotes activation of an innate immune response, and then triggers antigen-specific adaptive immunity (1-4). In the immunotherapy against malignant diseases, it was suggested that the induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs) is most important for eliminating tumor cells, and most immuno-oncologists have discussed how to induce adaptive immunity against cancer. However, the precedent activation of the innate immune system is essential for the subsequent induction of antigen-specific immunity. TLR-mediated activation of innate immunity should be important for the establishment of an effective anti-cancer immune response. Recently, several studies proposed the significance of TLR signaling in the induction of anti-cancer immunity. In this review, we present the recent progress of these studies including our investigation, focused on the involvement of TLR signaling in anti-cancer immunity.

TLRs, and their ligands
The toll gene controls dorsoventral pattern formation during the early embryonic development of Drosophila melanogaster (5). Interestingly, toll participates in anti-microbial immune responses upon infection in adult Drosophila (6). Recently, several mammalian homologues of the Drosophila Toll receptor protein (Toll-like receptors:TLRs) were identified. TLRs are transmembrane proteins and represent a newly recognized family of vertebrate pattern recognition receptors in the innate immune system. A prerequisite for the development of an effective host defense is the recognition of pathogens. TLRs are involved in this first step (2-4).
Eleven members of the TLR family (TLR1 to TLR10 and RP105)(7-12), co-factors (CD14, MD-1 and MD-2)(13-15) and their ligands have been reported (12, 16-46) as shown in Table 1. The well-characterized TLRs are TLR2, TLR4 and TLR9. TLR4 recognizes Gram-negative bacteria-derived lipopolysaccharide (LPS)(32-34) as well as Gram-positive bacteria-derived lipoteichoic acid (LTA)(17, 35). It was also reported that TLR4 mediates LPS-mimetic signal transduction by an anti-cancer agent Taxol, a plant-derived diterpene, in mice but not in humans (36). In addition, TLR4 recognizes host-derived proteins, heat shock protein (HSP) 60 (38, 39) and fibronectin fragment (40). In the antigen recognition by TLR4, MD-2 plays an essential role. MD-2 is physically associated with TLR4 on the cell surface and the TLR4/MD-2 complex confers responsiveness to bacterial components (15). TLR2 recognizes peptidoglycan (PGN)(17, 18), lipoprotein (19-24) and lipoarabinomannan (LAM)(25, 26) derived from Gram-positive bacteria, mycobacteria or mycoplasma. Several studies reported that Gram-positive bacteria-derived LTA is recognized by TLR2 (27-29). TLR9 recognizes bacterial unmethylated CpG DNA and is the receptor that distinguishes bacterial DNA from self-DNA (46).
Subsequent to pathogen-associated molecular pattern engagement, TLRs initiate the signaling via sequential recruitment of myeloid differentiation protein (MyD) 88, IL-1R-associated kinase (IRAK) and TNFR-associated factor (TRAF)6, which in turn activate downstream mediators such as nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) (47, 48). In addition, experiments using MyD88-deficient (MyD88-/-) mice revealed that TLR4 mediates the signaling in an MyD88-independent fashion in addition to an MyD88-dependent fashion (49). Recently, it was reported that newly identified molecule, Toll-interleukin 1 receptor (TIR) domain-containing adapter protein (TIRAP), associates with TLR4, and manages MyD88-independent signal transduction (2, 50). On the other hand, Kawai et al. reported that a transcription factor, interferon (IFN)-regulatory factor (IRF) 3, translocated into the nucleus in response to LPS in MyD88-/-mice (51). It was strongly suggested that IRF3 activation contributes to the MyD88-independent pathway. However, it remains uncertain whether TIRAP is involved in IRF3 activation (Fig. 1). Furthermore, most recent studies have demonstrated findings strongly suggesting that TIRAP mediates the signaling via TLR1, TLR2 and TLR6 in addition to TLR4, and is involved not only in the MyD88-independent but also in the MyD88-dependent signaling pathway (52, 53). The downstream molecular events of TLRs are significant to determine what type(s) of host response(s) are induced against different kinds of, and different doses of pathogens. Progress of this clarification is strongly expected.
TLR-mediated signaling stimulates the maturation of DCs, which migrate to the regional lymph nodes, where they stimulate T cells by presentation of antigen-major histocompatibility (MHC) complex in addition to costimulatory molecules such as CD80 and CD86. TLR signaling acts to trigger adaptive immunity by enhancing expression of MHC molecules in addition to these costimulatory molecules. Furthermore, TLR signaling frequently enhances the production of IL-12, a major helper-T cell 1 (Th1)-inducing cytokine, on APCs (2-4, 54). Thus, it was strongly suggested that the DCs matured by TLR stimulation may induce T-cell differentiation toward Th1 by presenting antigens to the T cells while promoting a Th1-leading situation in the local environment. Therefore, it is possible that the ligands of TLRs are able to be effective immunoadjuvants for cancer therapy. In the next sections, we review the recent progress focused on the TLR ligands as applications for cancer therapy.

Involvement of TLR2, TLR4, and TLR9 in anti-cancer immunity induced by the bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) or unmethylated CpG-DNA
1) BCG-CWS-induced anti-cancer host response via TLR2 and TLR4
Heat-killed mycobacterial cells suspended in mineral oil are potent immunoadjuvants to induce both cell-mediated and humoral immunity, and the CWS fraction of the cells of mycobacteria is the active immunoadjuvant component (55-58). It was reported that BCG-CWS enhances the cytotoxic activity of T cells and macrophages against cancer cells, and elicits an anti-tumor effect in mice and in rats bearing transplantable and authochthonous tumors (59, 60). Further, clinical trials with BCG-CWS were performed in patients with several types of malignancies, and it was demonstrated that BCG-CWS was effective in prolongation of survival of patients especially those with gastric cancer and lung cancer (61-67).
Recently, Seya and his co-investigators reported findings strongly suggesting that BCG-CWS augments the anti-cancer host response through TLR2 and TLR4 (Fig. 1). It was reported that BCG-CWS activates and matures DCs (58). Tsuji et al. reported that BCG-CWS induces tumor necrosis factor (TNF)-α secretion from DCs via both TLR2 and TLR4, and that the secreted TNF-α induces the maturation of DCs (68). The distribution profile of TLR2 and TLR4 matches the response profile of cells for BCG-CWS, and further investigation by Tsuji et al. suggested that the PGN portion of BCG-CWS is an active center for cytokine induction and DC maturation via Toll signaling (58). It was also reported that TLR2 mediates mycobacteria-induced proinflammatory signaling in macrophages (25). These findings strongly suggest that the signaling via TLRs is closely involved in BCG-CWS-induced anti-tumor immunity. However, Azuma and Seya suggested that TLRs are not merely the receptor for establishment of the BCG-CWS-induced anti-tumor host response, and proposed the “two receptor theory". Two types of receptors expressing on APCs, which consist of signal transducing receptor related to maturation and activation of APCs such as TLRs, and phagocytosis-related receptor to induce antigen presentation to T cells, may be essential for the establishment of antigen-specific, adaptive immunity, and BCG-CWS may augment the anti-tumor host response by activating both types of receptors (58, 69). As demonstrated previously, BCG-CWS consists of mycolic acid, arabinogalactan and PGN (57, 59, 70), and distinct portions may play significant roles in the binding and activation of the two receptors on macrophages and DCs. Identification of the phagocytosis receptor(s) is currently under investigation by Seya et al. (58).
2) Bacterial unmethylated CpG-DNA-induced host response via TLR9
The specific immunostimulatory effect of bacterial genomic DNA was first reported by Tokunaga et al., who demonstrated that bacterial DNA activates natural killer (NK) cells and induces IFN production in addition to tumor regression in some mouse models, but vertebrate DNA does not (71-73). In 1995, Krieg et al. demonstrated that CpG motifs in bacterial DNA trigger direct B-cell activation (74, 75). They also reported that CpG content and methylation distinguish vertebrate and bacterial DNAs. Genomic DNA from vertebrates but not from bacteria contains very few CpG dinucleotide motifs (76). Further, CpGs are commonly methylated in vertebrates, while the CpGs are not methylated in bacteria and viruses. This suggestes the possibility that the immune system may have evolved a defense mechanism based on the recognition of unmethylated CpG-DNAs, which could be a sign of foreign DNA (76).
The immune response of synthesized oligodeoxynucleotides (ODN) with CpG motifs was examined. Many studies demonstrated that unmethylated CpG-ODN strongly activates immunocompetent cells such as DCs, macrophages, NK cells, T cells, and B cells, and induces the Th1-like T-cell response including IFN-&ganma; production and CTL induction, in in vitro and in vivo models (74). Roman et al. reported that co-injection of CpG-ODN with a protein antigen greatly enhances the T- and B-cell response to this antigen (77). Significantly, this response is strongly biased toward the generation of a Th1-dependent immunity with all its ramifications, for example a preference for IgG2 immunoglobulin subclasses. It can be expected as an immunotherapeutic agent for cancer, allergy and infectious diseases as a potent Th1 inducer. The efficacy of CpG-DNA in preventing or treating tumor development or metastasis in mice has been examined in several experimental models. In CpG-DNA monotherapy, systemic or local administration of CpG-DNA protected 80% of syngeneic C57BL/6 mice from a lethal challenge of B16 melanoma. Further, SCID mice were also protected against tumor challenge by CpG-DNA, which suggests that neither B nor T cells are required (78). The potential of the CpG-DNA as an adjuvant for cancer vaccines was also examined. CpG-DNA-induced activation of DCs creates a Th1-like cytokine and chemokine environment in the secondary lymphoid organs that promotes cross-priming with strong IFN-&ganma;-secreting CTLs and antibody responses to peptides and protein antigens derived from tumors, and elicits marked anti-tumor activity (79, 80). Despite its promising clinical use, the molecular mechanism by which CpG-DNA activates immune cells has remained unclear.
In 2000, Hemmi et al. discovered the receptor molecule recognizing bacterial DNA (46). The identified protein, TLR9, recognizes the unmethylated CpG motif in bacterial DNA, and mediates an innate immune response (Fig. 1). They generated TLR9-deficient (TLR9-/-) mice and examined the immune effect of CpG-ODN using those mice (46). TLR9-/- mice did not show any response to CpG-DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of DCs. TLR9-/-mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated Th1 response was also abolished in TLR9-/-mice. Thus, it was clarified that the signaling via TLR9 plays an important role in CpG-DNA-induced host response.
Early-stage clinical trials of CpG-DNA as an immunotherapeutic agent for cancers and as an anti-allergic agent are currently on going, and preliminary findings from these trials appear to be encouraging (81).

Involvement of TLR4/MD-2 signaling in anti-cancer immunity induced by an LTA-related molecule, an effective component of OK-432, or by a plant-derived 55-kDa protein
Recently, we obtained 2 molecules that can induce the Th1-dominant state and elicit an anti-cancer effect. One is an LTA-related molecule isolated from a Streptococcus-derived anti-cancer agent OK-432, and another is a 55-kDa protein from Aeginetia indica L (AIL), a parasitic plant, and we obtained findings strongly suggesting that these molecules enhance anti-cancer immunity via TLR4/MD-2 complex. In this section, we present the recent progress of our basic and translational research focused on the involvement of TLR4/MD-2 signaling in anti-cancer immunity induced by these molecules.
1) Isolation of an effective component responsible for OK-432-induced anti-cancer effect
OK-432, a penicillin-killed and lyophilized preparation of a low-virulence strain (Su) of Streptococcus pyogenes (group A) (Chugai Pharmaceutical Co., Ltd., Tokyo, Japan), was developed by Okamoto et al. in 1967 (82), and has been successfully used as an immunotherapeutic agent in many types of malignancies (83-87). We also reported that OK-432-based immunotherapy exhibits a marked therapeutic effect in patients with oral squamous cell carcinoma (88, 89). Previous studies helped to clarify the cellular mechanism of OK-432-induced anti-cancer immunity. Namely, OK-432 elicits anti-tumor effects by stimulating immunocompetent cells such as macrophages, T cells and NK cells, and by inducing multiple cytokines including IL-1, IL-2, IL-6, TNF-α, and IFN-&ganma; (90-92). In addition, OK-432 induces IL-12 and polarizes the T-cell response to a Th1 dominant state (93). However, there has been limited progress in elucidating the molecular mechanism, i.e., in the identification of the effective molecule(s) for inducing anti-cancer immunity in whole bacterial preparation OK-432 and their molecular target(s), such as receptors and signal transducers on immunocompetent cells. Recently, we succeeded in isolating the LTA-related molecule that is most responsible for the anti-cancer effect of OK-432. This molecule designated as OK-PSA was isolated from a butanol extract of OK-432 using a CNBr-activated Sepharose 4B affinity column bound with the monoclonal antibody TS-2, which neutralizes the IFN-&ganma;-inducing activity of OK-432 (94, 95). We previously reported that OK-PSA is a more potent inducer of Th1 cytokines and killer cell activities on human peripheral blood mononuclear cells (PBMCs) than original OK-432, and showed a marked anti-tumor activity in tumor-bearing mice (94, 96-101)
2) OK-PSA-induced anti-cancer immunity via TLR4 signaling
We examined the role of TLR4 in the anti-tumor effect of OK-PSA using C3H/HeJ mice in which TLR4 is mutated and its function is impaired (34). C3H/HeN mice, which have the wild-type TLR4 gene, were used as control animals. In in vitro experiments, the spleen cells derived from C3H/HeN and C3H/HeJ mice were stimulated with OK-PSA for 48 h, then the cytokines in the supernatants were measured. Although Th1-type cytokines such as IFN-&ganma;, IL-12 and IL-18 were significantly induced by OK-PSA stimulation in the splenocytes derived from C3H/HeN mice, these cytokines were not induced in the splenocytes from C3H/HeJ (Fig. 2A). TNF-α and IL-2 were also induced by OK-PSA in the splenocytes from C3H/HeN but not from C3H/HeJ (data not shown). Furthermore, when the expression vector including mouse TLR4 cDNA was transfected into C3H/HeJ-derived splenocytes, the splenocytes acquired the responsiveness to OK-PSA to produce Th1 cytokines (Fig. 2B). Next, we evaluated the anti-tumor effect of OK-PSA in vivo. C3H/HeN and C3H/HeJ mice bearing syngeneic SCCVII tumors were treated with OK-PSA. The peritumoral injection of OK-PSA resulted in significant inhibition of tumor growth and lung metastasis in SCCVII-bearing C3H/HeN mice;however, no effect of OK-PSA was observed in C3H/HeJ mice (Fig. 2C). The cytolytic activities of tumor-infiltrating lymphocytes (TIL) and draining lymph node (LN) cells derived from SCCVII-bearing mice that were administered OK-PSA were also analyzed. The cytolytic activities of TIL and LN cells against SCCVII were markedly increased by OK-PSA administration in C3H/HeN but not in C3H/HeJ (Fig. 2D). These findings strongly suggest that TLR4 signaling is involved in regulating OK-PSA-induced anti-cancer immunity (102, 103). As described above, several studies reported that Gram-positive bacteria-derived LTA is recognized by TLR2 (27-29). Recent studies by Hartung et al. have demonstrated that the butanol-extracted LTA in addition to synthetic LTA from Staphylococcus aureus induce cytokines through TLR2 but not through TLR4 (28, 29). Further, it was also reported that LTA from Bacillus subtilis and from Staphylococcus aureus induced the maturation of murine DCs via TLR4 (27). Since TS-2 mAb recognizes LTAs similar to OK-PSA, it is suggested that OK-PSA has a certain chemical structure that LTAs share in common (94, 95), while the active structure of OK-PSA may not be LTA itself. Furthermore, recent evidence in LPS recognition suggests that there are structural and functional differences among LPS molecules from different bacteria. An LPS with a conical shape (e.g. from Escherichia coli) induces cytokines via TLR4, while a more cylindrical LPS (e.g. from Porphyromonas gingivalis) induces a different set of cytokines via TLR2 (104). It is possible that OK-PSA, a ligand for TLR4, may be a member of the LTA family with a different structure.
3) OK-PSA-induced DC maturation via TLR4 signaling
To evaluate the role of TLR4 in OK-PSA-induced maturation of human DCs, we performed a neutralizing test using anti-human TLR4 mAb (HTA125, 10 µg/ml;provided from Drs. Miyake and Akashi, The Institute of Medical Science, The University of Tokyo). When monocyte-derived immature DCs (iDCs) from healthy donors were stimulated by OK-PSA for 48 h, the increased expression of surface markers such as MHC class II, CD80 and CD86, and enhancement of the production of IL-12 and IL-18 were observed. The expression of these markers increased by OK-PSA was almost completely inhibited by the addition of anti-human TLR4 mAb. OK-PSA-induced cytokine production was also inhibited significantly by anti-TLR4 (Table 2). We next used the monocyte-derived iDCs from patients with oral cancer. In semiquantitative RT-PCR analysis, TLR4 mRNA was strongly expressed in peripheral blood monocytes both from patient 1 and from patient 2. MD-2 mRNA was clearly detected in patient 1-derived monocytes, while it was not detected in those from patient 2 in the current RT-PCR condition (data not shown). OK-PSA stimulation increased the expression of MHC class II, CD80 and CD86 on iDCs derived from patient 1. The expression of these cell surface antigens was also increased by OK-PSA on patient 2-derived DCs. Although DCs derived both from patient 1 and from patient 2 produced IL-12 by OK-PSA treatment, IL-12 secretion by patient 1-derived DCs was greater than that by patient 2-derived DCs. Next, these OK-PSA-treated or untreated DCs were irradiated, then cocultured with allogeneic T cells (DC:T=1:20) for 5 days. IFN-&ganma; in the supernatants from the cocultivation was markedly increased when T cells were cocultured with patient 1-derived DCs treated with OK-PSA compared with patient 2-derived DCs. Futher, allo-specific CTL activity of the T cells harvested from the above culture was also examined. CTL activity was significantly increased only when the T cells were cocultured with patient 1-derived DCs activated by OK-PSA but not with DCs from patient 2 (Table 2) (Okamoto and Sato, manuscript in preparation).
4) Requirement of both TLR4 and MD-2 genes in IFN-&ganma; induction by OK-432 administration in oral cancer patients
We evaluated the relation between the expression of TLR4 and MD-2 genes and IFN-&ganma; induction in response to OK-432 in 28 oral cancer patients. Nineteen of 20 patients (95%) who showed TLR4(+) and MD-2(+), demonstrated an increase in serum IFN-&ganma; protein by peritumoral administration of OK-432. Serum IFN-&ganma; protein was not detected after OK-432 administration in six of eight (75%) patients who showed TLR4(-) or MD-2(-). We detected a significant relation between increased IFN-&ganma; protein levels in the sera of patients administered OK-432 and expression of TLR4 and MD-2 genes (P=0.0005 in Fisher's exact test, Table 3). Both TLR4 and MD-2 were apparently required for IFN-&ganma; induction by OK-432 in patients with oral cancer (105). All of the patients examined in that study received therapy with OK-432 and UFT, an oral fluoropyrimidine formulation combining tegafur and uracil in a 1:4 ratio (Taiho Pharmaceutical Co., Tokyo, Japan) in combination with radiotherapy. Among these patients, 10 of 20 TLR4(+)MD-2(+) patients (50%) became histopathologically tumor-free after the therapy, and without surgical resection. In contrast, all eight patients who were TLR4(-) or MD-2 (-) became tumor-free only after having their tumors surgically resected after the therapy (105). We also clarified the requirement of TLR4 signaling for OK-432-induced anti-cancer immunity in a mouse model using TLR4-/- mice (105). These basic and clinical findings described above suggest that the target receptor molecule of original OK-432 in addition to its component OK-PSA is TLR4/MD-2 complex, and that the expression of TLR4 and MD-2 may be a useful marker to discriminate between responders and nonresponders to OK-432-based immunotherapy. Furthermore, these findings strongly support our opinion that the molecule which makes the largest contribution to the OK-432-induced anti-cancer immunity, is OK-PSA.
5) Isolation of a 55-kDa protein and its anti-tumor effect via TLR4 signaling
Aeginetia indica L. (AIL), a plant parasitic on roots of Japanese pampa grasses or sugar canes, has been used as a tonic and an anti-inflammatory herb agent in China and Japan. We previously reported that the butanol extract from seeds of AIL mediates potent anti-tumor immunity in tumor-bearing mice (106-108). We recently isolated a 55-kDa protein from the seed extract of the plant, and designated AILb-A. We reported that AILb-A was the protein with a molecular weight of 55-kDa not containing any carbohydrate determinants and markedly induced Th1-type cytokines and apoptosis-inducing factors such as TNF-α, TNF-β, Fas ligand, TNF-related apoptosis-inducing ligand (TRAIL) and perforin on human PBMCs in vitro (108, 109), and that AILb-A induced Th1-dominant state and elicited marked anti-tumor effects in syngeneic Meth-A tumor-bearing BALB/c mice in which the Th2 response is genetically dominant. It is strongly suggested that AILb-A may be a useful immunotherapeutic agent for patients with malignancies (110). We examined the role of TLR4-mediated signaling in AILb-A-induced anti-tumor immunity. In the luciferase assay using NF-κB-dependent reporter plasmid, AILb-A induced NF-κB activation in the cells transfected with the plasmid containing TLR4 gene in a dose-dependent manner. In the cells transfected both with TLR4 and with MD-2 genes, higher activity of NF-κB was observed by AILb-A stimulation than that in the cells expressing only TLR4. These cells transfected with TLR4 and/or MD-2 genes were provided by Drs. Miyake and Akashi, The Institute of Medical Science, The University of Tokyo. AILb-A did not induce cytokines (TNF-α and IL-12) in the peritoneal macrophages derived from TLR4-deficient mice (provided by Drs. Akira and Takeuchi, Research Institute for Microbial Diseases, Osaka University), while cytokines were markedly produced by AILb-A-stimulated macrophages from wild-type and TLR2-deficient mice (provided by Drs. Akira and Takeuchi). Further, in wild-type and TLR2-deficient mice bearing syngeneic LL/2 tumor, AILb-A treatment resulted in marked inhibition of tumor growth, but AILb-A was not effective in LL/2-bearing TLR4-deficient mice (Okamoto and Sato, manuscript in preparation). These findings suggest that AILb-A induces anti-cancer immunity via TLR4 signaling.

DISCUSSION AND FUTURE PERSPECTIVES
TAlthough the major therapies for malignancies are surgical resection, chemotherapy and radiotherapy, the accumulated evidence demonstrates that the host immune response is essential for eliminating cancer cells completely. Augmentation of anti-cancer immunity in patients is significant to cure the diseases in addition to increase the quality of life of patients with cancer. Recent studies strongly suggest that TLR ligands are useful applications for immunotherapy for cancer patients. The schema of TLR ligand-induced anti-cancer immunity is shown in Fig. 3. In tumor tissues, bacterial components such as BCG-CWS, CpG-DNA and OK-PSA, in addition to a plant-derived protein, AILb-A, activate APCs which have captured tumor antigens, via TLRs. TLR-mediated signaling stimulates the maturation of DCs. The matured DCs in which expression of MHC and costimulatory molecules is increased by TLR stimulation, migrate to the regional lymph nodes, then present antigen(s) to T cells. TLR-stimulated DCs a lso enhanced the producing ability of cytokines such as IL-12 and IL-18, potential Th1-inducing cytokines. Therefore, TLR-stimulated DCs may effectively induce tumor-antigen specific Th1 and CTL by presenting antigens to CD4+ and CD8+T cells while promoting a Th1-leading situation. It is possible that some ligands of TLRs are able to be effective immunotherapeutic agents for patients with cancer. Based on the results of our and other studies with regard to the role of TLR ligands on DC function as described above, TLR ligands may be useful applications as adjuvants in DC-based cancer immunotherapy. Clinical trials are expected. As described by many investigators and oncologists, the therapy to increase the host response in cancer patients should be effective, while it is a critical problem for clinical use of the immunoadjuvants that the molecular mechanism by which the immunotherapeuitic agents activate immune cells, has remained uncertain. Discovery of TLRs as immunoadjuvant receptors is a great progress to use the immunoadjuvants to treat human cancer. Furthermore, when TLR ligands are used in therapeutic applications, the expression of TLRs in the patients may be a useful marker to discriminate between responders and nonresponders to the therapy using the agents. For example, if a patient does not express TLR4/MD-2, CpG-DNA but not OK-PSA should be selected as a therapeutic application. CpG-DNA should not work in patients in whom TLR9 is not expressed. In addition, stimulation of both TLR4 and TLR9 by the combination therapy using OK-PSA and CpG-DNA may be more effective to cure cancers in patients expressi ng both TLR4/MD-2 and TLR9. The residual problem is that all TLR ligands and all types of signaling mediated by TLRs do not induce the Th1-type T-cell response. The next objective of these studies is to clarify the Th1-inducing mechanism(s) via TLRs, in addition to find Th1-inducing ligand(s) of TLRs. Establishment of the methodology to specifically induce the Th 1 response by utilizing TLR signaling is expected for future immunotherapy against cancers, allergic diseases in addition to infectious diseases. Further, if TLR ligand(s) that can selectively induce the Th2-type T-cell response will be found, it may be effective for the treatment of Th1-associated diseases such as autoimmune diseases. With regard to our work, both the OK-432-derived component OK-PSA and plant-derived 55-kDa protein AILb-A augment anti-cancer immune response by acting as potent Th1 inducers mediated by TLR4/MD-2 signaling. In an attempt to apply these agents for the treatment of human cancers, the determination of the chemical structure of OK-PSA and the isolation of the gene encoding AILb-A should be completed. It is currently under investigation in our laboratories. We believe that OK-PSA as well as AILb-A will be useful immunotherapeutic agents for patients with malignant diseases.

REFERENCES
1.Medzhitov R, Preston-Hurlburt P, Janeway CA Jr:A human homolog of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394-397, 1997
2.Kaisho T, Akira S:Toll-like receptors as adjuvant receptors. Biochim Biophys Acta 1589:1-13, 2002
3.Akira S, Takeda K, Kaisho T:Toll-like receptors:critical proteins linking innate and acquired immunity. Nat Immunol 2:675-680, 2001
4.Aderem A, Ulevitch RJ:Toll-like receptors in the induction of the innate immune response. Nature 406:782-787, 2000
5.Morisato D, Anderson KV:The spaetzle gene encodes a component of the extracellular signaling pathway establishing the dorsal-ventral pattern of the Drosophila embryo. Cell 76:677-688, 1994
6.Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA:Cloning and characterization of a sub-family of human toll-like receptors:hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw 11:372-378, 2000
7.Rock FL, Hardiman G, Timans JC, Kasteleins RA, Bazann JF:A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci USA 95:588-593, 1998
8.Takeuchi O, Kawai T, Sanjo H, Copeland NG, Gilbert DJ, Jenkins NA, Takeda K, Akira S:TLR6:A novel member of an expanding toll-like receptor family. Gene 231:59-65, 1999
9.Du X, Poltorak A, Wai Y, Beutler B:Three novel mammalian toll-like receptors:gene structure, expression, and evolution. Eur Cytokine Network 11:362-371, 2000
10.Chuang TH, Ulevitch RJ:Cloning and characterization of a sub-family of human toll-like receptors:hTLR7, hTLR8 and hTLR9. Eur Cytokine Network 11:372-378, 2000
11.Chuang TH, Ulevitch RJ:Identification of hTLR10:A novel human Toll-like receptor preferentially expressed in immune cells. Biochim Biophys Acta 1518:157-161, 2001
12.Miyake K, Yamashita Y, Ogata M, Sudo T, Kimoto M:RP105, a novel B cell surface molecule implicated in B cell activation, is a member of the leucine-rich repeat protein family. J Immunol 154:3333-3340, 1995
13.Means TK, Yoshimura A, Wang S, Golenbock DT, Fenton ML:The CD14 ligands lipoarabinomannan and lipopolysaccharide differ in their requirement for Toll-like receptors. J Immunol 163:6748-6755, 1999
14.Miura Y, Shimazu R, Miyake K, Akashi S, Ogata H, Yamashita Y, Narisawa Y, Kimoto M:RP105 is associated with MD-1 and transmits an activation signal in human B cells. Blood 92:2815-2822, 1998
15.Shimazu R, Akashi S, Ogawa H, Nagai Y, Fukudome K, Miyake K, Kimoto M:MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 189:1777-1782, 1999
16.Wyllie DH, Kiss-Toth E, Visintin A, Smith SC, Boussouf S, Segal DM, Duff GW, Dower SK:Evidence for accessory protein function for Toll-like receptor 1 in anti-bacterial responses. J Immunol 165:7125-7132, 2000
17.Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, Akira S:Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11:443-451, 2001
18.Schwandner R, Dziarski R, Wesche H, Rothe M, Kirschning CJ:Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem 274:17406-17409, 1999
19.Yoshimura A, Lien E, Ingalls RR, Tuomanen E, Dziarski R, Golenbock DT:Recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J Immunol 163:1-5, 1999
20.Underhill DM, Ozinsky A, Hajjar AM, Stevens A, Wilson CB, Bassetti M, Aderem A:The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature401:811-815, 1999
21.Aliprantis AO, Yang RB, Mark MR, Suggett S, Devaux B, Radolf JD, Klimpel GR, Godowski P, Zychlinski A:Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science 285:736-739, 1999
22.Brightbill HD, Libraty DH, Krutzik SR, Yang BR, Belisle JT, Bleharski JR, Maitland M, Norgard MV, Plevy SE, Smale ST, Brennan PJ, Bloom BR, Godowski PJ, Modlin RL:Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors. Science 285:732-736, 1999
23.Hirschfeld M, Kirschning CJ, Schwandner R, Wesche H, Weis JH, Wooten RM, Weis JJ:Inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by toll-like receptor 2. J Immunol 163:2382-2386, 1999
24.Lien E, Sellati TJ, Yoshimura A, Flo TH, Rawadi G, Finberg RW, Carroll JD, Espevik T, Ingalls RR, Radolf JD, Golenbock DT:Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem 274:33419-33425, 1999
25.Underhill DM, Ozinsky A, Smith KD, Aderem A:Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages. Proc Natl Acad Sci USA 96:14459-14463, 1999
26.Means TK, Yoshimura A, Wang S, Golenbock DT, Fenton ML:The CD14 ligands lipoarabinomannan and lipopolysaccharide differ in their requirement for Toll-like receptors. J Immunol 163:6748-6755, 1999
27.Michelsen KS, Aicher A, Mohaupt M, Hartung T, Dimmeler S, Kirschning CJ, Schumann RR:The role of Toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCs). Peptydoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2. J Biol Chem 276:25680-25686, 2001
28.Morath S, Geyer A, Hartung T:Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus. J Exp Med 193:393-397, 2001
29.Morath S, Stadelmaier A, Geyer A, Schmidt RR, Hartung T:Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release. J Exp Med 195:1635-1640, 2002
30.Ozinsky A, Underhill DM, Fontenot JD, Hajjar AM, Smith KD, Wilson CB, Schroeder L, Aderem A:The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc Natl Acad Sci USA 97:13766-13771, 2000
31.Alexopoulou L, Holt AC, Medzhitov R, Flavell RA:Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413:732-738, 2001
32.Poltorak A, He X, Smirnova I, Liu MY, Huffel CV, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B:Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice:Mutations in Tlr4 gene. Science 282:2085-2088, 1998
33.Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore KJ, Gros P, Malo D:Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J Exp Med 189:615-625, 1999
34.Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira, S:Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide:Evidence for TLR4 as the Lps gene product. J Immunol 162:3749-3752, 1999
35.Yang S, Tamai R, Akashi S, Takeuchi O, Akira S, Sugawara S, Takada H:Synergistic effect of muramyldipeptide with lipopolysaccharide or lipoteichoic acid to induce inflammatory cytokines in human monocytic cells in culture. Infect Immun 69:2045-2053, 2001
36.Kawasaki K, Akashi S, Shimazu R, Yoshida T, Miyake K, Nishijima M:Mouse Toll-like receptor 4?MD-2 complex mediates lipopolysaccharide-mimetic signal transduction by Taxol. J Biol Chem 275:2251-2254, 2000
37.Kurt-Jones E, Popova L, Kwinn L, Haynes LM, Jones LP, Tripp RA, Walsh EE, Freeman MW, Golenbock DT, Anderson LJ, Finberg RW:Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 1:398-401, 2000
38.Ohashi K, Burkart V, Flohe S, Kolb H:Heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol164:558-561, 2000
39.Vabulas RM, Ahmed-Nejab P, da Costa C, Miethke T, Kirschning CJ, Hacker H, Wagner H:Endocytosed HSP60s use Toll-like receptor 2 (TLR2) and TLR4 to activate the Toll/Interleukin-1 receptor signaling pathway in innate immune cells. J Biol Chem 276:31332-31339, 2001
40.Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, Rose J, Chow JC, Strauss III JF:The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 276:10229-10233, 2001
41.Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A:The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410:1099-1103, 2001
42.Takeuchi O, Kawai T, Muhlradt PF, Morr M, Radolf JD, Zychlinsky A, Takeda K, Akira S:Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol 13:933-940, 2001
43.Hajjar AM, O'Mahony DS, Ozinsky A, Underhill DM, Aderem A, Klebanoff SJ, Wilson CB:Functional interactions between Toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J Immunol 166:15-19, 2001
44.Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, Horiuchi T, Tomizawa H, Takeda K, Akira S:Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 3:196-200, 2002
45.Jurk M, Heil F, Vollmer J, Schetter C, Krieg AM, Wagner H, Lipford G, Bauer S:Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol 3:499, 2002
46.Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S:A Toll-like receptor recognizes bacterial DNA. Nature 408:740-745, 2000
47.Medzhitov R, Preston-Hurlburt P, Kopp E, Stadlen A, Chen C, Ghosh S, Janeway CA Jr:MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. Mol Cell 2:253-258, 1998
48.Muzio M, Polentarutti N, Bosisio D, Manoj Kumar PP, Mantovani A:Toll-like receptor family and signalling pathway. Biochem Soc Trans 28:563-566, 2000
49.Kaisho T, Takeuchi O, Kawai T, Hoshino K, Akira S:Endotoxin-induced maturation of MyD88-deficient dendritic cells. J Immunol 166:5688-5694, 2001
50.Horng T, Barton GM, Medzhitov R:TIRAP:an adapter molecule in the Toll signaling pathway. Nat Immunol 2:835-841, 2001
51.Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, Sato S, Hoshino K, Akira S:Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 167:5887-5894, 2001
52.Yamanoto M, Sato S, Hemmi H, Sanjo H, Uematsu S, Kaisho T, Hoshino K, Takeuchi O, Kobayashi M, Fujita T, Takeda K, Akira S:Essential role for TIRAP in activation of the signaling cascade shared by TLR2 and TLR4. Nature 420:324-329, 2001
53.Horng T, Barton GM, Flavell RA, Medzhitov R:The adaptor molecule TIRAP provides signaling specificity for Toll-like receptors. Nature 420:329-333, 2002
54.Melief CJM, van der Burg SH, Toes REM, Ossendorf F, Offringa R:Effective therapeutic anticancer vaccines based on precision guiding of cytolytic T lymphocytes. Immunol Review 188:177-182, 2002
55.Azuma I, Kishimoto S, Yamamura Y, Petit JF:Adjuvancity of mycobacterial cell wall. Jpn J Microbiol 15:193-197, 1971
56.Freund J:The mode of action of immuno-pharmacological adjuvants. Adv Tuberc Res1:130-148, 1956
57.Azuma I, Kanetsuna F, Taniyama T, Yamamura Y, Hori M:Adjuvant activity of microbacterial fractions:I. Purification and in vivo adjuvant activity of cell wall skeletons of Mycobacterium bovis BCG, Nocardia asteroids 131 and Corynebacterium diphtheriae PW8. Biken J 18:1-13, 1975
58.Azuma I, Seya T:Development of immunoadjuvants for immunotherapy of cancer. Int Immunopharmacol 1:1249-1259, 2001
59.Azuma I, Yamamura Y:Immunotherapy of cancer with BCG cell-wall skeleton and related materials. In:Sugimura T, Endo H, Ono T, Sugano H, eds. Progress in Cancer Biochemistry. Gann Monogr. Cancer Res. 24, Japan Scientific Societies Press, Tokyo, 1979, pp. 122-141
60.Azuma I:Immunologic and biochemical properties of bacterial fractions and related compounds with special reference to BCG cell wall skeleton and Nocardia rubra cell wall skeleton. In:Yamamura Y, Azuma I, eds. Molecular and cellular networks for cancer therapy. Experta Media, Tokyo, 1989, pp. 83-104
61.Yamamura Y, Sakatani M, Ogura T, Azuma I:Adjuvant immunotherapy of lung cancer with BCG cell-wall skeleton (BCG-CWS). Cancer 43:1314-1319, 1979
62.Yasumoto K, Manabe H, Yanagawa E, Nagano N, Ueda H, Hirota N, Ohta M, Nomoto K, Azuma I, Yamamura Y:Nonspecific adjuvant immunotherapy on lung cancer with cell wall skeleton of Mycobacteria bovis, Bacillus Calmette-Guerin. Cancer Res 39:3262-3267, 1979
63.Ohno R, Ueda R, Imai K, Kato Y, Watanabe E, Morishima Y, Yokomaku S, Kobayashi M, Takeyama H, Ezaki K, Kawashima K, Hirano M, Ohara K, Kosaki T, Yoshikawa S, Yamada K:A clinical trial of cell-wall skeleton of BCG in chemoimmunotherapy of acute leukemia. Jpn J Cancer Res 69:179-186, 1978
64.Ochiai T, Sato H, Hayashi R, Asano T, Sato H, Yamamura Y:Postoperative adjuvant immunotherapy of gastric cancer with BCG-cell wall skeleton. Cancer Immunol Immunother 14:167-171, 1983
65.Hayashi A, Doi O, Azuma I, Toyoshima K:Immunofriendly use of BCG-CWS-cell wall skeleton remarkably improves the survival rate of various cancer patients. Proc Jpn Acad 74:50-55, 1998
66.Hayashi A:Interferon as a marker for the effective cancer immunotherapy with BCG-CWS wall skeleton. Proc Jpn Acad 70:205-209, 1994
67.Matsumoto M, Seya T, Kikkawa S, Tsuji S, Shida K, Nomura M, Kurita-Taniguchi M, Ohigashi H, Yokouchi H, Takami H, Hayashi A, Azuma I, Masaoka T, Kodama K, Toyoshima K:Interferon gamma-producing ability in blood lymphocytes of patients with lung cancer through activation of the innate immune system by BCG cell wall skeleton. Int Immunopharmacol 1:1559-1569, 2001
68.Tsuji S, Matsumoto M, Takeuchi O, Akira S, Azuma I, Hayashi A, Toyoshima K, Seya T:Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guerin:Involvement of Toll-like receptors. Infect Immun 68:6883-6890, 2000
69.Seya T, Matsumoto M, Tsuji S, Begum NA, Nomura M, Azuma I, Hayashi A, Toyoshima K:Two receptor theory in innate immune activation:studies on the receptors for bacillus Calmette-Guerin-cell wall skeleton. Arch Immunol Ther Exp 49:S13-21, 2001
70.Azuma I, Ribi EE, Meyer TJ, Zbar B:Biological active components from mycobacterial cell walls:I. Isolation and composition of cell wall skeleton component P3. J Natl Cancer Inst 52:95-101, 1974
71.Tokunaga T, Yamamoto H, Shimada S, Abe H, Fukuda T, Fujisawa Y, Furutani Y, Tano O, Kataoka T, Sudo T, Makiguchi N, Suganuma T:Antitumor activity of Deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity. J Natl Cancer Inst 72:955-962, 1984
72.Yamamoto S, Kuramoto E, Shimada S, Tokunaga T. In vitro augmentation of natural killer cell activity and production of interferon-α/β and -&ganma; with deoxyribonucleic acid fraction from Mycobacterium bovis BCG. Jpn J Cancer Res 79:866-873, 1988
73.Yamamoto S, Yamamoto T, Shimada S, Kuramoto E, Yano O, Kataoka T, Tokunaga T:DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth. Microbiol Immunol 36:983-997, 1992
74.Krieg AM:CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 20:709-760, 2002
75.Krieg AM, Yi AK, Matson, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman DM.:CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374 : 546-549, 1995
76.Bird AP:CpG islands as gene markers in the vertebrate nucleus. Trend Genet 3:342-347, 1987
77.Roman M, Martin-Orozco E, Goodman JS, Nguyen MD, Sato Y, Ronaghy A, Kornbluth RS, Richman DD, Carson DA, Raz E:Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat Med 3:849-854, 1997
78.Ballas Z, Krieg AM, Warren TL, Rasmussen WL, Davis HL, Waldschmidt M, Weiner GJ:Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs. J Immunol 167:4878-4886, 2001
79.Lipford GB, Sparwasser T, Zimmermann S, Heeg K, Wagner H:CpG-DNA-mediated transient lymphadenopathy is associated with a state of Th1 predisposition to antigen-driven responses. J Immunol 165:1228-1235, 2000
80.Sparwasser T, Vabulas RM, Villmow B, Lipford GB, Wagner H:Bacterial CpG-DNA activates dendritic cells in vivo:T helper cell-independent cytotoxic T cell responses to soluble proteins. Eur J Immunol30:3591-3597, 2000
81.Agrawal S, Kandimalla ER:Medicinal chemistry and therapeutic potential of CpG DNA. Trend Mol Med 8:114-121, 2002
82.Okamoto H, Shoin S, Koshimura S, Shimizu R:Studies on the anticancer and streptolysin S-forming abilities of hemolytic streptococci. Jpn J Microbiol 11:323-336, 1967
83.Torisu M, Katano M, Kimura Y, Itoh H, Takesue M:New approach to management of malignant ascites with a streptococcus preparation, OK-432. I. Improvement of host immunity and prolongation of survival. SURGERY 93:357-364, 1983
84.Watanabe Y, Iwa T:Clinical value of immunotherapy with the streptococcal preparation OK-432 in non-small cell lung cancer. J Biol Response Mod 6:169-180, 1987
85.Kikkawa F, Kawai M, Oguchi H, Kojima M, Ishikawa H, Iwata M, Maeda O, Tomoda Y, Arii Y, Kuzuya K:Randomised study of immunotherapy with OK-432 in uterine cervical carcinoma. Eur J Cancer 29:1542-1546, 1993
86.Maehara Y, Okuyama T, Kakeji Y, Baba H, Furusawa M, Sigimachi K:Postoperative immunochemotherapy including streptococcal lysate OK-432 is effective for patients with gastric cancer and serosal invasion. Am J Surg 168:36-40, 1994
87.Kitahara S, Ikeda M, Inoue T, Matsunaga T, Yamaguchi K, Takayama E, Healy GB, Tsukuda M:Inhibition of head and neck metastatic and/or recurrent cancer by local administration of multi-cytokine inducer OK-432. J Laryngol Otol 110:449-453, 1996
88.Sato M, Yoshida H, Yanagawa T, Yura Y, Urata M, Atsumi M, Takegawa Y:Effect of intradermal administration of streptococcal preparation OK-432 on interferon and natural killer cell activities in patients with oral cancer. Int J Oral Surg 13:7-15, 1984
89.Sato M, Harada K, Yoshida H, Yura Y, Azuma M, Iga H, Bando T, Kawamata H, Takegawa Y:Therapy for oral squamous cell carcinoma by tegafur and streptococcal agent OK-432 in combination with radiotherapy:Association of the therapeutic effect with differentiation and apoptosis in the cancet cells. Apoptosis 2:227-238, 1997
90.Oshimi K, Kano S, Takaku F, Okumura K:Augmentation of mouse natural killer cell activity by a streptococcal preparation, OK-432. J Natl Cancer Inst 65:1265-1269, 1980
91.Saito M, Ebina T, Koi M, Yamaguchi T, Kamade Y, Ishida N:Induction of interferon-&ganma; in mouse spleen cells by OK-432, a preparation of Streptococcus pyogenes. Cell Immunol 68:187-192, 1982
92.Misaki T, Watanabe Y, Iida Y, Hidaka A, Kasagi K, Fukushima H, Konishi J:Recruitment of T lymphocytes and induction of tumor necrosis factor in thyroid cancer by a local immunotherapy. Cancer Immunol. Immunother 35:92-96, 1992
93.Fujimoto T, Duda RB, Azilvasi A, Chen X, Mai M, O'Donnell MA:Streptococcal preparation OK-432 is a potent inducer of IL-12 and a T helper cell 1 dominant state. J Immunol 158:5619-5626, 1997
94.Okamoto M, Kaji R, Kasetani H, Yoshida H, Moriya Y, Saito M, Sato M:Purification and characterization of interferon-&ganma;-inducing molecule of OK-432, a penicillin-killed streptococcal preparation, by monoclonal antibody neutralizing interferon-&ganma;-inducing activity of OK-432. J Immunother 13:232-242, 1993
95.Takada H, Kawabata Y, Arakaki R, Kusumoto S, Fukase K, Suda Y, Yoshimura T, Kokeguch S, Kato K, Komuro T, Tanaka N, Saito M, Yoshida T, Sato M, Kotani, S:Molecular and structural requirements of a lipoteichoic acid from Enterococcus hirae ATCC 9790 for cytokine-inducing, antitumor, and antigenic activities. Infect Immun 63:57-65, 1995
96.Okamoto M, Gohda H, Ohe G, Yoshida H, Matsuno T, Saito M, Sato M:Cytokine-inducing activity and anti-tumor effect of liposome-incorporated interferon-&ganma;-inducing molecule derived from OK-432, a streptococcal preparation. J. Immunother 23:94-103, 2000
97.Okamoto M, Ohe G, Oshikawa T, Nishikawa H, Furuichi S, Yoshida H, Matsuno T, Saito M, Sato M:Induction of Th1-type cytokines by lipoteichoic acid-related preparation isolated from OK-432, a penicillin-killed streptococcal agent. Immunopharmacol 49:363-376, 2000
98.Okamoto M, Ohe G, Oshikawa T, Furuichi S, Nishikawa H, Tano T, Ahmed SU, Yoshida H, Moriya Y, Saito M, Sato M:Enhancement of anti-cancer immunity by a lipoteichoic-acid-related molecule isolated from a penicillin-killed group A Streptococcus. Cancer Immunol Immunother 50:408-416, 2001
99.Okamoto M, Oshikawa T, Ohe G, Furuichi S, Nishikawa H, Tano T, Bando T, Yoshida H, Matsubara S, Matsuno T, Saito M, Sato M:Comparison of cytokine-inducing activity in a lipoteichoic acid-related molecule isolated from a penicillin-killed group A Streptococcus and untreated bacteria. Int Immunopharmacol 1:1957-1968, 2001
100.Okamoto M, Ohe G, Furuichi S, Nishikawa H, Oshikawa T, Tano T, Ahmed SU, Yoshida H, Moriya Y, Matsubara S, Ryoma Y, Saito M, Sato M:Enhancement of anti-tumor immunity by lipoteichoic acid-related molecule isolated from OK-432, a streptococcal agent, in athymic nude mice bearing human salivary adenocarcinoma:Role of natural killer cells. Anticancer Res 22:3229-3240, 2002
101.Oshikawa T, Okamoto M, Ohe G, Furuichi S, Nishikawa H, Ahmed SU, Yoshida H, Moriya Y, Matsubara S, Ryoma Y, Saito M, Sato M:Isolation of a Th1-inducing molecule from OK-432, a streptococcal preparation, by a monoclonal antibody TS-2 that neutralizes the interferon-g-inducing activity of OK-432:Comparison of the enhancement of anti-tumor immunity between the TS-2-binding and TS-2-unbinding fraction. Int Immunopharmacol (in press, 2003)
102.Okamoto M, Oshikawa T, Ohe G, Nishikawa H, Furuichi S, Tano T, Moriya Y, Saito M, Sato M:Severe impairment of anti-cancer Effect of lipoteichoic acid-related molecule isolated from a penicillin-killed Streptococcus pyogenes in toll-like receptor 4-deficient mice. Int Immunopharmacol 1:1789-1795, 2001
103.Okamoto M, Sato M:Effective molecule of a streptococcal preparation OK-432 and its molecular targets:Significance for cancer immunotherapy. Rec Res Dev Infect Immun (in press, 2002)
104.Netea MG, van Deuren M, Kullberg BJ, Cavaillon J-M, van der Meer JWM:Does the shape of lipid A determine the interaction of LPS with Toll-like receptors. Trend Immunol 23:135-139, 2002
105.Okamoto M, Oshikawa T, Tano T, Ohe G, Furuichi S, Nishikawa H, Ahmed SU, Akashi S, Miyake K, Takeuchi O, Akira S, Moriya Y, Matsubara S, Ryoma Y, Saito M, Sato M:Involvement of Toll-like receptor 4 signaling in interferon-&ganma; production and anti-tumor effect by a streptococcal agent OK-432. J Natl Cancer Inst (in press, 2003)
106.Chai JG, Bando T, Kobashi S, Oka M, Nagasawa H, Nakai S, Maeda K, Himeno K, Sato M, Ohkubo S:An extract of seeds from Aeginetia indica L., a parasitic plant, induces potent antigen-specific antitumor immunity in Meth A-bearing BALB/c mice. Cancer Immunol Immunother 35:181-185, 1992
107.Chai JG, Bando T, Nagasawa H, Himeno K, Sato M, Ohkubo S:Seed extract of Aeginetia indica L. induces cytokine production and lymphocyte proliferation in vitro. Immunopharmacol 27:13-21, 1994
108.Chai JG, Okamoto M, Bando T, Nagasawa H, Hisaeda H, Sakai T, Himeno K, Sato M, Ohkubo S:Dissociation between the mitogenic effect and antitumor activity of seed extract from Aeginetia indica L. Immunopharmacol 30:209-215, 1995
109.Okamoto M, Ohe G, Oshikawa T, Nishikawa H, Furuichi S, Bando T, Yoshida H, Sakai T, Himeno K, Sato M, Ohkubo S:Purification and characterization of cytokine-inducing protein of seed extract from Aeginetia indica L., a parasitic plant. Immunopharmacol 49:377-389, 2000
110.Ohe G, Okamoto M, Oshikawa T, Furuichi S, Nishikawa H, Tano T, Uyama K, Bando T, Yoshida H, Sakai T, Himeno K, Sato M, Ohkubo S. Th1-cytokine induction and anti-tumor effect of 55 kD protein isolated from Aeginetia indica L, a parasitic plant. Cancer Immunol Immunother 50:251-259, 2001

Received for publication January 6, 2003;accepted January 16, 2003.

Address correspondence and reprint requests to Dr. Masato Okamoto, D.D.S., Ph.D., Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, Kuramoto-cho, Tokushima770-8504, Japan and Fax:+81-88-633-7462.