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Background Recently, the gut microbiota has been shown to play an important role in the response and resistance to chemotherapy. Although there is much knowledge about chemotherapy-induced changes in the gut microbiota, chemotherapy-associated changes in the oral microbiota remain unclear. Herein, we aimed to evaluate the changes in oral microbiota associated with the initiation of chemotherapy in patients with malignant hematopoietic tumors. Methods Oral samples were collected before and 8–20 days after the start of chemotherapy from 50 patients with malignant hematopoietic tumors who were starting chemotherapy for the first time. The 16S ribosomal RNA gene sequencing of bacterial DNA extracted from oral samples was performed to compare the oral microbiota before and after the initiation of chemotherapy. Results The richness or evenness of diversity in the ‘after start of chemotherapy’ group decreased significantly, compared with the ‘before start of chemotherapy’ group (alpha-diversity; observed operational taxonomic units (OTUs) index, p p p = 0.001; and weighted UniFrac distances, p = 0.003). Linear discriminant analysis effect size analysis demonstrated an increased abundance of species of certain genera, such as Staphylococcus, and decreased abundance of species of some genera, such as Streptococcus and Neisseria, in the ‘after-chemotherapy’ group, compared with those in the ‘before-chemotherapy’ group. The amounts and trends of change in the oral microbiota before and after the start of chemotherapy differed among the subjects. Of the 25 bacterial genera whose prevalence changed significantly before and after the start of chemotherapy, the proportion of oral commensals such as Streptococcus and Neisseria decreased in many subjects. In contrast, Staphylococcus and Pseudomonas were detected only in a few subjects, but their relative abundance increased significantly after the start of chemotherapy. Conclusions The oral microbiota of patients with hematopoietic tumors changed markedly after the initiation of chemotherapy. Our findings are expected to aid the elucidation of the pathogenesis of oral mucositis, which is an adverse event of chemotherapy, and the development of treatment methods for this condition.

Oral squamous cell carcinoma (OSCC) frequently metastasizes to cervical lymph nodes, which is the most known prognostic factor. Screening methods to identify sentinel lymph nodes (SLNs) are therefore of great interest for the management of potential neck metastasis. The purpose of this study was to evaluate the clinical benefit of double SLN mapping with indocyanine green (ICG) and 99m-technetium-tin colloid ((99m)Tc-tin colloid) for sentinel node navigation surgery (SNNS). Between 2007 and 2010, 16 patients diagnosed with OSCC were investigated by SLN biopsy using the double mapping method. (99m)Tc-tin colloid was injected into the peri-tumoural region on the preoperative day, and ICG was administered intraoperatively in the same position to assist in detecting nodes during surgery. Based on the gamma-ray signal and near-infrared (NIR) fluorescence of ICG, SLNs were identified and thereafter assessed pathologically and genetically for cancer involvement. Radio-guided detection was successful for all patients. ICG mapping identified a relatively larger number of nodes, suggesting that several non-SLNs were potentially involved. The double mapping method assisted surgeons to explore SLNs. Since the ICG fluorescence was shielded by the subcutaneous fatty tissue and the muscle layer including platysma and sternocleidomastoid, it was necessary to retract the tissue away from nodes.

Antitumor functions of the host immune system are frequently compromised in patients with malignancies. In the current study, we evaluated the relationship between expression ratio of mRNAs for the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax (the Bcl-2/Bax ratio) in peripheral blood mononuclear cells and clinical outcomes in patients with head and neck carcinomas. The overall survival (OS) time of patients with Bcl-2/Bax ratios ≥ 1.2 tended to be longer than that of patients with Bcl-2/Bax ratios < 1.2 but not significantly so (P = .084, n = 61). Disease-free survival (DFS) of patients with Bcl-2/Bax ratios ≥ 1.2 was statistically significantly longer than that of patients with Bcl-2/Bax ratios < 1.2 (P = .001, n = 76). All of the patients whose Bcl-2/Bax ratio is ≥ 2.0 were alive after 36 months and survived without any evidence of disease for 24 months (Bcl-2/Bax ≥ 2.0 versus Bcl-2/Bax < 2.0; P = .035, n = 61 in OS, P < .001, n = 76 in DFS, respectively). In 56 patients who received immunochemoradiotherapy using UFT and OK-432 in combination with radiotherapy, a statistically significant relationship between the Bcl-2/Bax ratio and the therapeutic effect estimated using Response Evaluation Criteria in Solid Tumors was observed, as well as a relation with interferon-γ (IFN-γ) induction in response to the therapy [P = .002 in complete response versus partial response + stable disease; P = .046 in IFN-γ(+) versus IFN-γ(-)]. In addition, there were significant correlations of the Bcl-2/Bax ratio with both the absolute number of CD4+ T cells and the rate of CD4+ T cell and natural killer cell activity. These findings strongly suggest that the balance of expression of Bcl-2 and Bax genes in circulating immune cells has a high prognostic value in head and neck cancer patients.

Objective Id (inhibitor of DNA binding or differentiation) proteins are dominant-negative regulators of basic helix-loop-helix transcription factors that control malignant cell behavior in many different tissues. The study aim was to investigate the effects of Id on human oral squamous cell carcinoma (OSCC) cells. Materials and methods The expression spectrum of Id1 and Id2 was examined in 6 different OSCC cell lines. Next, antisense vector infection or siRNA-mediated gene silencing was used to knockdown Id1 and Id2 expression in the SAS and HSC-2 cell lines (only Id1) to determine the effects on proliferation and invasion in vitro. Results The poorly differentiated SAS cell line and the differentiated HSC-2 cell line abundantly expressed Id1, whereas the other 4 cell lines showed little or undetectable Id1 expression. Id2 was generally expressed at a lower level than Id1 in the SAS cell line but showed little to no expression in the other OSCC cell lines. The knockdown of Id1 significantly repressed cell proliferation and invasion and decreased telomerase activities in the SAS and HSC-2 cell lines, whereas the knockdown of Id2 in the SAS cell lines showed no effect on proliferation and invasion. Conclusion We conclude that Id1 plays a more important role than Id2 in the proliferation and invasion of human OSCC cells.

Acinic cell carcinoma usually arises in the parotid gland and rarely occurs in the minor salivary glands. We report a case of acinic cell carcinoma of papillary cystic type arising in the buccal mucosa. The patient was a 45-year-old woman who had a painless swelling in the right buccal mucosa. The tumor measured 18×15mm and was elastic hard in consistency. The tumor was excised under local anesthesia. Histologically, the tumor appeared to be encapsulated, and the tumor cells were arranged in papillary and partially microcystic patterns. Immunohistochemical examination showed that the tumor cells were positive for S-100 protein, lysozyme, α-antit rypsin, and AE1/AE3 and partially positive for periodic acid-Schiff (PAS). The histopathological diagnosis was acinic cell carcinoma of papillary cystic type. The postoperative course was uneventful, and there was no evidence of recurrence or metastasis 20 months after operation.

We examined the role of nitric oxide (NO) induced by OK-432, a streptococcal immunotherapeutic agent, in anti-tumor effects of the OK-432 by in vitro and in vivo experiments using an NO synthase inhibitor, N-monomethyl-l-arginine acetate (NMA). The in vitro treatment of mouse splenocytes with OK-432 increased the expression of inducible NO synthase (iNOS) gene and NO production in a dose-dependent manner. Although it is well known that OK-432 induces cytokines such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, both of which are known to be potent NO inducers, we observed only a partial reduction of OK-432-induced NO production with the addition of anti-IFN-gamma and/or anti-TNF-alpha neutralizing antibodies. The cytotoxicity of the splenocytes increased by in vitro OK-432 stimulation was almost completely inhibited by the treatment with NMA. OK-432 administration resulted in a marked prolongation of survival and a significant inhibition of tumor growth in syngeneic tumor-bearing mice, whereas NMA significantly inhibited the anti-tumor effects of OK-432. Although the increased cytotoxicity of adherent splenocytes derived from OK-432-treated tumor-bearing mice was almost completely inhibited by NMA, only partial inhibition by NMA was observed in the cytotoxicity of the nonadherent splenocytes. These findings strongly suggest that the iNOS/NO induced by OK-432 is intimately involved in the anti-tumor effects of OK-432.

OK-432 is a Streptococcus-derived immunotherapeutic agent for malignancies. Our group has tried to identify the effective components of OK-432 and has succeeded in isolating a lipoteichoic acid-related preparation designated as OK-PSA, which is a strong inducer of T helper 1 (T(H)1) cells, and elicits an anticancer effect via Toll-like receptor (TLR) 4. Conversely, bacterial DNA with unmethylated CpG motifs can stimulate a T(H)1-type host response via TLR9. The unmethylated CpG DNA contained in OK-432 may play a role in its anticancer effect. In the current study, we investigated the effect of OK-432-derived DNA (OK-DNA) in augmenting the anticancer immune response. Analysis of OK-DNA with the restriction enzymes Hpa II and MspI revealed that OK-DNA contained unmethylated CpG motifs. OK-DNA induced TH1-type cytokines such as interferon-gamma, tumor necrosis factor-alpha, interleukin (IL)-12, and IL-18 and augmented killer cell activities in vitro on human peripheral blood mononuclear cells, whereas the methylated OK-DNA did not. Cytokines were also produced by OK-DNA-stimulated splenocytes derived from wild-type mice but not from TLR9-deficient mice. In the in vivo study, peritumoral administration of OK-DNA resulted in a significant inhibition of tumor growth in syngeneic tumor-bearing wild-type and TLR4-deficient mice but not in TLR9-deficient mice. The antitumor effect of OK-432 in TLR9-deficient mice was significantly but partially reduced compared with that in wild-type mice, whereas the effect of OK-432 was almost completely eliminated in TLR4-deficient mice. These findings suggest that unmethylated CpG DNA in OK-432 functions as an active component in OK-432-induced anticancer immunity via TLR9.

It has previously been reported by our group that Toll-like receptor (TLR) 4 is involved in anticancer immunity induced by OK-432, a Streptococcus-derived immunotherapeutic agent. However the detailed mechanism of the OK-432-induced immune response via TLR4 remained uncertain, because it may not be possible for OK-432, which consists of whole bacterial bodies, to bind directly to TLR4. In the current study, we conducted in vitro and in vivo experiments to investigate the hypothesis that OK-432 may first be captured and dissolved by phagocytes and that the active components released by the cells may then induce host responses via TLR4. TS-2 monoclonal antibody, which recognizes an active component of OK-432 designated OK-PSA was used in the current study. First, it was observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited in vitro by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining using TS-2 demonstrated that OK-432 was captured and dissolved by phagocytes. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by enzyme-linked immunosorbent assay using TS-2. Supernatants from OK-432-treated DC culture increased nuclear factor (NF)-kappaB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-kappaB in these cells. In in vivo experiments, the anticancer effect of OK-432 was significantly inhibited by suppression of phagocytosis activity by cytochalasin B. In this case, the amount of OK-PSA, an active component of OK-432, in the sera was also reduced by cytochalasin B. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the immune effect of OK-432.

We investigated in the current study the effect of TX-1877, a bifunctional hypoxic cell radiosensitizer, in augmenting anticancer host response. In the syngeneic squamous cell carcinoma-bearing mouse model, a single administration of TX-1877 significantly inhibited the primary tumor growth as well as lung metastasis. TX-1877 administration resulted in a significant infiltration of immune cells, such as CD4+T, CD8+T cells, macrophages and dendritic cells (DCs), and an increased expression of chemokines for cytotoxic T lymphocytes (CTLs), helper T-cell 1 (Th1) cells, monocytes/macrophages and DCs, in tumor tissues. Nitric oxide (NO) production and the expression of inducible NO synthase (iNOS) and interferon-γ, a major Th1 cytokine that plays a major role in anticancer immunity, were also enhanced. Furthermore, neutralization of NO by N-monomethyl-L-arginine acetate resulted in a marked inhibition of the antitumor effect of TX-1877. In tumor-draining lymph nodes, MHC class I-restricted CD8+ memory CTLs specific for inoculated cancer cells were induced by TX-1877. In in vitro experiments, TX-1877 induced chemokines and iNOS/NO in several types of culture cells. These findings strongly suggested that TX-1877 induces migration of CD8+CTLs, CD4+Th1 cells, macrophage/monocytes and dendritic cells into the tumor site, and that this migration is mediated by chemokine induction. In addition, it was suggested that NO produced by several types of cells stimulated by TX-1877 in the tumor sites plays a major role in the anticancer effect of TX-1877. TX-1877 was thus shown to be an effective immunopotentiator as well as a hypoxic cell radiosensitizer. © 2005 Wiley-Liss, Inc.

A 55-kDa protein named AILb-A, isolated from the seed extract of Aeginetia indica L., a parasitic plant, induces a Th1-type T-cell response and elicits a marked antitumor effect in tumor-bearing mice. In the present study, we examined the role of Toll-like receptors (TLRs), which have been implicated in pathogen-induced cell signaling, in AILb-A-induced immune responses. In the luciferase assay using a nuclear factor (NF)-κB-dependent reporter plasmid, AILb-A induced NF-κB activation in the cells transfected with TLR4, but not with those transfected with the TLR2 gene, in a dose-dependent manner. TLR4-mediated NF-κB activation induced by AILb-A but not by lipopolysaccharide (LPS) was also observed under serum-free conditions. In in vitro experiments using human peripheral blood mononuclear cells, AILb-A-induced cytokine production was markedly inhibited by anti-TLR4 but not by anti-CD14 antibody, while LPS-induced, TLR4-mediated cytokine production was inhibited by anti-CD14 as well as anti-TLR4 antibodies. Cytokine production, killer cell activities, maturation of dendritic cells, phosphorylation of mitogen-activated protein kinases, and nuclear translocation of interferon-regulatory factor 3 induced by AILb-A were severely impaired in TLR4-deficient but not TLR2-deficient mice. Transfection of TLR4-deficient mouse-derived macrophages with the TLR4 expression plasmid led AILb-A to induce cytokines. Finally, the antitumor effect of AILb-A was also impaired in TLR4-deficient and TLR4-mutated mice. These findings suggest that TLR4 mediates antitumor immunity induced by the plant-derived protein AILb-A.

Purpose We investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in patients with oral squamous cell carcinoma (OSCC). Experimental Design Fifty OSCC patients who received radical resection of their tumor(s) were enrolled. Preoperative sera were measured for IL-8 by ELISA. Expression of IL-8 and the infiltration of immune cells in tumor tissues were analyzed by an immunohistochemical staining of surgical specimens. Results We found that disease-free survival (DFS) was significantly longer in the Stage I/II OSCC patients with low serum IL-8 levels compared to those with high levels (p = 0.001). The tumor expression of IL-8, i.e., IL-8(T) and the density of CD163-positive cells in the tumor invasive front, i.e., CD163(IF) were correlated with the serum IL-8 level (p = 0.033 and p = 0.038, respectively), and they were associated with poor clinical outcome (p = 0.007 and p = 0.002, respectively, in DFS) in all patients. A multivariate analysis revealed that N status, IL-8(T) and CD163(IF) significantly affected the DFS of the patients. Further analysis suggested that combination of N status with serum IL-8, IL-8(T) or CD163(IF) may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Interestingly, the in vitro experiments demonstrated that IL-8 enhanced generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10. Conclusions These findings indicate that IL-8 may be involved in poor clinical outcomes via generation of CD163-positive M2 macrophages, and that these factors in addition to N status may have prognostic value in patients with resectable OSCSS.

We isolated the lipoteichoic-acid-related molecule (OK-PSA) from OK-432, a streptococcal preparation, by affinity chromatography on CNBr-activated Sepharose-4B-bound monoclonal antibody TS-2, which neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. We have previously reported that OK-PSA is a potent inducer of Th1-type cytokines in human peripheral blood mononuclear cells in vitro. In this study, we conducted an animal experiment to examine whether OK-PSA exhibits an anti-tumor effect in vivo by acting as a Th1 inducer in syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. It was found that OK-PSA induced Th1-type cytokines [IFN-gamma, tumor necrosis factor-alpha, interleukin (IL)-2, IL-12 and IL-18] in BALB/c mice bearing Meth-A tumor and caused a marked anti-tumor effect. Although it was suggested by an in vitro study. using spleen cells derived from the animals, that IL-18 plays the greatest role in the induction of the Th1-dominant state and tumor cell killing induced by OK-PSA, the in vivo experiments demonstrated that both IL-12 and IL-18 are essential in the anti-tumor effect exhibited by OK-PSA. These findings strongly suggest that OK-PSA is a major effector molecule of OK-432 and may be a useful immunotherapeutic agent, as a potent Th1 inducer, for cancer patients with a Th2-dominant state.

Toll-like recetor (TLR) 4遺伝子発現と免疫療法剤 (OK-432およびその活性成分OK-PSA) の効果との関連につき検討した。OK-PSAの抗腫瘍免疫活性はTLR4の発現に依存して発揮される事が示された。OK-432局所投与された口腔癌患者の血清中IFN-γ上昇と末梢血単核球におけるTLR4とその co-factor MD2発現に関連性が認められた。さらに, 抗癌剤 Taxol および Taxotere がTLR4/MD2を介してNFκB活性化シグナルが伝達される事が明らかになった。菌体由来免疫療法剤の効果発現にTLR4/MD2シグナル伝達が重要である事, さらに抗癌剤でもTLR4/MD2を介したシグナルが伝わるものがある事が示唆された。

Summary Toll-like receptor 4 (TLR4) plays a significant role in cancer therapy as receptors of bacteria-derived immunotherapeutic agents such as OK-432, a streptococcal immunotherapeutic agent. In addition, recent reports demonstrated that TLRs, including TLR4, are also expressed in cancer cells as well as in immunocompetent cells. It is a problem in cancer therapy that the immunoadjuvant may activate survival signals such as nuclear factor (NF)-κB or mitogen-activated protein kinases (MAPKs) in cancer cells via TLRs. In the current study, we investigated responsiveness of human head and neck cancer cell lines against TLR4 ligands, OK-PSA, an active component of OK-432, and a lipopolysaccharide (LPS). Stimulation with LPS or OK-PSA resulted in the activation of NF-κB in these cell lines expressing TLR4 and MD-2 that is a significant coreceptor for TLR4 signaling. Interestingly, OK-PSA induced cell-growth inhibition, while LPS enhanced the proliferation of the cancer cells. OK-PSA induced NF-κB activation more slowly than that induced by LPS. In addition, phosphorylation of p38 MAPK by OK-PSA was only slight compared with that by LPS. OK-PSA also induced apoptosis of the cancer cells mediated by the activation of caspase 1, 3 and 8 in a p53-independent manner. These findings strongly suggest that active components of OK-432 may elicit anti-cancer effects via enhancing host immunity as well as via directly inducing the growth inhibition and apoptosis of head and neck cancer cells through TLR4 signal.

We have investigated the relationship between gene expression of Bcl 2 and Bax and the therapeutic effect in oral cancer patients. A significant correlation between Bcl-2/Bax gene expression ratio in the peripheral blood mononuclear cells (PBMCs) from the patients, and the therapeutic effect of radiation therapy in combination with UFT and OK-432, as well as survival rate, was observed. In addition, the statistically significant correlation was also observed between Bcl-2/Bax ratio and IFN-gamma and NK activity induced with OK-432. These findings suggest that Bcl-2 and Bax gene expression in PBMCs may be useful as a prognostic factor in oral cancer patients.

OK-PSA, an active component of OK-432, induces anti-tumor immunity via Toll-like receptor (TLR) 4/MD-2 complex. In the current study, we evaluated the effect of the OK-PSA on human head and neck cancer cell lines. Twelve cancer cell lines including 7 squamous cell carcinoma (SCC) cell lines and 5 salivary gland cancer (SGC) cell lines were examined. The quantitative real-time PCR analysis revealed that TLR4 mRNA was expressed in all 12 cell lines, and that MD-2 mRNA was expressed in 5 cell lines. OK-PSA stimulation resulted in the activation of NF-kappaB in the 4 SCC cell lines which express both TLR4 and MD-2 genes, and in 5 SGC cell lines which express at least TLR4 gene independently of MD-2 expression. In these OK-PSA-responsive cell lines, OK-PSA activated caspase-1, caspase-3 and caspase-8, and induced apoptosis. OK-PSA-induced apoptosis were observed even in a SGC cell line in which p53 is mutated and its function is impaired. These findings strongly suggest that OK-PSA induces apoptosis by the activation of caspases through p53-independent pathway via TLR4 signaling in head and neck cancer cells.

We have tried to identify the effective components of OK-432, a Streptococcus-derived anti-cancer immunotherapeutic agent. In the current study, we investigated the effect of OK-432-derived DNA (OK-DNA) in augmenting anti-cancer immune response. Analysis of OK-DNA with the restriction enzymes Hpa II and Msp I revealed that OK-DNA contained unmethylated CpG motifs. OK-DNA induced Th1-type cytokines, such as IFN-gamma and IL-12, and augmented killer cell activities in vitro on human peripheral blood mononuclear cells, whereas the methylated OK-DNA did not. Cytokines were also produced by OK-DNA-stimulated splenocytes derived from wild-type mice but not from TLR9-deficient mice. In the in vivo study, a peritumoral administration of OK-DNA resulted in a significant inhibition of tumor growth in syngeneic tumor-bearing wild-type and TLR4-deficient mice but not in TLR9-deficient mice. Anti-tumor effect of OK-432 in TLR9-deficient mice was significantly but partially reduced as compared with that in wild-type mice, while the effect of OK-432 was almost completely eliminated in TLR4-deficient mice. These findings suggest that unmethylated CpG-DNA in OK-432 functions as an active component in OK-432-induced anti-cancer immunity via TLR9, at least in part.

Although we have reported that Toll-like receptor (TLR) 4 is involved in OK-432-induced anti-cancer immunity, its detailed mechanism remained uncertain. We hypothesized that OK-432 may first be captured, dissolved by phagocytes, and then active components released from the cells may stimulate TLR4. This hypothesis was examined by the current in vitro experiments. We used TS-2 MoAb which recognizes OK-PSA, an active component of OK-432. First, we observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining by using TS-2 clearly demonstrated that OK-432 was captured and dissolved by these cells. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by ELISA with TS-2. The supernatants from OK-432-treated DC culture, but not from untreated DC culture, increased NF-kappaB activity in TLR4-expressing cells. The increased NF-kappaB activity was inhibited by TS-2. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the OK-432 action.

We investigated the in vivo anti-tumor effect of intratumoral administration of dendritic cells (DCs) after chemotherapy using TS-1, and followed by immunopotentiator OK-432. Both in Meth-A-bearing BALB/c and in SCCV II-bearing C3H/HeN mice, one week of oral administration of TS-1 affected a partial eradication of established tumors. TS-1 followed by DCs and OK-432 resulted in a marked inhibition in tumor growth, and also contributed to a greater prolongation of survival. Cytotoxic activities of tumor-infiltrating lymphocytes and draining lymph node cells against inoculated tumor cells were significantly increased by the therapy. Cytotoxic memory T cells were also induced. The same therapy was also applied to SCCV II-bearing C3H/HeJ mice in which the Toll-like receptor (TLR) 4 is mutated; no immunotherapeutic effect was observed in the mice. These findings suggest that local DC therapy in combination with TS-1 and OK-432 may well be a useful strategy for the treatment of solid tumors, and that TLR4 signaling is involved in the success of this therapy.

A lipoteichoic acid-related molecule OK-PSA is an active component of OK-432. In the in vitro experiments, OK-PSA enhanced expression of MHC class II, CD80 and CD86, as well as IL-12 production on dendritic cells (DCs) were derived from wild-type mice, but not from TLR4-deficient (TLR4-/-) mice. Next we examined the in vivo anti-cancer effect of intratumoral administration of syngeneic DCs followed by OK-PSA against established tumors in mice. Although OK-PSA augmented anti-tumor effect of DC administration in tumor-bearing wild-type mice, anti-tumor effect of DCs and OK-PSA was not significant in TLR4-/- mice. Interestingly, an administration of wild-type mice-derived DCs followed by OK-PSA exhibited a marked anti-tumor effect even in TLR4-/- mice. These findings suggest that OK-PSA may be a potential adjuvant for local DC therapy, and that DC therapy followed by OK-PSA is able to elicit anti-cancer activity even in TLR4-deficient host when TLR4 is expressed only in DCs injected intratumorally.

The authors investigated the in vivo anti-tumor effect of intratumoral administration of bone marrow-derived dendritic cells (DCs) after chemotherapy using an oral fluoropyrimidine anti-cancer drug TS-1, and followed by immunotherapeutic agent OK-432, in two syngeneic tumor-bearing mouse models. Both in Meth-A fibrosarcoma-bearing BALB/c mice and in SCCVII-bearing C3H/HeN mice, 1 week of oral administration of TS-1 effected partial eradication of established tumors. Intratumoral injection of DCs and OK-432 caused only slight inhibition of the tumor growth. However, TS-1 administration followed by DCs and OK-432 resulted in a marked inhibition in the tumor growth and also contributed to a greater prolongation of survival. By the injection of DCs and OK-432 after TS-1 administration, a significant infiltration of immune cells, especially CD8 + T cells, was observed. Furthermore, the cytotoxic activities of tumor-infiltrating lymphocytes and draining lymph node cells against inoculated tumor cells were significantly increased by the therapy, while activities against nonspecific target cells were not. Cytotoxic memory T cells were also induced; the main effectors were MHC class 1-restricted, CD8 + T cells. The same therapy was also applied to SCCVII-bearing C3H/HeJ mice in which the Toll-like receptor (TLR) 4 is mutated and its function impaired; no immunotherapeutic effect was observed in the TLR4-deficient mouse model. These findings suggest that the local DC therapy in combination with TS-1 and OK-432 may be a useful strategy for the treatment of solid tumors, and that TLR4 signaling is involved in the success of this therapy.

Background: The streptococcal agent OK-432 has been used for immunotherapy of head and neck cancer, among other malignancies, but its mechanism of action is unknown. Because the Toll-like receptor 4 (TLR4)/MD-2 complex is important in enabling the mammalian immune system to recognize bacterial components, we investigated whether expression of the TLR4 and MD-2 genes is associated with OK-432-induced anticancer immunity. Methods: Peripheral blood mononuclear cells (PBMCs) from 28 patients with head and neck cancer were analyzed for TLR4 and MD-2 mRNA expression by reverse transcription–polymerase chain reaction (RT–PCR) analysis. PBMCs were treated in vitro with OK-432 or with OK-PSA (a lipoteichoic-acidrelated molecule that is an active component of OK-432), and interferon-gamma (IFN-) mRNA expression, an immune response measure, was analyzed by RT–PCR. Patient sera collected 24 hours after OK-432 administration were examined for IFN- protein using an enzyme-linked immunosorbent assay. Lewis lung carcinoma-bearing wild-type C57BL/6 and TLR4-deficient mice (four mice per group) received intraperitoneal injections of OK-432, and tumor volumes and sera IFN- levels were measured over time. All statistical tests were two-sided. Results: Twenty patients expressed both TLR4 and MD-2. Expression of TLR4 and MD-2 genes was associated with the in vivo IFN- induction in 19 patients administered OK-432 (Fisher’s exact test P

OK-PSA, a lipoteichoic acid (LTA)-related molecule isolated from a streptococcal agent OK-432, enhances anti-tumor immunity as a potent inducer of Th1-type cytokines. Recently, we obtained the data suggesting that natural killer (NK) cells may play a significant role for OK-PSA-induced cytokine production in vitro.We conducted the animal experiments using athymic nude mice bearing human salivary adenocarcinoma to examine the role of NK cells in OK-PSA-induced anti-tumor immunity. OK-PSA was peritumorally injected into the mice. Cytokines in the sera were analyzed by ELISA. mRNAs for cytokines were detected by RT-PCR. 51Cr release test was performed to measure killer cell activities.OK-PSA markedly increased the amounts of IFN-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18 that are generally called "Th1-type cytokines" in the sera derived from tumor-bearing nude mice, and also accelerated the killing activities of tumor-infiltrating lymphocytes as well as of draining lymph node cells. Furthermore, OK-PSA administration resulted in significant inhibition of tumor growth, but the effect of OK-PSA was almost completely inhibited by the deletion of NK cells using anti-asialo GM1 antibody.These findings strongly suggested that NK cells are closely involved in OK-PSA-mediated anti-tumor immunity.

We previously generated a monoclonal antibody, TS-2, that neutralizes the interferon (IFN)-gamma-inducing activity of OK-432, a penicillin-killed streptococcal preparation [J. Immunother. 13 (1993) 232]. Expression of the TS-2-binding antigen was markedly higher in the cell wall of the penicillin-treated Streptococcus pyogenes (OK-432) than in the untreated bacteria (Su-BBM). We here isolated the antigens from OK-432 and Su-BBM, designated OK-PSA and Su-PSA, respectively. OK-432 markedly induced IFN-gamma and interleukin (IL)-18 as compared with Su-BBM in human peripheral blood mononuclear cells (PBMC). Furthermore, all of the Thl-type and Th1-inducing cytokines tested [IFN-gamma, tumor necrosis factor (TNF)-alpha, IL-12 and IL-18] were secreted by OK-PSA-stimulated PBMC far better than by Su-PSA-treated PBMC. In addition, the cytolytic activities of the PBMC were accelerated by the stimulation with OK-432 or OK-PSA far better than by the stimulation with Su-BBM or Su-PSA. These findings strongly suggested that OK-PSA is a highly important molecule of OK-432 and may be a useful immunotherapeutic agent for the patients with malignant diseases as a potent Th inducer. It was also shown that penicillin treatment effectively enhances OK-PSA-induced anti-cancer immunity.

A lipoteichoic acid-related molecule (OK-PSA) isolated from OK-432, a penicillin-killed Streptococcus pyogenes, is a potent inducer of Th1 cytokines, and elicits anti-cancer effect in tumor-bearing mice. Toll-like receptor (TLR) 4 is a member of the recently identified toll-like receptor family of proteins that has been implicated in lipopolysaccharide-induced cell signaling. In the present study, we have examined the role of TLR4 for OK-PSA-induced Th1-cytokine production and anti-tumor effect by using C3H/HeJ mice in which TLR4 function is impaired. Although OK-PSA strikingly induced Th1 cytokines [interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18] in the splenocytes derived from control animals (C3H/HeN), OK-PSA did not induce the cytokines in the splenocytes from C3H/HeJ. Furthermore, C3H/HeJ-derived splenocytes acquired the responsiveness to OK-PSA stimulation by overexpression of TLR4 gene. Finally, OK-PSA administration significantly inhibited the tumor growth and lung metastasis of syngeneic squamous cell carcinoma cells in C3H/HeN; however, no effect of OK-PSA was observed in C3H/HeJ. These findings strongly suggest that TLR4 signaling is involved in regulating OK-PSA-induced anti-cancer immunity.

We have isolated a 55 kDa protein from the seed extract of Aeginetia indica L. (AIL), a parasitic plant, by affinity chromatography on an N-hydroxysuccinimide-activated Sepharose High Performance column bound with F3, a monoclonal antibody that neutralizes the cytokine-inducing and anti-tumor effect of AIL. In the present study, we examined this protein (AILb-A) for cytokine induction and anti-tumor effects by animal study, using syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. AILb-A administration resulted in markedly increased levels of Th1 cytokines [interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18] in the sera derived from Meth-A-bearing mice. The in vitro re-stimulation with AILb-A of splenocytes derived from AILb-A-primed mice also selectively induced Th1-type cytokines and antigen-specific killer cell activity. The neutralizing test using cytokine-specific antibodies revealed that AILb-A-induced IL-18 plays a most significant role for and killer cell-inducing activities. Furthermore, IL-12 and IL-18 induced by AILb-A inhibited specifically IL-10 and IL-4 production, respectively. Finally, we examined the anti-tumor effect of AILb-A in both Meth-A-bearing BALB/c mice and Meth-A-bearing nude mice with BALB/c background. AILb-A exhibited a striking anti-tumor effect in normal BALB/c mice inoculated with Meth-A cells. In athymic nude mice, the anti-tumor effect of AILb-A was relatively weak. These findings strongly suggested that AILb-A is a potent Th1 inducer and may be a useful immunotherapeutic agent for patients with malignant diseases.

Purpose: Dendritic cells (DCs) are professional antigen-presenting cells that play a central role in initiating adaptive and innate immune responses. DC-based vaccination is widely tested as a promising therapeutic cancer vaccine in multiple clinical trials. Programed cell death 1(PD-1)/PD-1 Ligands (PD-Ls) pathway are involved in the negative regulation of T cell-functions. PD-1 is expressed on T cells, and PD-Ls are expressed on DCs as well as some cancer cells. More recent studies have indicated that certain chemotherapeutic agents might enhance the cancer vaccine-mediated immune responses. In the current study, we conducted the in vitro experiments to examine whether or not chemotherapeutic drugs which are used for patients with head and neck cancer, may enhance T cell-stimulating ability of DCs via regulation of the expression of PD-Ls. Methods: Healthy volunteer-derived human peripheral blood monocyte were cultured with IL-4 and GM-CSF for 5 days to generate immature DCs, and then the DCs were matured by the stimulation with OK-432 which is a streptococcal immune adjuvant, for 24 hours, followed by the treatment with each chemotherapeutic drug such as 5-FU, CDDP, docetaxel or gemcitabine for 24 hours. Viability of DCs was assessed by WST-8. IL-10 and IL-12 in the supernatants from the DC cultures were measured by ELISA. Expression of mRNAs and proteins for PD-Ls was analyzed by quantitative real-time PCR and flow cytometry, respectively. DCs were co-cultured with allogeneic T cells (E/T ratio=1/20, 1/200 or 1/2000), then IFN-γ in the supernatants were measured by ELISA to evaluate T cell-stimulating ability of the DCs. Results: IL-12 production by DCs was significantly enhanced by OK-432 stimulation. Although CDDP, docetaxel and gemcitabine inhibited the OK-432-induced IL-12 production on the DCs, 5-FU did not. 5-FU and gemcitabine significantly inhibited the IL-10 production by OK-432-stimulated DCs. Expression of PD-L1 and PD-L2 genes as well as of their proteins on the DCs which was increased by OK-432 stimulation, was significantly suppressed by the treated with 5-FU or docetaxel. OK-432-stimulated DCs-treated with 5-FU strongly enhanced both proliferation and IFN-γ production of allogeneic T cells as compared with DCs stimulated only with OK-432. Conclusions: The findings obtained from the current study suggested the possibility that 5-FU may enhance the T cell-stimulating ability of dendritic cells via regulation of the expression of PD-L1 and PD-L2, and that the combination therapy of DC-based cancer vaccine with 5-FU may elicit better anti-tumor efficacy than only vaccination. Citation Format: Tomoyuki Tano, Masato Okamoto, Hiroyuki Goda, Yohei Fujita, Koh-ichi Nakashiro, Hiroyuki Hamakawa. Enhancement of the T cell-stimulating ability of dendritic cells by 5-FU via regulation of the expression of ligands for programed cell death 1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4722. doi:10.1158/1538-7445.AM2013-4722

In the current study, we have evaluated the anti-cancer effect of OK-432 in combination with UFT, an oral preparation combining the 5-FU pro-drug tegafur and uracil in a 1:4 ratio, and radiation therapy (RT). RT+UFT+OK-432 therapy elicited better clinical response and longer survival time than only RT+UFT. Next, we conducted the in vitro study to examine the effect of 5-FU and X-ray irradiation in the OK-432-induced cytokines. Human peripheral blood mononuclear cells (PBMCs) were stimulated with OK-432, 5-FU or X-ray for 24 hr. OK-432 induced Th1-type cytokines (IFN-γ, IL-12, IL-18) as well as IL-10 and TGF-β. When PBMCs were stimulated by 5-FU and/or X-ray in combination with OK-432, production of IL-10 and TGF-β was significantly inhibited. Gene expression of negative regulators, SOCS-1 and -3, was augmented by OK-432, however, this was also inhibited by 5-FU and X-ray irradiation. OK-432-induced IL-10 and TGF-β, but not Th1-type cytokines, was significantly reduced by addition of antisense oligonucleotide for SOCS-1 or that for SOCS-3. Radiation and 5-FU induce Th1-dominant state by inhibiting the OK-432-induced production of IL-10 and TGF-β mediated by regulation of negative regulators, SOCS-1 and -3. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1804. doi:10.1158/1538-7445.AM2011-1804