Biomedical subjects

Ichiro Azuma

Publications and source records attributed to Ichiro Azuma.

10 recordsLinked to original sources

PubMed2006

Enhancement of immunologic tumor regression by intratumoral administration of dendritic cells in combination with cryoablative tumor pretreatment and Bacillus Calmette-Guerin cell wall skeleton stimulation.

PURPOSE: We developed an effective immunotherapy, which could induce antitumor immune responses against shared and unique tumor antigens expressed in autologous tumors. EXPERIMENTAL DESIGN: Intratumoral administration of dendritic cells is one of the individualized immunotherapies; however, the antitumor activity is relatively weak. In this study, we attempted to enhance the antitumor efficacy of the i.t. dendritic cell administration by combining dendritic cells stimulated with Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) additionally with cryoablative pretreatment of tumors and analyzed the therapeutic mechanisms. RESULTS: These two modifications (cryoablation of tumors and BCG-CWS stimulation of dendritic cells) significantly increases the antitumor effect on both the treated tumor and the untreated tumor, which was distant at the opposite side, in a bilateral s.c. murine CT26 colon cancer model. Further analysis of the augmented antitumor effects revealed that the cryoablative pretreatment enhances the uptake of tumor antigens by the introduced dendritic cells, resulting in the induction of tumor-specific CD8(+) T cells responsible for the in vivo tumor regression of both treated and remote untreated tumors. This novel combination i.t. dendritic cell immunotherapy was effective against well-established large tumors. The antitumor efficacy was further enhanced by depletion of CD4(+)CD25(+)FoxP3(+) regulatory T cells. CONCLUSIONS: This novel dendritic cell immunotherapy with i.t. administration of BCG-CWS-treated dendritic cells following tumor cryoablation could be used for the therapy of cancer patients with multiple metastases.

Adjuvants, Immunologic↗

PubMed2005

Dendritic cell maturation induced by muramyl dipeptide (MDP) derivatives: monoacylated MDP confers TLR2/TLR4 activation.

6-O-acyl-muramyldipeptides (MDP) with various lengths of fatty acid chains were examined for their dendritic cell (DC) maturation activity expressed through TLRs. Judging from anti-TLR mAb/inhibitor-blocking analysis, MDP derivatives with a single octanoyl or stearoyl fatty acid chain were found to activate TLR2 and TLR4 on human DCs, although intact and diacylated MDP expressed no ability to activate TLRs. Human DC activation profiles by the monoacylated MDP were essentially similar to those by Calmette-Guerin (BCG)-cell wall skeleton (CWS) and BCG-peptidoglycan (PGN) based on their ability to up-regulate costimulators, HLA-DR, beta(2)-microglobulin, and allostimulatory MLR. Monoacylated MDP induced cytokines with similar profiles to BCG-CWS or -PGN, although their potency for induction of TNF-alpha, IL-12p40, and IL-6 was less than that of BCG-CWS or -PGN. The MDP derivatives initiated similar activation in normal mouse macrophages, but exhibited no effect on TLR2/4-deficient or MyD88-deficient mouse macrophages. Mutation of d-isoGln to l-isoGln in monoacylated MDP did not result in loss of the DC maturation activity, suggesting marginal participation of nucleotide-binding oligomerization domain 2, if any, in monoacyl MDP-dependent DC maturation. These results define the adjuvant activity of 6-O-acyl MDP compounds at the molecular level. They target TLR2/TLR4 and act through the MyD88-dependent pathway in DCs and macrophages. Hence, the unusual combined activation of TLR2 and TLR4 observed with Mycobacterium tuberculosis is in part reflected in the functional properties of monoacylated MDP compounds. These findings infer that the essential minimal requirement for TLR2/4-mediated adjuvancy of BCG lies within a modified MDP.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed2004

WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone.

A Wilms' tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer. WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models. We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a "prophylactic" model). However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a "therapeutic" setting. In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a "therapeutic" model) compared with WT1 peptide vaccination alone. An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells. These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.

Animals↗

PubMed2004

Adjuvant-mediated tumor regression and tumor-specific cytotoxic response are impaired in MyD88-deficient mice.

The Mycobacterium bovis bacillus Calmette-Guérin cell-wall skeleton (BCG-CWS) activates Toll-like receptor (TLR) 2 and TLR4, but unlike the typical TLR4 agonist bacterial lipopolysaccharide barely induces type 1 IFN. BCG-CWS has been used for adjuvant immunotherapy for patients with cancer. We investigated the adjuvant potential of BCG-CWS for induction of CTLs subsequent to TLR-mediated dendritic cell (DC) maturation, using a syngeneic mouse tumor model (B16 melanoma in C57BL/6). We evaluated the retardation of tumor growth and cytotoxic response in wild-type and MyD88-/- mice immunized with tumor debris and/or BCG-CWS. Delays in tumor growth and cytotoxic response were induced by immunization with a mixture of BCG-CWS emulsion and the tumor. BCG-CWS was capable of activating DCs ex vivo by the criteria of CD80/CD86 up-regulation and cytokine (interleukin-12, tumor necrosis factor-alpha) induction. Efficient tumor suppression and ex vivo cytokine induction did not occur in MyD88-deficient mice and cells, suggesting that the MyD88 adapter is crucial for induction of tumor cytotoxicity. Because TLR4 is involved in both MyD88-dependent and -independent pathways and the latter affects DC maturation, our findings indicate that both pathways cooperate to induce CTL-based tumor immunity.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Mycobacterium bovis BCG cell wall-specific differentially expressed genes identified by differential display and cDNA subtraction in human macrophages.

We have analyzed the gene expression profile of monocytes in response to a highly purified cell wall fraction of Mycobacterium bovis BCG, a clinically approved adjuvant known as BCG cell wall skeleton (BCG-CWS). It is composed of mycolic acid, arabinogalactan, and peptidoglycan and confers Toll-like receptor 2 (TLR2)- and TLR4-dependent signaling that induces monocytes to differentiate into antigen-presenting cells (APCs). Here we report differential gene expression analysis with BCG-CWS-stimulated versus nonstimulated monocytes. BCG-CWS exerted massive induction of genes regulated by TLR signaling. Marked gene regulatory characteristics in BCG-CWS-stimulated cells compared to lipopolysaccharide (LPS)-stimulated cells follow. (i) Spliced mRNAs encoding soluble forms of TREM-1 and TREM-2 (recently discovered inflammatory-signal-amplifying receptors) were regulated by BCG-CWS, resulting in their differential expression. (ii) The genes for zinc-iron transporter protein (ZIP)-like family proteins HKE-1 and LIV-1 were induced exclusively by BCG-CWS. (iii) Interleukin-23 (IL-23), rather than IL-12p70, was induced by BCG-CWS, while interferon-inducible genes were induced only by LPS. By Northern and reverse transcription-PCR analyses, we confirmed the differential expression of more than 30 BCG-CWS regulatory genes, and their expression was compared with that of LPS and other known TLR ligands. A battery of genes responded rapidly and for a short time to LPS but for a long time to BCG-CWS. Structural analysis of the identified novel or hypothetical proteins revealed that some are potential candidates as signaling mediators or transcriptional regulators. Hence, BCG-CWS may profoundly modulate APC responses in a way distinct from that of LPS, leading to possible advantages for its adjuvant-active therapeutic potential.

Adjuvants, Immunologic↗

PubMed2003

Simultaneous blocking of human Toll-like receptors 2 and 4 suppresses myeloid dendritic cell activation induced by Mycobacterium bovis bacillus Calmette-Guérin peptidoglycan.

The Mycobacterium bovis bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-alpha) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-alpha production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-kappa B activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-kappa B activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-kappa B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.

Animals↗

PubMed2003

Erythrocyte-replaced mouse model for Haemoparasite studies: comparison of NOD/shi-scid and C.B-17/Jcl-scid mouse upon acceptability of human erythrocytes.

The erythrocyte-exchanged chimera mouse model has become to be a significant tool for studying animal and human (hu) protozoan haemoparasites, though the usefulness of this model varies depending primarily on the acceptability of xenogeneic red blood cells (RBC). To find a superior recipient in comparison with C.B-17/Jcl mouse with severe combined immuno-deficiency (scid) mutation, we examined in this report the non-obese diabetes (NOD)/shi-scid mouse, a recently available strain of SCID. When 2.5 x 10(8) of fluorescent dye-labeled hu-RBCs were transfused, C.B-17scid mouse eliminated them logarithmically by a simple linear regression, while NOD-scid mouse eradicated hu-RBCs by a unique two-step fashion, i.e., a potent but only briefly functioning RBC eradication followed by a weak steadily functioning step. The means of regression line constance +/- their standard deviations (SD) of 205 C.B-17scid and of 213 NOD-scid mice for their short- and long-lasting steps were -0.73 +/- 0.63, -0.53 +/- 0.25 and -0.16 +/- 0.10, respectively. Hu-RBC half-lives determined from these means of C.B-17scid mice and of NOD-scid mice for the short- and long-living steps were 3.6, 4.9 and 16.3 hr, respectively. Higher hu-RBC acceptability of NOD-scid mouse, especially at their long-lasting step, was also demonstrated under at an activated state of mouse innate immunity. Treatment with 1.0 mg heat-killed Candida cells caused an acceleration of hu-RBC elimination in both mouse strains but the magnitudes for the short- and long-living steps of NOD-scid mice evaluated by "stimulation index" were only 1/2.6 and 1/7.6 of C.B-17scid mice, respectively.

Animals↗

PubMed2002

Inhibitory effect of BCG cell-wall skeletons (BCG-CWS) emulsified in squalane on tumor growth and metastasis in mice.

The antimetastatic effect of BCG-CWS, which was emulsified in an oil-in-water form with either Drakeol 6VR mineral oil (BCG-CWS/DK) or squalane (BCG-CWS/SQA), on lung metastasis produced by highly metastatic murine tumor cells, Colon26-M3.1 carcinoma cells and B16-BL6 melanoma cells, was investigated in syngeneic mice. An intravenous (i.v.) administration of BCG-CWS (100 mg/mouse) 1 day after tumor inoculation significantly inhibited tumor metastasis of both Colon26-M3.1 carcinoma and B16-BL6 melanoma cells in experimental lung metastasis models. No differences in the antitumor activity of the two oil-based formulations (BCG-CWS/DK and BCG-CWS/SQA) were obverved. However, BCG-CWS/SQA administered through subcutaneous (s.c.) route was shown to be effective only when it was consecutively injected (3 times) after tumor inoculation. An in vivo analysis for tumor-induced angiogenesis showed that a single i.v. administration of BCG-CWS/SQA inhibited the number of tumor-induced blood vessels and suppressed tumor growth. Furthermore, the multiple administration of BCG-CWS/SQA given at on week intervals led to a significant reduction in spontaneous lung metastasis of B16-BL6 melanoma cells in a spontaneous metastasis model. These results suggest that BCG-CWS emulsified with squalane is a potent inhibitory agent of lung metastasis, and that the antimetastatic effect of BCG-CWS is related to the suppression of tumor growth and the inhibition of tumor-induced angiogenesis.

Angiogenesis Inhibitors↗

PubMed2002

Structural-functional relationship of pathogen-associated molecular patterns: lessons from BCG cell wall skeleton and mycoplasma lipoprotein M161Ag.

The innate immune system senses microbial components by signaling receptors and induces phagocytosis by uptake receptors. The Toll-like receptor represents the signaling receptors that cause maturation of dendritic cells, while phagocytosis is supported by other receptor families. We identify the structural signatures of microbial components recognized by these receptors to establish the two-receptor hypothesis in innate immunity.

Adjuvants, Immunologic↗

PubMedDate not supplied

Role of toll-like receptors and their adaptors in adjuvant immunotherapy for cancer.

The potentiation of immune responses to tumor-associated antigen (Ag) is a pivotal issue in immunotherapy for cancer and thus requires the use of adjuvants, which are involved in efficient antibody (Ab) production and killer cell induction. The efficacy for tumor regression of a number of adjuvants that have been applied to immunotherapy in humans and tumor-bearing animal models has been tested without understanding of the function of adjuvants. Recent findings on the function of Toll-like receptors (TLRs) and their adaptors facilitated the elucidation of the molecular basis of adjuvant activity. TLR signaling was found to induce interferons (IFNs), chemokines and proinflammatory cytokines and mature dendritic cells (DCs) for enhanced efficiency in antigen presentation. The mediators then play a crucial role in the organization of acquired immunity and, together with matured DCs, activate cytotoxic T cells (CTL) and NK cells. These TLR outputs vary among adjuvants, which may depend on adjuvant-specific selection of appropriate sets of TLRs and their adaptors. Here we review how a variety of host immune responses are induced by an individual adjuvant to confer an adjuvant-specific anti-tumor immunity. We elaborate specifically on two adjuvants, BCG-cell wall skeleton and double-stranded RNA (dsRNA). The former activates TLR2/4 on DCs and induces tumor-specific CTL allowing general application to patients with surgically dissected cancer and improving prognosis, while the latter activates TLR3 on DCs to release type 1 IFN that induces tumor cell apoptosis and NK-mediated tumor cytotoxicity.

Adjuvants, Immunologic↗