Yui Harada

Sustained and NK/CD4+ T Cell-Dependent Efficient Prevention of Lung Metastasis Induced by Dendritic Cells Harboring Recombinant Sendai Virus

We recently demonstrated efficient antitumor immunity against murine tumors using dendritic cells (DCs) activated by recombinant Sendai viruses (rSeVs), and proposed a new concept, “immunostimulatory virotherapy,” for cancer immunotherapy. However, there has been little information on the efficacy of this method in preventing metastatic diseases. In this study, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV (rSeV/dF) using a murine model of lung metastasis. Bolus and i.v. administration of DCs harboring rSeV/dF-expressing GFP without pulsation of tumor Ag (DC-rSeV/dF-GFP) 2 days before tumor inoculation showed efficient prevention against lung metastasis of c1300 neuroblastoma, but not of RM-9 prostatic cancer. We found that the timing of DC therapy was critical for the inhibition of pulmonary metastasis of RM-9, and that the optimal effect of DCs was seen 28 days before tumor inoculation. Interestingly, the antimetastatic effect was sustained for over 3 mo, even when administered DCs were already cleared from the lung and organs related to the immune system. Although NK cell activity had already declined to baseline at the time of tumor inoculation, Ab-mediated depletion studies revealed that CD4+ cells as well as the presence of, but not the activation of, NK cells were crucial to the prevention of lung metastasis. These results are the first demonstration of efficient inhibition of lung metastasis via bolus administration of virally activated DCs that was sustained and NK/CD4+ cell-dependent, and may suggest a potentially new mechanism of DC-based immunotherapy for advanced malignancies.

Carbon-ion beam treatment induces systemic antitumor immunity against murine squamous cell carcinoma.

Carbon‐ion beam (CIB) treatment is a powerful tool for controlling primary tumors in the clinical setting. However, to date, few clinical or experimental studies have investigated the effects of CIB treatment on tumor recurrence and antitumor immunity.

Cytokine-based high log-scale expansion of functional human dendritic cells from cord-blood CD34-positive cells

Dendritic cells (DCs) play a crucial role in maintaining the immune system. Though DC-based cancer immunotherapy has been suggested as a potential treatment for various kinds of malignancies, its clinical efficacies are still insufficient in many human trials. Issues that limit the clinical efficacy of DC-based immunotherapy, as well as the difficulty of the industrial production of DCs, are largely due to the limited number of autologous DCs available from each patient. We here established a possible breakthrough, a simple cytokine-based culture method to expand the log-scale order of functional human DCs. Floating cultivation of cord-blood CD34+ cells under an optimized cytokine cocktail led these progenitor cells to stable log-scale proliferation and to DC differentiation. The expanded DCs had typical features of conventional myeloid DCs in vitro. Therefore, the concept of DC expansion should contribute significantly to the progress of DC immunotherapy.

Antagonism of VEGF by genetically engineered dendritic cells is essential to induce antitumor immunity against malignant ascites

Malignant ascitis (MA) is a highly intractable and immunotherapy-resistant state of advanced gastrointestinal and ovarian cancers. Using a murine model of MA with CT26 colon cancer cells, we here determined that the imbalance between the VEGF-A/vascular permeability factor and its decoy receptor, soluble fms-like tryrosine kinase receptor-1 (sFLT-1), was a major cause of MA resistance to dendritic cell (DC)-based immunotherapy. We found that the ratio of VEGF-A/sFLT-1 was increased not only in murine but also in human MA, and F-gene–deleted recombinant Sendai virus (rSeV/dF)-mediated secretion of human sFLT-1 by DCs augmented not only the activity of DCs themselves, but also dramatically improved the survival of tumor-bearing animals associated with enhanced CTL activity and its infiltration to peritoneal tumors. These findings were not seen in immunodeficient mice, indicating that a VEGF-A/sFLT-1 imbalance is critical for determining the antitumor immune response by DC-vaccination therapy against MA. Mol Cancer Ther; 10(3); 540–9. ©2011 AACR.

Cytokine-based log-scale expansion of functional murine dendritic cells

Background Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies. Methodology/Principal Findings Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b+/CD11c+ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines. Conclusions/Significance The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.

BubR1 Insufficiency Inhibits Neointimal Hyperplasia Through Impaired Vascular Smooth Muscle Cell Proliferation in Mice

Objective—BubR1, a cell cycle–related protein, is an essential component of the spindle checkpoint that regulates cell division. Mice with BubR1 expression reduced to 10% of the normal level display a phenotype characterized by progeria; however, the involvement of BubR1 in vascular diseases is still unknown. We generated mice in which BubR1 expression was reduced to 20% (BubR1L/L mice) of that in wild-type mice (BubR1+/+) to investigate the effects of BubR1 on arterial intimal hyperplasia. Approach and Results—Ten-week-old male BubR1L/L and age-matched wild-type littermates (BubR1+/+) were used in this study. The left common carotid artery was ligated, and histopathologic examinations were conducted 4 weeks later. Bone marrow transplantation was also performed. Vascular smooth muscle cells (VSMCs) were isolated from the thoracic aorta to examine cell proliferation, migration, and cell cycle progression. Severe neointimal hyperplasia was observed after artery ligation in BubR1+/+ mice, whereas BubR1L/L mice displayed nearly complete inhibition of neointimal hyperplasia. Bone marrow transplantation from all donors did not affect the reconstitution of 3 hematopoietic lineages, and neointimal hyperplasia was still suppressed after bone marrow transplantation from BubR1+/+ mice to BubR1L/L mice. VSMC proliferation was impaired in BubR1L/L mice because of delayed entry into the S phase. VSMC migration was unaffected in these BubR1L/L mice. p38 mitogen–activated protein kinase–inhibited VSMCs showed low expression of BubR1, and BubR1-inhibited VSMCs showed low expression of p38. Conclusions—BubR1 may represent a new target molecule for treating pathological states of vascular remodeling, such as restenosis after angioplasty.

Ex Vivo Generation of Highly Purified and Activated Natural Killer Cells from Human Peripheral Blood

Adoptive immunotherapy using natural killer (NK) cells has been a promising treatment for intractable malignancies; however, there remain a number of difficulties with respect to the shortage and limited anticancer potency of the effector cells. We here established a simple feeder-free method to generate purified (>90%) and highly activated NK cells from human peripheral blood-derived mononuclear cells (PBMCs). Among the several parameters, we found that CD3 depletion, high-dose interleukin (IL)-2, and use of a specific culture medium were sufficient to obtain highly purified, expanded (∼200-fold) and activated CD3(-)/CD56(+) NK cells from PBMCs, which we designated zenithal-NK (Z-NK) cells. Almost all Z-NK cells expressed the lymphocyte-activated marker CD69 and showed dramatically high expression of activation receptors (i.e., NKG2D), interferon-γ, perforin, and granzyme B. Importantly, only 2 hours of reaction at an effector/target ratio of 1:1 was sufficient to kill almost all K562 cells, and the antitumor activity was also replicated in tumor-bearing mice in vivo. Cytolysis was specific for various tumor cells, but not for normal cells, irrespective of MHC class I expression. These findings strongly indicate that Z-NK cells are purified, expanded, and near-fully activated human NK cells and warrant further investigation in a clinical setting.

Genetic heterogeneity of the cytolethal distending toxin B (cdtB) gene locus among isolates of Campylobacter lari

coli, are curved Gram-negative bacteria that are the recognised cause of acute bacterial diarrhoea around the world. C. lari is a relatively recently discovered thermophilic Campylobacter species first isolated from mammalian and avian species, particularly seagulls of the genus Larus. C. lari has also been shown to be a cause of clinical infection. An atypical group of isolates of urease-positive thermophilic campylobacters (UPTC) was first isolated from the natural environment in England in 1985. Thereafter, these organisms were described as a biovar or variant of C. lari. Subsequent reports described four human isolates in France. Some additional isolates of UPTC have also been reported in Ireland, in The Netherlands and in Japan. The possible association of UPTC with human disease remains unclear. Two representative taxa, namely ureasenegative (UN) C. lari and UPTC, occur within the species of C. lari. Although several Campylobacter species’ cytotoxins have been identified, only the cytolethal distending toxin (CDT) has been characterised in detail. The cdt genes of C. jejuni have been cloned and characterised by Pickett et al. However, in relation to the cdt genes, no reports have yet appeared for C. lari. Therefore, the aim of the present study is to clone, sequence and analyse the cdtB gene of C. lari isolates and compare the sequences obtained with those of other thermophilic campylobacters. Twenty-four isolates of C. lari (UN C. lari [n=16] and UPTC [n=8]) were used in the present study (Table 1), together with three reference strains (JCM2530T, NCTC12892 and NCTC12893). The test organisms were isolated from different sources in several countries. The organisms were cultured on blood agar containing defibrinated horse blood (Nippon Bio-Test, Tokyo, Japan) and supplemented with campylobacter-selective medium (Nissui, Tokyo, Japan), under microaerophilic conditions at 37 ̊C for two days. Template DNA was prepared by boiling in water at 95 ̊C for five minutes. The PCR mixture contained 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 400 μmol each dNTP, 1 μmol each primer, and 1 unit of Thermus aquaticus (Taq) DNA polymerase (Takara Bio, Shiga, Japan). A schematic representation of the cdtB gene and its genetic loci for C. lari RM2100 (GenBank Accession No. AAFK00000000) including the locations of a primer pair for the cdtB (JCB common up and JCB common down [Asakura primer]) employed in the present study for PCR amplification is shown in Figure 1. This primer pair was designed to generate a product of approximately 700 bp (equivalent to a 90% segment of the cdtB structural gene of C. lari RM2100, AAFK00000000) of the cdtB gene with C. jejuni, C. coli and C. fetus isolates. The polymerase chain reaction (PCR) was performed in 50-μL reaction volumes, for 30 cycles at 94 ̊C for 30 sec, 55 ̊C for 30 sec, 72 ̊C for 45 sec, followed by a final extension of 72 ̊C for 5 min. Amplified PCR products were separated by 1.0% (w/v) agarose gel electrophoresis in 0.5x TBE at 100 V and detected by ethidium bromide staining. PCR products amplified by the constructed primer pair for the partial cdtB gene were purified using a QIA quick PCR purification kit (Qiagen, CA, USA) and inserted in the pGEM-T vector using the pGEM-T Easy Vector System (Promega, Tokyo, Japan). Sequencing of the cloned cdtB gene fragment was performed (Hitachi DNA autosequencers SQ-5500L and SQ5500EL) after a dideoxy nucleotide sequencing reaction, using a Thermo Sequenase premixed cycle sequencing kit (Amersham Pharmacia Biotech, Tokyo, Japan). Sequence analysis of the PCR amplicons was carried out using the GENETYX-MAC (version 9) computer software. Genetic heterogeneity of the cytolethal distending toxin B (cdtB) gene locus among isolates of Campylobacter lari

Phospholipase C-related, but catalytically inactive protein (PRIP) up-regulates osteoclast differentiation via calcium-calcineurin-NFATc1 signaling

Phospholipase C-related, but catalytically inactive protein (PRIP) was previously identified as a novel inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ but lacking phospholipase activity. We recently showed that PRIP gene knock-out (KO) in mice increases bone formation and concomitantly decreases bone resorption, resulting in increased bone mineral density and trabecular bone volume. However, the role of PRIP in osteoclastogenesis has not yet been fully elucidated. Here, we investigated the effects of PRIP on bone remodeling by investigating dynamic tooth movement in mice fitted with orthodontic devices. Morphological analysis indicated that the extent of tooth movement was smaller in the PRIP-KO mice than in wild-type mice. Histological analysis revealed fewer osteoclasts on the bone-resorption side in maxillary bones of PRIP-KO mice, and osteoclast formation assays and flow cytometry indicated lower osteoclast differentiation in bone marrow cells isolated from these mice. The expression of genes implicated in bone resorption was lower in differentiated PRIP-KO cells, and genes involved in osteoclast differentiation, such as the transcription factor NFATc1, exhibited lower expression in immature PRIP-KO cells initiated by M-CSF. Moreover, calcineurin expression and activity were also lower in the PRIP-KO cells. The PRIP-KO cells also displayed fewer M-CSF-induced changes in intracellular Ca2+ and exhibited reduced nuclear localization of NFATc1. Up-regulation of intracellular Ca2+ restored osteoclastogenesis of the PRIP-KO cells. These results indicate that PRIP deficiency impairs osteoclast differentiation, particularly at the early stages, and that PRIP stimulates osteoclast differentiation through calcium-calcineurin-NFATc1 signaling via regulating intracellular Ca2+.

BubR1 Insufficiency Results in Decreased Macrophage Proliferation and Attenuated Atherogenesis in Apolipoprotein E‐Deficient Mice

Background Budding uninhibited by benzimidazole‐related 1 (BubR1), a cell cycle–related protein, is an essential component of the spindle checkpoint that regulates cell division. BubR1 insufficiency causes early aging‐associated vascular phenotypes. We generated low‐BubR1‐expressing mutant (BubR1 L/L) and apolipoprotein E‐deficient (ApoE −/−) mice (BubR1 L/L ‐ApoE −/− mice) to investigate the effects of BubR1 on atherosclerosis. Methods and Results Eight‐week‐old male BubR1 L/L ‐ApoE −/− mice and age‐matched ApoE −/− mice were used in this study. Atherosclerotic lesion development after being fed a high‐cholesterol diet for 12 weeks was inhibited in BubR1 L/L ‐ApoE −/− mice compared with ApoE −/− mice, and was accompanied by decreased accumulation of macrophages. To address the relative contribution of BubR1 on bone marrow–derived cells compared with non‐bone marrow–derived cells, we performed bone marrow transplantation in ApoE −/− and BubR1 L/L ‐ApoE −/− mice. Decreased BubR1 in bone marrow cells and non‐bone marrow–derived cells decreased the atherosclerotic burden. In vitro assays indicated that decreased BubR1 expression impaired proliferation, but not migration, of bone marrow–derived macrophages. Conclusions BubR1 may represent a promising new target for regulating atherosclerosis.