Investigation of a new implant surface modification using phosphorylated pullulan.

Various implant surface treatment methods have been developed to achieve good osseointegration in implant treatment. However, some cases remain impossible to treat with implants because osseointegration is not obtained after implantation, and the implants fail. Thus, this study focused on phosphorylated pullulan because of its adhesiveness to titanium (Ti) and bone, high biocompatibility, and early replacement with bone. In this study, the response of bone-related cells to phosphorylated pullulan was evaluated to develop a new surface treatment method. Saos-2 (human osteosarcoma-derived osteoblast-like cells), MC3T3-E1 (mouse calvaria-derived osteoblast-like cells), and RAW264.7 (mouse macrophage-like cells) were used. In evaluating cellular responses, phosphorylated pullulan was added to the culture medium, and cell proliferation and calcification induction tests were performed. The proliferation and calcification of cells on the surface of Ti disks coated with phosphorylated pullulan were also evaluated. In addition, bone morphogenetic protein-2 (BMP-2), an osteogenic factor, was used to evaluate the role of phosphorylated pullulan as a drug carrier in inducing calcification on Ti disks. Phosphorylated pullulan tended to promote the proliferation of osteoblast-like cells and the formation of calcification on Ti disks coated with phosphorylated pullulan. Ti disks coated with phosphorylated pullulan loaded with BMP-2 enhanced calcification. Phosphorylated pullulan inhibited osteoclast-like cell formation. These results are due to the properties of phosphorylated pullulan, such as adhesiveness to titanium and drug-loading function. Therefore, phosphorylated pullulan effectively promotes bone regeneration when coated on titanium implants and is useful for developing a new surface treatment method.

Transcriptome analysis of long non-coding RNAs in Mycobacterium avium complex-infected macrophages.

Mycobacterium avium complex (MAC) is a non-tuberculous mycobacterium widely distributed in the environment. Even though MAC infection is increasing in older women and immunocompromised patients, to our knowledge there has been no comprehensive analysis of the MAC-infected host-cell transcriptome-and particularly of long non-coding RNAs (lncRNAs). By using in vitro-cultured primary mouse bone-marrow-derived macrophages (BMDMs) and Cap analysis of gene expression, we analyzed the transcriptional and kinetic landscape of macrophage genes, with a focus on lncRNAs, during MAC infection. MAC infection of macrophages induced the expression of immune/inflammatory response genes and other genes similar to those involved in M1 macrophage activation, consistent with previous reports, although Nos2 (M1 activation) and Arg1 (M2 activation) had distinct expression profiles. We identified 31 upregulated and 30 downregulated lncRNA promoters corresponding respectively to 18 and 26 lncRNAs. Upregulated lncRNAs were clustered into two groups-early and late upregulated-predicted to be associated with immune activation and the immune response to infection, respectively. Furthermore, an Ingenuity Pathway Analysis revealed canonical pathways and upstream transcription regulators associated with differentially expressed lncRNAs. Several differentially expressed lncRNAs reported elsewhere underwent expressional changes upon M1 or M2 preactivation and subsequent MAC infection. Finally, we showed that expressional change of lncRNAs in MAC-infected BMDMs was mediated by toll-like receptor 2, although there may be other mechanisms that sense MAC infection. We identified differentially expressed lncRNAs in MAC-infected BMDMs, revealing diverse features that imply the distinct roles of these lncRNAs in MAC infection and macrophage polarization.

Epithelial-mesenchymal transition in oral cancer cells induced by prolonged and persistent Fusobacterium nucleatum stimulation.

OBJECTIVES: Several studies have reported the effects of Fusobacterium nucleatum stimulation on oral cancer cells. However, given that these studies typically span a stimulation period of three days to eight days, the in vitro studies conducted to date may not fully mimic the oral cancer environment, which involves constant exposure to oral commensal bacteria. This study aimed to elucidate the effects of prolonged and persistent Fusobacterium nucleatum infection on oral cancer cells. METHODS: Human tongue squamous cell carcinoma (SCC) cells were continuously stimulated with Fusobacterium nucleatum for two or four weeks, then experimentally evaluated. RESULTS: Prolonged, persistent Fusobacterium nucleatum stimulation increased the cells' proliferative, invasive, and migratory capacities, decreased their expression of epithelial markers, and increased their expression of mesenchymal markers progressively with time. The cells also adopted a spindle-shaped morphology and cell-to-cell contact dependence was progressively lost, suggesting time-dependent occurrence of epithelial-mesenchymal transition. Furthermore, mRNA levels of CD44, a cancer stem cell marker, were time-dependently upregulated. When SCC cells were stimulated with Fusobacterium nucleatum for four weeks in the presence of dexamethasone, Fusobacterium nucleatum induced epithelial-mesenchymal transition was inhibited. CONCLUSIONS: Epithelial-mesenchymal transition in human tongue SCC cells was time-dependently induced by prolonged, persistent Fusobacterium nucleatum stimulation and inhibited by dexamethasone. Routine decontamination of the oral cavity may be crucial for controlling tumor invasion and metastasis.

Inflammatory Cytokines and Chemokines Are Synergistically Induced in a ROS-Dependent Manner by a Co-Culture of Corneal Epithelial Cells and Neutrophil-like Cells in the Presence of Particulate Matter.

Ocular exposure to particulate matter (PM) causes local inflammation; however, the influence of neutrophils on PM-induced ocular inflammation is still not fully understood. In this study, we constructed a system to investigate the role of PM in ocular inflammation using a co-culture of human corneal epithelial cells (HCE-T) and differentiation-induced neutrophils (dHL-60). To investigate whether HCE-T directly endocytosed PM, we performed a holographic analysis, which showed the endocytosis of PM in HCE-T. The cytokines and chemokines produced by HCE-T were measured using an ELISA. HCE-T treated with PM produced IL-6 and IL-8, which were inhibited by N-Acetyl-L-cysteine (NAC), suggesting the involvement of ROS. Their co-culture with dHL-60 enhanced their production of IL-6, IL-8, and MCP-1. This suggests an inflammatory loop involving intraocular corneal epithelial cells and neutrophils. These cytokines and chemokines are mainly regulated by NF-κB. Therefore, this co-culture system was examined in the presence of an IKK inhibitor known to downregulate NF-κB activity. The IKK inhibitor dramatically suppressed the production of these factors in co-culture supernatants. The results suggest that the inflammatory loop observed in the co-culture is mediated through ROS and the transcription factor NF-κB. Thus, the co-culture system is considered a valuable tool for analyzing complex inflammations.

Innovative submucosal injection solution for endoscopic resection with phosphorylated pullulan: a preclinical study.

BACKGROUND AND AIMS: A submucosal injection solution is used to assist in endoscopic surgery. The high viscosity of current solutions makes them difficult to inject. In the present study, we developed an extremely low-viscosity, easy-to-use submucosal injection solution using phosphorylated pullulan (PPL). METHODS: The PPL solutions were prepared at different concentrations, and their viscosities were measured. The mucosal elevation capacity was evaluated using excised porcine stomachs. Controls included 0.4% sodium hyaluronate (SH), 0.6% sodium alginate (SA), and saline. To evaluate the practicality, the catheter injectability of 0.7% PPL was measured, and EMR and endoscopic submucosal dissection (ESD) were performed using the stomach and colorectum of live pigs. As controls, 0.4% SH and saline were used. RESULTS: The PPL solutions were of extremely low viscosity compared to the solutions of 0.4% SH and 0.6% SA. Nevertheless, the mucosal elevation capacity of PPL solutions for up to 0.7% concentration was similar to that of 0.4% SH, and 0.7% PPL was less resistant to catheter infusion than 0.4% SH and 0.6% SA. In live pig experiments with endoscopic mucosal resection and ESD, snaring after submucosal injection of 0.7% PPL was easier than with 0.4% SH, ESD with 0.7% PPL produced less bubble formation than with 0.4% SH, and the procedure time tended to be shorter with 0.7% PPL than with 0.4% SH because of the shorter injection time. CONCLUSIONS: The PPL solution is an innovative and easy-to-use submucosal injection solution.

ATP Consumption Is Coupled with Endocytosis in Exudated Neutrophils.

Neutrophil energy metabolism during phagocytosis has been previously reported, and adenosine triphosphate (ATP) plays a crucial role in endocytosis. Neutrophils are prepared by intraperitoneal injection of thioglycolate for 4 h. We previously reported a system established for measuring particulate matter endocytosis by neutrophils using flow cytometry. In this study, we utilized this system to investigate the relationship between endocytosis and energy consumption in neutrophils. A dynamin inhibitor suppressed ATP consumption triggered by neutrophil endocytosis. In the presence of exogenous ATP, neutrophils behave differently during endocytosis depending on ATP concentration. The inhibition of ATP synthase and nicotinamide adenine dinucleotide phosphate oxidase but not phosphatidylinositol-3 kinase suppresses neutrophil endocytosis. The nuclear factor kappa B was activated during endocytosis and inhibited by I kappa B kinase (IKK) inhibitors. Notably, IKK inhibitors restored endocytosis-triggered ATP consumption. Furthermore, data from the NLR family pyrin domain containing three knockout mice suggest that inflammasome activation is not involved in neutrophil endocytosis or concomitant ATP consumption. To summarize, these molecular events occur via endocytosis, which is closely related to ATP-centered energy metabolism.

Can a low dosage of recombinant human bone morphogenetic protein-2 loaded on collagen sponge induce ectopic bone?

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is one of the growth factors that may induce the formation of new bone. The aim was to determine the efficacy of low doses of rhBMP-2 for bone regeneration using a collagen sponge as a carrier. Three doses of rhBMP-2 (1.167, 0.117, and 0.039 mg/mL) were combined with an absorbable collagen sponge (ACS) as a delivery vehicle. The rhBMP-2/ACS implants were placed in the subcutaneous tissues of rat backs. X-ray microcomputed tomography (micro-CT) and histological analysis were used to evaluate bone formation. The samples treated with 1.167 mg/mL of rhBMP-2 showed greater bone formation than the samples treated with 0.117 mg/mL of rhBMP-2 four weeks after surgery. However, there was no evidence of bone formation in the samples that were treated with 0.039 mg/mL of rhBMP-2. It was found that rhBMP-2 was osteogenic even at one-tenth of its manufacturer's recommended concentration (1.167 mg/mL), indicating its potential for clinical use at lower concentrations.

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm.

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Pentacyclic triterpenoid ursolic acid induces apoptosis with mitochondrial dysfunction in adult T-cell leukemia MT-4 cells to promote surrounding cell growth.

We investigated the antitumor effects of oleanolic acid (OA) and ursolic acid (UA) on adult T-cell leukemia cells. OA and UA dose-dependently inhibited the proliferation of adult T-cell leukemia cells. UA-treated cells showed caspase 3/7 and caspase 9 activation. PARP cleavage was detected in UA-treated MT-4 cells. Activation of mTOR and PDK-1 was inhibited by UA. Autophagosomes were detected in MT-4 cells after UA treatment using electron microscopy. Consistently, mitophagy was observed in OA- and UA-treated MT-4 cells by confocal microscopy. The mitochondrial membrane potential in MT-4 cells considerably decreased, and mitochondrial respiration and aerobic glycolysis were significantly reduced following UA treatment. Furthermore, MT-1 and MT-4 cells were sorted into two regions based on their mitochondrial membrane potential. UA-treated MT-4 cells from both regions showed high activation of caspase 3/7, which were inhibited by Z-vad. Interestingly, MT-4 cells cocultured with sorted UA-treated cells showed enhanced proliferation. Finally, UA induced cell death and ex vivo PARP cleavage in peripheral blood mononuclear cells from patients with adult T-cell leukemia. Therefore, UA-treated MT-4 cells show caspase activation following mitochondrial dysfunction and may produce survival signals to the surrounding cells.

Osteoclast formation from mouse bone marrow cells on micro/nano-scale patterned surfaces.

OBJECTIVES: Osteoclasts can sense the surface topography of materials. However, it is difficult to identify the structural factors that affect osteoclast formation and its function. Furthermore, we hypothesized that the type of osteoclast precursor cells also affects osteoclastogenesis in the materials. In this study, we investigated the effects of defined micro/nanoscale patterns on osteoclastogenesis from bone marrow cells (BMCs). METHODS: Various cyclo-olefin polymer (COP) patterns were prepared using nanoimprinting. The effects of shape, size, and height of the patterns, and the wettability of the patterned surfaces on osteoclastogenesis from BMCs were evaluated in vitro. RESULTS: Osteoclast formation was promoted on pillars (diameter, 1 µm or 500 nm; height, 500 nm). Notably, osteoclastogenesis from BMCs was better promoted on hydrophobic pillars than on hydrophilic pillars. In contrast, decreased osteoclast formation was observed on the nanopillars (diameter, 100 nm; height, 200 nm). CONCLUSIONS: We demonstrated the promotion of osteoclast formation from BMCs on hydrophobic pillars with diameters of 1 µm and 500 nm. Some cellular behaviors in the patterns were dependent on the type of osteoclast precursor cells. The designed patterns are useful for designing the surface of dental implants or bone replacement materials with a controllable balance between osteoblast and osteoclast activities.

Expression of T-Type Voltage-Gated Calcium Channel in the Cilia of Human Nasal Epithelial Cells.

INTRODUCTION: The mucociliary transport function of the airway epithelium is largely dependent on ciliary beating. The control signal of ciliary beating is thought to be intracellular Ca2+. We herein investigated the expression of T-type voltage-gated calcium channel (VGCC), a generator of intracellular Ca2+ oscillation, in the human nasal mucosa. METHODS: The inferior turbinate was collected from patients with chronic hypertrophic rhinitis. The expression of T-type VGCC α1 subunits was examined by immunohistochemistry, transmission immunoelectron microscopy, Western blot, and real-time reverse transcription-polymerase chain reaction (RT-PCR). Participation of T-type VGCC in the ciliary beat regulation was examined by pharmacological inhibition tests using specific blockers of T-type VGCC in ex vivo measurements of the ciliary beat frequency (CBF) and ATP release and in intracellular Ca2+ imaging of isolated ciliated cells. RESULTS: Immunohistochemical staining showed the expressions of T-type VGCC α1 subunits, Cav3.1 and Cav3.3, on the surface of the epithelial cells. At the ultrastructural level, immunoreactivity for Cav3.1 was localized on the surface of the cilia, and that for Cav3.3 was localized in the cilia and at the base of the cilia. The existence of Cav3.1 and Cav3.3 was confirmed at the protein level by Western blot and at the transcriptional level by real-time RT-PCR. Specific blockers of T-type VGCC, mibefradil and NNC 55-0396, significantly inhibited CBF. These blockers also inhibited a CBF increase induced by 8-bromo-cAMP/8-bromo-cGMP and significantly lowered the intracellular Ca2+ level of isolated ciliated cells in a time-dependent manner. On the other hand, the ATP release from the nasal mucosa was not changed by mibefradil or NNC 55-0396. CONCLUSION: These results indicate that T-type VGCC α1 subunits, Cav3.1 and Cav3.3, exist at the cilia of the nasal epithelial cells and participate in the regulation of ciliary beating and that these channels act downstream of cAMP/cGMP.

Decreased CFTR/PPARγ and increased transglutaminase 2 in nasal polyps.

OBJECTIVE: Transglutaminase (TGM)2 and peroxisome proliferator-activated receptor (PPAR)γ are thought to participate in the pathogenesis of nasal polyp formation in cystic fibrosis (CF). We herein investigated expressions of cystic fibrosis transmembrane conductance regulator (CFTR), TGM2, PPARγ and isopeptide bonds, a reaction product of TGM, in non-CF nasal polyps. METHODS: Nasal polyps and inferior turbinates were collected from chronic rhinosinusitis patients without CF during transnasal endoscopic sinonasal surgery. Expressions of CFTR, TGM2, isopeptide bonds and PPARγ were examined by fluorescence immunohistochemistry and quantitative RT-PCR. Expression of CFTR was also analyzed by Western blot. RESULTS: Immunohistochemical fluorescence of the nasal polyp was significantly lower for CFTR and PPARγ, and significantly higher for TGM2 and isopeptide bonds than that of the turbinate mucosa. Lower expression of CFTR in the nasal polyp than in the turbinate mucosa was also observed in Western blot. Expression of PPARG mRNA was significantly lower in the nasal polyp than in the turbinate mucosa, whereas expressions of CFTR mRNA or TGM2 mRNA did not differ between the two tissues. Immunohistochemical fluorescence for CFTR showed significant negative correlation with that for TGM2 and isopeptide bonds, and significant positive correlation with that for PPARγ. The fluorescence for TGM2 was positively correlated with that for isopeptide bonds and negatively correlated with that for PPARγ. The fluorescence for isopeptide bonds tended to be negatively correlated with that for PPARγ. CONCLUSIONS: These results suggest a possible role of the CFTR-TGM2-PPARγ cascade in the pathogenesis of nasal polyp formation in non-CF patients as in CF patients.

Senescence-associated secretory phenotype and activation of NF-κB in splenocytes of old mice exposed to irradiation at a young age.

DNA damage-induced cellular senescence is involved in aging. We reported previously that p53+/- mice subjected to irradiation at a young age exhibited an increased number of splenic lymphocytes in the S and G2/M phases. However, the detailed nature of splenic disorders in these mice is not fully understood. In this study, we investigated the effects on molecules in splenocytes, especially on senescence factors after early exposure of mice to radiation. Mice, 8- (young) or 17-, 30-, and 41-week-old (old) p53+/- were subjected to 3-Gy whole-body irradiation. Splenocytes were prepared at 56 weeks of age. Immunoblot showed that irradiation at 8 weeks enhanced the expression and phosphorylation of p53, cyclin-dependent kinase 2, cell division cycle 6, and the MDM2 proto-oncogene in splenocytes. However, these molecules were not affected by irradiation at 17, 30, and 41 weeks of age. Similarly, irradiation at 8, but not 17, 30, or 41 weeks, induced phosphorylation of IKKα, NF-κB inhibitor alpha, and p65. Electrophoretic mobility shift assay demonstrated that active forms of NF-κB were increased. In addition, enzyme-linked immunosorbent assay showed that lipopolysaccharide-induced IL-6 production was enhanced in splenocytes of mice irradiated at 8 weeks. ATP levels were increased in splenocytes of mice irradiated at 8, but not 17, 30, or 41 weeks. CDK2 expression and p65 phosphorylation were induced in CD45R/B220+ cells from irradiated mice. Overall, irradiation induced a NF-κB-related immune response in the spleen with an increase in senescence marker proteins, such as CDKs and IL-6, which are known to be typical senescence-associated secretory phenotype factors related to stresses, such as DNA damage.

Mitogen-activated protein kinases are involved in cucurbitacin D-induced antitumor effects on adult T-cell leukemia cells.

Adult T cell leukemia (ATL) is an aggressive and malignant blood disease. We previously reported that steroid-structured cucurbitacin D (CuD) induces apoptosis in ATL cells. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) signaling inhibitors on CuD-induced cell death in peripheral blood lymphocytes (PBLs) isolated from ATL/acute lymphoblastic leukemia (ALL) patients and two human leukemia cell lines (MT-1 and MT-4). PBLs were isolated from an ATL/ALL patient as well as from a healthy donor. Cell surface markers were examined using flow cytometry. Serum cytokine levels were estimated using LEGENDplex or analyzed at the Center for Clinical and Translational Research of Kyushu University Hospital. Cell proliferation was assessed using the Cell Titer-Glo luminescent cell viability assay. Protein expression was determined by western blotting. PBLs from patients highly expressed CD4 and CD5. Serum from the patient contained high levels of interleukin (IL)-8, IL-10, IL-18, and interferon-γ compared to the healthy donor. CuD-induced cell death was enhanced by the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor U0126. However, a c-Jun N-terminal kinase (JNK) inhibitor prevented CuD-induced cell death. Immunoblot analyses revealed that CuD reduced the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and JNK, and co-treatment with CuD and U0126 did not affect the phosphorylation of ERK. MEK1/2 and p38 inhibitors enhanced CuD-induced cell death, and U0126 enhanced the CuD-induced de-phosphorylation of ERK in MT-1 and MT-4 cells. We conclude that CuD reduces ERK activation, resulting in enhanced antitumor effects on leukemic cells.

Long-term survival of thoracoscopic surgery compared with open surgery for clinical N0 adenocarcinoma.

BACKGROUND: Early stage non-small cell lung cancer (NSCLC) is good candidate for video-assisted thoracoscopic surgery (VATS). Long-term outcome compared between VATS and open surgery remains unclear. The aim of this study was to assess the long-term outcome of VATS in early stage adenocarcinoma. METHODS: A retrospective study was performed in 546 patients which were operated between January 2006 and December 2010 in our institute and of those, 240 (220 lobectomies, and 20 segmentectomies) were clinical N0 adenocarcinoma. One hundred and thirty-five patients underwent VATS and 105 patients for open surgery. Long-term oncological outcomes were compared in both groups. RESULTS: There were significant differences in age, gender, Blinkman index, clinical T factor and tumor size between two groups. VATS group showed statistically longer operation time (P=0.01), less blood loss (P=0.005), shorter length of stay (P=0.001), and less dissected number of lymph nodes (P<0.001) compared with open surgery. Disease-free survival in VATS was significantly better than open surgery (5- and 10-year survival; VATS, 91.4%, 79.0%; open, 85.1%, 73.6%; respectively, P=0.04). Overall survival in VATS was not different from open (P=0.58). Propensity matched disease-free and overall survival was not significantly different between two groups. Multivariate Cox regression analysis showed that age [P=0.04, 95% confidence interval (CI): (1.02-6.81)] in overall and T factor [P=0.01, 95% CI: (1.41-17.3)] in disease-free survival was prognostic significant after propensity matching. CONCLUSIONS: Our study demonstrated that long-term outcome in VATS for early stage adenocarcinoma was equivalent to open surgery. VATS may be a treatment of choice for promising long-term prognosis.

In vivo immune activation of splenocytes following exposure to tar from Asian sand dust.

Air pollution, especially that initiated by particulate matter (PM), has been implicated as a risk factor for several inflammatory diseases. Previously, it was reported that PM enhances immune responses. PM includes the tar fraction that contains polycyclic aromatic hydrocarbons (PAHs), which produce adverse health effects in exposed individuals. However, the influence of the tar fraction (as a component of PM) on splenocytes is not fully understood. The aim of this study was to determine the effects of the tar fraction extracted from PM collected from the atmosphere in Fukuoka, Japan, on mouse splenocytes. ICR mice were administered tar (1 or 5 µg/mouse) intratracheally 4 times at 2-week intervals, and splenocytes from the tar-treated mice were extracted and examined. The parameters determined were proliferation, cytokine concentrations and transcription factors activation. Following tar treatment, splenocyte proliferation increased relative to controls. Concanavalin A (ConA)-induced interleukin (IL)-2 formation and ConA- or lipopolysaccharide (LPS)-induced interferon-γ production were elevated in splenocytes from tar-exposed mice. However, the production of tumor necrosis factor-α and IL-6 induced by LPS was not markedly changed following tar treatment. Further, nuclear factor of activated T cells, but not nuclear factor-κB, was enhanced in splenocytes of tar-exposed mice. Data indicate that tar-activated splenocytes and PM-bound PAHs might contribute to T cell activation in the spleen.

Nuclear factor of activated T cells as a marker of in vivo low-dose dibenzo[a,h]anthracene exposure.

We previously demonstrated that particulate matter ≤2.5 µm (PM2.5) suppresses the immune response in the spleen in vivo. Although PM2.5 includes the polycyclic aromatic hydrocarbon (PAH) such as dibenzo[a,h]anthracene (DBA), it is unclear whether PAH has a direct effect on the responses of splenocytes. In our study, the concentration of DBA used was approximately 0.8 µm, which is much lower than concentrations used in other toxicological studies of DBA. Although exposure to high concentrations of DBA is implicated in carcinogenesis, the effects of low doses of DBA on immune cells in vivo remain unclear. Here, we investigated the effects of low DBA doses on mouse splenocytes in vivo. Mice were administered dimethyl sulfoxide or DBA (0.4 or 0.8 µm) intratracheally. Twenty-four hours after treatment, the mice were killed and their splenocytes were collected. DBA treatment enhanced mitogen-induced cell proliferation and cytokine production in the mouse splenocytes. Furthermore, DBA enhanced splenic CD4+ and CD8+ cell proliferation and cytokine production. The nuclear factor of activated T cells (NFAT) was activated in CD4+ cells. DBA also activated nuclear factor-kappa B and CCAAT enhancer-binding protein pathways in CD11b+ cells. DBA-enhanced splenocyte activation was Toll-like receptor 2-, 4-, 9- and MyD88-independent. These results suggest that NFAT represents a promising marker for evaluation of the effects of DBA on T cells and T-cell-dependent antibody responses.

Novel Reporter System Monitoring IL-18 Specific Signaling Can Be Applied to High-Throughput Screening.

Very recently, the immunotherapies against cancer, autoimmune diseases, and infection have been feasible and promising. Thus, we have examined the possibility whether or not human gamma delta T cells can be applied for the novel immunotherapies. We previously established the cells stably maintaining NFkB-driven human secreted embryonic alkaline phosphatase (SEAP) expression. The cells can be used to determine the transcription activity of NFkB with high-standard dynamic range and accuracy. Because IL-18 is a kind of cytokines that enhances cytotoxicity and activity of human gamma delta T cells through NFkB activation, we have focused on the activity and signaling of IL-18. In this study, we modified the previous reporter cell that can determine the transcription activity of NFkB to express two subunits consisted of human IL-18 receptor. The modified cells secreted SEAP in response to treatment with human recombinant IL-18 in a concentration-dependent manner. We also observed the concentration-dependently enhancement of NFkB activity in the cells treated with mouse recombinant IL-18 although the affinity was lower compared to human recombinant IL-18. We also previously established the cells stably expressing and secreting human recombinant IL-18 and then validated whether or not the conditioned medium from the cells activate NFkB transcription activity using this assay. Our university has kept collecting many extracts from over 18,000 marine bacteria in our local sea around Omura bay-fungi, plants for Chinese herbal medicine, and so on-and also have kept gathering synthetic compounds from many Japanese chemists as drug libraries. Finally, in order to identify drugs mimicking IL-18 biological activity or possessing inhibitory effects on IL-18-induced NFkB, we demonstrated drug screening using number of extracts derived from marine bacteria and synthetic compounds.

Proliferation of Saos-2 and Ca9-22 cells on grooved and pillared titanium surfaces.

BACKGROUND: Surface nanostructures in titanium (Ti) oral implants are critical for rapid osseointegration. OBJECTIVE: The purpose of this study was to evaluate the growth of osteoblast-like (Saos-2) and epithelial-like (Ca9-22) cells on nanopatterned Ti films. METHODS: Ti films with 500 nm grooves and pillars were fabricated by nanoimprinting, and seeded with Saos-2 and Ca9-22 cells. Cell viability and morphology were assessed by cell proliferation assay and scanning electron microscopy, respectively. RESULTS: As assessed after 1 hour, proliferation of Saos-2 cells was most robust on grooved films than on pillared and smooth films, in this order. These cells approximately doubled on grooved and pillared substrates in 24 hours and after 5 days, but not on smooth surfaces. In contrast, Ca9-22 cells favored smooth surfaces, followed by grooved and pillared films. Indeed, cells sparsely adhered to pillared films over 5 days of incubation (p < 0.05). CONCLUSIONS: The data show that Saos-2 and Ca9-22 cells respond differently to different nanostructures, and highlight the potential use of nanopatterns to promote bone regeneration or to prevent epithelial downgrowth at the implant-bone interface.

Establishment of Novel Cells Stably Secreting Various Human IL-18 Recombinant Proteins.

BACKGROUND: The immunotherapies against cancer, autoinmmune diseases or infection are remarkable development. These days programmed cell death (PD)-1 antibody-induced immune checkpoint blockade or chimeric antigen receptor-T cells (CAR-T) have been shown to have eminent therapeutic effects on tumor development. We have focused on adoptive transfer with human gamma delta T cells for novel immunotherapies. Additionally, IL-18 is one of the cytokines that enhances cytokine secretion and cytotoxicity of human gamma delta T cells. METHOD: Thus, we established novel cell lines stably expressing and secreting various types of human recombinant IL-18 proteins to their culture supernatants using episomal vector. We also differentiated primary cultured human gamma delta T cells from peripheral blood mononuclear leukocytes to validate biological activity of the IL-18 proteins using measuring IFN-γ by ELISA. RESULTS AND CONCLUSION: Finally, we demonstrated that the supernatant could activate human gamma delta T cells using monitoring interferon gamma in culture medium.