Ephrin type-A receptor 2 on tumor-derived exosomes enhances angiogenesis through the activation of MAPK signaling.

Exosomes are potent players in the development of metastases and they play an important role in cancer angiogenesis and exacerbation. However, it is unclear how proteins on exosomes affect development of blood vessel networks. In this study, we focused on relationships between membrane proteins on exosomes and angiogenesis using human umbilical vein endothelial cells (HUVEC). Lung tumor cell-derived exosomes induced tube formation and growth of endothelial cells in vitro in a dose-dependent manner involving MAPK activation, but this was not seen in normal lung epithelial cells. Ephrin type-A receptor 2 (EphA2) was identified by proteomic analysis and an inhibition assays showed it is a major MAPK activator on exosomes. Thus EphA2 on exosomes participates in angiogenesis as a ligand of the ephrin signaling pathway. These results support the development of novel therapeutic strategies such as blockade of remote cancer communications through exosomes.

High-dose cutaneous exposure to mite allergen induces IgG-mediated protection against anaphylaxis.

BACKGROUND: The relationship among natural allergen exposure, induction of blocking antibody and the occurrence of atopic allergy-particularly in the presence of IgE production-is debatable. OBJECTIVE: To clarify the relationship between the dose of cutaneous exposure to dust mite allergen and susceptibility to the IgE-mediated allergic response in relation to IgG production. METHODS: NC/Nga mice were epicutaneously exposed to various doses of Dermatophagoides pteronyssinus allergen to induce atopic dermatitis-like skin lesions. We then evaluated the skin lesions, induction of mite-specific immune responses, and susceptibility to anaphylaxis. RESULTS: Dose-dependent exacerbation of atopic dermatitis-like skin lesions and increases in mite-specific IgG and IgE production were observed. However, mice exposed to relatively low doses of mite allergen showed hypersusceptibility to mite allergen-specific anaphylaxis. We also showed that adoptive transfer of total IgG from Dp-sensitized mice rescued mice from the hypersusceptibility seen in those exposed to low doses of mite allergen. CONCLUSIONS AND CLINICAL RELEVANCE: High-dose cutaneous exposure to dust mites induced effective blocking IgG production, even if accompanied by IgE production. Our data might support the concept that an increase in IgG titre, not a decrease in IgE titre, is a marker of clinical improvement in allergen-specific immunotherapy.

Wavelength-selective light-triggered strand exchange reaction.

We prepared an oligodeoxynucleotide (ODN) bearing two 4-hydroxy-2-mercaptobenzimidazole nucleobase analogues (SB(NV) and SB(NB)) modified with different photolabile groups. This ODN enabled a light-triggered strand exchange reaction in a wavelength-selective manner.

Generation of a sensitive TNFR2-specific murine assays system.

Tumor necrosis factor (TNF)/TNF receptors (TNFR1/TNFR2) are considered to be potential drug targets to treat refractory diseases, including autoimmune diseases and malignant tumors. However, their specific functions, especially in the case of TNFR2, are poorly understood. In this study, we constructed a mouse TNFR2 (mTNFR2)-mediated biological assay system that shows no effects of mouse TNFR1 (mTNFR1) in order to screen mTNFR2-selective stimulating agents. Mouse TNFR1(-/-)R2(-/-) preadipocytes were transfected with the gene encoding the mTNFR2/mouse Fas (mFas) chimeric receptor in which the extracellular and transmembrane domains of mTNFR2 were fused to the intracellular domain of mFas. Our results demonstrated that this cell line exhibits highly sensitive mTNFR2-mediated cytotoxic effects. We propose that this mTNFR2-mediated biological assay system would be a useful tool to screen for mTNFR2-selective stimulating agents.

Circulating Tumor Cells as an Independent Predictor of Survival in Advanced Gastric Cancer.

BACKGROUND: When the indication for surgery of highly advanced gastric cancer is considered, careful selection of the patients is important. In addition to tumor-node-metastasis factors and peritoneal lavage cytology (CY), which are important predictors of prognosis, detection of circulating tumor cells (CTCs) could be another potential marker. METHODS: This study prospectively evaluated CTCs using a semi-automated immunomagnetic separation system (CellSearch) for 136 patients with advanced gastric cancer to determine the frequency of CTC positivity. For 123 patients who also had their CY evaluated, the significance of both CTC and CY, was investigated as a potential biomarker to predict progression-free survival (PFS) or to monitor the therapeutic effect. RESULTS: In 25 patients (18.4 %), CTCs were positive. Positive CTC counts were more common for tumors with diffuse histologic type and distant metastasis. The PFS of CTC-positive patients was significantly shorter than that of CTC-negative patients (hazard ratio 2.03; P = 0.016). A multivariate analysis of 123 patients showed that CTC and CY as well as performance status and macroscopic distant metastasis were independent factors for PFS. When both CTC and CY were converted to negative values by therapeutic interventions, long-term PFS was achieved. CONCLUSIONS: Detection of CTCs was an independent predictor of a shorter PFS in advanced gastric cancer. For selecting patients who require intensive treatment, CTCs could be a valuable biomarker. The combined status of CTC and CY would be useful in selecting patients for radical surgery. Further investigation with a larger number of patients is necessary to establish the importance of CTCs.

C5-azobenzene-functionalized locked nucleic acid uridine: isomerization properties, hybridization ability, and enzymatic stability.

Oligonucleotides (ONs) modified with a locked nucleic acid (LNA) are widely used in the fields of therapeutics, diagnosis, and nanotechnology. There have been significant efforts towards developing LNA analogues bearing modified bridges to improve their hybridization ability, nuclease resistance, and pharmacokinetic profiles. Moreover, nucleobase modifications of LNA are useful strategies for the functionalization of ONs. Modifications of the C5-position of pyrimidine nucleobases are particularly interesting because they enable predictable positioning of functional groups in the major groove of the duplex. Here we report the synthesis of C5-azobenzene-functionalized LNA uridine (LNA-U(Az)) and properties of LNA-U(Az)-modified ONs, including isomerization properties, hybridization ability, and enzyme stability. LNA-U(Az) in ON is photo-isomerized effectively and reversibly by irradiation at 365 nm (trans to cis) and 450 nm (cis to trans). LNA-U(Az)-modified ONs show RNA-selective hybridization ability despite the large hydrophobic azobenzene moiety extending into the major groove of the duplex. The enzymatic stability of LNA-U(Az)-modified ONs is higher than that of natural and LNA-modified ONs with or without photo-irradiation. Our results indicate that LNA-U(Az) holds promise for RNA targeting and photo-switchable technologies.

Epidermal growth factor receptor localized to exosome membranes as a possible biomarker for lung cancer diagnosis.

Detection of drug-target proteins and biomarkers that are expressed in cancer tissue has significant potential for both diagnosis and treatment of cancer. However, current immuno-histochemical and cytogenetic analyses of biopsy specimens for pre-operational diagnosis are highly invasive and often difficult to apply to lung cancer patients. The purpose of this study was to evaluate the possible utility of determining epidermal growth factor receptor (EGFR) expression on exosomal membranes using a targeted ELISA with an anti-CD81 antibody as a capture antibody for lung cancer diagnosis. While soluble EGFR (sEGFR) levels in plasma were not remarkably different between lung cancer patients and normal controls, significantly higher exosomal EGFR expression levels were observed in 5/9 cancer cases compared to normal controls. These results suggest that measurement of exosomal protein levels could be useful for in vitro diagnosis, and that exosomal EGFR is a possible biomarker for characterization of lung cancer.

Biochemical and hematologic effects of polyvinylpyrrolidone-wrapped fullerene C60 after oral administration.

The fullerene C60 is used in consumer products such as cosmetics owing to its antioxidative effects and is being developed for nanomedical applications. However, knowledge regarding the safety of fullerene C60, especially after oral administration, is sparse. Here, we examined the safety of fullerene C60 in mice after 7 d of exposure to orally administered polyvinylpyrrolidone (PVP)-wrapped fullerene C60 (PVP-fullerene C60). Mice treated with PVP-fullerene C60 showed few changes in the plasma levels of various markers of kidney and liver injury and experienced no significant hematologic effects. Furthermore, the histology of the colon of PVP-fullerene C60-treated mice was indistinguishable from that of control mice. These results suggest that PVP-fullerene C60 lacks toxicity after high-dose oral administration and indicate that PVP-fullerene C60 can be considered safe for oral medication. These data provide basic information that likely will facilitate the production of safe and effective forms of fullerene C60.

Rho GDP-dissociation inhibitor alpha is associated with cancer metastasis in colon and prostate cancer.

Since metastasis is one of the most important prognostic factors in colorectal cancer, development of new methods to diagnose and prevent metastasis is highly desirable. However, the molecular mechanisms leading to the metastatic phenotype have not been well elucidated. In this study, a proteomics-based search was carried out for metastasis-related proteins in colorectal cancer by analyzing the differential expression of proteins in primary versus metastasis focus-derived colorectal tumor cells. Protein expression profiles were determined using a tissue microarray (TMA), and the results identified Rho GDP-dissociation inhibitor alpha (Rho GDI) as a metastasis-related protein in colon and prostate cancer patients. Consequently, Rho GDI may be useful as a diagnostic biomarker and/or a therapeutic to prevent colon and prostate cancer metastasis.

Longitudinal changes in life-space mobility and the factors influencing it among chronic community-dwelling post-stroke patients.

PURPOSE: To identify longitudinal changes in life-space mobility and the factors influencing it among chronic, stable post-stroke patients. MATERIALS AND METHODS: This prospective study included Japanese post-stroke patients who received day-care rehabilitation services and could undergo three life-space mobility assessments (at baseline, 12, and 24 months) for over 2 years, using the Life-Space Assessment (LSA) tool. Physical function, cognitive function, and activities of daily living were assessed by self-selected comfortable gait speed, Mini-Mental State Examination (MMSE), and Functional Independence Measure Motor subscale (FIM motor) scores, respectively, in addition to age, sex, time from onset, stroke type, and comorbidities. A multivariable linear mixed-effects model was used to examine the longitudinal changes in LSA scores and associated factors. RESULTS: A total of 89 participants were enrolled. At baseline, the median age was 74 years, 33% were women, and median time from onset was 75 months. The LSA scores significantly declined over the two-year period. In the multivariate linear mixed-effects model adjusted for clinical characteristics, comfortable gait speed and age were significantly associated with changes in the LSA score, independent of FIM motor scores and MMSE scores. CONCLUSIONS: Life-space mobility may persistently decline, and gait function may be a determinant influencing these changes in community-dwelling chronic post-stroke patients.Implications for RehabilitationLimited life-space mobility leads to less frequent participation in social activities and an increased risk of adverse health outcomes such as hospitalization.Changes in life-space mobility should be considered in the rehabilitation care plan for chronic post-stroke patients.Life-space mobility may decline persistently in stable post-stroke patients, even if they have periodically received day-care rehabilitation services.Since gait speed is a predominant factor affecting life-space mobility, regular assessment of gait function and appropriate strategies are needed to prevent deterioration of gait speed in chronic post-stroke patients.

Size-dependent immune-modulating effect of amorphous nanosilica particles.

The immune-modulating effect following intradermal injection of various-sized amorphous silica particles was analyzed in terms of induction of ovalbumin-specific CD8+ T cells in vivo. IFN-gamma ELISPOT assays revealed that only nanosilica particles with a diameter of less than 100 nm significantly enhanced CD8+ T cell responses against ovalbumin. These results indicate that the size of nanomaterials is a critical determinant in terms of their safe use.

Assessment of oral midazolam limited sampling strategies to predict area under the concentration time curve (AUC) during cytochrome P450 (CYP) 3A baseline, inhibition and induction or activation.

UNLABELLED: A previous study reported a 2- and 3-timepoint limited sampling strategy (LSS) model accurately predicted oral midazolam area under the concentration time curve (AUC), and thus cytochrome P450 (CYP) 3A activity. OBJECTIVE: This study evaluated whether the LSS models predict midazolam AUC during CYP3A baseline, inhibition and induction/activation. MATERIALS AND METHODS: Plasma midazolam concentrations from 106 healthy adults from 6 published studies were obtained where oral midazolam was co-administered alone or with ketoconazole, double-strength grapefruit juice, Ginkgo biloba extract, pleconaril, or rifampin. Observed and predicted midazolam AUCs were determined. Bias and precision of the LSS models were determined. RESULTS: Contrasting results were observed for the 2- and 3-timepoint LSS models in accurately predicting midazolam AUC during baseline CYP3A conditions. With the exception of 1 study (single dose, double-strength grapefruit juice), the 2- and 3-timepoint LSS models did not accurately predict midazolam AUC during conditions of CYP3A inhibition and induction/activation. CONCLUSION: The previously reported 2- and 3-timepoint oral midazolam LSS models are not applicable to the evaluated conditions of CYP3A baseline, inhibition, and induction/ activation.

Concomitant expression of HER2 and HIF-1alpha is a predictor of poor prognosis in uterine cervical carcinoma treated with concurrent chemoradiotherapy: prospective analysis (KGROG0501).

BACKGROUND: In previously reported retrospective analyses of uterine cervical carcinoma cases, HER2 was correlated with poor radiation sensitivity and poor treatment outcomes and HIF-1alpha was found to be an indicator of poor prognosis. To date, no prospective studies have been performed to evaluate the radiation sensitivity and treatment outcomes of patients with uterine cervical carcinoma relative to HER2 and HIF-1alpha expressions. We conducted a prospective evaluation of HER2 and HIF-1alpha in cases of locally advanced uterine cervical carcinoma treated with concurrent chemoradiotherapy. METHODS: Between June 2005 and April 2008, 25 patients with locally advanced uterine cervical carcinoma were registered in this study, KGROG0501. Their clinical stages were Ib2/IIb/IIIb/IVa in 1/2/22/1 cases, respectively. Nineteen cases had squamous cell carcinoma and six had adenocarcinoma. HER2 expression and HIF-1alpha expression were analyzed using an immunohistochemical kit on pretreatment biopsied specimens. HIF-1alpha expression was studied using another commercial immunohistochemical kit on pretreatment biopsied specimens. The survival rates were compared between patients with and without positive HER2 and HIF-1alpha expressions. RESULTS: The 20-month survival of HER2(-) and HIF-1alpha(-) cases (n = 6) was 100% and that of HER2(+) and HIF-1alpha(+) cases (n = 4) was 37.5% (p = 0.0032). CONCLUSIONS: In this first prospective analysis of patients with uterine cervical carcinoma treated with concurrent chemoradiotherapy, concomitant expression of HER2 and HIF-1alpha was suggested to be a strong indicator of poor prognosis. A novel therapy including molecular targeted therapy such as anti-HER2 and anti-HIF-1alpha may be worth considering in patients with concomitant expression of HER2 and HIF-1alpha.

Initial analysis of relationship between plasma platinum concentration and hematological adverse reaction associated with weekly chemotherapy using nedaplatin in combination with radiotherapy for cervical carcinoma.

PURPOSE: Established therapeutic guidelines for cervical carcinoma recommend concurrent chemo- and radiotherapy as standard treatment for locally advanced cervical carcinoma. Nedaplatin (CDGP) is a platinum agent developed in Japan that is less nephrotoxic than cisplatin (CDDP), but with equivalent antitumor potency. In the standard dosage regimen for cervical carcinoma, CDGP is administered once every four weeks (monthly regimen). We investigated the efficacy and safety of a new dosage regimen, in which CDGP was administered once weekly for five weeks (weekly regimen). METHODS: We measured plasma platinum concentration of patients after administration of CDGP, and analyzed the relationship between plasma platinum concentration and hematological adverse reactions such as thrombocytopenia and leucopenia. RESULTS: The relative rates of change in platelet and white blood cell counts tended to increase as the plasma concentration of platinum increased. Furthermore, the rate of change in platelet counts in relation to the area under the curve was greater for the monthly regimen as compared to weekly. On the other hand, the relative rates of change in WBC were nearly the same between the regimens. CONCLUSIONS: These findings indicate that when using chemotherapy with CDGP for a patient with a cervical carcinoma, a weekly regimen might reduce the severity of thrombocytopenia, while still exhibiting the same therapeutic efficacy as the monthly regimen.

Cytotoxicity of amorphous silica particles against macrophage-like THP-1 cells depends on particle-size and surface properties.

Recent studies have indicated that amorphous silica particles (SPs) show cytotoxicity against various types of cells, including macrophages. However, the mechanism of cell death has not been determined, and systematic investigations of the relationship between particle characteristics and cytotoxicity are still quite limited. Here, we compared the cytotoxicity of SPs of various sizes (30-1000 nm) and surface properties against differentiated THP-1 human macrophage-like cells. We found that 300 and 1000 nm SPs showed cytotoxicity against THP-1 cells, whereas 30, 50, and 70 nm SPs did not induce cell death. We demonstrated that 1000 nm SP showed strong cytotoxicity that depended on reactive oxygen species but was independent of caspases. Furthermore, we showed that surface modification of 1000 nm SPs dramatically suppressed their cytotoxicity. Our results suggest that systematic evaluation of the association between particle characteristics and biological effects is necessary for the creation of safe SPs.

Mutant TNF-alpha, mTNF-K90R, is a novel candidate adjuvant for a mucosal vaccine against HIV.

The development of a safe and effective mucosal vaccine adjuvant is a crucial step for the development of vaccines against human immunodeficiency virus type-1 (HIV). We have previously reported that a mutant tumor necrosis factor-alpha (TNF-alpha), mTNF-K90R, possessed strong mucosal vaccine adjuvant activities in mice. Here, we evaluated the potential of mTNF-K90R as a mucosal vaccine adjuvant for the induction of systemic and mucosal immune responses against HIV. Nasal immunization of BALB/c mice with 5 microg of an HIV gp120 env protein immunogen together with mTNF-K90R induced higher serum anti-HIV gp120 protein immunoglobulin G (IgG) responses than gp120 alone. Furthermore, mTNF-K90R induced anti-gp120 IgA responses in nasal as well as vaginal washes from immunized mice, although these were not administration sites. Again, responses with mTNF-K90R were higher than with gp120 alone. These results indicate that mTNF-K90R may be applicable as amucosal adjuvant for HIV vaccination to induce both systemic and mucosal immune responses.

Arsenic trioxide induces down-regulation of gp46 via protein oxidation: proteomics analysis of oxidative modified proteins in As2O3-treated HTLV-1-infected cells.

Adult T-cell leukemia (ATL) is a severe chemotherapy-resistant malignancy associated with prolonged infection by the human T cell-lymphotropic virus 1 (HTLV-1) retrovirus. Epidemiology studies strongly indicate that an increase in HTLV-1 virus load is an important factor during the onset of ATL. Therefore, inhibition of the growth/transmission of HTLV-1 infected cells is a promising strategy in preventing the disease. In our previous study, we revealed that arsenic trioxide (As2O3), a drug used to treat acute promyelocytic leukemia (APL), exerts an inhibitory effect on syncytium formation between HTLV-1 infected cells and HeLa cells via suppression of HTLV-1 envelope protein gp46 expression at low concentrations. In this study, we analyze the mechanism of action of As2O3 using a proteomics approach. Our results suggest that down-regulation of gp46 might be related to As2O3-induced oxidation of the 71-kDa heat shock cognate protein (HSC70) and the 78-kDa glucose-regulated protein (BiP/GRP78). We postulate that AS2O3 exerts an inhibitory effect on HTLV-1 virus transmission via down-regulation of gp46-production, which might be caused by oxidative modification of various proteins such as chaperones.

Size-dependent cytotoxic effects of amorphous silica nanoparticles on Langerhans cells.

Amorphous silica nanoparticles (nSPs), are widely used in medicines, cosmetics and food. However, due to their reduced particle size they are suspected to pose new risks induced by changes in biological reactivity and kinetics, which differ from those of bulk materials. In a previous study, we showed that silica particles with a diameter of 70 nm penetrated the stratum corneum (SC) of mouse skin and were taken up by living cells such as keratinocytes and Langerhans cells. To clarify the relationship between particle size, distribution and cellular response, we have evaluated size-dependent intracellular localization and cytotoxicity of silica particles, using the mouse epidermal Langerhans cell line XS52. On treatment with silica particles of diameters 70, 300, and 1000 nm, cellular uptake and cytotoxicity increased with reduction in particle size. These results suggest that smaller sized silica particles induced greater cytotoxicity against Langerhans cells, which was correlated with the quantity of particle uptake into the cells.

Creation of an improved mutant TNF with TNFR1-selectivity and antagonistic activity by phage display technology.

Tumor necrosis factor-alpha (TNF), which binds two types of TNF receptors (TNFR1 and TNFR2), regulates the onset and exacerbation of autoimmune diseases such as rheumatoid arthritis and Crohn's disease. In particular, TNFR1-mediated signals are predominantly related to the induction of inflammatory responses. We have previously generated a TNFR1-selective antagonistic TNF-mutant (mutTNF) and shown that mutTNF efficiently inhibits TNFR1-mediated bioactivity in vitro and attenuates inflammatory conditions in vivo. In this study, we aimed to improve the TNFR1-selectivity of mutTNF This was achieved by constructing a phage library displaying mutTNF-based variants, in which the amino acid residues at the predicted receptor binding sites were substituted to other amino acids. From this mutant TNF library, 20 candidate TNFR1-selective antagonists were isolated. Like mutTNF, all 20 candidates were found to have an inhibitory effect on TNFR1-mediated bioactivity. However, one of the mutants, N7, displayed significantly more than 40-fold greater TNFR1-selectivty than mutTNF. Therefore, N7 could be a promising anti-autoimmune agent that does not interfere with TNFR2-mediated signaling pathways.