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.

Hepatotoxicity, nephrotoxicity, and drug/chemical interaction toxicity of platinum nanoparticles in mice.

Nanomaterials are frequently used in microelectronics, cosmetics, and sunscreens. Platinum reagents are commonly used in disease diagnosis, cosmetics, and the food industry. Although research into the development of nanomaterialbased drug delivery systems has yielded promising results, the toxicity of these materials is not fully understood. We investigated the toxicity and drug interactions of 1- and 8-nm diameter platinum nanoparticles (nPt1 and nPt8, respectively) in mice. Acute hepato-renal toxicity of intravenously administered platinum nanoparticles was evaluated biochemically and histologically. Dose-dependent increases in serum markers of hepato-renal function (serum aminotransferases and blood urea nitrogen) were observed following administration of nPt1, whereas nPt8 had no effect, even at 20 mg/kg. Moreover, nPt1 induced interleukin (IL)-6 and IL-1ß production 3 and 6 hours after administration. The effect of nPts on drug-induced toxicity was evaluated in mice injected intraperitoneally with carbon tetrachloride or cisplatin, with or without intravenous administration of platinum nanoparticles. All treatments in the absence of nanoparticles were non-lethal and resulted in moderate toxicity. However, exacerbated toxicity was observed in mice injected with carbon tetrachloride or cisplatin together with nPt1, but not in mice co-injected with nPt8. We found that nPt1 cause hepato-renal damage, and the effect is enhanced by chemical inducers of hepatotoxicity and nephrotoxicity. This is the first report demonstrating that nPt1 not only are hepatotoxic and nephrotoxic but also exacerbate drug toxicity. These findings will be useful for future nanotechnology and nanoscience research.

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.

IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer.

BACKGROUND: PD-L1 (programmed cell death 1 ligand 1) on tumour cells suppresses host immunity through binding to its receptor PD-1 on lymphocytes, and promotes peritoneal dissemination in mouse models of ovarian cancer. However, how PD-L1 expression is regulated in ovarian cancer microenvironment remains unclear. METHODS: The number of CD8-positive lymphocytes and PD-L1 expression in tumour cells was assessed in ovarian cancer clinical samples. PD-L1 expression and tumour progression in mouse models under conditions of altering IFN-γ signals was assessed. RESULTS: The number of CD8-positive cells in cancer stroma was very high in peritoneally disseminated tumours, and was strongly correlated to PD-L1 expression on the tumour cells (P<0.001). In mouse models, depleting IFNGR1 (interferon-γ receptor 1) resulted in lower level of PD-L1 expression in tumour cells, increased the number of tumour-infiltrating CD8-positive lymphocytes, inhibition of peritoneal disseminated tumour growth and longer survival (P=0.02). The injection of IFN-γ into subcutaneous tumours induced PD-L1 expression and promoted tumour growth, and PD-L1 depletion completely abrogated tumour growth caused by IFN-γ injection (P=0.01). CONCLUSIONS: Interferon-γ secreted by CD8-positive lymphocytes upregulates PD-L1 on ovarian cancer cells and promotes tumour growth. The lymphocyte infiltration and the IFN-γ status may be the key to effective anti-PD-1 or anti-PD-L1 therapy in ovarian cancer.

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.

Dermal absorption of amorphous nanosilica particles after topical exposure for three days.

The skin penetration and cellular localization of well-dispersed amorphous nanosilica particles (nSPs) with a diameter of 70 nm was analyzed in mice. Our results suggest that after topical exposure for three days the particles penetrate the skin barrier and are transported to the lymph nodes. These findings underscore the need to examine biological effects following dermal exposure to nSPs for the development of safer use of nSPs.

Amorphous nanosilica particles induce ROS generation in Langerhans cells.

Generation of total intracellular reactive oxygen species (ROS) was measured in XS52 cells, a Langerhans cell-like line, treated with different sized amorphous silica particles. The results suggested that exposure to amorphous nanosilica particles (nSPs) with a particle size of 70 nm induced a higher level of ROS generation than did exposure to micron-sized amorphous silica particles. This finding means that it is essential to examine the biological effects of ROS generated after exposure to nSPs, which will provide useful information for hazard identification as well as the design of safer nanomaterials.

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.

Detection of titanium dioxide particles on frozen tissue sections using synchrotron radiation X-ray fluorescence analysis.

Recent studies into the in vivo absorption and biological influence of particulate matter, especially nanomaterials (NMs), have raised worldwide concerns over their safety. However, it is often technically difficult to conduct these studies because NMs are too small to be observed by optical microscopy. Here, we attempted to establish a new method to visually detect NMs on tissue samples. Specifically, we have analyzed titanium dioxide particles with a diameter of 5 microm, which are widely used in cosmetics, using frozen tissue sections by synchrotron radiation X-ray fluorescence analysis.

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.

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.

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.

Systemic administration of a PEGylated adenovirus vector with a cancer-specific promoter is effective in a mouse model of metastasis.

Cancer gene therapy by adenovirus vectors (Advs) for metastatic cancer is limited because systemic administration of Adv produces low therapeutic effect and severe side effects. In this study, we generated a dual cancer-specific targeting vector system by using PEGylation and the telomere reverse transcriptase (TERT) promoter and attempted to treat experimental metastases through systemic administration of the vectors. We first optimized the molecular size of PEG and modification ratios used to create PEG-Ads. Systemic administration of PEG-Ad with 20-kDa PEG at a 45% modification ratio (PEG[20K/45%]-Ad) resulted in higher tumor-selective transgene expression than unmodified Adv. Next, we examined the effectiveness against metastases and side effects of a TERT promoter-driven PEG[20K/45%]-Ad containing the herpes simplex virus thymidine kinase (HSVtk) gene (PEG-Ad-TERT/HSVtk). Systemic administration of PEG-Ad-TERT/HSVtk showed superior antitumor effects against metastases with negligible side effects. A cytomegalovirus (CMV) promoter-driven PEG[20K/45%]-Ad also produced antimetastatic effects, but these were accompanied by side effects. Combining PEG-Ad-TERT/HSVtk with etoposide or 5-fluorouracil enhanced the therapeutic effects with negligible side effects. These results suggest that modification with 20-kDa PEG at a 45% modification ratio is the optimal condition for PEGylation of Adv, and PEG-Ad-TERT/HSVtk is a prototype Adv for systemic cancer gene therapy against metastases.

Metastasis of carcinoma ex pleomorphic adenoma to the brain without previous metastasis to the lungs or bones: a case report.

Carcinoma ex pleomorphic adenoma is a rare type of cancer of the salivary gland that involves the malignant transformation of a primary or recurrent pleomorphic adenoma, which often metastasises to the lungs or bones, or both. To the best of our knowledge, however, nobody has reported a distant metastasis of this lesion to the brain without such previous metastasis. We report a case in a 64-year-old man.

Pathological factors involved in local failure in squamous cell carcinoma of the oral cavity: retrospective study and proposal of a new clinical classification.

The control of local failure (LF) is essential to improve outcomes in patients with squamous cell carcinoma of the oral cavity (OSCC). In this study, LF of OSCC was classified into three clinical types: deep recurrence (type 1R), adjacent superficial recurrence (type 2R), and distant primary tumour (type 3R). LF was analyzed after surgical resection of OSCC to determine the validity and usefulness of this classification system. Of 257 patients with OSCC, 58 experienced LF; 21 had type 1R, 23 had type 2R, and 20 had type 3R. Clinical factors influencing LF were analyzed by log-rank test and Cox test. Type 1R was significantly related to the TN classification, resection margin status, and invasive pattern. Type 2R was strongly associated with the grade of epithelial dysplasia at the surgical margins. Type 1R rarely developed more than 1year after surgery, whereas type 2R did not develop within 2 years. Type 1R may be caused by residual cancer cells in the deep margins, and type 2R by precancerous cells remaining in the marginal epithelium and gradually becoming invasive cancer. Type 3R may be considered an independent tumour. The newly proposed clinical classification is convenient and roughly reflects the causes and mechanisms of relapse.

Comparative study on transduction and toxicity of protein transduction domains.

BACKGROUND AND PURPOSE: Protein transduction domains (PTDs), such as Tat, antennapedia homeoprotein (Antp), Rev and VP22, have been extensively utilized for intracellular delivery of biologically active macromolecules in vitro and in vivo. There is little known, however, about the relative transduction efficacy, cytotoxicity and internalization mechanism of individual PTDs. EXPERIMENTAL APPROACH: We examined the cargo delivery efficacies of four major PTDs (Tat, Antp, Rev and VP22) and evaluated their toxicities and cell internalizing pathways in various cell lines. KEY RESULTS: The relative order of the transduction efficacy of these PTDs conjugated to fluorescein was Rev>Antp>Tat>VP22, independent of cell type (HeLa, HaCaT, A431, Jurkat, MOLT-4 and HL60 cells). Antp produced significant toxicity in HeLa and Jurkat cells, and Rev produced significant toxicity in Jurkat cells. Flow cytometric analysis demonstrated that the uptake of PTD-fluorescein conjugate was dose-dependently inhibited by methyl-beta-cyclodextrin, cytochalasin D and amiloride, indicating that all four PTDs were internalized by the macropinocytotic pathway. Accordingly, in cells co-treated with 'Tat-fused' endosome-disruptive HA2 peptides (HA2-Tat) and independent PTD-fluorescent protein conjugates, fluorescence spread throughout the cytosol, indicating that all four PTDs were internalized into the same vesicles as Tat. CONCLUSIONS AND IMPLICATIONS: These findings suggest that macropinocytosis-dependent internalization is a crucial step in PTD-mediated molecular transduction. From the viewpoint of developing effective and safe protein transduction technology, although Tat was the most versatile carrier among the peptides studied, PTDs should be selected based on their individual characteristics.