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.

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.

Cathepsin K inhibitor causes changes in crystallinity and crystal structure of newly-formed mandibular bone in rats.

Cathepsin K inhibitors are new drugs with the potential for the treatment of osteoporosis because they sustain bony remodelling better than bone resorption inhibitors such as bisphosphonates. The treatment of osteoporosis with inhibitors of bony resorption is associated with osteonecrosis of the jaw, as the deterioration in bony quality that they induce is thought to be one of its causes. The quality of bone is delineated by structural and material characteristics (which include the degree and quality of mineralisation, and depends on the content of proteoglycan and the structural integrity of the bony collagen).1,2 Animal and clinical studies have shown that cathepsin K inhibitors improve the mineral density and structural characteristics of bone, but their effect on the rest remains unknown. We therefore hypothesised that these inhibitors will affect the material characteristics of newly-formed mandibular bone. To verify our hypothesis, we used Raman microspectroscopy to examine such bone in rats that were given a cathepsin K inhibitor, and found unusual crystallinity and an increased substitution of carbonate (CO32-) in its crystal structure.

Molecular alterations of newly formed mandibular bone caused by zoledronate.

Bone quality is defined by structural and material characteristics. Most studies on the mandible have focused on the analysis of structural characteristics, with insufficient investigation of material characteristics. This study tested whether zoledronate affects the material characteristics of newly formed mandibular bone. Thirty-six female Wistar rats were assigned to three groups: sham-ovariectomized rats (SHAM, n=12), ovariectomized rats (OVX, n=12), and ovariectomized rats treated with zoledronate (ZOL, n=12). The left side of the mandibular ramus of all rats was drilled bicortically. Twenty-eight days after surgery, all surviving rats were euthanized and all mandibles were removed. Raman microspectroscopy was performed, and five spectra per specimen of newly formed mandibular bone were analysed. Compared with OVX rats, the mineral/matrix ratio in ZOL rats was significantly increased (5.43±1.88 vs. 7.86±2.05), while crystallinity (0.055±0.002 vs. 0.050±0.002), relative proteoglycan content (0.43±0.10 vs. 0.31±0.05), and collagen structural integrity (1.16±0.21 vs. 0.72±0.06) were significantly decreased. These changes in material characteristics may explain why rats that received zoledronate exhibited peculiar biological phenomena such as bisphosphonate-related osteonecrosis of the jaw.

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.

Comparison of the prognosis of bisphosphonate-related osteonecrosis of the jaw caused by oral and intravenous bisphosphonates.

Bisphosphonates (BPs) have been used in medical practice for the treatment of osteoporosis, bone metastasis, and multiple myeloma. Although many studies have been published, the treatment and prognosis of bisphosphonate-related osteonecrosis of the jaw (BRONJ) remain unclear. This study included 59 patients with BRONJ: 29 had taken oral BPs and 30 had taken intravenous (IV) BPs. All received conservative treatments. When separated sequestra were seen, a sequestrectomy was performed. Segmental mandibular resection was performed when pathological fractures were diagnosed. The outcomes of treatments were compared between groups. For patients treated with oral rinses or mandibular resection, the number in whom clinical healing was observed did not differ between the oral BP and IV BP groups. With regard to sequestrectomy, 94% of patients in the oral BP group showed improvement with this treatment compared to 50% in the IV BP group. The number of patients in whom clinical healing of BRONJ was achieved was statistically better in the oral BP group than in the IV BP group after 6 months of treatment (P<0.001). The results showed that >90% of patients treated with oral BPs could be cured. However, 50% of patients treated with IV BPs did not show an improvement. Additional research is needed to further increase the therapeutic efficacy for the resolution of BRONJ.

Comparison between diffuse infrared and acoustic transmission over the human skull.

Skull-induced distortion and attenuation present a challenge to both transcranial imaging and therapy. Whereas therapeutic procedures have been successful in offsetting aberration using from prior CTs, this approach impractical for imaging. In effort to provide a simplified means for aberration correction, we have been investigating the use of diffuse infrared light as an indicator of acoustic properties. Infrared wavelengths were specifically selected for tissue penetration; however this preliminary study was performed through bone alone via a transmission mode to facilitate comparison with acoustic measurements. The inner surface of a half human skull, cut along the sagittal midline, was illuminated using an infrared heat lamp and images of the outer surface were acquired with an IR-sensitive camera. A range of source angles were acquired and averaged to eliminate source bias. Acoustic measurement were likewise obtained over the surface with a source (1MHz, 12.7mm-diam) oriented parallel to the skull surface and hydrophone receiver (1mm PVDF). Preliminary results reveal a positive correlation between sound speed and optical intensity, whereas poor correlation is observed between acoustic amplitude and optical intensity.

A short-wavelength infrared emitting multimodal probe for non-invasive visualization of phagocyte cell migration in living mice.

For the non-invasive visualization of cell migration in deep tissues, we synthesized a short-wavelength infrared (SWIR) emitting multimodal probe that contains PbS/CdS quantum dots, rhodamine 6G and iron oxide nanoparticles. This probe enables multimodal (SWIR fluorescence/magnetic resonance) imaging of phagocyte cell migration in living mice.

Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway.

Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis.

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.

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.

Postoperative changes in cerebral metabolites associated with cognitive improvement and impairment after carotid endarterectomy: a 3T proton MR spectroscopy study.

BACKGROUND AND PURPOSE: Cognitive function can improve or decline after carotid endarterectomy. Proton MR spectroscopy can be used evaluate cerebral metabolites, such as N-acetylaspartate, choline, and creatine, in vivo. The purpose of the present study was to determine whether postoperative changes in cerebral metabolites measured by using 3T proton MR spectroscopy were associated with changes in cognitive function after CEA. MATERIALS AND METHODS: In 100 patients undergoing CEA for ipsilateral cervical internal carotid artery stenosis (≥70%), brain proton MR spectroscopy was performed before and after surgery. NAA/Cr and Cho/Cr ratios were measured in regions of interest placed in the centrum semiovale of both cerebral hemispheres. Neuropsychological testing was also performed preoperatively and 1 month postoperatively. Multivariate statistical analysis of factors related to postoperatively changed cognition was performed, and odds ratios with 95% confidence intervals were calculated. RESULTS: On the basis of the neuropsychological assessments, 10 (10%), 80 (80%), and 10 (10%) patients were defined as having postoperatively improved, unchanged, and impaired cognition, respectively. A positive and high ΔNAA/Cr ratio (postoperative value-preoperative value) in the cerebral hemisphere ipsilateral to the operative site was significantly associated with postoperatively improved cognition (95% CI, 13.3-21.3; P = .0016). Negative and high absolute values of the ΔNAA/Cr ratio (95% CI, 0.018-0.101; P = .0039) and ΔCho/Cr ratio (95% CI, 0.042-0.135; P = .0046) in the ipsilateral cerebral hemisphere were significantly associated with postoperatively impaired cognition. CONCLUSIONS: Postoperative changes in cerebral metabolites measured by using proton MR spectroscopy were associated with changes in cognitive function after CEA.

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.

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.

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.