Pancreatic Cancer Cell Fraction Estimation in a DNA Sample.

OBJECTIVE: Pancreatic cancers are characterized by dense stroma. To estimate the degree of interference by coexisting noncancer cells in molecular analyses, we aimed to develop a DNA methylation marker that assesses a cancer cell fraction in DNA samples. METHODS: The microarray data of 22 pancreatic cancer tissues from the The Cancer Genome Atlas database and 9 noncancer tissues were used for genome-wide screening. Thirty-one surgical tumor samples (10 intraductal papillary mucinous neoplasms [IPMNs] and 21 pancreatic cancers), 4 normal, and 26 nontumor samples were used for validation. Gene-specific methylation analysis was conducted by bisulfite pyrosequencing. RESULTS: Genome-wide screening isolated SIM1, MIR129-2, NR1I2, and HOXB-AS4, as specifically methylated in pancreatic cancer cells. Bisulfite pyrosequencing validated that one or more of three genes (SIM1, MIR129-2, and NR1I2) were methylated in 22 (71.0%) tumor samples (8 IPMNs and 14 cancers), and all showed low levels of methylation in 26 (86.7%) normal and nontumor samples. Therefore, the three genes collectively constituted one marker for a pancreatic cancer cell fraction. The cancer cell fraction estimated by the marker was highly correlated with that estimated using the KRAS mutant allele frequency (R = 0.79). CONCLUSION: The DNA methylation marker is useful to estimate the pancreatic cancer cell fraction in DNA samples.

Influence of the difference of breastfeeding volume on a rat model of oxygen-induced retinopathy.

This study aimed to investigate the effects of abundant breast milk intake on rats model of oxygen-induced retinopathy (OIR). Neonatal Sprague-Dawley rats were randomly assigned to expand litters of 7 pups/litter (7-rats group) and 14 pups/litter (14-rats group). They were exposed to 80% oxygen from postnatal day (P) 0 to P12. Body weights were measured daily. At P13 and 18, rats were sacrificed, and the blood and eyes were collected. Retinal neovascularization (NV) score, total retinal area (TRA), avascular area (AVA), and vascularized area (VA) were measured in ADPase stained retinas. Retinal vascular endothelial growth factor (VEGF) and serum insulin-like growth factor (IGF-1) were measured using ELISA. Body weight gain was significantly greater in 7-rats group from P2. Serum IGF-1 levels at P13 and 18 were significantly higher in 7-rats group. Retinal VEGF and TRA at P18 were significantly larger in 7-rats group. NV score at P18 tended to be higher in 7-rats group. There was no significant difference in VA between the 2 groups at P13 and 18. Excess breast milk intake in OIR rat pups caused body weight gain and retinal development, whereas there was less effect on retinal vascularization in our study.

Dual-fluorescent RNA probes with an extremely large stokes shift.

Fluorescent probes are powerful and indispensable tools for imaging RNA in vivo and in vitro. To simultaneously visualize multiple RNA targets in a cell, it is necessary to develop probes which emit fluorescence with different colors by excitation at a single wavelength. We synthesized OMUpy1 and OMUpy2 in this study with a cyanine dye respectively conjugated at their 5' ends. A fluorescent analysis revealed these probes to have yellow or pink fluorescence derived from the cyanine dyes with an extremely large Stokes shift. Three color-coded fluorescent images were also obtained in the presence of target RNAs.

RNA-based diagnosis in a multicellular specimen by whole mount in situ hybridization using an RNA-specific probe.

Recent RNA research has revealed the close involvement of various RNAs in cellular functions. RNAs are becoming the inevitable target molecules for research into details of gene expression. RNA and its related complexes are also promising targets for disease diagnosis. Multi cellular specimens such as organ tissues, histopathological specimens, and embryos are among the possible targets of RNA-based diagnostic techniques. In this report, we focused on a method that would provide such spatial and temporal information. We demonstrated that an RNA-specific probe (OMUpy2) was not only applicable to the detection of a specific mRNA in Drosophila embryos in a temporal and spatial manner but was also relatively quick and easy to use. The probe, OMUpy2, could be applied to other multi cellular systems for RNA-based diagnosis and research. The promising results of this manuscript show the great potential of RNA-based detection for both biological research and diagnostic medicine.

Clinical outcome of amrubicin therapy according to the prior chemotherapy sensitivities of extensive small cell lung cancer.

BACKGROUND: Amrubicin (AMR) is an active agent for relapsed small cell lung cancer (SCLC). However, the activity of AMR in refractory relapsed patients is controversial. The objective of this retrospective analysis was to evaluate the efficacy and safety of AMR as second-line chemotherapy in SCLC, especially refractory relapsed SCLC. METHODS: Between July 2003 and February 2009, a total of 27 patients were treated with AMR at a dosage of 40 mg x m(-2) x day(-1) on days 1-3 every 3 weeks. Safety was assessable for all patients. Efficacy was evaluated in 26 patients (one patient was not assessable for response), in 12 patients with chemotherapy-sensitive relapse and 14 patients with chemotherapy-refractory relapse. Sensitive relapse means that a first-line response lasted more than 90 days. Refractory relapse means that either did not respond to first-line chemotherapy or responded initially but relapsed within 90 days. RESULTS: Thirteen patients (50%, 95% CI, 31% to 69%) had partial response, including 6 (50%) of the 12 patients with chemotherapy-sensitive relapse and 7 (50%) of 14 patients with chemotherapy-refractory relapse. Median survival times of patients with chemotherapy-sensitive and -refractory relapse were 9.7 months and 8.4 months, respectively, showing significant difference (p = 0.0337). Adverse events were observed in all 27 patients. Grade 3 and 4 neutropenia was seen in 8 patients (29.6%) and 15 patients (55.5%), respectively. Grade 3 and 4 thrombocytopenia occurred in 10 patients (37.0%) and 2 patients (7.4%). Non-hematologic toxicities were generally mild, except for febrile neutropenia. Febrile neutropenia was seen in 6 patients (22.2%). No treatment-related deaths occurred. CONCLUSIONS: AMR is an active agent for the treatment of relapsed SCLC, especially chemotherapy-refractory relapse SCLC, with predictable and manageable toxicities.

Eye damage control by reduced blue illumination.

The aim of this study was to demonstrate that a blue light and ultraviolet cut-off filter (blue filter) could reduce short-wavelength retina/RPE damage threshold by a continuous spectrum source. Sixteen normal eyes of two rhesus monkeys and six cynomolgus monkeys were subjected to macular irradiation of 20, 24, 27.4, 30, 35, 45, 50 and 60 J/cm(2) energy densities. The values of energy density were measured before the blue filter. Lesions were measured before and at 2 and 30 days after irradiation of a 2.8 mm diameter region within the macular arcade. Measures were fundoscopy, fluorescein angiography and long wavelength scanning by the Heidelberg Retinal Tomograph (HRT) unit. The lesions, which were produced, were scored and compared to irradiant energy density of the blue LED (NSPB500S, Nichia, Tokushima, Japan). The exposure at the 20 J/cm(2) produced no detectable result at 2 or 30 days. Exposure at 35 J/cm(2) showed definite lesion production without blue filter. With the filter added there was one indication of minor change. At 60 J/cm(2) there was extensive heavy, enduring damage without the filter and with the filter damage was present but was significantly attenuated. These results strongly support the conclusion that the blue filter attenuation reduces the frequency of damage by exposure. This experimental system is a useful model for normal human eye aging and continuous spectrum environment irradiance.

Zymographical techniques for detection of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases associated with extracellular matrix degradation, cellular migration, tissue remodeling, and angiogenesis. The activity of MMPs is regulated by the tissue inhibitors of metalloproteinases (TIMPs). Zymography and reverse zymography are useful to detect MMPs and TIMPs activities from various samples, for example vitreous, retina, plasma, and so on. Sample proteins are separated in substrate containing polyacrylamide gel by electrophoresis. The gel is incubated and then stained with Coomassie Blue. MMPs' activities are detected as clear bands.

[The effect of theanine on oxygen-induced retinal neovascularization in the neonatal rat].

PURPOSE: This study was aimed to investigate the preventive effects of theanine treatment on a rat model of oxygen-induced ischemic retinopathy (OIR). METHODS: OIR was induced by maintaining the Sprague-Dawley neonatal rats in 80% oxygen. The rats were treated once daily with gastric gavage of theanine (5 or 50 mg/kg) or distilled water (DW) from postnatal days 6 to 17. The retinal neovascularization (NV) was scored and avascular areas(AVAs) were measured as a % of total retinal area (% AVAs) at day 18. RESULTS: The % AVAs in 5 mg/kg theanine (13.2 +/- 2.8%) and 50 mg/kg theanine (9.4 +/- 2.2%, p < 0.05) treatment were lower than those in DW (18.9 +/- 2.9 %). The NV scores with 5 mg/kg theanine(4.2 +/- 0.5) or 50 mg/kg theanine (3.4 +/- 0.6) treatment were lower than those with DW (4.7 +/- 0.6). CONCLUSION: Theanine treatment suppresses the neovascularization in a rat model of OIR. These results suggest that theanine may prevent retinopathy of prematurity.

Effects of green tea fractions on oxygen-induced retinal neovascularization in the neonatal rat.

This study aimed to investigate the preventive effects of green tea fractions (GTFs) on rat model of oxygen-induced retinopathy (OIR). Neonatal Sprague-Dawley rats were exposed to daily cycles of 80% oxygen (20.5 h), ambient air (0.5 h), and progressive return to 80% oxygen (3 h) until postnatal day 12 (P12), then the rats were placed in ambient air until P18. The green tea was fractionated by DM-A50, DM-W, M-B, and M-W. The rats were treated once daily from P6 to P17 by gastric gavage of GTFs (0.05 or 0.01 g/ml) or distilled water (DW) at 50 microl/10 g body weight. On P18, the rats were sacrificed and the retinal samples were collected. The retinal neovascularization (NV) was scored and avascular areas (AVAs) were measured as a % of total retinal area (%AVAs) in ADPase stained retinas. The NV scores in 0.01 g/ml M-W were significantly lower than those in DW. The %AVAs in 0.05 g/ml DM-A50 and in 0.05 g/ml and 0.01 g/ml M-W were significantly lower than those in DW. There were less catechins, and less caffeine in M-W fraction compared with other GTFs, suggesting components of green tea except for catechins and caffeine might suppress the neovascularization in rat model of OIR.

Activation of AP-1 and increased synthesis of MMP-9 in the rabbit retina induced by lipid hydroperoxide.

PURPOSE: We identified the temporal expression of activator protein-1 (AP-1) and matrix metalloproteinases (MMPs) after linoleic acid hydroperoxide (LHP) induction of retinal neovascularization. METHODS: After injection of LHP into the vitreous of rabbits, samples were collected for AP-1 binding activity and mRNA for MMP-9 and MMPs activity. AP-1 binding activity was measured by electrophoretic mobility shift assay. MMP-9 activity was measured by zymography and mRNA by quantitative RT-PCR. RESULTS: AP-1 binding activity was increased at 1-3 hr. MMP-9 mRNA levels were increased at 3 hr in the neural retina and by 12 hr in the retinal pigment epithelium (RPE) layer. MMP-9 proteolytic activity was elevated within the neural retina and within the vitreous and in the RPE-interphotoreceptor matrix (IPM) at 12 hr and peaked at 24 hr or 4 days. CONCLUSIONS: LHP increases the transcription factor AP-1 which in turn may regulate retinal MMP-9 synthesis during neovascularization.

Impaired contact hypersensitivity reaction and reduced production of vascular endothelial growth factor in tumor necrosis factor-alpha gene-deficient mice.

Tumor necrosis factor (TNF)-alpha is an important proinflammatory cytokine in contact hypersensitivity (CHS) reactions. Previous efforts to assay CHS in TNF-alpha gene-deficient (-/-) mice have demonstrated a significant reduction in ear skin weight at 24 h following challenge by oxazolone, although the mechanisms of this suppression have not been examined. To further characterize the impaired CHS during evolution of the elicitation phase in TNF-alpha -/- mice and to clarify its mechanisms, focusing on the roles of TNF-alpha and vascular endothelial growth factor (VEGF), we used an established method of CHS assay-sensitization and challenge by trinitrochlorobenzene (TNCB)- in TNF-alpha -/- and wild-type mice. We compared the histopathology of the sequential evolution of CHS between the two groups of mice and assessed both the extent of inflammatory cell infiltration and the degree of dilatation in dermal vessels labeled with CD31. We quantified the production of VEGF in the epidermis at specific time points by using a murine VEGF ELISA kit. The CHS reaction was markedly suppressed in TNF-alpha -/- mice at all time points of the elicitation phase. Histologically, in TNF-alpha -/- mice we observed diminished vascular permeability, reduced numbers of infiltrating inflammatory cells, neutrophils at 12 h, mononuclear cells and eosinophils at 24 h, and a decreased area of dilatation of vessels labeled with CD31. The level of epidermal VEGF in wild type mice increased rapidly after challenge and peaked at 24 h, paralleling the peak of ear swelling. In contrast, the peak level of epidermal VEGF in TNF-alpha -/- mice was significantly reduced. These results suggest that TNF-alpha plays an enhancing role in the elicitation phase of the CHS reaction. Diminished degrees of vascular permeability, dilatation of CD31+ vessels, and inflammatory cell infiltration in TNF-alpha -/- mice are likely to be the result of a lack of TNF-alpha and reduced production of epidermal VEGF.

Lipid hydroperoxide-induced tumor necrosis factor (TNF)-alpha, vascular endothelial growth factor and neovascularization in the rabbit cornea: effect of TNF inhibition.

Lipid hydroperoxides (LHP) at high concentrations are cytotoxic, but at sublethal concentration, they induce synthesis of cytokine vascular growth factors. Intracorneal injections of 30 µg LHP placed 5 mm from the superior limbus stimulated early vasodilation of limbal vasculature and a rapidly developing, sustained neovascularization. Under these conditions, vessels grew at the rate of 0.3 mm/day to a total length of 7.5 mm, 25 days after injection. Cholesterol peroxides were less effective. Developing vessels were oriented towards the stimulus. Around the developing vessel there was dissolution of the stromal extracellular matrix. The most distal endothelial cells displayed prominent endoplasmic reticulum, a lack of basement membrane or tight junction complexes and leakage of fluorescein dye. Both the injection site and superior quadrant showed increased levels of tumor necrosis factor (TNF)-alpha and vascular endothelial growth factor after exposure to LHP. The neovascular response was inhibited by simultaneous administration of TNF-alpha antibody or pentoxifylline, an inhibitor of TNF-alpha synthesis. This corneal model of peroxide-induced neovascularization should prove useful for temporal studies of events in the initiation and propagation of signals leading to neovascularization, and for evaluating effects of treatment on neovascular growth.

Lipid hydroperoxide induced corneal neovascularization in hyperglycemic rabbits.

PURPOSE: To compare the effect of hyperglycemia on corneal neovascularization (NV) induced by linoleic acid hydroperoxide (LHP) in a rabbit model. METHODS: Male New Zealand rabbits received 80 mg/kg alloxan i.v. and subsequently developed hyperglycemia. Four weeks later, 10 microl of LHP (40 mM) was injected into corneal stroma 5 mm from the superior limbus with a 30 gauge needle. Vessel growth area from the limbal vasculature was measured over a period of 2 weeks and was correlated with plasma levels of insulin, HbA(1c), and corneal vascular endothelial growth factor (VEGF). RESULTS: Two days after alloxan, blood glucose was increased from 97 +/- 4 mg/dl in the untreated control group to 413 +/- 3 mg/dl. At 24 and 72 hours after LHP injection, VEGF in cornea of hyperglycemic rabbits was elevated 2 to 4 times above that of normoglycemic rabbits. At 14 days after LHP injection, the normoglycemic rabbits vessel growth area measured 2.42 +/- 0.31 mm(2), but in the hyperglycemic group, vessel growth area was significantly increased to 7.96 +/- 2.26 mm(2) (p < 0.05). At the end of the experimental period, HbA(1c) was elevated from 3.9 +/- 0.8 % to 8.4 +/- 0.6 % and insulin was decreased from 440 +/- 123.9 pg/ml to 24 +/- 11.0 pg/ml. CONCLUSIONS: These data suggest that hyperglycemia may sensitize corneal and vascular endothelial cells, perhaps by glucose derived radicals, which enhance production of additional LHP through endogenous propagation reactions, and raise in turn the concentration of VEGF levels to induce an enhanced, sustained NV response.

Ultraviolet action spectrum for cell killing of primary porcine lens epithelial cells.

OBJECTIVES: The aim of this study was to determine ultraviolet (UV) action spectra for cell killing of primary porcine lens epithelial cells (LECs) that can be used to establish guidelines for evaluation of the hazard of cataract due to UV exposure in the workplace. METHODS: Primary porcine LECs were exposed to different doses (radiant exposure) of UV at 17 different wavelengths from 235 nm to 311 nm. At 2 days after exposure, cell viability was assessed by measuring crystal violet staining of the cells and lactate dehydrogenase release into the culture medium. The exposure dose required to kill 50% of cells (LD(50)) was determined from the dose-effect relationship obtained at each wavelength and was used to construct action spectra. RESULTS: The action spectra had a broad minimum in the approximate range of 250-280 nm, indicating that UV is most hazardous to porcine LECs within this wavelength range. The spectra rose steeply at both longer and shorter wavelengths. These action spectra are consistent with the in vivo action spectra for opacities in the rabbit lens and for light scattering in the rat lens, taking the transmittance of the ocular media into account. CONCLUSIONS: These results will help to determine a UV hazard function for cataract formation, which can be used to draft guidelines for evaluation of the hazard of cataract due to UV exposure in the workplace.

Moxibustion at mingmen reduces inflammation and decreases IL-6 in a collagen-induced arthritis mouse model.

The purpose of this study was to compare the effectiveness of moxibustion (MOX) treatment at the GV4 and CV12 acupoints, and to determine the correlations between MOX treatment and interleukin (IL)-6 and corticosterone levels in a collagen-induced arthritis (CIA) mouse model. CIA mice were immunized twice intradermally over a 3-week interval with bovine type II collagen. After the second immunization (day 21), MOX was applied to the mouse equivalent of the GV4 and CV12 acupoints with a 1mg moxa cone five times/day. Clinical symptoms of CIA were observed three times/week until day 35. The concentrations of IL-6 and corticosterone in the blood samples were measured by immunoassay kits. At day 35, the incidence of CIA was significantly decreased in mice treated with MOX at the GV4 acupoint (78%, n=23, p<0.05), compared to untreated CIA mice (100%) and mice treated with MOX at the CV12 acupoint (100%). IL-6 and corticosterone levels were significantly increased by immunization. IL-6 levels significantly decreased in mice treated with MOX at the GV4 acupoint. These results suggest that MOX treatment suppressed CIA at the GV4 acupoint, not at the CV12 acupoint, possibly through inhibition of IL-6 production.

Changes of blood fluidity in the early hepatopathy in rats.

The present study was designed to examine the influence of the early stage hepatopathy on blood fluidity by using a rat experimental system. F344 male rats, 4 weeks of age, were fed chow containing 3'-methyl-4-dimethylaminoazobenzene (DAB) at 0.06%. These rats were autopsied 8, 12, 16 and 20 weeks after starting DAB feeding. Blood samples were collected from the inferior vena cava under pentobarbital anesthesia and blood fluidity and platelet aggregation activity were examined by a Micro Channel Array Flow Analyzer and a platelet aggregometer, respectively. We also examined histological changes in the liver after staining with hematoxylin-eosin. Histological observation of the liver revealed early-stage hepathopathy when the organs were obtained from rats that fed DAB for more than 16 weeks. Although DAB-feeding of rats for 8 and 12 weeks barely affected blood fluidity, long-term intake (>16 weeks) caused decrease in fluidity. On the other hand, platelet aggregation activity was increased when rats were fed DAB for more than 16 weeks. The present results suggest that assaying for blood fluidity may be useful for the assessment of the degree of hepatopathy.

Real-time monitoring of mRNAs with fluorescence-modified RNA probes in living cells.

In our previous study, we reported that bispyrene-modified RNA probes (OMUpy2) were useful for RNA detection in homogeneous physiological media(1-2). The aim of this study is to establish in situ monitoring system to detect mRNAs in living cells by OMUpy2. We chose c-fos mRNA as a target RNA which is known as one of the immediate-early genes. The real-time moni- toring of mRNAs was carried out in living C4II cells. After transfection of OMUpy2 into the cells which had been incubated with serum-free medium, cells were stimulated by media with serum and the resulting expression of mRNA was monitored by a fluorescence microscope. In the case of OMUpy2-CF3S, which was complementary to c-fos mRNA, fluorescence emission around 480 nm was observed obviously. These results suggest that mRNA expressed by the serum stimulation was successfully visualized in a real-time manner.

Effect of a dihydrobenzofuran derivative on lipid hydroperoxide-induced rabbit corneal neovascularization.

The aim of this study was to investigate the effect of A-3922, a dihydrobenzofuran derivative, on linoleic acid hydroperoxide (LHP)-induced corneal neovascularization (NV) in a rabbit model. Male New Zealand rabbits received intraperitoneal (i.p.) injections of 10 or 30 mg/kg per day A-3922 or its vehicle as control for 3 days. One day after i.p. injections, LHP was injected with a 30-gauge needle into the corneal stroma of the superior quadrant 4.5-mm below the limbus. Photographs of the vessels were taken for digital analysis with a surgical microscope. Vascular endothelial growth factor (VEGF) was measured using an immunoassay kit, and matrix metalloproteinase (MMP)-9 was measured by gelatin zymography in corneal samples. At 7 days post-LHP injection, the total vessel length was 26.7 +/- 3.8 mm in the control animals (n = 8), 16.1 +/- 0.8 mm in the A-3922 (10 mg/kg)-treated group (n = 5), and 11.4 +/- 2.1 mm in the 30 mg/kg group (n = 8, P<0.01 vs control), respectively. After LHP injection, the content of VEGF and MMP-9 activity were increased in the superior cornea, but these were not influenced by A-3922 treatments. These results indicate that LHP-induced corneal NV is inhibited by treatment with A-3922 and therefore may represent a potential pharmacological intervention for ocular neovascularization disorders.

Effect of lipid-hydroperoxide-induced oxidative stress on vitamin E, ascorbate and glutathione in the rabbit retina.

BACKGROUND: It is possible that oxidative stress causes several retinal diseases. However, the natural biogenic role of antioxidants in the retina is not clear. PURPOSE: This study investigates the change in concentration of vitamin E (VE), ascorbate and glutathione (GSH) in the retina following vitreous injection of 600 mug 18:2 linoleic acid hydroperoxide (LHP) in male New Zealand rabbits. METHOD: LHP was injected above the retinal surface. The animals were sacrificed and the eyes enucleated before LHP injection, 1, 3, 6, 12, 24 h and 4 and 7 days after LHP injection. Retinas were removed, VE and ascorbate measured by HPLC, and GSH determined by a fluorometric method. RESULTS: The concentration of VE in the retina decreased from pretreatment levels of 154.6 +/- 29.7 nmol/g wet weight (n = 7) and was lowest at 6 h (61.1 +/- 18.1 nmol/g wet weight, n = 4, p < 0.05), then increased gradually, returning slowly to pre-LHP levels by 7 days. The concentration of ascorbate in control retinas decreased at 6 h from pretreatment levels of 7.33 +/- 0.93 micromol/g wet weight (n = 7) to 2.74 +/- 0.16 micromol/g wet weight (n = 4, p < 0.05) and returned to pretreatment levels rapidly by 24 h after injection. The concentration of GSH in retinas decreased from baseline levels of 109.53 +/- 8.19 microg/g wet weight (n = 9), was lowest at 12 h (72.40 +/- 11.17 microg/g wet weight, n = 5, p < 0.05) and returned to pretreatment levels by 7 days. CONCLUSION: The results suggest that intravitreous LHP injection is a contributor to oxidative stress in the rabbit retina by causing a reduction in antioxidant capacity.