Kouji Matsumura

CD47 is an adverse prognostic factor and a therapeutic target in gastric cancer.

CD47 is an antiphagocytic molecule that acts via ligation to signal regulatory protein alpha on phagocytes; its enhanced expression and therapeutic targeting have recently been reported for several malignancies. However, CD47 expression in gastric cancer is not well documented. Immunohistochemical expression of CD47 in surgical specimens was investigated. Expression of CD47 and CD44, a known gastric cancer stem cell marker, were investigated in gastric cancer cell lines by flow cytometry. MKN45 and MKN74 gastric cancer cells were sorted by fluorescence‐activated cell sorting according to CD44 and CD47 expression levels, and their in vitro proliferation, spheroid‐forming capacity, and in vivo tumorigenicity were studied. In vitro phagocytosis of cancer cells by human macrophages in the presence of a CD47 blocking monoclonal antibody (B6H12) and the survival of immunodeficient mice intraperitoneally engrafted with MKN45 cells and B6H12 were compared to experiments using control antibodies. Immunohistochemistry of the clinical specimens indicated that CD47 was positive in 57 out of 115 cases, and its positivity was an independent adverse prognostic factor. Approximately 90% of the MKN45 and MKN74 cells expressed CD47 and CD44. CD47hi gastric cancer cells showed significantly higher proliferation and spheroid colony formation than CD47lo, and CD44hiCD47hi cells showed the highest proliferation in vitro and tumorigenicity in vivo. B6H12 significantly enhanced in vitro phagocytosis of cancer cells by human macrophages and prolonged the survival of intraperitoneal cancer dissemination in mice compared to control antibodies. In conclusion, CD47 is an adverse prognostic factor and promising therapeutic target in gastric cancer.

Comparison of DNA Copy Numbers in Original Oral Squamous Cell Carcinomas and Corresponding Cell Lines by Comparative Genomic Hybridization

We analyzed regional DNA copy numbers in 4 oral squamous cell carcinomas (SCCs) by using comparative genomic hybridization, and compared them with those in cell lines derived from the SCCs. In the original tumors, DNA copy number increases were observed on chromosomes 5p (4/4 cases), 8q (4/4), 20p (3/4), 3q (2/4), 5q (2/4), 7p (2/4), 7q (2/4), 1ip (2/4), 11q (2/4) and 13q (2/ 4). Although most of these changes have been described previously for SCC tumors in the head and neck, the incidence of increases in 8q and 20p was much higher in the present study; this may be important in relation to cell line establishment, since 8q contains c‐myc, which is involved in immortalization. No common chromosomal region with DNA copy number decreases was observed, except for 18q (2/4). When the original tumors and the cell lines were compared, their profiles were essentially similar with one exception. Further, there was no region that commonly changed in the cell lines, but not in the original tumors, suggesting that the DNA copy number changes observed in the cell lines mostly represent those of the original tumors.

Ramatroban, a thromboxane A2 receptor antagonist, prevents macrophage accumulation and neointimal formation after balloon arterial injury in cholesterol-fed rabbits

Macrophage infiltration appears to play an important role in restenosis after arterial intervention. Monocyte chemoattractant protein-1 (MCP-1) is a major chemotactic factor for macrophages. We have previously shown that ramatroban, a thromboxane A2 (TXA2) receptor antagonist, diminished the expression of MCP-1 in human vascular endothelial cells. The aim of this study was to evaluate whether, after balloon angioplasty of atherosclerotic arteries, ramatroban would reduce MCP-1 expression, macrophage accumulation, and neointimal formation. New Zealand white rabbits were fed a cholesterol-rich diet for 4 weeks, and the abdominal aorta of the rabbits were injured by a 2-French Fogarty catheter. They were randomized to receive 1 or 5 mg/kg daily of ramatroban (n = 7 or n = 8) or saline (n = 6). At 4 weeks after balloon angioplasty, the intimal hyperplasia and the macrophage-positive area in the intima by the ramatroban treatment was significantly reduced. Monocyte chemoattractant protein-1 gene expression in injured aortas of the ramatroban-treated group was significantly less evident than in the vehicle-treated group. Thromboxane A2 receptor blockade by ramatroban for 4 weeks after balloon angioplasty in the atherosclerotic rabbits prevented macrophage infiltration through MCP-1 downregulation and neointimal formation.

The non-contact monitoring of heart and respiratory rates using laser irradiation: an experimental simultaneous monitoring with and without clothes during biochemical hazards

The purpose of this study is to develop a non-contact method to evaluate the heart and respiratory rates simultaneously using a single optical sensor which can be used without the removal of clothes before a decontamination procedure in biochemical hazards. We measured the heart and respiratory rates with and without clothes to assess the vital sign monitoring before decontamination. In order to monitor the heart and respiratory rates of rabbits simultaneously, the respiratory and cardiac peaks were separated using fast Fourier transform from a 5 mW helium-neon laser (wavelength 632.8 nm) reflected off the chest walls of rabbits. A cloth (50 mm 2 50 mm, 2 mm thick) was placed on the chest of the rabbits to simulate the vital sign monitoring with clothes. The heart rate measured using this method agreed with the rate derived from an electrocardiogram ( r = 0.82, p <0.05). The respiratory rate correlated with the manually measured respirator rate ( r = 0.93, p <0.05). This method appears promising as a non-contact method for monitoring the heart and respiratory rates of patients under biochemically hazardous conditions.

Defining a common region of DNA amplification at 22q11.2–12 in head and neck squamous cell carcinomas by quantitative FISH analysis

By comparative genomic hybridization (CGH) with 17 head and neck squamous cell carcinoma (HNSCC) cell lines, we previously detected an amplified region as a distinct peak at 22q11.2–12 in 3 cell lines. Because the possible presence of an oncogene was strongly suggested, the region was mapped in more detail by defining the minimum region that was commonly amplified by using fluorescence in situ hybridization (FISH) with a series of cosmids. Eighteen cosmid clones on 22q11.2–12 were assigned to their locations as a fractional length map and hybridized to cells from three HNSCC cell lines. The three cosmid clones, cHKA‐118, cHKAD‐26, and D22S938, showed the highest levels of amplification, and the size of the amplicon was calculated to be ∼ 1.7 Mb in the OM1 and HSC6 cell lines. Several genes related to oncogenesis, including PRKM1, map to this locus. Thus, the definition of the common region with the highest level of copy number increases by FISH provides a starting point for identifying the gene that may play an important role in the development of HNSCC.

Development of a continuous temperature mapping system using a deep body thermometer

To determine continuous body temperature distribution, an inexpensive temperature mapping system was developed using a deep body thermometer adopting the finite-element method. A stripe with 16 thermocouples was wrapped around the waist of rats to measure body surface temperatures (the boundary conditions). The abdominal deep temperature of the rats was measured from the dorsum using the thermal compensation probe of a deep body thermometer. The abdominal temperature of the rats was mapped by solving a heat conduction equation using surface and deep temperatures obtained in real time. The temperature measured with a thermocouple inserted into the abdominal centre of the rats correlated well with the calculated temperature ( r = 0.93, p < 0.01 ). The system is low cost and simple to use compared with the magnetic resonance temperature mapping system. Our temperature mapping system could potentially result in improved management of patients in critical care medicine.

A novel ferromagnetic thermo-stent for plaque stabilization that self-regulates the temperature

The purpose of this study is to investigate the vascular wall with a thermally self-regulating, cylindrical stent made of a low Curie temperature ferromagnetic alloy. Physiologic saline was circulated in the silicone model vessel implanted with the stent. The stent-temperature remained nearly constant for variable saline flows, saline temperatures, and magnetic flux densities. Stent implants of this type in human blood vessels could potentially enable thermotherapy and temperature determination without catheterization.

Determining the temperature distribution of swine aorta with simulated atheromatous plaque under pulsed laser irradiation: an experimental attempt to detect the vulnerability of atherosclerosis.

We developed a method to determine the temperature distribution of swine aortas with simulated atheromatous plaques in order to measure the temperature of atherosclerotic lesions. The inflammation associated with temperature elevation is considered to be one of the aggravating mechanisms of atherosclerosis resulting in fissuring or rupture of atheromatous plaques. The temperature distribution of plaques covered by fibrous caps cannot be measured by conventional thermistors. Indocyanine green (ICG) solution was injected into the subintima of swine aorta to simulate the light absorption coefficient of human atheromatous plaques. The temperature distribution was calculated from measured temperature changes of the aortic intima under pulsed laser irradiation. The aorta was heated from the adventitial side with a halogen lamp to simulate the temperature elevation derived from inflammation. The temperature distribution of the aorta was obtained by solving the heat transfer equation using the surface layer thickness (corresponding to the fibrous cap thickness). The surface layer thickness can be calculated using the following working formula: D( µm)=1363-398 – T +35 – T 2, where s s – T s denotes intimal surface temperature change under pulsed laser irradiation. The calculated temperature of the ICG layer (corresponding to the atheromatous core) correlated well with the measured temperature (r=0.97, p<0.0001).We developed a method to determine the temperature distribution of swine aortas with simulated atheromatous plaques in order to measure the temperature of atherosclerotic lesions. The inflammation associated with temperature elevation is considered to be one of the aggravating mechanisms of atherosclerosis resulting in fissuring or rupture of atheromatous plaques. The temperature distribution of plaques covered by fibrous caps cannot be measured by conventional thermistors. Indocyanine green (ICG) solution was injected into the subintima of swine aorta to simulate the light absorption coefficient of human atheromatous plaques. The temperature distribution was calculated from measured temperature changes of the aortic intima under pulsed laser irradiation. The aorta was heated from the adventitial side with a halogen lamp to simulate the temperature elevation derived from inflammation. The temperature distribution of the aorta was obtained by solving the heat transfer equation using the surface layer thickness (corresponding to the fibrous cap thickness). The surface layer thickness can be calculated using the following working formula: D(microm)=1363-398DeltaTs+35DeltaTs(2), where AT, denotes intimal surface temperature change under pulsed laser irradiation. The calculated temperature of the ICG layer (corresponding to the atheromatous core) correlated well with the measured temperature (r=0. 97, p<0.0001).

Significance of angioscopic morphology for the estimation of macrophage infiltration and vascular physiology.

Objective: The aim of the present study was to determine whether direct visual morphology can predict vascular physiology, macrophage infiltration and plaque architecture of atherosclerotic lesions. Methods: Twenty male New Zealand white rabbits weighing 2.5–2.8 kg (mean, 2.7 ± 0.2 kg) were used. We fed rabbits a 1% cholesterol diet after creating an injury in the middle abdominal aorta using a 2 Fr Fogarty balloon catheter. After 8 weeks, the morphology of plaque lesions was evaluated by angioscopy, and was classified as protruding or lining. Vascular endothelial function (VEF) was evaluated using a Doppler guidewire, and was expressed as ratio of basal to peak velocity (cm/s) in 30 s occlusion using a balloon catheter. Macrophages obtained from abdominal sections were stained with monoclonal antibody against rabbit macrophages (RAM-11). Density of total macrophage cell infiltration was quantified as absolute area of RAM-11 staining. Results: Macrophage density and intima/media (I/M) ratio were significantly higher in the protruding group than in the lining group: macrophage density, 40 ± 10 vs. 5 ± 10%, p < 0.01; I/M ratio, 1.2 ± 1.4 vs. 0.2 ± 4, p < 0.05. Vascular flow reserve (VFR) was lower in the protruding group than in the lining group (1.8 ± 0.7 vs. 2.2 ± 0.5, p < 0.05). There was significant negative correlation between VEF and macrophage cell density (r=−0.593, p < 0.01), whereas there was no significant correlation between VEF and I/M ratio (r=−0.332, p= 0.330). Conclusion: The present findings suggest that protruding lesions revealed by angioscopy are rich in macrophages and have a higher I/M ratio and lower VFR.

Development of the hyperspectral cellular imaging system to apply to regenerative medicine

Regenerative medicine by the transplantation of differentiated cells or tissue stem cells has been clinically performed, particularly in the form of cell sheets. To ensure the safety and effectiveness of cell therapy, the efficient selection of desired cells with high quality is a critical issue, which requires the development of a new evaluation method to discriminate cells non-invasively with high throughput. There were many ways to characterize cells and their components, among which the optical spectral analysis has a powerful potential for this purpose. We developed a cellular hyperspectral imaging system, which captured both spatial and spectral information in a single pixel. Hyperspectral data are composed of continual spectral bands, whereas multispectral data are usually composed of about 5 to 10 discrete bands of large bandwidths. The hyperspectral imaging system which we developed was set up by a commonly-used inverted light microscope for cell culture experiments, and the time-lapse imaging system with automatic focus correction. Spectral line imaging device with EMCCD was employed for spectral imaging. The system finally enabled to acquire 5 dimensional (x, y, z, time, wavelength) data sets and cell-by-cell evaluation. In this study, we optimized the protocol for the creation of cellular spectral database under biological understanding. We enabled to confirm spectrum of autofluorescence of collagen, absorption of specific molecules in the cultural sample and increase of scattering signal due to cell components although detail spectral analyses have not been performed.