Yasuhiro Miura

DNA ploidy analysis by laser scanning cytometry (LSC) in colorectal cancers and comparison with flow cytometry

We evaluated laser scanning cytometry (LSC) by comparing nuclear DNA ploidy determined by LSC and by flow cytometry (FCM) in 77 samples of human colorectal cancer from 48 patients. Both methods revealed an aneuploid peak in 30 (62.5%) of the cases, although two samples that were aneuploid by LSC were diploid by FCM and two others were diploid by LSC and aneuploid by FCM. The concordance rate for nuclear DNA ploidy was 91.7% in the 48 patients and 87.0% for the 77 samples. The DNA index was also highly correlated between two methods (r2 = 0.97, P < 0.001). We concluded that LSC provides DNA histograms equivalent to FCM for surgical specimens and has potential clinical application in pathology.

Cartilage Destruction Is Partly Induced by the Internal Proteolytic Enzymes and Apoptotic Phenomenon of Chondrocytes in Relapsing Polychondritis

Objective. We analyzed 9 cases by immunohistochemical studies in order to elucidate the mechanisms of cartilage destruction in relapsing polychondritis (RP), which often involves the external auricle and respiratory tract through immunological disorder. Methods. Cartilage tissues were obtained during surgical operations. Cell species in the granulation tissues, especially near the cartilage, were identified by cell-surface markers [CD3, CD4, CD8, CD20, CD45 (LCA), and CD68]. The proteolytic enzymes expressed in the cells in the perichondral granulation and in chondrocytes themselves were analyzed by immunohistochemical studies using anti-matrix metalloproteinase (MMP) -1, -3, -8, -9, and -13, and cathepsin D, K, L, and elastase antibodies. Apoptosis and nitric oxide (NO), an apoptosis-related factor, were also examined using ApopTag and antinitrotyrosine antibody, respectively. Results. Among cell species that infiltrated in perichondral granulation, LCA, CD68 (monocytes/macrophages), and CD4 cells were dominant in number; MMP-8, MMP-9, and elastase were expressed only in the perichondral granulation; whereas MMP-3 and cathepsin K and L were detected in both chondrocytes and granulations. Out of 9 cases examined, 6 revealed apoptotic cells in excess of 50% of chondrocytes. There was a strong correlation between the number of apoptotic cells and the number of MMP-3-positive (r = 0.83) and cathepsin K-positive cells (r = 0.92). Abundant NO-expressing cells were observed in the chondrocytes in degenerated cartilage, similar to apoptosis. Conclusion. Cartilage destruction in polychondritis is induced not only by perichondral inflammation, but also by intrinsic factors expressed in chondrocytes themselves, including certain kinds of proteolytic enzymes and apoptosis.

Histological analysis of esophageal muscular layers from 27 autopsy cases with mixed connective tissue disease (MCTD)

Esophageal symptoms in mixed connective tissue disease (MCTD) have been investigated radiologically. We investigated the esophageal lesions in MCTD histopathologically, and analyzed relationships between these lesions and autoantibodies extracted from the serum of MCTD patients. Esophageal tissues from 27 MCTD patients submitted to autopsy were examined. We compared histopathological features of the esophagus in different wall layers from the mucosa, submucosa, and muscular layer to the adventitia, and in the upper, middle, and lower portions of esophagus. The most striking change observed was severe atrophy and occasional loss of smooth muscle cells in the muscular layer, followed by fibrosis. These muscular changes were particularly prominent in the inner layer of the lower esophagus. Immunohistochemically, degenerated muscular tissues of the esophagus were positive for anti-IgG and anti-C3 antibodies, but not for anti-IgM antibodies. IgG fractions extracted from three MCTD patients were immunohistochemically used to examine whether some antibodies in MCTD patients showed reactivity for esophageal components. The IgG fractions isolated from MCTD patients reacted with smooth muscle from non-connective tissue disease cases, suggesting that some serum antibodies may trigger esophageal changes. These findings suggest that esophageal lesions associated with clinical dysphagia in MCTD may be related to autoantibodies.

Systemic sclerosis presented as congestive heart failure: an autopsy case

We report the autopsy results of a patient with systemic sclerosis with myositis lesions in the skeletal muscles and myocardium. A 69-year-old Japanese woman developed congestive heart failure and died due to respiratory failure with restrictive hypoventilation. The heart at autopsy showed dilated ventricular hypertrophy, and histopathology of the heart exhibited diffuse replacement fibrosis resembling the lesion of ischemic heart diseases in addition to patchy fibrosis around myocardial fibers suggesting post-myocarditis-like fibrosis.

A case of acute hepatitis E associated with multidrug hypersensitivity and cytomegalovirus reactivation

A 65‐year‐old Japanese man was hospitalized because of acute hepatitis and severe cholestasis due to hepatitis E virus (HEV) infection combined with a drug reaction to a cold preparation. He died of disseminated intravascular coagulation and severe intestinal bleeding due to systemic cytomegalovirus reactivation following the development of severe eruptions with marked eosinophilia due to drug hypersensitivity to taurine and ursodeoxycholate preparations. The close interaction between viral infection or reactivation and drug hypersensitivity was considered as a pathophysiology in this case, which emphasizes the need for further study of the immunological mechanism of the interaction.

Flow Cytometric Analysis of p53 Expression during the Cell Cycle

Mutant p53 expressed in many types of carcinoma lacks an inhibitory function on cell growth, but its role has been unclear. We performed two-parameter flow cytometry (FCM) to elucidate the relationship between the expression of p53 and the cell cycle in A431 cells. Fluorescence in situ hybridization proved that an A431 cell had two p53 genes whereas chromosome 17 was tetraploid. FCM showed that A431 cells expressed constantly high levels of p53 during the cell cycle. Under conditions of both serum deprivation and presence of hydroxyurea, p53 expression was decreased throughout the cell cycle, and the bivariate DNA/p53 distribution pattern during the cell cycle did not change. The expression of p53 was reduced to 60% for the first 4 h after the addition of cycloheximide, and showed no significant changes at least for 20 h. Treatment with Triton X-100 increased p53 immunoreactivity throughout the cell cycle. These results indicate that mutant p53 differs from proliferative markers such as PCNA, Ki-67 and DNA polymerase-alpha, and that there are no links between the expression of p53 and the cell cycle in A431 cells.

Terahertz spectroscopic imaging of liver cancer using ring cavity THz-wave parametric oscillator

Recently, research of THz spectroscopic imaging has been conducted widely for biological and medical studies using monochromatic THz-wave sources, or THz-TDS [1–5]. This paper describes a THz spectroscopic imaging system that we developed using a ring cavity THz-wave parametric oscillator [6]. The experimental setup of a ring cavity TPO spectrometer is shown schematically in Fig. 1. The THz-waves were generated from a MgO:LiNbO3 crystal pumped using a Q-switched Nd:YAG pulse laser (1.064 μm, 24 mJ/pulse, 25 ns (FWHM), 50 Hz). The maximum THz-wave output energy was about 100 pJ/pulse at 1.5 THz. The spectral resolution was ca. 1.0 cm (30 GHz) and the duration was 15 ns. The tuning range was 35–80 cm (1.01–2.4 THz).

Development of sub-THz TUNNETT diode for biomedical imaging

TUNNETT diodes are low-noise oscillators based on transit-time delay on tunneling injection of electrons. We have fabricated TUNNETTs and demonstrated sub-THz imaging using a continuous-wave (CW), fundamental-mode generator in the frequency up to 706 GHz. Some biomedical applications have been demonstrated using the feature of TUNNETT as predominant stability of the power. A scanning sub-THz imaging has been demonstrated for an inspection of tissues from internal organs containing cancer cells, and skin surfaces.

Application of THz imaging for medical diagnostics

A paraffinized pathologic sample was measured using a THz 2D-EO imaging system. The structure of the sample was successfully identified in the spectroscopic image as lines that corresponds to the interface between cancer and normal tissues. Introduction Recently, terahertz (THz) imaging is attracting much attention because of potential use in the medical and the biological applications. One of the most interesting application is the diagnosis of biological tissues [1]. Because THz electromagnetic wave is strongly absorbed by water, in vivo biological tissues containing a significant level of water are difficult to observe. However, we consider even observation of in vitro (dried or paraffinized) biological tissues still has an importance if information on the structure of the tissues and/or that of diseases, such as skin cancers, can be extracted. In this paper, we report an observation for a paraffinized pathologic sample with a THz two-dimensional electro-optic (2D-EO) imaging system. Experimental We used an amplified femtosecond laser operated at 1 kHz (λ~800 nm, δt ~150 fs) as the pump source. Because an optical chopper was synchronized to one-half of the laser repetition (500Hz), pump laser pulses were chopped alternately. THz radiation, generated by pumping a (110)-cut ZnTe crystal (1.5-mm thick), was expanded with two off-axis parabolic mirrors. Two polyethylene lenses were placed between the sample and the detector ZnTe (3-mm thick) to focus the THz image on the EO detector. The probe laser beam was combined collinearly with the THz beam by a high-resistivity silicon beam splitter. The averaged pump power on the emitter ZnTe and probe beam power on the detector ZnTe were ~200 mW and ~60 mW, respectively. The probe beam was prepared to be linearly polarized in a direction exactly orthogonal to the axis of a linear polarizer placed after the EO sampling crystal so that a zero phase bias in the EO sampling is achieved (see Fig.1). The CMOS camera (Model C8201 from Hamamatsu Photonics K. K.) is able to obtain the image for each laser shot. The camera had 128 x 128 image pixels on an area of 5.12 mm x 5.12 mm, and can be operated at 1kHz frame rate. Because the observed images include THz signal information alternately, the dynamic subtraction technique was used for noise reduction [2-4]. For measurements of images at various time-delays (or 2D THz waveforms), the optical delay line Fig.1: The block diagram of 2D EO imaging system. fs-laser amp. Delay Line Emitter (ZnTe) Detector (ZnTe) Poly Lens Polarizer

Dual-energy X-ray computed tomography system using a cadmium telluride detector and its application to gadolinium imaging

To obtain two tomograms with two different photon energy ranges simultaneously, we have performed dual-energy Xray photon counting using a cadmium telluride (CdTe) detector, two comparators, two frequency-voltage converters (FVCs), and an analog digital converter (ADC). X-ray photons are detected using the CdTe detector with an energy resolution of 1% at 122 keV, and the event pulses from a shaping amplifier are sent to two comparators simultaneously to regulate two thresholds of photon energy. The logical pulses from a comparator are sent to an FVC consisting of two integrators, a microcomputer, and a voltage-voltage amplifier. The smoothed outputs from the two FVCs are input to the ADC to carry out dual-energy imaging. To observe contrast variations with changes in threshold energy, we performed energy-dispersive computed tomography utilizing the dual-energy photon counting at a tube voltage of 100 kV and a current of 8.7 μA. Two tomograms were obtained simultaneously at two energy ranges of 34.0-50.2 keV and 50.2-100 keV. The photon-count subtraction was carried out using a computer program. The maximum count rate was 5.4 kilocounts per second with energies of 10.0-100 keV, and the exposure time for tomography was 10 min.