Kiyomi Takahashi

Neuronal apoptosis and inflammatory responses in the central nervous system of a rabbit treated with Shiga toxin-2.

BackgroundShiga toxins (Stxs) are the major agents responsible for hemorrhagic colitis and hemolytic-uremic syndrome (HUS) during infections caused by Stx-producing Escherichia coli (STEC) such as serotype O157:H7. Central nervous system (CNS) involvement is an important determinant of mortality in diarrhea associated-HUS. It has been suggested that vascular endothelial injuries caused by Stxs play a crucial role in the development of the disease. The current study investigates the relationship between the cytotoxic effects of Stxs and inflammatory responses in a rabbit brain treated with Stx2.MethodsIn a rabbit model treated with purified Stx2 or PBS(-), we examined the expression of the Stx receptor globotriaosylceramide (Gb3)/CD77 in the CNS and microglial activation using immunohistochemistry. The relationship between inflammatory responses and neuronal cell death was analyzed by the following methods: real time quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) to determine the expression levels of pro-inflammatory cytokines, and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method to detect apoptotic changes.ResultsGb3/CD77 expression was detected in endothelial cells but not in neurons or glial cells. In the spinal cord gray matter, significant levels of Gb3/CD77 expression were observed. Severe endothelial injury and microvascular thrombosis resulted in extensive necrotic infarction, which led to acute neuronal damage. Conversely, in the brain, Stx receptor expression was much lower. The observed neuropathology was less severe. However, neuronal apoptosis was observed at the onset of neurological symptoms, and the number of apoptotic cells significantly increased in the brain at a later stage, several days after onset. Microglial activation was observed, and tumor necrosis factor (TNF)-α and interleukin (IL)-1β mRNA in the CNS parenchyma was significantly up-regulated. There was significant overexpression of TNF-α transcripts in the brain.ConclusionThis study indicates that Stx2 may not directly damage neural cells, but rather inflammatory responses occur in the brain parenchyma in response to primary injury by Stx2 in vascular endothelial cells expressing Gb3/CD77. These findings suggest that neuroinflammation may play a critical role in neurodegenerative processes during STEC infection and that anti-inflammatory intervention may have therapeutic potential.

Energy-Discriminating Gadolinium K-Edge X-ray Computed Tomography System

An energy-discriminating K-edge X-ray computed tomography (CT) system is useful for increasing the contrast resolution of a target region utilizing contrast media and for reducing the absorbed dose for patients. The CT system is of the first-generation type of detector using cadmium telluride (CdTe). CT is performed by repeated translations and rotations of an object. Penetrating X-ray photons from the object are detected by a CdTe detector, and event signals of X-ray photons are produced using charge-sensitive and shaping amplifiers. Both photon energy and energy width are selected out using a multichannel analyzer, and the number of photons is counted by a countercard. To perform energy discrimination, a low-dose-rate X-ray generator for photon counting was developed. Its maximum tube voltage and minimum tube current were 110 kV and 1 µA, respectively. In energy-discriminating CT, the tube voltage and tube current were 100 kV and 20 µA, respectively, and the X-ray intensity was 2.98 µGy/s at a distance of 1.0 m from the source and a tube voltage of 100 kV. The demonstration of enhanced gadolinium K-edge X-ray CT was carried out by selecting photons with energies just beyond the gadolinium K-edge energy of 50.3 keV.

Current HIV type 1 molecular epidemiology profile and identification of unique recombinant forms in Jakarta, Indonesia.

HIV infection is a major problem in Indonesia. The number of people living with HIV has been increasing from year to year, especially among injecting drug users (IDUs). Since there were only limited data about molecular epidemiology profiles of HIV/AIDS in Indonesia, a cross-sectional study involving 208 HIV-1-seropositive individuals was conducted in 2007 in Jakarta. The majority of participants were 16-30 years of age (64.9%) and 74.5% were male. The most frequent risk factor was injecting drug use (IDU) (45.7%) followed by heterosexual transmission (34.1%). Phylogenetic analysis of gag (p17 and p6) and env C2V3 regions showed 200 (96.2%) of 208 DNA samples were CRF01_AE and only 3 (1.4%) were subtype B. Five samples (2.4%) indicated discordant subtypes between the three aforementioned regions: three of them showed unique CRF01_AE/B recombination patterns in 2.3-kbp nucleotide sequences (from p17 to part of RT), including one sample showing similarity to CRF33_01B, reported previously in Malaysia. This study shows the current predominant subtype is CRF01_AE in every risk group, with a decreasing number of pure subtype B, and the first identification of CRF01_AE/B recombinant forms among HIV-1-seropositive Indonesians.

A Silkworm Larvae Plasma Test for Detecting Peptidoglycan in Cerebrospinal Fluid Is Useful for the Diagnosis of Bacterial Meningitis

The silkworm larvae plasma (SLP) test has been established based on a cascade reaction triggered by either peptidoglycan or (1, 3)‐β‐D‐glucan to produce melanin. We applied this test to the diagnosis of bacterial meningitis. Cerebrospinal fluid (CSF) obtained from patients with bacterial meningitis due to gram‐positive bacteria, gram‐negative bacteria, or fungi, showed positive reactions to the test. In contrast, CSF from patients with viral meningitis or noninfectious illnesses gave negative reactions. Therefore, this test seems to be useful for diagnosis of bacterial and fungal meningitis. When this test was used together with two types of limulus tests, an endotoxin‐specific test, and a conventional test, meningitis was further characterized as gram‐positive, gram‐negative or fungal meningitis. The SLP test requires a computerized instrument for quantitative colorimetric measurement. A qualitative alternative of this test also can be accomplished by visually observing the darkening color. Thus, this method can be applied for simple and rapid diagnosis of meningitis.

Co-administration of cholera toxin and apple polyphenol extract as a novel and safe mucosal adjuvant strategy

Although native cholera toxin (CT) is an extremely effective adjuvant, its toxicity prevents its use in humans. We report here that apple polyphenol extract (APE), obtained from unripe apples, reduces CT-induced morphological changes and cAMP accumulation. Based upon this finding, we have attempted to design a novel, effective and safe mucosal vaccine by using CT with several dosages of APE as nasal adjuvants. Mice nasally immunized with OVA plus CT and an optimal dosage of APE showed significantly reduced levels of inflammatory responses as well as total and OVA-specific IgE antibodies when compared with mice given without APE. However, levels of both mucosal and systemic OVA-specific antibody responses were maintained. Further, APE significantly down-regulated accumulation of CT in the olfactory nerves and epithelium. In summary, an optimal dosage of APE would take full advantage of mucosal adjuvanticity of native CT without any toxicity for application in humans.

Weakly ionized plasma flash x-ray generator and its distinctive characteristics

In the plasma flash x-ray generator, a high-voltage main condenser of approximately 200 nF is charged up to 50 kV by a power supply, and electric charges in the condenser are discharged to an x-ray tube after triggering the cathode electrode. The flash x-rays are then produced. The x-ray tube is a demountable triode that is connected to a turbo molecular pump with a pressure of approximately 1 mPa. As electron flows from the cathode electrode are roughly converged to a rod copper target of 3.0 mm in diameter by the electric field in the x-ray tube, weakly ionized linear plasma, which consists of copper ions and electrons, forms by target evaporation. At a charging voltage of 50 kV, the maximum tube voltage was almost equal to the charging voltage of the main condenser, and the peak current was about 15 kA. When the charging voltage was increased, the linear plasma formed, and the K-series characteristic x-ray intensities increased. The K-series lines were quite sharp and intense, and hardly any bremsstrahlung rays were detected. The x-ray pulse widths were approximately 700 ns, and the time-integrated x-ray intensity had a value of approximately 30 μC/kg at 1.0 m from the x-ray source with a charging voltage of 50 kV.

Tunable narrow-photon-energy x-ray source using a silicon single crystal

A preliminary experiment for producing narrow-photon-energy cone-beam x-rays using a silicon single crystal is described. In order to produce low-photon-energy x-rays, a 100-µm-focus x-ray generator in conjunction with a (111) plane silicon crystal is employed. The x-ray beams from the source are confined by an x-y diaphragm, and monochromatic cone beams are formed by the crystal and three lead plates. The x-ray generator consists of a main controller and a unit with a high-voltage circuit and a 100-µm-focus x-ray tube. In this experiment, the maximum tube voltage and current were 35 kV and 0.50 mA, respectively, and the x-ray intensity of the microfocus generator was 343 μGy/s at 1.0 m from the source with a tube voltage of 30 kV and a current of 0.50 mA. The effective photon energy is determined by Braggs angle, and the photon-energy width is regulated by the angle delta. Using this generator in conjunction with a computed radiography system, quasi-monochromatic radiography was performed using a cone beam with an effective energy of approximately 15.5 keV.

Magnification Embossed Radiography Utilizing Image-Shifting Subtraction Program

We developed an image-shifting subtraction program and carried out magnification embossed radiography (MER) utilizing single- and dual-energy subtractions. In particular, dual-energy subtraction was carried out to decrease the absorption contrast of unnecessary regions. The contrast resolution of the target region was increased by the use of subtraction software and a linear-contrast system in a flat-panel detector (FPD). The X-ray generator had a 100-µm-focus tube, and the subtractions were performed at tube voltages of 40 and 70 kV, a tube current of 0.50 mA, and an X-ray exposure time of 5.0 s. MER images with threefold magnification were obtained using the FPD with a pixel size of 48×48 µm2, and the shifting dimensions of the imaged object in the horizontal and vertical directions ranged from 48 to 192 µm. At a shifting distance ranging from 48 to 144 µm, the spatial resolutions in the horizontal and vertical directions measured with a lead test chart were both 50 µm. In the MER of nonliving animals, we obtained high-contrast embossed images of fine bones, gadolinium oxide particles in blood vessels, and iodine-based microspheres in coronary arteries of approximately 100 µm diameter.

High-speed photon-counting x-ray computed tomography system utilizing a multipixel photon counter

High-speed photon counting is useful for discriminating photon energy and for decreasing absorbed dose for patients in medical radiography, and the counting is usable for constructing an x-ray computed tomography (CT) system. A photon-counting x-ray CT system is of the first generation type and consists of an x-ray generator, a turn table, a translation stage, a two-stage controller, a multipixel photon counter (MPPC) module, a 1.0-mm-thick LSO crystal (scintillator), a counter card (CC), and a personal computer (PC). Tomography is accomplished by repeating the linear scanning and the rotation of an object, and projection curves of the object are obtained by the linear scanning using the detector consisting of a MPPC module and the LSO. The pulses of the event signal from the module are counted by the CC in conjunction with the PC. The lower level of the photon energy is roughly determined by a comparator circuit in the module, and the unit of the level is the photon equivalent (pe). Thus, the average photon energy of the x-ray spectra increases with increasing the lower-level voltage of the comparator. The maximum count rate was approximately 20 Mcps, and energy-discriminated CT was roughly carried out.

Demonstration of enhanced K-edge angiography utilizing a samarium x-ray generator

The samarium-target x-ray tube is useful in order to perform cone-beam K-edge angiography because K-series characteristic x-rays from the samarium target are absorbed effectively by iodine-based contrast media. This generator consists of the following components: a constant high-voltage power supply, a filament power supply, a turbomolecular pump, and an x-ray tube. The x-ray tube is a demountable diode which is connected to the turbomolecular pump and consists of the following major devices: a samarium target, a tungsten hairpin cathode (filament), a focusing (Wehnelt) electrode, a polyethylene terephthalate x-ray window 0.25 mm in thickness, and a stainless-steel tube body. In the x-ray tube, the positive high voltage is applied to the anode (target) electrode, and the cathode is connected to the tube body (ground potential). In this experiment, the tube voltage applied was from 50 to 70 kV, and the tube current was regulated to within 0.10 mA by the filament temperature. The exposure time is controlled in order to obtain optimum x-ray intensity. The electron beams from the cathode are converged to the target by the focusing electrode, and bremsstrahlung x-rays were absorbed using a 50-µm-thick tungsten filter. The x-ray intensity was 1.04 μGy/s at 1.0 m from the x-ray source with a tube voltage of 60 kV and a tube current of 0.10 mA, and angiography was performed using a computed radiography system and iodine-based microspheres 15 µm in diameter. In angiography of non-living animals, we observed fine blood vessels of approximately 100 µm with high contrasts.