Predicting carotid plaque characteristics using quantitative color-coded T1-weighted MR plaque imaging: correlation with carotid endarterectomy specimens.

BACKGROUND AND PURPOSE: MR plaque imaging is used to evaluate the risk of embolic complications during carotid endarterectomy and carotid artery stent placement. However, its performance for characterizing intraplaque components has varied across studies and is generally suboptimal. Hence, we correlated MR imaging results with histologic findings to determine whether a combination of high-contrast T1-weighted imaging and quantitative image analysis could readily determine plaque characteristics. MATERIALS AND METHODS: We prospectively examined 40 consecutive patients before carotid endarterectomy by using a 1.5T scanner and axial T1-weighted spin-echo images under optimized scanning conditions. The percentage areas of intraplaque fibrous tissue, lipid/necrosis, and hemorrhage were calculated automatically by using the software with previously reported cutoff values and were compared with those of the specimens. The thickness of the fibrous cap was also measured manually. RESULTS: The percentage areas of fibrous, lipid/necrotic, and hemorrhagic components were 5.7%-98.7%, 1.3%-65.7%, and 0%-82.0%, respectively, as determined by the MR images, whereas the corresponding values were 4.8%-92.3%, 7.0%-93.8%, and 0%-70.4%, respectively, as determined by histologic examination. Significant positive correlation and agreement were observed between MR images and histologic specimens (r = 0.92, 0.79, and 0.92; intraclass correlation coefficients = 0.91, 0.67, and 0.89; respectively). Thickness of the fibrous caps on MR images (0.21-0.87 mm) and in the specimens (0.14-0.83 mm) also showed positive correlation and agreement (r = 0.61, intraclass correlation coefficient = 0.59). CONCLUSIONS: Quantitative analysis of high-contrast T1-weighted images can accurately evaluate the composition of carotid plaques in carotid endarterectomy candidates.

A compact and continuously driven supersonic plasma and neutral source.

A compact and repetitively driven plasma source has been developed by utilizing a magnetized coaxial plasma gun (MCPG) for diagnostics requiring deep penetration of a large amount of neutral flux. The system consists of a MCPG 95mm in length with a DN16 ConFlat connection port and an insulated gate bipolar transistor (IGBT) inverter power unit. The power supply consists of an array of eight IGBT units and is able to switch the discharge on and off at up to 10 kV and 600 A with a maximum repetitive frequency of 10 kHz. Multiple short duration discharge pulses maximize acceleration efficiency of the plasmoid. In the case of a 10 kHz operating frequency, helium-plasmoids in the velocity range of 20 km/s can be achieved.

Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.

Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen/non-allergen, especially using THP-1 cells. These results suggest that our method, human Cell Line Activation Test (h-CLAT), using human cell lines THP-1 and U-937, but especially THP-1 cells at 24h treatment, may be a useful in vitro skin sensitization model to predict various contact allergens.

Development of an in vitro skin sensitization test using human cell lines: the human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol.

The aim of this study is to optimize the experimental conditions for an in vitro skin sensitization test using the human cell lines THP-1 and U-937. As regards pre-culturing time, the expression of CD86 on DNCB-treated THP-1 cells tended to be higher after 48h and 72h pre-culture compared with other time points evaluated. Next, we investigated the effect of chemical treatment time, and found that induction of CD86 expression on THP-1 cells by DNCB reached a plateau after 24h. Augmentation of CD86 expression is often observed when cells are treated with a subtoxic dose of allergens. To determine the appropriate dose of test samples, the cytotoxicity of test samples to THP-1 and U-937 cells was assessed with MTT assay, and the 50% inhibitory concentration (IC50) of each test sample was calculated. Based on the cytotoxicity assay data, four concentrations in the range between toxic and non-toxic were selected (0.1x, 0.5x, 1x and 2x IC50). Several kinds of antibodies were tested for staining THP-1 and U-937 cells treated with allergens/non-allergens (e.g., DNCB, Ni/SLS), and suitable antibodies for staining CD86 and CD54 were selected. We confirmed that the working dilutions of the selected CD86 and CD54 antibodies were appropriate for use in our method. The effect of an FcR blocking procedure was also evaluated. The mean fluorescence intensity (MFI value) was decreased by the FcR blocking procedure, which indicated that non-specific staining was blocked. Therefore, this procedure should be included in the method. Based on our findings, the protocol for this assay was optimized and the experimental conditions to be used in a future validation study were identified. We propose to call this kind of in vitro skin sensitization test h-CLAT, which is short for human Cell Line Activation Test.

Effect of peripheral blood progenitor cell dose on hematopoietic recovery: identification of minimal progenitor cell requirements for rapid engraftment.

Repeated high-dose chemotherapy (HDC) with stem cell support is advocated for curative treatment of epithelial ovarian cancer patients, requiring large quantities of progenitor cell harvest. Although the switchover to peripheral blood stem cell transplantation has generally made possible the harvest of large quantities of progenitor cells, the minimum threshold is still pertinent for planning the safe conduct of HDC. However, as the minimum threshold for safe peripheral blood stem cell transplantation (PBSCT) is not yet established, this study was designed to clarify the minimum amount of progenitor cells required for prompt recovery of hematopoietic. Retrospective analysis was performed on 52 HDCs administered in 37 ovarian cancer patients. After autologous bone marrow aspiration (10 patients) or peripheral blood stem cell harvest (27 patients), colony-forming unit granulocyte macrophage (CFU-GM) were enumerated prior to cryopreservation. Numbers of CFU-GM were again calculated before reinfusion and the patients were divided into eight groups: 0.13-<0.4, 0.4-<0.7, 0.7-<1.0, 1.0-<3.5, 3.5-<5.0, 5.0-<10.0, 10.0-<20.0 and >20.0 (x 10(5)/kg). The minimum CFU-GM threshold (x 10(5)/kg) was found to be 1.0-<3.5 for platelets and 3.5-<5.0 for white blood cells. Higher infusion doses did not lead to significant benefits in hematopoietic reconstruction. These results indicate that preservation of a minimum of 7-10 x 10(5)/kg CFU-GM is recommended for the safe conduct of tandem HDCs.

The olfactory responses of the antenna and maxillary palp of the fleshfly, Neobellieria bullata (Diptera: Sarcophagidae), and their sensitivity to blockage of nitric oxide synthase.

The relative sensitivities of the olfactory receptors in the antenna and maxillary palp of the fleshfly, Neobellieria bullata, were assessed using simultaneous electroantennograms (EAGs) and electropalpograms (EPGs). In general, the antennae and maxillary palps were more sensitive to odors related to animals (blood extract and saturated carboxylic acid) than to odors that were plant-derived (citral, hexenol, hexenal). In addition, the maxillary palps were relatively less sensitive to plant-derived odorants than the antennae, perhaps related to their anatomical position. Scanning electron microscopy was also used to assess the types of sensilla found on the two organs. In addition, NADPH-diaphorase histochemistry was used in an attempt to localize the enzyme nitric oxide synthase (NOS) in the antenna and the maxillary palps. We found evidence of NADPH-diaphorase staining in both organs, with localized staining in the antennal cells and more general staining in the maxillary palps. When NOS was selectively blocked using the antagonist L-NAME, the amplitude of the EAGs and EPGs to odorants fell by 30-50%. In contrast, application of the inactive enantiomer, D-NAME, did not change the amplitude of the EAGs or the EPGs. Our results indicate that NOS is involved in the function of olfactory receptor cells in the fleshfly.

Evaluation of CD86 expression and MHC class II molecule internalization in THP-1 human monocyte cells as predictive endpoints for contact sensitizers.

The aim of this study was to explore the usefulness of a human monocyte cell line in the development of in vitro models for predictive testing of contact sensitizers. Several studies have shown that contact sensitizers induce CD86 expression and enhanced internalization of MHC class II molecules in dendritic cells (DCs). We used THP-1, a human monocyte cell line, as a replacement for DCs for evaluation of these phenotypical alterations as predictive endpoints for contact sensitizers. Known sensitizers and irritants were evaluated. After 24-h exposure to samples, the expression of CD86 on THP-1 cells was measured by flow cytometry. Sensitizers such as dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), eugenol, p-phenylenediamine (PPDA) and ammonium tetrachloroplatinate (Pt) enhanced CD86 expression on THP-1 cells, while nickel sulfate, cobalt sulfate and irritants such as methylsalicylate (MS), sodium dodecyl sulfate (SDS) and dimethyl sulfoxide (DMSO) did not augment CD86 expression. A synergistic effect was observed when DNCB and IFN-alpha were added simultaneously to a culture of THP-1 cells. Furthermore, internalization of MHC class II molecules was observed when the cells were treated with some of sensitizers for 2 h. The inducing effects of chemicals on the two phenotypical alterations were the same. These results suggest that these test systems can be used to predict contact-sensitizing ability of chemicals as an in vitro sensitization assay.

Sarcocystis sp. from cattle slaughtered in Japan.

Sarcocystis sp. was detected from cattle slaughtered in Saitama Prefecture, Japan. The cysts were 3,400-4,400 x 198-238 microm in size and had the thick cyst wall which was 7 to 10 microm thick and provided with finger-like villar protrusions. The protrusions were 8-9.5 x 2-2.5 microm in size and had microtubules in the core.

New technique for producing hybridoma by using laser radiation.

BACKGROUND AND OBJECTIVE: With conventional methods, cell fusion rate is extremely low, and fusion of two specific cells is not possible. We developed a new method for inducing cell fusion under the microscope by using a microprocessing device by laser. STUDY DESIGN/MATERIALS AND METHODS: Under a microscope, the target cells were irradiated with laser beams for trapping. Then, the trapped cells were transferred and placed in contact with the corresponding cells, which were also fixed by laser beam. The pulse laser beams are focused on the contact surface to cut small perforations for mutual communication of the cytoplasms. RESULTS: The fusion rate of mouse myeloma cells was 38%. The rate of hybridoma production of myeloma cell and lymphocyte was 2%. We confirmed the proliferation of the newly formed hybridoma in HAT medium and the production of immunoglobulin G. CONCLUSION: This new cell fusion method is characterized by production of hybridomas of target cells, lower cell toxicity, and a high rate of hybrid production.

Peripheral and central structures involved in insect gustation.

Studies in insect gustation have a long history in general physiology, particularly with work on fly labellar and tarsal sensilla and in the general field of insect-plant interactions, where work on immature Lepidoptera and chrysomelid beetles has been prominent. Much more emphasis has been placed on the physiological characteristics of the sensory cells than on the central cellular mechanisms of taste processing. This is due to the fairly direct access for physiological experimentation presented by many taste sensilla and to the obvious importance of tastants in insect feeding and oviposition behaviour. In some of the insect models used for gustatory studies, advances have been made in understanding the basic morphology of the central neuropils involved in the first stages of taste processing. There is much less known about the physiology of interneurons involved. In this review, we concentrate on four insect models (Manduca sexta, Drosophila melanogaster, Neobellieria bullata (and other large flies), and Apis mellifera) to summarize morphological knowledge of peripheral and central aspects of insect gustation. Our views of current interpretations of available data are discussed and some important areas for future research are highlighted.

Induction of tumor necrosis factor alpha and nitric oxide in rabbits inoculated with a cyst extract of Sarcocystis cruzi.

Rabbits develop a toxic reaction similar to endotoxemia following inoculation with a Sarcocystis cruzi cyst extract. To analyze the pathophysiology of the reaction, serum tumor necrosis factor alpha (TNFalpha), nitric oxide (NO) and lipid-lipoprotein profiles were investigated in rabbits given the cyst extract and lipopolysaccharide (LPS) by subcutaneous inoculation. In animals given the cyst extract, overproduction of TNFalpha was detected, together with an increase in NO. The animals developed derangement of lipid metabolism, which was considered to have resulted from TNFalpha induction, consisting of elevated triglyceride and very low density lipoprotein levels and decreased high density lipoprotein (HDL) levels. Serum interleukin-6-like activity also increased transiently in the animals. Capability of the cyst extract to induce TNFalpha, NO and the lipid profile derangement were completely lost by boiling. In animals given LPS, TNFalpha was induced and HDL decreased moderately, without inactivation of those activities of LPS by boiling. These results indicated that S. cruzi cyst extract is a potent, but thermolabile inducer of TNFalpha and NO for rabbits. It is likely that TNFalpha and NO play important roles as mediators in the reaction associated with toxicity of the cyst extract in rabbits.

First isolation of Sarcocystis hominis from cattle in Japan.

Sarcocystis hominis was first isolated from slaughtered cattle raised in Japan. Cysts were 1,220-4,460 x 80-384 microns in size and their wall was 3 to 6 microns thick and appeared radially striated in the histopathological sections because of the presence of palisade-like villar protrusions on the surface. The protrusions were 3.1-4.3 x 0.7-1.1 microns in size and had many microtubules in the core. Two cynomolgus monkeys, Macaca fascicularis, fed with the Sarcocystis cysts began to pass sporocysts, which measured a size of 14.3-15 x 9.5-10 microns, in the feces 10 days after ingestion.

Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system.

Cell numbers limit the widespread clinical use of cord blood (CB) for gene therapy and marrow replacement in adults; a simple and effective method for ex vivo expansion of CB primitive progenitor cells (PPC) is required. Recently, the combination of thrombopoietin (TPO) and Flk-2/Flt-3 ligand (FL-2) was reported to support slow proliferation of CB-PPC in stroma-free liquid culture. We established a novel culture system in which the murine stromal cell line HESS-5 dramatically supports the rapid expansion of cryopreserved CB-PPC in synergy with TPO/FL-2. Furthermore, while HESS-5 cells directly adhered to human progenitors during culture, the cultured human cells could easily be harvested without contamination by HESS-5 cells. Within 7 days of culture, a 100-fold increase in CD34bright/CD38dim cells was obtained in serum-containing culture. When HESS-5 cells were physically separated from human progenitor cells in the presence of TPO/FL-2, synergy was blocked, suggesting that HESS-5 cells support proliferation of PPC by direct cell-to-cell interaction. The hematopoietic-supportive effects of this xenogeneic coculture system were then assessed in a very short-term (5 days) serum-free culture. Expansion was further enhanced by addition of stem cell factor (SCF) or interleukin-3 (IL-3). As a result, a 50- to 100-fold increase in CD34bright/CD38dim cells was noted. Colony-forming units in culture (CFU-C) and mixed colonies (CFU-GEMM) were enhanced by 10- to 30-fold and 10- to 20-fold, respectively. Moreover, generation of long-term-culture-initiating cells (LTC-IC) from CD34bright/CD38dim cells was amplified by 25-fold. The severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. These results indicate that this xenogeneic coculture system, in combination with human cytokines, can rapidly generate PPC from cryopreserved CB.

Higher-order brain function analysis by trans-cranial dynamic near-infrared spectroscopy imaging.

Near-infrared spectroscopy is discussed from the viewpoint of human higher-order brain function analysis. Pioneering work in this field is reviewed; then we describe our concept of noninvasive trans-cranial dynamic optical topography and its instrumentation. Also, the validity of its functional images is assessed from both physical and physiological viewpoints. After confirming the validity of this method, we have applied it to a wide variety of fields such as clinical medicine, cognitive science, and linguistics in collaboration with researchers at several other institutes. Further application possibilities and the future of trans-cranial dynamic optical topography are also discussed. © 1999 Society of Photo-Optical Instrumentation Engineers.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (3) Evaluation of the haemolysis test.

The haemolysis test using sheep red blood cells (RBC) was evaluated as an alternative method to the Draize rabbit eye irritation test (Draize test) by six to nine laboratories. The participating laboratories performed the test according to the standard operating procedure (SOP). Thirty-eight cosmetic ingredients and isotonic sodium chloride solution were used as test substances in this validation study. The concentrations of the test substances that induced 50% haemolysis (HC(50) value) was obtained to serve as a toxicological index and compared with in vivo Draize scores. HC(50) values were not obtained for coloured or water-insoluble (turbid) substances. Three acids caused denaturation of haemoglobin leaked from RBC and consequently interfered with the determination of the HC(50) value. Interlaboratory reproducibility was relatively good except in the case of water-insoluble substances. The average values of coefficient of variation (CV) was 37%. The correlation coefficient and Spearman's rank correlation between the HC(50) value and maximum average Draize total score (MAS) were -0.631 and 0.641, respectively. The equivalence ratio between the haemolysis test and MAS was 70.0% when MAS 15 was set as the in vivo cut-off point. On the other hand, strong irritants (MAS50) could be correctly classified by this method. These results suggest that the haemolysis test might be applied to cosmetic ingredients as a screening method to distinguish strong irritants that directly affect the cell membrane permeability and do not disturb spectrophotometrical determination of haemoglobin. In order to evaluate the potential for eye irritation of cosmetic ingredients, a combination of haemolysis with other methods based on different mechanism should be employed to improve the predictability.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (8) Evaluation of cytotoxicity tests on SIRC cells.

Two common assays, the neutral red uptake assay (SIRC-NRU) and the crystal violet staining assay (SIRC-CVS), were evaluated as alternatives to the Draize eye irritation test (Draize test).The cytotoxicity of thirty-eight cosmetic ingredients as well as a physiological saline solution was determined on SIRC cells at five to seven laboratories. SIRC-NRU and SIRC-CVS were performed according to the common standard operating procedure (SOP). The 50% effective concentration (EC(50)) was determined for each ingredient. The EC(50) of SIRC-CVS was similar to that of SIRC-NRU, showing a strong correlation (r=0.995). The coefficient of variation (CV) of EC(50) which represents the interlaboratory reproducibility of SIRC-NRU was 32.1%, whereas that of SIRC-CVS was 32.8%. The logarithmically transformed EC(50) values showed a strong correlation with the maximal average Draize total score (MAS) (SIRC-NRU: r=-0.816 (n=30), SIRC-CVS: r=-0.805 (n=29)). Both methods could be applied to water-insoluble substances and dyes. However, strong acids, alkanolamines and alcohols had a tendency to deviate from the linear regression lines which were obtained from the in vivo and in vitro data for both methods in the present study. These results suggest that cytotoxicological testing on SIRC cells may provide an alternative method to the Draize test for cosmetic ingredients.

Sarcocystis suihominis detected for the first time from pigs in Japan.

Sarcoystis suihominis was detected for the first time in Japan from the heart and diaphragm of 5 out 600 older culled breeding pigs slaughtered in Saitama Prefecture, Japan. Fresh cysts were 1,080-2,040 x 106-170 microns in size. Bradysoites measured 15 x 4 microns on average. The cyst wall was usually observed thick, 4-6 microns, and striated, but occasionally thin and smooth according to the difference in sectioning angle and in portion of cysts. Scanning electron microscopy showed that many palisade-like villar protrusions, 6-6 x 0.3-0.5 microns in size, were closely folded onto the surface of cyst. A small number of microtubules were seen in the core of protrusion. No dogs nor domestic cats fed with 20 fresh cysts each excreted oocysts or sporocysts in the feces throughout the experimental period of 30 days.

Soil factors influencing the distribution of Oncomelania quadrasi, the intermediate host of Schistosoma japonicum, on Bohol Island, Philippines.

Soil conditions essential to the survival of Oncomelania quadrasi on Bohol Island in the Philippines were examined to clarify the factors limiting distribution of the snail and to develop a method for breeding large numbers of the snail in the laboratory. Soil samples in and around snail habitats were analysed and used for breeding experiments in the laboratory. Experiments using paddy soil derived from different parent materials revealed that the numbers of juvenile snails hatched varied widely between several soil samples. The best soils for reproduction generally had a pH of 5.6-7.9 and > 200 mg of available CaO/100 g. These soil factors, in addition to shade and moisture, determine the optimum conditions for the breeding of O. quadrasi in the field as well as in the laboratory. The determination of the optimum conditions for laboratory breeding of O. quadrasi and other intermediate snail hosts should facilitate detailed study of the hosts and the development of better methods to control or eradicate schistosomiasis and other snail-transmitted diseases.

[Development of new cell fusion technique by laser device and application to bio-medical field].

We developed a new cell fusion method which was sterile, noncontact, and selective technique under the microscope, using the micro-processing device by LASER. By this technique, we succeeded in fusing myeloma cell (SP2) and lymphocyte in mouse. We also defined the proliferation of the fused cells in HAT medium and the function of the fused cells in the Ouchterlony method, i.e. production of IgG. This method enable us to make hybridomas from very small number of cells and to fuse target cells selectively. This method is applicable to fuse cells which are difficult to be fused by conventional methods.

[Functional MRI using interleaved EPI].

One of main purposes of functional MRI is to identify functional map on human brain. We considered spatial resolution was a important factor and investigated influences of it on signal changes and activated region size. Functional images were acquired with Echo-Planar imaging (EPI) and interleaved EPI (IEPI). IEPI is superior in spatial resolution but inferior in signal to noise ratio (SNR) to EPI. As a result of Student's t-test analysis, activated region sizes of IEPI reduced to less than half those of EPI while IEPI had larger signal increase than EPI. We confirmed that the reduction was caused by low SNR and our estimation analysis could prevent it.