Synchrotron-based XRD from rat bone of different age groups.

Synchrotron-based XRD spectra from rat bone of different age groups (w, 56 w and 78w), lumber vertebra at early stages of bone formation, Calcium hydroxyapatite (HAp) [Ca10(PO4)6(OH)2] bone fill with varying composition (60% and 70%) and bone cream (35-48%), has been acquired with 15keV synchrotron X-rays. Experiments were performed at Desy, Hamburg, Germany, utilizing the Resonant and Diffraction beamline (P9), with 15keV X-rays (λ=0.82666 A0). Diffraction data were quantitatively analyzed using the Rietveld refinement approach, which allowed us to characterize the structure of these samples in their early stages. Hydroxyapatite, received considerable attention in medical and materials sciences, since these materials are the hard tissues, such as bone and teeth. Higher bioactivity of these samples gained reasonable interest for biological application and for bone tissue repair in oral surgery and orthopedics. The results obtained from these samples, such as phase data, crystalline size of the phases, as well as the degree of crystallinity, confirm the apatite family crystallizing in a hexagonal system, space group P63/m with the lattice parameters of a=9.4328Å and c=6.8842Å (JCPDS card #09-0432). Synchrotron-based XRD patterns are relatively sharp and well resolved and can be attributed to the hexagonal crystal form of hydroxyapatite. All the samples were examined with scanning electron microscope at an accelerating voltage of 15kV. The presence of large globules of different sizes is observed, in small age groups of the rat bone (8w) and lumber vertebra (LV), as distinguished from, large age groups (56 and 78w) in all samples with different magnification, reflects an amorphous phase without significant traces of crystalline phases. Scanning electron microscopy (SEM) was used to characterize the morphology and crystalline properties of Hap, for all the samples, from 2 to 100µm resolution.

Brownian dynamics simulation of a model simple electrolyte in solvents of low dielectric constant.

Brownian dynamics simulations are performed to investigate the ionic transport of model simple electrolytes, in which ions are interacting with each other through the repulsive core and Coulombic interactions. The equivalent conductivity and self-diffusion coefficient show minima as the function of the number density of ions when the dielectric constant of the solvent is low. Although the minimum of the former is in harmony with various experiments, no experiment has ever been reported on that of the latter. The analysis of time-dependent transport coefficients reveals that the presence of the minima is ascribed to the slow dynamics, rather than to static association models. The inclusion of a model function that resembles the short-range part of the potential of mean force induced by solvent affects the transport coefficients qualitatively, which suggests the importance of solvent-induced potential of mean force in the conduction mechanism of electrolytes in solvents of low dielectric constant.

Investigation of the distribution of elements in snail shell with the use of synchrotron-based, micro-beam X-ray fluorescence spectrometry.

In this study, synchrotron-based micro-beam was utilized for elemental mapping of a small animal shell. A thin X-ray spot of the order of approximately 10microm was focused on the sample. With this spatial resolution and high flux throughput, the X-ray fluorescent intensities for Ca, Mn, Fe, Ni, Zn, Cr and Cu were measured using a liquid-nitrogen-cooled 13-element energy-dispersive HpGe detector. The sample is scanned in a 'step-and-repeat' mode for fast elemental mapping and generated elemental maps at 8, 10 and 12keV. All images are of 10microm resolution and the measurement time was 1s per point. The accumulation of trace elements was investigated from the soft-tissue in small areas. Analysis of the small areas will be better suited to establish the physiology of metals in specific structures like small animal shell and the distribution of other trace elements.

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells treated with an anthrax toxin fusion protein.

As a novel and safe vaccine strategy, the anthrax toxin-mediated antigen delivery system composed of lethal factor (LF) fusion protein and protective antigen (PA) has been studied to prime hepatitis C virus (HCV) core-specific cytotoxic T lymphocytes (CTLs) in vivo. The core epitope fused to LF (LF-core) together with PA induces a negligible core-specific CTL response in mice, whereas core-specific CTL are effectively primed in mice by injecting dendritic cells (DCs) treated in vitro with LF-core and PA. These findings imply that LF fusion protein plus PA in combination with dendritic cells may be useful for a novel T cell vaccine against HCV infection.

Preliminary experiment of fluorescent X-ray computed tomography to detect dual agents for biological study.

The simultaneous observation of various information, such as blood flow, tissue metabolism and distribution of receptors, is quite important in order to understand the functional state of biomedical objects. The simultaneous detectability of contrast agents by fluorescent X-ray computed tomography (FXCT) with synchrotron radiation is examined in this study. The system consisted of a silicon (111) double-crystal monochromator, an X-ray slit system, a scanning table, a PIN diode, a highly purified germanium detector and an X-ray charge-coupled device (CCD) camera. The monochromatic X-ray beam energy was adjusted to 37.0 keV and collimated into a pencil beam of 1 x 1 mm. The fluorescent spectra of the K alpha lines for iodine and xenon were detected simultaneously. FXCT could image the distribution of both iodine and xenon agents in a phantom clearly and the contrast ratio was significantly better than that of transmission X-ray computed tomography images.

Introduction of tapasin gene restores surface expression of HLA class I molecules, but not antigen presentation of an HIV envelope peptide in a hepatoma cell line.

A hepatoma cell line, Hep G2, reveals the diminished HLA class I surface expression and the reduced expression of LMP2, LMP7, and tapasin transcripts, suggesting that the reduced expression of these transcripts may be associated with the low expression of HLA class I molecules. Introduction of tapasin gene dramatically up-regulates the surface expression of HLA class I molecules on Hep G2 cells, and unexpectedly, enhances the expression of LMP2 and LMP7 transcripts as well. Unlike Hep G2, these tapasin-transfected Hep G2 cells are recognized by allo-specific CTL. However, the transfectant is unable to endogenously present an HIV envelope peptide to an HIV-specific CTL clone, suggesting that a proteasome-independent antigen processing pathway exists and still remains defective in the transfectant. These data may provide significant evidence that the nonproteasomal antigen processing pathway as well as the proteasomal pathway may be impaired in tumor cells to escape immune surveillance performed by CTL.

Loss of ErbB2 expression in pulmonary metastatic lesions in osteosarcoma.

OBJECTIVES: The c-erbB2 protooncogene is located on human chromosome 17 and encodes a 185-kilodalton transmembrane glycoprotein (ErbB2). Elevated ErbB2 expression or gene amplification has been shown to be associated with a poor prognosis in many cancers. Recently, it has been demonstrated that overexpression of the ErbB2 protein in osteosarcoma is associated with the presence of pulmonary metastasis and decreased survival. To further investigate the role of ErbB2 overexpression in pulmonary metastasis of osteosarcoma, its expression in the primary and metastatic lesions of the same osteosarcoma patients was compared. METHODS: We compared the expression levels of ErbB2 receptor protein between the biopsy samples and pulmonary metastatic lesions in each of 19 patients with osteosarcoma who had not presented with metastasis at diagnosis. All archival materials from patients were retrieved and stained with monoclonal antibody CB11 to detect ErbB2 protein. RESULTS: The rate of overexpression was significantly lower in the pulmonary metastatic tumors than in the biopsy samples (11 versus 42%; p = 0.03). Among 8 patients who had shown increased levels of ErbB2 in the biopsy samples, 7 exhibited complete absence of ErbB2 in the pulmonary metastatic lesions. Overall loss of ErbB2 expression was noted in 14 of 19 patients as the initial tumor became metastatic. CONCLUSIONS: It is suggested that overexpression of ErbB2 decreases within individual osteosarcomas as they become metastatic. Overexpression of ErbB2 may not play an important role in the development of pulmonary metastases of osteosarcoma. Further data are needed before ErbB2 can be used in making clinical decisions for osteosarcoma patients.

New chimpanzee MHC class I alleles cloned by polymerase chain reaction.

We describe polymerase chain reaction (PCR) cloning of full-length Patr-A, -B and -Cw locus alleles from a chimpanzee using one sense/antisense primer pair. Of the six alleles cloned here, two have not previously been described. Comparison of nucleotide sequences between the two novel Patr alleles and various HLA alleles suggests that the new Patr-A allele belongs to the A1/A3/A11 family, whereas the new Patr-Cw allele is not closely related to any known HLA-Cw allele.

Development of dengue IgM-capture enzyme-linked immunosorbent assay with higher sensitivity using monoclonal detection antibody.

An IgM-capture enzyme-linked immunosorbent assay (IgM-ELISA) is used widely for serodiagnosis of dengue. A dengue IgM-ELISA with higher sensitivity has been developed. In the new ELISA, anti-dengue IgM antibody, which had been captured on the solid phase, was reacted with tetravalent dengue viral antigens, and detected by a flavivirus group specific monoclonal antibody, D1-4G2-4-15 (4G2). Reaction of 4G2 to viral antigens was similar to that of dengue patients' IgG. Non-specific reaction of 4G2 to the control antigen, which was prepared from uninfected cell culture fluid of mosquito C6/36 cells, was much lower than that of patients' IgG. Thus, specificity of the ELISA with 4G2 was much higher than that with patients' IgG, and lower levels of specific IgM was detected in the serum samples. These results suggest that the modified dengue IgM-ELISA with monoclonal antibody 4G2 has many advantages over the original "in-house" ELISA.

High expression of HLA-A2 on an oral squamous cell carcinoma with down-regulated transporter for antigen presentation.

Using the reverse transcription PCR, we evaluated expression levels of various antigen presentation-related genes, including LMP2, LMP7, MECL-1, PA28alpha, PA28beta, TAP1, TAP2, and tapasin, in two oral squamous cell carcinoma cell lines, HSC5 and HSC7. Expression levels of LMP2, MECL-1, TAP1, and TAP2 transcripts are reduced in both cell lines in comparison with a normal epithelial cell line. Further, HSC5 and HSC7 show diminished expression of LMP7/tapasin, and PA28alpha/beta, respectively. Surface expression of HLA-B alleles is down-regulated in both lines presumably due to low expression of TAP1/2. However, HLA-A2 surface expression is not significantly down-regulated in HSC5 cells, suggesting an involvement of signal-sequence derived peptides in the TAP-independent pathway. The current study would contribute to our understanding of significance of abnormalities in the antigen presentation machinery of oral squamous cell carcinoma, and provide meaningful information in the design of CTL-based tumor vaccines by intra-cellular delivery system.

[Examination of eye position after strabismus surgery in Kawasaki Medical School Hospital].

PURPOSE: To examine the factors which affect changes in the position of the eyes following the strabismus surgery. METHODS: The postoperative position of 240 eyes with concomitant strabismus was examined. The factors which seemed to have an effect on the position of the eyes were enumerated, and a logistic regression analysis of the results was done. RESULTS: In comparison with esotropia, in exotropia the eyes showed a tendency to return gradually to a normal position. There was no remarkable improvement in stereoscopic vision following strabismus surgery. Based on the standards of the Japanese Association of Strabismus and Amblyopia [the cure standard for strabismus], the results of strabismus surgery were as follows: Grade 1(cosmetically satisfactory) was mainly observed for esotropia, and Grade 3(good) for exotropia. The factors which had a significant effect on the postoperative position of the eyes were as follows: esotropia amblyopia, muscle movement, and operative methods; exotropia: retinal correspondence, operative methods, and the necessity of inferior oblique muscle surgery. CONCLUSIONS: There was little correction of the position of the eyes. In individual cases, the factors which affect position of eyes were examined, and it was considered necessary to increase the amount of correction. Age is a factor which has an effect, but the effect on esotropia and exotropia is small.

Serotype-cross-reactive immunoglobulin M responses in dengue virus infections determined by enzyme-linked immunosorbent assay.

We developed immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assays (ELISAs) with four monovalent dengue virus antigens. We attempted to determine whether IgM responses in dengue virus infections are serotype specific or serotype cross-reactive. Serum samples from 14 confirmed dengue cases were examined. In these 14 cases, which consisted of 12 Japanese and 2 non-Japanese patients, infecting dengue virus serotypes were defined by reverse transcription-PCR. Thirteen of the 14 cases were IgM positive in ELISA. IgM responses were serotype cross-reactive in these 13 cases but were highest against infecting dengue virus serotype in 9 of the 13 cases. These results indicate that IgM responses are generally dengue serotype cross-reactive but that IgM levels are highest against the infecting serotype in most dengue cases.

New types of X-ray computed tomography (CT) with synchrotron radiation: fluorescent X-ray CT and phase-contrast X-ray CT using interferometer.

New types of X-ray computed tomography (CT), fluorescent X-ray CT and phase-contrast X-ray CT are being developed for biomedical research. While fluorescent scanning X-ray CT (FXCT) can detect specific contrast elements, or endogenous iodine, at very low content (less than 400 pg iodine of tissue in a volume of 8 x 10(-6) ml), the phase-contrast X-ray CT (PCCT) is a highly sensitive imaging technique to differentiate between different biological tissue types (based on their specific gravity variation) without the use of a contrast agent. Therefore, we can expect functional diagnosis with FXCT, and high contrast, high resolution biological imaging with PCCT. In this paper, a human thyroid gland imaged by FXCT, and a metastatic human cancerous lesion depicted using PCCT are presented. The latter method used a newly manufactured, large, monolithic, X-ray interferometer, which is described in this paper in detail.

Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B.

The tight junction serves as a paracellular gate to seal the paracellular space of apposing cells and as a molecular fence to prevent diffusion of membrane proteins and lipids in epithelial cells. Although involvement of the actin cytoskeleton has been considered to be important in these two functions, it remains to be elucidated whether both functions are regulated in a coupled manner or differentially by actin. Treatment of highly polarized MDCK cells with mycalolide B (MB), a recently developed actin-depolymerizing reagent, induced a decrease of transepithelial resistance in a dose- and time-dependent manner with reversibility when the reagent was washed out. Changes in cytoskeletal actin, such as a reduction of cortical actin, irregularity of stress fibers, and punctated actin aggregates, were observed after MB treatment. However, the fence function, as studied by diffusion of apically labeled sphingomyelin/BSA complex, remained intact in the MB-treated MDCK cells. Localization of junctional molecules and apical marker proteins such as E-cadherin, ZO-1, and 114-kDa protein was shown to be unaffected. Furthermore, freeze-fracture study showed apparent tight junction strands. Collectively, MB treatment abolished the paracellular gate but not the fence function of MDCK cells, suggesting that cytoskeletal actin may play differential roles in the gate and fence functions of the tight junction.

Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis.

To investigate the clinical significance of vascular endothelial growth factor (VEGF) in osteosarcoma, we immunohistochemically stained biopsy specimens of 27 primary osteosarcomas using an antibody against VEGF and evaluated the correlation between the expression of VEGF and local density of CD34-positive microvessels, clinicopathological variables, and survival of patients. VEGF staining was positive in 17 tumors (63.0%) in which the density of CD34-positive microvessels was significantly higher than that in VEGF-negative 10 tumors (P < 0.05). In terms of clinicopathological variables, there was no correlation between the expression of VEGF and histological subtype, stage, or response to neoadjuvant chemotherapy, or, strikingly, to the development of pulmonary metastasis (89% of VEGF-positive tumors versus 10% of VEGF-negative tumors; P < 0.0003). Moreover, patients with a VEGF-positive tumor were poorer in both disease-free survival (P < 0.001) and overall survival (P < 0.03) compared to those with a VEGF-negative tumor. These findings strongly suggest that VEGF expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis in patients who underwent aggressive therapy and also provide the basis for a therapeutic strategy targeting angiogeneic property of osteosarcoma.

Molecular analyses of five new chimpanzee MHC class I alleles: implications for differences between evolutional mechanisms of HLA-A, -B, and -C loci.

In order to study the origin of the polymorphism of MHC class I molecules, we have cloned and sequenced five new Patr-A, -B, and -C loci alleles from two chimpanzees. Previous studies of sequence comparison between Patr and HLA class I alleles revealed that many of the sequence motifs were shared and the origin of class I molecules predated the divergence of chimpanzees and humans. These findings are confirmed by our current study. Additionally, our data suggest significant differences between mechanisms of evolution of the A, B, and C loci: (1) The B locus is characterized by frequent nucleotide substitutions, whereas the A and C loci are relatively more conserved; (2) However, unlike the A locus, the alpha2 domains of the C locus sequenced appear to produce MHC polymorphism between these species. These differences might imply the distinctive contributions of each locus during the evolutionary history.

T cell recognition of hypervariable region-1 from hepatitis C virus envelope protein with multiple class II MHC molecules in mice and humans: preferential help for induction of antibodies to the hypervariable region.

Hypervariable region-1 (HVR1) from the hepatitis C virus (HCV) envelope protein is thought to be a target for neutralizing Abs. To explore HVR1 recognition by helper T cells, and their role in Ab responses, we attempted to generate helper T cells specific for HVR1 in mice of three MHC types, and with PBMC from HCV-infected HLA-diverse humans. In both species, HVR1 was presented by >1 class II MHC molecule to CD4+ helper T cells and showed surprising interisolate cross-reactivity. The epitope for two DR4+ patients was mapped to a more conserved C-terminal sequence containing a DR4 binding motif, possibly accounting for cross-reactivity. Strikingly, Abs to patients' own HVR1 sequences were found only in patients with T cell responses to HVR1, even though all had Abs to envelope protein, suggesting that induction of Abs to HVR1 depends on helper T cells specific for a sequence proximal to the Ab epitope. Thus, helper T cells specific for HVR1 may be functionally important in inducing neutralizing Abs to HCV. These results may be the first example of "T-B reciprocity," in which proximity of a helper T cell epitope determines Ab epitope specificity, in a human disease setting.

Enhanced in vitro potency and in vivo immunogenicity of a CTL epitope from hepatitis C virus core protein following amino acid replacement at secondary HLA-A2.1 binding positions.

Since the natural immune response to hepatitis C virus (HCV) is often unable to clear the infection, to enhance immunogenicity we studied substituted peptides from an HCV cytotoxic T lymphocyte (CTL) epitope (C7A2) from a conserved region of the HCV core protein (DLMGYIPLV) recognized by CTL lines from HLA-A2.1(+) HCV-infected patients and HLA-A2.1 transgenic mice. HLA-A2.1 binding, human and murine CTL recognition, and in vivo immunogenicity (using mice transgenic for human HLA-A2 in lieu of immunizing humans) were analyzed to define peptides with enhanced immunogenicity. Peptides substituted at position 1 showed enhanced HLA-A2 binding affinity, but paradoxically poorer immunogenicity. A peptide with Ala substituted at position 8 (8A) showed higher HLA-A2 binding affinity and CTL recognition and was a more potent in vivo immunogen in HLA-A2-transgenic mice, inducing higher CTL responses with higher avidity against native C7A2 than induced by C7A2 itself. These results suggest that peptide 8A is a more potent in vitro antigen and in vivo immunogen than C7A2 and may be useful as a vaccine component. They provide proof of principle that the strategy of epitope enhancement can enhance immunogenicity of a CTL epitope recognized by human CTL.

Enhancement of laser CT image contrast by correction of artifacts due to surface effects.

Mismatched boundary conditions such as air-sample interface introduce artifacts that obscure internal information in the reconstructed laser computed tomographic (CT) images. Here, we demonstrate enhancement of target structure in the laser CT images by correcting the projection data using the experimentally determined angle dependence of sample surface attenuation. The images reconstructed with the corrected projection data are shown to have improved image contrast. Our proposed correction to laser CT reconstruction is effective for visualizing internal structure with small variations in the attenuation coefficients that would otherwise be masked by the dominant surface attenuation.

Medical applications with synchrotron radiation in Japan.

In Japan, various medical applications of synchrotron X-ray imaging, such as angiography, monochromatic X-ray computed tomography (CT), radiography and radiation therapy, are being developed. In particular, coronary arteriography (CAG) is quite an important clinical application of synchrotron radiation. Using a two-dimensional imaging method, the first human intravenous CAG was carried out at KEK in May 1996; however, further improvements of image quality are required in clinical practice. On the other hand, two-dimensional aortographic CAG revealed canine coronary arteries as clearly as those on selective CAG, and coronary arteries less than 0.2 mm in diameter. Among applications of synchrotron radiation to X-ray CT, phase-contrast X-ray CT and fluorescent X-ray CT are expected to be very interesting future applications of synchrotron radiation. For actual clinical applications of synchrotron radiation, a medical beamline and a laboratory are now being constructed at SPring-8 in Harima.