Graphic and movie illustrations of human prenatal development and their application to embryological education based on the human embryo specimens in the Kyoto collection.

Morphogenesis in the developing embryo takes place in three dimensions, and in addition, the dimension of time is another important factor in development. Therefore, the presentation of sequential morphological changes occurring in the embryo (4D visualization) is essential for understanding the complex morphogenetic events and the underlying mechanisms. Until recently, 3D visualization of embryonic structures was possible only by reconstruction from serial histological sections, which was tedious and time-consuming. During the past two decades, 3D imaging techniques have made significant advances thanks to the progress in imaging and computer technologies, computer graphics, and other related techniques. Such novel tools have enabled precise visualization of the 3D topology of embryonic structures and to demonstrate spatiotemporal 4D sequences of organogenesis. Here, we describe a project in which staged human embryos are imaged by the magnetic resonance (MR) microscope, and 3D images of embryos and their organs at each developmental stage were reconstructed based on the MR data, with the aid of computer graphics techniques. On the basis of the 3D models of staged human embryos, we constructed a data set of 3D images of human embryos and made movies to illustrate the sequential process of human morphogenesis. Furthermore, a computer-based self-learning program of human embryology is being developed for educational purposes, using the photographs, histological sections, MR images, and 3D models of staged human embryos.     

Biological properties and gene expression associated with metastatic potential of human osteosarcoma

Lung metastasis has a great influence on the prognosis of patients with osteosarcoma. We previously established two high-metastatic sublines, M112 and M132, from the HuO9 human osteosarcoma cell line by in vivo selection. In this study, we newly isolated a high-metastatic subline, H3, and three low-metastatic sublines, L6, L12 and L13, from HuO9 by the dilution plating method. Three high-metastatic sublines produced more than 200 metastatic nodules in the lung, while three low-metastatic sublines produced no or few nodules after injection of 2 × 10⁶ cells into the tail vein of nude mice. There were significant differences in the motility and invasiveness between high- and low-metastatic sublines, whereas the growth rates in vitro and the tumorigenicity in vivo showed no correlation with their metastatic abilities. Early adherence to culture plates was significantly lower in two of three low-metastatic sublines, which occupied smaller surface areas on the culture plates than other sublines did. Comparison of the expression of 637 cancer-related genes by cDNA microarray revealed that seven genes were differentially expressed between high- and low-metastatic sublines. Among them, five genes (AXL, TGFA, COLL7A1, WNT5A, and MKK6) were associated with adherence, motility, and/or invasiveness. These results suggest that the differences in motility/invasiveness and adhesive abilities are key determinants of lung metastasis in osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5' region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.     

Antifouling blood purification membrane composed of cellulose acetate and phospholipid polymer

The ideal surface of an artificial blood purification membrane needs hemocompatibility and durability of high performance; it should not adsorb any proteins or cells but should still have high permeability in the desired range of solute size. To improve the anti-fouling property of cellulose acetate (CA) membranes, a CA membrane blended with poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (PMB30) was designed as a blood purification membrane. The polymer solutions for preparing the membrane were prepared using a solvent mixture composed of N, N-dimethylformamide, acetone, 2-propanol or water. The CA and CA/PMB30 blend membranes with an asymmetric and porous structure were prepared by a phase inversion process.The characteristics of the CA/PMB30 blend membrane, such as structural properties, mechanical properties, and solute permeability were examined with attention to changes in the preparation conditions of the membrane. The CA/PMB30 blend membrane had good water and solute permeability and a sharp molecular weight cut-off property. Moreover, the amount of proteins adsorbed on the CA/PMB30 blend membrane surface was less than that of the original CA membrane and a conventional polysulfone membrane. Adhesion and activation of platelets on the CA/PMB30 blend membrane were reduced compared with that on a CA membrane. In addition, the CA/PMB30 blend membrane showed good permselectivity and an antifouling property during a long time ultrafiltration experiment with protein solutions.     

The frequency of type 2 CD8+ T cells is increased in peripheral blood from patients with psoriasis vulgaris

Studies have suggested that psoriasis vulgaris is mediated by type 1 T cells. In this study, we examined both chemokine receptor expression and intracellular cytokine production by circulating T cells to check the type 1/type 2 balance in psoriasis. CCR4⁺ and CXCR3⁺ T cells predominantly produced interleukin-4 and interferon-, respectively. The frequency of interferon--producing cells and that of CXCR3⁺ cells in circulating CD4⁺ T cells were similar for psoriatic patients and healthy control subjects. By contrast, the frequency of CCR4⁺CD8⁺ T cells and CCR4/CXCR3 ratio in circulating CD8⁺ T cells were significantly higher in psoriatic patients than in healthy control subjects. Analysis of intracellular cytokine production also indicated relative increase of type 2 CD8⁺ T (Tc2) cells in peripheral blood from psoriatic patients. The frequency of circulating Tc2 cells directly correlated with Psoriasis Area and Severity Index. Immunohistochemical analysis showed that not only CXCR3⁺CD8⁺ T cells but also a similar number of CCR4⁺CD8⁺ T cells infiltrated the psoriatic epidermis and dermis. Our findings suggest an increase in Tc2 cell number in blood from psoriatic patients, and the association of Tc2 cells with inflammation in psoriasis.     

Gorlin syndrome with ulcerative colitis in a Japanese girl

We present the case of a 14-year-old Japanese girl who had both Gorlin syndrome and ulcerative colitis. She had complained of blood stools for 6 months and severe scoliosis from her infancy. Physical examination revealed multiple nevi, palmar and plantar pits, jaw cysts, and calcification of the falx cerebri, leading to the diagnosis of Gorlin syndrome. Total colonoscopy revealed an edematous and spotty bleeding mucosa extending from the anus to the transverse colon. Histological examination was also compatible with ulcerative colitis. Thus, we diagnosed her as having Gorlin syndrome with ulcerative colitis. Gene analysis revealed a mutation, 1247InsT, in the human patched gene (PTCH), resulting in the truncation of PTCH protein. Since Gorlin syndrome and ulcerative colitis are rare disorders in childhood, this association is interesting, suggesting a correlation between the hedgehog signaling and intestinal disorders.     

Primary cutaneous B-cell lymphoma in Japanese patients

Primary cutaneous B-cell lymphoma (PCBCL) is a rare group of lymphoproliferative disorders. There have been few reports of Japanese patients with PCBCL, so the present study investigated the clinicopathological and immunological features and Bcl-2 gene rearrangement and protein expression in 28 Japanese patients with PCBCL. According to the Revised European-American Lymphoma (REAL) classification, there were 25 diffuse large B-cell lymphomas (DLBCL), one Burkitt type lymphoma, one lymphoblastic lymphoma and one marginal zone cell lymphoma. Of the 25 DLBCL, 17 were in males and eight in females, with an average age of 69.4 years. Follow-up data were available in 19 cases of DLBCL of which seven died and 12 were alive. The overall 5-year survival rate was 61%. Cases of DLBCL involving the legs were found to have poorer clinical outcomes; two of four cases with leg lesions died, with a mean survival of 13 months. Of 14 cases with non-leg lesions, four died, and the mean survival was 38.9 months. Only one case of Burkitt type lymphoma was CD10 positive. Bcl-2 rearrangement was not observed in 13 cases studied by polymerase chain reaction. Bcl-2 expression was observed in nine of 13 cases studied. All five cases with leg lesions exhibited Bcl-2 expression, but four of six cases with non-leg lesions also expressed the protein. These results show that DLBCL is the most frequent subtype of PCBCL in Japanese patients and that the prognosis of Japanese patients with DLBCL is worse than that of reported European cases. The study also found that PCBCL was frequently associated with Bcl-2 expression, which was not site-confined, and that there was no evidence for a follicular center origin of PCBCL.     

Infrequent genetic alterations of the PTEN gene in Japanese patients with sporadic prostate cancer

Prostate cancer is a major cause of cancer death among elderly men in America, Europe, and Japan. However, the molecular mechanism of carcinogenesis is not yet well characterized. Frequent loss of heterozygosity (LOH) on chromosome 10q was reported in prostate cancer, and a candidate tumor suppressor gene, PTEN, was isolated on chromosome band 10q23.3. To investigate the genetic alterations of PTEN, we examined 45 primary prostate cancer specimens. LOH at the PTEN locus was observed in two (11.1%) of 18 tumors. However, no mutations were observed in any of the primary prostate cancers. These data suggest that mutation of the PTEN gene does not play a major role in prostate carcinogenesis of Japanese patients. Received: February 6, 1998 / Accepted: July, 3, 1998     

Blood Flow and Leukocyte Adhesiveness Are Reduced in the Microcirculation of a Peritoneal Disseminated Colon Carcinoma

Dynamic behavior of leukocytes in the microcirculation of solid tumor tissue was visualized using a fluorescent labeling technique combined with the use of a real-time confocal laser-scanning microscope (CLSM) system. Colon tumor cells (RCN-9) were inoculated into the peritoneal cavity of male Fischer 344 rats. Tumor-free rats were similarly injected with physiological saline (intraperitoneally). Ten days after tumor inoculation, the mesentery was exteriorized and subjected to vital microscopic observation under the CLSM system. Leukocytes were labeled with rhodamine 6G (100 g kg^–1, intravenously), and their behavior within the microvessels (10–30 m in diameter) was analyzed both in the solid tumor tissues and the normal mesentery. Wall shear rate was calculated from the measured values of vessel diameter and erythrocyte flow velocity. In tumor microvasculature of tumor-bearing rats, the centerline erythrocyte velocity (0.73 ± 0.58 mm s^–1, mean±standard deviation) and wall shear rate (210 ± 151 s^–1 were significantly lower than those of the tumor-free rats (1.27 ± 0.83 mm s^–, 344 ± 236 s^–1, respectively). Despite such reduced flow conditions, flux of the rolling leukocytes as well as density of the adhered leukocytes both decreased significantly in tumor microvasculature as compared with normal controls. The methods developed in this work show promise in improving our understanding of tumor biology and pathophysiology. © 1998 Biomedical Engineering Society. PAC98: 8722Fy, 8745Hw, 8745Ft, 8764-t, 4262Be