Generation of a considerable number of functional mast cells with a high basal level of FcepsilonRI expression from cord blood CD34+ cells by co-culturing them with bone marrow stromal cell line under serum-free conditions

The number of mast cells (MC) that can be obtained from tissue is limited, making it difficult to study the role of MC. Cultured MC derived from cord blood (CB)-CD34(+) cells proliferate well compared with those derived from adult CD34(+) cells; however, they have been reported to be phenotypically or functionally immature regardless of culture system. For example, very few cells express FcepsilonRI. To resolve this problem, we addressed the effect of human bone marrow stromal cell line on the development of cultured MC. CB-CD34(+) (1 x 10(4)) cells were cultured for 8 weeks in a serum-free medium containing rhIL-6 and rhSCF with or without a human bone marrow stromal cell line, namely, co-culture and liquid culture, and were compared in various regards. MC were basically determined by metachromatic staining of granules. The number of MC obtained (60.3 +/- 15.8 x 10(5) versus 2.0 +/- 1.0 x 10(5)), percentage of FcepsilonRI(+) cells (29.3 +/- 9.4% versus 1.9 +/- 0.8%), histamine content (9.7 +/- 2.8 pg/cell versus 5.8 +/- 2.3 pg/cell), and IgE-mediated histamine release (46 +/- 10% versus 17 +/- 7%) were higher (P < 0.01 and P < 0.05) in the co-culture than in the liquid culture. When CB-CD34(+) cells were developed in liquid culture with the co-culture supernatant (CM), a significant increase (P < 0.01) in the percentage of FcepsilonRI(+) cells and in cell number was observed but these values were lower than those of co-cultured MC. We concluded that this co-culture system was useful for obtaining a considerable number of mature MC with a high basal level of functional FcepsilonRI expression from CB-CD34(+) cells. Yet unknown humoral factors in CM may partly mediate this effect.     

Multiple gastrointestinal stromal tumors in neurofibromatosis type 1 treated with laparoscopic surgery

Gastrointestinal stromal tumors (GISTs) have been reported to occasionally occur in patients with neurofibromatosis type 1 (NF-1), and many cases have had multiple lesions predominantly involving the small intestine. We report herein a case of multiple GISTs associated with NF-1 from whom laparoscopic surgery was beneficial. In a 79-year-old female admitted with anemia and melena, the abdominal computed tomography revealed a tumor arising from the small intestine. Laparoscopic surgery was performed, and another small tumor was revealed during laparoscopic observation. Extracorporeal partial and wedge resection of the small intestine were undertaken. Both lesions were diagnosed as typical GISTs of low risk. Laparoscopic surgery would be useful for examination and a minimally invasive approach to tumors of the small intestine, especially on cases with the possibility of multiple tumors.     

Optimization for fast-scanning irradiation in particle therapy

In three-dimensional irradiation with pencil beam scanning, an extra dose is inevitably delivered to the irradiated site due to the finite reaction times of the beam delivery system, and it causes a severe distortion of the dose distribution in the target region. Since the amount of the extra dose is proportional to the beam intensity, the dose uniformity deteriorates as the beam intensity is increased in order to shorten the treatment time. In order to overcome this problem and shorten the treatment time, we have developed an optimization method in which the extra dose is integrated into the optimization process of the best weighting matrix. The effectiveness and applicability of the optimization method for spot and raster scanning irradiation were confirmed with computer simulations and also confirmed using irradiation experiments for spot scanning irradiation. The treatment time could be shortened to about one sixth of the time needed without taking the extra dose into account while obtaining the same degree of dose homogeneity in the target volume. A typical treatment time with the proposed method is about 15 s for the irradiation of a spherical target with an 80 mm diameter at 3 GyE.     

Clinical significance of small, dense LDL cholesterol in patients with hyperlipidemia and type 2 diabetes

We investigated the serum levels of small, dense LDL-cholesterol (sd LDL-C) in patients with hyperlipidemia and type 2 diabetes. An analytical assay was used to determine the levels of sd LDL-C, employing a filter method using a separating agent of polyanion and divalent cation natures. Reference intervals of sd LDL-C in normal healthy subjects (n=113) ranged from 8.0 to 42.0 mg/dl. We found a strong correlation between the levels of sd LDL-C and both the ratio of LDL-C/apolipoprotein B and the LDL migration index. The LDL migration index was analyzed using polyacrylamide gel disk electrophoresis. The levels of sd LDL-C in patients with types IIa, IIb and IV hyperlipidemia were significantly higher than those in normal subjects and in patients with normal lipidemia. The levels of sd LDL-C in patients with type IIb were higher than those with types IIa and VI. Examination of patients with polydisperse LDL showed that the levels of nodular and disrupted type sd LDL-C were higher than the levels of symmetry type sd LDL-C. Moreover, the levels of sd LDL-C in patients with type 2 diabetes were higher than those in normal subjects. A high level of sd LDL-C in patients with type 2 diabetes was found to be an indicator of possible complications of hyperlipidemia and lessly related to glycemic control. Therefore, the determination of sd LDL-C levels can be useful in the diagnosis of patients with hyperlipidemia and polydisperse LDL and in patients with type 2 diabetes with complications of hyperlipidemia and atherosclerosis.     

Fibrosis markers in heavy alcohol drinkers

Alcoholic intake has increased in society in recent years. gamma-GTP is used as a marker of liver damage by alcohol intake, but there is no reliable marker of pancreatic fibrosis. We used animal experiments and clinical data to identify a new reliable marker of early-stage pancreatic fibrosis. Pancreatic fibrosis is induced by intra-peritoneal injection of diethyldithiocarbamate. Pancreas tissue was extracted and measured. Human pure pancreatic juice was collected by endoscopic procedures. Prolyl hydroxylase in pancreas tissue is increased in the early stage of pancreatic fibrosis. Secretion of matrix metalloproteinase from pancreatic stellate cells is increased by diethyldithiocarbamate stimulation. Pancreatic stellate cells, prolyl hydroxylase and a tissue inhibitor of metalloproteinase in human pure pancreatic juice is increased in heavy alcohol drinkers and normalized in former alcohol drinkers. Active matrix metalloproteinase 2 is detected in pure pancreatic juice of chronic pancreatitis patients. Treatment with oral camostat increases pancreatic secretory trypsin inhibitor in chronic pancreatitis patients. Experimental and clinical data indicated that matrix metalloproteinase 2 and prolyl hydroxylase are candidates as markers of early-stage pancreatic fibrosis. Clinical data showed that tissue inhibitor of metalloproteinase and pancreatic secretory trypsin inhibitor in pure pancreatic juice had potential as markers of early-stage pancreatic fibrosis.     

Erythrophagocytosis by liver macrophages (Kupffer cells) promotes oxidative stress, inflammation, and fibrosis in a rabbit model of steatohepatitis: implications for the pathogenesis of human nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. Here, we report a molecular aspect of this disease elucidated using rabbits fed a cholesterol-rich high-fat diet and exhibiting insulin resistance. The liver in this model showed steatohepatitis with fibrosis and high mRNA expression for some cytokines, heme oxygenase-1, transforming growth factor-beta1, and collagen alpha1(I). Erythrocytes isolated from the model showed marked fragility and the externalization of phosphatidylserine (PS) on the outer leaflet of the membrane and were frequently engulfed by Kupffer cells/macrophages in the hepatic sinusoids. Expression of milk fat globule-epidermal growth factor (EGF)-factor 8, a PS-binding protein, was augmented in the liver. In culture, RAW 264.7 cells engulfed erythrocytes oxidized by tert-butyl hydroperoxide, a process that was inhibited by anti-milk fat globule-EGF-factor 8 antibody. In addition, PS-positive erythrocytes appeared entrapped in the model liver in ex vivo perfusion experiments. Finally, in specimens from NASH patients, the aggregation of erythrocytes in inflammatory hepatic sinusoids was notable. These results indicate that the engulfment of PS-externalized, apoptotic signal-positive, erythrocytes by hepatic macrophages may lead to the deposition of iron derived from hemoglobin in the liver and be involved in the pathogenesis of steatohepatitis.     

Control of 3D limb dynamics in unconstrained overarm throws of different speeds performed by skilled baseball players

This study investigated how the human CNS organizes complex three-dimensional (3D) ball-throwing movements that require both speed and accuracy. Skilled baseball players threw a baseball to a target at three different speeds. Kinematic analysis revealed that the fingertip speed at ball release was mainly produced by trunk leftward rotation, shoulder internal rotation, elbow extension, and wrist flexion in all speed conditions. The study participants adjusted the angular velocities of these four motions to throw the balls at three different speeds. We also analyzed the dynamics of the 3D multijoint movements using a recently developed method called "nonorthogonal torque decomposition" that can clarify how angular acceleration about a joint coordinate axis (e.g., shoulder internal rotation) is generated by the muscle, gravity, and interaction torques. We found that the study participants utilized the interaction torque to generate larger angular velocities of the shoulder internal rotation, elbow extension, and wrist flexion. To increase the interaction torque acting at these joints, the ball throwers increased muscle torque at the shoulder and trunk but not at the elbow and wrist. These results indicates that skilled ball throwers adopted a hierarchical control in which the proximal muscle torques created a dynamic foundation for the entire limb motion and beneficial interaction torques for distal joint rotations.     

Dynamic changes in conjunctival dendritic cell numbers, anatomical position and phenotype during experimental allergic conjunctivitis

Dendritic cells (DCs) are a subset of antigen-presenting cells (APCs) that are involved in the initiation and control of the immune response to antigens present at the interface with the environment. A limited number of groups have studied DCs in human and animal conjunctiva but no data is available concerning the different DC subsets present in the conjunctival tissue. The aims of this study are to characterize the phenotypes and numbers of DCs present in the murine model of allergic conjunctivitis using the technique of immunohistochemistry so as to aid the understanding of the mechanisms involved in allergic eye disease. A double immunofluorescence method was used to analyze the phenotypic distribution and density of DC subsets in the mouse conjunctival tissues of the allergic model using a panel of antibodies: CD11c, as a general marker of DCs, coupled with another DC subset marker such as Langerin for Langerhans cells (LCs), CD11b for myeloid DCs (mDCs) and mPDCA-1 for plasmacytoid DCs (pDCs). In the na?ve conjunctiva, mDCs were consistently detected in the subepithelial layer and substantia propria. In the epithelium and the subepithelial layer, very few LCs and virtually no pDCs were observed. Following allergen challenge, there was a marked influx of mDCs and pDCs, but no LCs, into the subepithelial layer and throughout the substantia propria. These results indicate that conjunctival DC subsets may play an important role in the immune-regulatory processes involved in the inflammatory component of allergic conjunctivitis.     

Bile Acid as an Effective Absorption Enhancer for Oral Delivery of Epidermal Growth Factor Receptor–Targeted Hybrid Peptide

The aim of this study was to improve the oral absorption of epidermal growth factor receptor–targeted hybrid peptide using bile acid as an absorption enhancer. The oral formulation of this peptide was formed through electrostatic interactions between the cationic peptide and anionic bile acid. Comparative studies of in vitro cell permeability and in vivo antitumor effects of peptide and peptide/bile acid complex were performed in Caco-2 cells and in a xenograft mouse model of human gastric cancer. The in vitro permeability of peptide/bile acid complex across Caco-2 cell monolayers was significantly enhanced to about 5.0-fold over those of peptide alone. Furthermore, in vivo mouse xenograft model treated with peptide/bile acid complex showed a 1.6-fold reduction in the mean tumor volume as compared with the peptide alone. A preliminary safety evaluation of blood cells counts, liver enzyme levels, and histopathology of gastrointestinal tissues and main organs showed that the peptide/bile acid complex did not induce any acute toxicity. These results suggest that bile acid is an effective absorption enhancer for improving the oral bioavailability and bioactivity of epidermal growth factor receptor–targeted hybrid peptide.