Aperture maneuver with compelled breath (AMC) for moving tumors: a feasibility study with a moving phantom

Respiration causes target motion, which is known to be one of the technical bottlenecks in radiotherapy, especially for stereotactic radio-surgery and intensity modulated radiotherapy (IMRT). To overcome this problem, aperture maneuver with compelled breath (AMC) has been developed. In order to simulate compelled respiratory motion, a moving phantom using a ventilator was designed. As the air flow was forced to the bellows, which simulates the lungs, by a ventilator, a film connected to the ventilator moved like the respiratory target motion. A software was developed to transfer multileaf collimator motion from breathless to actual periodic breathing conditions. Static fields as well as step-and-shoot IMRT fields were modified in accordance with moving shapes to follow the target position, using the software with the controlled breathing information. Film dosimetry for a small field and for IMRT fields with a moving phantom was performed. To evaluate clinical implementation, five healthy volunteers were tested to breathe through a ventilator, and all of them could adapt the compelled breath without any difficulties. Additive margins for a moving target with AMC were not larger than 3 mm for respiratory organ motions up to 18 mm, while those with the static beam were 9 mm. For IMRT fields, large discrepancies were present between a static target and a moving target with the static beam, while they coincided well with AMC. Clinical acceptable differences between the dose distributions from a static target with the static beam and from a moving target with AMC revealed that this technique could be applied clinically.     

Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.     

Hydrogen peroxide-induced phospholipase D2 activation in lymphocytic leukemic L1210 cells

Extracellular hydrogen peroxide (H2O2) has been implicated in the activation of phospholipase D (PLD). However, it was still unclear how this activation occurs and what the molecular identity of the H2O2-stimulated PLD isozyme is. This study shows that H2O2 potently increases the PLD activity in mouse lymphocytic leukemic L1210 cells, which contain exclusively PLD2. In addition, H2O2 increased PLD activity only in PLD2-transfected COS-7 cells and not in PLD1-transfected cells. This suggests that PLD2 is selectively activated by H2O2. Depletion of extracellular Ca2+ with EGTA completely blocked the H2O2-induced PLD activation, indicating that Ca2+ influx is required. Moreover, pretreatment of the cells with the protein kinase C (PKC) inhibitors GF-109203X and RO-31-8220 and down-regulation of PKCalpha by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate inhibited the H2O2-stimulated PLD2 activity, which points to the involvement of PKCalpha. Based on these new findings we suggest that PLD2 activity is specifically up-regulated by H2O2 and that the H2O2-induced PLD2 activation is mediated by Ca2+ influx and PKCalpha activation.     

Evaluation of surface and build-up region dose for intensity-modulated radiation therapy in head and neck cancer

Despite much development, there remains dosimetric uncertainty in the surface and build-up regions in intensity-modulated radiation therapy treatment plans for head and neck cancers. Experiments were performed to determine the dosimetric discrepancies in the surface and build-up region between the treatment planning system (TPS) prediction and experimental measurement using radiochromic film. A head and neck compression film phantom was constructed from two semicylindrical solid water slabs. Treatment plans were generated using two commercial TPSs (PINNACLE3 and CORVUS) for two cases, one with a shallow (approximately 0.5 cm depth) target and another with a deep (approximately 6 cm depth) target. The plans were evaluated for a 54 Gy prescribed dose. For each case, two pieces of radiochromic film were used for dose measurement. A small piece of film strip was placed on the surface and another was inserted within the phantom. Overall, both TPSs showed good agreement with the measurement. For the shallow target case, the dose differences were within +/- 300 cGy (5.6% with respect to the prescribed dose) for PINNACLE3 and +/- 240 cGy (4.4%) for CORVUS in 90% of the region of interest. For the deep target case, the dose differences were +/- 350 (6.5%) for PINNACLE3 and +/- 260 cGy (4.8%) for CORVUS in 90% of the region of interest. However, it was found that there were significant discrepancies from the surface to about 0.2 cm in depth for both the shallow and deep target cases. It was concluded that both TPSs overestimated the surface dose for both shallow and deep target cases. The amount of overestimation ranges from 400 to 1000 cGy (approximately 7.4% to 18.5% with respect to the prescribed dose, 5400 cGy).     

Detection and genotyping of Mycobacterium species from clinical isolates and specimens by oligonucleotide array

Identification of pathogenic Mycobacterium species is important for a successful diagnosis of mycobacteriosis. The purpose of this study was to develop an oligonucleotide array which could detect and differentiate mycobacteria to the species level by using the internal transcribed spacer (ITS) sequence. Using a genus-specific probe and 20 species-specific probes including two M. avium-intracellulare complex (MAC)-specific probes, we have developed an ITS-based oligonucleotide array for the rapid and reliable detection and discrimination of M. tuberculosis, MAC, M. fortuitum, M. chelonae, M. abscessus, M. kansasii, M. gordonae, M. scrofulaceum, M. szulgai, M. vaccae, M. xenopi, M. terrae, M. flavescens, M. smegmatis, M. malmoense, M. simiae, M. marinum, M. ulcerans, M. gastri, and M. leprae. All mycobacteria were hybridized with a genus-specific probe (PAN-03) for detection of the genus Mycobacterium. Mycobacterial species were expected to show a unique hybridization pattern with species-specific probes, except for M. marinum and M. ulcerans, which were not differentiated by ITS-based probe. Among the species-specific probes, two kinds of species-specific probes were designed for MAC in which there were many subspecies. The performance of the oligonucleotide array assay was demonstrated by using 46 reference strains, 149 clinical isolates, and 155 clinical specimens. The complete procedure (DNA extraction, PCR, DNA hybridization, and scanning) was carried out in 4.5 h. Our results indicated that the oligonucleotide array is useful for the identification and discrimination of mycobacteria from clinical isolates and specimens in an ordinary clinical laboratory.     

Xanthii Fructus Inhibits Inflammatory Responses in LPS-Stimulated Mouse Peritoneal Macrophages

Xanthii Fructus (XF) is an herb widely used in medicine for the treatment of a variety of inflammatory pathologies. In this study, using mouse peritoneal macrophages, we have examined whether XF affects nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-12p40 production induced by interferon (IFN)-γ and lipopolysaccharide (LPS). XF inhibits IFN-γ and LPS-induced NO production in a dose dependent manner. The decrease in NO synthesis was reflected as a decreased amount of inducible NO synthase protein. Furthermore, we also found that XF inhibits pro-inflammatory cytokine TNF-α production. However, treatment of XF in peritoneal macrophages had no effect on IL-12p40 production. These findings suggest that XF may be used in controlling macrophages-mediated inflammatory diseases.     

Malignant fibrous histiocytoma: an ultrastructural perspective

Malignant fibrous histiocytoma is a frequent diagnosis, but the relationship of the tumors to histologically similar soft tissue neoplasms is controversial. In this study, 157 examples representing the 4 main subtypes were reviewed by light microscopy and each tumor was studied with the electron microscope. Immunohistochemical stains were performed on 77 tumors. Electron micrographs on 100 fibrosarcomas were reviewed for comparison. Malignant fibrous histiocytomas often closely resemble fibrosarcomas at the ultrastructural level and differences between the two are generally of degree only. Evidence for true histiocytic differentiation was not found. The immunohistochemical results did not contradict the authors' impression from electron microscopy that malignant fibrous histiocytoma forms part of the histologic spectrum of tumors of fibroblasts.     

Cutaneous bronchogenic cyst of the abdominal wall

We report a case of the unusual location of a cutaneous bronchogenic cyst on the abdominal wall. The patient was a 9-month-old boy who had presented with a 1.5 cm-sized polypoid mass, present since birth. Pathological examination of the excised mass revealed multiple small cystic structures surrounded by the fibroadipose tissue. The lining epithelium consisted of either pseudostratified ciliated columnar epithelium with goblet cells or a single layer of ciliated or non-ciliated cuboidal to columnar cells. The cystic walls contained a well-developed smooth muscle bundle, mucous glands and hyaline cartilage plate. This lesion was adherent to the peritoneum, but there was no direct communication with the abdominal cavity. Cutaneous bronchogenic cyst located in the abdominal wall has not been described in the English literature. The present case suggests a possible origin from a downward migration, from the sequestered bud of a tracheobronchial tree primordium along the midline of the body surface, during embryonic development.     

Cloning, expression and characterization of a murine-human chimeric antibody with specificity for pre-S2 surface antigen of hepatitis B virus

The cloning, expression and characterization of a murine-human chimeric antibody with specificity for the pre-S2 surface antigen (Ag) of hepatitis B virus (HBV) is described. The heavy and light chain variable region (VH and VL) genes encoding the murine monoclonal antibody (mAb) were isolated and combined with human γ 1 and κ constant region genes, respectively. The expression vectors containing the chimeric heavy and light chain genes were sequentially electroporated into murine Sp2/0 hybridoma cells and transfectomas secreting chimeric antibody were isolated. The chimeric antibody was purified and characterized by ELISA, Western analysis and competition immunoassay, demonstrating that the transfectoma functionally express and secrete murine-human chimeric antibody which retained the specificity and affinity of the parental murine mAb.