Neuroblastoma is a vascularized pediatric tumor derived from neural crest stem cells that displays vasculogenic mimicry and can express a number of stemness markers, such as SOX2 and NANOG. Tumor relapse is the major cause of succumbing to this disease, and properties attributed to cancer stem-like cells (CSLC), such as drug-resistance and cell plasticity, seem to be the key mechanisms. However, the lack of controllable models that recapitulate the features of human neuroblastoma limits our understanding of the process and impedes the development of new therapies. In response to these limitations, we engineered a perfusable, vascularized in vitro model of three-dimensional human neuroblastoma to study the effects of retinoid therapy on tumor vasculature and drug-resistance.

Next-generation sequencing (NGS) diagnostics have shown clinical utility in predicting survival benefits in patients with certain cancer types who are undergoing targeted drug therapies. Currently, there are no guidelines or recommendations for the use of NGS in patients with metastatic cancer from an Asian perspective. In this article, we present the Asia-Pacific Oncology Drug Development Consortium (APODDC) recommendations for the clinical use of NGS in metastatic cancers.

A 53-year-old female with peritoneal metastasis of breast cancer was treated with hyperthermia with chemotherapy. One course consisted of THP 60 mg and FT 400 mg/iv (day 1), CPM 100 mg and MPA 800 mg/daily/po) with RF heating of hyperthermia monthly. After completion of 10 courses of this regimen, ascites completely disappeared and peritoneal metastases were not found observed in any peritoneal cavity with abdominal CT and ultrasound. Peritoneal metastasis of breast cancer was one of the worst reactions with any therapy. The patient has been living for about four years without any signs of recurrence, with the tumor markers within the normal range after surgery. Thermochemotherapy seems a very promising treatment modality for peritoneal metastasis of breast cancer.

A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light meromyosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosin-antibody complexes has localized the antibody binding site to LMM at a point approximately 92 nm from the C-terminus of the myosin heavy chain. Since this epitope in native thick filaments is accessible to monoclonal antibodies, we used this antibody as a high affinity ligand to analyze the packing of LMM along the backbone of the thick filament. By immunofluorescence microscopy, MF20 was shown to bind along the entire A-band of chicken pectoralis myofibrils, although the epitope accessibility was greater near the ends than at the center of the A-bands. Thin-section, transmission electron microscopy of myofibrils decorated with MF20 revealed 50 regularly spaced, cross-striations in each half A-band, with a repeat distance of approximately 13 nm. These were numbered consecutively, 1-50, from the A-band to the last stripe, approximately 68 nm from the filament tips. These same striations could be visualized by negative staining of native thick filaments labeled with MF20. All 50 striations were of a consecutive, uninterrupted repeat which approximated the 14-15-nm axial translation of cross-bridges. Each half M-region contained five MF20 striations (approximately 13 nm apart) with a distance between stripes 1 and 1', on each half of the bare zone, of approximately 18 nm. This is compatible with a packing model with full, antiparallel overlap of the myosin rods in the bare zone region. Differences in the spacings measured with negatively stained myofilaments and thin-sectioned myofibrils have been shown to arise from specimen shrinkage in the fixed and embedded preparations. These observations provide strong support for Huxley's original proposal for myosin packing in thick filaments of vertebrate muscle (Huxley, H. E., 1963, J. Mol. Biol., 7:281-308) and, for the first time, directly demonstrate that the 14-15-nm axial translation of LMM in the thick filament backbone corresponds to the cross-bridge repeat detected with x-ray diffraction of living muscle.

Although the introduction of tyrosine kinase inhibitors (TKIs) has improved overall survival of patients with chronic myeloid leukemia (CML), about half of the patients eventually relapse after cessation of TKIs. In contrast, the remainder of the patients maintain molecular remission without TKIs, indicating that the patients' immune system could control proliferation of TKI-resistant leukemic stem cells (LSCs). However, the precise mechanism of immunity against CML-LSCs is not fully understood. We have identified a novel immune target, CXorf48, expressed in LSCs of CML patients. Cytotoxic T cells (CTLs) induced by the epitope peptide derived from CXorf48 recognized CD34+CD38- cells obtained from the bone marrow of CML patients. We detected CXorf48-specific CTLs in the peripheral blood mononuclear cells from CML patients who have discontinued imatinib after maintaining complete molecular remission for more than 2 years. Significantly, the relapse rate of CXorf48-specific CTL-negative patients was 63.6%, compared to 0% in CXorf48-specific CTL-positive patients. These results indicate that CXorf48 could be a promising therapeutic target of LSCs for immunotherapy to obtain durable treatment-free remission in CML patients.

In this immunohistochemical examination, the expression of Runx2, Notch1, Delta and Osteopontin peptides were detected in neoplastic cells in 10 Japanese cases of osteosarcoma. Immunohistochemically, Runx2 peptide expression appeared in the cytoplasm of almost all neoplastic cells of the 10 cases examined. However, Notch1 peptide expression appeared in the cytoplasm of neoplastic cells in the localized and comparatively well-differentiated area of osteosarcoma, which osteoblastic and chondroblastic containing osteoid and/or chondroid tissues. No expression of Notch1 peptide was detected in the fibroblastic and poorly differentiated areas. Delta peptide appearance was nearly the same pattern of Notch1 peptide. Expression of Osteopontin peptide appeared in almost all cells and the strength expression was shown in the area of comparatively well-differentiated tissues. Therefore, these results suggest that Runx2, Notch1, and Delta peptides are closely related to cytological differentiation or acquisition of tissue specific characteristics in neoplastic cells in osteosarcomas.

Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-beta (TGF-beta) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-beta receptor (TbetaR) phosphorylation inhibitor on the invasiveness of gastric cancer cells.

Most of the sounds of human speech are produced by vibration of the vocal folds, yet the biomechanics and control of these vibrations are poorly understood. In this study the muscle within the vocal fold, the thyroarytenoid muscle (TA), was examined for the presence and distribution of slow tonic muscle fibers (STF), a rare muscle fiber type with unique contraction properties. Nine human TAs were frozen and serially sectioned in the frontal plane. The presence and distribution pattern of STF in each TA were examined by immunofluorescence microscopy using the monoclonal antibodies (mAb) ALD-19 and ALD-58 which react with the slow tonic myosin heavy chain (MyHC) isoform. In addition, TA muscle samples from adjacent frozen sections were also examined for slow tonic MyHC isoform by electrophoretic immunoblotting. STF were detected in all nine TAs and the presence of slow tonic MyHC isoform was confirmed in the immunoblots. The STF were distributed predominantly in the medial aspect of the TA, a distinct muscle compartment called the vocalis which is the vibrating part of the vocal fold. STF do not contract with a twitch like most muscle fibers, instead, their contractions are prolonged, stable, precisely controlled, and fatigue resistant. The human voice is characterized by a stable sound with a wide frequency spectrum that can be precisely modulated and the STF may contribute to this ability. At present, the evidence suggests that STF are not presented in the vocal folds of other mammals (including other primates), therefore STF may be a unique human specialization for speech.

Leukotriene B(4) (LTB(4)) is a potent chemoattractant and activator of both granulocytes and macrophages. The actions of LTB(4) appear to be mediated by a specific G protein-coupled receptor (GPCR) BLT1, originally termed BLT (Yokomizo, T., T. Izumi, K. Chang, Y. Takuwa, and T. Shimizu. 1997. Nature. 387:620-624). Here, we report the molecular cloning of a novel GPCR for LTB(4), designated BLT2, which binds LTB(4) with a Kd value of 23 nM compared with 1.1 nM for BLT1, but still efficiently transduces intracellular signaling. BLT2 is highly homologous to BLT1, with an amino acid identity of 45.2%, and its open reading frame is located in the promoter region of the BLT1 gene. BLT2 is expressed ubiquitously, in contrast to BLT1, which is expressed predominantly in leukocytes. Chinese hamster ovary cells expressing BLT2 exhibit LTB(4)-induced chemotaxis, calcium mobilization, and pertussis toxin-insensitive inhibition of adenylyl cyclase. Several BLT1 antagonists, including U 75302, failed to inhibit LTB(4) binding to BLT2. Thus, BLT2 is a pharmacologically distinct receptor for LTB(4), and may mediate cellular functions in tissues other than leukocytes. BLT2 provides a novel target for antiinflammatory therapy and promises to expand our knowledge of LTB(4) function. The location of the gene suggests shared transcriptional regulation of these two receptors.

The crystal structure of a DNA-binding domain of PHO4 complexed with DNA at 2.8 A resolution revealed that the domain folds into a basic-helix-loop-helix (bHLH) motif with a long but compact loop that contains a short alpha-helical segment. This helical structure positions a tryptophan residue into an aromatic cluster so as to make the loop compact. PHO4 binds to DNA as a homodimer with direct reading of both the core E-box sequence CACGTG and its 3'-flanking bases. The 3'-flanking bases GG are recognized by Arg2 and His5. The residues involved in the E-box recognition are His5, Glu9 and Arg13, as already reported for bHLH/Zip proteins MAX and USF, and are different from those recognized by bHLH proteins MyoD and E47, although PHO4 is a bHLH protein.

Pathogenic amyloid-β peptide precursor (APP) mutations clustered around position 693 of APP-position 22 of the Aβ sequence--are commonly associated with congophilic amyloid angiopathy (CAA) and intracerebral hemorrhages. In contrast, the Osaka (E693Δ) intra-Aβ APP mutation shows a recessive pattern of inheritance that leads to AD-like dementia despite low brain amyloid on in vivo positron emission tomography imaging. Here, we investigated the effects of the Osaka APP mutation on Aβ accumulation and deposition in vivo using a newly generated APP transgenic mouse model (E22ΔAβ) expressing the Osaka mutation together with the Swedish (K670N/M671L) double mutation. E22ΔAβ mice exhibited reduced α-processing of APP and early accumulation of intraneuronal fibrillar Aβ oligomers associated with cognitive deficits. In line with our in vitro findings that recombinant E22Δ-mutated Aβ peptides form amyloid fibrils, aged E22ΔAβ mice showed extracellular CAA deposits in leptomeningeal cerebellar and cortical vessels. In vitro results from thioflavin T aggregation assays with recombinant Aβ peptides revealed a yet unknown antiamyloidogenic property of the E693Δ mutation in the heterozygous state and an inhibitory effect of E22Δ Aβ42 on E22Δ Aβ40 fibrillogenesis. Moreover, E22Δ Aβ42 showed a unique aggregation kinetics lacking exponential fibril growth and poor seeding effects on wild-type Aβ aggregation. These results provide a possible explanation for the recessive trait of inheritance of the Osaka APP mutation and the apparent lack of amyloid deposition in E693Δ mutation carriers.

Gestational choriocarcinoma is a malignant trophoblastic tumor that usually occurs in the uterus after pregnancy. The tumor is curable with advanced chemotherapy, but the molecular mechanism of choriocarcinoma tumorigenesis remains unclear. This is partly because the low incidence makes it difficult to obtain clinical samples for investigation and because an appropriate choriocarcinoma cell model to study the tumorigenesis has not been developed. We have established a new choriocarcinoma cell line, induced choriocarcinoma cell-1 (iC(3)-1), that possesses unique characteristics compared to other choriocarcinoma cell lines, including production of tumors that consist of the two types of cells commonly found in choriocarcinoma and mimicking of the clinical pathology. Existing trophoblast cell lines utilized in previous choriocarcinoma studies have had significantly dissimilar gene expression profiles. Therefore, it is important to choose an appropriate cell line for a particular study based on the characteristics of the cell line. In this study, to clarify the genetic characteristics of iC(3)-1 and to explore the tumorigenesis mechanism, we examined the gene profile of iC(3)-1 compared to those of existing cell lines and normal placental tissue. Bioinformatics analysis showed that several characteristic genes, IGF1R, CHFR, MUC3A, TAF7, PARK7, CDC123 and PSMD8, were significantly upregulated in iC(3)-1 compared to BeWo and JEG3 cells. Interestingly, HAS2, CD44 and S100P were significantly upregulated in iC(3)-1 compared to parental HTR8/SVneo cells and normal third trimester placenta. Choriocarcinoma samples also showed immunoreactivity to HAS2, CD44 and S100. In summary, the gene expression profile of iC(3)-1 suggests that studies using this cell line can make an important contribution to improved understanding of choriocarcinoma tumorigenesis.

The multidrug resistance-1 (MDR1) gene encodes an ATP-dependent efflux transporter that is highly expressed in the colon. In mice, loss of MDR1 function results in colitis with similarities to human inflammatory bowel diseases (IBD). Here, we show that MDR1 has an unexpected protective role for the mitochondria where MDR1 deficiency results in mitochondrial dysfunction with increased mitochondrial reactive oxygen species (mROS) driving the development of colitis. Exogenous induction of mROS accelerates, while inhibition attenuates colitis in vivo; these effects are amplified in MDR1 deficiency. In human IBD, MDR1 is negatively correlated to SOD2 gene expression required for mROS detoxification. To provide direct evidential support, we deleted intestinal SOD2 gene in mice and showed an increased susceptibility to colitis. We exploited the genome-wide association data sets and found many (∼5%) of IBD susceptibility genes with direct roles in regulating mitochondria homeostasis. As MDR1 primarily protects against xenotoxins via its efflux function, our findings implicate a distinct mitochondrial toxin+genetic susceptibility interaction leading to mitochondrial dysfunction, a novel pathogenic mechanism that could offer many new therapeutic opportunities for IBD.

Genes were isolated using the suppression subtractive hybridization method by stimulation of pro/pre B cells with anti-CD40 and interleukin (IL)-4 to mature S mu-Sepsilon-switched cells. One of the strongly upregulated genes encodes a novel murine CC chemokine we have named ABCD-1. The ABCD-1 gene has three exons separated by 1. 2- and 2.7-kb introns. It gives rise to a 2.2-kb transcript containing an open reading frame of 276 nucleotides. Two polyadenylation sites are used, giving rise to cDNAs with either 1550 or 1850 bp of 3' untranslated regions. The open reading frame encodes a 24 amino acid-long leader peptide and a 68 amino acid-long mature protein with a predicted molecular mass of 7.8 kD. ABCD-1 mRNA is found in highest quantities in activated splenic B lymphocytes and dendritic cells. Little chemokine mRNA is present in lung, in unstimulated splenic cells, in thymocytes, and in lymph node cells. No ABCD-1 mRNA is detected in bone marrow, liver, kidney, or brain, in peritoneal exudate cells as well as in the majority of all unstimulated B lineage cells tested. It is also undetectable in Concanavalin A-activated/IL-2-restimulated splenic T cells, and in bone marrow-derived IL-2-induced natural killer cells and IL-3-activated macrophages. Recombinant ABCD-1 revealed a concentration-dependent and specific migration of activated splenic T lymphoblasts in chemotaxis assays. FACS(R) analyses of migrated cells showed no preferential difference in migration of CD4(+) versus CD8(+) T cell blasts. Murine as well as human T cells responded to ABCD-1. Freshly isolated cells from bone marrow, thymus, spleen, and lymph node, IL-2-activated NK cells, and LPS-stimulated splenic cells, all did not show any chemotactic response. Thus, ABCD-1 is the first chemokine produced in large amounts by activated B cells and acting selectively on activated T lymphocytes. Therefore, ABCD-1 is expected to play an important role in the collaboration of dendritic cells and B lymphocytes with T cells in immune responses.

Serum levels of eosinophil cationic protein (ECP), myeloperoxidase (MPO), tryptase, total IgE and differential blood cell counts were studied in atopic children with: 1) moderate to severe asthma using inhaled steroids and symptom-free for the last 3 weeks (n = 13), 2) mild asthma with sporadic symptoms, using only inhaled beta 2-agonists < 3 times/week (n = 15), 3) acute asthmatic attacks admitted to hospital (n = 12), 4) mild to moderate atopic dermatitis (n = 14). Fifteen children without any history of atopy served as controls. ECP, MPO, tryptase and IgE were measured in serum by radioimmunoassays (RIA). The symptom-free children with inhaled steroids had similar median ECP and MPO values as the controls, 8.0 and 360 micrograms/l, vs. 9.0 and 310 micrograms/l, while both ECP and MPO were significantly (p < 0.001) increased in the symptom-free children without anti-inflammatory treatment, 32 and 887 micrograms/l and in those with acute asthma, 28 and 860 micrograms/l. The children with atopic dermatitis had increased ECP but normal MPO levels, 16.0 and 455 micrograms/l. Tryptase in serum was not measurable in any patient. All groups except the control group had significantly elevated total IgE levels. The results indicate that in atopic children serum ECP is a good marker of ongoing asthma or atopic dermatitis. The normal levels of ECP and MPO in the children with asthma using inhaled steroids seem to reflect successful anti-inflammatory treatment. The increased levels of ECP and MPO in the children with mild asthma and no anti-inflammatory treatment may indirectly reflect airway inflammation.

A case of benign paraganglioma arising in the middle mediastinum was reported. 201Tl SPECT showed high accumulation in tumor on early images at 15 min and reduced on late images at 3 hours after infusion. The patient was a 57 year old female. In the contrast enhanced CT, 3 x 4 cm tumor with intensive enhancement was recognized at the right middle mediastinum. Under the radiological images, the tumor was surgically removed. The pathologic diagnosis was a low atypical nonfunctioning aortico-pulmonary paraganglioma. This report was suggested that 201Tl SPECT was useful for differential diagnosis of benign neurogenic mediastinal neoplasms.

Leukotriene B(4) (LTB(4)) is a lipid mediator that activates leukocytes and is involved in host defense and inflammation. BLT1, a high-affinity receptor for LTB(4) (originally termed BLT), is expressed exclusively in inflammatory cells and is inducible in macrophages upon activation. The mechanisms of tissue-specific expression and induction of BLT1 are important for the understanding of mechanism of onset and the potential treatment of inflammatory disorders. Here, we report the genomic structure and a promoter analysis of the human BLT1 gene, with an emphasis on the mechanism of cell-specific transcription. No TATA or CAAT elements exist around the transcription initiation sites, but a GC-rich sequence is observed in this region. A reporter gene assay revealed that a region approximately 80 basepair upstream from the initiator sequence is required for the basal transcription of the BLT1 gene. Sp1 was found to be a major activator of basal transcription by electrophoretic mobility shift assays and site-directed mutagenesis. The CpG sites of the BLT1 promoter region were highly methylated in BLT1-nonexpressing cells, but not methylated in BLT1-expressing cells. Further, methylation of this region in vitro inhibited the promoter activity to approximately 15% of the control. Thus, methylation at CpG sites in the promoter region is important for cell-specific transcription of the BLT1 gene. The promoter region of the BLT1 gene is localized within the open reading frame (ORF) of the BLT2 gene, which encodes a low-affinity receptor for LTB(4) (Yokomizo, T., K. Kato, K. Terawaki, T. Izumi, and T. Shimizu. 2000. J. Exp. Med. 192:421-431). To our knowledge, this is the first example of "promoter in ORF" in higher eukaryotes.

To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone.

Processes of restenosis, following arterial injury, are complex involving different cell types producing various cytokines and enzymes. Among those enzymes, smooth muscle cell-derived matrix metalloproteinases (MMPs) are thought to take part in cell migration, degrading of extracellular matrix, and neointima formation. MMP-9, also known as gelatinase B, is expressed immediately after vascular injury and its expression and activity can be inhibited by statins. Using an established in vivo model of vascular injury, we investigated the effect of the HMG-CoA reductase inhibitor rosuvastatin on MMP-9 expression and neointima formation.

Bisphosphonates (BPs) and teriparatide (TPTD) are both effective treatments for osteoporosis, but BP treatment prior to daily TPTD treatment has been shown to impair the effect of TPTD in some clinical studies. In contrast, the loss of bone mineral density (BMD) that occurs after withdrawal of TPTD can be prevented by BP treatment. Although various studies have investigated the combination and/or sequential use of BP and TPTD, there have been no clinical studies investigating sequential treatment with zoledronic acid (ZOL) and TPTD (or vice versa). In this study, we evaluated the effects of sequential treatment with TPTD followed by ZOL, and ZOL followed by TPTD, using ovariectomized (OVX) rats. Two months after OVX, osteopenic rats were treated with ZOL, TPTD, or vehicle for a period of 4 months (first treatment period), and then the treatments were switched and administered for another 4 months (second treatment period). The group treated with ZOL followed by TPTD showed an immediate increase in BMD of the proximal tibia and greater BMD and bone strength of the lumbar vertebral body, femoral diaphysis, and proximal femur than the group treated with ZOL followed by vehicle. Serum osteocalcin, a marker of bone formation, increased rapidly after switching to TPTD from ZOL. The group treated with TPTD followed by ZOL did not lose BMD in the proximal tibia after TPTD was stopped, while the group treated with TPTD followed by vehicle did lose BMD. The BMD and bone strength of the lumbar vertebral body, femoral diaphysis, and proximal femur were greater in the group treated with TPTD followed by ZOL than in the group treated with TPTD followed by vehicle. The increase in serum osteocalcin and urinary CTX after withdrawal of TPTD was prevented by the switch from TPTD to ZOL. In conclusion, our results demonstrate that switching from ZOL to TPTD resulted in a non-attenuated anabolic response in the lumbar spine and femur of OVX rats. In addition, switching from TPTD to ZOL caused BMD to be maintained or further increased. If these results can be reproduced in a clinical setting, the sequential use of ZOL followed by TPTD or vice versa in the treatment of osteoporosis patients would contribute to increases in BMD that, hopefully, would translate into a corresponding decrease in the incidence of vertebral and non-vertebral fractures.