Development of a quantitative analysis method for assessing patient body surface deformation using an optical surface tracking system.

This study aimed to develop a new method to quantitatively analyze body shape changes in patients during radiotherapy without additional radiation exposure using an optical surface tracking system. This method's accuracy was evaluated using a cubic phantom with a known shift. Surface images of three-dimensionally printed phantoms, which simulated the head and neck shapes of real patients before and after treatment, were used to create a deformation surface area histogram. The near-maximum deformation value covering an area of 2 cm2 in the surface image (Def-2cm2) was calculated. A volumetric modulated arc therapy (VMAT) plan was also created on the pre-treatment phantom, and the dose distribution was recalculated on the post-treatment phantom to compare the dose indices. Surface images of four patients were analyzed to evaluate Def-2cm2 and examine whether this method can be used in clinical cases. Experiments with the cubic phantom resulted in a mean deformation error of 0.08 mm. With head and neck phantoms, the Def-2cm2 value was 17.5 mm, and the dose that covered 95% of the planning target volume in the VMAT plan decreased by 11.7%, indicating that deformation of the body surface may affect the dose distribution. Although analysis of the clinical data showed no clinically relevant deformation in any of the cases, slight skin sagging and respiratory changes in the body surface were observed. The proposed method can quantitatively and accurately evaluate the deformation of a body surface. This method is expected to be used to make decisions regarding modifications to treatment plans.

Emerging roles of hypoxia-inducible factors and reactive oxygen species in cancer and pluripotent stem cells.

Eukaryotic organisms require oxygen homeostasis to maintain proper cellular function for survival. During conditions of low oxygen tension (hypoxia), cells activate the transcription of genes that induce an adaptive response, which supplies oxygen to tissues. Hypoxia and hypoxia-inducible factors (HIFs) may contribute to the maintenance of putative cancer stem cells, which can continue self-renewal indefinitely and express stemness genes in hypoxic stress environments (stem cell niches). Reactive oxygen species (ROS) have long been recognized as toxic by-products of aerobic metabolism that are harmful to living cells, leading to DNA damage, senescence, or cell death. HIFs may promote a cancer stem cell state, whereas the loss of HIFs induces the production of cellular ROS and activation of proteins p53 and p16(Ink4a), which lead to tumor cell death and senescence. ROS seem to inhibit HIF regulation in cancer cells. By contrast, controversial data have suggested that hypoxia increases the generation of ROS, which prevents hydroxylation of HIF proteins by inducing their transcription as negative feedback. Moreover, hypoxic conditions enhance the generation of induced pluripotent stem cells (iPSCs). During reprogramming of somatic cells into a PSC state, cells attain a metabolic state typically observed in embryonic stem cells (ESCs). ESCs and iPSCs share similar bioenergetic metabolisms, including decreased mitochondrial number and activity, and induced anaerobic glycolysis. This review discusses the current knowledge regarding the emerging roles of ROS homeostasis in cellular reprogramming and the implications of hypoxic regulation in cancer development.

Effects of a cysteinyl leukotriene dual 1/2 receptor antagonist on antigen-induced airway hypersensitivity and airway inflammation in a guinea pig asthma model.

BACKGROUND: Little is known about the role of the cysteinyl leukotriene (cysLT) 2 receptor in the pathophysiology of asthma. The aim of this study is to investigate the effects of a cysLT1 receptor antagonist (montelukast) and a dual cysLT1/2 receptor antagonist (BAY-u9773) on airway hypersensitivity and airway inflammation induced by antigen challenge in ovalbumin (OVA)-sensitized guinea pigs. METHODS: Male Hartley guinea pigs sensitized with OVA were intraperitoneally administered 0.1, 1, or 10 mg/kg of montelukast or 0.1 mg/kg of BAY-u9773 and then challenged with inhaled OVA. Airway reactivity to acetylcholine, inflammatory cells in bronchoalveolar lavage (BAL) fluid, and eosinophil infiltration in airway walls after OVA challenge were evaluated. RESULTS: Pretreatment with 1 or 10 mg/kg, but not 0.1 mg/kg, of montelukast significantly suppressed airway hypersensitivity and eosinophil infiltration into the BAL fluid. Moreover, 0.1 mg/kg of BAY-u9773 significantly suppressed the development of these markers. The suppressive effects of BAY-u9773, although not significantly different, trended toward being greater than those of montelukast. Although all of the doses of montelukast tested and 0.1 mg/kg of BAY-u9773 significantly suppressed eosinophil infiltration in airway walls, the suppressive effect of BAY-u9773 was significantly greater than that of 0.1 mg/kg of montelukast. CONCLUSION: Signaling may contribute to the pathophysiology of asthma via the cysLT1/2 receptor.

Impact of implanted bone marrow progenitor cell composition on limb salvage after cell implantation in patients with critical limb ischemia.

OBJECTIVE: The aim of this study is to identify which factors influence limb salvage after bone marrow mononuclear cell implantation (BMI) in patients with chronic critical limb ischemia (CLI). METHODS: Thirteen no-option CLI patients treated with BMI were enrolled in the present study. Limb ischemia was assessed using the ankle-brachial index (ABI), transcutaneous oxygen tension (TcO(2)), and rest pain score. The cell populations among the implanted cells were determined by May-Giemsa staining and flow cytometry. RESULTS: Major lower extremity amputations after BMI were performed in seven patients. Before implantation, there were no significant differences between the amputation group (n=7) and the salvage group (n=6) in clinical characteristics, the ABI, the TcO(2) level, or the rest pain score. After implantation, there were no differences between the groups in the serum levels of angiogenic or inflammatory cytokines. The number of implanted BM cells was the same in the two groups, but the cells implanted in the limb salvage group were composed of significantly higher numbers of hematopoietic progenitors (erythroblasts and myeloblasts) and lymphocytes (p<0.05, respectively). The number of CD34-positive cells was somewhat greater in the salvage group than in the amputation group (p=0.09) and was positively associated with the number of erythroblasts (r(2)=0.29, p=0.06) and the number of myeloblasts (r(2)=0.59, p<0.01). CONCLUSIONS: The cellular composition of the BM cells injected may affect limb salvage after the implantation in patients with severe CLI. The favorable effects of BMI appear to reflect the impact of the progenitor cell doses.

Inhaled montelukast inhibits cysteinyl-leukotriene-induced bronchoconstriction in ovalbumin-sensitized guinea-pigs: the potential as a new asthma medication.

Oral cysteinyl-leukotriene (LT) receptor antagonists such as montelukast are used for reducing airway inflammation and exacerbations. However, inhaled therapy using LT receptor antagonists has not been studied. In the present study, the effect of inhaled montelukast was investigated on airway hyperresponsiveness measured by cysteinyl-LT induced bronchoconstriction in an animal model of asthma. Bronchoconstriction responses were induced by inhaled LTC4 and LTD4 (0.2 microg/ml each) or three doses of intravenous LTC4 and LTD4 (0.3, 1, 3 microg/kg) in ovalbumin (OVA)-sensitized Hartley male guinea-pigs. The response was measured by the change in peak pressure of airway opening (Pao). The effect of montelukast was evaluated by the comparison of bronchoconstriction responses between the groups of animals pre-treated with 15-min inhalation of 10mg/ml montelukast and saline. To evaluate the tissue injury which might be caused by montelukast inhalation, lung tissues were examined for the histology. The broncoconstriction responses induced by inhaled LTC4 and LTD4 were enhanced by OVA sensitization in the guinea-pigs. In sensitized animals, the significant increases in peak Pao were 18.5+/-2.1 cmH(2)O by LTC4 inhalation and 25.0+/-1.6 cmH(2)O by LTD4 inhalation on average. Prior treatment of inhaled montelukast potently suppressed the peak Pao increases induced by both inhaled and intravenous LTC4 and LTD4 (all P<0.01 vs. saline control). Moreover, the suppression of inhaled montelukast against LTD4-induced bronchoconstriction was observed for at least up to 24h. According to the histological examination, montelukast inhalation produced no injury to the lung tissue. Inhaled montelukast, a cysteinyl-LT receptor antagonist, was effective in inhibiting cysteinyl-LT-induced acute bronchoconstriction, and may have the potential for clinical use as a new asthma drug.

Time-course of changes in the levels of interleukin 6 in acutely decompensated heart failure.

BACKGROUND: Although cytokine elevation has been demonstrated in chronic heart failure, little attention has been focused on cytokine levels during the acute stage. We examined the changes of cytokine levels in patients with acutely decompensated heart failure to investigate their relationship with severity of heart failure. METHODS: Patients with acutely decompensated heart failure (73 patients; 72+/-2 years) were included. Blood samples were taken from the peripheral vein on admission before the start of drug therapy, at 12, 24, 48 and 72 h as well as 1, 2 and 4 weeks after admission. Control data were obtained from age-matched normal patients who had no cardiovascular disease. Serum IL-6, IL-1beta and TNF-alpha levels were measured using the ELISA method. RESULTS: Mean IL-6, IL-1beta and TNF-alpha levels on admission were significantly higher than those in the control patients (p<0.001). IL-6 peaked at 12 h and declined thereafter, whereas IL-1beta and TNF-alpha remained unchanged throughout the duration of the study. Peak IL-6 significantly correlated with pulmonary wedge pressure on admission (r=0.332, p=0.0041). % change of IL-6 levels between peak (12 h after admission) and 24 h was significantly correlated with that of pulmonary wedge pressure between peak (on admission) and 24 h (r=0.308, p=0.0081). Peak IL-6 in patients treated with mechanical ventilation on admission was significantly higher than that in patients who underwent no mechanical ventilation (p<0.05). CONCLUSIONS: IL-6 levels possibly reflect the severity of heart failure and thus may be useful for the evaluation of disease status in acutely decompensated heart failure.

Concomitant expression of adrenomedullin and its receptor components in rat adipose tissues.

Adrenomedullin (AM) expressed by and secreted from a variety of cells plays pluripotent roles in an autocrine/paracrine fashion. The present study was undertaken to explore the expression of AM and its receptor genes in adipose tissues, their changes during the development of obesity, and the process of preadipocyte differentiation. Both mature adipocytes and stromal vascular cells constituting adipose tissue expressed AM transcript. AM and its receptor component [calcitonin receptor-like receptor and receptor activity-modifying protein-2 (CRLR/RAMP2)] mRNAs were expressed in a variety of rat adipose tissues, including epididymal, mesenteric, retroperitoneal, and subcutaneous adipose tissue. AM mRNA levels in rat and human epididymal adipose tissue were about one-tenth of those in the kidney. Steady-state mRNA levels of AM and CRLR/RAMP2 in epididymal, mesenteric, and retroperitoneal adipose tissues in rats fed a high-fat diet for 4 wk were far greater than those in rats with normal diet accompanied by increased plasma AM levels, whereas steady-state AM mRNA levels conversely decreased in other organs, such as kidney and liver. AM mRNA expressed in a mouse preadipocyte cell line (3T3-L1) transiently decreased by day 3, returned to basal level by day 6, and then increased by day 9 during preadipocyte differentiation, which paralleled AM secretion from the cells. However, the addition of either exogenous AM or AM receptor antagonist calcitonin gene-related peptide-(8-37), to block endogenous AM did not affect lipid droplet accumulation during preadipocyte differentiation. The present study demonstrates for the first time that AM and its receptor component (CRLR/RAMP2) mRNAs were concomitantly expressed in various adipose tissues, whose tissue-specific upregulation was induced during the development of obesity. These data suggest that AM may act as a new member of adipokines, although its functional role, as well as its pathophysiological significance in obesity, remains to be determined.

High molecular weight form insulin-like growth factor II-producing mesenteric sarcoma causing hypoglycemia.

An 81-year-old woman presented with frequent episodes of hypoglycemia. Her serum level of insulin was normal, but her serum insulin-like growth factor (IGF)-II level was high. She was found to have a spindle cell sarcoma originated from the mesentery of the sigmoid colon, which was completely resected. Postoperatively, hypoglycemia ameliorated with concomitant reduction in serum IGF-II levels. Immunohistochemical study revealed positive immunostaining for IGF-II in tumor cells, and the abundant expression of IGF-II mRNA was demonstrated by RT-PCR. The presence of high molecular weight (HMW) form IGF-II in patient's serum was confirmed by immunoblotting. This is the first report of a patient with HMW form IGF-II-producing mesenteric sarcoma causing hypoglycemia.

Ectodomain shedding of SHPS-1 and its role in regulation of cell migration.

SHPS-1 is a transmembrane protein whose cytoplasmic region undergoes tyrosine phosphorylation and then binds the protein-tyrosine phosphatase SHP-2. Formation of the SHPS-1-SHP-2 complex is implicated in regulation of cell migration. In addition, SHPS-1 and its ligand CD47 constitute an intercellular recognition system that contributes to inhibition of cell migration by cell-cell contact. The ectodomain of SHPS-1 has now been shown to be shed from cells in a reaction likely mediated by a metalloproteinase. This process was promoted by activation of protein kinase C or of Ras, and the released ectodomain exhibited minimal CD47-binding activity. Metalloproteinases catalyzed the cleavage of a recombinant SHPS-1-Fc fusion protein in vitro, and the primary cleavage site was localized to the juxtamembrane region of SHPS-1. Forced expression of an SHPS-1 mutant resistant to ectodomain shedding impaired cell migration, cell spreading, and reorganization of the actin cytoskeleton. It also increased the tyrosine phosphorylation of paxillin and FAK triggered by cell adhesion. These results suggest that shedding of the ectodomain of SHPS-1 plays an important role in regulation of cell migration and spreading by this protein.

Antioxidant effect of adrenomedullin on angiotensin II-induced reactive oxygen species generation in vascular smooth muscle cells.

Recent adrenomedullin (AM) gene-targeting studies have proposed a novel concept that AM plays a protective role against oxidative stress in vivo. The present study was undertaken to explore the underlying molecular mechanism of the putative antioxidant action of AM against angiotensin II (Ang II)induced reactive oxygen species (ROS) generation in rat vascular smooth muscle cells (VSMCs). Intracellular ROS levels were measured by dichlorofluoroscein fluorescence. Redox-sensitive c-Jun amino-terminal kinase (JNK) and ERK1/2 activation and gene expression induced by Ang II in VSMCs were also studied. AM dose-relatedly (10(-8)-10(-7) m) inhibited intracellular ROS generation stimulated by Ang II (10(-7) m), as mimicked by dibutyl-cAMP, the effect of which was inhibited by the pretreatment with N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride, a protein kinase A inhibitor, and calcitonin gene-related peptide(8-37), an AM/calcitonin gene-related peptide receptor antagonist. Ang II induced JNK and ERK1/2 activation via a redox-sensitive manner, whereas AM inhibited JNK, but not ERK1/2, activation by Ang II. Furthermore, AM inhibited Ang II-induced redox-sensitive gene expression (plasminogen activator inhibitor-1 and monocyte chemoattractant protein-1) in the same manner as N-acetyl-l-cysteine, a potent antioxidant. AM also inhibited Ang II-induced up-regulation of Nox1, a critical membrane-bound component of reduced nicotinamide adenine dinucleotide phosphate oxidase in VSMCs, in the same degree as N-acetyl-l-cysteine. Our study demonstrates for the first time that AM directly inhibits intracellular ROS generation via an AM receptor-mediated and c-AMP-protein kinase A-dependent mechanism in VSMCs and that AM with its potent antioxidant action inhibits redox-sensitive JNK activation and gene expression induced by Ang II. These data suggest that AM plays a protective role as an endogenous antioxidant in Ang II-induced vascular injury.

Regulation of multiple functions of SHPS-1, a transmembrane glycoprotein, by its cytoplasmic region.

SHPS-1 is a receptor-type transmembrane glycoprotein, which contains four tyrosine residues in its cytoplasmic region, and the phosphorylation of these tyrosine residues serves the binding sites for SHP-2 protein-tyrosine phosphatase. Its extracellular region interacts with another membrane protein, CD47, thereby constituting a cell-cell communication system. We analyzed this ligand-receptor interaction using Chinese hamster ovary (CHO) cells expressing wild-type (WT) or mutant SHPS-1. The binding affinity of an SHPS-1 mutant such as deltaCyto, that lacked most of cytoplasmic region, or 4F, in which all four tyrosine residues in cytoplasmic region were substituted with phenylalanine, for a recombinant CD47-Fc was greater than that of WT. In addition, oligomerization of deltaCyto or 4F mutant by binding of CD47-Fc was greater than WT. Chemical cross-linking of SHPS-1 indicated that SHPS-1 formed a cis-dimer. Furthermore, WT cells exhibited a less polarized cell shape with decreased formation of actin stress fibers, compared with parental CHO cells and mutant SHPS-1 expressing cells. Prominent lamellipodium formation and membrane ruffling were also observed at leading edges of migrating WT cells but not at those of other mutant SHPS-1 expressing cells. These results suggest that the binding affinity of SHPS-1 to CD47, clustering ability of SHPS-1, and cytoskeletal reorganization are regulated by the cytoplasmic region of SHPS-1.

Octreotide-sensitive ectopic ACTH production by islet cell carcinoma with multiple liver metastases.

We report a 21-year-old woman with ectopic ACTH syndrome due to islet cell carcinoma with multiple liver metastases. On admission, she showed Cushingoid appearance (moon face, central obesity etc.) and had acute respiratory distress syndrome due to pneumocystis carinii pneumonia. Laboratory examination revealed marked elevations of plasma ACTH (735 pg/ml) and cortisol (145 microg/dl) with a profound hypokalemia (2.0 mEq/l). She was found to have multiple masses in the liver and a solid mass in the tail of pancreas by abdominal computerized tomography scanning. Treatment with octreotide successfully reduced elevated plasma ACTH and cortisol levels, and she received frequent transhepatic arterial embolization and chemotherapy. The primary pancreatic tumor was surgically removed, revealing islet cell carcinoma which contained high content of ACTH (100 microg/g wet weight) and abundantly expressed proopiomelanocortin and somatostatin receptor subtype-2 mRNAs as determined by Northern blot analysis. Postoperatively, she was free from symptoms for almost one year. However, progressive enlargement of multiple liver metastases refractory to chemotherapy led her to decide on total hepatectomy and liver transplantation from her father. After liver transplantation, she remained almost free from symptoms for almost one year. However, metastases developed to the mediastinal and paraaortic lymph nodes as detected by 111[In] pentetreotide scintigraphy. Eleven months after liver transplantation, she was again treated with octreotide and, 16 months after, with metyrapone, both of which were effective in reducing ACTH and cortisol levels, respectively, until she died of acute respiratory failure. This case of a young female patient with ectopic ACTH-producing islet cell carcinoma of the pancreas was quite unique in that she survived for 5 years despite the acute onset and rapid progression of the multiple liver metastases at least in part due to the long-lasting favorable response to octreotide and living-related liver transplantation.

Role of the CD47-SHPS-1 system in regulation of cell migration.

SHPS-1 is a transmembrane protein whose extracellular region interacts with CD47 and whose cytoplasmic region undergoes tyrosine phosphorylation and there by binds the protein tyrosine phosphatase SHP-2. Formation of this complex is implicated in regulation of cell migration by an unknown mechanism. A CD47-Fc fusion protein or antibodies to SHPS-1 inhibited migration of human melanoma cells or of CHO cells overexpressing SHPS-1. Overexpression of wild-type SHPS-1 promoted CHO cell migration, whereas expression of the SHPS-1-4F mutant, which lacks the phosphorylation sites required for SHP-2 binding, had no effect. Antibodies to SHPS-1 failed to inhibit migration of CHO cells expressing SHPS-1-4F. SHPS-1 ligands induced the dephosphorylation of SHPS-1 and dissociation of SHP-2. Antibodies to SHPS-1 also enhanced Rho activity and induced both formation of stress fibers and adoption of a less polarized morphology in melanoma cells. Our results suggest that engagement of SHPS-1 by CD47 prevents the positive regulation of cell migration by this protein. The CD47- SHPS-1 system and SHP-2 might thus contribute to the inhibition of cell migration by cell-cell contact.

Circulating nuclear matrix protein in Graves' disease.

The Fas/Fas ligand system induces apoptosis, while soluble Fas (sFas) blocks the system and soluble Fas ligand (sFasL) functions to induce apoptosis. The assay of nuclear matrix protein (NMP) released from dead or dying cells can be used to quantitate cell death. Therefore, we evaluated the relationship among serum levels of NMP, sFas, and sFasL in patients with Graves' disease. We measured serum levels of sFas, sFasL, NMP, thyroid hormones and TSH receptor antibody in 20 normal control subjects (5 men, 15 women; mean age, 44.3 years), 32 patients with untreated Graves' disease (4 men, 28 women; mean age, 44.1 years), and 10 patients with Graves' disease treated by methimazole (3 men, 7 women; mean age 39.2 years). Serum NMP was significantly lower (10.4 +/- 4.3 IU/ml, p < 0.02) in patients with untreated Graves' disease than in patients with treated Graves' disease (16.4 +/- 7.3 IU/ml) and control subjects (15.3 +/- 8.9 IU/ml). Serum sFas and sFasL were significantly higher in patients with untreated Graves' disease than in patients with treated Graves' disease and in control subjects. In the patient groups with Graves' disease, serum NMP was negatively correlated with sFas (r = -0.612, p < 0.001) and serum sFas was positively correlated with FT4 (r = 0.360, p < 0.05) and TRAb (r = 0.384, p < 0.05). Serum NMP was correlated with sFas. These results suggest that serum NMP is decreased in patients with untreated Graves' disease, and that cell death or apoptosis in patients with Graves' disease is affected by soluble Fas under the influence of thyroid function.

Accelerated production of nucleosome in cultured human mononuclear cells in untreated Graves' disease.

The apoptosis of lymphocytes, which occurs in autoimmune diseases, is usually induced by the Fas/Fas ligand system. As the assay of nucleosomes produced by apoptotic cells can be used to quantitate apoptosis, we evaluated nucleosome and soluble Fas ligand (sFasL) levels of cultured mononuclear cells to clarify the apoptosis of mononuclear cells in patients with autoimmune thyroid diseases by enzyme-linked immunosorbent assay. Nucleosome levels of cultured mononuclear cells in patients with untreated Graves' disease were significantly higher (3.27+/-2.90 U/ml) than those of control subjects (1.39+/-0.24 U/ml) and euthyroid patients with treated Graves' disease (1.53+/-0.33 U/ml). Nucleosome levels of cultured mononuclear cells were positively correlated with sFasL levels (r=0.544, p<0.01). It is therefore likely that increased sFasL levels elicit apoptosis of these cells in untreated Graves' disease.