Cytokine marker measurement in human neuroblastoma cells with supersensitive and multiplex assay: MUSTag technology.

BACKGROUND: Recently, various sets of protein -biomarkers have been discovered in important diseases such as cancers, brain stroke and heart attack. However, clinical validation is difficult and time-consuming by individual assays or because of very low concentrations at early stages of the diseases. We have developed assay technology through an innovative modification of the immuno-PCR method for the super-sensitive and multiplex detection of target biomarkers. METHODS: In the assay technology, each different oligo-tag simultaneously detects multiplex protein targets with extremely high-level sensitivity in a dose-dependent manner by qRT-PCR (maximum: three plexes). In this study, we measured specific secreted protein concentrations in the culture supernatant of a 24-h culture of transfected SH-SY5Y cells with MUSTag. RESULTS: There was a significant increase in the protein level of tumor necrosis factor (TNF)-α measured with extremely high-level sensitivity (≥10 pg/mL). Compared with negative controls, the levels of TNF-α increased from 16.9 to 28.1 pg/mL (p = 0.011). CONCLUSION: We suggest that our assay technology might be of clinical value in treating patients with cancer, cerebral ischemia, or patients who need a prompt and predictive diagnosis for adequate treatment.

The asparaginyl endopeptidase legumain after experimental stroke.

Various proteases in the brain contribute to ischemic brain injury. We investigated the involvement of the asparaginyl endopeptidase legumain after experimental stroke. On the basis of gene array studies and in situ hybridizations, we observed an increase of legumain expression in the peri-infarct area of rats after transient occlusion of the middle cerebral artery (MCAO) for 120 mins with a maximum expression at 24 and 48 h. Immunohistochemical analyses revealed the expression of legumain in Iba1(+) microglial cells and glial fibrillary acidic protein-positive astrocytes of the peri-infarct area in mice after MCAO. Post-stroke recovery was also studied in aged legumain-deficient mice (45 to 58 weeks old). Legumain-deficient mice did not show any differences in physiologic parameters compared with respective littermates before, during MCAO (45 mins), and the subsequent recovery period of 8 days. Moreover, legumain deficiency had no effect on mortality, infarct volume, and the neurologic deficit determined by the rotating pole test, a standardized grip strength test, and the pole test. However, a reduced number of invading CD74(+) cells in the ischemic hemisphere indicates an involvement in post-stroke inflammation. We conclude that legumain is not essential for the functional deficit after MCAO but may be involved in mechanisms of immune cell invasion.

[One-lung ventilation using a new original connector: with a bronchial blocker and a single lumen tracheal tube].

We experienced 22 consecutive anaesthetic managements requiring one-lung ventilation for thoracotomy. We used our original connector, which enabled us to insert a bronchial blocker cut from Univent tube and fibreoptic bronchoscope into the single lumen tracheal tube without air leakage and instability. The bronchial blocker was easily introduced into the mainstem bronchus of the non-dependent lung. The pulmonary alveoli were well collapsed through air-vent inside the bronchial blocker, and we could offer good surgical field. When one-lung ventilation was finished during surgery, we removed the bronchial blocker with connector, and could insert a larger fibreoptic bronchoscope and a suction catheter into the single lumen tracheal tube. We could detach or exchange the bronchial blocker, when they are not functioning well. This method was excellent in the airway management for usual thoracotomy.