Gas-Selective Catalytic Regulation by a Newly Identified Globin-Coupled Sensor Phosphodiesterase Containing an HD-GYP Domain from the Human Pathogen Vibrio fluvialis.

Globin-coupled sensors constitute an important family of heme-based gas sensors, an emerging class of heme proteins. In this study, we have identified and characterized a globin-coupled sensor phosphodiesterase containing an HD-GYP domain (GCS-HD-GYP) from the human pathogen Vibrio fluvialis, which is an emerging foodborne pathogen of increasing public health concern. The amino acid sequence encoded by the AL536_01530 gene from V. fluvialis indicated the presence of an N-terminal globin domain and a C-terminal HD-GYP domain, with HD-GYP domains shown previously to display phosphodiesterase activity toward bis(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates numerous important physiological functions in bacteria, including in bacterial pathogens. Optical absorption spectral properties of GCS-HD-GYP were found to be similar to those of myoglobin and hemoglobin and of other bacterial globin-coupled sensors. The binding of O2 to the Fe(II) heme iron complex of GCS-HD-GYP promoted the catalysis of the hydrolysis of c-di-GMP to its linearized product, 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), whereas CO and NO binding did not enhance the catalysis, indicating a strict discrimination of these gaseous ligands. These results shed new light on the molecular mechanism of gas-selective catalytic regulation by globin-coupled sensors, with these advances apt to lead to a better understanding of the family of globin-coupled sensors, a still growing family of heme-based gas sensors. In addition, given the importance of c-di-GMP in infection and virulence, our results suggested that GCS-HD-GYP could play an important role in the ability of V. fluvialis to sense O2 and NO in the context of host-pathogen interactions.

Establishment of a novel NFAT-GFP reporter platform useful for the functional avidity maturation of HLA class II-restricted TCRs.

CD4+ T cells that recognize antigenic peptides presented on HLA class II are essential for inducing an optimal anti-tumor immune response, and adoptive transfer of tumor antigen-specific TCR-transduced CD4+ T cells with high responsiveness against tumor is a promising strategy for cancer treatment. Whereas a precise evaluation method of functional avidity, an indicator of T cell responsiveness against tumors, has been established for HLA class I-restricted TCRs, it remains unestablished for HLA class II-restricted TCRs. In this study, we generated a novel platform cell line, CD4-2D3, in which GFP reporter was expressed by NFAT activation via TCR signaling, for correctly evaluating functional avidity of HLA class II-restricted TCRs. Furthermore, using this platform cell line, we succeeded in maturating functional avidity of an HLA class II-restricted TCR specific for a WT1-derived helper peptide by substituting amino acids in complementarity determining region 3 (CDR3) of the TCR. Importantly, we demonstrated that transduction of an avidity-maturated TCR conferred strong cytotoxicity against WT1-expressing leukemia cells on CD4+ T cells, compared to that of its original TCR. Thus, CD4-2D3 cell line should be useful not only to evaluate TCR functional avidity in HLA class II-restricted TCRs but also to screen appropriate TCRs for clinical applications such as cancer immunotherapy.

Menaquinone-4 Suppresses Lipopolysaccharide-Induced Inflammation in MG6 Mouse Microglia-Derived Cells by Inhibiting the NF-κB Signaling Pathway.

The overactivation of microglia is known to trigger inflammatory reactions in the central nervous system, which ultimately induce neuroinflammatory disorders including Alzheimer's disease. However, increasing evidence has shown that menaquinone-4 (MK-4), a subtype of vitamin K2, can attenuate inflammation in the peripheral system. Whereas it was also observed at high levels within the brain, its function in this organ has not been well characterized. Therefore, we investigated the effect of MK-4 on microglial activation and clarified the underlying mechanism. Mouse microglia-derived MG6 cells were exposed to lipopolysaccharide (LPS) either with or without MK-4 pretreatment. Cell responses with respect to inflammatory cytokines (Il-1ß, Tnf-α, and Il-6) were measured by qRT-PCR. We further analyzed the phosphorylation of TAK1, IKKα/ß, and p65 of the NF-κB subunit by Western blotting. We observed that in LPS-induced MG6 cells, MK-4 dose-dependently suppressed the upregulation of inflammatory cytokines at the mRNA level. It also significantly decreased the phosphorylation of p65, but did not affect that TAK1 and IKKα/ß. Furthermore, the nuclear translocation of NF-κB in LPS-induced MG6 cells was inhibited by MK-4. These results indicate that MK-4 attenuates microglial inflammation by inhibiting NF-κB signaling.

Establishment of a novel platform cell line for efficient and precise evaluation of T cell receptor functional avidity.

Adoptive T-cell therapy with T cell receptor (TCR) -engineered T cells is an attractive strategy for cancer treatment and the success in this therapy is dependent on the functional avidity of the transduced TCRs against targeted tumor antigens. Therefore, the establishment of the methodology of the efficient and precise evaluation of TCR functional avidity has been awaited. Here, we show a novel platform cell line, named 2D3, which enables the functional avidity of transduced TCRs to be evaluated efficiently and precisely. In the 2D3, the precise TCR functional avidity of transduced TCRs is easily evaluable by the expression of green fluorescent protein (GFP) reporter gene driven by nuclear factor of activated T cells (NFAT) activation via TCR signaling. Four different TCRs of HLA-A*24:02-restricted Wilms' tumor gene 1 (WT1)-specific CD8+ cytotoxic T lymphocytes (CTLs) were transduced into 2D3 cells and the functional avidities of these four TCRs were evaluated. The evaluated functional avidity of these TCRs positively correlated with cell proliferation, cytokine production, and WT1-specific cytotoxicity of the TCR-transduced CD8+ T cells in response to WT1 antigen. These results showed that 2D3 cell line was a novel and stable tool useful for the efficient and precise evaluation of the functional avidity of isolated and transduced TCRs in developing TCR-based immunotherapy.

Chemical and Genetic Study of two Ligularia Hybrids in Shangrila County, Yunnan Province, China.

The composition of root chemicals was examined for samples of L. cyathiceps x L. duciformis, L. duciformis x L. yunnanensis, and L. yunnanensis. Various furanoeremophilanes were isolated from a sample of L. cyathiceps x L. duciformis and found to be very similar to those isolated from L. cyathiceps. Lupeol and docosyl ferulate were isolated from L. yunnanensis, L. duciformis, and L. duciformis x L. yunnanensis.

[Changes in Appetite, Nutrition, and Oxidative Stress Reaction of Cancer Patients during Chemotherapy].

Weight loss during chemotherapy is a result of malnutrition and metabolism abnormality. A few reports have focused on the treatment and prevention of weight and appetite loss in patients with advanced cancer. We evaluated the relationship between weight and appetite loss during chemotherapy by studying the meal intake of patients. In addition, we also investigated whether anorexia is associated with the level of 8-hydroxy-2' -deoxyguanosine (8-OHdG), an oxidation stress marker. The weight loss rate in patients who lack energy intake was significantly higher than that in patients with adequate energy intake. Moreover, the pre-chemotherapy total energy intake, measured according to the hospital meal consumption of patients, was lower among those with than among those without anorexia. The 8-OHdG levels in the patients with anorexia were significantly higher. In conclusion, nutritional management is important for patients even before chemotherapy is started. The 8-OHdG level can be used as an index to evaluate appetite.

Transduction of a novel HLA-DRB1*04:05-restricted, WT1-specific TCR gene into human CD4+ T cells confers killing activity against human leukemia cells.

BACKGROUND/AIM: Wilms' tumor gene 1 (WT1) product is a pan-tumor-associated antigen. We previously identified WT1 protein-derived promiscuous helper peptide, WT1332. Therefore, isolation and characterization of the WT1332-specific T-cell receptors (TCRs) are useful to develop broadly applicable TCR gene-based adoptive immunotherapy. MATERIALS AND METHODS: A novel HLA-DRB1*04:05-restricted WT1332-specific TCR gene was cloned and transduced into human CD4+ T-cells by using a lentiviral vector. RESULTS: The WT1332-specific TCR-transduced CD4+ T-cells showed strong proliferation and Th1-cytokine production in an HLA-DRB1*04:05-restricted, WT1332-specific manner. Furthermore, the WT1332-specific TCR-transduced CD4+ T-cells could lyse HLA-DRB1*04:05-positive, WT1-expressing leukemia cells in vitro. CONCLUSION: The novel TCR gene cloned here should be a promising tool to develop adoptive immunotherapy of WT1332-specific TCR-transduced CD4+ T-cells for the treatment of WT1-expressing cancer, such as leukemia.

HLA-DPB1*05: 01-restricted WT1332-specific TCR-transduced CD4+ T lymphocytes display a helper activity for WT1-specific CTL induction and a cytotoxicity against leukemia cells.

Wilms tumor gene 1 (WT1) is overexpressed in various malignant neoplasms, and has been demonstrated as an attractive target for cancer immunotherapy. We previously reported the identification of a WT1 protein-derived, 16-mer helper peptide WT1332 that could elicit Th1-type CD4+ T-cell response and bind to multiple HLA class II molecules. In this study, we examined the feasibility of adoptive therapy using CD4+ T cells that were transduced an HLA-DPB1*05:01-restricted, WT1332-specific T-cell receptor (TCR). HLA-DPB1*05:01-restricted, WT1332-specific TCR-transduced CD4+ T cells were successfully generated using lentiviral vector and exhibited strong proliferative response and Th1-type cytokine production in response to WT1332 peptide, WT1 protein, or WT1-expressing tumor cell lysate. Furthermore, the WT1332-specific TCR-transduced CD4+ T cells lysed HLA-DPB1*05:01-positive, WT1-expressing human leukemia cells through granzyme B/perforin pathway. Furthermore, stimulation of peripheral blood mononuclear cells with both HLA-A*24:02-restricted cytotoxic T lymphocytes-epitope peptide (modified 9-mer WT1235 peptide, WT1235m) and WT1332 helper peptide in the presence of WT1332-specific TCR-transduced CD4+ T cells strikingly enhanced the induction of WT1235m-specific cytotoxic T lymphocytes. Thus, these results demonstrated the feasibility of immunotherapy based on adoptive transfer of WT1332-specific TCR-transduced CD4+ T cells for the treatment of leukemia.

A noninvasive partial carbon dioxide rebreathing technique for measurement of pulmonary capillary blood flow is also a useful oxygenation monitor during one-lung ventilation.

STUDY OBJECTIVES: To test the hypothesis that effective pulmonary capillary blood flow can be a useful indicator for estimating appropriate oxygenation and ventilation during one-lung ventilation in lung surgery. DESIGN: Prospective data analysis. SETTING: A 770-bed general teaching hospital. PATIENTS: 15 ASA physical status II and III patients undergoing elective lung surgery. INTERVENTIONS: Patients received general and thoracic epidural anesthesia and underwent lung operation with one-lung ventilation. MEASUREMENTS: We measured effective pulmonary capillary blood flow by a partial CO2 rebreathing method and oxygenation parameters during two-lung ventilation before surgery, during one-lung ventilation, and during two-lung ventilation after lung surgery. MAIN RESULTS: The effective pulmonary capillary blood flow index significantly decreased by 31.6%, which was associated with a significant decrease in arterial oxygen tension (PaO2). The pulmonary shunt fraction increased to 46.3% during one-lung ventilation. During two-lung ventilation, after chest closure, effective pulmonary capillary blood flow index divided by heart rate (i.e., effective pulmonary blood stroke flow index) was still significantly lower than that seen during two-lung ventilation before thoracotomy. There were significant correlations between effective pulmonary capillary blood flow, pulmonary blood stroke flow index, and PaO2. CONCLUSIONS: Effective pulmonary capillary blood flow index and effective pulmonary blood stroke flow index are useful indicators for determining appropriate oxygenation therapy during one-lung ventilation.