In vitro expansion of CD34(+)CD38(-) cells under stimulation with hematopoietic growth factors on AGM-S3 cells in juvenile myelomonocytic leukemia.

Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34(+) cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34(+) cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34(+) cells on AGM-S3 cells. The expansion potential of CD34(+) cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34(+) cells were negative for CD38 and cryopreservable. Cultured JMML CD34(+)CD38(-) cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34(+) cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.

Loss of interleukin-2-dependency in HTLV-I-infected T cells on gene silencing of thioredoxin-binding protein-2.

The transition from interleukin-2 (IL-2)-dependent to IL-2-independent growth is considered one of the key steps in the transformation of human T-cell leukemia virus type-I (HTLV-I)-infected T cells. The expression of thioredoxin-binding protein-2 (TBP-2) is lost during the transition of HTLV-I-infected T-cell lines. Here, we analysed the mechanism of loss of TBP-2 expression and the role of TBP-2 in IL-2-dependent growth in the in vitro model to investigate multistep transformation of HTLV-I. CpGs in the TBP-2 gene are methylated in IL-2-independent but not in IL-2-dependent cells. Sequential treatment with 5-aza-2'-deoxycytidine and a histone deacetylase inhibitor augmented histone acetylation and TBP-2 expression, suggesting that loss of TBP-2 expression is due to DNA methylation and histone deacetylation. In IL-2-dependent cells, a basal level of TBP-2 expression was maintained by IL-2 associated with cellular growth, whereas TBP-2 expression was upregulated on deprivation of IL-2 associated with growth suppression. Overexpression of TBP-2 in IL-2-independent cells suppressed the growth and partially restored responsiveness to IL-2. Knockdown of TBP-2 caused the IL-2-dependent cells to show partial growth without IL-2. These results suggested that epigenetic silencing of the TBP-2 gene results in a loss of responsiveness to IL-2, contributing to uncontrolled IL-2-independent growth in HTLV-I-infected T-cell lines.

Redox control of cellular function by thioredoxin; a new therapeutic direction in host defence.

Compelling evidence has suggested that oxidative stress mediates various cellular responses, and control of reduction/oxidation (redox) is important in maintaining the homeostasis of an organism. The thioredoxin (TRX) system, as well as the glutathione system, is one of the key systems in controlling cellular redox status. TRX is a small ubiquitous protein with the redox-active site sequence -Cys-Gly-Pro-Cys-. It has been demonstrated to be a multifunctional protein, which has regulatory roles in cellular signaling and gene transcription in addition to cytoprotective activities through the quenching of reactive oxygen species. Various oxidative stimuli, such as UV irradiation, cytokines and some chemicals, promptly induce the expression of TRX. Overexpression of TRX correlates with a wide variety of oxidative stress conditions and, in some cases, TRX has shown promising effects for clinical use, for instance in the attenuation of tissue injury in ischemia reperfusion models. The modulation of TRX functions in association with other redox-regulatory molecules should give us a new therapeutic strategy in the treatment of oxidative stress-mediated disorders and diseases.

Circulating thioredoxin suppresses lipopolysaccharide-induced neutrophil chemotaxis.

Thioredoxin (Trx), a redox enzyme with a conserved active site (Cys-32-Gly-Pro-Cys-35), is induced and secreted into circulation in response to inflammation. Studies here demonstrate that elevating Trx levels in circulation either by i.v. injection of recombinant Trx or stimulating Trx release in Trx-transgenic mice dramatically blocks lipopolysaccharide (LPS)-stimulated neutrophil migration in the murine air pouch chemotaxis model. Furthermore, we show that leukocyte recruitment induced by the murine chemokines KC/GROalpha, RANTES (regulated upon activation, normal T cell expressed and secreted), and monocyte chemoattractant protein-1 (MCP-1) is suppressed also in Trx-transgenic mice. Addressing the mechanism responsible for this suppression, we show that circulating Trx blocks (i) the LPS-stimulated in vitro activation of neutrophil p38 mitogen-activated protein kinase, (ii) the normal down-regulation of CD62L on neutrophils migrating into the LPS-stimulated air pouch, and (iii) the in vitro adhesion of LPS-activated neutrophils on endothelial cells. However, as we also show, Trx does not alter the expression of endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD62P, and CD62E) within 3 h. Collectively, these findings indicate that elevated levels of circulating Trx interfere with chemotaxis by acting directly on neutrophils. We discuss these findings in the context of recent studies reporting beneficial effects of acutely elevated Trx in ischemic injury and negative effects associated with chronically elevated Trx in HIV disease.

Regulatory roles of thioredoxin in oxidative stress-induced cellular responses.

Thioredoxin (TRX) is a small ubiquitous and multifunctional protein having a redox-active dithiol/disulfide within the conserved active site sequence -Cys-Gly-Pro-Cys-. TRX is induced by a variety of oxidative stimuli, including UV irradiation, inflammatory cytokines and chemical carcinogens, and has been shown to play crucial roles in the regulation of cellular responses such as gene expression, cell proliferation and apoptosis. Overexpression of TRX protects cells from cytotoxicity elicited by oxidative stress in both in vitro and in vivo models. The regulatory mechanism of TRX expression and activity is also being elucidated. Recently, TRX binding protein-2 (TBP-2)/vitamin D3 up-regulated protein 1 (VDUP1) was identified as a negative regulator of TRX. The analysis of TRX promoter region has revealed putative regulatory elements responsible for oxidative stress. Thus, the modulation of TRX functions may be a new therapeutic strategy for the treatment of oxidative stress-mediated diseases.

Redox regulation by thioredoxin and thioredoxin-binding proteins.

Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys- and constitutes a major thiol reducing system, the thioredoxin system. Thioredoxin plays multiple roles in cellular processes such as proliferation or apoptosis. It also promotes DNA binding of transcription factors such as NF-kappaB, AP-1, p53, and PEBP2. Overexpression of thioredoxin suppresses the degradation of IkappaB and the transactivation of NF-kappaB, whereas overexpression of nuclear-targeted thioredoxin exhibits the enhancement of NF-kappaB-dependent transactivation. ASK1, a MAP kinase kinase kinase mediating the TNF-alpha signal has been identified as a thioredoxin binding protein. Thioredoxin shows an inhibitory effect on the TNF-alpha induced activation of ASK1 and p38 MAP kinase pathway. We identified p40phox as the thioredoxin binding protein-1 (TBP-1) and vitamin D3 up-regulated protein 1 (VDUP1) as the thioredoxin binding protein-2 (TBP-2) by yeast two-hybrid system. TBP-2/VDUP1 negatively regulates the expression and reducing activity of thioredoxin. Thioredoxin interacting proteins may be involved in thioredoxin-mediating redox regulation.

Redox control of EBV infection: prevention by thiol-dependent modulation of functional CD21/EBV receptor expression.

CD21 serves as a receptor for the Epstein-Barr virus (EBV). In this report, surface expression of CD21 on B and T cells was shown to be suppressed by a thiol-antioxidant, N-acetylcysteine (NAC), in a dose- and time-dependent manner. In contrast, expression of other surface markers, CD25 and CD4 for T cells and CD19 and surface IgM for B cells, was not affected by NAC. When an EBV-negative B-cell line B104 was treated with NAC, the cells were not susceptible to infection with B95-8-derived EBV. The effect of NAC was shown to be irrelevant to the transcriptional levels of CD21 mRNA and the intracellular glutathione levels. Immunoprecipitation study revealed that NAC causes a loss of anti-CD21 monoclonal antibody (HB5) binding to both membrane and soluble CD21, suggesting that NAC modulates the structure of CD21. Other thiol-antioxidants, such as 2-mercaptoethanol, pyrrolidine dithiocarbamate, and glutathione, showed similar effect to NAC on CD21 expression. These results suggest the possible modulation of EBV infection via thiol-dependent redox control of CD21, and thiol-antioxidants may be good candidates for controlling EBV infection.

Expression of thioredoxin and glutaredoxin, redox-regulating proteins, in pancreatic cancer.

Pancreatic cancer (pancreatic ductal carcinoma) is one of the most malignant solid tumors with poor prognosis. There is accumulating evidence that cellular reduction/oxidation (redox) status is deeply involved in the growth promotion and drug resistance of cancer cells. We therefore investigated the expression of redox-regulating proteins, such as thioredoxin (TRX) and glutaredoxin (GRX) in surgically resected pancreatic tissues and cis-diamminedichloroplatinum (CDDP)-resistant cells. Plasma levels of TRX were also measured in subjects with pancreatic diseases. Pancreatic ductal carcinoma tissues were immunohistochemically more positive for TRX (24/32 cases) and GRX (29/32 cases) than pancreatic cystadenocarcinoma or normal pancreas tissues. Plasma levels of TRX (mean +/- SD) measured by ELISA were significantly higher in patients with pancreatic ductal carcinoma (54.8 +/- 37.6 ng/ml, n = 60) than in healthy controls (24.4 +/- 12.9 ng/ml, n = 81). CDDP-resistant subclones of HeLa cells, HeLa-CP5 cells, had higher expression of TRX (1.5 fold) and GRX (1.6 fold) compared with parental HeLa cells by immunoblotting. These results indicate the possible association of TRX and GRX with malignant potential of pancreatic ductal carcinoma.

Structural and immunochemical characterization of a Neisseria gonorrhoeae epitope defined by a monoclonal antibody 2C7; the antibody recognizes a conserved epitope on specific lipo-oligosaccharides in spite of the presence of human carbohydrate epitopes.

Lipo-oligosaccharides (LOS) produced by Neisseria gonorrhoeae are important antigenic and immunogenic components of the outer membrane complex. Previously, we showed that murine monoclonal antibody (mAb) 2C7 did not cross-react with human glycosphingolipids but identified the LOS epitope that is widely expressed in vivo and in vitro (Gulati, S., McQuillen, D. P., Mandrell, R. E., Jani, D. B., and Rice, P. A. (1996) J. Infect. Dis. 174, 1223-1237). In the present study, we analyzed the structure of gonococcal strain WG LOS containing the 2C7 epitope and investigated the structural requirements for expression of the epitope. We determined that the WG LOS components are Hep[1]-elongated forms of 15253 LOS that have a lactose on both Hep[1] and Hep[2] (Yamasaki, R., Kerwood, D. E., Schneider, H., Quinn, K. P., Griffiss, J. M., and Mandrell, R. E. (1994) J. Biol. Chem. 269, 30345-30351). In addition, we found that expression of the 2C7 epitope within the LOS is blocked when the Hep[2]-lactose is elongated. Based on the structural data of these LOS and the results obtained from immunochemical analyses, we conclude the following: 1) mAb 2C7 requires both the 15253 OS minimum structure and the N-linked fatty acids in the lipoidal moiety for expression of the epitope; 2) mAb 2C7 binds to the LOS that elongates the lactose on Hep[1] of the 15253 OS, but not the one on Hep[2]; and 3) the 2C7 epitope is expressed on gonococcal LOS despite the presence of human carbohydrate epitopes such as a lactosamine or its N-acetylgalactosaminylated (globo) form. Our study shows that the conserved epitope defined by mAb 2C7 could potentially be used as a safe site for the development of a vaccine candidate.

IgM anti-ganglioside antibodies induced by melanoma cell vaccine correlate with survival of melanoma patients.

Melanoma cells express ganglioside antigens GM3, GD3, GM2, and GD2 on their surface. This study examined whether immunization with a melanoma cell vaccine induced anti-ganglioside antibody responses in melanoma patients and whether these responses were correlated with survival. Sixty-six patients who had received melanoma cell vaccine immunotherapy after surgical removal of regional metastatic melanoma were identified. Cryopreserved serum samples from these patients were used in an enzyme-linked immunsorbent assay to determine the IgM antibody levels to GM2, GD2, GM3, and GD3 prior to melanoma cell vaccine treatment and 4 wk after the first melanoma cell vaccine immunization. All antibody levels significantly increased by week 4 (p < 0.001 for all four antibodies) and all increases were significantly associated with survival (anti-GD2, p < 0.001; anti-GM2, p = 0.001; anti-GD3, p < 0.001; anti-GM3, p < 0.001). Anti-tumor activity of these antibodies was proved using five representative antibody-positive sera in a complement-dependent cytotoxicity assay with cultured melanoma cell lines. These studies suggest that GM2, GD2, GM2, and GD3 expressed by melanoma cells can induce specific IgM antibodies and that high levels of these antibodies might have a beneficial impact on survival.

707-AP peptide recognized by human antibody induces human leukocyte antigen A2-restricted cytotoxic T lymphocyte killing of melanoma.

We recently identified a tumor-associated antigen that was recognized by human monoclonal antibody L94. The antibody-reactive 707-AP sequence RVAALARDAP, cloned from a melanoma cDNA library, was also found to be recognized by peripheral blood lymphocytes (PBLs) from melanoma patients. In this study, 707-AP was used to stimulate melanoma patients' PBLs for the establishment of peptide-specific CTL cell lines. CTL cell lines derived from 258 melanoma patients of different human leukocyte antigen (HLA)-A and HLA-B allele expressions were assessed by a 51Cr cytotoxicity assay against the peptide-pulsed autologous B lymphoblastoid cells and T2 HLA-A2 antigen-presenting cells and autologous and allogeneic melanoma cell lines. The analysis of 707-AP CTL activity demonstrated that only HLA-A2 patients' PBLs could be stimulated with 707-AP. 707-AP CTLs were able to specifically lyse HLA-A2 autologous and allogeneic melanoma cell lines. This verified the endogenous processing and presentation of 707-AP by melanoma cells. 707-AP CTL cytotoxicity against peptide-pulsed autologous HLA-A2 B lymphoblastoid cells and T2 HLA-A2 cells was also demonstrated. The killing activity of HLA-A2 707-AP CTL cell lines (CD8+ CD3+) was inhibited by anti-HLA class and anti-HLA-A2 monoclonal antibodies. The amino acid substitution or deletion analysis of the 707-AP sequence in CTL stimulation and recognition confirmed that position 2, amino acid V and position 9, amino acid A were essential. Both positions are known as supermotif anchors for HLA-A2 peptide sequences. Our studies demonstrated that 707-AP is a potent stimulator of CTLs that can induce peptide-specific HLA-A2 melanoma cell killing. The recognition of 707-AP by both antibody and CTLs suggests its potential significance as a peptide immunotherapeutic.

Development of a human monoclonal antibody to ganglioside G(M2) with potential for cancer treatment.

A human B-lymphoblastoid cell clone, L55-81, that produces human monoclonal antibody (MAb) to ganglioside G(M2) was established from peripheral blood B lymphocytes of a melanoma patient. L55-81 secretes IgMkappa light chain antibody in a serum-free medium. G(M2) specificity of the antibody was tested by immune adherence assay, TLC immunostaining, and ELISA. Anti-G(M2) antibody was shown to have the ability to kill the G(M2)-rich human melanoma cell line M14 in the presence of human or rabbit complement. A purified L55-81 MAb (>99.5% purity in protein concentration) was biotinylated and tested for its reactivity to various histological-type biopsied tumor and normal tissues in an avidin-biotin detection system. L55-81 MAb (20 microg/ml) reacted with several types of tumor tissues such as melanoma (7 of 10), colon carcinoma (4 of 5), ovary carcinoma (4 of 5), breast carcinoma (1 of 5), kidney carcinoma (1 of 5), and prostate carcinoma (1 of 5). None of the normal tissues derived from 24 different organs and adjacent normal tissues surrounding the cancerous tissues were stained. Production of the antibody in a serum-free medium, the cytotoxic potential with human complement, the inability to react to normal tissues, and the ability to target antigen-specific target cells make L55-81 a potential therapeutic agent for the treatment of cancers expressing ganglioside G(M2).

Correlation between the exercise-induced increase in left ventricular filling pressure and the extent of ischemic or infarcted myocardium.

We investigated the correlation between left ventricular filling pressure and the extent of ischemic or infarcted myocardium in 39 patients with coronary artery disease: 25 with angina pectoris (group A) and 14 with old myocardial infarction but without overt transient myocardial ischemia (group B). Hemodynamic parameters were measured at rest and during exercise. The extent and severity scores of ischemia or infarct were calculated using thallium-201 (201Tl) myocardial single-photon emission computed tomography. In group A, the extent and severity scores of ischemia were strongly correlated with pulmonary artery wedge pressure at peak exercise (r = 0.71, p < 0.001, r = 0.62, p < 0.01, respectively). In group B, the extent and severity scores of the infarct were significantly correlated with left ventricular ejection fraction (r = -0.81, p < 0.001, r = -0.77, p < 0.01, respectively), but were not correlated with pulmonary artery wedge pressure. Since no relationship was found between the extent of infarct and left ventricular filling pressure, dynamic exercise appears to elicit a different compensatory mechanisms in nonischemic myocardium for exercise-induced transient ischemia and in noninfarcted myocardium for old infarction. The compensatory mechanism in patients with old myocardial infarction may be affected by ventricular remodeling.

Improvement in exercise-induced left ventricular dysfunction by infusion of alpha-human atrial natriuretic peptide in coronary artery disease.

The effects of recombinant alpha-human atrial natriuretic peptide (alpha-hANP) infusion an acute left ventricular dysfunction provoked by exercise were examined in 14 men with coronary artery disease. Patients performed symptom-limited, graded exercise on a supine bicycle ergometer. Plasma alpha-hANP and guanosine 3',5'-monophosphate (cyclic GMP) concentrations as well as hemodynamic variables were measured at rest, during and after exercise. In 14 patients whose pulmonary artery wedge pressure was > 20 mm Hg at peak exercise, the same exercise protocol was repeated at 30 minutes after starting intravenous alpha-hANP infusion (0.05 microgram.kg-1.min-1). In 8 of these patients, a Webster thermodilution catheter was advanced into the coronary sinus for measurement of coronary sinus blood flow. From the control exercise test, plasma alpha-hANP concentration increased from 86 +/- 20 pg/ml at rest to 188 +/- 32 pg/ml at peak exercise (p < 0.001), and plasma cyclic GMP concentration increased from 4.8 +/- 1.9 pmol/ml at rest to 7.2 +/- 2.9 pmol/ml at peak exercise (p < 0.001). Both plasma alpha-hANP and cyclic GMP concentrations showed a significant positive correlation with pulmonary artery wedge pressure during control exercise. With alpha-hANP infusion, systolic and diastolic pulmonary artery pressures and pulmonary artery wedge pressure were significantly decreased at all time points during exercise testing. Heart rate was increased and systolic blood pressure was significantly decreased at rest and at 3 minutes of exercise. Diastolic blood pressure, systemic vascular resistance, and pulmonary vascular resistance were significantly decreased at rest.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic effects of the calcium channel blocker pranidipine on exercise-induced angina.

Hemodynamic effects of a newly developed calcium channel blocker, pranidipine, on dynamic exercise-induced angina were investigated. Ten patients with stable effort angina pectoris underwent symptom-limited bicycle ergometer exercise testings before and after a single oral administration of pranidipine, and effects of pranidipine on systemic, cardiac and coronary hemo-dynamics induced by dynamic exercise were evaluated invasively. Pranidipine administration reduced systemic vascular resistance (from 1,764 +/- 109 to 1,115 +/- 60 dynes.sec/cm5; p < 0.01 at test, and from 1,120 +/- 102 to 795 +/- 62 dynes.sec/cm5; p < 0.05 at peak exercise) and mean arterial pressure (from 93 +/- 5 to 76 +/- 3 mmHg; p < 0.01 at test, and from 85 +/- 7 to 72 +/- 6 mmHg; p < 0.05 at peak exercise) with the increase in heart rate and cardiac index throughout exercise. Pranidipine also decreased coronary vascular resistance from 1.29 +/- 0.21 to 0.89 +/- 0.17 mmHg/ml/min (p < 0.05) at resting condition. At peak exercise, rate-pressure product and myocardial oxygen consumption decreased (from 237 +/- 21 to 215 +/- 18 x 10(2); p < 0.05, and from 31.3 +/- 7.5 to 21.7 +/- 3.9 ml/min; p < 0.05, respectively), while coronary vascular resistance did not change significantly. Furthermore, pranidipine mitigated ST-segment depression and elevation of pulmonary artery wedge pressure at peak exercise (from 0.20 +/- 0.03 to 0.13 +/- 0.02 mV; p < 0.01, and from 25 +/- 3 to 11 +/- 2 mmHg; p < 0.01, respectively). These results suggest that the major therapeutic effects of pranidipine for dynamic exercise-induced angina would be to reduce myocardial oxygen demand by improving peripheral circulation and reducing preload and afterload.(ABSTRACT TRUNCATED AT 250 WORDS)

[Myocardial metabolism during dynamic exercise].

The development of Webster catheterization has enabled us to make clinical measurement of coronary sinus blood flow and to estimate the kinetics in myocardial substrate uptake. In this study, exercise tolerance test using supine multistage bicycle ergometer test was performed and exercise-induced hemodynamic alteration was evaluated in 18 patients with ischemic heart disease (16 males and 2 females, average age 56.3 years). The change in substrate kinetics in myocardial metabolism was also examined in terms of two indexes; myocardial uptake rate and myocardial uptake. The following results were obtained. 1) The myocardial uptake rates of glutamate and free fatty acid were significantly decreased by exercise, while those of glucose and lactate showed no significant change. 2) The myocardial uptakes of glutamate, glucose and lactate were significantly increased by exercise, but that of free fatty acid did not change significantly. 3) A significant negative correlation (r = -0.52, p < 0.05) was observed between the change in myocardial glutamate uptake and the change in pulmonary artery wedge pressure induced by exercise, suggesting that patients with ischemic heart disease might fail in glutamate uptake induced by exercise. The difference in the kinetics of myocardial uptake rate and myocardial uptake for glutamate, alanine, glucose, free fatty acid and lactate is observed. This difference occurs from the decrease in uptake rate due to the increase in coronary sinus blood flow. Myocardial uptake, which directly reflects myocardial energy metabolism is regarded as a more useful index of myocardial metabolism than myocardial uptake rate.

Abnormal postexercise systolic blood pressure response is a good indicator of impaired left ventricular filling during supine cycle ergometer exercise in patients with coronary artery disease.

To determine whether the postexercise systolic blood pressure (SBP) response is a useful marker of left ventricular filling abnormalities, supine leg exercise testing was conducted in 14 control subjects and 70 patients with coronary artery disease (CAD). An abnormal postexercise SBP response (the ratio of SBP after 3 min of recovery to the peak exercise SBP) was defined as 0.85 or more, which represented the cutoff point with the highest sensitivity and specificity for prediction of pulmonary artery wedge pressure (PAWP) of at least 20 mmHg at peak exercise in CAD patients. There was a significant difference between the SBP ratios of the two groups (Control, 0.72 +/- 0.05; CAD, 0.86 +/- 0.13; p < 0.01). There was no significant difference between the PAWP of the two groups at rest, but the PAWP at peak exercise was significantly higher in the CAD group (20.2 +/- 8.9 mmHg) than in the control group (11.5 +/- 4.0 mmHg)(p < 0.01). PAWP at peak exercise was > or = 20 mmHg in 35 (50%) of the 70 CAD subjects. The SBP ratio was significantly correlated with PAWP at peak exercise (r = 0.67, p < 0.01) in the CAD group, but not in the control group. An SBP ratio of > or = 0.85 showed a sensitivity of 80% and a specificity of 80% for predicting a peak exercise PAWP of > or = 20 mmHg in CAD patients.(ABSTRACT TRUNCATED AT 250 WORDS)

A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells.

A sensitive chemiluminescence method for measuring the production of superoxide anion (O2-) by activated EoL-1 cells (human eosinophilic leukemia cell line) is described. Recently, we succeeded in synthesizing a new chemiluminescence probe, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012). In the presence of L-012, activated EoL-1 cells which produce reactive oxygen species generated a marked chemiluminescence with negligible background. The L-012-dependent chemiluminescence was completely abolished by 100-300 U/ml superoxide dismutase, indicating that the main reactive oxygen species detected in this reaction was O2-. The light intensity and the sensitivity of L-012 to O2- were higher than those of other chemiluminescence probes such as luminol and Cypridina luciferin analog (MCLA). Thus, L-012 would provide an improved chemiluminescence method for measuring O2- from cells.