ABSTRACT
In this study, we describe the creation of three interferon-alpha (IFN-alpha)8 mutants with markedly higher antiviral and antiproliferative activities in comparison with those of the wild-type (wt)IFN-alpha8, wtIFN-alpha2, and IFN-con1 using a phage display system. Sequence analysis showed that three out of the six hot-spot amino acid residues of wtIFN-alpha8 known to be important for the interaction with the IFN-alpha receptor-2 (IFNAR-2)-binding sites were substituted to other amino acids and the others remained. Although affinity analysis revealed that the dissociation constant (K(D)) of IFN-alpha8 mutants was almost the same with that of wtIFN-alpha8, furthermore, the rates of association (k(a)) and dissociation (k(d)) were relatively lower. These results suggest that changes in the surface electronic charge of amino acid residues lead to changes in binding affinity and kinetics (prolonged dissociation time) toward the IFNAR-2, resulting in the modification of the biological activity. Moreover, our results demonstrate that the molecular engineering of the IFN-alpha8 provides important insight into action of IFN and also it would be useful in the development of therapeutically prominent IFN preparations than those used in clinical practice.
Subject(s)
Amino Acid Substitution , Interferon-alpha/genetics , Interferon-alpha/metabolism , Receptor, Interferon alpha-beta/metabolism , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites/genetics , Binding, Competitive , Cell Line, Tumor , Cell Proliferation/drug effects , Growth Inhibitors/chemistry , Growth Inhibitors/pharmacology , Hep G2 Cells , Humans , Interferon Type I/genetics , Interferon Type I/metabolism , Interferon Type I/pharmacology , Interferon-alpha/pharmacology , Kinetics , Models, Molecular , Mutation , Peptide Library , Protein Binding , Protein Conformation , Recombinant Proteins , Sindbis Virus/drug effects , Surface Plasmon Resonance , U937 Cells , Vesicular stomatitis Indiana virus/drug effectsABSTRACT
BACKGROUND: Visual or quantitative assessment of coronary angiography may not exactly predict the physiological significance of intermediate (40-70%) coronary stenosis. Coronary flow reserve is a well-established marker of the functional significance of coronary stenosis. OBJECTIVES: The aim of this study was to compare the coronary flow velocity reserve (CFVR) using contrast-enhanced transthoracic Doppler echocardiography (CE-TTDE) with thallium-201 imaging in assessment of intermediate lesions in the left anterior descending coronary artery (LAD). METHODS: A consecutive series of 50 patients with intermediate stenosis in the LAD underwent pharmacological stress thallium-201 imaging and CFVR measured by CE-TTDE. RESULTS: CFVR could be measured in 49 of 50 patients by the present method. A CFVR <2.0 predicted the presence of a stress thallium defect in 12 of 14 patients (agreement=90%, kappa=0.76, P < 0.001). The sensitivity and specificity of CFVR for stress thallium-201 results were 86 and 91%, respectively. In contrast, significant stenosis (>50% by diameter) showed fair agreement for stress thallium defects (agreement=59%, kappa=0.28, P < 0.05). CONCLUSIONS: In the evaluation of intermediate lesions in the LAD, CFVR as assessed by CE-TTDE could accurately predict the presence of ischemia on stress thallium imaging, whereas angiographic stenosis did not yield reliable results.
Subject(s)
Coronary Stenosis/diagnosis , Echocardiography , Image Enhancement , Adenosine Triphosphate , Aged , Arteries/diagnostic imaging , Arteries/physiopathology , Blood Flow Velocity/physiology , Coronary Angiography , Coronary Circulation/physiology , Coronary Stenosis/epidemiology , Coronary Stenosis/physiopathology , Coronary Vessels/diagnostic imaging , Coronary Vessels/physiopathology , Echocardiography, Doppler/methods , Female , Follow-Up Studies , Humans , Infusions, Intravenous , Male , Middle Aged , Observer Variation , Sensitivity and Specificity , Severity of Illness Index , Thallium Radioisotopes , Tomography, Emission-Computed, Single-PhotonABSTRACT
BACKGROUND: This study assessed the feasibility of transthoracic Doppler scan echocardiography (TTDE) combined with echo-contrast agent in measuring coronary flow velocity (CFV) and coronary flow velocity reserve (CFVR) in the left anterior descending artery. METHODS: In 68 consecutive patients who underwent cardiac angiography, TTDE was recorded before and after induction of a hyperemic condition with intravenous administration of adenosine triphosphate (0.14 mg/kg/min). After CFV values returned to baseline, the same measurements were repeated while an echo-contrast agent was continuously infused. CFVR was assessed as the ratio of hyperemic to basal CFV. The pulsed wave Doppler scan quality was graded from 1 to 3 (TTDE score: 1, no signal detection; 2, poor definition of outline; 3, optimal outline definition). RESULTS: Before enhancement, CFVR could not be measured in 20 patients. Prevalence of delayed stenosis (Thrombolysis In Myocardial Infarction [TIMI] II grade flow) in these patients (30%) was significantly greater than in those whose CFVRs could be measured without enhancement (2%; P <.01). TTDE scores both at baseline and after hyperemia were significantly improved with contrast enhancement (before, 2.8 +/- 0.6; after, 3.0 +/- 0.3; P <.01; before, 2.6 +/- 0.7; after, 3.0 +/- 0.3; P <.01; respectively). Overall contrast enhancement increased the rate of successful CFVR measurement from 70% to 97% (P <.01). Sensitivity and specificity of significant left anterior descending artery stenosis detection with CFVR of <2.0 were 94.4% and 87.8%, respectively. CONCLUSION: These data suggest that administration of echo-contrast agent improves pulse wave Doppler scan quality and thus the feasibility of measuring CFVR.
