Cloning and biological characterization of human single-chain Fv fragments that mediate neutralization of HIV-1.

OBJECTIVE: To develop recombinant single-chain Fv fragments against HIV-1 gp120. METHODS: A panel of human monoclonal antibody Fv fragments were generated against the HIV-1 gp120 by affinity selection from an antibody library expressed on the surface of filamentous phage. The library was prepared from peripheral blood lymphocytes of an asymptomatic HIV-1-infected mother with a high neutralization titer. This mother did not transmit HIV-1 to her offspring (non-transmitter). Heavy and light chains were initially amplified separately and combined by splicing by overlap extension to generate Fv fragments. RESULTS: Several clones expressing single-chain Fv fragments bind strongly to HIV-1 gp120 and several were found to neutralize cell-free HIV-1IIIB. Gross epitope mapping suggest that different clones bound to different functional regions on the envelope. The clones also exhibited sequence diversity. CONCLUSIONS: This strategy of cloning resulted in the development of functional human-derived antibody reagents with different anti-HIV-1 biological properties in vitro. These recombinant Fv fragments have potential utility as immune reagents, as well as in the design of potential immunotherapeutics. In addition, these antibody reagents may provide information on the relationship between humoral immunity and maternal-fetal (vertical) HIV-1 transmission.

The effect of a preceptorship on nursing performance.

The purpose of this quasi-experimental study was to determine the effectiveness of a preceptorship program in increasing the nursing performance of the new graduate. Before and immediately after participating in a 5-week preceptorship the students completed a background data sheet and Schwirian's Six Dimension Scale of Nursing Performance questionnaire. The findings indicated that in all areas measuring frequency of experiences, the students perceived themselves as having performed nursing activities more frequently as a result of the preceptorship. There were no significant changes in the students' perception of their previous preparation. They were equally satisfied about their academic background before and after the preceptorship. Only one nursing indicator, teaching/collaboration, measured a decrease in the quality of performance. All other indicators showed that, from the students point of view, there was an increase in the quality of their nursing performance. The positive trend of the results indicates to the investigators that there was an increase in the effectiveness of the students nursing performance following the preceptorship experience.

Impassable urethral strictures: percutaneous transvesical catheterization and balloon dilatation.

Urethral strictures that are impassable in retrograde fashion present a special problem to the urologist because they cannot be managed by standard dilatation techniques or direct-vision internal urethrotomy. Open urethroplasty is usually required. We describe a technique to catheterize the urethra in an antegrade fashion and dilate the strictures. Our study involved six patients with impassable urethral strictures that were successfully traversed and dilated. Percutaneous transvesical antegrade catheterization and balloon dilatation are beneficial for patients with bladder outlet obstruction.

Functional characterization of left ventricular segmental responses during the initial 24 h and 1 wk after experimental canine myocardial infarction.

Characterization of the temporal evolution of resting segmental function and inotropic reserve after coronary occlusion may be important in evaluating attempts to salvage ischemic but non-necrotic myocardium. Accordingly, we chronically implanted up to six pairs of pulse-transit piezoelectric crystals in the left ventricular myocardium of dogs to measure segmental wall thickness. Segments were separated into groups according to the loss of net systolic thickening (NET) at 5 min postocclusion of the left anterior descending coronary artery in awake, unsedated dogs. Group 1 included segments with NET values of 67--100+ (percent control); group 2 between 67 and 0; and group 3 less than 0 (paradoxical motion). 5 min after coronary occlusion, group 1 NET was 92 +/- 5% (SEM) although significant decreases occurred in NET in group 2 (36 +/- 4%) and group 3 segments (-33 +/- 5%). Between 5 min and 24 h after coronary occlusion, no further significant changes occurred in NET in groups 1, 2, and 3 crystals. Some segments underwent further functional deterioration between 24 h and 1 wk after left anterior descending coronary artery occlusion, although no overall change occurred in segments with mild to moderate ischemic dysfunction. Segments with NET less than 0 at 24 h, on the other hand, exhibited a reduction in aneurysmal bulging between 24 h and 1 wk from -41 +/- 10 to -23 +/- 11% (n = 12, P = 0.02). Inotropic reserve was assessed with postextrasystolic potentiation (PESP) in 14 dogs, and with infusions of dopamine (11 dogs), and isoproterenol (13 dogs). PESP was the most potent intervention and produced a significant augmentation in NET in group 2 crystals at 1, 2, 4, 6,8, and 24 h after coronary occlusion but only at 1 and 2 h in NET in group 3 crystals. Thus, following experimental coronary occlusion, the evolution of ischemic segmental dysfunction is dynamic and variable. A significant degree of inotropic reserve, as assessed by PESP, dopamine, and isoproterenol, exists in segments with moderate ischemic dysfunction for 24 h but for only 2 h after coronary occlusion in those segments with the most severe ischemic dysfunction. In addition, at least some segmental sites with mild to moderate ischemic dysfunction at 24 h deteriorate further between 24 h and 1 wk after experimental coronary occlusion.

Use of chronically-implanted ultrasonic crystals in the intact heart of the cat.

Methodology has been developed for utilising ultrasonic crystals in closed chest cats to measure alterations in regional left ventricular function. The technique is a modification of one previously utilised to monitor regional left ventricular length and thickness changes in closed chest dogs. The ultrasonic crystals we have utilised in the cat heart can monitor regional left ventricular epicardial length changes chronically and are able to identify changes in inotropic state produced by the administration of either isoprenaline or propranolol. These ultrasonic crystals should be useful in the acute and chronic monitoring of left ventricular function in closed chest and relatively small experimental animals that might be studied in a variety of different protocols.

Abnormal myocardial fluid retention as an early manifestation of ischemic injury.

Fifty-seven isolated, blood perfused, continuously weighed canine hearts have been utilized to study the development of abnormal myocardial fluid retention during early myocardial ischemic injury. Inflatable balloon catheters were positioned around the left anterior descending coronary arteries (LAD) of 54 hearts or the proximal left circumflex coronary arteries of three hearts for study of the following intervals of coronary occlusion: a) 10 minutes followed by 20 minutes of reflow, b) 40 minutes followed by either no reflow or by 20 minutes of reflow, and c) 60 minutes without reflow. After 60 minutes of fixed coronary occlusion, histologic and ultrastructural examination revealed mild swelling of many ischemic cardiac muscle cells in the absence of interstitial edema, cardiac weight gain, and obvious structural defects in cell membrane integrity. After 40 minutes of coronary occlusion and 20 minutes of reflow, significant cardiac weight gain occurred in association with characteristic alterations in the ischemic region, including widespread interstitial edema and focal vascular congestion and hemorrhage and swelling of cardiac muscle cells. Focal structural defects in cell membrane integrity were also noted. The development of abnormal myocardial fluid retention after 40 minutes of LAD occlusion occurred in association with a significant reduction in sodium-potassium-ATPase activity in the ischemic area, but with no significant alteration in either creatine phosphokinase or citrate synthase activity in the same region. Despite the abnormal myocardial fluid retention in these hearts, it was possible pharmacologically to vasodilate coronary vessels with adenosine and nitroglycerin infusion to maintain a consistently high coronary flow following release of the coronary occlusion after 40 minutes and to even exceed initial hyperemic flow values following release of the occlusion when adenosine and nitroglycerin infusion was delayed until 15 minutes after reflow. Thus, the data indicate that impaired cell volume regulation and interstitial fluid accumulation and focal structural defects in cell membrane integrity are early manifestations of ischemic injury followed by reflow, but fail to establish a major role for the abnormal fluid retention in altering coronary blood flow prior to the development of extensive myocardial necrosis. In contrast, fixed coronary occlusion for 60 minutes results in mild intracellular swelling but no significant interstitial edema and no obvious structural defects in cell membrane integrity.