Tetramer binding and secretion of interferon-gamma in response to antigenic stimulation are compatible with a range of affinities of MHC:TCR interaction and distinct programs of cytotoxic T-lymphocyte activation

Tetramer staining and detection of IFN-gamma secretion in response to specific stimulation are widely used to quantify and isolate specific T-cells. However, it remains unclear how these assays reflect different functional outcomes of T-cell triggering with MHC:peptide ligands. An immunogenic EBV-derived A11-restricted CTL peptide epitope and its partially agonistic analogue trigger different programs of activation induced cell death (AICD) in specific CTLs. In this study we analysed a panel of CTL clones, bulk CTL cultures and PBMCs isolated from HLA A11-positive EBV-infected individuals for their ability to bind tetrameric complexes assembled with either of the two peptides and correlated tetramer binding with the activity of the peptides in functional assays. This analysis demonstrates that specific tetramer staining and secretion of IFN-gamma are compatible with at least two activation programs in CTLs. One of these programs corresponds to full-scale CTL activation and death of a proportion of activated T-cells in a Fas-dependent manner. In contrast, the alternative program is characterized by selective induction of IFN-gamma and TNF-alpha, absence of proliferative response and Fas-independent cell death. These findings may have important implications for the evaluation of data obtained with MHC:peptide tetramers and IFN-gamma secretion assays, especially in experimental systems with extensive antigenic variability.     

The use of on-line and off-line chromatographic extraction techniques coupled with mass spectrometry for support of in vivo and in vitro assays in drug discovery

We have investigated various sample chromatographic extraction and sample preparation methods for liquid chromatography mass spectrometry analysis in order to increase the throughput of various in vivo and in vitro assays in support of drug discovery. The results indicated that direct plasma injection, although certainly faster than conventional protein precipitation for sample preparation, had problems associated with column longevity and overall robustness. Frequently a single study could not be completed without column replacement. On-line solid phase extraction, on the other hand, compared well with off-line solid phase extraction, using our LC extraction column design, as contamination of the extraction column was minimized by back flushing using the Gilson syringe pump. Finally, on-line solid phase extraction for support of Caco-2 permeability studies worked very well for both single components and mixtures as the matrix was much simpler, presenting fewer contamination problems.     

Respiratory function after repair of congenital diaphragmatic hernia

Respiratory function studies were carried out in 22 infants who had successful repair of diaphragmatic herniae of the Bochdalek type. Thoracic gas volume was initially reduced in only 3 of these, but subsequent studies showed that improvement occurred. There were no consistent abnormalities in either dynamic compliance or mean pulmonary conductance. This is evidence that there is rapid adaptation which compensates for any alteration in the parenchymatous tissue in the lungs or abnormalities in the bronchial tree in infants soon after the repair of congenital diaphragmetic herniae. Further studies are necessary to determine the changes in these lungs with growth.     

Characterization of EBV-carrying B-cell populations in healthy seropositive individuals with regard to density, release of transforming virus and spontaneous outgrowth

Peripheral or tonsil lymphocyte populations of EBV-seropositive donors give rise to EBV-carrying LCLs upon in vitro explantation. Such lines can arise either by a 2-step mechanism, namely release of virus from some of the explanted cells followed by infection of previously uninfected B cells, or by direct outgrowth of virus-harboring B cells (Rickinson et al., 1974; Dalens et al., 1975; Hinuma and Katsuki 1978; Katsuki et al., 1979). We observed that cells responsible for both the 2-step mechanism and for direct outgrowth are found in the purified B-cell compartment. Virus release was more frequent than direct outgrowth. The majority of virus-releasing cells were found in the low-density fraction that contains large, activated B blasts. Cells that were capable of spontaneous outgrowth in the presence of the viral inhibitor PFA and of virus-neutralizing antibody gave rise to cell lines that carried the sex chromosome marker of the original donor, rather than that of admixed cord blood lymphocyte of the opposite sex. Such cells were found in both the low- and the high-density fractions. The majority of the EBV-carrying B cells in vivo are thus low-density blasts. Rare small B cells of high density harboring EBV were capable of spontaneous outgrowth. This may be indicative of a host control mechanism that is removed upon cultivation in vitro.     

Postbiopsy renal transplant arteriovenous fistulas: color Doppler US characteristics

Color Doppler ultrasound (US) with point-spectral analysis was performed on eight patients with postbiopsy renal transplant arteriovenous fistulas. Waveform analysis of the supplying artery documented decreased resistive indices in all cases and increased flow velocities in seven. The peak-systolic flow velocity in the arteries supplying the fistulas ranged from 55 to 180 cm/sec (mean, 92 cm/sec), while the range in normal arteries was 20-52 cm/sec (mean, 32 cm/sec). The resistive indices of the arteries supplying the fistulas ranged from 0.31 to 0.50 (mean, 0.45), while the resistive indices of the normal arteries ranged from 0.60 to 0.92 (mean, 0.74). Arterialization of the venous waveform from the draining vein was also documented in all cases. In six cases, the increased flow velocities resulted in increased color saturation toward white in the supplying artery (n = 2) or in both the artery and the draining vein (n = 4), which was detectable on the realtime image. In six cases, flow turbulence resulted in localized tissue vibration, which appeared as random color assignment in extravascular renal parenchyma adjacent to the fistula. Knowledge of these imaging and Doppler characteristics should aid in the identification of renal transplant arteriovenous fistulas with color Doppler US.     

Pace-Termination and Pacing for Prevention of Atrial Tachyarrhythmias: Results from a Multicenter Study with an Implantable Device for Atrial Therapy

INTRODUCTION: Patients with bradycardia requiring permanent pacing frequently suffer from additional atrial tachyarrhythmias (ATs). This study evaluated the safety and efficacy of atrial antitachycardia pacing (ATP) and the performance of pacing for AT prevention implemented into a new pacemaker. METHODS AND RESULTS: In patients with conventional indications for permanent pacing, an investigational DDDRP pacemaker (Medtronic AT500, model 7253) was implanted. The primary study objectives were to determine the safety of overall device functioning and its efficacy in terminating spontaneous AT. A secondary endpoint was to determine the reliability of AT detection. Pacemaker memory functions were used to analyze the impact of dedicated pacing algorithms on AT prevention. In 33 European and Canadian centers, 325 patients were enrolled (mean follow-up 2.3+/-1.3 months). Complication-free survival at 3 months was 88%. In 2,145 episodes stored with atrial electrograms, AT detection was confirmed in 97%. The algorithm for continuous overdrive pacing increased the percentage of atrial pacing to 97%. After ATP activation, 16,683 of 52,468 AT episodes were treated (120 patients). Of these, 8,903 episodes (53%) were terminated successfully by ATP. No proarrhythmic effect of preventive pacing or atrial ATP was observed. Preventive pacing algorithms increased the median percentage of atrial pacing from 62% to 97%. However, the number of AT/AF (atrial fibrillation) episodes (4.1 vs 4.1 per patient per day) and the time in AT/AF (13.7% vs 12.8%) was not significantly different before and after activation of preventive pacing. CONCLUSION: DDDRP pacing with a new system for AT therapy was safe and associated with successful pace-termination of AT in 53% of episodes. Preventive pacing and atrial ATP algorithms represent two new functions that can be implemented safely into pacemaker systems for nonpharmacologic treatment of ATs in patients requiring pacemaker therapy.     

Ossicular erosions in the dry ear: CT diagnosis

Erosions of the ossicular chain that occurred as a complication of noncholesteatomatous chronic otitis media were studied with computed tomography (CT) in 55 patients. The incus (particularly the long and lenticular processes) was the ossicle most commonly involved (50 cases). Coronal and axial CT sections were complementary in the diagnosis of these erosions. Fibrous replacement of the incudostapedial articulation was diagnosed in four cases on axial CT scans when an unusually wide joint was present.