In vivo radiolabel quantification in small-animal models

Current developments in emission tomography especially designed for small-animal imaging are presented. Adaptations of the human tomography principles take advantage of the smaller field of view to achieve about 2-mm usable resolution. Some evaluations in rat tomography are presented, and the problems of limiting resolution of PET and SPECT systems are discussed. Finally, a new approach that more specifically takes into account the parameters induced by in vivo quantification in rodents is presented.     

Reproducibility of color Doppler flow quantification of aortic regurgitation

The preferred method for quantification of aortic regurgitation severity with color Doppler echocardiography is the assessment of the ratio of jet diameter to left ventricular outflow tract diameter and jet area to left ventricular outflow tract area. However, the reproducibility of these measurements is not known and may limit its clinical application. This study was performed to identify sources of variability and reproducibility of the echocardiographic data. We examined 62 color Doppler echocardiographic examinations of patients showing isolated aortic regurgitation after human tissue valve implantation. The mean differences with standard deviations between paired measurements were calculated. The interobserver, intraobserver, and interframe variability showed a close agreement for the jet diameter and left ventricular outflow tract diameter measurements. The agreement for jet area and left ventricular outflow tract area measurements showed a small bias, but a large variance. The reproducibility of jet-left ventricular outflow tract diameter is better than the jet-left ventricular outflow tract area measurement and is more accurate to assess the severity of aortic regurgitation from color Doppler images.     

Tissue Doppler imaging and the quantification of myocardial function

Tissue Doppler imaging (TDI) has recently been introduced in clinical echocardiography. Most widely used are tissue velocity maps, in which the velocity of moving tissue is calculated relative to the transducer from the Doppler shift and displayed as colour-encoded velocity maps in either M-mode or two-dimensional image formats (Doppler velocity mode). This allows detection and quantification of dyssynergic areas of the myocardium. Additionally, the velocities may be studied with pulsed wave-tissue Doppler sampling (PW-TDS) which displays the velocity of a selected myocardial region versus time with high temporal resolution. Less often used, are tissue acceleration maps which display acceleration or velocity change of subsequent frames as different colours (Doppler acceleration mode). These maps may find application in clinical electrophysiology. Another TDI modality is tissue energy imaging, which is based on the integration of the power spectrum of the Doppler signals from the tissue. This technique provides maps of Doppler energy which are represented as colour brightness. Such maps offer potential for the study of myocardial perfusion. TDI modalities have promise to become clinically useful for quantifying myocardial function.     

Pharmacokinetic studies of different echo-contrast agents in the cerebral circulation of dogs

Ultrasound contrast agents (UCAs) are improving the signal-to-noise ratio of the reflected ultrasound so that Doppler frequency spectra can be recorded even under poor sonographic conditions. This is of particular diagnostic value in transcranial Doppler sonography. Depending on specific pharmacologic properties, echo-contrast agents may cause different increase and duration in Doppler signal amplitude. We used a dog model to study the temporal profile of Doppler signal amplitude after application of a phospholipid-containing echo-contrast agent (BY 963). In addition, we compared it with a contrast agent containing albumin (Albunex). Spherosome suspension BY 963 resulted in a dose-dependent increase in the duration of ultrasound amplification (Doppler enhancement of 1 mL, 3 mL and 10 mL of BY 963 per animal: 62.4 +/- 8.4, 72.8 +/- 9.2 and 67.7 +/- 4.2 s, respectively, n = 6). Detection of BY 963 in the extracranial jugular vein demonstrated that the UCA passes through the cerebral microcirculation (cerebral transit time was 2.2 +/- 0.2 s, n = 4). The signal enhancement lasted longer using the spherosome suspension compared with the albumin-containing solution (77.2 +/- 11.6 and 31.1 +/- 3.7 s, respectively, n = 6), and the maximum increase in intensity was more pronounced (27.0 +/- 2.0 and 17.5 +/- 2.2 dB, respectively, n = 6).     

An integrated approach to the quantification of aortic regurgitation by Doppler echocardiography

BACKGROUND: Although different Doppler methods have been proposed for the quantification of aortic regurgitation, no study has prospectively compared these methods with each other and their correlation with angiography. The aim of this study was to prospectively analyze the usefulness of different Doppler echocardiography parameters by testing all such parameters in each patient. METHODS: Fifty-one patients with aortic regurgitation underwent 2-dimensional and Doppler echocardiographic studies and catheterization. The following Doppler indexes were analyzed and compared with aortography. Color Doppler: (1) jet color height/left ventricular outflow tract height in parasternal long-axis view, and (2) jet color area/left ventricular outflow tract area in short-axis view. Continuous Doppler: (3) regurgitant flow pressure half-time, (4) regurgitant flow time velocity integral (in centimeters), and (5) regurgitant flow time velocity integral (in centimeters)/diastolic period (in milliseconds). Pulsed Doppler in thoracic and abdominal aorta: (6) time velocity integral of diastolic reverse flow (in centimeters), (7) time velocity integral of systolic anterograde flow/integral of diastolic reverse flow, (8) (time velocity integral of diastolic reverse flow/diastolic period) x 100, and (9) diastolic reverse flow duration/diastolic period (as a percentage). We compared these parameters with severity of regurgitation measured by angiography and classified as mild, moderate, or severe. RESULTS: The most useful parameters were (1) jet color height/left ventricular outflow tract height (correctly classified 42 of 49 patients), (2) (time velocity integral of diastolic reverse flow/diastolic period) x 100 in the thoracic aorta (correctly classified 41 of 46 patients), and (3) (time velocity integral of diastolic reverse flow/diastolic period) x 100 in the abdominal aorta (correctly classified 42 of 49 patients). Sequential integration of these 3 parameters correctly classified 96% of patients (44 of 46 patients) and was achieved in 90% of cases. CONCLUSION: An integrated combination of several Doppler parameters can quickly and accurately classify the degree of aortic regurgitation as determined by angiography.     

Influence of transducer frequency on Doppler microemboli signals in an in vivo model

The purpose of this study was to apply virtual reality technology to spiral computed tomographic (CT) angiogram images in a rabbit model of atherosclerosis and to correlate the images with histopathologic evaluation of the aorta. Image data were transferred to the virtual endoscope system in a graphics workstation. "Virtualized angioscopy" includes an interactive graphic user interface, which controls the viewpoint, the direction of the observation, and rendering and navigation functions. The virtual angioscopy system demonstrated irregularities of the luminal surface of the ascending aorta and a smooth luminal surface in the descending aorta. These observations were correlated with histopathologic findings. The results of this study indicate that the potential and real benefits of virtualized angioscopy far outweigh several technical limitations.     

Scan optimization of gadolinium contrast-enhanced three-dimensional MRA of peripheral arteries with multiple bolus injections and in vitro validation of stenosis quantification

OBJECTIVE: Development of a method for semiautomated preparation of purified, representative and conventionally stained monolayer smears from bronchial secretions suitable for subjective and/or automated cytodiagnosis. STUDY DESIGN: Bronchial secretions from 50 patients with and 48 without carcinoma cells of different types were collected in Saccomanno's fixative. After routine pick-and-smear processing, residual material was subjected to a mucolytic agent (ammonium thioglycolate). Separation of cells was performed by differential centrifugation through aqueous sucrose. The pellet was automatically processed by the AutoCyte PREP system. RESULTS: Slides revealed well-preserved, slightly shrunken, homogeneously distributed cells devoid of mucus, cellular debris and bacteria in monolayer arrangement nearly without overlap. Granulocytes were eliminated to a large extent. Comparison with pick-and-smear specimens showed more tumor cells per square centimeter of slide surface in 100% of AutoCyte PREP slides. The number of tumor cells per AutoCyte PREP slide was higher in 46% and lower in 54%. Selecting slides at random and requiring at least 10 abnormal cells to establish a tumor diagnosis were achieved in 82.7% if only one, in 88.0% if two and 94.0% if seven or eight AutoCyte PREP slides were investigated. CONCLUSION: The semiautomated method yielded conventionally stained, purified monolayer smears from bronchial secretions with cellular morphology suitable for evaluation by cytologists and screening machines. Representativity of AutoCyte PREP monolayers was superior to that of pick-and-smear slides.     

Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific

Recent experiments have demonstrated that the behavior of the interphase microtubule array is cell-type specific: microtubules in epithelial cells are less dynamic than microtubules in fibroblasts (Pepper-kok et al., 1990; Wadsworth and McGrail, 1990). To determine which parameters of microtubule dynamic instability behavior are responsible for this difference, we have examined the behavior of individual microtubules in both cell types after injection with rhodamine-labeled tubulin subunits. Individual microtubules in both cell types were observed to grow, shorten, and pause, as expected. The average amount of time microtubules remained within the lamellae of CHO fibroblasts, measured from images acquired at 10-s intervals, was significantly shorter than the average amount of time microtubules remained within lamellae of PtK1 epithelial cells. Further analysis of individual microtubule behavior from images acquired at 2-s intervals reveals that microtubules in PtK1 cells undergo multiple brief episodes of growth and shortening, resulting in little overall change in the microtubule network. In contrast, microtubules in lamellae of CHO fibroblasts are observed to undergo fewer transitions which are of longer average duration, resulting in substantial changes in the microtubule network over time. A small subset of more stable microtubules was also detected in CHO fibroblasts. Quantification of the various parameters of dynamic instability behavior from these sequences demonstrates that the average rates of both growth and shortening are significantly greater for the majority of microtubules in fibroblasts than for microtubules in epithelial cells (19.8 +/- 10.8 microns/min, 32.2 +/- 17.7 microns/min, 11.9 +/- 6.5 microns/min, and 19.7 +/- 8.1 microns/min, respectively). The frequency of catastrophe events (1/interval between catastrophe events) was similar in both cell types, but the frequency of rescue events (1/time spent shrinking) was significantly higher in PtK1 cells. Thus, individual microtubules in PtK1 lamellae undergo frequent excursions of short duration and extent, whereas most microtubules in CHO lamellae undergo more extensive excursions often resulting in the appearance or disappearance of microtubules within the field of view. These observations provide the first direct demonstration of cell-type specific behavior of individual microtubules in living cells, and indicate that these differences can be brought about by modulation of the frequency of rescue. These results directly support the view that microtubule dynamic instability behavior is regulated in a cell-type specific manner.     

In vivo cell tracking by scanning laser ophthalmoscopy: quantification of leukocyte kinetics

PURPOSE: To image peripheral blood leukocyte traffic in the normal retinal and choroidal vasculature and to quantify the differences in the circulation dynamics between normal and concanavalin A (ConA)-activated leukocytes. METHODS: Normal or ConA-activated splenocytes were fluorescently labeled in vitro with 6-carboxyfluorescein diacetate (CFDA) and reinfused in vivo where they were tracked in the retinal and choroidal circulations of syngeneic rats by means of a scanning laser ophthalmoscope (SLO). Simultaneous digital and video images were captured for as long as 30 minutes, and the initial 15 seconds of image sequences and leukocyte dynamics were analyzed from digitized images by recording the velocity of trafficking cells and the number of stationary cells that accumulated with time, using a customized software package. RESULTS: Mean velocity (+/-SD) was 29.8 +/- 15.3 mm/sec in the retinal arteries, 14.7 +/- 7.2 mm/sec in the retinal veins, and 3.0 +/- 3.6 mm/sec in the retinal capillaries. Mean velocity in the choroidal vessels was 6.1 +/- 6.0 mm/sec. No significant difference in leukocyte velocity was found between activated and normal leukocytes in any of the vessel systems. However, activated leukocytes were observed to accumulate more within the choroidal vasculature (P < 0.001) and the retinal capillaries (P < 0.001) than in control animals, but not in larger retinal vessels. CONCLUSIONS: A technique to measure the kinetics of circulating leukocytes in vivo has been developed. Although leukocyte activation itself is insufficient to cause slowing of leukocyte velocity, the data indicate that leukocyte adherence to endothelium can be induced in the absence of local or systemic activating stimuli.     

Coronary stents: In vitro aspects of an angiographic and ultrasound quantification with in vivo correlation

BACKGROUND: The validity of quantitative coronary angiography (QCA) after stent placement has been questioned because the optical density of a metallic stent, added to the density of a contrast-filled lumen, could affect border definition. METHODS and RESULTS: We deployed 3.0- and 4.0-mm Palmaz-Schatz, Wiktor, Multilink, NIR, and InStent stents in precision-cast phantoms. Central lumens of 2.0 mm were created. There was no difference between the "true" diameters of any stented lumen by both QCA and quantitative ultrasonic (QCU) measurement poststenting. QCA systematic error (SE) varied from 0.01 for the Wiktor stents to 0.14 mm for the Palmaz-Schatz stents; the random error (RE) was 0.03 to 0.14 mm. QCU SE varied from 0.05 to 0.11 mm, and RE ranged from 0.01 to 0.07 mm. At the next stage, 4.0-mm Wiktor and Palmaz-Schatz stents were deployed into the phantom lumens; 1.5-, 2.0-, 2.5- and 3.0-mm lumens were created inside the stents. QCA and QCU measurements of 1.5- to 2.5-mm residual lumens were overestimated by 0.1 to 0.3 mm. In the 3. 0-mm residual lumen within the Wiktor stent, QCA underestimated the luminal size by -0.1 mm. There was no QCA inaccuracy for a 3.0-mm lumen within the Palmaz-Schatz stent. In patients, in 25 stented segments in both the Palmaz-Schatz and Wiktor groups, there was no difference between QCA and QCU diameters. CONCLUSIONS: QCU is sufficiently precise for the assessment of the coronary lumen after stenting. QCA can be used as an accurate method of poststent assessment, except when a very mild recurrence within a highly opaque stent is measured. In that instance, QCA may underestimate the luminal diameter.     

In vivo quantification of endotoxin-induced nitric oxide production in pigs from Na15NO3-infusion

1. In this investigation the NO production rate is quantified in the pig during normotensive endotoxin-induced shock with increased cardiac output and during subsequent treatment with the NO synthase inhibitor N omega-monomethy-L-arginine (L-NMMA). NO production rate was derived from the plasma isotope-enrichment of 15N-labelled nitrate (15NO3-). 2. Three groups of animals (control, n = 5; endotoxin, n = 6; endotoxin + L-NMMA, n = 6) were anaesthetized and instrumented for the measurement of systemic and pulmonary haemodynamics. Each animal received a primed-continuous infusion of stable, non-radioactively labelled Na15 NO3 (bolus 30 mg, infusion rate 2.1 mg h-1). Arterial blood samples were taken 5, 10, 15, 30, 60 and 90 min later and every 90 minutes until the end of the experiment. 3. Continuous i.v. infusion of endotoxin was incrementally adjusted until mean pulmonary artery pressure (PAP) reached 50 mmHg and subsequently titrated to keep mean PAP approximately 35 mmHg. Hydroxyethylstarch was administered as required to maintain mean arterial pressure (MAP) > 60 mmHg. Six hours after the start of the endotoxin continuous i.v. L-NMMA (1 mg kg-1 h-1) was administered to the endotoxin + L-NMMA group. Haemodynamic data were measured before as well as 9 h after the start of the endotoxin. 4. After conversion of NO3- to nitro-trimethoxybenzene and gas chromatography-mass spectrometry analysis the total NO3- pool, basal NO3- production rate and the increase per unit time in NO3- production rate were calculated from the time-course of the 15NO3- plasma isotope-enrichment. A two compartment model was assumed for the NO3- kinetics, one being an active pool in which newly generated NO3- appears and from which it is eliminated, the other being an inactive volume of distribution in which only passive exchange takes place with the active compartment. 5. Although MAP did not change during endotoxin infusion alone, cardiac output (CO) increased by 42 +/- 40% (P < 0.05 versus baseline) by the end of the experiment due to a significant (P < 0.05 versus baseline) fall in systemic vascular resistance (SVR) to 65 +/- 25% of the baseline value. L-NMMA given with endotoxin did not change MAP, and both CO and SVR were maintained close to the pre-shock levels. 6. Baseline plasma NO3- concentrations were 43 +/- 13 and 40 +/- 10 mumol l-1 in the control and endotoxin animals, respectively, and did not differ at the end of the experiment (39 +/- 8 and 44 +/- 15 mumol l-1, respectively). The mean NO3- pool and basal NO3- production rate were 1155 +/- 294 mumol and 140 +/- 32 mumol h-1, respectively, without any intergroup difference. Endotoxin significantly increased NO3- production rate (23 +/- 10 mumol h-2, P < 0.05 versus control (6 +/- 7 mumol h-2) and endotoxin + L-NMMA groups). L-NMMA given with endotoxin (-1 +/- 2 mumol h-2, P < 0.05 versus control and endotoxin groups) had no effect. 7. Analysis of the time course of the 15NO3- plasma isotope enrichment during primed-continuous infusion of Na15NO3 allowed us to quantify the endotoxin-induced increase in NO3- production rate independently of total NO3- plasma concentrations. Low-dose L-NMMA blunted the increase in NO3- production rate while maintaining basal NO3- formation.     

In vivo correlation of Doppler waveform analysis with arterial input impedance parameters

Previous studies have confirmed that Doppler waveform analysis (DWA) offers a valid reflection of changes in peripheral vascular resistance. However, the ability of the pulsatility index (PI), a parameter of DWA, to reflect the dynamic components of the circulation, as assessed by arterial input parameters, remains uncertain. In addition, the state of the central circulation is considered an important factor influencing the accuracy of this technique. This study evaluated the ability of the aortic PI to reflect alterations of input impedance in a chronically instrumented lamb model that was subjected to pharmacologic alteration of the circulation. Pressure, volumetric flow and continuous-wave Doppler frequency shift measurements were recorded from the infrarenal abdominal aorta. The parameters of input impedance, peripheral vascular resistance (Zpr), characteristic impedance (Zo) and reflection coefficient (Rc), were determined and then correlated with changes in the aortic PI. Initially, perturbations of the circulatory state were created with a vasodilator, hydralazine (HY) and a vasoconstrictor, phenylephrine (PE). During a second set of experiments, the effect of the reflex heart rate (HR) responses on the PI was evaluated. This was accomplished by inhibiting reflex HR responses to these vasoactive agents with either trimethophan (TM) or atropine methyl bromide (AMB). In response to HY and HY with TM, significant decreases in the PI and impedance parameters occurred. Administration of PE and PE with AMB resulted in significant increases in PI and each of the impedance parameters. HY and PE induced changes in PI correlated significantly with changes in volumetric flow (r = 0.82, 0.80; p < 0.001), mean arterial blood pressure (r = 0.64, 0.70; p < 0.001) and Zpr (r = 0.77, 0.80; p < 0.001), but not with Zo (r = 0.34, 0.36) and Rc (r = 0.26, 0.31). However, when reflex HR responses were inhibited during the administration of the vasoactive agents, HY with TM and PE with AMB, induced changes in PI correlated significantly with Zo (r = 0.93, 0.89; p < 0.001) and Rc (r = 0.84, 0.83; p < 0.001), and the correlation with mean arterial pressure (r = 0.78, 0.87; p < 0.05) and Zpr (r = 0.92, 0.91; p < 0.05) was significantly greater. These findings indicate that the PI accurately assesses pharmacologically induced changes in the downstream arterial input impedance. The accuracy of this assessment is enhanced further when central circulatory factors such as changes in HR are considered.     

Absolute metabolite quantification by in vivo NMR spectroscopy: II. A multicentre trial of protocols for in vivo localised proton studies of human brain

We have performed a multicentre trial to assess the performance of three techniques for absolute quantification of cerebral metabolites using in vivo proton nuclear magnetic resonance (NMR). The techniques included were 1) an internal water standard method, 2) an external standard method based on phantom replacement, and 3) a more sophisticated method incorporating elements of both the internal and external standard approaches, together with compartmental analysis of brain water. Only the internal water standard technique could be readily implemented at all participating sites and gave acceptable precision and interlaboratory reproducibility. This method was insensitive to many of the experimental factors affecting the performance of the alternative techniques, including effects related to loading, standing waves and B1 inhomogeneities; and practical issues of phantom positioning, user expertise and examination duration. However, the internal water standard method assumes a value for the concentration of NMR-visible water within the spectroscopic volume of interest. In general, it is necessary to modify this assumed concentration on the basis of the grey matter, white matter and cerebrospinal fluid (CSF) content of the volume, and the NMR-visible water content of the grey and white matter fractions. Combining data from 11 sites, the concentrations of the principal NMR-visible metabolites in the brains of healthy subjects (age range 20-35 years) determined using the internal water standard method were (mean+/-SD): [NAA]=10.0+/-3.4 mM (n=53), [tCho]=1.9+/-1.0 mM (n=51), [Cr + PCr]=6.5+/-3.7 mM (n=51). Evidence of system instability and other sources of error at some participating sites reinforces the need for rigorous quality assurance in quantitative spectroscopy.     

In vivo quantification of brain volumes in subcortical vascular dementia and Alzheimer''s disease. An MRI-based study

Ibuprofen is one of the most effective and widely used non-steroidal analgesic and anti-inflammatory agent. It is marketed as a racemic mixture though it is known that the pharmacological activity resides in the (S)-(+)-enantiomer only. Several direct/indirect liquid chromatographic methods involving a variety of chiral/achiral phases along with their possible role in resolution, chiral and achiral agents used for derivatisation have been discussed with special reference to ibuprofen, and mentioning their application to the resolution of other 2-aryl-propionic acids/profens.     

Validation of S-1'-[18F]fluorocarazolol for in vivo imaging and quantification of cerebral beta-adrenoceptors

S-1'-[18F]fluorocarazolol (S-(-)-4-(2-hydroxy-3-(1'-[18F]fluoroisopropyl)-aminopropoxy)carba zole, a non-subtype-selective beta-adrenoceptor antagonist) has been investigated for in vivo studies of beta-adrenoceptors. Previous results indicated that uptake of this radioligand in heart and lung can be inhibited by beta-adrenoceptor agonists and antagonists. In the present study, blocking, displacement and saturation experiments were performed in rats, in combination with metabolite analysis to investigate the suitability of this radioligand for in vivo positron emission tomography (PET) imaging and quantification of beta-adrenoceptors in the brain. The results demonstrate that, (i) the uptake of S-1'-[18F]fluorocarazolol reflects specific binding to beta-adrenoceptors, (ii) binding of S-1'-[18F]fluorocarazolol to atypical or non-beta-adrenergic sites is negligible, (iii) uptake of radioactive metabolites in the brain is less than 25% of total radioactivity, 60 min after injection, (iv) in vivo measurements of receptor densities (Bmax) in cortex, cerebellum, heart, lung and erythrocytes are within range of densities determined from in vitro assays, (v) binding of S-1'-[18F]fluorocarazolol can be displaced. In conclusion, S-1'-[18F]fluorocarazolol seems to possess the appropriate characteristics to visualize and quantify beta-adrenoceptors in vivo in the central nervous system using PET.     

Intra-articular glucocorticoid injections in joint diseases

Intra-articular glucocorticosteroid injections are widely used in mono- or oligoarticular flares of patients with rheumatoid arthritis and other aseptic inflammatory joint diseases, as well as in osteoarthritis. Rapid and pronounced, but usually temporary, suppression of local joint inflammation may be achieved with only minor systemic effect. In osteoarthritis the effect is brief and transient. Triamcinolone hexacetonide provides the longest clinical effect, but since this drug may cause local tissue necrosis when injected outside a synovial cavity it should be used only by experienced clinicians. The risk of glucocorticoid-induced cartilage damage is discussed. The risk is probably less than that of untreated joint inflammation. Nevertheless, it is recommended that injections into the same joint are limited, for instance to one injection every six weeks and no more than three or four in one year. Furthermore, indications and contraindications should be carefully considered prior to each injection. Intra-articular glucocorticoid therapy may be of considerable clinical value in the management of aseptic arthritis, if administered on correct indications using a correct technique.     

Local tolerance of subcutaneous injections

Human insulin-like growth factor I (hIGF-I) has several possible clinical applications. Because subcutaneous administration of the drug can cause pain, local tolerance to injection of different formulations with or without hIGF-I has been investigated in man using isotonic saline solution as reference. The formulations, made isotonic with NaCl, ranged in pH from 6 to 7 with phosphate buffer concentrations of 5 to 50 mM. The local tolerance after injection was assessed as injection pain on a visual analogue scale, pain duration and local tolerance (redness, paleness and oedema). The discomfort at the injection site was lowest with 10 mM phosphate, pH 7. Injection of buffer at pH 6 (50 mM phosphate) caused significantly more pain than using 10 mM phosphate, whereas the pain at pH 6 using 10 mM phosphate did not differ significantly from that experienced on injection of the solution at pH 7 using either 10 or 50 mM phosphate. hIGF-I itself did not seem to cause pain. We concluded that for subcutaneous injections at non-physiological pH, the buffer strength should be kept as low as possible to avoid pain upon injection. We also hypothesize that when a non-physiological pH must be used for stability reasons, a lower buffer strength enables more rapid normalization of the pH at the injection site.     

Tolerant CD8 T cells induced by multiple injections of peptide antigen show impaired TCR signaling and altered proliferative responses in vitro and in vivo

The mechanisms responsible for peripheral CD8 T cell tolerance to foreign Ags remain poorly understood. In this study we have characterized the state of CD8 T cell tolerance induced in F5 TCR transgenic mice by multiple peptide injections in vivo. The tolerant state of CD8 T cells is characterized by impaired proliferative responses, increased sensitivity to cell death, and failure to acquire cytotoxic effector function after in vitro antigenic challenge. In vivo monitoring of CD8 T cell proliferation using 5-carboxyfluorescein diacetate succinimidyl ester showed that a large subset of the tolerant T cell population failed to divide in response to peptide. TCR down-regulation could not account for this loss of responsiveness to Ag since recombination-activating gene-1 (RAG-1)-/-F5 CD8 T cell responses were similar to those of RAG-1(-/-)F5 x RAG-1(-/-)F1 T lymphocytes, which express lower levels of the transgenic TCR. Analysis of early signal transduction in tolerant CD8 T cells revealed high basal levels of cytoplasmic calcium as well as impaired calcium mobilization and tyrosine phosphorylation after cross-linking of CD3epsilon and CD8alpha. Together these data indicate that repeated exposure to soluble antigenic peptide in vivo can induce a state of functional tolerance characterized by defective TCR signaling, impaired proliferation, and increased sensitivity to cell death.     

The regurgitation of large vitreous injections

The increasing prevalence of tuberculosis (TB) and human immunodeficiency virus (HIV), particularly in Africa and Asia, led us to investigate the prevalence of HIV infection in immigrants with pulmonary TB at the time of arrival in the UK. We performed anonymous HIV testing of stored sera from 39/65 immigrants referred to our unit between January 1991 and December 1994, who had culture-positive pulmonary TB. None of the 39 patients tested was positive for either HIV-1 or HIV-2, and the characteristics of the 26 patients for whom no serum was available were similar to those of the tested group. Despite the need to consider concomitant HIV infection in any patient with TB, particularly those from an area of HIV endemnicity, the present data do not suggest that recently arrived refugees, asylum seekers, immigrants or long-term visitors to the UK constitute a group in whom dual infection of HIV and Mycobacterium tuberculosis is common.