Overexpression of Batf induces an apoptotic defect and an associated lymphoproliferative disorder in mice.

Activator protein-1 (AP-1) is a dimeric transcription factor composed of the Jun, Fos and Atf families of proteins. Batf is expressed in the immune system and participates in AP-1 dimers that modulate gene expression in response to a variety of stimuli. Transgenic (Tg) mice overexpressing human BATF in T cells were generated using the human CD2 promoter (CD2-HA (hemagglutinin antigen) - BATF). By 1 year of age, over 90% of the mice developed a lymphoproliferative disorder (LPD). The enlarged lymph nodes characteristic of this LPD contain a polyclonal accumulation of T cells with a CD4(+) bias, yet efforts to propagate these tumor cells in vitro demonstrate that they do not proliferate as well as wild-type CD4(+) T cells. Instead, the accumulation of these cells is likely due to an apoptotic defect as CD2-HA-BATF Tg T cells challenged by trophic factor withdrawal in vitro resist apoptosis and display a pro-survival pattern of Bcl-2 family protein expression. As elevated levels of Batf expression are a feature of lymphoid tumors in both humans and mice, these observations support the use of CD2-HA-BATF mice as a model for investigating the molecular details of apoptotic dysregulation in LPD.

A critical role for vesicle-associated membrane protein-7 in exocytosis from human eosinophils and neutrophils.

BACKGROUND: Granulocyte exocytosis is proposed to be critically dependent on the interaction of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) located on granules/vesicles (v-SNAREs) and plasma membrane (t-SNAREs). Previous studies indicated that the v-SNARE, vesicle-associated membrane protein (VAMP)-2, as well as t-SNAREs (SNAP-23, syntaxin-4 and -6) are implicated in exocytosis from human granulocytes. Vesicle-associated membrane proteins-7 and -8 have been implicated in endosome/lysosome trafficking, however, their role in granulocyte exocytosis remains obscure. OBJECTIVE: We sought to investigate the expression and functional role of SNARE isoforms in the secretion of different granule-derived mediators in human eosinophils and neutrophils. METHODS: The expression of SNAREs was determined by subcellular fractionation and flow cytometry. SNARE-specific antibodies were examined for their ability to impair mediator release from permeabilized eosinophils and neutrophils. RESULTS: Vesicle-associated membrane proteins-7 and -8 were localized to granule and membrane-enriched fractions in eosinophils and neutrophils, whereas syntaxin-6 was not detectable. In permeabilized cells, anti-VAMP-7, but not anti-VAMP-8, antibody impaired the secretion of all mediators examined (in eosinophils, eosinophil peroxidase and eosinophil-derived neurotoxin; in neutrophils, myeloperoxidase, lactoferrin and matrix metalloprotease-9) in a dose-dependent manner. In contrast, anti-VAMP-2 modestly and selectively impaired secretion from small granules and vesicles. Syntaxin-4, but not syntaxin-6, was found to interact with SNAP-23 and was partially involved in mediator secretion from multiple compartments. CONCLUSION: Our observations indicate for the first time a critical role for VAMP-7 in both eosinophil and neutrophil mediator release.

Soil inactivation of DNA viruses in septic seepage.

AIMS: To generate field-relevant inactivation data for incorporation into models to predict the likelihood of viral contamination of surface waters by septic seepage. METHODS AND RESULTS: Inactivation rates were determined for PRD1 bacteriophage and Adenovirus 2 in two catchment soils under a range of temperature, moisture and biotic status regimes. Inactivation rates presented for both viruses were significantly different at different temperatures and in different soil types (alpha = 0.05). Soil moisture generally did not significantly affect virus inactivation rate. Biotic status significantly affected inactivation rates of PRD1 in the loam soil but not the clay-loam soil. Adenovirus 2 was inactivated more rapidly in the loam soil than PRD1 bacteriophage. CONCLUSIONS: Virus inactivation rates incorporated into models should be appropriate for the climate/catchment in question with particular regard to soil type and temperature. Given that PRD1 is similar in size to adenoviruses, yet more conservative with regard to inactivation in soil, it may be a useful surrogate in studies of Adenovirus fate and transport. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the factors that govern virus fate and transport in catchments would facilitate the design of barrier measures to prevent viral contamination of surface waters by septic seepage.

Fusion protein vesicle-associated membrane protein 2 is implicated in IFN-gamma-induced piecemeal degranulation in human eosinophils from atopic individuals.

BACKGROUND: Exocytosis is an integral event during IFN-gamma-induced piecemeal degranulation in eosinophils. In many tissues soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), including vesicle-associated membrane protein (VAMP), act as specific intracellular receptors to allow granule fusion with the membrane during degranulation. However, the mechanisms underlying eosinophil piecemeal degranulation induced by IFN-gamma are not well understood. OBJECTIVE: We sought to assess whether eosinophils express the vesicular SNARE protein VAMP-2 and to determine the involvement of VAMP-2 in IFN-gamma-induced piecemeal degranulation. METHODS: Human peripheral blood eosinophils (> or =97%) from atopic subjects were subjected to RT-PCR and sequence analysis with specific primers for VAMP-2 mRNA. Western blotting and flow cytometric analysis were carried out to confirm the identity of VAMP-2 and its susceptibility to cleavage by tetanus toxin. Confocal laser scanning microscopy imaging was conducted on double-labeled cytospin preparations of eosinophils at 0, 5, 10, 30, and 60 minutes and 16 hours of IFN-gamma (500 U/mL) stimulation. RESULTS: Eosinophils expressed VAMP-2 mRNA (n = 4 donors), which exhibited 100% homology with human VAMP-2 cDNA on sequencing. Eosinophils were also found to express tetanus toxin-sensitive VAMP-2 protein. RANTES and VAMP-2 immunofluorescence were observed to colocalize to similar intracellular structures by means of confocal imaging. IFN-gamma induced a rapid translocation of VAMP-2(+) organelles toward the cell membrane in correlation with RANTES. CONCLUSIONS: These findings suggest that exocytosis in human eosinophils is regulated by SNAREs, with a specific role indicated for VAMP-2 in piecemeal degranulation.

Jejunal brush border microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and interleukin-6.

Intestinal colonization with the protozoan Giardia causes diffuse brush border microvillous alterations and disaccharidase deficiencies, which in turn are responsible for intestinal malabsorption and maldigestion. The role of T cells and/or cytokines in the pathogenesis of Giardia-induced microvillous injury remains unclear. The aim of this study was to assess the role of T cells and interleukin-6 (IL-6) in the brush border pathophysiology of acute murine giardiasis in vivo. Athymic nude (nu(-)/nu(-)) CD-1 mice and isogenic immunocompetent (nu(+)/nu(+)) CD-1 mice (4 weeks old) received an axenic Giardia muris trophozoite inoculum or vehicle (control) via orogastric gavage. Weight gain and food intake were assessed daily. On day 6, segments of jejunum were assessed for parasite load, brush border ultrastructure, IL-6 content, maltase and sucrase activities, villus-crypt architecture, and intraepithelial lymphocyte (IEL) infiltration. Despite similar parasitic loads on day 6, infected immunocompetent animals, but not infected nude mice, showed a diffuse loss of brush border microvillous surface area, which was correlated with a significant reduction in maltase and sucrase activities and a decrease in jejunal IL-6 concentration. In both athymic control and infected mice, jejunal brush border surface area and disaccharidases were high, but levels of tissue IL-6 were low and comparable to the concentration measured in immunocompetent infected animals. In both immunocompetent and nude mice, infection caused a small but significant increase in the numbers of IELs. These findings suggest that the enterocyte brush border injury and malfunction seen in giardiasis is, at least in part, mediated by thymus-derived T lymphocytes and that suppressed jejunal IL-6 does not necessarily accompany microvillous shortening.

Changes in end-expiratory lung volume on induction of anaesthesia with thiopentone or propofol.

To determine the effects of induction agent and obesity on the change in functional residual capacity caused by induction of anaesthesia, we measured the change in end-expiratory respiratory volume (EERV) during induction of anaesthesia with either thiopentone or propofol. Female patients breathed via a face mask from a closed circle system connected to a water spirometer. The spirometer tracing was used to measure the decrease in EERV. The median decrease was 200 (95% confidence limits 90-280) ml after thiopentone and 285 (200-425) ml after propofol. The decrease in each group was significant (P < 0.01) but there was no difference between the two groups. The change in EERV, expressed as a proportion of the predicted functional residual capacity, was not related to obesity (estimated as the ratio of actual to predicted body weight).

Cranio-caudal movement of the sternum on induction of anaesthesia.

The cranio-caudal movement of the sternum was studied by the technique of video magnification during induction of anaesthesia with thiopentone 2-4 mg kg-1 i.v. in 20 patients. Anaesthesia produced no consistent change in end-expiratory position of the sternum; there was a range of movement from 3.1 mm cephalad to 5.4 mm caudad. There was a significant relationship between movement and degree of obesity of the patient (P less than 0.01), with the sternum tending to caudal movement in the obese patient.

The effects of injected solution temperature on intravenous regional anaesthesia.

Ten healthy volunteers received three standard Bier's blocks. Prilocaine 0.5%, 40 ml was injected at a solution temperature of 0 degrees C, 22 degrees C or 37 degrees C. Recordings were made of sensory block, motor block, intravenous pressure, limb temperature and pain on injection. There were no differences between the three treatments in the rate of development or in the quality of block but there was a significant difference in the comfort of injection. Cold solutions caused most, and warm solutions least discomfort.

Spinal anesthesia and lumbar lordosis.

Hyperbaric bupivacaine 0.5% (3.0 ml) was injected intrathecally in two groups of 20 patients. Both groups of patients lay in the lateral position with their hips flexed at 90 degrees. In group F, the hip flexion was maintained for 5 minutes after turning supine. In group S, the hips were straightened before the patients were turned to the supine position. The technique of hip flexion to reduce the lumbar lordosis did not significantly limit the height of anesthetic blockade. The distribution of height of anesthetic blockade showed marked bimodality (P less than 0.05) in both groups, in group F at T4 and T9 and in group S at T3 and T9. Cardiovascular side effects were minimal and equal in both groups.

Stereophotogrammetric analysis of changes in body volume associated with the induction of anaesthesia.

Changes in the volume of the trunk were measured in seven supine patients, when anaesthesia was induced with thiopentone i.v. Stereo-photograms were taken before and after induction and measurements of volume calculated from contour plots of a reconstructed stereo image. The changes in volume of the visible part of the trunk were between -846 and +505 ml. No systematic change in total or lower trunk ("abdominal") volume was evident, but there was a significant negative linear relationship (r = -0.87) between upper trunk ("ribcage") volume change and obesity (expressed as actual weight/expected weight). Changes in volume were more evident in the upper parts of the supine subject, in both ribcage and abdomen (P less than 0.05).

Single-shot i.v. anaesthesia for outpatient dental surgery. Comparison of 2,6 di-isopropyl phenol and methohexitone.

A single i.v. bolus dose of propofol 3 mg kg-1 was compared with methohexitone 2 mg kg-1 as the sole anaesthetic agent for simple dental extraction in outpatients. Induction of anaesthesia was smoother with propofol, with a lower incidence of excitatory phenomena. Pain on injection was a common complication of both drugs and related to the site of injection. The Leeds Psychomotor Tester was used to assess psychomotor performance during the recovery. Rate of recovery from anaesthesia was similar with both agents, and there was little residual impairment of psychomotor function 40 min after induction.

Changes in diaphragmatic position in association with the induction of anaesthesia.

Images of a saggital section of the right hemidiaphragm were obtained using an ultrasound sector scanner in 20 patients in the supine position immediately before, and after, the induction of anaesthesia (with thiopentone). In the awake patient, the mean excursion of the part of the diaphragm that showed the greatest tidal movement was 1.56 +/- 0.52 (SD) cm. The end-expiratory position of this part of the diaphragm was noted before and after induction. Craniad movement of this position was seen in 10 patients. In a further eight, the end-expiratory position did not change, and in two patients it moved caudally. The mean movement was 0.36 +/- 0.52 cm in a cranial direction, which was statistically significant (P less than 0.01) but was only 23% of the movement associated with quiet breathing. The extent or direction of movement was not related to the weight of the patient (expressed as a proportion of the expected weight). The findings do not support the hypothesis that the reduction of lung volume on induction of anaesthesia is caused solely by movement of the diaphragm.

Plain bupivacaine: an unpredictable spinal anaesthetic agent.

Plain bupivacaine 0.5% or 0.75% was injected intrathecally in four groups of 10 patients. Group A received 0.75% bupivacaine 2.7 ml at L3/4, group B 0.5% bupivacaine 4 ml at L3/4, group C 0.75% bupivacaine 2.7 ml at L2/3 and group D 0.75% bupivacaine 2 ml at L3/4. A very wide range of height of block was found in each group and because of this there were few statistically significant differences between the groups. The mean height of anaesthetic blockade was significantly higher when 20 mg (group C) was injected compared with 15 mg (group D). Decreasing the volume of injection from 4 ml (group B) to 2.7 ml (group A) did not decrease this variability. By injecting at L2/3 spinal space (group C) the mean maximum level of anaesthesia (T7) was four segments higher than group A (T11) injected at L3/4, and this was reflected in a greater incidence of arterial hypotension.

Effect of added epinephrine on spinal anesthesia with lidocaine.

The effect of adding 0.1, 0.2, or 0.3 ml of 1:1000 epinephrine to 1.5 ml of 5% lidocaine in 7.5% dextrose for spinal anesthesia was assessed in 40 patients in a double-blind study. The addition of epinephrine produced little or no clinically useful prolongation of block. There was a small reduction in the speed of decay of nerve block, but this only reached statistical significance with regard to total recovery, which took 40 to 50 minutes longer with the epinephrine-containing solutions. No difference could be seen between the three doses of epinephrine.

Occurrence of 5-hydroxy-3-indoleacetic acid in urine from several animal species.

1. The normal levels of 5-hydroxy-3-indoleacetic acid (5HIAA) were determined in the urine of four species of laboratory animals (dog, rabbit, rat and guinea-pig). 2. When 5HIAA excretion was calculated as mg/24 h, the output in the four species was in the order of rabbit greater than dog greater than guinea-pig greater than rat; but when it was calculated as mu/mg of creatinine, the order was guinea-pig greater than rabbit greater than rat greater than dog. 3. There were significant differences in the excretion of urinary 5HIAA between males and females within two species: rats and dogs.