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Notes: Abstract. Objective and Design: To investigate the role of tyrosine kinases (TK) in IgE-mediated signal transduction in human lung mast cells (HLMC) and basophils.¶Materials: Peripheral blood basophils (n ≥ 4) and human lung mast cells (n ≥ 6).¶Treatment: Cells were preincubated with TK inhibitor for 15 min at 37 °C, before the addition of anti-IgE.¶Methods: Histamine release (HR) was assayed using a fluorimetric technique. Results were compared using non-parametric statistics.¶Results: Piceatannol and ST638 significantly (p ≤ 0.05) inhibited anti-IgE induced HR from HLMCs and basophils whilst lavendustin C had no effect in either cell type. Herbimycin A also significantly (p ≤ 0.05) inhibited anti-IgE induced HR from both cell types, an effect which was dose dependent but did require a 16 h preincubation with drug.¶Conclusions: In summary, HLMCs and basophils exhibit distinct inhibitory profiles in the presence of various inhibitors of TK.

Notes: Background Raised peripheral blood mononuclear cells (PBMCs) proliferative responses to food allergens have been demonstrated in children with established atopic dermatitis.Objective In this report we investigate the PBMC proliferative responses to inhalant and food allergens from babies at birth, 6 months and 1 year of age, born to atopic and non-atopic parents.Methods PBMCs, separated by density gradient centrifugation. were cultured for 6 days with autologous plasma and a range of allergens (house dust mite [HDM], cat, grass pollen, tree pollen, betalactoglobulin and ovalbumin). Proliferative responses were measured by the uptake of [3H] thymidine added for the final 18 h of culture.Results At birth, infants born to atopic parents who developed allergic disease by 1 year of age had significantly more positive responses (stimulation index ± 2 with a value of ± 1000 cpm above background) to HDM (P = 0.0091), betalactoglobulin (P= 0.0166) and ovalbumin (P = 0.0035) than newborns who did not develop allergy. Tnfants who developed allergy also had significantly more positive responses to HDM (P - 0.03) and ovalbumin (P = 0.0057) than babies, born to non-atopic parents, who did not develop allergies. At 6 months of age a significant fall in response to HDM (P = 0.003) and cat fur extract (P = 0.006) was seen in infants who developed allergic disease by 1 year of age. A similar pattern was seen for proliferative responses to betalactoglobulin and ovalbumin (P = 0.0006. P= 0.004). Conversely, proliferations to grass and tree pollen extracts increased at 6 months (P = 0.04. P = NS) and 1 year (P= NS. P= 0.01) compared with birth which was significant for infants who did not develop allergic disease.Conclusion Proliferative responses to seasonal allergens increased over the first year of life whilst those to perennial allergens, both inhalant and food, fell. This suggests either the induction of a systemic immune tolerance by perennial exposure to antigens or movement of sensitized cells to target organs where allergen exposure occurs. This process may be independent of the development allergic disease.

Notes: This study was set up to evaluate the food panel of a multiple specific IgE antibody assay in 67 atopic asthmatic children by comparing it to the conventional radioallergosorbent test (RAST) and skin-prick tests (SPT) and then comparing the results of these investigations with the parents perceptions of food related problems.Fifteen food specific IgE antibodies were measured using the multiple chemiluminescence assay (MAST-CLA). IgE antibodies to five of these food allergens were also measured by conventional RAST and SPTs were performed in 43 using 11 standardized food extracts matched to the multiple allergosorbent chemiluminescent assay (MASTCLA) profile. SPT and MAST-CLA results showed good agreement with one another, range 688-96.7% (average 87%), with significant correlation for most allergens tested. MAST-CLA was discrepant with RAST and/or SPTs in 58/210 (27 6%).A questionnaire was sent to the parents to determine their perception of food related symptoms. Sixty-two (92%) questionnaire replies were received, of which 56% reported symptoms with food. The most frequent symptom perceived to be due to food intolerance was behavioural disturbance. The commonest foods implicated were additives (39%), egg (27%), milk (26%), chocolate (23%) and orange (15%). History, SPT, MAST-CLA and RAST were compared for live allergens in 42 patients (210 values). In 14/210 (6·7%), all the tests were negative despite reported symptoms. Conversely in 49/210 (23·3%) at least one test was positive without symptoms.This study did not support a benefit of multiple antibody testing instead of individually selected RASTs or SPTs. The frequent perception of food related symptoms in these asthmatic children was often not supported by SPT, RAST and/or MAST-CLA. This may be a reflection of the current public concern about food, or of non-IgE mechanisms. These uncertainties can only be resolved by double blind placebo controlled food challenge. The inclusion of food specific IgE antibodies together with inhalant antibodies in a multiple test system for use in atopic asthmatics may be misleading.

Notes: Background Human basophils undergo anaphylactic degranulation, characterized by extrusion of membrane-free granules, and piecemeal degranulation, characterized by progressive removal of granule contents in the absence of granule extrusion. F-Met peptide stimulates a degranulation continuum in human basophils that includes both forms of secretion. Charcot-Leyden crystal protein is stored in tbe granules of unstimulated human basopbils.Objective The objective of this study was to determine the subcellular localization of the Charcot-Leyden crystal protein in individual morphological basophil phenotypes that are stimulated by f-Met peptide and are associated with secretion.Methods A post-embedding immunogold analysis was used to detect changes in the subcellular sites of Charcot-Leyden crystal protein in human basophils stimulated with f-Met peptide. Human basophils from nonnal donors were purified by countercurrcnt centrifugal elutriation and Percoll density gradients, stimulated to degranulate with 1 μm f-Met peptide (or incubated in buffer controls), and recovered for histamine assay, electron microscopy and immunogold labelling. Specificity controls Included omission of the primary antibody and substitution of the primary antibody with non-immune normal rabbit IgG or with Charcot-Leyden crystal protein-Sepharose-absorbed primary antibody.Results The results showed new sites of labelling and different densities of labelling for Charcot-Leyden crystal protein in distinctive basophil phenotypes stimulated by f-Met peptide. New sites for Charcot-Leyden crystal protein included nucleus, cytoplasm, degranulation channel, degranulation channel membrane, plasma membrane, and a newly recognized granule population similar to primary granules in eosinophils. These new sites, as well as previously documented sites of Charcot-Leyden crystal protein (granules, intragranular Charcot-Leyden crystals, cytoplasmic vesicles) showed variahic labelling when analysed by phenotype. Other sites (besides intragranular Charcot-Leyden crystals) of formed Charcot-Leyden crystals included cytoplasm, degranulation channel, extracellular space and rarely, nucleus. Analysis of cytoplasmic vesicles, total granules and altered granules, and gold particles in subcellular compartments in seven identifiable phenotypes revealed that f-Met peptide stimulated human basophils to empty their granules by transporting Charcot-Leyden crystal protein in vesicles to the plasma membrane in the absence of granule extrusion in cells exbibiting piecemeal degranulation. In cells exhibiting anaphylactic degranulation. gold-labelled Charcot-Leyden crystals were extruded to the cells' exterior in concert with granule particles and concentric dense membranes contained within granules. Completely degranulated cells had a high density of plasma membrane gold label that was associated with numerous Correspondence: Ann M. Dvorak MD, Department of Pathology, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215, USA. gold-laden endocytotic cytoplasmic vesicles. Basophils reconstituted their main granule population, within which Charcot-Leyden crystals resided, in part by endocytosis of previously released plasma membrane-bound Charcot-Leyden crystal protein. Completely recovered cells displayed decreased Charcot-Leyden crystal protein labelling of the plasma membrane and vesicle compartments, the presence of a highly labelled new granule subset that resembled Charcot-Leyden crystal protein-containing primary granules in eosinophils, and the highest density of granule and intragranular Charcot-Leyden crystal gold labelling of all phenotypes tbat developed after stimulation.Conclusion Seven individual f-Met peptide-activated human basophil phenotypes labelled by an ultrastructural immunogold method to detect subcellular sites of Charcot-Leyden crystal protein showed changing distributions of this protein which document the capability of human basopbils to undergo complex release and recovery reactions that may be pertinent to the functions of Charcot-Leyden crystal protein and the capabilities of these cells in vivo in a variety of diseases.

Notes: In 1985 and 1990 postal questionnaires were sent to anaesthetic senior registrars in training in the United Kingdom to determine the extent of higher specialist training in chronic pain management. There were wide variations in training and experience amongst senior registrars. Overall there was little change between 1985 and 1990. In particular the number of anaesthetic senior registrars who felt equipped to undertake a consultant post with an interest in chronic pain management had not increased.

Notes: Background Lung function tests, including forced expiratory volume in one second (FEV1), forced expiratory flow at 25–75% of vital capacity (FEF25–75%) and provocation concentrations of histamine which reduce FEV] by 20% (PC20), are used as indicators of airway form and function in bronchial asthma. Recently, markers of eosinophil activation in bronchial lavage and serum have been suggested as a measure of eosinophil mediated inflammation in the airways. These include eosinophil cationic protein (ECP), eosinophil protein X (EPX) (also known as eosinophil derived neuro-toxin) and eosinophil peroxidase (EPO). Similarly, serum tryptase has been used as a marker of mast cell activation in systemic anaphylaxis.Objectives We measured both sets of indices in a group of children with moderately severe asthma to assess the contribution of eosinophil and mast cell mediated events to airflow limitation and bronchial hyperresponsiveness.Methods Forty-eight children aged 5–10 years had spirometric assessments, histamine challenges and blood sampling on the same occasion. After analysis of sera, the indices were compared.Results The eosinophil markers ECP and EPX correlated very well with each other. They showed a moderate negative correlation with PC20 for histamine. EPX was also found to negatively correlate with FEV, and FEF25–75%. Serum tryptase levels showed no such correlates with airway function.Conclusion These results suggest that serum markers of eosinophil activation correlate with airway function in childhood asthma, and may be of value in assessing the severity of the disease. It further supports the notion that childhood asthma has a similar immunopathology to that occurring in adults, with predominance of eosinophil mediated inflammation.

Notes: Backgrottnd Basophils arc circulaling, secretory grunulocytes that are generally considered to be end-stage cells. In one species of guinea-pigs, basophilic leucocytes have been shown to recover from stimulated secretion in short-term cultures. Similar studies have not been done using human basophils.Objective The purpose of this study was to examine human hasophils in short-term recovery intervals following stimulation of secretion to determine whether visual evidence of recovery occurred.Methods We examined the ultrastrucural morphology of early recovery (l0 min-6h) of human basophils following secretion stimulated by formyl-mcthionyl-lcucyl-phenyl-alanine (FMLP). A combined technique for electron microscopy consisted of postfixation exposure to cationizcd ferritin and reduced osmium, providing maximum quality images and allowing identification of intracellular spaces/organelles that opened to the cell surface, often out of the plane of section.Results The ultrastructural evaluation revealed that control basophils (0 time–6h) did not undergo regulated secretion or develop the morphologies associated with recovery following secretion. FMLP-stimulated basophils underwent an overlapping continuum of piecemeal degranulation - anaphylactic degranutation (0 time- I min), producing vesicle- and granule-free, completely degranulated, viable, mature basophils with polylobed nuclei. The early recovery period (10 min-h) following FMLP stimulation was characterized by reconstitution of granules. Morphological mechanisms for granule reconstitution included a mixture of conservation, condensation, and synthetic events.Coticlttsion Human basophils, like guinea pig basophils, have the polential to recover from regulated secretion.

Notes: Abstract. Peripheral blood mononuclear cell proliferative responses and interferon-γ product ion to anti-CD3, cat fur extract, betalactoglobulin and ovalbumin were determined al birth in a group of 34 babies born to families where at least one parent was; atopic. The development of atopic eczema with positive allergy skin-prick tests to cows’ milk and egg at 1 year of age, where the symptoms improved on an egg and milk-free diet, was significantly associated with raised proliferative responses and defective IFN-γ production to stimulation with betalactoglobulin with a trend for similar responses to ovalbumin. This was not observed in those who did not develop atopic eczema or those with atopic eczema not associated with foods. Responses to cat fur extract were not significantly different between those with and without atopic eczema. This has important implications for the prediction at birth, not only of the probability of being allergic, but also of the specific allergens which will cause problems. The implementation of specific targeted allergen avoidance during the critical period in infancy, therefore, should be more easily applied and should facilitate attempts to prevent disease.