Mast cells are key effector cells of the allergic response. When stimulated by specific allergen through the high-affinity IgE receptors or through other stimuli, these cells release a number of potent mediators of inflammation. Amongst these are the serine proteases tryptase and chymase. In humans, tryptase is the most abundant mediator stored in mast cells. Chymase is present in more moderate amounts in a subpopulation of mast cells (MC(TC)). This subtype of mast cells predominates in connective tissue, whereas the other major subtype, the MC(T), predominates in mucosal tissue. Both proteases have been shown to act on specific extracellular proteins and peptides, as well as to alter the behavior of various cell types. Inhibitors of tryptase have been found to be efficacious in animal and human models of asthma, and both proteases are currently being investigated as potential targets for therapeutic intervention. Such pharmacological, physiological, and biochemical studies require the availability of purified tryptase and chymase. In this chapter, we shall describe procedures for the purification of tryptase and chymase from human tissues and provide protocols for monitoring purification and characterization of the final product. The preparation of recombinant proteases will not be covered, though some of the procedures described may be readily adapted for their purification from recombinant expression systems. The procedures described here have been developed for the purification of the human proteases and will require some modification if applied to purify mast cell proteases from the tissues of other species.

Background: Basogranulin, the novel basophil granule protein recognized by the monoclonal antibody BB1, can be released by stimulation with anti-IgE antibody or calcium ionophore. However, the kinetics and regulation of its secretion are unknown.Objective: We quantified basogranulin and histamine release in response to a range of stimuli to assess whether basogranulin secretion is a reliable marker of basophil activation.Methods: Isolated peripheral blood basophils were stimulated with anti-IgE antibody, calcium ionophore, N-formyl-Met-Leu-Phe, and complement C5a. The released basogranulin and histamine were quantified by dot blotting with BB1 and a fluorometric method, respectively. Basogranulin localization was confirmed by flow cytometry.Results: Both basogranulin and histamine displayed a bell-shaped response curve when basophils were challenged with anti-IgE. Half-maximal release occurred within 30 seconds. Basogranulin levels were maximal by 15 minutes, whereas those for histamine continued increasing to 30 minutes. Wortmannin, a PI3-K inhibitor, suppressed the release of both mediators. Basophils from donors with the "nonreleaser" phenotype secreted neither mediator in response to anti-IgE. Non-IgE-dependent stimuli released both mediators in parallel in a concentration-dependent manner. The correlation between the relative amounts of each mediator released was highly significant (r = .901, P < .0001, n = 87). Flow cytometry revealed that some of the secreted basogranulin adhered to the cell surface.Conclusions: Basogranulin is secreted along with histamine in response to both Fc epsilon R I-related and unrelated stimuli. It is therefore a valid marker of basophil activation and could provide the basis for an immunoassay that distinguishes between basophil and mast cell activation.

Eosinophil, basophil, and T helper 2 (TH2) cell recruitment into tissues is a characteristic feature of allergic diseases. These cells have in common the expression of the chemokine receptor CCR3, which may represent a specific pathway for their accumulation in vivo. Although animal models of allergic reactions are available, findings cannot always be extrapolated to man. To overcome these limitations, we have developed a humanized mouse model of allergic cutaneous reaction using severe combined immunodeficiency mice engrafted with skin and autologous peripheral blood mononuclear cells from allergic donors. Intradermal injection of the relevant allergen into human skin xenografts from allergic individuals induced a significant recruitment of human CD4(+) T cells, basophils, and TH2-type cytokine mRNA-expressing cells, as well as murine eosinophils. Human skin xenografts, atopic status, and autologous peripheral blood mononuclear cell reconstitution were all mandatory to induce the allergic reaction. Next, we addressed the role of CCR3 in the endogenous mechanisms involved in the inflammatory cell recruitment in this experimental model of allergic cutaneous reaction. In vivo administration of an anti-human CCR3-blocking antibody selectively reduced accumulation of eosinophils but not that of CD4(+) cells, basophils, or cells expressing mRNA for TH2-type cytokines. These findings establish a new in vivo model of humanized allergic reaction and suggest that eosinophil migration is mediated mainly through CCR3. Finally, these results suggest that this model might be useful to test human-specific antiallergic modulators.

Auto-immunity 76 Bacteria 131 Bacteroides vulgatus 131 Bimosiamose 297 Bone resorption 26 Breast cancer 324 Bronchus-associated lymphoid tissue 262 C57BL/6 mice 215 Cancer-host relationship 348 Cardiac fibroblast 191 Cardiomyocytes 69 CARP 197 CD1a 324 CD14 103 -, soluble 18 CD34 11 CD34+ 333 CD44 variant isoforms 184 CD62L+ CD4+ T cells 170 CD156 47 Cell cycle 1 CFTR 89 Chaperonin 83 ß-Chemokines 229 Cholesterol 215 Chronic bronchitis 277 -obstructive pulmonary disease model 284 Chymase 303 Citrate buffer 98 Classification 121 Colitis 131, 156 -impedance analysis 139 Collagen type IIa 333 Collagenase 98 Collectins 287 Colon cancer 348 Comparative genomic hybridization 204 Computational chemistry 297 Confocal laser scanning microscopy 11 Cowden disease 34 CR3 177

There is an accumulation of evidence to suggest that mast cells may play a key role in gastrointestinal inflammation. We have investigated the numbers and heterogeneity in staining properties of mast cells in biopsies of the duodenum of normal subjects (n = 10), and of normal duodenum from patients with Crohn’s disease of the ileum and/or colon (n = 7) or with Helicobacter-associated gastritis of the antrum/corpus (n = 6). In normal donors, two subsets of mast cells, one located in the duodenal mucosa and the other in the submucosa, were clearly distinguished by their morphology and dye-binding properties. Whereas submucosal mast cells stained metachromatically with Toluidine Blue after neutral formalin fixation and emitted a yellow fluorescence after staining with Berberine sulphate, those in the mucosa were invisible using these stains. In patients with gastritis or Crohn’s disease, there were marked changes in the numbers of mucosal mast cells compared with control subjects, even though the duodenal biopsies were from apparently uninvolved tissue. Gastritis was associated with increased mucosal mast cell numbers (controls: 187 ± 23 cells mm−2; gastritis: 413 ± 139 cells mm−2; p = 0.0004), but mean mucosal mast cell counts in the uninvolved duodenum of Crohn’s patients were actually decreased (34 ± 30 cells mm−2, p = 0.0147). The clear differentiation between mucosal and submucosal mast cells on the basis of metachromasia with Toluidine Blue was not seen in biopsies from the patients with gastritis or Crohn’s disease. Previous studies which have suggested that there are no distinct mucosal and submucosal mast cell subsets in the human intestine may, therefore, have been affected by the use of tissue from diseased subjects. Heterogeneity in the expression of mast cell tryptase and chymase was seen by immunohistochemistry using specific antibodies, but the relative numbers of mast cell subsets were critically dependent on the methods used. Using a sensitive staining procedure, the majority of mucosal mast cells stained positively for chymase as well as for tryptase, an observation confirmed by immunoelectron microscopy and immunoabsorption studies. Our findings suggest that early stages in intestinal inflammation may be reflected in changes in mast cell numbers and in their staining properties, and call for a reappraisal of mast cell heterogeneity in the human intestinal tract

Methanol-extractable material from flush shoot tissue of cocoa ( Theobroma cacao ) inhibited the germination of basidiospores of the witches' broom pathogen Crinipellis perniciosa . In more dilute extracts, abnormally short, swollen germ-tubes emerged from the basidiospores. The anti-fungal factor responsible for these effects was retained by dialysis tubing, and bound to erythrocytes and to protein. These properties were characteristic of the major component of the dialysed extracts, namely polymeric procyanidin (condensed tannin). Highly purified polymeric procyanidin produced effects on C. perniciosa basidiospores that were identical to those produced by crude extracts. The implications of these results for the involvement of procyanidin in host-pathogen interactions are discussed.