Staphylococcus aureus internalization in mast cells in nasal polyps: Characterization of interactions and potential mechanisms.

BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps is a common chronic condition. The exact cause of nasal polyps remains unknown. Recently, we made the novel observation of intracellular localization of Staphylococcus aureus within mast cells in nasal polyps. OBJECTIVE: This follow-up study aimed to further characterize interactions between S aureus and mast cells in this setting and elucidate potential internalization mechanisms with particular emphasis on the role of staphylococcal enterotoxin B (SEB). METHODS: A prospective study was performed using an explant tissue model with ex vivo inferior turbinate mucosa obtained from patients with chronic rhinosinusitis with nasal polyps (n = 7) and patients without CRS (n = 5). Immunohistochemistry was used to characterize S aureus uptake into mast cells and investigate the effects of SEB on this process. An in vitro cell-culture model was used to investigate mast cell-S aureus interactions by using a combination of fluorescent in situ hybridization, confocal laser scanning microscopy, scanning electron microscopy, transmission electron microscopy, and proliferation assays. RESULTS: S aureus was captured by extracellular traps and entered mast cells through phagocytosis. Proliferating intracellular S aureus led to the expansion and eventual rupture of mast cells, resulting in release of viable S aureus into the extracellular space. The presence of SEB appeared to promote internalization of S aureus into mast cells. CONCLUSION: This study provides new insights into the interactions between S aureus and mast cells, including the internalization process, and demonstrates a prominent role for SEB in promoting uptake of the bacteria into these cells.

Eosinophils, basophils and type 2 immune microenvironments in COPD-affected lung tissue.

Although elevated blood or sputum eosinophils are present in many patients with COPD, uncertainties remain regarding the anatomical distribution pattern of lung-infiltrating eosinophils. Basophils have remained virtually unexplored in COPD. This study mapped tissue-infiltrating eosinophils, basophils and eosinophil-promoting immune mechanisms in COPD-affected lungs.Surgical lung tissue and biopsies from major anatomical compartments were obtained from COPD patients with severity grades Global Initiative for Chronic Obstructive Lung Disease stages I-IV; never-smokers/smokers served as controls. Automated immunohistochemistry and in situ hybridisation identified immune cells, the type 2 immunity marker GATA3 and eotaxins (CCL11, CCL24).Eosinophils and basophils were present in all anatomical compartments of COPD-affected lungs and increased significantly in very severe COPD. The eosinophilia was strikingly patchy, and focal eosinophil-rich microenvironments were spatially linked with GATA3+ cells, including type 2 helper T-cell lymphocytes and type 2 innate lymphoid cells. A similarly localised and interleukin-33/ST2-dependent eosinophilia was demonstrated in influenza-infected mice. Both mice and patients displayed spatially confined eotaxin signatures with CCL11+ fibroblasts and CCL24+ macrophages.In addition to identifying tissue basophilia as a novel feature of advanced COPD, the identification of spatially confined eosinophil-rich type 2 microenvironments represents a novel type of heterogeneity in the immunopathology of COPD that is likely to have implications for personalised treatment.

Mast cell chymase impairs bronchial epithelium integrity by degrading cell junction molecules of epithelial cells.

BACKGROUND: An increased degree of mast cell (MC) degranulation and damage to the epithelial lining are prominent features of bronchial asthma. In asthmatic airways, it seems likely that epithelial cells will be exposed to increased concentrations of proteases from MC, though their actions on the epithelium are still not very clear. METHODS: Bronchial rings from human lung tissue or 16HBE cell monolayer were incubated with MC chymase in different doses or various inhibitors. The sections of paraffin-embedded tissue were haematoxylin-eosin stained and computerized by image analysis for epithelial damage-scale-evaluation; the cell viability, proliferation, adhesion and lactate dehydrogenase activity release were assayed; the expressions of gelatinases, cell junction molecules and structure proteins of 16HBE were examined. RESULTS: Mast cell chymase was found to provoke profound changes in the morphology of bronchi epithelial layer. Following incubation with chymase, there was 40% reduction in the length of epithelium that was intact, with detachment of columnar epithelial cells and basal cells. Chymase reduced epithelial cell proliferation and induced cell detachment, which were associated with the changes in secretion and activation of matrix metalloproteinase-2/9. In intact epithelial cell layers, immunocytochemistry study revealed that chymase reduced the expressions of occludin, claudin-4, ZO-1, E-cadherin, focal adhesion kinase and cytokeratin. Overall data of this study indicated that MC chymase can influence tissue remodelling, disrupt epithelial cell junctions, inhibit wound healing and impair the barrier function of epithelium, resulting in dysfunction of airway wall and ECM remodelling in pathogenesis of asthma. CONCLUSION: Mast cell chymase plays a key role in inducing the damage to bronchial epithelium in asthma.

Repeated Amblyomma testudinarium tick bites are associated with increased galactose-α-1,3-galactose carbohydrate IgE antibody levels: A retrospective cohort study in a single institution.

BACKGROUND: Alpha-gal syndrome is a hypersensitivity reaction to red meat mediated by IgE antibody specific to galactose-α-1,3-galactose carbohydrate (alpha-gal). Amblyomma tick bites are associated with this condition, but the pathophysiology is not understood. OBJECTIVE: To clarify the mechanism of development of alpha-gal syndrome after tick bites. METHODS: We compared alpha-gal antibody levels between patients with and without a history of tick bites and examined histologic stainings of tick bite lesions between patients with and without detectable alpha-gal IgE antibody. RESULTS: Patients who had ≥2 tick bites had higher levels of alpha-gal IgE antibody compared with those with only 1 tick bite or healthy individuals. On histologic investigation, greater numbers of basophils and eosinophils, but not mast cells, were observed infiltrating lesions of patients with ≥2 tick bites compared with those with 1 tick bite. Type 2 cytokine-producing T-cell infiltration was predominantly observed in such patients. LIMITATIONS: The study was conducted at a single institution in Japan. CONCLUSION: In Amblyomma tick bite lesions, basophils; eosinophils; and type 2, cytokine-producing T cells infiltrate the skin and alpha-gal IgE antibodies are produced. These findings provide a potential mechanistic connection between Amblyomma bites and red meat hypersensitivity.

The regulatory role of Dipeptidyl peptidase I on the activation of immune granulocytes.

Dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, required for activation of serine proteases of granulocytes including mast cells (MCs), neutrophils (NPs) and others, which were found in synovial tissue of patients with rheumatoid arthritis (RA). But, the role of DPPI associated with those cells in RA development is unclear. In this study, the collagen-induced-arthritis (CIA) rat-model was employed to investigate the expression and activity levels of DPPI and its association with RA progress. Primary granulocytes were freshly extracted from bone-marrows of normal or CIA rats, human mast cell line LAD-2 and primary neutrophils, human-recombinant-DPPI, DPPI-inhibitor Gly-Phe-CHN2 , LTB4, anti-IgE antibody, calcium ionophore were used to study the regulatory role of DPPI in cell activations. The increased DPPI activities in synovial fluids, serum, and bone-marrow homogenates of CIA rats associated with RA severities progress were observed after injections. MMP2/9 expressions in SFs and bone-marrow were in different patterns. Regular-Blood-Tests have shown the high leveled DPPI activities associated with granulocytes differentiations in-vivo in blood of CIA rats. In-vitro cell models, DPPI up-regulated the proliferation of primary bone-marrow granulocytes of normal rats, but inhibited that of CIA rats. DPPI up-regulated and Gly-Phe-CHN2 down-regulated MCs intracellular DPPI and chymase activities. Gly-Phe-CHN2 also inhibited the LTB4 -activated-NPs and NP-elastase activities. Following stimulation of calcium ionophore, the net-releases of DPPI and ß-hexosaminidase from MCs were increased over a time-course, while Gly-Phe-CHN2 down-regulated MCs and NPs activation. Our findings demonstrate the role of DPPI in regulating MCs and NPs activation, and modulating proteolysis in the process of RA.

A mechanistic multicentre, parallel group, randomised placebo-controlled trial of mesalazine for the treatment of IBS with diarrhoea (IBS-D).

INTRODUCTION: Immune activation has been reported in the mucosa of IBS patients with diarrhoea (IBS-D), and some small studies have suggested that mesalazine may reduce symptoms. We performed a double-blind, randomised placebo-controlled trial of 2 g mesalazine twice daily versus placebo for 3 months in patients with Rome III criteria IBS-D. Primary outcome was daily average stool frequency during weeks 11-12; secondary outcomes were abdominal pain, stool consistency, urgency and satisfactory relief of IBS symptoms. METHODS: Participants were randomised after a 2-week baseline stool diary. All participants completed a 12-week stool diary and at the end of each week recorded the presence of 'satisfactory relief of IBS symptoms'. RESULTS: 136 patients with IBS-D (82 women, 54 men) were randomised, 10 patients withdrew from each group. Analysis by intention to treat showed the daily average stool frequency during weeks 11 and 12 were mean (SD), 2.8 (1.2) in mesalazine and 2.7 (1.9) in the placebo group with no significant group difference, (95% CI) 0.1 (-0.33 to 0.53), p=0.66. Mesalazine did not improve abdominal pain, stool consistency nor percentage with satisfactory relief compared with placebo during the last two-weeks follow-up. CONCLUSIONS: This study does not support any clinically meaningful benefit or harm of mesalazine compared with placebo in unselected patients with IBS-D. More precise subtyping based on underlying disease mechanisms is needed to allow more effective targeting of treatment in IBS. TRIAL REGISTRATION NUMBER: NCT01316718.

NADPH oxidase-independent formation of extracellular DNA traps by basophils.

Basophils are primarily associated with a proinflammatory and immunoregulatory role in allergic diseases and parasitic infections. Recent studies have shown that basophils can also bind various bacteria both in the presence and the absence of opsonizing Abs. In this report, we show that both human and mouse basophils are able to produce mitochondrial reactive oxygen species and to form extracellular DNA traps upon IL-3 priming and subsequent activation of the complement factor 5 a receptor or FcεRI. Such basophil extracellular traps (BETs) contain mitochondrial, but not nuclear DNA, as well as the granule proteins basogranulin and mouse mast cell protease 8. BET formation occurs despite the absence of any functional NADPH oxidase in basophils. BETs can be found in both human and mouse inflamed tissues, suggesting that they also play a role under in vivo inflammatory conditions. Taken together, these findings suggest that basophils exert direct innate immune effector functions in the extracellular space.

Innate and adaptive T cells in asthmatic patients: Relationship to severity and disease mechanisms.

BACKGROUND: Asthma is a chronic inflammatory disease involving diverse cells and mediators whose interconnectivity and relationships to asthma severity are unclear. OBJECTIVE: We performed a comprehensive assessment of TH17 cells, regulatory T cells, mucosal-associated invariant T (MAIT) cells, other T-cell subsets, and granulocyte mediators in asthmatic patients. METHODS: Sixty patients with mild-to-severe asthma and 24 control subjects underwent detailed clinical assessment and provided induced sputum, endobronchial biopsy, bronchoalveolar lavage, and blood samples. Adaptive and invariant T-cell subsets, cytokines, mast cells, and basophil mediators were analyzed. RESULTS: Significant heterogeneity of T-cell phenotypes was observed, with levels of IL-13-secreting T cells and type 2 cytokines increased at some, but not all, asthma severities. TH17 cells and γδ-17 cells, proposed drivers of neutrophilic inflammation, were not strongly associated with asthma, even in severe neutrophilic forms. MAIT cell frequencies were strikingly reduced in both blood and lung tissue in relation to corticosteroid therapy and vitamin D levels, especially in patients with severe asthma in whom bronchoalveolar lavage regulatory T-cell numbers were also reduced. Bayesian network analysis identified complex relationships between pathobiologic and clinical parameters. Topological data analysis identified 6 novel clusters that are associated with diverse underlying disease mechanisms, with increased mast cell mediator levels in patients with severe asthma both in its atopic (type 2 cytokine-high) and nonatopic forms. CONCLUSION: The evidence for a role for TH17 cells in patients with severe asthma is limited. Severe asthma is associated with a striking deficiency of MAIT cells and high mast cell mediator levels. This study provides proof of concept for disease mechanistic networks in asthmatic patients with clusters that could inform the development of new therapies.

Alveolar T-helper type-2 immunity in atopic asthma is associated with poor clinical control.

Real-world evaluation studies have shown that many patients with asthma remain symptomatic despite treatment with inhaled corticosteroids (ICSs). As conventional ICSs have poor access to the peripheral airways, the aim of the present paper was to study the relationship between peripheral airway inflammation and clinical control in allergic asthma. Consequently, bronchial and transbronchial biopsies were obtained from patients with poorly controlled asthma [n=12, asthma control test (ACT) score<20], patients with well-controlled asthma (n=12, ACT score≥20) and healthy controls (n=8). Tissue sections were immunostained to assess multiple leucocyte populations. To determine the degree of T-helper type-2 (Th2) immunity, the logarithmic value of the ratio between Th2 cells/mm2 and Th1 cells/mm2 was used as a surrogate score for Th2-skewed immunity. In the bronchi, the leucocyte infiltration pattern and the Th2-score were similar between patients with well-controlled asthma and those with poorly controlled asthma. In contrast, in the alveolar parenchyma, the expression of T-helper cells was significantly higher in patients with poorly controlled asthma than in patients with well-controlled asthma (P<0.01). Furthermore, the alveolar Th2-score was significantly higher in patients with poorly controlled asthma (median 0.4) than in the controlled patients (median -0.10, P<0.05). In addition, in contrast with bronchial Th2-score, the alveolar Th2-score correlated significantly with ACT score (rs=-0.62, P<0.01) in the pooled asthma group. Collectively, our data reveal an alveolar Th2-skewed inflammation, specifically in asthmatic patients who are poorly controlled with ICSs, and suggest that pharmacological targeting of the peripheral airways may be beneficial in this large patient category.

Cutaneous basophil infiltration in atopic dermatitis is associated with abundant epidermal infiltration of helper T cells: A preliminary retrospective study.

Atopic dermatitis (AD) is a heterogenous inflammatory skin disorder. Our previous study revealed that basophil infiltration in skin is observed in approximately 60% of AD cases. However, the clinical and histological characteristics of AD associated with basophil infiltration remain unclear. We examined basophil infiltration by immunohistochemical staining of 38 specimens from 34 patients who underwent skin biopsies to diagnose AD from April 2016 to September 2021 at Tokyo Medical and Dental University Hospital. The patients/specimens were divided into two groups, 17 patients/21 specimens associated with little or no basophil infiltration (basophil-low group) and 17 patients/17 specimens associated with marked basophil infiltration (basophil-high group). The clinical characteristics of the patients (age, sex, complications, blood biomarkers, skin symptoms, and treatment) and histological features of the specimens were compared between the groups. Basophil-high patients were significantly younger than basophil-low patients. Blood basophil counts were higher in basophil-high patients than in basophil-low patients. CD4+ T-cell infiltration was more marked in basophil-high specimens than in basophil-low specimens. CD4+ T cells infiltrated into the dermis as well as into the epidermis only in the basophil-high specimens. Thus, basophil-high AD can be characterized by skin lesions associated with abundant helper T-cell infiltration in younger patients.

Modulation of basophil activity: a novel function of the neuropeptide α-melanocyte-stimulating hormone.

BACKGROUND: Little is known about the effect of neuropeptides on basophils, which are important effector cells in immune and allergic responses. OBJECTIVE: This study aimed at revealing the role of α-melanocyte-stimulating hormone (α-MSH) on basophil function. METHODS: Expression of melanocortin receptors and proopiomelanocortin (POMC) was analyzed by means of RT-PCR, Western immunoblotting, fluorescence-activated cell sorting, and double-immunofluorescence analysis. Signal transduction studies included cyclic AMP and Ca(2+) mobilization assays. Basophil activity was assessed based on CD63 surface expression and cytokine release. RESULTS: MC-1R expression was detectable in basophils isolated from human peripheral blood, as well as in basophils within nasal tissue. In isolated basophils from human blood, truncated POMC transcripts were present, but there was no POMC protein. Treatment of basophils with α-MSH increased intracellular Ca(2+) but not cyclic AMP levels. α-MSH at physiologic doses potently suppressed basophil activation induced by N-formyl-methionyl-leucyl-phenylalanine, phorbol 12-myristate 13-acetate, or grass pollen allergen in whole blood of healthy or allergic subjects, respectively. The effect of α-MSH on basophil activation was MC-1R mediated (as shown by blockade with a peptide analogue of agouti-signaling protein) and imitated by adrenocorticotropic hormone but not elicited by the tripeptides KPV and KdPT, both of which lack the central pharmacophore of α-MSH. Moreover, α-MSH at physiologic doses significantly suppressed secretion of 3 proallergic cytokines, IL-4, IL-6, and IL-13, in basophils stimulated with anti-IgE, N-formyl-methionyl-leucyl-phenylalanine, or phorbol 12-myristate 13-acetate. CONCLUSION: Our findings highlight a novel functional activity of α-MSH, which acts as a natural antiallergic basophil-response modifier. These findings might point to novel therapeutic strategies in treating allergic diseases.

Decreased peripheral basophil counts in urticaria and mouse model of oxazolone-induced hypersensitivity, the latter suggesting basopenia reflecting migration to skin.

A decrease in the number of basophils in the peripheral blood, or basopenia, has been noted, reflecting the activity of chronic spontaneous urticaria (CSU). Infiltration of basophils into the skin has also been reported, but the mechanism of basopenia in CSU has not been clarified. The phenomenon of basopenia during the active phase of urticaria was confirmed, and basophil numbers increased following symptom improvement in 15 out of 17 patients treated with omalizumab and in 13 of 15 patients treated with antihistamines. Our examination by immunostaining also revealed basophil infiltration of the CSU lesions, as in previous reports, but since most of our patients were already taking oral steroids, it was not considered appropriate to examine the relationship between basophil numbers in tissue and peripheral blood. Then, we used mouse model of contact hypersensitivity with a single application of oxazolone, which is known to stimulate basophil infiltration, and investigated basophil counts in the skin, peripheral blood, and bone marrow. In this model, a decrease in peripheral blood basophil numbers was observed one day after challenge, but not after 2 days, reflecting supplementation from the bone marrow. Indeed, when cultured basophils expressing GFP were transplanted into the peripheral blood, GFP-positive basophil numbers in the peripheral blood remained low even after 2 days of challenge. Despite differences among species and models, these results suggest that one reason for the decrease of basophils in the peripheral blood in CSU may involve migration of circulating basophils into the skin.

Mast cells from different molecular and prognostic subtypes of systemic mastocytosis display distinct immunophenotypes.

BACKGROUND: Systemic mastocytosis (SM) is a heterogeneous group of disorders with distinct clinical and biological behavior. Despite this, little is known about the immunophenotypic features of the distinct diagnostic categories of SM. OBJECTIVE: To analyze the immunophenotypic characteristics of bone marrow (BM) mast cells (MCs) of different subtypes of SM. METHODS: Bone marrow samples from 123 patients with different subtypes of SM and 92 controls were analyzed for a broad panel of immunophenotypic markers by flow cytometry. RESULTS: Three clearly different maturation-associated immunophenotypic profiles were found for BMMCs in SM. These different profiles were associated with both genetic markers of the disease and its clinical behavior. BMMCs from poor-prognosis categories of SM (aggressive SM and MC leukemia) typically showed an immature phenotype with clonal involvement of all myeloid lineages by the D816V stem cell growth factor receptor gene (KIT) mutation. In turn, a mature activated versus resting BMMC immunophenotype was commonly found among patients with good-prognosis subtypes of SM depending on whether they carried (indolent SM and clonal MC activation disorders) or not (well differentiated SM) the D816V KIT mutation. CONCLUSION: Bone marrow MCs from SM show 3 different maturation-related immunophenotypic profiles that are associated with both the genetic markers of the disease and its clinical behavior.

Effect of honey and its major royal jelly protein 1 on cytokine and MMP-9 mRNA transcripts in human keratinocytes.

Honey has been used since ancient times as a remedy in wound healing. However, even though the results from randomized clinical trials document that honey accelerates wound healing, no study dealing with its influence on human skin cells (epidermal keratinocytes and dermal fibroblast) has been performed. We demonstrate that keratinocytes, which are known to be involved in wound healing, are responsible for elevated production of mediators including cytokines (TNF-alpha, IL-1beta and TGF-beta) and matrix metalloproteinase-9 (MMP-9) after incubation with honey. Real-time PCR was performed for the quantification of mRNA level of selected cytokines and MMP-9. Furthermore, we show that the increased level of MMP-9 in the epidermis following incubation with honey leads to degradation of type IV collagen in the basement membrane. These data indisputably demonstrate that honey activates keratinocytes and support the findings that honey may accelerate wound healing process.

Detection of Mast Cells and Basophils by Immunohistochemistry.

Staining cells or tissues with basic dyes was the mainstay of mast cell and basophil detection methods for more than a century following the first identification of these cell types using such methods. These techniques have now been largely supplanted by immunohistochemical procedures with monoclonal antibodies directed against unique constituents of these cell types. Immunohistochemistry with antibodies specific for the granule protease tryptase provides a more sensitive and discriminating means for detecting mast cells than using the classical histochemical procedures, and using antibodies specific for products of basophils (2D7 antigen and basogranulin) has allowed detection of basophils that infiltrate into tissues. The application of immunohistochemistry to detect more than one marker in the same cell has underpinned concepts of mast cell heterogeneity based on differential expression of chymase and other proteases. The double labeling procedures employed have also provided a means for investigating the expression of cytokines and a range of other products. Protocols are here set out that have been used for immunohistochemical detection of mast cells and basophils and their subpopulations in human tissues. Consideration is given to pitfalls to avoid and to a range of alternative approaches.

Proof of concept pilot study: prevalence of grass virus infection and the potential for effects on the allergenic potency of pollen.

BACKGROUND: Wild plants harbour a variety of viruses and these have the potential to alter the composition of pollen. The potential consequences of virus infection of grasses on pollen-induced allergic disease are not known. METHODS: We have collected pollen from Dactylis glomerata (cocksfoot; a grass species implicated as a trigger of allergic rhino-conjunctivitis) from Wytham Wood, Oxfordshire UK. Extracts were prepared from pollen from uninfected grass, and from grass naturally infected by the Cocksfoot streak potyvirus (CSV). Preparations of pollen from virus-infected and non-infected grasses were employed in skin testing 15 grass pollen-allergic subjects with hayfever. Allergen profiles of extracts were investigated by Western blotting for IgE with sera from allergic subjects. RESULTS: The prevalence of CSV infection in cocksfoot grasses sampled from the study site varied significantly over an eight-year period, but infection rates of up to 70% were detected. Virus infection was associated with small alterations in the quantities of pollen proteins detected by polyacrylamide gel electrophoresis, and in the patterns of allergens identified by Western blotting with IgE from grass pollen allergic subjects. For individual subjects there were differences in potencies of standardised extracts of pollen from virus-free and virus-infected plants as assessed by skin testing, though a consistent pattern was not established for the group of 15 subjects. CONCLUSION: Infection rates for CSV in cocksfoot grass can be high, though variable. Virus-induced alterations in components of grass pollen have the potential to alter the allergenic potency.

Identification of mast cells and mast cell subpopulations.

Mast cells generate mediators of inflammation which are stored in granules and secreted on activation either by allergen crosslinking of membrane-bound IgE or through other stimuli. Most methods for mast cell identification rely on the histochemical detection of constituents of the secretory granules. Although staining for mast cells with histochemical stains can be rapid and relatively inexpensive, it is not always possible to distinguish reliably between mast cells and basophils in tissues. A further problem with the staining of mast cells with commonly used basic dyes is that the reagents employed to fix the tissues can influence the results, leading to confusion regarding the numbers of mast cells present in various tissues. Recognition that there is considerable heterogeneity between mast cell populations in the degree to which staining properties are lost with formalin fixation has led to mast cell subsets being defined on this basis. The development and application of procedures for identifying mast cell proteases has led to important advances in our understanding of the role of mast cells and in the nature of heterogeneity in man. The techniques described here should allow the reliable detection of mast cells and mast cell subsets in a range of tissues and cell preparations. There will be a continuing need for validation, for consideration of potential sources of error, and for the development of new and more reliable techniques for mast cell identification.

Purification and characterization of mast cell tryptase and chymase from human tissues.

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.