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This proof-of-principle trial showed that imatinib treatment reduced mast-cell activation and improved airway responsiveness in patients with severe refractory asthma. Many patients with severe asthma do not have adequate disease control despite the use of high-dose inhaled or systemic glucocorticoids. 1 Severe asthma is associated with airway hyperresponsiveness — that is, an exaggerated response to a bronchoconstrictor stimulus — and airway inflammation, both of which persist despite high-dose glucocorticoid therapy. 2 , 3 Increased airway hyperresponsiveness is associated with a progressive loss of lung function, 4 and, among patients with moderate-to-severe asthma, those with airway hyperresponsiveness have a poorer quality of life than those without this trait. 5 In addition, studies have shown that treatment targeting airway hyperresponsiveness leads to more effective control of asthma . . .

BackgroundMast cell activation is thought to be involved in visceral hypersensitivity, one of the main characteristics of the irritable bowel syndrome (IBS). A study was therefore undertaken to investigate the effect of the mast cell stabiliser ketotifen on rectal sensitivity and symptoms in patients with IBS.Methods60 patients with IBS underwent a barostat study to assess rectal sensitivity before and after 8 weeks of treatment. After the initial barostat, patients were randomised to receive ketotifen or placebo. IBS symptoms and health-related quality of life were scored. In addition, mast cells were quantified and spontaneous release of tryptase and histamine was determined in rectal biopsies and compared with biopsies from 22 age- and gender-matched healthy volunteers.ResultsKetotifen but not placebo increased the threshold for discomfort in patients with IBS with visceral hypersensitivity. This effect was not observed in normosensitive patients with IBS. Ketotifen significantly decreased abdominal pain and other IBS symptoms and improved quality of life. The number of mast cells in rectal biopsies and spontaneous release of tryptase were lower in patients with IBS than in healthy volunteers. Spontaneous release of histamine was mostly undetectable but was slightly increased in patients with IBS compared with healthy volunteers. Histamine and tryptase release were not altered by ketotifen.ConclusionsThis study shows that ketotifen increases the threshold for discomfort in patients with IBS with visceral hypersensitivity, reduces IBS symptoms and improves health-related quality of life. Whether this effect is secondary to the mast cell stabilising properties of ketotifen or H1 receptor antagonism remains to be further investigated.Trial Registration NumberNTR39, ISRCTN22504486.

Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of inflammatory responses in asthma, allergy and other diseases. Here we report an anti-β-tryptase antibody with a superior mechanism of action compared to others since it not only inhibits tetrameric β-tryptase, but also completely inhibits monomeric β-tryptase activity. The antibody binds to an exosite that causes tetramer dissociation as either an IgG or Fab and, in addition, allosterically alters the substrate binding cleft on monomers, thus preventing substrate binding and proteolysis. We solve the cryoEM structure of the complex, generate biochemical data and engineer point mutations to elucidate the allosteric path of inhibition. This ultimately reveals a single Asp to Gly mutation in CDR-L3 that only slightly impacts binding affinity, but completely eliminates inhibitory activity. Finally, we improve antibody inhibitory potency up to 4.7-fold by structure-based design creating new charge-charge interactions. This antibody may have enhanced efficacy and potential to assess the relevance of β-tryptase, including monomers, in biological and clinical settings. The authors report an anti-β-tryptase antibody as a potential therapeutic for asthma with a superior mechanism of action that inhibits both tetrameric and monomeric β-tryptase activity. Structural analysis reveals that the molecular path of allosteric inhibition is due to a single antibody residue.

PRG4 is an extracellular matrix protein that maintains homeostasis through its boundary lubricating and anti-inflammatory properties. Altered expression and function of PRG4 have been associated with joint inflammatory diseases, including osteoarthritis. Here we show that mast cell tryptase β cleaves PRG4 in a dose- and time-dependent manner, which was confirmed by silver stain gel electrophoresis and mass spectrometry. Tryptase-treated PRG4 results in a reduction of lubrication. Compared to full-length, cleaved PRG4 further activates NF-κB expression in cells overexpressing TLR2, −4, and −5. In the destabilization of the medial meniscus model of osteoarthritis in rat, tryptase β and PRG4 colocalize at the site of injury in knee cartilage and is associated with disease severity. When human primary synovial fibroblasts from male osteoarthritis patients or male healthy subjects treated with tryptase β and/or PRG4 are subjected to a quantitative shotgun proteomics and proteome changes are characterized, it further supports the role of NF-κB activation. Here we show that tryptase β as a modulator of joint lubrication in osteoarthritis via the cleavage of PRG4. Altered expression and function of the extracellular matrix protein PRG4 have been associated with osteoarthritis. Here, the authors show that mast cell tryptase β cleaves PRG4, resulting in a reduction of lubrication and activation of inflammation in this context.

In the current classification of the World Health Organization (WHO), bone marrow mastocytosis (BMM) is a provisional variant of indolent systemic mastocytosis (ISM) defined by bone marrow involvement and absence of skin lesions. However, no additional diagnostic criteria for BMM have been proposed. Within the registry dataset of the European Competence Network on Mastocytosis, we compared characteristics and outcomes of 390 patients with BMM and 1175 patients with typical ISM. BMM patients were significantly older, predominantly male, had lower tryptase and lower burden of neoplastic mast cells, and displayed a higher frequency of allergic reactions, mainly triggered by Hymenoptera, than patients with typical ISM. The estimated 10-year progression-free survival of BMM and typical ISM was 95.9% and 92.6%, respectively. In BMM patients defined by WHO-based criteria, the presence of one B-Finding and tryptase level ≥125 ng/mL were identified as risk factors for progression in multivariate analyses. BMM patients without any of these risk factors were found to have better progression-free survival (p < 0.05) and better overall survival (p < 0.05) than other ISM patients. These data support the proposal to define BMM as a separate SM variant characterized by SM criteria, absence of skin lesions, absence of B-Findings, and tryptase levels <125 ng/mL.

Rosacea is a chronic inflammatory skin disease whose pathophysiological mechanism is still unclear. However, it is known that mast cell (MC) numbers are increased in the dermis of rosacea patients. MC proteases not only recruit other immune cells, which amplify the inflammatory response, but also cause vasodilation and angiogenesis. MCs are also one of the primary sources of cathelicidin LL-37 (Cath LL-37), an antimicrobial peptide that has been shown to be an enabler of rosacea pathogenesis. Here, we demonstrate that MCs are key mediators of cathelicidin-initiated skin inflammation. After Cath LL-37 injection into the dermis, MC-deficient B6.Cg−KitW−sh/HNihrJaeBsmJ (KitW−sh) mice did not develop rosacea-like features. Conversely, chymase (P<0.001), tryptase, and Mmp9 (P<0.01) mRNA levels were significantly higher in C57BL/6 wild-type (WT) mice. Treating WT mice with an MC stabilizer significantly decreased the expressions of Mmp9 and Cxcl2 (P<0.01). Our data were confirmed on erythematotelangiectatic rosacea subjects who showed a decrease in matrix metalloproteinase activity (P<0.05), after 8 weeks of topical cromolyn treatment. We conclude that MCs have a central role in the development of inflammation subsequent to Cath LL-37 activation and that downregulation of activated MCs may be a therapy for rosacea treatment.

Mast cells (MC) are resident tissue cells found primarily at the interphase between tissues and the environment. These evolutionary old cells store large amounts of proteases within cytoplasmic granules, and one of the most abundant of these proteases is tryptase. To look deeper into the question of their in vivo targets, we have analyzed the activity of the human MC tryptase on 69 different human cytokines and chemokines, and the activity of the mouse tryptase (mMCP-6) on 56 mouse cytokines and chemokines. These enzymes were found to be remarkably restrictive in their cleavage of these potential targets. Only five were efficiently cleaved by the human tryptase: TSLP, IL-21, MCP3, MIP-3b, and eotaxin. This strict specificity indicates a regulatory function of these proteases and not primarily as unspecific degrading enzymes. We recently showed that the human MC chymase also had a relatively strict specificity, indicating that both of these proteases have regulatory functions. One of the most interesting regulatory functions may involve controlling excessive TH2-mediated inflammation by cleaving several of the most important TH2-promoting inflammatory cytokines, including IL-18, IL-33, TSLP, IL-15, and IL-21, indicating a potent negative feedback loop on TH2 immunity.

Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1β, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable.

Tryptase is a serine protease that is predominantly produced by tissue mast cells (MCs) and stored in secretory granules together with other pre-formed mediators. MC activation, degranulation and mediator release contribute to various immunological processes, but also to several specific diseases, such as IgE-dependent allergies and clonal MC disorders. Biologically active tryptase tetramers primarily derive from the two genes TPSB2 (encoding β-tryptase) and TPSAB1 (encoding either α- or β-tryptase). Based on the most common gene copy numbers, three genotypes, 0α:4β, 1α:3β and 2α:2β, were defined as “canonical”. About 4–6% of the general population carry germline TPSAB1-α copy number gains (2α:3β, 3α:2β or more α-extra-copies), resulting in elevated basal serum tryptase levels. This condition has recently been termed hereditary alpha tryptasemia (HαT). Although many carriers of HαT appear to be asymptomatic, a number of more or less specific symptoms have been associated with HαT. Recent studies have revealed a significantly higher HαT prevalence in patients with systemic mastocytosis (SM) and an association with concomitant severe Hymenoptera venom-induced anaphylaxis. Moreover, HαT seems to be more common in idiopathic anaphylaxis and MC activation syndromes (MCAS). Therefore, TPSAB1 genotyping should be included in the diagnostic algorithm in patients with symptomatic SM, severe anaphylaxis or MCAS.

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2.