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BACKGROUND: We conducted a prospective, observational study to assess the serum chymase level, a mast cell derived protease, as a predictor of dengue severity. METHODS: NS1-positive non-severe dengue patients of age > 14 years with duration of fever < 4 days were included in the study. At the time of admission, the serum sample was taken for chymase estimation. Patients were followed up to four days after they became afebrile to find out the final diagnosis. RESULTS: Total of 338 non-severe dengue patients were recruited (mean age - 29.15 years, male - 66%). On follow-up, 26 patients (7.8%) developed severe dengue. Only chymase level (adjusted odds ratio, aOR-1.787; 95%CI:1.309-2.440) and platelet count at admission (aOR-0.981; 95%CI:0.968-0.993) were able to predict the severity after adjustment for all variables. But, for prediction of severe dengue, the area under Receiver's Operating Curve of chymase was 0.835 (95%CI:0.765-0.905), which was significantly higher than that of the platelet count at admission (0.760, 95%CI:0.650-0.870) (p < 0.001). Patients who developed severe dengue in due course of illness had significantly higher serum chymase level at admission as compared to the rest of the patients. Similar findings were noted across all age-groups. At an optimum cut-off value of 1.35 ng/mL, chymase had a positive likelihood ratio (LR) of 3.5 and a negative LR of 0.15, for predicting severe dengue. CONCLUSION: This study demonstrated the potential ability of serum chymase levels at admission, as a biomarker for prediction of severe dengue in due course of illness. This article is protected by copyright. All rights reserved.

Background Amino acid‐based formulas (AAFs) are used for the dietary management of cow's milk allergy (CMA). Whether AAFs have the potential to prevent the development and/or symptoms of CMA is not known. Objective The present study evaluated the preventive effects of an amino acid (AA)‐based diet on allergic sensitization and symptoms of CMA in mice and aimed to provide insight into the underlying mechanism. Methods C3H/HeOuJ mice were sensitized with whey protein or with phosphate‐buffered saline as sham‐sensitized control. Starting 2 weeks before sensitization, mice were fed with either a protein‐based diet or an AA‐based diet with an AA composition based on that of the AAF Neocate, a commercially available AAF prescribed for the dietary management of CMA. Upon challenge, allergic symptoms, mast cell degranulation, whey‐specific immunoglobulin levels, and FoxP3+ cell counts in jejunum sections were assessed. Results Compared to mice fed with the protein‐based diet, AA‐fed mice had significantly lower acute allergic skin responses. Moreover, the AA‐based diet prevented the whey‐induced symptoms of anaphylaxis and drop in body temperature. Whereas the AA‐based diet had no effect on the levels of serum IgE and mucosal mast cell protease‐1 (mMCP‐1), AA‐fed mice had significantly lower serum IgG2a levels and tended to have lower IgG1 levels (P = .076). In addition, the AA‐based diet prevented the whey‐induced decrease in FoxP3+ cells. In sham‐sensitized mice, no differences between the two diets were observed in any of the tested parameters. Conclusion This study demonstrates that an AA‐based diet can at least partially prevent allergic symptoms of CMA in mice. Differences in FoxP3+ cell counts and serum levels of IgG2a and IgG1 may suggest enhanced anti‐inflammatory and tolerizing capacities in AA‐fed mice. This, combined with the absence of effects in sham‐sensitized mice indicates that AAFs for the prevention of food allergies may be an interesting concept that warrants further research., This study demonstrates that an amino acid‐based diet can at least partially prevent clinical symptoms of cow's milk allergy (CMA) in mice. Differences in FoxP3+ cell counts and serum levels of IgG2a and IgG1 suggest enhanced anti‐inflammatory and tolerizing capacities in mice fed the amino acid‐based diet.

5-Fluorouracil (5-FU) is a front-line cytotoxic therapy. However, intestinal mucositis is a well-known adverse event of 5-FU, which limits its therapeutic use. Indeed, thymol, which is a monoterpene component of the essential oil derived from thymus, has a potential anti-inflammatory and immunomodulatory activity. Therefore, this study aimed to investigate the potential chemoprotective effect of thymol against 5-FU-induced intestinal mucositis. Rats were either exposed to two doses of 5-FU (150 mg/kg, ip) and/or treated with thymol (60 or 120 mg/kg). Oxidative stress and inflammatory markers, as well as pathological changes, were assessed. 5-FU-induced severe intestinal damages as were evidenced by histopathological changes as well as oxidative and inflammatory responses. Thymol pretreatment inhibited 5-FU-induced oxidative stress by reducing lipid peroxidation and increasing intestinal levels of antioxidant systems. Moreover, inflammatory response markers, such as interleukin-6, prostaglandin E2, and COX-2 were also improved. The immunoblotting analysis also showed that thymol significantly inhibited the 5-FU-induced expression of nuclear factor-κB, tumor necrosis factor-α, and transforming growth factor β-1 (TGF-β1), in addition to the suppression of p38 and phosphorylated c-Jun N-terminal kinases (p-JNK) mitogen-activated protein kinase proteins' expressions. Our study is the first to demonstrate the promising protective effect of thymol against 5-FU-induced intestinal mucositis through inhibition of oxidative, inflammatory pathways, and suppression of TGF-β/p38/p-JNK signaling.

The chymase inhibitor fulacimstat is developed as a first-in-class treatment option for the inhibition of adverse cardiac remodeling in patients with left ventricular dysfunction (LVD) after acute myocardial infarction (MI). The aim of the study was to examine the safety and tolerability of fulacimstat in patients with LVD after remote MI. A multicenter, multinational randomized, placebo-controlled study was performed in clinically stable patients (40–79 years of age, left ventricular ejection fraction ≤ 45% because of MI in medical history) who were on stable evidence-based standard-of-care therapies for LVD post-MI including an angiotensin converting enzyme inhibitor or angiotensin receptor blocker at doses of at least half the recommended target dose. Patients were treated for 2 weeks with either placebo (n = 12) or 4 different doses of fulacimstat (5 mg twice daily, n = 9; 10 mg twice daily, n = 9; 25 mg twice daily, n = 10; 50 mg once daily, n = 9). Fulacimstat was safe and well tolerated at all examined doses. There were no clinically relevant effects on vital signs or potassium levels compared with placebo treatment. Mean plasma concentrations of fulacimstat increased with the administered dose and reached exposures predicted to be therapeutically active. The safety profile and the absence of effects on blood pressure or heart rate in a chronic patient population having similar comorbidities and receiving similar comedication as patients after acute MI support future clinical trials with fulacimstat in patients after acute MI.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is linked with inflammatory disorders and the development of oxidative stress in extreme cases. Therefore, anti-inflammatory and antioxidant drugs may alleviate these complications. Ginkgo biloba L. folium extract (EGb) is a herbal medicine containing various active constituents. This review aims to provide a critical discussion on the potential role of EGb in the management of coronavirus disease 2019 (COVID-19). The antiviral effect of EGb is mediated by different mechanisms, including blocking SARS-CoV-2 3-chymotrypsin-like protease that provides trans-variant effectiveness. Moreover, EGb impedes the development of pulmonary inflammatory disorders through the diminution of neutrophil elastase activity, the release of proinflammatory cytokines, platelet aggregation, and thrombosis. Thus, EGb can attenuate the acute lung injury and acute respiratory distress syndrome in COVID-19. In conclusion, EGb offers the potential of being used as adjuvant antiviral and symptomatic therapy. Nanosystems enabling targeted delivery, personalization, and booster of effects provide the opportunity for the use of EGb in modern phytotherapy.

A novel coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), or 2019 novel coronavirus] has been identified as the pathogen of coronavirus disease 2019. The main protease (Mpro, also called 3‐chymotrypsin‐like protease) of SARS‐CoV‐2 is a potential target for treatment of COVID‐19. A Mpro homodimer structure suitable for docking simulations was prepared using a crystal structure (PDB ID: https://doi.org/10.2210/pdb6Y2G/pdb; resolution 2.20 Å). Structural refinement was performed in the presence of peptidomimetic α‐ketoamide inhibitors, which were previously disconnected from each Cys145 of the Mpro homodimer, and energy calculations were performed. Structure‐based virtual screenings were performed using the ChEMBL database. Through a total of 1 485 144 screenings, 64 potential drugs (11 approved, 14 clinical, and 39 preclinical drugs) were predicted to show high binding affinity with Mpro. Additional docking simulations for predicted compounds with high binding affinity with Mpro suggested that 28 bioactive compounds may have potential as effective anti‐SARS‐CoV‐2 drug candidates. The procedure used in this study is a possible strategy for discovering anti‐SARS‐CoV‐2 drugs from drug libraries that may significantly shorten the clinical development period with regard to drug repositioning., Here, I describe virtual screenings for compounds targeting main protease of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) to predict potential anti‐SARS‐CoV‐2 drugs and lead compounds. The procedure described herein may be a suitable drug repositioning strategy for the discovery of anti‐SARS‐CoV‐2 drugs from drug libraries.

Chymase released from mast cells produces pro‐fibrotic, inflammatory, and vasoconstrictor agents. Studies were performed to test the hypothesis that chronic chymase inhibition provides a renal protective effect in type 2 diabetes. Diabetic (db/db) and control mice (db/m) were chronically infused with a chymase‐specific inhibitor or vehicle for 8 weeks. Baseline urinary albumin excretion (UalbV) averaged 42 ± 3 and 442 ± 32 microg/d in control (n = 22) and diabetic mice (n = 27), respectively (p, The most significant finding of the present study is the identification that chronic inhibition of chymase in the db/db type II diabetic mouse results in renoprotection as reflected by a significant attenuation of urinary albumin excretion and mesangial expansion/glomerular injury. These studies suggest that chymase inhibition may represent a novel therapeutic target in cardiovascular and renal disease.

The relatively low efficacy of ACE-inhibitors in the treatment of heart failure in women after estrogen loss may be due to their inability to reach the intracellular sites at which angiotensin (Ang) II is generated and/or the existence of cell-specific mechanisms in which ACE is not the essential processing pathway for Ang II formation. We compared the metabolic pathway for Ang II formation in freshly isolated myocytes (CMs) and non-myocytes (NCMs) in cardiac membranes extracted from hearts of gonadal-intact and ovariectomized (OVX) adult WKY and SHR rats. Plasma Ang II levels were higher in WKY vs. SHR (strain effect: WKY: 62 ± 6 pg/mL vs. SHR: 42 ± 9 pg/mL; P

A hallmark of parasite infection is the accumulation of innate immune cells, notably granulocytes and mast cells, at the site of infection. While this is typically viewed as a transient response, with the tissue returning to steady state once the infection is cleared, we found that mast cells accumulated in the large-intestinal epithelium following infection with the nematode Trichuris muris and persisted at this site for several months after worm expulsion. Mast cell accumulation in the epithelium was associated with the induction of type-2 immunity and appeared to be driven by increased maturation of local progenitors in the intestinal lamina propria. Furthermore, we also detected increased local and systemic levels of the mucosal mast cell protease MCPt-1, which correlated highly with the persistent epithelial mast cell population. Finally, the mast cells appeared to have striking consequences on epithelial barrier integrity, by regulation of gut permeability long after worm expulsion. These findings highlight the importance of mast cells not only in the early phases of infection but also at later stages, which has functional implications on the mucosal tissue.

Chymase has been identified as an angiotensin II-forming enzyme found in cardiovascular tissues. Angiotensin II is involved not only in the regulation of blood pressure, but also in the progression of cardiovascular remodelling. Interestingly, chymase inhibitors prevent cardiovascular remodelling without lowering blood pressure. The reason why chymase inhibitors do not affect blood pressure may depend on the localization of chymase in vivo. In normal tissues, chymase is stored in mast cell granules and has no enzymatic function; whereas, in damaged tissues, chymase exhibits enzymatic activity immediately following its release from the granules. Chymase also activates transforming growth factor-β and matrix metalloproteinase-9, both of which are involved in cardiovascular remodelling, and their enzymatic functions are also observed only in damaged tissues. In animal models of hypertension, diabetes and hypercholesterolaemia, chymase inhibitors improve cardiovascular remodelling without a general circulatory effect, including blood pressure. Thus, it is proposed that chymase is a potentially important target for preventing cardiovascular diseases.

Mast cell chymase can have a pro-inflammatory or an immunosuppressive function in psoriasis, but the outcome may depend on the level of chymase activity. Therefore, mast cells showing chymase activity (Chyact ) and immunoreactivity (Chyprot ) were studied during the Köbner reaction (0 days, 2 h, 1 day, 3 days and 7 days) of psoriasis induced by the tape-stripping technique. Also, the effect of recombinant human chymase (rh-chymase) or human LAD2 mast cells (LAD2) on the (3) H-thymidine uptake of psoriatic peripheral blood mononuclear cells (PBMC) or total T cells was studied. The Chyact /Chyprot ratio tended to be higher in all time-point biopsies in the Köbner-negative (n = 10) than -positive (n = 8) group (P = 0.073), although chymase activity decreased significantly at 2 h to 1 day only in the Köbner-negative group. rh-chymase (0.05-0.5 μg/mL) stimulated to a varying extent PBMC in eight out of nine cultures, but in all cultures 5 μg/mL rh-chymase turned the stimulation towards inhibition. The effect of rh-chymase on T cells varied from stimulation to inhibition, but in 11 of 15 cultures rh-chymase, at least at 5 μg/mL, produced a change to inhibition. In co-cultures, LAD2 inhibited PBMC in the absence of soybean trypsin inhibitor (SBTI). In the presence of SBTI, LAD2 stimulated PBMC in the majority of seven cultures. In summary, the psoriatic immunopathogenesis may be promoted at low, but controlled at high, activity status of chymase.

NKp46 (natural cytotoxic receptor 1/CD335) is expressed on natural killer cells and Th2-type innate lymphocytes. However, NKp46 expression in human mast cells has not yet been reported. Here, we explored the expression of, and possible role played by, NKp46 in such cells. NKp46 protein was expressed in human mast cells in urticaria pigmentosa principally of the tryptase-positive/chymase-negative type (MCT), but not in human non-neoplastic skin mast cells of the tryptase-positive/chymase-positive (MCTC) type. NKp46 expression was also evident in the human neoplastic mast cell line HMC1.2. NKp46 knockdown changed the phenotype of this cell line from MCT to MCTC and downregulated GrB production, but did not influence IL-22 production. An agonistic anti-NKp46 antibody upregulated production of GrB and IL-22, but did not change the MCT-like phenotype of HMC1.2 cells. NKp46 was thus involved in the production of serine proteases and IL-22 in human mast cells.

Scope Glycation of allergens via Maillard reaction or chemical conjugation has been shown to influence susceptibility to food-induced allergies. It is hypothesized that mucosal immune response bias can be favorably altered by orally administering various forms of glycated ovalbumin (OVA). Methods and results Groups of Balb/c mice (n = 10) were orally sensitized to OVA and administered various forms of glycated OVA (glucose, mannose, glucomannan, galactomannan, and a mixture containing OVA and glucomannan). Outcomes post oral challenge were measured as clinical allergic signs, serum histamine, mouse mast cell protease 1 (MMCP-1), antibody activity, type-1/2 cytokines, percentage of T-regulatory cells (T-regs) and in vitro dendritic cell, and T-cell-related mechanisms. Clinical signs and specific IgE were decreased (p ≤ 0.05), and T-reg cell percentage was increased in the mannose and glucomannan treated groups. The OVA-mannose group also had less histamine, MMCP-1, specific IgG, IL-4 and IL-17, and more IL-12p70 (p ≤ 0.05). Other parameters measured did not differ significantly among groups. Also, OVA-glycated mannose reduced maturation and uptake by dendritic cells. Less activation of T cells and type-2 cytokine response in DC–T-cell cocultures were observed with OVA-glycated mannose stimulation. Conclusion This study validates, for the first time, the use of OVA-glycated mannose and glucomannan for potential beneficial dietary interventions for allergy.

Mast cells (MCs) are found abundantly in the brain and the meninges and play a complex role in neuroinflammatory diseases, such as stroke and multiple sclerosis. Here, we show that MC-deficient Kit/Kit mice display increased neurodegeneration in the lesion area after brain trauma. Furthermore, MC-deficient mice display significantly more brain inflammation, namely an increased presence of macrophages/microglia, as well as dramatically increased T-cell infiltration at days 4 and 14 after injury, combined with increased astrogliosis at day 14 following injury. The number of proliferating Ki67 macrophages/microglia and astrocytes around the lesion area is more than doubled in these MC-deficient mice. In parallel, MC-deficient Kit mice display increased presence of macrophages/microglia at day 4, and persistent astrogliosis at day 4 and 14 after brain trauma. Further analysis of mice deficient in one of the most relevant MC proteases, i.e., mouse mast cell protease 4 (mMCP-4), revealed that astrogliosis and T-cell infiltration are significantly increased in mMCP-4-knockout mice. Finally, treatment with an inhibitor of mMCP-4 significantly increased macrophage/microglia numbers and astrogliosis. These data suggest that MCs exert protective functions after trauma, at least in part via mMCP-4, by suppressing exacerbated inflammation via their proteases.

Mucosal mast cells (MMCs) have an important role in allergic inflammation, and effective antagonists are required for their regulation. To discover a possible mechanism of controlling the activation of MMCs, we investigated the expression and function of syntaxin4, one of the soluble membrane N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, in RBL-2H3 cells, which is a rat mucosal mast cell line. Syntaxin4 silencing was induced by transfection of short interfering RNAs (siRNAs). Syntaxin4 was knocked down in mast cells at both the mRNA and protein levels. The release of granule contents that are involved in inflammation, such as histamine and hexosaminidase, was significantly suppressed by the gene silencing of syntaxin4. Silencing of this gene was also induced in the trachea and bronchi of rats by intratracheal application of the siRNAs using an atelocollagen delivery system. The activation of MMCs, which was monitored by the level of rat mast cell protease-II (RMCPII) in the bronchoalveolar lavage fluid (BALF), was inhibited, and asthmatic airway constriction was prevented by administration of the syntaxin/atelocollagen complex. These results indicate that siRNAs targeting syntaxin4 can stabilize mucosal mast cells and may have beneficial therapeutic effects on the asthmatic response.

Deposition of Schistosoma mansoni eggs in the intestinal mucosa is associated with recruitment of mucosal mast cells (MMC) expressing mouse mast cell protease-1 (mMCP-1). We investigated the involvement of mMCP-1 in intestinal barrier disruption and egg excretion by examining BALB/c mice lacking mMCP-1 (Mcpt-1−/−). Tissue and faecal egg counts from 6 weeks until 12 weeks post-infection (w p.i.) revealed no differences between wild type (WT) and Mcpt-1−/−mice. Using chamber experiments on ileal tissue revealed that at 8 w p.i., the epithelial barrier and secretory capacity were severely impaired, whereas no difference was found between WT and Mcpt-1−/−mice in this respect. However, a fragmented distribution of the tight junction (TJ) protein occludin, but not of claudin-3 or ZO-1, was observed in WT mice at 8 w p.i., while no changes in TJ integrity were seen in Mcpt-1−/−mice. Therefore, we conclude that in contrast to the situation in Trichinella spiralis-infected mice, in schistosomiasis, mMCP-1 is not a key mediator in egg excretion or impairment of the intestinal barrier. The marked decrease in ileal secretory capacity during S. mansoni egg excretion suggests that the mechanisms facilitating the passage of schistosoma eggs through the gut wall are directed more particularly at the epithelial cells.

BACKGROUND Lupin has been introduced as a new food ingredient in an increasing number of European countries, resulting in reports of allergic reactions mostly due to cross-reactions in peanut-allergic individuals. Some cases of primary lupin allergy have also been reported. OBJECTIVE The aim of our study was to develop a food allergy model of lupin in mice with anaphylaxis as the endpoint and further, to develop an approach to estimate the allergen dose inducing maximal sensitization using a statistical design requiring a limited number of animals. METHODS Mice were immunized by intragastric gavage using cholera toxin as an adjuvant. A two-compartment response surface design with IgE as the main variable was used to estimate the maximal sensitizing dose of lupin in the model. This estimated dose was further used to evaluate the model. The mice were challenged with a high dose of lupin and signs of an anaphylactic reaction were observed. Antibody reactions (IgE and IgG2a), serum mast cell protease [mouse mast cell protease-1 (MMCP-1)] and ex vivo production of cytokines (IL-4, IL-5 and IFN-gamma) by spleen cells were measured. An immunoblot with regard to IgE binding was also performed. RESULTS The dose that elicited the maximal sensitization measured as IgE was 5.7 mg lupin protein per immunization. Mice that received this dose developed anaphylactic reactions upon challenge, IgE against several proteins in the lupin extract, and high levels of MMCP-1, and showed a general shift towards a T-helper type 2 response. Post-challenge serum MMCP-1 levels corresponded to the seriousness of the anaphylactic reactions. CONCLUSION We have established a mouse model with clinical symptoms of lupin allergy, with an optimized dose of lupin protein. A statistical design that can be used to determine an optimal immunization dose with the use of a minimum of laboratory animals is described.

Melatonin (N-acetyl-5-methoxytryptamine) is an efficient free radical scavenger and antioxidant, both in vitro and in vivo. The role of melatonin as an immunomodulator is, in some cases, contradictory. In this study we have investigated the therapeutic efficacy of melatonin in rats subjected to Pelagia noctiluca crude venom (of the familia Pelaguiidae; and genus Pelagia) induced acute paw inflammation. In particular, injection of the venom into the paw of rats elicited an acute inflammatory response characterized by accumulation of fluid containing a large number of polymorphonuclear neutrophils in the paw and subsequent lipid peroxidation. Furthermore, the venom promoted an expression of iNOS, nitrotyrosine and the activation of the nuclear enzyme poly (ADP-ribose) polymerase as determined by immunohistochemical analysis of paw tissues. Administration of melatonin 30 min, 1 and 6 hr after the challenge with the venom, caused a significant reduction in all the parameters of inflammation measured. Thus, based on these findings we propose that melatonin may be useful a treatment of local acute inflammation induced by P. noctiluca crude venom.

Summary Background Secretory leucocyte protease inhibitor (SLPI), which is present in many physiological fluids including saliva, sputum and nasal discharge, is the most effective inhibitor of chymase. Previously, we demonstrated that chymase is able to cleave SLPI and that the cleaved portion, cSLPI, is a biomarker of chymase activity. Objective We investigated the potential of cSLPI as a biomarker of chymase activity in subjects with allergic rhinitis (AR) and asthmatic airway disease. Methods Baseline sputum samples were collected from atopic asthmatics and healthy controls (HC). Nasal lavages (NAL) were performed in subjects with AR both at baseline and following a nasal challenge with allergen or placebo. Levels of cSLPI and chymase were determined by Western analysis, and tryptase and α-2 macroglobulin were measured by immunoassay. Results As compared with HC, asthmatics showed a significant increase in baseline cSLPI/total SLPI ratios and an increase in chymase levels. There was a high correlation of cSLPI/SLPI ratios to chymase levels in normal individuals and untreated asthmatics. In the NAL of patients with AR, as compared with placebo, allergen challenge increased inflammatory biomarkers, including cSLPI/SLPI ratios, chymase levels, tryptase levels and α2-macroglobulin levels. Correlations were observed between cSLPI/SLPI ratios and chymase levels and cSLPI/SLPI ratios and α2-macroglobulin levels; no correlation was seen between cSLPI/SLPI ratios and tryptase levels. Conclusion Our data indicate that cSLPI reflects chymase activity in AR and asthma. Hence, cSLPI may serve as a biomarker for disease activity and for monitoring the efficacy of novel anti-inflammatory treatments in chymase-mediated diseases.

Summary The microphthalmia-associated transcription factor (MITF) gene encodes a basic helix-loop-helix and leucin zipper protein. In this study, we identified a novel MITF isoform, MITF-CM, which possesses a unique amino terminus. Exon 1CM is located 84 kb upstream of the exon encoding the B1b domain. MITF-CM was expressed in the human mast cell line HMC-1, the human basophilic cell line KU812, and CB-derived mast cells cultured for 10 weeks as well as bone marrow mononuclear cells. Transient transfection of MITF-CM cDNA in COS-7 cells resulted in the expression of a 64-kDa protein, detected by Western blotting, and nuclear localization of the protein, detected by immunostaining. The transient cotransfection of a luciferase construct under the control of the tyrosinase promoter and MITF-CM cDNA increased luciferase activity threefold. In contrast, none of the MITF isoforms transactivated both the tryptase and chymase gene promoters, indicating differences in the gene transactivation system between humans and mice.

Obesity and diabetes are frequently associated with cardiovascular disease. When a normal heart is subjected to brief/sublethal repetitive ischemia and reperfusion (I/R), adaptive responses are activated to preserve cardiac structure and function. These responses include but are not limited to alterations in cardiac metabolism, reduced calcium responsiveness, and induction of antioxidant enzymes. In a model of ischemic cardiomyopathy inducible by brief repetitive I/R, we hypothesized that dysregulation of these adaptive responses in diet-induced obese (DIO) mice would contribute to enhanced myocardial injury. DIO C57BL/6J mice were subjected to 15 min of daily repetitive I/R while under short-acting anesthesia, a protocol that results in the development of fibrotic cardiomyopathy. Cardiac lipids and candidate gene expression were analyzed at 3 days, and histology at 5 days of repetitive I/R. Total free fatty acids (FFAs) in the cardiac extracts of DIO mice were significantly elevated, reflecting primarily the dietary fatty acid composition. Compared with lean controls, cardiac fatty acid oxidation capacity of DIO mice was significantly higher, concurrent with increased expression of fatty acid metabolism gene transcripts. Following 15 min of daily repetitive I/R for 3 or 5 days, DIO mice exhibited increased susceptibility to I/R and, in contrast to lean mice, developed microinfarction, which was associated with an exaggerated inflammatory response. Repetitive I/R in DIO mice was associated with more profound significant downregulation of fatty acid metabolism gene transcripts and elevated FFAs and triglycerides. Maladaptive metabolic changes of fatty acid metabolism contribute to enhanced myocardial injury in diet-induced obesity.

The Cre-loxP technology allows the introduction of somatic gene alterations in a tissue and/or cell type specific manner. The development of transgenes that target Cre expression to specific cell types is a critical component in this system. Here, we describe the generation and characterization of transgenic mouse lines expressing Cre recombinase under the control of the baboon alpha-chymase promoter, designated Chm:Cre, in order to direct Cre expression specifically to mouse mast cells. Chm:Cre expression was detected in mast cells in lung and colon tissue. Cre-mediated recombination in these mice identified a population of mature tissue resident mast cells using ROSA26R reporter mice. No Cre-expression and Cre-mediated recombination was induced in in vitro generated bone marrow derived mast cells or mast cells isolated from the peritoneal cavity indicating that Cre-expression under the control of the alpha-chymase promoter is solely activated in tissue resident mast cells. These Chm:Cre transgenic mice represent a useful tool to specifically inactivate genes of interest in mast cells of these tissues.

Background In basal cell carcinoma (BCC), mast cells accumulate in the peritumoral stroma. The serine proteinases tryptase and chymase are the major mediators in mast cell granules and they may exert their enzymatic activity in the BCC lesion by inducing matrix remodeling and epithelial cell detachment. Objective To analyse the numbers of mast cells showing tryptase enzyme activity, chymase enzyme activity and chymase immunoreactivity as well as the presence of chymase inhibitors α 1 -antichymotrypsin ( α 1 -AC), α 1 -proteinase inhibitor ( α 1 -PI) and squamous cell carcinoma antigen-2 (SCCA-2) in BCC. Methods Eleven biopsies were taken from the lesion and healthy-looking skin of 10 patients with superficial spreading BCC. The frozen biopsies were analysed enzyme- and immunohistochemically, and a sequential double-staining method was applied. Results In the BCC lesion, the number of mast cells with tryptase activity and chymase immunoreactivity was significantly increased by 2.2- to 2.3-fold. Practically all tryptase-immunopositive cells contained tryptase activity although occasional tryptase-immunopositive cells (about 1% of total) revealed no activity. However, the ratio of cells with chymase activity to those with chymase immunoreactivity was significantly decreased from 49 ± 19% in the healthy skin to 33 ± 19% in the BCC lesion. Instead, the percentage of mast cells displaying α 1 -AC or α 1 -PI immunoreactivity was significantly increased by 1.7-fold in the BCC lesion. SCCA-2 expression was strongly increased in the malignant BCC epithelium but mostly in the suprabasal layers. Conclusions Tryptase- and chymase-positive mast cells (MC TC ) increased in the BCC lesion. However, chymase is partially inactivated, possibly by the effective chymase inhibitors α 1 -AC and α 1 -PI. SCCA-2 increased in BCC, but was localized mostly to the suprabasal layers, and thus it seems not to be crucial in inhibiting chymase.

Proteinase-activated receptor-2 (PAR-2) is a G-protein coupled receptor. Tryptic proteases cleave PAR-2 exposing a tethered ligand (SLIGKV), which binds and activates the receptor. Although PAR-2 is highly expressed by cultured keratinocytes and is an inflammatory mediator, its precise localization in the normal and inflamed human skin is unknown, and the proteases that activate PAR-2 in the skin have not been identified. We localized PAR-2 in human skin by immunohistochemistry, examined PAR-2 expression by RT-PCR and RNA blotting, and investigated PAR-2 activation by mast cell tryptase. PAR-2 was localized to keratinocytes, especially in the granular layer, to endothelial cells, hair follicles, myoepithelial cells of sweat glands, and dermal dendritic-like cells. PAR-2 was also highly expressed in keratinocytes and endothelial cells of inflamed skin. PAR-2 mRNA was detected in normal human skin by RT-PCR, and in cultured human keratinocytes and dermal microvascular endothelial cells by Northern hybridization. Trypsin, tryptase and a peptide corresponding to the tethered ligand (SLIGKVNH2) increased [Ca2+]i in keratinocytes, measured using Fura-2/AM. Although tryptase-containing mast cells were sparsely scattered in the normal dermis, they were numerous in the dermis in atopic dermatitis, and in the dermis, dermal-epidermal border, and occasionally within the lower epidermis in psoriasis. Tryptase may activate PAR-2 on keratinocytes and endothelial cells during inflammation.

Previous studies on the frequency of mast cells (MCs) in recurrent aphthous ulcers (RAU) have yielded conflicting results. Monoclonal antibodies specific for tryptase (AA1) and anti-IgE (polyclonal antibody) were used to identify density and distribution of MCs in an immunohistochemical study of RAU (n=15), induced oral traumatic ulcers (TUs) (n=9), and control clinically healthy oral mucosa (n=15). Results were quantified by means of a VIDAS image analyzer. In all sections studied, IgE-positive cells showed similar frequency and distribution to tryptase-positive MCs. In RAU lesions, numerous tryptase-positive MCs were found in the sub-epithelial lamina propria, but MC numbers in the epithelium were low and present only in some RAU biopsies. MCs were also more numerous in RAU-inflammatory infiltrates (118+/-31 cells/mm2) than those seen in TU-inflammatory infiltrates (75+/-18 cells/mm2, P

Mast cell numbers are increased significantly in oral lichen planus (OLP). In other inflammatory conditions, mast cells frequently adhere to extracellular matrix proteins such as laminin. The aim of this study, therefore, was to determine whether the distribution of mast cells in OLP is related topographically to laminin in vascular and epithelial basement membranes. Monoclonal antibodies for tryptase, laminin and the alpha6beta1 CD49f laminin-binding integrin were used to identify mast cells, basement membranes (blood vessels and basal epithelium) and the "classical" laminin adhesion receptor, respectively. A double-labelling immunoperoxidase technique was employed to examine and compare mast cell-laminin relationships in OLP (n=19) and normal buccal mucosa (NBM, n=13). In both OLP and NBM, the majority of mast cells were located close to vascular basement membranes. Quantitative studies revealed that the number of mast cells associated with the laminin of vascular basement membranes (distance1 microm) was two-fold and three-fold higher, respectively, in the superficial and deep layers in OLP compared with NBM (P0.001). The frequency distribution of mast cells associated with basal epithelium was not statistically different in both groups (P0.05). The association of mast cells with laminin may be an important determinant of mast cell density in OLP During OLP lesion formation and progression, the preferential distribution of mast cells in the immediate perivascular region provides an ideal situation for mast cell-derived mediators to influence the vascular endothelium.

Aims Mast cells (MCs) are associated with fibrosis in various diseases. MCs comprise two phenotypes: the MC(TC) phenotype contains tryptase and chymase, whereas the MC(T) phenotype contains tryptase. Interleukin (IL)-4 promotes the development of MC(TC) from the MC(T) phenotype. The aim of this study was to determine the relationship between MC phenotypes and fibrosis in diffuse large B-cell lymphoma (DLBCL). Methods and results We examined the distribution and density of MCs in 50 DLBCL and 20 reactive lymph nodes, and evaluated MC phenotypes and IL-4-expressing cells. To detect MCs, immunohistochemistry for tryptase and chymase was performed. The 50 DLBCLs were histologically divided into three groups: no fibrosis (32 cases), reticular type (eight cases) showing reticular fibrosis, and bundle type (10 cases) showing collagenous bundles. The density of tryptase-positive MCs was higher than that of chymase-positive MCs. The densities of tryptase-positive and chymase-positive MCs in fibrotic areas were significantly higher than those in the cellular areas in the reticular and bundle groups. Double immunostaining revealed that MCs in DLBCL comprised MC(T) and MC(TC) phenotypes. Chymase-positive MCs and T lymphocytes expressed IL-4. Although there were few chymase-positive MCs in reactive lymph nodes, the density of tryptase-positive MCs was not different from that in the 'no fibrosis' group. Conclusions Tryptase-positive and chymase-positive MCs are associated with fibrosis in DLBCL.

SUMMARY It is possible to induce eosinophilia in congenitally athymic rats by infection with the parasite Ascaris suum. Athymic bronchial eosinophilia is associated with increased expression of IL-5 and eotaxin mRNA, and with the presence of residual T cells and mast cells. Anamnestic mastocytosis is particularly pronounced and in this study we examine the relationship between mast cell degranulation and IgE production in athymic rats following infection. Incubation of peritoneal mast cells from athymic rats with anti-IgE induced dose-dependent degranulation, as measured by histamine release. However, the failure of mast cells from infected athymic rats to degranulate following incubation with all but one of the parasite antigens selected confirms the absence of a specific IgE response. In contrast, all agonists induced degranulation in euthymic rats. The only parasite-derived factor to induce histamine release in all mast cells was Ascaris body fluid, which contains an element capable of inducing IgE-independent degranulation. Furthermore, increased levels of rat mast cell protease II in athymic bronchoalveolar lavage fluid suggest degranulation of mast cells by IgE and allergen independent means in vivo. We believe that the development of eosinophilic lung inflammation in the absence of specific IgE makes this a prototype for investigating the immunological mechanisms underlying nonatopic asthma.

Human mast cells (MC) exhibit two distinct phenotypes based on the protease content of their secretory granules. MC(TC) cells express tryptase, chymase, cathepsin G, and mast cell carboxypeptidase, while MC(T) cells express only tryptase. Both mast cell phenotypes were assessed near regions of osteolysis in uninfected failed joint implants by immunohistochemistry with antibodies to tryptase and chymase, and by in situ hybridization with anti-sense RNA probes for the respective mRNA molecules. Specimens from the interface membrane of 32 aseptically loosened total hip implants were studied, 28 of which had mast cells of the MC(TC) type. Most of these mast cells were aligned along the bone-prosthesis interface adjacent to loosened implants, suggesting involvement in the chronic inflammatory response that leads to bone resorption. Further ultrastructural evidence of mast cells in tissues from failed joint interface membranes was shown by transmission electron microscopy, and detection by staining after magnetic activated cell sorting. The presence of MC at the periprosthetic interface of eroded bone suggests MC degranulation and activity are associated with osteolysis in failed joint prostheses.

Background and Objectives Chymase is expressed in mast cells and induces angiogenesis via activation of angiotensin II and matrix metalloproteinase-9. However, it has been unclear whether chymase is involved in the pathophysiology of angiogenesis in gastric cancer. To clarify the contribution of chymase to angiogenesis in gastric cancer, we assessed the relationship between chymase-positive cells and tumor angiogenesis. Methods We evaluated chymase-positive cells and microvessels using anti-human chymase and anti-CD34 antibodies in 168 cases of gastric cancer, respectively. Results Chymase-positive cells in gastric tumor region were significantly higher than the cells in normal region. The number of chymase-positive cells in the undifferentiated type of gastric tumor region was significantly higher than the one in the differentiated type. Specimens from patients with advanced histological stages of disease had more chymase-positive cells than those with early-stage disease. There was a significant positive correlation between chymase-positive cells and microvessels in gastric cancer specimens. Postoperative survival curves revealed that patients with a high number of chymase-positive cells had a poor prognosis. Conclusions These results suggest that accumulation of chymase-positive cells in gastric cancer may lead to an increase of tumor angiogenesis, and may contribute to tumor growth and progression. J. Surg. Oncol. 2006;93:36–42. © 2005 Wiley-Liss, Inc.

Summary Background Mast cells (MCs) arise from haematopoietic stem cells. We have recently reported that CD34+ progenitors derived from human bone marrow (BM) develop into tryptase+, chymase+ MCs when cultured in the presence of recombinant human stem cell factor (rhSCF) and recombinant human IL-6 (rhIL-6). In an experiment for the expression of chymase during differentiation, chymase+ cells were detected in human BM, but tryptase+ cells were not found. Objective The purpose of this study was to show the appearance of chymase+ cells in CD34+ cells with an origin different from MC differentiation. Methods CD34+ cells from human BM were sorted with anti-CD117 monoclonal antibody (mAb), and cytospins of CD34+, CD34+CD117+, or CD34+CD117− were prepared. These cells were cultured with rhSCF+rhIL-6 for 12 weeks. Some of the cells were subjected to either histological stain with Wright–Giemsa or immunocytochemistry with anti-chymase mAb. Real-time RT-PCR was also performed to compare the transcriptional level of chymase from each cell preparation. Results Chymase was expressed in CD34+ cells as well as human MCs by immunocytochemistry. Substantial CD34+CD117− cells, but not CD34+CD117+ cells, were stained immunocytochemically with anti-chymase mAb. For 1 week culture with rhSCF+rhIL-6, no cells expressed chymase in any preparation. Real-time RT-PCR revealed positivity for chymase mRNA in CD34+ cells, but it reduced at 1 week of culture, and increased as cells developed into MCs. Chymase mRNA in CD34+CD117+ cells was negligible compared with that in CD34+CD117−. Tryptase mRNA was below the detectable level in CD34+ cells, and increased along with MC differentiation. After 12 weeks of culture, CD34+CD117+ developed predominantly into MCs, whereas CD34+CD117− developed into monocytes/macrophages. Conclusion Our findings suggested that chymase is present not only in MCs but also in CD34+CD117− BM progenitors, but that its origin is different from the MC lineage.

Background: Actinic cheilitis (AC) is a pre-malignant lesion caused by ultraviolet (UV) radiation and characterized by epithelial and connective tissue alterations. Mast cells (MCs), key contributors to solar elastosis in murine UV-irradiated skin, were characterized in order to assess their potential contribution to connective tissue degeneration in AC. Methods: Actinic cheilitis (n = 15) and normal lip (n = 8) biopsies were stained immunohistochemically for tryptase and enzymehistochemically for chymase to determine MC density and protease content. MC subpopulations (i.e. MCT containing only tryptase, and MCTC containing chymase and tryptase) and their distribution were also determined. Results: Mast cells and their proteases were increased in AC as compared with normal lip (P

The role of Stat6 (signal transducers and activators of transcription) in the recruitment and activation of eosinophils has been studied in detail in asthma and other allergic diseases. In this study, we demonstrated that eosinophil responses occur in a Stat6-independent manner in mice infected with the intestinal nematode, Nippostrongylus brasiliensis. Stat6-deficient (Stat6(-/-)) mice cannot expel N. brasiliensis and establish chronic infections. Prominent blood and intestinal eosinophilia were induced after day 14 postinfection (p.i.) and maintained at this level in Stat6(-/-) mice, whereas in wild-type mice eosinophil responses reached a peak on day 10 p.i. and declined thereafter. The introduction of a secondary infection of N. brasiliensis into wild-type mice induced rapid and exaggerated eosinophilia, whereas secondary infection in Stat6(-/-) mice resulted in almost the same eosinophil responses as those of the primary infection, suggesting a lack of memory responses. Blood eosinophilia was also induced in Stat6(-/-) mice implanted with N. brasiliensis in the small intestine, suggesting that intestinal exposure to parasitic antigen is sufficient to induce eosinophil responses. Furthermore, this prominent eosinophil response of Stat6(-/-) mice after day 14 was closely associated with an increase of interleukin (IL)-5 production in serum and intestine. Neither IL-4 nor eotaxin were significantly induced in Stat6(-/-) mice after infection with N. brasiliensis. We also found that mRNA for IL-5, GATA-3 and eosinophil peroxidase (EPO) are induced in the intestine of Stat6(-/-) mice on day 14 p.i. Taken together, these results provide evidence for Stat6-independent IL-5 production and subsequent eosinophil responses after chronic infection with N. brasiliensis.

Background Mast cells are specialized secretory cells of the immune system. Through exocytosis of their secretory lysosomes and secretory granules, mast cells release biologically active substances such as histamine and proteases. Mast cell secretory granules have been studied extensively but much less attention has been given to secretory lysosomes. Studies on mast cell secretory lysosomes are limited by the lack of selective markers and the difficulty to isolate this organelle from conventional lysosomes. Our goal was to develop better tools to study secretory lysosomes. Methods We engineered a rat mast cell line over expressing a rat mast cell protease (RMCP) tagged with a red fluorescent protein (RMCP-DsRed). We used single organelle flow analysis (SOFA) to detect fluorescently labeled secretory lysosomes. The labeled organelles were then sorted using the fluorescence-assisted organelle sorting (FAOS) method. Results We show that the RMCP-DsRed fusion protein selectively localizes to the lysosomal compartment and is exocytosed upon activation, confirming its localization in secretory lysosomes. Lysosomal fractions from cells expressing the RMCP-DsRed fusion were analyzed by SOFA and a specific population of secretory lysosome was identified. Finally, we sorted secretory lysosomes and showed that the sorted material had a higher specific activity for the compartment marker hexosaminidase than a sample obtained by conventional methods. Conclusions Our work further demonstrates the usefulness of flow cytometry to study cellular organelles, and provides new tools to better understand the physiology of secretory lysosomes. Cytometry Part A 55A:94–101, 2003. © 2003 Wiley-Liss, Inc.

SUMMARY An experiment was carried out to compare the parasitological and immunological responses of SWR and CBA mice to trickle (repeated) infection with Heligmosomoides polygyrus. Male mice were given 125 L3 once per week and were killed in groups, together with naive control mice, weekly until week 8. Worm burdens accumulated in CBA, stabilizing in week 5 in excess of 400 worms and remaining high until week 8. In contrast in SWR worm burdens peaked in week 3 at a mean worm burden of 129 and then fell sharply so that by week 6, despite continuing re-infection, no more worms were recovered from these mice. SWR mice showed a marked mast cell and mMCP-1 response, peaking in weeks 2–3, whereas in CBA mice these responses were slower, and even at their height in week 8 still less intense than those in SWR mice. Both strains responded initially with a very similar goblet cell response, which declined in SWR mice as worms were eliminated, but was sustained in CBA mice until week 8. Serum TNFα concentrations were higher in SWR mice throughout the experiment. Infection elicited strong serological responses against adult and L4 antigens in both SWR and CBA mice, involving all the isotypes tested (IgG1, IgA and IgE). Anti-L3 responses were examined only for IgG1. However, only two responses differed significantly between the strains: the IgE response to L4 antigens was more intense in SWR mice, and interestingly and unexpectedly, the IgG1 response to adult worm antigens was more intense in CBA mice. These results reflect the activation of predominantly Th2-driven effector mechanisms, that may be associated with host-protective immunity developing under the trickle infection protocol exploited in these experiments.

This paper reports the results of a genome-wide search for quantitative trait loci (QTL) influencing immunological responses to infection with the gastro-intestinal nematode parasite Heligmosomoides polygyrus in an F2 population created by crossing the resistant SWR and the susceptible CBA inbred mouse strains. Following infections, intestinal granuloma score at post mortem, mucosal mast cell protease 1, and IgE and IgG1 titres were recorded. The susceptible CBA mice had significantly higher IgG1, but significantly lower IgE, mucosal mast cell protease 1 and granuloma scores than SWR mice. Significant QTL were mapped to chromosomes 4, 11, 13 and 17 for granuloma score; chromosomes 12 and 17 for IgE; chromosome 10, 17 and 18 for IgG1 and chromosomes 1, 9, 10, 11, 17 and 18 for mucosal mast cell protease 1. Chromosomes 10, 11, 17 and 18 had QTL affecting more than one trait, and these are most likely to represent single QTL with multiple effects rather than multiple QTL. Some of these QTL map to regions known to harbour genes responsible for the induction of immunological responses to intestinal worms.

We investigated the influence of vitamin E on mediator activity and release in a canine mastocytoma cell line (C2) as a model for canine atopic dermatitis. Cells were incubated without and with vitamin E (100 microm) for 24 h. The histamine and prostaglandin D2 (PGD2) release as well as the chymase and tryptase activity were measured. To stimulate the PGD2 and histamine release, cells were incubated with the wasp venom peptide mastoparan (50 microm) for 30 or 45 min. Nonstimulated as well as mastoparan-stimulated histamine and PGD2 release was reduced significantly in vitamin E-treated cells. The activity of chymase tended to decrease, but the tryptase activity of C2 cells was not influenced by vitamin E. These results indicate that vitamin E decreased the production and release of inflammatory mediators in C2 cells, suggesting that vitamin E might have a possible beneficial effect in inflammatory diseases.

Serine proteinases with trypsin-like (tryptase) and chymotrypsin-like (chymase) properties are major constituents of mast cell granules. Several tetrameric tryptases with differing specificities have been characterized in humans, but only a single chymase. In other species there are larger families of chymases with distinct and narrow proteolytic specificities. Expression of chymases and tryptases varies between tissues. Human pulmonary and gastrointestinal mast cells express chymase at lower levels than tryptase, whereas rodent and ruminant gastrointestinal mast cells express uniquely mucosa-specific chymases. Local and systemic release of chymases and tryptases can be quantified by immunoassay, providing highly specific markers of mast cell activation. The expression and constitutive extracellular secretion of the mucosa-specific chymase, mouse mast cell proteinase-1 (mMCP-1), is regulated by transforming growth factor-beta1 (TGF-beta1) in vitro, but it is not clear how the differential expression of chymases and tryptases is regulated in other species. Few native inhibitors have been identified for tryptases but the tetramers dissociate into inactive subunits in the absence of heparin. Chymases are variably inhibited by plasma proteinase inhibitors and by secretory leucocyte protease inhibitor (SLPI) that is expressed in the airways. Tryptases and chymases promote vascular permeability via indirect and possibly direct mechanisms. They contribute to tissue remodelling through selective proteolysis of matrix proteins and through activation of proteinase-activated receptors and of matrix metalloproteinases. Chymase may modulate vascular tissues through its ability to process angiotensin-I to angiotensin-II. Mucosa-specific chymases promote epithelial permeability and are involved in the immune expulsion of intestinal nematodes. Importantly, granule proteinases released extracellularly contribute to the recruitment of inflammatory cells and may thus be involved in innate responses to infection.

Mast cells are involved in the pathogenesis of both allergies to food and inflammatory bowel disorders. In addition, there are several lines of evidence suggesting that mucosal mast cells also respond to intraluminal stimuli. Our aim was to identify neuroendocrine stimuli that could modify mucosal mast cell activity in the rat. Anaesthetized rats were prepared for duodenal perfusion and mast cell activation was measured by analysis of RMCP II concentration in the duodenal perfusate. Either buffered saline solution or a 5% ovalbumin hydrolysate (OVH) solution was infused into the duodenum. Subdiaphragmatic vagotomy or afferent ablation by intraluminal treatment with capsaicin diminished RMCP II concentration in basal conditions and significantly reduced the response to OVH, which in control animals induced a three-fold increase of the protease. The noradrenergic blockers phentholamine and propranolol significantly diminished RMCP II concentration in basal conditions and completely blocked the response to OVH. Intravenous infusion of cholecystokinin-related peptides also induced a response of mast cells. However, the response was different depending on the peptide. CCK-8 induced a slight increase of RMCP II, whereas both CCK-33 and gastrin induced a significant decrease in mast cell activity. These results show that intraluminal content modulates mucosal mast cell activity by complex mechanisms involving both nervous and endocrine pathway.

Mast cell chymase plays important roles in inflammation and tissue remodeling. Here we show that mast cell chymase also functions as an enhancer of immunoglobulin production. In the culture of murine spleen cells stimulated with lipopolysaccharide and interleukin-4, purified rat chymase (rat mast cell protease-I; RMCP-I), at physiological concentrations, enhanced immunoglobulin E (IgE) and IgG1 syntheses but not IgG3 synthesis. The enhancement was also evident when spleen cells depleted of T cells and macrophages were employed as responding cells. Enzymatic activity of RMCP-I was required to enhance IgE and IgG1, because two inhibitors for chymotryptic enzymes, chymostatin and Y-40613, a novel chymase inhibitor, suppressed the enhanced immunoglobulin production, and phenylmethylsulphonyl fluoride, an irreversible inhibitor for serine proteases, totally abolished the enhancing effect. Furthermore, a specific inhibitor for Zn2+-dependent metalloproteases, GI 129471, could also completely inhibit the production of IgE and IgG1 that was enhanced by RMCP-I, suggesting that a metalloprotease also played an essential role in the immunoglobulin production. Our results together with others show that proteases from mast cell granules have important function not only in the efferent phase but also in the afferent phase of immune responses.

We recently characterized a heparin-deficient mouse strain generated by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2-/- mice show severe defects in their organization of mast cell (MC) secretory granules, with an almost total absence of the various heparin-binding MC proteases. In the present report we have studied the consequences of heparin/MC protease deficiency for extravascular coagulation and fibrinolysis. Addition of prothrombin to peritoneal cells-a mixture of macrophages, lymphocytes, and MCs-resulted in formation of thrombin but the accumulation of thrombin occurred faster in the NDST-2-/-cells than in normal controls. Further, the generated thrombin was subsequently inactivated in the NDST-2+/+ cell cultures but not in the NDST-2-/- cells. Plasminogen was activated to plasmin at an apparently higher rate in peritoneal cells from NDST-2 null mice than in the normal controls. Similar to thrombin, the generated plasmin was inactivated by NDST-2+/+ but not by the NDST-2-/- cells. Subsequent experiments with normal cells showed that cell surface-associated MC chymase, in a strongly heparin-dependent manner, was responsible for both the thrombin-inactivating- and plasmin-inactivating activities. These results show that MC chymase-heparin complexes have the potential to regulate extravascular coagulation processes, as well as the plasminogen activator/plasmin system.

It is well known that the destrobilation and later expulsion are characteristics of multiple Hymenolepis diminuta infections in rats. This process is suggested to be mediated by a variety of host cellular responses. It has also been suggested that immunoglobulin (Ig) E may have a beneficial role for some cestodes including H. diminuta. We examined the intestinal mast cell and serum IgE responses to a 10-H. diminuta infection in three different rat strains. Tapeworm infection induced no increased mast cell and IgE responses in F344 rats in which neither worm biomass nor worm burden decreased during 6 weeks of observation. The number of mast cells and amounts of serum rat mast cell protease (RMCP) II and IgE markedly increased from 3 weeks postinfection (p.i.) in BN rats. The worm biomass in BN rats was significantly lower than that in F344 rats, but worm burden was not different from that in F344 rats at 3 or 6 weeks p.i. In DA rats, the number of mast cells and levels of serum RMCP II and IgE increased at 6 weeks but not at 3 weeks p.i. Although numbers of mast cells and serum RMCP II and IgE levels were lower in DA rats than in BN rats, smaller and fewer worms were recovered in DA rats than in F344 and BN rats at from 3 and 6 weeks p.i. Worms were recovered from all of F344 and BN rats, while only 40% of DA rats harboured worms at 6 weeks p.i. These results suggested that the worm biomass was related to mast cell and IgE responses, but these responses were not required for worm expulsion during low dose H. diminuta infection in rats.

Background: Because the types of mast cells present in the bladder of patients with interstitial cystitis (IC) have not been elucidated, we have used immunohistochemical techniques to determine which of the mast cell types is present in biopsy specimens. Methods: For all patients diagnosed with IC (n = 10; female) their symptoms satisfied the criteria proposed by the National Institutes of Health criteria of IC and six patients suffering from bladder tumors were selected as control patients. Adjacent sections of paraffin-embedded tissues that had been fixed in Carnoy's solution were reacted with either antitryptase or antichymase antibodies. Results: In detrusor and in mucosa, the number of tryptase-positive and chymase-negative mast cells (MCT) was 146 ± 25 and 81 ± 31 cells/mm2, respectively, and the number of tryptase-positive and chymase-positive mast cells (MCTC) was 124 ± 50 and 54 ± 20 cells/mm2, respectively. These numbers were significantly greater than those of the control group. A significant negative correlation (P < 0.005; R = 0.943) was observed between the number of MCTC and the bladder capacity. The number of mast cells obtained by toluidine blue staining in detrusor and in mucosa was 95 ± 68 and 71 ± 39 cells/mm2, respectively, suggesting that staining with toluidine blue underestimated the number of mast cells. Conclusions: Mast cells were significantly increased in number in both the mucosa and detrusor of bladder specimens from IC patients compared with those from control. The MCTC may be the type of mast cell dominantly present in the bladder of IC patients. The MCTC in detrusor increased with the progression of contracted bladder.

BACKGROUND Angiogenesis in individuals with various solid tumors has provided useful information on tumor progression and prognosis, and many examples of mast cell (MC) accumulation coupled with angiogenesis can be found in the literature. METHODS Utilizing monoclonal antibodies for tryptase that are specific markers for MC and for the endothelial surface marker CD34, the authors quantified MC infiltration in 180 patients with pulmonary adenocarcinoma who underwent curative tumor resection, to investigate the relation between mast cell density (MCD) and microvessel density (MVD), clinicopathologic factors, and prognosis. RESULTS A significant association was found between MCD and MVD (P < 0.0001). The MC count was significantly related to tumor progression, involving N classification and stage (P = 0.0002 for N classification and P = 0.0015 for stage). A significant difference in the rate of patient survival was detected between patients whose tumors had an MCD defined as high and those whose tumors had an MCD defined as low (P < 0.0001). Multivariate analysis also showed that MCD was significantly related to survival (P = 0.0378). CONCLUSIONS These data indicate that MC infiltration may contribute to tumor angiogenesis and tumor progression, and that MCD is a useful prognostic marker in pulmonary adenocarcinoma. Cancer 2000;88:2686–92. © 2000 American Cancer Society.

Bronchial eosinophilic inflammation and bronchial hyperresponsiveness (BHR) are the main features of allergic asthma (AA), but they have also been demonstrated in allergic rhinitis (AR), suggesting a continuity between both diseases. In spite of not fully reproducing natural allergenic exposure, the allergen bronchial provocation test (A-BPT) has provided important knowledge of the pathophysiology of AA. Our aim was to verify the existence of a behavior of AA and AR airways different from the allergen bronchial challenge-induced airway eosinophilic inflammation and BHR changes. We studied a group of 31 mild and short-evolution AA and 15 AR patients, sensitized to Dermatophagoides pteronyssinus. The A-BPT was performed with a partially biologically standardized D. pteronyssinus extract, and known quantities of Der p 1 were inhaled. Peripheral blood (eosinophils and ECP) and induced sputum (percentage cell counts, ECP, albumin, tryptase, and interleukin [IL]-5) were analyzed, before and 24 h after A-BPT. Methacholine BHR, assessed before and 32 h after the A-BPT, was defined by M-PD20 values and, when possible, by maximal response plateau (MRP). The A-BPT was well tolerated by all the patients. AA presented a lower Der p 1 PD20 and a higher occurrence of late-phase responses (LPR). M-PD20 values decreased in AA, but not in AR, patients. MRP values increased in both groups. Eosinophils numbers and ECP levels increased in blood and sputum from both AA and AR, but only the absolute increment of sputum ECP levels was higher in AA than AR patients (P=0.025). The A-BPT induced no change in sputum albumin, tryptase, or IL-5 values. We conclude as follows: In spite of presenting a lower degree of bronchial sensitivity to allergen, AR patients responded to allergen inhalation with an eosinophilic inflammation enhancement very similar to that observed among AA. MRP levels increased in both AA and AR patients after allergen challenge; however, M-PD20 values significantly changed only in the AA group, suggesting that the components of the airway response to methacholine were controlled by different mechanisms. It is possible that the differences between AR and AA lie only in the quantitative bronchial response to allergen inhalation.

Based on the peculiar spatial array of the active sites in the internal chamber of the multicatalytic proteasome, as derived from the X-ray structure of yeast proteasome, homo- and heterobivalent inhibitors were designed and synthesized to exploit the principle of multivalency for enhancing inhibition potency. Peptidic bis-aldehyde compounds of the octapeptide size were synthesized to address adjacent active sites, whilst a PEG spacer with a statistical length distribution of 19-25 monomers was used to link two identical or different tripeptide aldehydes as binding heads. These bis-aldehyde compounds were synthesized applying both methods in solution and solid phase peptide synthesis. Bivalent binding was observed only for the PEG-spaced inhibitors suggesting that binding from the primed side prevents hemiacetal formation with the active site threonine residue.

Hepatolithiasis and primary sclerosing cholangitis (PSC) are intractable chronic biliary diseases. In hepatolithiasis, bilirubin-calcium stones are packed in multiple irregularly dilated intrahepatic bile ducts. In PSC, small bilirubin-calcium stones develop terminally. The progressive periductal fibrosis with dilated and stenotic bile ducts in these two diseases may play a role in their incurability. This immunohistochemical study has investigated the expression of some factors that might be involved in fibrogenesis in hepatolithiasis and PSC. Many mast cells positive for c-kit were found in the periductal and ductal fibrosis around the intrahepatic large bile ducts and also around the proliferative peribiliary glands. These mast cells also expressed basic fibroblast growth factor and/or tumour necrosis factor-alpha, which are known as fibrogenetic factors. It was of interest that the aberrant expression of stem cell factor (SCF), a ligand of c-kit, was demonstrated on biliary epithelia of the dilated and stenotic bile ducts showing periductal fibrosis and inflammation and also of the proliferated peribiliary glands in hepatolithiasis and PSC, while no such expression was seen in non-affected bile ducts in hepatolithiasis or in the bile ducts in normal livers. Some of the infiltrating mononuclear cells around the SCF-expressing bile ducts were also positive for SCF. It seems likely that aberrantly expressed SCF on biliary epithelial cells accumulates and stimulates mast cells via the c-kit receptor and that these up-regulated mast cells induce progressive periductal and portal fibrosis by displaying fibrogenetic factors in hepatolithiasis and PSC.

Different subpopulations of mast cells are characterized by their abundant contents of either tryptase or in addition chymase. These two neutral proteases are found in mast cells and may thus hold a key to the understanding of mast cell dependent reactions. Such studies are however hampered by the lack of readily available supplies of chymase. We have therefore studied the simultaneous purification of both proteases from hairless moro hr/hr mouse skin, using a sequence of salt extractions and chromatographic separations. After three steps of extraction, a 13-fold purification with an 82% yield was obtained for tryptase and a 15-fold purification with a 90% yield for chymase. Further one step purification on conventional sephadex, sephacryl and octyl sepharose columns was unsatisfactory because of further protein contamination of the fractions. Heparin affinity chromatography caused a high loss of tryptase and residual protein contamination. Gradient elution on a benzamidine sepharose 6B column resulted however in a single, low yield (17.9%) tryptase peak and a broader, high yield (>90%) chymase peak, and a 34% yield high purity fraction. The proteases thus purified exhibited their typical inhibitor profile. On Western blot analysis and on autoradiography in the presence of the serine protease inhibitor diisopropylfluorophosphate (DFP), only one 28 kD molecule with chymase activity was identified, whereas a broad 32-38 kD band of tryptase monomers was noted. Taken together, these data show that, after salt extraction and a single benzamidine affinity chromatography step, both mast cell chymase and tryptase can be separated and in case of chymase also highly purified, allowing thus for the study of biological activities of this molecule.

Tryptase-like activities of T and NK cells contribute to the induction of target cell apoptosis, but only granzyme A (GzmA) has been shown to exhibit Z-Lys-SBzl esterase activity in murine T cells. GzmA-deficient mice exhibit residual Z-Lys-SBzl hydrolyzing activity and almost normal levels of lymphocyte-mediated cytotoxicity. Here we report the cloning and biochemical characterization of recombinant mouse granzyme K (GzmK). The purified murine protein shows Z-Lys-SBzl hydrolyzing activity and is inhibited by bikunin, the light chain of inter-α-trypsin inhibitor, like the human homolog. We conclude that GzmK expressed by GzmA-deficient T cells accounts for the remaining Z-Lys-SBzl activity. Functional similarities between GzmA and GzmK may explain the subtle immunological deficits observed in GzmA-deficient mice.