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PubMed2001

Synthesis and kinetic evaluation of peptide alpha-keto-beta-aldehyde-based inhibitors of trypsin-like serine proteases.

New, synthetic peptide analogues bearing a C-terminal basic alpha-keto-beta-aldehyde moiety were prepared as novel inhibitors of the trypsin-like serine proteases. The compounds, Ac-Leu-Leu-Arg-COCHO, Ac-Arg-Gln-Arg-COCHO and Boc-Val-Leu-Lys-COCHO were evaluated kinetically against trypsin and three other trypsin-like serine proteases, tryptase, plasmin and thrombin, all of which are implicated as mediators of important disease processes. Results illustrate that alpha-keto-beta-aldehydes are potent inhibitors, with similar potency to comparable peptide aldehydes, and intriguingly, appearto act, in some instances, by a novel mechanism of action. Ac-Leu-Leu-Arg-COCHO, an analogue of the natural product leupeptin, is a potent, tight-binding inhibitor of trypsin (Ki(final) = 1.9 microM), plasmin (Ki(final) = 4.9 microM) and tryptase (Ki(final) = 1.2 microM) and an irreversible inactivator of thrombin (k2nd 4,500 M(-1).min(-1)). Boc-Val-Leu-Lys-COCHO was found to be a tight-binding inhibitor of its target protease plasmin (Ki(final) = 3.1 microM) and was inactive against thrombin. Ac-Arg-Gln-Arg-COCHO was a slow-binding inhibitor of tryptase (Ki(final) = 1.6 microM) and also irreversibly inactivated trypsin (k2nd = 8,920 M(-1) min(-1)). Peptides or peptidomimetics with a C-terminal basic alpha-keto-beta-aldehyde function thus provide a useful new molecular template for the development of new therapeutic agents against a wide range of disorders, such as coagulopathies and asthma, which may be mediated by the aberrant activity of trypsin-like serine proteases.

Aldehydes↗
PubMed1993

Effects of synthetic trypsin inhibitors on the cell cycle of synchronized HeLa cells.

HeLa cells were synchronized by a double-thymidine block. After removal of thymidine, the cells immediately caused the uptake of [3H]thymidine into DNA and reached a half-maximum. The duration of the cell cycle was 23 h, and definite changes in cell density were observed between 12 and 13 h and between 35 and 36 h after removal of thymidine. Thus, the initiation time of S phase could be fixed. A trypsin-like proteinase appearing at around 17 h 17 min after removal of thymidine and correlated with the onset of the second S phase, tryptase 17:17 [cf., M. Muramatu et al., Biochim. Biophys. Acta, 1087, 87 (1990)], was obtained. 4-tert-Butylphenyl and 4-biphenyl esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA) and amidinopiperidine-4-alkanoic acids, trypsin inhibitors, strongly inhibited the tryptase activity, and these esters exert different effects on the cell cycle of HeLa cells at concentrations showing complete inhibition or maximal inhibitory effect on the tryptase. Both esters of GMCHA elongated the onset of the second S phase for 3 h. Esters of amidinopiperidine-4-acetic and 4-propionic acids showed a similar effect at lower concentrations than GMCHA esters. 4-tert-Butylphenyl esters of amidinopiperidine-4-propionic acid and butyric acids strongly suppressed the second S and M phases by probably affecting the G1 late phase, since they have no effect on the first S and M phases. The addition of amidinopiperidine-4-carboxylic acid 4-tert-butylphenyl ester 0 min after removal of thymidine into the cells completely suppressed the first S and M phases.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidines↗
PubMed2003

Pathogenesis of aspirin-exacerbated respiratory disease.

The underlying respiratory disease is activated by unknown mechanism and results in an intense infiltration of mast cells and eosinophils into the entire respiratory mucosa. These cells synthesize leukotrienes (LTs) at a very high rate and mast cells also release histamine and tryptase and synthesize PGD(2) a vasodilator and bronchoconstrictor. Furthermore, AERD patients under synthesize from arachidonic acid (AA) a peculiar product called lipoxins, which opposes inflammation generated by leukotrienes. Finally, cysLT1 receptors are over expressed and highly responsive to LTE(4), further augmenting the underlying inflammatory disease. This inflammatory condition is partly inhibited by synthesis of PGE(2) through COX-1. PGE(2) partially inhibits 5-lipogygenase conversion of AA to LTA(4) and blocks release of histamine and tryptase from mast cells. When COX-l is inhibited by ASA or NSAIDs, PGE(2) synthesis stops and an enormous release of histamine and synthesis of LTs occurs. The upper respiratory reaction is mediated by both histamine and LTs but the bronchospastic reaction is mediated by LTs. The systemic effects of flush, gastric pain and hives are mediated by histamine. Aspirin desensitization can not be explained by disappearance of LT synthesis since urine LTE(4) levels are still elevated at acute ASA desensitization. However, mast cell products such as histamine, tryptase and PGD(2) are no longer released or synthesized at acute desensitization. It is more likely that a diminution in number or function of cysLT receptors accounts for the diminished inflammatory response found in ASA desensitization.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2004

Mast cell subpopulations in gingival overgrowth induced by immunosuppressive and nifedipine medication.

BACKGROUND: An immunohistochemical study was conducted to compare distributions of mast cell subpopulations in normal human gingiva and in gingival overgrowth induced by nifedipine and immunosuppressive medication. METHODS: Gingival samples were collected from 12 triple-medicated organ transplant recipients (immunosuppression group), 11 triple-medicated organ transplant recipients taking nifedipine (immunosuppression plus nifedipine group), 11 nifedipine-medicated cardiac outpatients (nifedipine group), and 20 generally healthy individuals (control group). Cryostat sections were stained with mAbs for tryptase and chymase, and an avidin-biotin enzyme complex method was used to detect tryptase-positive mast cells (MC(T)), tryptase- and chymase-positive mast cells (MC(TC)), and chymase-positive mast cells (MC(C)). Total numbers of labeled cells were determined in connective tissue beneath the sulcular epithelium, connective tissue beneath the oral epithelium, and middle connective tissue. Statistical analyses were conducted using the Kruskal-Wallis test, the Mann-Whitney U-test, and Pearson's correlation test. RESULTS: In the three counting zones combined, numbers of MC(TC) cells and MC(C) cells were lower (P = 0.001 and P = 0.048, respectively) in the immunosuppression group than in the control group. The difference in numbers of MC(TC) cells was most marked in the middle connective tissue. Nifedipine medication had no effect on numbers of the mast cell subclasses. CONCLUSIONS: Immunosuppressive medication without concomitant nifedipine decreases the numbers of MC(TC) and MC(C) in overgrown gingiva. Chymase-positive mast cells may play a role in formation of gingival overgrowth, especially in patients receiving cyclosporin A (CsA) medication with no concomitant nifedipine. In this respect, nifedipine and CsA are different.

Adult↗
PubMed1989

Roles of mast cell proteases in airways.

The mast cell proteases tryptase and chymase have long been known to constitute one-fifth of the total protein in mast cells. However, their biological functions have not been easy to study because of the difficulty in obtaining sufficient amounts of the enzymes to study their biological functions. Recently, we have been fortunate to have available a permanent line of dog mastocytoma cells to purify both enzymes to homogeneity, and we have used the purified enzymes in two ways. First, in a series of biological studies, we have discovered unique and potent actions of the enzymes that may provide important insights into the pathogenesis of diseases such as asthma and cystic fibrosis. Important biological activities are also likely to exist in other tissues. Because of their structures, mast cell proteases are likely to act in proximity to their sites of release. Thus, the presence and amounts of tryptase and chymase in specific loci may play important roles in tissue responses. In diseases such as asthma and cystic fibrosis, there is evidence that the expression of these mast cell enzymes changes, and these changes have important pathogenetic implications. Second, we have begun to perform structural studies of the enzymes. The recent cloning of tryptase by our group should assist in the better understanding of its functions. Crystallography of the pure proteins should provide further insights and could be the basis of rational development of potent and selective drugs that will inhibit their actions.

Animals↗
PubMed1996

Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping.

Mast cells and blood basophils are distinct hemopoietic cells. They can be distinguished from each other and from all other lymphohemopoietic cells using antibodies against surface receptors or stored cytoplasmic molecules. In patients with myelodysplastic syndromes (MDS) or myeloproliferative syndromes (MPS), an elevation of metachromatically granulated cells (MCS) is frequently seen. These cells can be classified as basophils or mast cells using monoclonal antibodies (mAbs) against leukocyte antigens, including mast cell tryptase, c-kit (= mast cell growth factor [MGF] receptor), interleukin-3 receptor alpha chain (IL-3R alpha = CD123), and CD11b (C3biR). In a stable phase of MDS or MPS, the circulating MCS usually are basophils (histamine+, tryptase-, c-kit-, IL-3R alpha +, CD11b+). In an accelerated or terminal phase of disease, however, mast cell lineage involvement and circulating mast cell precursors (histamine+, tryptase+, c-kit+, IL-3R alpha-, CD11b-) are found in a subset of patients. The use of mAbs against mast cell antigens and granulocyte antigens is diagnostic in these patients.

Basophils↗
PubMed2004

Key role of mast cells and their major secretory products in inflammatory bowel disease.

Historically, mast cells were known as a key cell type involved in type I hypersensitivity. Until last two decades, this cell type was recognized to be widely involved in a number of non-allergic diseases including inflammatory bowel disease (IBD). Markedly increased numbers of mast cells were observed in the mucosa of the ileum and colon of patients with IBD, which was accompanied by great changes of the content in mast cells such as dramatically increased expression of TNFalpha, IL-16 and substance P. The evidence of mast cell degranulation was found in the wall of intestine from patients with IBD with immunohistochemistry technique. The highly elevated histamine and tryptase levels were detected in mucosa of patients with IBD, strongly suggesting that mast cell degranulation is involved in the pathogenesis of IBD. However, little is known of the actions of histamine, tryptase, chymase and carboxypeptidase in IBD. Over the last decade, heparin has been used to treat IBD in clinical practice. The low molecular weight heparin (LMWH) was effective as adjuvant therapy, and the patients showed good clinical and laboratory response with no serious adverse effects. The roles of PGD2, LTC4, PAF and mast cell cytokines in IBD were also discussed. Recently, a series of experiments with dispersed colon mast cells suggested there should be at least two pathways in man for mast cells to amplify their own activation-degranulation signals in an autocrine or paracrine manner. The hypothesis is that mast cell secretogogues induce mast cell degranulation, release histamine, then stimulate the adjacent mast cells or positively feedback to further stimulate its host mast cells through H1 receptor. Whereas released tryptase acts similarly to histamine, but activates mast cells through its receptor PAR-2. The connections between current anti-IBD therapies or potential therapies for IBD with mast cells were discussed, implicating further that mast cell is a key cell type that is involved in the pathogenesis of IBD. In conclusion, while pathogenesis of IBD remains unclear, the key role of mast cells in this group of diseases demonstrated in the current review implicates strongly that IBD is a mast cell associated disease. Therefore, close attentions should be paid to the role of mast cells in IBD.

Cell Degranulation↗
PubMed2002

Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway.

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 micro g/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.

Allergens↗
PubMed2000

Recruitment, distribution and phenotypes of mast cells in interstitial cystitis.

PURPOSE: Interstitial cystitis (IC) is a chronic disabling condition of unknown etiology. One of its major characteristics is an increase in mast cells (MC) showing signs of activation. It has been suggested that the proteinase content defines two MC types: MC(TC), containing chymase and tryptase, and MC(T), which contains tryptase but lacks chymase. Here, we investigated the MC distribution and the MC proteinase expression in IC together with the tissue expression of the major MC growth factors, stem cell factor (SCF) and interleukin-6 (IL-6). MATERIALS AND METHODS: MC were enumerated in bladder specimens from patients with classic IC, nonulcer IC and controls. MC were visualized in terms of metachromasia, reflecting glycosaminoglycan content, and immunohistochemically, visualizing tryptase, chymase and IL-6 as well as the surface markers CD117 and SCF. RESULTS: Classic IC displayed a 6 to 10-fold increase of MC identified by proteinase content while in nonulcer IC there were twice as many MC as in controls. In contrast to nonulcer IC and controls, classic IC displayed an abundance of epithelial MC. Fewer CD117+ than proteinase+ MC were detected in IC but not in controls. Classic IC coexpressed SCF and IL-6 in the epithelium and displayed numerous SCF and IL-6+ cells in the mucosa and detrusor muscle, many of which were MC. CONCLUSIONS: Redistribution of MC into the epithelium and a high bladder wall MC density distinguish classic IC from nonulcer IC. Our findings suggest an SCF/IL-6-driven MC response in IC. They also indicate a downregulation of the SCF receptor in IC.

Aged↗
PubMed1992

Characterization of human mast cells developed in vitro from fetal liver cells cocultured with murine 3T3 fibroblasts.

Cocultures of dispersed human fetal liver cells with murine Swiss 3T3 fibroblasts resulted in the development of human mast cells after 1 to 4 weeks of culture. Mast cells were detected by immunohistochemistry using a murine monoclonal anti-tryptase antibody, before metachromasia appeared with toluidine blue. When subjected to double immunohistochemistry using murine monoclonal anti-chymase and anti-tryptase antibodies, 94% +/- 10% (SD) of the mast cells seen at day 30 of culture were of the MCT type. These results contrast with those obtained with human mast cells derived from cord blood mononuclear cells cocultured with murine 3T3 fibroblasts which are comprised of substantially greater numbers of MCTC cells, averaging 48% +/- 31% (SD) at day 30 of culture. Mast cells developed in vitro from fetal liver cells or cord blood mononuclear cells contained similar amounts (+/- SD) of histamine (0.9 +/- 0.5 pg/cell and 1.1 +/- 1 pg/cell, respectively) and tryptase (1.7 +/- 0.4 pg/cell and 1.9 +/- 1.2 pg/cell, respectively) on day 30 of culture. Fetal-liver-derived mast cells from a 30-day-old culture were identified by immunoelectron microscopy using gold-labelled antitryptase antibody. Typically, these mast cells appeared immature as they had large nuclear to cytoplasmic ratio and a small number of ill-formed cytoplasmic granules. For both fetal-liver- and cord-blood-derived mast cells, there was no evidence of conversion of the MCT type into the MCTC type provided by this study. These results suggest that commitment to develop as an MCT or MCTC type of mast cell may have occurred in mast cell precursors present in fetal liver and cord blood mononuclear cells, prior to granulation.

Animals↗
PubMed2004

Immunohistochemical analysis of the interstitial mast cells in acute rejection of human renal allografts.

BACKGROUND: Morphometric investigations were performed to determine whether mast cells correlate with tubulointerstitial fibrosis in acute renal transplant rejection (ARTR) and to examine the relationship between mast cells, interstitial alpha-smooth muscle actin (alpha-SMA) expression, and interstitial infiltrates. MATERIAL/METHODS: Twenty-four renal allograft biopsy specimens from patients with ARTR were examined quantitatively by means of a computer image analysis system. As a control 11 allograft biopsy specimens were used from patients with no signs of rejection. RESULTS: The morphometric study revealed that the mean values of the interstitial tryptase positive cells, expression of alpha-SMA, interstitial volume, CD 68+, CD 43+ and CD 20+ cells were significantly increased in ARTR patients in comparison to controls. In the ARTR group there were significant positive correlations between interstitial tryptase positive cells and interstitial expression of alpha-SMA, interstitial volume, CD 43+ and CD 68+. Moreover, CD 68 positive cells significantly correlated with interstitial volume (r=0.51, p<0.02). The correlations between interstitial tryptase positive cells and CD 20+ cells as well as between CD 43+ and CD 20+ cells and interstitial volume were positive, but did not reach statistical significance. CONCLUSIONS: Our findings demonstrate that mast cells are one of the constitutive cell types in the interstitium in ARTR. Although the significant positive correlations between interstitial mast cell count and relative interstitial volume, as well as interstitial expression of alpha-SMA, suggest that these cells have a role in the development of early interstitial fibrosis in renal allografts, these relationships need further investigation.

Actins↗
PubMed2005

Physical exercise does not favour adverse reactions to allergen immunotherapy by the sublingual route.

It is generally recommended in consensus documents on allergen immunotherapy to avoid any kind of physical exercise in the 24 hours following the administration of the allergen extract but such recommendation is not supported by scientific evidence. We evaluated the risk of developing adverse reactions in a group of patients submitted to sublingual immunotherapy by performing a controlled exercise test. Eleven patients were included in the study, 8 treated with grass pollen and 3 with Parietaria pollen extract by Staloral300 (Stallergénes, Antony, France), with the build-up phase in 11 days suggested by the manufacturer and a top dose of 300 IR. At the first maintenance dose, in all patients were measured heart rate, blood pressure, FEV1, and tryptase in blood. Then the maintenance dose was assumed and the physical exercise performed by cycloergometer maintaining a heart rate around 100 b/min and measuring again tryptase, blood pressure and FEV1. The results showed that no patient had adverse reaction following physical exercise, neither showed significant changes in FEV1, blood pressure, or tryptase level, which was 5.57 +/- 4.54 mcg/l before and 5.81 +/- 4.34 mcg/l after exercise. This suggests that there is no reason to advise against physical exercise, and particularly sporting activity, in patients treated with sublingual immunotherapy thus avoiding to interfere with the subject's lifestyle and consequently with his quality of life.

Abdominal Pain↗
PubMed1991

A novel membrane-bound serine esterase in human T4(+)-lymphocytes is a binding protein of envelope glycoprotein gp120 of HIV-1.

A novel membrane-bound serine esterase, named tryptase TL2, which is immunologically reactive with the antibody inhibiting induction of syncytia by human immunodeficiency virus-1 (HIV-1) (HATTORI, T., KOITO, A., TAKATSUKI, K., KIDO, H., and KATUNUMA, N., 1989, FEBS Lett., 248, 48-52), has been purified from a human T4+ lymphocyte clone. The enzyme has a molecular mass of 198 +/- 15 kDa, and is composed of two subunits of 32 kDa and four subunits of 28 kDa. The enzyme was strongly inhibited by the envelope glycoprotein gp120 of HIV-1, by synthetic peptides of V3 domains of gp120 s with the sequence GPGR in their center, which correspond to the principal neutralizing epitopes of the gp120s of various HIV-1 strains, by Kunitz-type inhibitors with the sequence GPCR in their active site, such as trypstatin, H130, and [Arg15, Glu52] aprotinin and by the microbial inhibitors leupeptin and antipain. This enzyme was specifically bound to the inhibitor V3 domain of gp120 of HIV-1, and this binding was blocked by the inhibitors of tryptase TL2, with a central motif GPCR or GPGR sequence in their center, but not by leupeptin and antipain without the motif. These findings suggest that tryptase TL2 is important in target site recognition and binding of HIV-1 in co-operation with CD4 receptor in the initial process of HIV-1 infection.

Alpha-Globulins↗
PubMed1997

Cloning and expression of the dog mast cell alpha-chymase gene.

Chymases are chymotrypsin-like serine proteinases secreted by mast cells. Alpha- and beta-chymases differ in structure, function, and mast cell subset- and species-specific expression. Seeking genetic regulatory elements shared by alpha-chymases, we sequenced the dog alpha-gene. Extensive homology was found in intronic and flanking sequences of the dog, human, and mouse alpha-chymase genes, but little in corresponding beta-chymase sequences. Repetitive elements probably derived from retroposons are unique features of the dog flank. DNA blots suggest that the dog alpha-gene, like its human counterpart, may be the genome's sole chymase, unlike in rodents, in which beta-chymases predominate. Nuclear runoff studies predict that transcriptional mechanisms explain differences in steady state chymase and tryptase mRNA levels between mastocytoma and non-mast cells. In dog BR mastocytoma cells incubated with phorbol ester, high steady state levels of alpha-chymase mRNA drop dramatically with little change in tryptase mRNA, whereas dexamethasone decreases expression of both mRNAs. Portions of the dog or human gene 5' flank transfected into BR cells drive expression of a reporter gene and define regions with active promoters. Thus, BR cells express high levels of alpha-chymase mRNA regulated independently of tryptase and support transcription using dog or human promoters. These studies reinforce the alphabeta-chymase dichotomy and suggest the utility of BR cells in probing regulation of alpha-chymase expression.

Animals↗
PubMed2003

Safety of cyclooxygenase 2 inhibitors and increased leukotriene synthesis in chronic idiopathic urticaria with sensitivity to nonsteroidal anti-inflammatory drugs.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) exacerbate various forms of urticaria by a nonallergic mechanism involving inhibition of cyclooxygenases. OBJECTIVES: To assess safety of cyclooxygenase inhibitors in patients with chronic idiopathic urticaria (CIU) and NSAID sensitivity and to evaluate a role of cysteinyl leukotriene metabolism and mast cell activation in sensitivity to NSAIDs in CIU. DESIGN: Aspirin challenge test followed by randomized, prospective, double-blind, placebo-controlled crossover trial with cyclooxygenase 2 inhibitors. SETTING: Tertiary referral center of a university hospital. PATIENTS: Thirty-six patients with CIU. INTERVENTIONS: Aspirin challenge test (up to 500 mg); randomized trial with rofecoxib (up to 37.5 mg) and celecoxib (up to 300 mg) in aspirin-sensitive patients. After completion of the trial, 7 patients received naproxen sodium (500 mg) as a positive control. MAIN OUTCOME MEASURES: Standardized skin examination, skin biopsy with mast cell count, urinary levels of leukotriene E4 (LTE4), and serum levels of mast cell tryptase. RESULTS: Aspirin induced skin eruption in 18 patients. Rofecoxib or celecoxib did not elicit skin eruption in any of the aspirin-sensitive patients. Patients with CIU had higher urinary excretion of LTE4 than healthy control subjects. Basal urinary levels of LTE4 and serum mast cell tryptase were increased in aspirin-sensitive compared with aspirin-tolerant patients. Severity and duration of aspirin-induced urticaria showed a positive correlation with urinary LTE4 excretion. Naproxen precipitated urticaria in 5 of 7 aspirin-sensitive patients and caused further increase in urinary LTE4. CONCLUSIONS: Cyclooxygenase 2 inhibitors do not induce urticaria in patients with CIU sensitive to NSAIDs. Sensitivity to NSAIDs in CIU is associated with overproduction of cysteinyl leukotrienes and mast cell activation and most likely depends on inhibition of cyclooxygenase 1.

Adolescent↗
PubMed1997

Human synovial mast cells. II. Heterogeneity of the pharmacologic effects of antiinflammatory and immunosuppressive drugs.

OBJECTIVE: To evaluate the in vitro effects of 4 antiinflammatory and 5 immunosuppressive agents on the release of preformed and de novo-synthesized mediators from human synovial mast cells (HSyMC) activated by immunologic and nonimmunologic stimuli. METHODS: The effects of antiinflammatory and immunosuppressive agents were evaluated on the in vitro release of histamine and tryptase and the de novo synthesis of prostaglandin D2 (PGD2) and leukotriene C4 (LTC4) by HSyMC challenged with anti-IgE and substance P. RESULTS: Nimesulide, a sulfonanilide nonsteroidal antiinflammatory drug (NSAID) chemically unrelated to other acidic NSAIDs (such as acetylsalicylic acid [ASA], diclofenac, and piroxicam) inhibited in a concentration-dependent manner the release of preformed (histamine and tryptase) mediators from HSyMC challenged with anti-IgE. In contrast, diclofenac and piroxicam had little or no effect on HSyMC activated by anti-IgE. ASA, diclofenac, piroxicam, and nimesulide caused a concentration-dependent inhibition of IgE-mediated PGD2 release from HSyMC. Nimesulide, but not diclofenac or piroxicam, also inhibited the de novo synthesis of LTC4 by HSyMC challenged with anti-IgE. Nimesulide, diclofenac, and piroxicam had no effect on HSyMC activated by substance P. Cyclosporin A (CSA) inhibited histamine release from HSyMC challenged with anti-IgE, whereas cyclosporin H (CSH) had no effect. FK-506 also inhibited histamine release from HSyMC activated by anti-IgE, whereas rapamycin had no effect. Neither CSA, CSH, FK-506, nor rapamycin inhibited the release of histamine from HSyMC induced by substance P. Methotrexate had no effect on the release of mediators from these cells, whereas adenosine (R-phenylisopropyl adenosine and 5'-N-ethylcarboxamide adenosine) enhanced histamine release from immunologically activated HSyMC in a concentration-dependent manner. CONCLUSION: Mast cells isolated from human synovia display 4 levels of pharmacologic heterogeneity with regard to 1) the inhibitory effects of 4 antiinflammatory drugs; 2) the capacity of different immunosuppressive drugs to exert antiinflammatory activity; 3) the inhibition of the release of different mediators; and 4) the capacity of antiinflammatory and immunosuppressive drugs to modulate HSyMC activated by different stimuli. This complexity of pharmacologic modulation of HSyMC in vitro might help explain the different activity of the compounds used to treat various pathophysiologic aspects of the inflammatory arthritides.

Adenosine↗
PubMed2004

Origin of the murine implantation serine proteinase subfamily.

The S1 serine protease family is one of the largest gene families known. Within this family there are several subfamilies that have been grouped together as a result of sequence comparisons and substrate identification. The grouping of related genes allows for the speculation of function for newly found members by comparison and for novel subfamilies by contrast. Analysis of the evolutionary patterns of genes indicates whether or not orthologs are likely to be identified in other species as well as potentially indicating that hypothesized orthologs are in fact not. Looking at subtle differences between subfamily members can reveal intricacies about function and expression. Previously, we have described genes encoding two novel serine proteinases, ISP1 and ISP2, which are most closely related to tryptases. The ISP1 gene encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching and invasion in vitro. Additionally both ISP1 and ISP2 are co-expressed in the endometrial gland during the time of hatching, suggesting that they may also both participate in zona lysis from within the uterine lumen. Here, we demonstrate that the ISPs are tandemly linked within the tryptase cluster on 17A3.3. We suggest that remarkable similarities within the 5'-untranslated and first intron regions of ISP1 and ISP2 may explain their intimate co-regulation in uterus. We also suggest that ISP genes have evolved through gene duplication and that the ISP1 gene has also begun to adopt an additional new function in the murine preimplantation embryo.

Amino Acid Sequence↗
PubMed1998

Inhibitory effect of adenosine on degranulation of human cultured mast cells upon cross-linking of Fc epsilon RI.

Adenosine is a potent bronchoconstricting agent that is released by activated mast cells and hypoxic lung tissue. However, both inhibition and stimulation of mediator release from human lung mast cells by adenosine have been described, and this discrepancy seems to be due to contaminating cells or the effects of enzymatic treatment. We, therefore, investigated the effects of adenosine and its receptor-specific analogues on human cultured mast cells (HCMC). Adenosine inhibited Fc epsilon RI-mediated tryptase release from HCMC in a dose-dependent manner, and this inhibitory effect was completely blocked by the A2a receptor antagonist ZM241385. The specific agonist of A2a adenosine receptors CGS21680 inhibited the release of tryptase more potently than A1 and A3 agonists, and A2a receptor mRNA was detected by RT-PCR, suggesting the involvement of A2a receptors in the inhibitory effects of adenosine. In addition, adenosine increased intracellular cAMP level in a dose-dependent manner and inhibited protein tyrosine phosphorylation including that of ERK-2. These results suggest that adenosine acts via A2 receptors to inhibit Fc epsilon RI-mediated mediator release from human mast cells.

Adenosine↗