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PubMed1997

Anaphylactoid shock--a common cause of death in heroin addicts?

We measured mast-cell tryptase in postmortem blood from 22 heroin addicts dying suddenly after injection. In 32%, the concentration of tryptase was elevated (> or = 10 micrograms/l), and the mean value of tryptase was significantly different from a control group dying from known, nonimmunologic causes (P < 0.05). The increased tryptase concentrations indicate that death was preceded by systemic mast-cell degranulation. All victims of drug deaths had morphine in blood, most below 0.2 microgram/ml. In 71% of the victims of drug-related deaths with tryptase values > or = 10 micrograms/l, the intermediate degradation product, 6-monoacetyl-morphine, was not found in blood, whereas this was the case in only two victims with values below that cutoff point. This indicates that those with high tryptase concentrations survived longer than those with lower values. No correlation was found between the IgE levels and tryptase in either group, supporting the hypothesis that tryptase release was not mediated by an allergic reaction. The well-known property of opiates to stimulate unspecifically the liberation of histamine and other constituents of mast-cell granules offers one explanation of our observations. The results suggest that many heroin fatalities are caused by an anaphylactoid reaction.

Adult↗
PubMed1999

Mast cell heterogeneity in the human uvea.

To identify chymase- and tryptase-positive mast cells in the human uvea, and to study their associations with different types of resident uveal cells, uveal specimens from 24 human donor eyes were cryosectioned in sagittal and tangential planes. Enzyme histochemical staining of chymase was combined with immunohistochemical staining for tryptase, detected with the APAAP method. Fluorescence immunohistochemistry was performed with antibodies against c-kit, alpha smooth muscle actin, protein gene product (PGP) 9.5, CD45, and HLA-DR. In different uveal compartments, the total amounts of mast cells were calculated and the distributions of chymase and tryptase were quantified. All uveal mast cells were c-kit and CD45 positive and HLA-DR negative. No association existed between mast cells and actin-containing cells. Only a few mast cells were in close association with PGP 9.5-labeled nerve fibers. In the choroid, most mast cells were located in the inner central part (mean density = 48.9/mm(2)), and contained both chymase and tryptase (96%). The ciliary muscle contained numerous mast cells (mean density = 33.7/mm(2)), many of them tryptase positive but chymase negative (63%). In the pars plana, a high number of chymase-positive, tryptase-negative mast cells were found (20%). In the iris only a few mast cells were present. Although the choroid contains the most common subtype of mast cells, a unique situation concerning the distribution of chymase and tryptase is present in the anterior uveal tissues. A possible role for these cells in the special immunological situation of the anterior eye chamber merits further investigation.

Actins↗
PubMed2003

Effects on inflammation parameters of a double-blind, placebo controlled one-year course of SLIT in children monosensitized to mites.

BACKGROUND: Clinical documentation about effects on local markers of inflammation of sublingual immunotherapy (SLIT) in children is still poor. METHODS: Twenty-four children (age range 4-16 years, average 8.5 years) monosensitized to house dust mites (HDMs) were randomized to receive active or placebo SLIT for this allergen according to a double-blind, placebo-controlled design. Before treatment and 10-12 months later the following parameters were checked: ECP and tryptase in sputum and nasal secretion, serum and nasal mite-specific IgE (sIgE), allergen-specific nasal challenge test (sNCT), nasal symptoms and tryptase after sNCT. RESULTS: Nasal tryptase and nasal IgE in basal conditions were unchanged in treated children but significantly increased in untreated children (P = 0.0156 and P = 0.0313, respectively). The threshold for sNCT was unchanged in both groups of children, but the symptom score after sNCT was unchanged in the placebo group and significantly decreased in the active group (P = 0.0084). The nasal tryptase after sNCT was unchanged in the active group and significantly increased in the placebo group (P = 0.0218). Intergroup comparison showed a significant difference in oral tryptase and nasal tryptase after sNCT in favour of the active group. CONCLUSIONS: These interim results after only 1 year of treatment show that SLIT in children monosensitized to HDMs is able to avoid the spontaneous increase in both nasal sIgE antibodies and in local allergic inflammation in basal conditions. These outcomes are confirmed and supported by the decrease of symptoms in the active group combined with the increase of nasal tryptase only in the control group in both cases after sNCT.

Administration, Sublingual↗
PubMed2006

Mast cells in diffuse large B-cell lymphoma; their role in fibrosis.

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.

Adult↗
PubMed2005

Activation of human tonsil and skin mast cells by agonists of proteinase activated receptor-2.

AIM: To investigate the effects of the agonists of proteinase activated receptor (PAR)-2, and histamine on degranulation of human mast cells. METHODS: Human mast cells were enzymatically dispersed from tonsil and skin tissues. The dispersed cells were then cultured with various stimuli, and tryptase and histamine levels in cell supernatants collected from challenge tubes were measured. RESULTS: PAR-2 agonist peptide SLIGKV provoked a dose-dependent release of histamine from skin mast cells. It also induced tryptase release from tonsil mast cells. tc-LIGRLO appeared less potent than SLIGKV in induction of release of histamine and tryptase. Trypsin was able to induce a bell shape increase in tryptase release from tonsil mast cells. It was also able to induce a dose-dependent release of histamine from both tonsil and skin mast cells. The actions of trypsin on mast cells were inhibited by soy bean trypsin inhibitor (SBTI) or alpha1-antitrypsin (alpha1-AT). Time course study revealed that both stimulated tryptase or histamine release initiated within 10 s and reached their peak release between 4 and 6 min. Pretreatment of cells with metabolic inhibitors or pertussis toxin reduced the ability of mast cells to release tryptase or histamine. CONCLUSION: It was demonstrated that the in vitro tryptase release properties of human tonsil and skin mast cells suggested a novel type of mast cell heterogeneity. The activation of mast cells by PAR-2 agonists indicated a self-amplification mechanism of mast cell degranulation.

Antibodies, Anti-Idiotypic↗
PubMed2003

Chymase expressing bone marrow mast cells in mastocytosis and myelodysplastic syndromes: an immunohistochemical and morphometric study.

BACKGROUND: Two cell specific neutral proteases, tryptase and chymase, are produced by human mast cells (MC). Tryptase is constitutively expressed by all MC, whereas chymase is found only in an MC subset. Very little is known about chymase expression in MC proliferative disorders (mastocytosis). AIMS AND METHODS: Routinely processed, formalin fixed, and paraffin wax embedded bone marrow trephine biopsy specimens obtained from patients with various subtypes of mastocytosis (n = 47) and myelodysplastic syndromes (MDS; n = 28) were immunostained with antibodies against chymase and tryptase. Normal/reactive bone marrow specimens with intact haemopoiesis (n = 31) served as controls. The numbers of chymase expressing (C+) and of tryptase expressing (T+) MC were assessed morphometrically using a computer assisted video camera system. RESULTS: In normal/reactive bone marrow, the numbers of C+ MC (median, 8/mm(2); maximum, 159/mm(2)) were in the same range as those of T+ MC (median, 4/mm(2); maximum, 167/mm(2)). Because normal MC express both chymase and tryptase, these findings indicate that the common phenotype of bone marrow MC in normal/reactive states is MC(TC) (MC expressing both tryptase and chymase). In contrast, in MDS and mastocytosis, the bone marrow exhibited far more T+ MC than C+ MC in almost all cases. CONCLUSIONS: According to these findings, the predominant MC type in the bone marrow in neoplastic states such as MDS and mastocytosis is MC(T) (MC expressing only tryptase). Although the pathophysiological basis of this apparent lack of chymase expression in most neoplastic MC in mastocytosis and MC involved in MDS remains unknown, this study has produced further evidence of the superior value of antitryptase antibodies in the diagnosis of mastocytosis.

Bone Marrow Cells↗
PubMed2002

Serum interleukin-6 reflects disease severity and osteoporosis in mastocytosis patients.

BACKGROUND: Systemic mastocytosis (SM) is a condition typically characterized by an increased number of mast cells in the bone marrow or in skin areas known as urticaria pigmentosa. Patients may present with flushing, itching, gastrointestinal symptoms, arrhythmias, headaches and osteoporosis. Some patients experience systemic symptoms indicative of SM in the absence of a positive bone marrow or skin biopsy, and are known as 'clinical mastocytosis', but are herein referred to as suspected of having systemic mastocytosis. Serum tryptase has been increasingly used as a biochemical marker of mastocytosis, but is not always elevated. OBJECTIVE: To investigate the association of serum levels of two key mast cell mediators, interleukin-6 (IL-6) and tryptase, to each other and with disease severity in patients with mastocytosis. METHODS: Patients responded to an announcement from the Systemic Mastocytosis Society (USA) and submitted frozen serum samples, but the precise diagnosis made by their own health providers was not known until after the assays were completed. There were 9 suspected systemic mastocytosis (SuSM), 3 cutaneous mastocytosis (CM), 11 indolent systemic mastocytosis (ISM), and 3 aggressive systemic mastocytosis (ASM). Five normal volunteers (3 females/2 males) also submitted samples, as did 33 cardiac patients without coronary artery disease. For 2 days prior to and during the collection period, mastocytosis patients were asked to abstain from any over-the-counter or food products containing biogenic amines, as well as drugs prescribed for this condition. Serum levels of IL-6 and tryptase were measured using established assays. RESULTS: Twenty-six patients (14 females/12 males) submitted serum samples. There were 9 cases of SuSM (6 females/3 males) in whom tryptase values were borderline normal; IL-6 values were slightly elevated with one being high. In 3 cases of CM (2 females/1 male), both tryptase and IL-6 were slightly elevated. In patients with ISM (5 females/6 males), only 6/11 had any tryptase elevated significantly as compared to 9/11 with elevated serum IL-6. Three patients with ASM had significant elevations of both IL-6 and tryptase. The most consistent finding was that of IL-6 elevations in 7/7 patients (3 females/4 males) who reported symptoms of osteoporosis and/or bone pain (1 SuSM, 3 ISM, 3 ASM) in the absence of any coexisting condition involving bone pathology. CONCLUSION: Serum IL-6 is elevated in mastocytosis patients and correlates with severity of symptoms and the presence of osteoporosis. High serum IL-6 may not only signify disease progression, but may also participate in the pathophysiology of mastocytosis.

Adult↗
PubMed2003

Correlation between T-cell and mast cell activity in patients with chronic urticaria.

BACKGROUND: The presence of autoantibodies reacting with the high affinity IgE receptor (FcepsilonRIalpha) usually indicates a more severe form of chronic urticaria (CU). Previously, we showed an increased lymphocyte reactivity in CU patients; however, the relation between enhanced cellular immunity and the presence of anti-FcepsilonRIalpha-specific autoantibodies has not been investigated. METHODS: Cellular and humoral immune reactivity of 50 CU patients and 28 healthy controls was studied. Serum sIL-2R, neopterin, and tryptase levels were measured to assess T-cell, monocyte/macrophage and mast cell activity, respectively. Helicobacter pylori (HP)-specific IgG antibody, and IgE levels were also tested. Anti-FcepsilonRIalpha-specific autoantibody was determined by Western blotting. In vivo histamine-releasing activity of patients' sera was assessed by the autologous serum skin test (AST). RESULTS: 17/50 CU patients, who both had IgG-type anti-FcepsilonRIalpha-antibodies by Western blotting and a positive AST response, were classified as autoimmune CU. All patients with CU had significantly higher serum sIL-2R and tryptase levels than healthy controls (p = 0.000257, p = 0.000166, respectively), indicating T-cell and mast cell activation. Patients with higher sIL-2R levels also had higher tryptase levels; the strongest correlation was shown in the autoimmune subgroup of patients (rho = 0.688, p = 0.002). There was a tendency towards higher tryptase levels in the autoimmune subgroup, as compared to the nonautoimmune CU patients. While the serum IgE was significantly lower in autoimmune than in nonautoimmune CU (p = 0.000836), there was no significant difference in their sIL-2R, neopterin and HP-specific IgG antibody levels. CU patients with a positive AST response (38/50) had significantly higher tryptase levels (p = 0.0107) when compared to the negative skin test group. CONCLUSIONS: The significant correlation between sIL-2R and tryptase levels in patients with CU indicates that T cell activation is proportional to mast cell degranulation in these patients. The increased level of tryptase in autoimmune CU may suggest more severe disease.

Adolescent↗
PubMed2004

Effector cells of anaphylaxis: mast cells and basophils.

Systemic anaphylaxis arises when mast cells, possibly along with other cell types, are provoked to secrete mediators that evoke a systemic response. Mast cells in perivascular, respiratory, gastrointestinal and cutaneous tissues are likely involved, regardless of whether IgE or non-IgE-dependent pathways are invoked. Alpha/beta tryptases are selectively and abundantly produced by mast cells. Tryptase levels in the circulation provide a precise indicator of mast cell involvement. Mature beta tryptase is stored in secretory granules and is released when the cells are activated to degranulate, as occurs in anaphylaxis. Alpha/beta pro/pro' tryptases are spontaneously secreted by mast cells. Consequently, mature tryptase levels in serum (normally 1 ng/ml) are elevated in systemic anaphylaxis. Total tryptase levels (mature plus precursor forms), normally 1-15 ng/ml in baseline serum samples, are elevated in patients with systemic mastocytosis (> 20 ng/ml), a disease that also predisposes one to anaphylactic reactions. The assessment of basophils in systemic anaphylactic reactions has been problematic, because an assay for a specific releasable marker from this cell type has not been developed. Nevertheless, in cases of anaphylaxis in which elevations of histamine, but not tryptase, have been detected, it is enticing to speculate that basophil-dependent anaphylaxis may have occurred.

Allergens↗
PubMed1999

The human mucus protease inhibitor and its mutants are novel defensive compounds against infection with influenza A and Sendai viruses.

Tryptase Clara, a trypsin-like protease localized exclusively in and secreted by Clara cells of the bronchial epithelium, is a prime host factor that processes viral envelope glycoproteins and determines the infectivity of influenza A and Sendai viruses (H. Kido, Y. Yokogoshi, K. Sakai, M. Tashiro, Y. Kishino, A. Fukutomi, and N. Katunuma, The Journal of Biological Chemistry, 1992, Vol. 267, pp. 13573-13579). We report here that human mucus protease inhibitor (MPI), a major inhibitor of granulocyte elastase in the lining fluid of the human respiratory tract, significantly inhibited induction of the infectivity of influenza A and Sendai viruses by tryptase Clara in vitro and multicycles of mouse-adapted influenza A virus replication in rat lungs in vivo. Recombinant MPI and the C- but not the N-terminal domain of MPI inhibited both the activity of tryptase Clara and the induction of virus infection by tryptase Clara. The 50% inhibitory concentrations of MPI and the C-terminal domain peptide (Pro50-Ala107) of MPI for tryptase Clara were 7.4 and 61.6 nM, respectively, with Sendai virus envelope glycoproteins as the substrate. Studies on deletion mutants of the C-terminal domain of MPI revealed that the minimal size of MPI required for the inhibition of tryptase Clara is the peptide Lys60-Ala107. Studies involving site-directed mutagenesis of the C-terminal domain of MPI indicated that the Leu72-Met73 site of MPI is the inhibitory site for tryptase Clara. Substitution of residue Leu72 with a basic amino acid significantly increased in the inhibitory activity of the C-terminal domain of MPI, but further substitution of residue Met73 with various amino acids in these mutants reduced the inhibitory activity. Since there is evidence suggesting that the concentration of MPI in respiratory fluid is insufficient for prevention of virus infection, the administration of MPI, the recombinant C-terminal domain of MPI, and their mutants, with residue Leu72 substituted with residues Arg72 and Lys72, may be useful for treatment of such pneumotropic virus infections.

Animals↗
PubMed2000

Regulation of chymase production in human mast cell progenitors.

BACKGROUND: Although mature tryptase-positive mast cells (MCs) and tryptase and chymase double-positive MCs are recognized using in situ staining and are preferentially distributed in different tissues, recent findings suggest that tryptase-positive MCs can give rise to tryptase and chymase double-positive MCs. OBJECTIVE: We investigated the regulation of chymase production in developing MCs. METHODS: Human cord blood or peripheral blood cells were cultured in the presence of stem cell factor and IL-6 with or without IL-4 in methylcellulose or liquid medium. Intracellular chymase and tryptase were determined with immunocytochemistry, flow cytometry, and ELISA. Chymase messenger RNA expression was examined with 3 different methods, such as Northern blotting. RESULTS: Flow cytometric analysis always showed a unimodal histogram of chymase-positive, as well as tryptase-positive, cells in the presence of various cytokines, even when chymase was not detected in some MCs with immunocytochemistry. The chymase protein expression increased by culture duration and was enhanced by cytokines, such as a high concentration of stem cell factor or IL-4. Chymase messenger RNA was expressed higher in immature MCs than mature chymase protein-rich MCs. We generated macroscopic MC colonies in methylcellulose by culturing CD34(+) cells for 10 weeks and measured cellular chymase, tryptase, and histamine. The chymase/histamine ratio widely varied (0.07-1.01) depending on MC colony, even under the same culture conditions, including IL-4, whereas the tryptase/histamine ratio was relatively constant (1.02-1.89). CONCLUSION: All human MCs in culture are capable of producing chymase, and the production is clonally regulated at their progenitors by cytokine-independent mechanisms, as well as being totally controlled by cytokine-dependent mechanisms accompanied by maturation.

Cells, Cultured↗
PubMed1995

Gastrin stimulates gastric mast cells in rabbits.

In a previous study we demonstrated that in human gastric mucosa tryptase was localized only in mast cells and that its levels were correlated with serum gastrin, suggesting a link between gastrin action and mucosal mast cell function. The aim of the present study was to discover whether pentagastrin injection could stimulate gastric mucosal mast cells in rabbits. Ten female rabbits (group S) were injected s.c. with pentagastrin (10 mu g/kg); another group of ten animals (group C) was injected s.c. with an equal volume of saline solution. One hour after the injection the rabbits were sacrificed and their stomachs removed. Antrum (A), corpus (C) and fundus (F) mucosal homogenates were assayed for total protein, tryptase, pepsinogen A (PGA), histamine and gastrin. Histamine tissue levels were significantly lower in group S than in group C in the antrum (Mann-Whitney test: U = 82, P < 0.01) and in the corpus (U = 83, P < 0.005). Tryptase levels were significantly higher in group S than in group C in all gastric areas (antrum: U = 95, P < 0.001; corpus: U = 85, P < 0.005 and fundus: U = 75, P < 0.05). Total protein, PGA and gastrin did not vary significantly between groups. In group C, no significant correlations were found among the five parameters. In group S, corpus tryptase was correlated with fundus tryptase (Spearman's r = 0.831, P < 0.01). The same relationship was observed for histamine (r = 0.672, P < 0.05). In group S, antrum gastrin was inversely correlated with antrum tryptase (r = -0.903, P < 0.001), and with corpus PGA (r = -0.806, P < 0.05). This study demonstrates that bolus pentagastrin administration stimulates gastric mucosal mast cells in the rabbit.

Animals↗
PubMed1998

Contribution of mast cells to the tubulointerstitial lesions in IgA nephritis.

BACKGROUND: Mast cells have never been extensively investigated in renal disease, particularly glomerulonephritis. Recent improvements in monoclonal antibody production to mast cell specific enzymes have made it possible to study mast cells in tissues more accurately and easily. Mast cells have been found to secrete basic fibroblast growth factor (bFGF) in human pulmonary fibrosis. METHODS: Mast cells in 67 cases of IgA nephritis were investigated. Toluidine blue (TB) stainings at pH 5.0 and pH 0.5 were employed histochemically, and anti-human mast cell tryptase and chymase monoclonal antibodies were used immunohistochemically. Anti-bFGF antibody was also used immunohistochemically. RESULTS: Mast cells were scattered in the interstitium including in fibrotic areas. TB pH 0.5-positive mast cells were more numerous than TB pH 5. 0-positive mast cells. Immunostaining with anti-tryptase monoclonal antibody detected more mast cells than the TB stainings. Mast cells in the interstitium of IgA nephritis had both tryptase and chymase. Immunoelectron microscopy showed that tryptase was exclusively localized in the specific granules of mast cells. The average number of tryptase positive mast cell in the interstitium of IgA nephritis was lower than that of T lymphocyte but more than that of macrophages. The average number of mast cells increased with the progression of interstitial fibrosis and had a significant correlation with 24-hour creatinine clearance. Using double labeled immunohistochemistry, some tryptase-positive mast cells had bFGF in their cytoplasm. Electron microscopy showed that mast cells were associated with fibroblasts and/or lymphocytes in the interstitium. CONCLUSION: Mast cells are one of the constitutive cells in the interstitium of IgA nephritis patients and affect renal function by contributing to the interstitial fibrosis.

Adolescent↗
PubMed1995

Is unrecognized anaphylaxis a cause of sudden unexpected death?

BACKGROUND: Serum tryptase levels reflect mast cell activation and correlate with anaphylactic reactions. Elevated post-mortem serum tryptase levels have been found in witnessed fatal anaphylaxis. OBJECTIVE: This study was designed to examine whether or not unwitnessed anaphylaxis may be a hitherto unrecognized cause of sudden unexplained death. METHODS: Mast cell tryptase was measured by immunoassay in 68 post-mortem sera remaining from a previous study which reported elevated venom-specific IgE antibodies in 22 (23%) of 94 victims of sudden unexpected death. Autopsies were performed in all cases. The cause of death was independently reported by pathologists unfamiliar with the nature of this study. RESULTS: Serum tryptase levels were elevated (> 10 ng/mL) in nine of 68 cases. The levels could not be predicted from the clinical circumstances surrounding death. Sera from four individuals contained both elevated tryptase and previously reported elevated venom-specific IgE. CONCLUSIONS: We conclude that mast cell activation may accompany up to 13% of sudden unexpected deaths in adults. Measurement of both tryptase and specific IgE antibody levels in post-mortem sera from persons experiencing sudden, unexpected death may identify a small subset of cases due to clinically unrecognized fatal anaphylaxis, including those due to insect stings.

Adolescent↗
PubMed1999

Purification of mast cell proteases from murine skin.

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.

Animals↗
PubMed2003

A novel influenza A virus activating enzyme from porcine lung: purification and characterization.

Proteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gin-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsin-type serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-alpha and -beta. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.

Amino Acid Sequence↗
PubMed2005

Mastin is a gelatinolytic mast cell peptidase resembling a mini-proteasome.

Mastin is a tryptic peptidase secreted by canine mast cells. This work reveals that mastin is composed of catalytic domain singlets and disulfide-linked dimers. Monomers unite non-covalently to form tryptase-like tetramers, whereas dimers aggregate with monomers into larger clusters stabilized by hydrophobic contacts. Unlike tryptases, mastin resists inactivation by leech-derived tryptase inhibitor, indicating a smaller central cavity, as confirmed by structural models. Nonetheless, mastin is strongly gelatinolytic while not cleaving native collagen or casein, suggesting a preference for denatured proteins threaded into its central cavity. Phylogenetic analysis suggests that mammalian mastins shared more recent ancestors with soluble alpha/beta/delta tryptases than with membrane-anchored gamma-tryptases, and diverged more rapidly. We hypothesize that gelatinase activity and formation of inhibitor-resistant oligomers are ancestral characteristics shared by soluble tryptases and mastins, and that secreted mastin is a mini-proteasome-like complex that breaks down partially degraded proteins without causing bystander damage to intact, native proteins.

Amino Acid Sequence↗
PubMed2000

Mast cell and myeloid marker expression during early in vitro mast cell differentiation from human peripheral blood mononuclear cells.

UNLABELLED: In order to characterize the phenotype of human mast cell precursors in the peripheral blood mononuclear fraction and its alterations during in vivo mast cell differentiation, cells were studied before and during culture with stem cell factor or stem cell factor-containing cell supernatants. Prior to culture, 86% of cells were immunoreactive for the monocytic marker CD14, slightly fewer for CD11b and CD64, < 10% expressed FcepsilonRIalpha, rare cells were CD34 + ( < 0,1%), and none stained for CD1, CD33, c-Kit, and tryptase. After 2 wk of culture, there was de novo expression of c-Kit (14% - 43% positive cells), tryptase (26% - 79%), CD33 (57%), and CD64 (64%), an upregulation of FcepsilonRIalpha (23% - 52%), CD11b (93%), and CD68 (95%), but no expression of CD34. Levels of mRNA for FcepsilonRIalpha and c-Kit were detectable prior to culture and increased during culture, together with de novo expression of tryptase. Double staining after 2 wk of culture showed that FcepsilonRIalpha-positive cells were mostly CD14 + (90%), CD64 + (82%), and CD68 + (52%) on flow cytometry. Intracellular tryptase activity was first detectable after 1 wk of culture, increased FcepsilonRIalpha expression was only detectable by week 2. Cultured cells acquired the ability to release histamine during IgE-dependent stimulation, and culture with the c-Kit antibody YB5.B8 resulted in a downregulation of tryptase and FcepsilonRIalpha, but not of c-Kit. These data show that human mast cells develop from c-Kit- and tryptase-negative precursors in the myelomonocytic fraction of peripheral blood and that they upregulate, maintain, and share many phenotypic characteristics of cells from the monocyte/macrophage lineage during early phases of in vitro differentiation. KEYWORDS: c-kit/FcepsilonRI/SCF/tryptase.

Biomarkers↗