Mast Cell Carboxypeptidase Research Articles

Certain Aspects of Mast Cell Carboxypeptidase A3 Involvement in the Pathogenesis of COVID-19

The objective: to determine the involvement of lung mast cell carboxypeptidase A3 (SPA3) in the pathogenesis of COVID-19. Subjects and Methods. Samples of autopsy specimens from the lungs of patients who died of severe COVID-19 and patients who died of external causes were examined. On day 2 after the patient's death, the autopsy was performed and autopsy specimens were collected. Histological samples were prepared to analyze the degranulation activity of CPA3-positive mast cells (MCs). Correlations between protease profile and clinical and laboratory parameters were analyzed.Results. In the patients died of COVID-19, the total numbers of CPA3-positive MCs, CPA3-positive MCs with signs of degranulation, and coadjacent CPA3-positive MCs (two CPA3-positive MCs sharing a common cytoplasm) were statistically significantly higher versus the patients from Control Group. Positive correlations were established between the total number of CPA3-positive MCs and CPA3-positive MCs with signs of degranulation with blood hemoglobin level (r = 0.491; p = 0.008; r = 0.521; p = 0.004), between the total number of CPA3-positive MCs degranulating CPA3-positive MCs and adjacent CPA3-positive MCs with total blood protein (r = 0.431, p = 0.020; r = 0.449, p = 0.015; r = 0.456, p = 0.013), and between CPA3-positive MCs with signs of degranulation and total number of CPA3-positive MCs with aPTT level (r = 0.428; p = 0.021; r = 0.374; p = 0.045). Negative correlations were established between the total number of CPA3-positive MCs and the level of INR (r = 0.812; p = 0.050), separately lying CPA3-positive MCs with the number of monocytes (r = 0.384; p = 0.044), jointly adjacent CPA3-positive Mcs with blood eosinophils (r = 0.420, p = 0.023).

Abstract A64: Mast cell expressed FcεR beta subunit (MS4A2) is prognostic in lung adenocarcinoma

Abstract Background: The context of immune cell infiltrates within the tumor is associated with cancer prognosis. The type of immune cells and their activation state can provide mediators that either promote or inhibit tumor growth. A growing body of evidence suggest the presence of innate immune cells play a role in shaping early tumor growth. In attempts to gain insight into the immune networks that regulate tumorigenesis, we used genome wide expression datasets of patients with resected early stage non-small cell lung cancer (NSCLC) to identify immune-related genes associated with patient survival. Methods: Gene expression analysis was conducted on microarray datasets from 128 early-stage NSCLC adenocarcinoma resected tumor samples. Limiting analysis to immune-related genes, we identified a minimum gene set that is prognostic for lung adenocarcinoma and validated the gene signature in additional published microarray NSCLC datasets. Using pathway analysis and immunohistochemistry (IHC), we identified a role for mast cell-expressed IgE receptor beta subunit, MS4A2, in lung adenocarcinoma progression. Results: From an immune-related gene list, we identified a minimal gene signature that was able to separate patients into high or low-risk survival groups. This gene signature was shown to be prognostic for NSCLC adenocarcinoma in nine validation datasets (n=1264, hazard ratio 2.07, 95% confidence interval 1.69-2.53, p<0.0001). Pathway analysis of differentially expressed genes within high or low-risk survival subgroups reveals gene ontologies and immune signatures that are enriched for IgE receptor signaling and mast cells in patients with favourable prognosis. Univariate analysis shows that the IgE receptor complex genes, as well as mast cell specific protease, mast cell carboxypeptidase A, are highly prognostic for survival. We are currently using IHC of matched patient samples to validate receptor localization. Conclusion: By limiting gene expression analysis to immune-related genes, we identified a minimal gene set that indicate an important role for IgE receptor signaling and mast cells in favourable lung cancer prognosis. Our data highlights the importance of innate immune cells in shaping lung cancer development. Citation Format: Dalam Ly, Chang-Qi Zhu, Michael Cabanero, Ming-Sound Tsao, Li Zhang. Mast cell expressed FcεR beta subunit (MS4A2) is prognostic in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A64.

Non-ionic iodinated contrast media related immediate reactions: A mechanism study of 27 patients

The underlying mechanism of non-ionic iodinated contrast media-related immediate reactions was evaluated in this study. Patients presenting at least grade II immediate reactions after non-ionic iodinated contrast media injection were enrolled. Basophil activation was evaluated by flow cytometry. The plasma concentration of human terminal complement complex SC5b-9, as well as concentrations of serum chymase, tryptase, human mast cell carboxypeptidase A3, human prostaglandin D2, and total IgE were measured by enzyme-linked immunosorbent assay. The basophil activation percentage was significantly higher in the study group than in the control group (17.94±21.06% vs 3.45±1.49%). The plasma concentration of human terminal complement complex SC5b-9 and concentrations of serum chymase, human mast cell carboxypeptidase A3, prostaglandin D2, tryptase, and total IgE were also significantly increased (236.99±318.21 vs 49.70±30.41ng/mL, 0.41±0.49 vs 0.09±0.06ng/mL, 1.17±0.67 vs 0.30±0.17ng/mL, 203.52±137.27 vs 102.28±48.72pg/mL, 3.81±0.22 vs 2.70±0.16ng/mL, 102.00±51.84 vs 19.97±2.75ng/mL, respectively). Both mast cells and basophils were activated in non-ionic iodinated contrast media to mediate immediate hypersensitivity, and mast cells may be involved. Different mechanisms, including IgE-dependent, complement-dependent, and direct membrane effects, contributed to mast cell and basophil activation. Individual patients may use a single or combined mechanism involving single or combined mast cells and basophils. Immediate reactions following non-ionic iodinated contrast media injection may be a mechanically heterogenous disease.

Pivotal role of mast cell carboxypeptidase A in mediating protection against small intestinal ischemia–reperfusion injury in rats after ischemic preconditioning

Aim of the studyMast cell (MC) degranulation contributes to the protection mediated by ischemic preconditioning (IPC); however, the precise mechanisms underlying this protection remain largely unknown. Mast cell carboxypeptidase A (MC-CPA) is released solely from MCs and plays a critical role in degrading toxins and endothelin 1 (ET-1). The present study sought to explore whether MC-CPA is involved in the process of IPC in a rodent model of small intestinal ischemia reperfusion (IIR) injury. Materials and methodsIIR injuries were induced in Sprague–Dawley rats by clamping the superior mesenteric artery for 60 min followed by reperfusion for 2 h. One cycle of 10 min intestinal ischemia and 10 min of reperfusion was used in the IPC group, and the MC stabilizer cromolyn sodium and MC potato carboxypeptidase inhibitor were administered before the start of IPC. At the end of experiment, intestine tissue was obtained for assays of the MC-CPA3, tumor necrosis factor-α, interleukin-6, and ET-1 contents and myeloperoxidase activities. Intestinal histologic injury scores and MC degranulation were assessed. Apoptosis indices and cleaved caspase- 3 protein expressions were quantified. ResultsIIR resulted in severe injury, as evidenced by significant increases in injury scores and MC-CPA3, tumor necrosis factor-α, interleukin-6, and ET-1 contents that were accompanied with concomitant elevations in cleaved caspase 3 expression, apoptosis indices, and myeloperoxidase activities. IPC induced a significant increase in MC-CPA3, induced MC degranulation, and attenuated IIR injury by downregulating IIR-induced biochemical changes, whereas cromolyn sodium and potato carboxypeptidase inhibitor abolished the IPC-mediated changes. ConclusionsThese data suggest that IPC protected against IIR injury via the MC degranulation-mediated release of MC-CPA.

Proteome-derived Peptide Libraries to Study the Substrate Specificity Profiles of Carboxypeptidases

Through processing peptide and protein C termini, carboxypeptidases participate in the regulation of various biological processes. Few tools are however available to study the substrate specificity profiles of these enzymes. We developed a proteome-derived peptide library approach to study the substrate preferences of carboxypeptidases. Our COFRADIC-based approach takes advantage of the distinct chromatographic behavior of intact peptides and the proteolytic products generated by the action of carboxypeptidases, to enrich the latter and facilitate its MS-based identification. Two different peptide libraries, generated either by chymotrypsin or by metalloendopeptidase Lys-N, were used to determine the substrate preferences of human metallocarboxypeptidases A1 (hCPA1), A2 (hCPA2), and A4 (hCPA4). In addition, our approach allowed us to delineate the substrate specificity profile of mouse mast cell carboxypeptidase (MC-CPA or mCPA3), a carboxypeptidase suggested to function in innate immune responses regulation and mast cell granule homeostasis, but which thus far lacked a detailed analysis of its substrate preferences. mCPA3 was here shown to preferentially remove bulky aromatic amino acids, similar to hCPA2. This was also shown by a hierarchical cluster analysis, grouping hCPA1 close to hCPA4 in terms of its P1 primed substrate specificity, whereas hCPA2 and mCPA3 cluster separately. The specificity profile of mCPA3 may further aid to elucidate the function of this mast cell carboxypeptidase and its biological substrate repertoire. Finally, we used this approach to evaluate the substrate preferences of prolylcarboxypeptidase, a serine carboxypeptidase shown to cleave C-terminal amino acids linked to proline and alanine.

High Serum Levels of Mast Cell Chymase and Carboxypeptidase in Mastocytosis

High Serum Levels of Mast Cell Chymase and Carboxypeptidase in Mastocytosis

Age-related Reduction Of S100A8/A9 In Chronic Rhinosinusitis Is Associated With Increased Production Of Soluble gp130

S A T U R D A Y 163 Mast Cells with a Unique Phenotype Are Highly Elevated in Chronic Rhinosinusitis with Nasal Polyps T. Takabayashi, A. Kato, A. T. Peter, L. A. Suh, R. Carter, J. Norton, L. C. Grammer, B. K. Tan, R. K. Chandra, D. B. Conley, R. C. Kern, S. Fujieda, R. P. Schleimer; The Division of Allergy and Immunology Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, The Division of Otorhinolaryngology Head and Neck Surgery, Department of Sensory and Locomotor Medicine, University of Fukui, Fukui, JAPAN, Department of Otolaryngology, Northwestern University Feinberg School of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL. RATIONALE: Although chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by Th2 inflammation, the role of mast cells is poorly understood. The objective of this study was to investigate the presence, localization and phenotype of mast cells in affected tissues from patients with chronic rhinosinusitis (CRS). METHODS: We collected nasal tissue and nasal lavage fluid from patients with CRS and control subjects. We analyzed mRNA for the mast cell proteases tryptase, chymase and carboxypeptidase A3 (CPA3), using real-time PCR, and assessed the presence of mast cell proteases using ELISA and immunohistochemistry. We also performed immunofluorescence to observe the pattern of colocalization of the mast cell proteases. RESULTS: Tryptase mRNA and protein were significantly increased in nasal polyps frompatientswithCRSwNPcomparedwith uncinate tissue from patients with CRS or healthy subjects. We made the striking observation that therewere abundantmast cells localizedwithinglands of nasalpolyps and that these mast cells expressed all three proteases. We also observed increased numbers of mast cells in epithelium but not elsewhere within the lamina propria in nasal polyps. The mast cells detected in epithelium in nasal polyps were characterized by expression of tryptase and CPA3 but not chymase. CONCLUSIONS: Herein we demonstrate increased mast cells in epithelium and glands of nasal polyp tissue, and show that nasal polyp mast cells have unique phenotypes that vary by tissue location. These diverse subsets of mast cells may contribute to the pathogenesis of CRSwNP.

Cre-Mediated Cell Ablation Contests Mast Cell Contribution in Models of Antibody- and T Cell-Mediated Autoimmunity

Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy.

Differential gene expression in nerve biopsies of inflammatory neuropathies

DNA microarray analysis is a powerful tool for simultaneous analysis and comparison of gene products expressed in normal and diseased tissues. We used this technique to identify differentially expressed genes (DEGs) in nerve biopsy samples of chronic inflammatory demyelinating polyneuropathy (CIDP) and vasculitic neuropathy (VAS) patients. We found novel previously uncharacterized genes of relevance to CIDP or VAS pathogenesis. Of particular interest in CIDP were tachykinin precursor 1, which may be involved in pain mediation, stearoyl-co-enzyme A (CoA) desaturase, which may be a marker for remyelination, HLA-DQB1, CD69, an early T-cell activation gene, MSR1, a macrophage scavenger receptor, and PDZ and LIM domain 5 (PDLIM5), a factor regulating nuclear factor (NF)-kappa B activity. Genes upregulated in VAS included IGLJ3, IGHG3, IGKC, and IGL, which all function in B-cell selection or antigen recognition of B cells. Other upregulated genes included chemokines, such as CXCL9 and CCR2, as well as CPA3, a mast cell carboxypeptidase. Allograft inflammatory factor-1 (AIF-1), a modulator of immune response was upregulated both in CIDP and VAS. Microarray-based analysis of human sural nerve biopsies showed distinct gene expression patterns in CIDP and VAS. DEGs might provide clues to the pathogenesis of the diseases and be potential targets for therapeutics.

Mast Cell Carboxypeptidase as a Confirmatory and Predictive Marker in Allergic Reactions to Drugs

Mast Cell Carboxypeptidase as a Confirmatory and Predictive Marker in Allergic Reactions to Drugs

Mast Cell Chymase: A Useful Serum Marker in Anaphylaxis

RATIONALE: The reliable diagnosis of anaphylaxis can present challenge. We have explored mast cell chymase as a potential marker for anaphylaxis. METHODS: A sandwich-based ELISA was developed with monoclonal antibodies we have prepared against chymase, and validated for application to serum samples. The enzymatic activity of chymase was determined in parallel using the chromogenic substrates suc-AAPF-pNA and RETF-4NA. Tryptase, carboxypeptidase and DPPI were measured by ELISA. Serum was collected from cases of anaphylaxis provoked by food, drug and insect stings (n=181); from healthy blood donors (n=123); and from patients allergic to food (n=76) or drugs (n=26). RESULTS: The ELISA allowed detection of chymase in all serum samples, and values obtained were unaffected by the presence of alpha-2-macroglobulin or other inhibitors. Chymase levels in serum collected from patients within 8h of anaphylaxis were greater than those in the control group (p=0.0069); and concentrations remained high at least 24h after onset of the reaction. Chymase levels in anaphylaxis correlated with levels of mast cell carboxypeptidase and with dipeptidyl peptidase 1 (DPPI), but not tryptase. Concentrations of chymase measured by ELISA were weakly associated with serum chymase activity. Addition of DPPI to serum increased the chymase activity in samples collected prior to allergen challenge (p=0.0008) consistent with the presence of pro-forms of chymase in the circulation. However, DPPI-induced increases in serum chymase activity were not observed in samples collected following a reaction (when serum DPPI concentrations were elevated). CONCLUSIONS: Measurement of mast cell chymase in serum should be useful as a means for laboratory confirmation of anaphylaxis.

A Role for Serglycin Proteoglycan in Mast Cell Apoptosis Induced by a Secretory Granule-mediated Pathway

Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin(-/-) cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis.

Adverse Reactions Associated With Therapeutic Cephalosporin And Sulfonamide Antibiotic Use After Penicillin Skin Testing

S U N D A Y 539 Mast Cell Chymase: A Useful Serum Marker in Anaphylaxis X. Zhou, H. S. Whitworth, E-Khedr M., T. A. Brown, R. Goswami, E. Eren, J. A. Lucas, M. D. S. Erlewyn-Lajeunesse, C. W. Summers, R. S. H. Pumphrey, G.Wild, J. Pedersen, A. F. Walls; University of Southampton, Southampton, UNITEDKINGDOM, Southampton University Hospital Trust, Southampton, UNITED KINGDOM, University of Manchester/Manchester Royal Infirmary, Manchester, UNITED KINGDOM, Northern General Hospital, Sheffield, UNITED KINGDOM, Unizyme Laboratories A/S, Horsholm, DENMARK. RATIONALE:The reliable diagnosis of anaphylaxis can present challenge. We have explored mast cell chymase as a potential marker for anaphylaxis. METHODS: A sandwich-based ELISA was developed with monoclonal antibodies we have prepared against chymase, and validated for application to serum samples. The enzymatic activity of chymase was determined in parallel using the chromogenic substrates suc-AAPF-pNA and RETF4NA. Tryptase, carboxypeptidase and DPPI were measured by ELISA. Serum was collected from cases of anaphylaxis provoked by food, drug and insect stings (n5181); from healthy blood donors (n5123); and from patients allergic to food (n576) or drugs (n526). RESULTS: The ELISA allowed detection of chymase in all serum samples, and values obtained were unaffected by the presence of alpha-2-macroglobulin or other inhibitors. Chymase levels in serum collected from patients within 8h of anaphylaxis were greater than those in the control group (p50.0069); and concentrations remained high at least 24h after onset of the reaction. Chymase levels in anaphylaxis correlated with levels of mast cell carboxypeptidase and with dipeptidyl peptidase 1 (DPPI), but not tryptase. Concentrations of chymase measured by ELISAwere weakly associatedwith serum chymase activity. Addition of DPPI to serum increased the chymase activity in samples collected prior to allergen challenge (p50.0008) consistent with the presence of pro-forms of chymase in the circulation. However, DPPI-induced increases in serum chymase activity were not observed in samples collected following a reaction (when serum DPPI concentrations were elevated). CONCLUSIONS:Measurement of mast cell chymase in serum should be useful as a means for laboratory confirmation of anaphylaxis.

Characterization of the Substrate Specificity of Human Carboxypeptidase A4 and Implications for a Role in Extracellular Peptide Processing

CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment.

Mast Cell Carboxypeptidase and Angiotensin-Converting Enzyme (ACE) as Serum Markers of Susceptibility to Severe Food Allergic Reactions

RATIONALE: Allergic sensitivity to food is widespread. Establishing a reliable diagnosis can represent a challenge, and it is not easy to predict who may be at risk of anaphylaxis. There is a need for better means of identifying those patients who may be susceptible to severe allergic reactions to food.

Deletion of Notch1 Converts Pro-T Cells to Dendritic Cells and Promotes Thymic B Cells by Cell-Extrinsic and Cell-Intrinsic Mechanisms

Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

Molecular mechanism of mast cell–mediated innate defense against endothelin and snake venom sarafotoxin

Mast cells are protective against snake venom sarafotoxins that belong to the endothelin (ET) peptide family. The molecular mechanism underlying this recently recognized innate defense pathway is unknown, but secretory granule proteases have been invoked. To specifically disrupt a single protease function without affecting expression of other proteases, we have generated a mouse mutant selectively lacking mast cell carboxypeptidase A (Mc-cpa) activity. Using this mutant, we have now identified Mc-cpa as the essential protective mast cell enzyme. Mass spectrometry of peptide substrates after cleavage by normal or mutant mast cells showed that removal of a single amino acid, the C-terminal tryptophan, from ET and sarafotoxin by Mc-cpa is the principle molecular mechanism underlying this very rapid mast cell response. Mast cell proteases can also cleave ET and sarafotoxin internally, but such “nicking” is not protective because intramolecular disulfide bridges maintain peptide function. We conclude that mast cells attack ET and sarafotoxin exactly at the structure required for toxicity, and hence sarafotoxins could not “evade” Mc-cpa's substrate specificity without loss of toxicity.

Risk assessment in anaphylaxis: Current and future approaches

Risk assessment of individuals with anaphylaxis is currently hampered by lack of (1) an optimal and readily available laboratory test to confirm the clinical diagnosis of an anaphylaxis episode and (2) an optimal method of distinguishing allergen-sensitized individuals who are clinically tolerant from those at risk for anaphylaxis episodes after exposure to the relevant allergen. Our objectives were to review the effector mechanisms involved in the pathophysiology of anaphylaxis; to explore the possibility of developing an optimal laboratory test to confirm the diagnosis of an anaphylaxis episode, and the possibility of improving methods to distinguish allergen sensitization from clinical reactivity; and to develop a research agenda for risk assessment in anaphylaxis. Researchers from the American Academy of Allergy, Asthma & Immunology and the European Academy of Allergology and Clinical Immunology held a PRACTALL (Practical Allergy) meeting to discuss these objectives. New approaches being investigated to support the clinical diagnosis of anaphylaxis include serial measurements of total tryptase in serum during an anaphylaxis episode, and measurement of baseline total tryptase levels after the episode. Greater availability of the test for mature beta-tryptase, a more specific mast cell activation marker for anaphylaxis than total tryptase, is needed. Measurement of chymase, mast cell carboxypeptidase A3, platelet-activating factor, and other mast cell products may prove to be useful. Consideration should be given to measuring a panel of mediators from mast cells and basophils. New approaches being investigated to help distinguish sensitized individuals at minimum or no risk from those at increased risk of developing anaphylaxis include measurement of the ratio of allergen-specific IgE to total IgE, determination of IgE directed at specific allergenic epitopes, measurement of basophil activation markers by using flow cytometry, and assessment of allergen-specific cytokine responses. Algorithms have been developed for risk assessment of individuals with anaphylaxis, along with a research agenda for studies that could lead to an improved ability to confirm the clinical diagnosis of anaphylaxis and to identify allergen-sensitized individuals who are at increased risk of anaphylaxis.

Anaphylactic Mechanisms in Sudden Unexpected Infant Death: Elevated Concentrations of Mast Cell Carboxypeptidase in the Serum

Anaphylactic Mechanisms in Sudden Unexpected Infant Death: Elevated Concentrations of Mast Cell Carboxypeptidase in the Serum

Preparation, Characterization and Epitope Mapping of Monoclonal Antibodies Specific to Human Mast Cell Carboxypeptidase

Human mast cell carboxypeptidase (hMC-CP) is a unique product of mast cells. Unlike tryptase and chymase, its potential function and expression in diseased conditions remain largely unknown. To develop an assay for hMC-CP, the recombinant fusion protein of hMC-CP and purified native skin hMC-CP was prepared, and two novel monoclonal antibodies against hMC-CP named CCP1 (IgG1 isotype) and CCP2 (IgM isotype) were raised in the present study. Epitope analysis shows that CCP1 and CCP2 antibodies recognize epitopes located in the region of amino acids 112-202 of hMC-CP, and hydrophilicity analysis implies that epitopes might be located in the amino acid residues 123-134 and 165-177. Furthermore, using a competition enzyme-linked immunosorbent assay, it was shown that the epitope recognized by CCP1 is close to that recognized by CCP2 or the two antibodies partially share the same epitope. Flow cytometry analysis shows that basophilic leukemia cell line KU812 reacts with both CCP1 and CCP2 antibodies, suggesting that this cell line expresses hMC-CP. In conclusion, although the two antibodies possess different isotypes, they may partially share the same epitope. These two antibodies will be valuable tools for the development of an assay to detect the levels of hMC-CP in the biological fluids in man.