Development of a specific immunoassay to selectively measure active tryptase in airway samples

Bioanalysis ◽  
2020 ◽  
Vol 12 (19) ◽  
pp. 1377-1388
Author(s):  
Gizette Sperinde ◽  
Meire Bremer ◽  
Henry R Maun ◽  
Amos Baruch ◽  
Robert A Lazarus ◽  
...  
Keyword(s):  
Mast Cells ◽  
Proteolytic Activity ◽  
Human Disease ◽  
Respiratory Diseases ◽  
Inflammatory Responses ◽  
Secretory Granules ◽  
Human Tissues ◽  
Important Mediator ◽  
Capture Antibody

Aim: Tryptase is a tetrameric trypsin-like serine protease contained within the secretory granules of mast cells and is an important mediator of allergic inflammatory responses in respiratory diseases. Detection of active tryptase in the airway may provide important information about asthma and other respiratory diseases. Materials & Methods: An activity based probe has been incorported within an immunoassay to allow for measurement of active tryptase in human tissues. Results: A specific Simoa immunoassay to measure active tryptase in nasosorption samples was developed and qualified using an activity-based probe label and a specific antitryptase capture antibody. Conclusion: The assay was capable of measuring active tryptase in human samples, which will enable evaluation of the role of tryptase proteolytic activity in human disease.

scholarly journals The Role of Mast Cells in Stroke

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 437 ◽  
Author(s):  
Edoardo Parrella ◽  
Vanessa Porrini ◽  
Marina Benarese ◽  
Marina Pizzi
Keyword(s):  
Central Nervous System ◽  
Immune Response ◽  
Nervous System ◽  
Mast Cells ◽  
Brain Edema ◽  
Hemorrhagic Stroke ◽  
Inflammatory Responses ◽  
Adult Brain ◽  
The Central Nervous System

Mast cells (MCs) are densely granulated perivascular resident cells of hematopoietic origin. Through the release of preformed mediators stored in their granules and newly synthesized molecules, they are able to initiate, modulate, and prolong the immune response upon activation. Their presence in the central nervous system (CNS) has been documented for more than a century. Over the years, MCs have been associated with various neuroinflammatory conditions of CNS, including stroke. They can exacerbate CNS damage in models of ischemic and hemorrhagic stroke by amplifying the inflammatory responses and promoting brain–blood barrier disruption, brain edema, extravasation, and hemorrhage. Here, we review the role of these peculiar cells in the pathophysiology of stroke, in both immature and adult brain. Further, we discuss the role of MCs as potential targets for the treatment of stroke and the compounds potentially active as MCs modulators.

scholarly journals The emerging role of mast cell proteases in asthma

2019 ◽  
Vol 54 (4) ◽  
pp. 1900685 ◽  
Author(s):  
Gunnar Pejler
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Current Knowledge ◽  
Secretory Granules ◽  
Cross Linking ◽  
Lysosomal Hydrolases ◽  
Ige Receptor ◽  
Deficient Mice ◽  
Lines Of Evidence

It is now well established that mast cells (MCs) play a crucial role in asthma. This is supported by multiple lines of evidence, including both clinical studies and studies on MC-deficient mice. However, there is still only limited knowledge of the exact effector mechanism(s) by which MCs influence asthma pathology. MCs contain large amounts of secretory granules, which are filled with a variety of bioactive compounds including histamine, cytokines, lysosomal hydrolases, serglycin proteoglycans and a number of MC-restricted proteases. When MCs are activated, e.g. in response to IgE receptor cross-linking, the contents of their granules are released to the exterior and can cause a massive inflammatory reaction. The MC-restricted proteases include tryptases, chymases and carboxypeptidase A3, and these are expressed and stored at remarkably high levels. There is now emerging evidence supporting a prominent role of these enzymes in the pathology of asthma. Interestingly, however, the role of the MC-restricted proteases is multifaceted, encompassing both protective and detrimental activities. Here, the current knowledge of how the MC-restricted proteases impact on asthma is reviewed.

scholarly journals ALIAmides Update: Palmitoylethanolamide and Its Formulations on Management of Peripheral Neuropathic Pain

2020 ◽  
Vol 21 (15) ◽  
pp. 5330 ◽  
Author(s):  
Ramona D’Amico ◽  
Daniela Impellizzeri ◽  
Salvatore Cuzzocrea ◽  
Rosanna Di Paola
Keyword(s):  
Nervous System ◽  
Neuropathic Pain ◽  
Mast Cells ◽  
Inflammatory Responses ◽  
Bioactive Lipids ◽  
Peripheral Neuropathic Pain ◽  
Novel Approach ◽  
Preclinical And Clinical Studies ◽  
Somatosensory Nervous System

Neuropathic pain results from lesions or diseases of the somatosensory nervous system and it remains largely difficult to treat. Peripheral neuropathic pain originates from injury to the peripheral nervous system (PNS) and manifests as a series of symptoms and complications, including allodynia and hyperalgesia. The aim of this review is to discuss a novel approach on neuropathic pain management, which is based on the knowledge of processes that underlie the development of peripheral neuropathic pain; in particular highlights the role of glia and mast cells in pain and neuroinflammation. ALIAmides (autacoid local injury antagonist amides) represent a group of endogenous bioactive lipids, including palmitoylethanolamide (PEA), which play a central role in numerous biological processes, including pain, inflammation, and lipid metabolism. These compounds are emerging thanks to their anti-inflammatory and anti-hyperalgesic effects, due to the down-regulation of activation of mast cells. Collectively, preclinical and clinical studies support the idea that ALIAmides merit further consideration as therapeutic approach for controlling inflammatory responses, pain, and related peripheral neuropathic pain.

VAMP-8 segregates mast cell–preformed mediator exocytosis from cytokine trafficking pathways

Blood ◽  
2008 ◽  
Vol 111 (7) ◽  
pp. 3665-3674 ◽  
Author(s):  
Neeraj Tiwari ◽  
Cheng-Chun Wang ◽  
Cristiana Brochetta ◽  
Gou Ke ◽  
Francesca Vita ◽  
...  
Keyword(s):  
Mast Cells ◽  
Inflammatory Responses ◽  
Secretory Granules ◽  
Snare Complex ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Syntaxin 4 ◽  
Deficient Mice ◽  
Snare Complex Formation ◽  
Vesicular Compartment

Abstract Inflammatory responses by mast cells are characterized by massive exocytosis of prestored granular mediators followed by cytokine/chemokine release. The vesicular trafficking mechanisms involved remain poorly understood. Vesicular-associated membrane protein-8 (VAMP-8), a member of the soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) family of fusion proteins initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. Here we show that in bone marrow–derived mast cells (BMMCs) VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. This was associated with increased SNARE complex formation with the target t-SNAREs, SNAP-23 and syntaxin-4. VAMP-8–deficient BMMCs exhibited a markedly reduced degranulation response after IgE+ antigen-, thapsigargin-, or ionomycin-induced stimulation. VAMP-8–deficient mice also showed reduced plasma histamine levels in passive systemic anaphylaxis experiments, while cytokine/chemokine release was not affected. Unprocessed TNF accumulated at the plasma membrane where it colocalized with a VAMP-3–positive vesicular compartment but not with VAMP-8. The findings demonstrate that VAMP-8 segregates secretory lysosomal granule exocytosis in mast cells from cytokine/chemokine molecular trafficking pathways.

Defective Regulated Exocytosis in Mast Cells from Synaptotagmin-2 Knockout Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3090-3090
Author(s):  
Leticia Sansores-Garcia ◽  
Ernestina Melicoff ◽  
Abigail H. Teich ◽  
Jayasimha Murthy ◽  
Shakeel M. Thakurdas ◽  
...  
Keyword(s):  
Mast Cells ◽  
Calcium Binding ◽  
Bacterial Infections ◽  
Transmembrane Domain ◽  
Secretory Granules ◽  
Surface Expression ◽  
Calcium Sensor ◽  
Normal Surface ◽  
Regulated Exocytosis

Abstract Synaptotagmins (Syts) are a family of membrane proteins present on a variety of intracellular organelles. They contain a short N terminus, a single transmembrane domain and two calcium binding C2 domains. Twelve Syt isoforms have been identified, in vertebrates. Syt-1 is the postulated calcium sensor in neuronal regulated exocytosis. Syt2 is functionally and structurally closely related to Syt1, it is the major Syt isoform in mast cells (MC) and it localizes on the MC secretory granules. MC are central effectors of allergic reactions and enhancers of host immune responses against bacterial infections. MC respond to a variety of stimuli with three effector mechanisms: secretion of preformed inflammatory mediators stored in their granules via regulated exocytosis, release of newly synthesized cytokines and chemokynes by constitutive exocytosis, and secretion of prostaglandins and leukotrienes by an exocytosis-independent mechanism. Based on the role of Syt1 in neuronal exocytosis, we postulate that Syt2 is the calcium sensor in MC regulated exocytosis. To investigate this possibility we generated a Syt2 knockout mouse using homologous recombination. Disruption of Syt2 results in low viability (around 14 days survival), growth impairment (approximately half size and weight of the control littermates) and severe ataxia and muscular weakness. We derived MC from bone marrow and lungs of 2 weeks-old Syt2−/− mice and control littermates (Syt2+/−, Syt2+/+). We found no morphological or developmental difference in Syt2−/− MC when compared with controls, and after 6 weeks in culture Syt2−/− MC have normal surface expression of FcεRI and c-kit as measured by flow cytometry. However, we found a 50% inhibition in β-hexosaminidase and histamine release upon stimulation by IgE crosslinking, compatible with a defect in degranulation. Furthermore, other effector responses seem not to be compromised, as there was no difference in stimulated LTC4 release when compared with controls. Our data proves the essential role of Syt2 in MC regulated exocytosis. We are now performing additional functional assays and single-cell electrophysiology.

Heparanase-mediated cleavage of macromolecular heparin accelerates release of granular components of mast cells from extracellular matrices

2014 ◽  
Vol 458 (2) ◽  
pp. 291-299 ◽  
Author(s):  
Nobuaki Higashi ◽  
Michihiko Waki ◽  
Mayumi Sue ◽  
Yusuke Kogane ◽  
Hiroaki Shida ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Connective Tissue ◽  
Cell Function ◽  
Secretory Granules ◽  
Extracellular Matrices ◽  
Regulatory Role ◽  
Tissue Type ◽  
Size Dependent

Connective tissue-type mast cells express heparin and heparanase in the secretory granules. Cleavage of granular heparin by heparanase accelerates release of granular components from collagen-based extracellular matrices. A size-dependent novel regulatory role of heparin for mast cell function is proposed.

scholarly journals New Pathways for the Skin's Stress Response: The Cholinergic Neuropeptide SLURP-1 Can Activate Mast Cells and Alter Cytokine Production in Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Christoph M. Ertle ◽  
Frank R. Rommel ◽  
Susanne Tumala ◽  
Yasuhiro Moriwaki ◽  
Jochen Klein ◽  
...  
Keyword(s):  
Mast Cells ◽  
Psychosocial Stress ◽  
Cytokine Production ◽  
Inflammatory Responses ◽  
Alpha7 Nicotinic Acetylcholine Receptor ◽  
Immune Parameters ◽  
And Oxidative Stress ◽  
Pro Inflammatory Cytokines ◽  
Inflammatory Effect

Background: The alpha7 nicotinic acetylcholine receptor (Chrna7) plays an essential anti-inflammatory role in immune homeostasis and was recently found on mast cells (MC). Psychosocial stress can trigger MC hyperactivation and increases pro-inflammatory cytokines in target tissues such as the skin. If the cholinergic system (CS) and Chrna7 ligands play a role in these cascades is largely unknown.Objective: To elucidate the role of the CS in the response to psychosocial stress using a mouse-model for stress-triggered cutaneous inflammatory circuits.Methods: Key CS markers (ACh, Ch, SLURP-1, SLURP-2, Lynx1, Chrm3, Chrna7, Chrna9, ChAT, VAChT, Oct3, AChE, and BChE) in skin and its MC (sMC), MC activation, immune parameters (TNFα, IL1β, IL10, TGFβ, HIF1α, and STAT3) and oxidative stress were analyzed in skin from 24 h noise-stressed mice and in cultured MC (cMC) from C57BL/6 or Chrna7-Knockout mice.Results: First, Chrna7 and SLURP-1 mRNA were exclusively upregulated in stressed skin. Second, histomorphometry located Chrna7 and SLURP-1 in nerves and sMC and demonstrated upregulated contacts and increased Chrna7+ sMC in stressed skin, while 5 ng/mL SLURP-1 degranulated cMC. Third, IL1β+ sMC were high in stressed skin, and while SLURP-1 alone had no significant effect on cMC cytokines, it upregulated IL1β in cMC from Chrna7-KO and in IL1β-treated wildtype cMC. In addition, HIF1α+ sMC were high in stressed skin and Chrna7-agonist AR-R 17779 induced ROS in cMC while SLURP-1 upregulated TNFα and IL1β in cMC when HIF1α was blocked.Conclusions: These data infer that the CS plays a role in the regulation of stress-sensitive inflammatory responses but may have a surprising pro-inflammatory effect in healthy skin, driving IL1β expression if SLURP-1 is involved.

scholarly journals TPC1 deficiency or blockade augments systemic anaphylaxis and mast cell activity

2020 ◽  
Vol 117 (30) ◽  
pp. 18068-18078 ◽  
Author(s):  
Elisabeth Arlt ◽  
Marco Fraticelli ◽  
Volodymyr Tsvilovskyy ◽  
Wiebke Nadolni ◽  
Andreas Breit ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Ex Vivo ◽  
Secretory Granules ◽  
Cell Activity ◽  
New Drugs ◽  
Pore Channel ◽  
Systemic Anaphylaxis

Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca2+) signals. Ca2+release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca2+homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca2+uptake and filling of endoplasmic reticulum Ca2+stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity.

scholarly journals iNOS Interacts with Autophagy Receptor p62 and is Degraded by Autophagy in Macrophages

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1255 ◽  
Author(s):  
Jing Wang ◽  
Ming-Yue Wu ◽  
Huanxing Su ◽  
Jinjian Lu ◽  
Xiuping Chen ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Degradation Process ◽  
No Production ◽  
Nervous System Diseases ◽  
Central Nervous System Diseases ◽  
Important Mediator ◽  
Autophagy Induction ◽  
High Level ◽  
Autophagy Inhibition

Nitric oxide (NO) is an important mediator of inflammation response and the production of NO has been linked to a variety of diseases, including tumors, inflammation and central nervous system diseases. In macrophages, a high level of NO is generated by iNOS during inflammatory responses triggered by cytokines or pathogens. Autophagy, a cellular bulk degradation process via lysosome, has been implicated in many disease conditions including inflammation. In this study, we have reported the previously unknown role of autophagy in regulating iNOS levels in macrophages, both under basal and Lipopolysaccharides (LPS)-induced conditions. Our data showed that iNOS levels accumulated upon autophagy inhibition and decreased upon autophagy induction. iNOS interacted and co-localized with autophagy receptor p62/SQSTM1, especially under LPS-stimulated condition in macrophages. Moreover, the immunostaining data revealed that iNOS also co-localizes with the autophagosome marker LC3 and lysosome marker LAMP1, especially under lysosomal inhibition conditions, indicating iNOS is an autophagy substrate. Finally, we showed that autophagy negatively regulated the generation of NO in macrophages, which is consistent with the changes of iNOS levels. Collectively, our study revealed a previously unknown mechanism by which autophagy regulates iNOS levels to modulate NO production during inflammation.

scholarly journals The Role of Mast Cell Specific Chymases and Tryptases in Tumor Angiogenesis

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Devandir Antonio de Souza Junior ◽  
Ana Carolina Santana ◽  
Elaine Zayas Marcelino da Silva ◽  
Constance Oliver ◽  
Maria Celia Jamur
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tumor Growth ◽  
Tumor Angiogenesis ◽  
Secretory Granules ◽  
Therapeutic Targets ◽  
Exact Role ◽  
Mast Cell Granule ◽  
Novel Therapeutic

An association between mast cells and tumor angiogenesis is known to exist, but the exact role that mast cells play in this process is still unclear. It is thought that the mediators released by mast cells are important in neovascularization. However, it is not known how individual mediators are involved in this process. The major constituents of mast cell secretory granules are the mast cell specific proteases chymase, tryptase, and carboxypeptidase A3. Several previous studies aimed to understand the way in which specific mast cell granule constituents act to induce tumor angiogenesis. A body of evidence indicates that mast cell proteases are the pivotal players in inducing tumor angiogenesis. In this review, the likely mechanisms by which tryptase and chymase can act directly or indirectly to induce tumor angiogenesis are discussed. Finally, information presented here in this review indicates that mast cell proteases significantly influence angiogenesis thus affecting tumor growth and progression. This also suggests that these proteases could serve as novel therapeutic targets for the treatment of various types of cancer.

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