Serum mast cell tryptase measurements: Sensitivity and specificity for a diagnosis of anaphylaxis in emergency department patients with shock or hypoxaemia.

OBJECTIVE: Clinical diagnosis of anaphylaxis is principally based on symptoms and signs. However, particularly for patients with atypical symptoms, laboratory confirmation of anaphylaxis would be useful. This study investigated the utility of mast cell tryptase, an available clinical biomarker, for differentiating anaphylaxis from other causes of critical illness, which can also involve mast cell activation. METHODS: Tryptase was measured (ImmunoCAP) in serum from patients with anaphylaxis and non-anaphylactic critical illness (controls) at ED arrival, and after 1-2, 3-4 and 12-24 h. Differences in both peak and delta (difference between highest and lowest) tryptase concentrations between groups were investigated using linear regression models, and diagnostic ability was analysed using Receiver Operating Characteristic curve analysis. RESULTS: Peak tryptase was fourfold (95% CI: 2.9, 5.5) higher in anaphylaxis patients (n = 67) than controls (n = 120) (P < 0.001). Delta-tryptase was 5.1-fold (95% CI: 2.9, 8.9) higher in anaphylaxis than controls (P < 0.001). Optimal test characteristics (sensitivity: 72% [95% CI: 59, 82] and specificity: 72% [95%CI: 63, 80]) were observed when peak tryptase concentrations were >11.4 ng/mL and/or delta-tryptase ≥2.0 ng/mL. For hypotensive patients, peak tryptase >11.4 ng/mL had improved test characteristics (sensitivity: 85% [95% CI: 65, 96] and specificity: 92% [95% CI: 85, 97]); the use of delta-tryptase reduced test specificity. CONCLUSION: While peak and delta tryptase concentrations were higher in anaphylaxis than other forms of critical illness, the test lacks sufficient sensitivity and specificity. Therefore, mast cell tryptase values alone cannot be used to establish the diagnosis of anaphylaxis in the ED. In particular, tryptase has limited utility for differentiating anaphylactic from non-anaphylactic shock.

Characterisation of subpopulations of myeloid cells in infantile haemangioma.

AIMS: Cells expressing markers of mast cells, macrophages and dendritic cells have previously been demonstrated within the interstitium of infantile haemangioma (IH). This study characterised these myeloid cellular subpopulations within IH. METHODS: Immunohistochemical staining was performed on proliferating and involuted IHs for the expression of Nanog, tryptase, CD163, DC-SIGN and CD45. The presence of mRNA corresponding to Nanog, tryptase α/ß-1, tryptase ß-2, CD163 and DC-SIGN was confirmed by NanoString and RT-PCR in snap-frozen IH tissues. RESULTS: Immunohistochemical staining showed expression of Nanog by interstitial phenotypical mast cells within proliferating IH, which were separate from the interstitial M2-polarised macrophages that also expressed DC-SIGN, a dendritic cell marker. These two myeloid cellular subpopulations in IH did not express the pan-haematopoietic marker, CD45. CONCLUSIONS: There are two interstitial subpopulations of myeloid cells within IH: phenotypical mast cells which also express Nanog, indicating a primitive phenotype; and M2-polarised macrophages which also express DC-SIGN.

High-sensitivity flow cytometric analysis of mast cell clustering in systemic mastocytosis: a quantitative and statistical analysis.

We characterized recently identified event clusters in systemic mastocytosis (SM) by calculating the coefficient of variation (CV, %) of CD117 and side scatter (SSC), based on the mean fluorescence intensity of mast cells using flow cytometry. Seventy-five samples were from patients with SM and 124 samples from patients negative for SM (non-SM). Discrete cluster formation seen in 50 cases correlated with significantly lower CV for SSC (46.1% vs. 61.0%, p < 0.0001) and CD117 (64.5% vs. 80.5%, p < 0.0001) for SM vs. non-SM samples. A combined CVCD117 + SSC of < 125 showed a sensitivity of 80% and specificity of 80% for SM with PPV of 67% and NPV of 80%. Probability scores of having SM, generated based on CVs for SSC and CD117, were significantly higher in patients with SM than non-SM (0.55 vs. 0.17, respectively; p < 0.001). Flow cytometric-based quantitative analysis of event clustering is a useful approach for diagnosing and monitoring patients with SM.

The role of mast cells in atherosclerosis.

Rupture of an atherosclerotic plaque is the major underlying cause of adverse cardiovascular events such as myocardial infarction or stroke. Therapeutic interventions should therefore be directed towards inhibiting growth of atherosclerotic lesions as well as towards prevention of lesion destabilization. Interestingly, the presence of mast cells has been demonstrated in both murine and human plaques, and multiple interventional murine studies have pointed out a direct role for mast cells in early and late stages of atherosclerosis. Moreover, it has recently been described that activated lesional mast cells correlate with major cardiovascular events in patients suffering from cardiovascular disease. This review focuses on the effect of different mast cell derived mediators in atherogenesis and in late stage plaque destabilization. Also, possible ligands for mast cell activation in the context of atherosclerosis are discussed. Finally, we will elaborate on the predictive value of mast cells, together with therapeutic implications, in cardiovascular disease.

Soluble CD14 is essential for lipopolysaccharide-dependent activation of human intestinal mast cells from macroscopically normal as well as Crohn's disease tissue.

Mast cells are now considered sentinels in immunity. Given their location underneath the gastrointestinal barrier, mast cells are entrusted with the task of tolerating commensal microorganisms and eliminating potential pathogens in the gut microbiota. The aim of our study was to analyse the responsiveness of mast cells isolated from macroscopically normal and Crohn's disease-affected intestine to lipopolysaccharide (LPS). To determine the LPS-mediated signalling, human intestinal mast cells were treated with LPS alone or in combination with soluble CD14 due to their lack of surface CD14 expression. LPS alone failed to stimulate cytokine expression in human intestinal mast cells from both macroscopically normal and Crohn's disease tissue. Upon administration of LPS and soluble CD14, there was a dose- and time-dependent induction of cytokine and chemokine expression. Moreover, CXCL8 and interleukin-1ß protein expression was induced in response to activation with LPS plus soluble CD14. Expression of cytokines and chemokines was at similar levels in mast cells from macroscopically normal and Crohn's disease-affected intestine after LPS/soluble CD14 treatment. In conclusion, human intestinal mast cells appear to tolerate LPS per se. The LPS-mediated activation in mast cells may be provoked by soluble CD14 distributed by other LPS-triggered cells at the gastrointestinal barrier.

Mast cells and IgE: from history to today.

Role of mast cells in allergy had remained undetermined until the discovery of IgE in 1966. Then, IgE purified from many Liters of plasma, which had been donated from a patient with fatal myeloma, was distributed to researchers all over the world, and thus accelerated exploring the mechanisms involved in allergic reactions, particularly about the role of mast cells and basophils in the IgE-mediated reactions. Identification of mast cells as a progeny of a bone marrow hematopoietic stem cell in 1977 led us to successful in vitro culture of human mast cells. Along with the development of molecular biological techniques, the structure of the high affinity IgE receptor (FcεRI) was determined in 1989. These findings and subsequent investigations brought deeper understanding of IgE-mediated allergic diseases in the past half century, especially where mast cells are involved. We have now even obtained the information about whole genome expression of FcεRI-dependently activated mast cells. In sharp contrast to our comprehension of allergic diseases where IgE and mast cells are involved, the mechanisms involved in non-IgE-mediated allergic diseases or non-IgE-mediated phase of IgE-mediated diseases are almost left unsolved and are waiting for devoted investigators to reveal it.

Macrophage imbalance (M1 vs. M2) and upregulation of mast cells in wall of ruptured human cerebral aneurysms: preliminary results.

BACKGROUND: M1 and M2 cells are two major subsets of human macrophages that exert opposite effects on the inflammatory response. This study aims to investigate the role of macrophage M1/M2 imbalance and mast cells in the progression of human cerebral aneurysms to rupture. METHODS: Ten patients with cerebral aneurysms (five ruptured and five unruptured) underwent microsurgical clipping. During the procedure, a segment of the aneurysm dome was resected and immunostained with monoclonal antibodies for M1 cells (anti-HLA DR), M2 cells (anti-CD 163), and mast cells (anti-tryptase clone AA). A segment of the superficial temporal artery (STA) was also removed and immunostained with monoclonal antibodies for M1, M2, and mast cells. RESULTS: All ten aneurysm tissues stained positive for M1, M2, and mast cells. M1 and M2 cells were present in equal proportions in unruptured aneurysms. This contrasted with a marked predominance of M1 over M2 cells in ruptured aneurysms (p = 0.045). Mast cells were also prominently upregulated in ruptured aneurysms (p = 0.001). Few M1 and M2 cells were present in STA samples. CONCLUSIONS: M1/M2 macrophages and mast cells are found in human cerebral aneurysms; however, M1 and mast cell expression seems to markedly increase in ruptured aneurysms. These findings suggest that macrophage M1/M2 imbalance and upregulation of mast cells may have a role in the progression of cerebral aneurysms to rupture.

Revisión acerca de la biología y las funciones inmunológicas de los mastocitos / Review of the biology and immunological functions of mast cells

Los mastocitos o células cebadas son células que se hallan ampliamente distribuidas en todos los tejidos, principalmente en la piel y en las superficies mucosas cerca de los vasos sanguíneos y linfáticos. Pueden ser activadas por diversos estímulos de origen inmunitario o no inmunitario, liberando un amplio espectro de mediadores que incluyen histamina, proteasas, citoquinas, factores de crecimiento y metabolitos del ácido araquidónico. Estas moléculas juegan un importante papel en numerosos procesos fisiológicos y patológicos. La función mejor conocida de los mastocitos es la defensa contra infestaciones parasitarias; sin embargo, son importantes en la defensa del huésped a través de su participación en la inmunidad innata y adaptativa. Median la respuesta inflamatoria, la remodelación de los tejidos y la angiogénesis. Intervienen en las reacciones de hipersensibilidad tipo I y tienen un papel contradictorio en la progresión tumoral. En esta revisión se analiza el origen, la distribución y las funciones de los mastocitos en condiciones fisiológicas y en distintas enfermedades humanas.

Mast cells and matrix metalloproteinase 9 expression in actinic cheilitis and lip squamous cell carcinoma.

OBJECTIVES: The aim of this study was to evaluate mast cell (MC) density and migration and their association with matrix metalloproteinase (MMP) 9 expression in squamous cell carcinoma (SCC) and actinic cheilitis (AC). STUDY DESIGN: Tryptase, c-Kit, and MMP-9 expression was evaluated in 20 cases of SCC, 20 cases of AC, and 7 cases of normal lip (control samples) by immunohistochemistry techniques. RESULTS: Tryptase(+) and c-Kit(+) MC densities were significantly higher in SCCs than in ACs and control samples (P < .001). However, no significant difference was found when comparing tryptase(+) and c-Kit(+) MC densities between ACs and control samples (P values .185 and .516, respectively). MMP-9 was strongly expressed in SCCs and moderately expressed in ACs and control samples. A highly significant association was found between tryptase(+) MC density and the expression of MMP-9 (P < .001). CONCLUSIONS: The increase in MC density associated with the strong expression of MMP-9 may favor SCC progression.

Phenotypical characterization, distribution and quantification of different mast cell subtypes in transmural biopsies from the gastrointestinal tract of cats with inflammatory bowel disease.

In this study subtypes, distribution and number of mast cells were investigated within mucosa and submucosa of the gastrointestinal tract of 24 cats with inflammatory bowel disease (IBD) in comparison to 11 control cats. Paraffin sections of formalin-fixed transmural gastrointestinal biopsies from stomach, duodenum, jejunum, ileum and colon were examined. Mast cells were phenotyped and quantified based on their chymase and tryptase content, by applying a combined enzyme-histochemical and immunohistochemical double-labeling technique and on their heparin content by a metachromatic staining method (kresylecht-violet, MC(KEV)). Mast cells containing both chymase and tryptase were not found in any of the samples examined. Furthermore, in the stomach neither chymase (MC(C)) nor tryptase (MC(T)) bearing mast cells were detected. In cats with lymphocytic-plasmacytic enteritis or enterocolitis elevated numbers of MC(T) or MC(C) were identified in comparison to controls mainly located in the inflamed segments. The highest quantity of MC(C) was found in cats with eosinophilic gastroenterocolitis or enterocolitis in comparison to other IBD forms, but only minor numbers of MC(T) were detected in these cases. In cats with fibrosing enteropathy (FE) a decrease of MC(C) and mast cells containing heparin was detected in affected segments, while increased numbers of MC(T) were detected in all locations. The elevation in the number of MC(T) was higher in unaffected areas than in fibrotic regions. Regarding all IBD cases higher counts of MC(C) were found especially in the inflamed locations, whereas in unaffected segments increased numbers of MC(T) were detected. The clear predominance of MC(C) and MC(T) within the mucosa and of MC(KEV) within the submucosa of all cats examined possibly represents differences of the cytokine milieu within the intestinal layers. In FE, mast cells are possibly pivotal for the containment of the inflammatory process because of their antiinflammatory properties. The results of this study indicate that mast cells and their mediators are involved in the pathogenesis of different IBD forms in cats.

Advances in mast cell biology: new understanding of heterogeneity and function.

Mast cells are classically viewed as effector cells of IgE-mediated allergic diseases. However, over the last decade our understanding has been enriched about their roles in host defense, innate and adaptive immune responses, and in homeostatic responses, angiogenesis, wound healing, tissue remodeling, and immunoregulation. Despite impressive progress, there are large gaps in our understanding of their phenotypic heterogeneity, regulatory mechanisms involved, and functional significance. This review summarizes our knowledge of mast cells in innate and acquired immunity, allergic inflammation and tissue homeostasis, as well as some of the regulatory mechanisms that control mast cell development, phenotypic determination, and function, particularly in the context of mucosal surfaces.

The fundamental contribution of William Bate Hardy to shape the concept of mast cell heterogeneity.

This review article acknowledges the pioneering contribution of William Bate Hardy in shaping the concept of mast cell heterogeneity. In two outstanding papers, published in 1894 and 1895, he focussed on the 'wandering cells' (the modern leucocytes) in different mammalian species and distinguished two types of granular basophil cells, i.e., the coarsely granular basophil cells and the splanchnic basophil cells. These corresponded to the populations of connective tissue-type and mucosal mast cells, respectively, described 70 years later by Enerbäck in rodents. Among the coarsely granular basophil cells, he also differentiated those cells which populated the serosal cavities - the so-called coelomic coarsely granular basophil cells - from the common coarsely granular basophil cells, which were localized in the connective tissues. He stated that the granular basophil cells presented with different morphological and histochemical characteristics in diverse animal species as well as at different anatomical sites. Remarkably, he performed a series of functional experiments on the basophil cells as well as the other wandering cells, and suggested the view that different granular basophil cells might express functional specializations.

Histochemical method for demonstrating quail mast cell types simultaneously.

The development and application of selective staining methods for routine detection of mast cells are of considerable interest, because these cells play an important role in health and disease. The composition of cytoplasmic mast cell granules depends on the species and type of mast cell. The study reported here was conducted to investigate the combined use of aldehyde fuchsin (AF) and the Alcian blue-critical electrolyte concentration (AB-CEC) (pH 5.8, 0.3 M MgCl(2)) techniques for differentiating avian mast cell subtypes. Tissue samples from skin, intestines, and lungs of six healthy adult quail and two control rats were fixed in Carnoy's solution and 10% formolin for routine histological processing. To determine the staining properties of sulfated glycosaminoglycans (GAGs), a three-step staining technique was applied using berberine sulfate, AF, and AB-CEC. In quail, AF positivity following application of the AB-CEC technique was found only in the lungs, mostly in cells that gave a berberine sulfate-positive reaction, and this positivity was determined to be localized particularly in the nucleus and perinuclear cytoplasm. In other regions, the pale AF staining of cells that did not emit fluorescence when stained with berberine sulfate was determined to be replaced by a blue color after application of AB-CEC. The AF/AB-CEC (pH 5.8, 0.3 M MgCl(2)) technique demonstrated that rat and quail mast cells varied in both GAG types and their distribution within the cell. Especially in avian species, this technique can be applied to distinguish mast cells according to their GAG content. It can be used as an alternative to the AB/safranin O staining procedure for differentiating mast cells that contain and lack heparin.

Novel site-specific mast cell subpopulations in the human lung.

BACKGROUND: Lung mast cells are stereotypically divided into connective tissue (MC(TC)) and mucosal (MC(T)) mast cells. This study tests the hypothesis that each of these subtypes can be divided further into site-specific populations created by the microenvironment within each anatomical lung compartment. METHODS: Surgical resections and bronchial and transbronchial biopsies from non-smoking individuals were obtained to study mast cells under non-inflamed conditions. Morphometric and molecular characteristics of mast cell populations were investigated in multiple lung structures by immunohistochemistry and electron microscopy. RESULTS: MC(T) and MC(TC) coexisted in all compartments, with MC(T) being the prevailing type in bronchi, bronchioles and the alveolar parenchyma and MC(TC) being more abundant in pulmonary vessels and the pleura. Each of the MC(TC) and MC(T) phenotypes could be further differentiated into site-specific populations. MC(TC) were significantly larger in pulmonary vessels than in small airway walls, while the reverse was observed for MC(T). Within each MC(TC) and MC(T) population there were also distinct site-specific expression patterns of the IgE receptor, interleukin-9 receptor, renin, histidine decarboxylase, vascular endothelial growth factor, fibroblast growth factor, 5-lipoxygenase and leukotriene C4 synthase (eg, bronchial MC(T) consistently expressed more histidine decarboxylase than alveolar MC(T)). Renin content was high in vascular MC(TC) but markedly lower in MC(TC) in other compartments. For both MC(TC) and MC(T), the IgE receptor was highly expressed in conducting airways but virtually absent in alveolar parenchyma. CONCLUSIONS: These findings demonstrate novel site-specific subpopulations of lung MC(TC) and MC(T) at baseline conditions. This observation may have important implications in the future exploration of mast cells in a number of pulmonary diseases.

Mastocytosis: a rare case of anaphylaxis in paediatric age and literature review

The term "mastocytosis" denotes a heterogeneous group of disorders characterised by abnormal growth and accumulation of mast cells (MC) in one or more organ systems. Symptoms result from MC chemical mediator's release, pathologic infiltration of neoplastic MC in tissues or both. Multiple molecular, genetic and chromosomal defects seem to contribute to an autonomous growth, but somatic c-kit D816V mutation is more frequently encountered, especially in systemic disease. We present a literature review of mastocytosis and a rare case report of an 18 month-old-girl with a bullous dermatosis, respiratory distress and anaphylaxis, as clinical manifestations of mastocytosis. The developments of accepted classification systems and novel useful markers allowed a re-evaluation and updating of the classification of mastocytosis. In paediatric age cutaneous forms of disease prevail and may regress spontaneously. SM is more frequently diagnosed in adults and is a persistent (clonal) disease of bone marrow. The clinical course in these patients is variable. Today diagnostic criteria for each disease variant are reasonably well defined. There are, however, peculiarities, namely in paediatric age, that makes the diagnostic approach difficult. Systemic disease may pose differential diagnostic problems resulting from multiple organ systems involvement. Coversly, the "unexplained" appearance of those symptoms with no skin lesions should raise the suspicion of MC disease. This case is reported in order to stress the clinical severity and difficult diagnostic approach that paediatric mastocytosis may assume

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Cutaneous mast cell receptors.

Two types of mast cells, MC(T) and MC(TC), exist in humans. MC(T) and MC(TC) are different in their granular neutral proteases, tissue localizations, and functions. This article describes the differences between the cutaneous mast cell receptors.

Characterization of mast cell numbers and subtypes in biopsies from the gastrointestinal tract of dogs with lymphocytic-plasmacytic or eosinophilic gastroenterocolitis.

It has been suggested but not proven that hypersensitivity type I reactions are involved in the pathogenesis of canine inflammatory bowel disease (IBD). The main effector cells in type I hypersensitivity reactions are mast cells (MCs). Canine MCs, as human MCs, can be subdivided into three subtypes according to their content of mast cell-specific proteases: tryptase (MCT), chymase (MCC), or tryptase and chymase bearing MCs (MCTC). In this study, numbers and subsets of mast cells were investigated in biopsies from the gastrointestinal tract of dogs with histopathologically confirmed lymphocytic-plasmacytic enteritis (LPE) (n=4), lymphocytic-plasmacytic colitis (LPC) (n=1) and eosinophilic gastroenterocolitis (EGE) (n=11). Paraffin sections of formalin-fixed samples from the stomach, small intestine (duodenum, jejunum, ileum) and colon were stained by using a metachromatic staining method (kresylecht-violet; KEV) and a combined enzyme histochemical and immunohistochemical technique for chymase and tryptase. Additionally, immunohistochemistry with antibodies against T cells (CD3), macrophages (myeloid/histiocyte antigen) and IgA, IgG and IgM bearing cells was conducted. Quantitative evaluation of mast cells and semiquantitative scoring of immunohistochemically stained cells were performed. Between the two histopathologically defined groups clear differences concerning mast cell numbers were detected. In most affected intestinal tissue locations of dogs with LPE/LPC a decrease in metachromatically (kresylecht-violet) stained granule-containing MCs and immunohistochemically stained MCT,C,TC was found. This reduction could be due to mast cell degranulation, a T helper cell 1 dominated reaction pattern or a "thinning out" due to increasing T cells, IgA and IgG bearing cells. Dogs with EGE displayed higher variability in mast cell numbers but most of the affected large and small intestinal locations had increased numbers of MCs. In these cases, T cells, IgA bearing cells and macrophages also increased. Increased numbers of MCs and eosinophils seen in the intestinal mucosa of dogs with EGE could indicate the presence of a type I hypersensitivity reaction (T helper cell 2 pattern) in response to dietary antigens. Changes in cell numbers occurred also in unaffected locations of dogs with LPE/LPC and EGE which showed reduced MCT,C,TC, increased KEV positive cells and partially increased leucocytes and macrophages.

Submucosal connective tissue-type mast cells contribute to the production of lysophosphatidic acid (LPA) in the gastrointestinal tract through the secretion of autotaxin (ATX)/lysophospholipase D (lysoPLD).

Lysophosphatidic acid (LPA) is involved in a broad spectrum of biological activities, including wound healing and cancer metastasis. Autotaxin (ATX), originally isolated from a melanoma supernatant as a tumor cell motility-stimulating factor, has been shown to be molecularly identical to lysophospholipase D (lysoPLD), which is the main enzyme in the production of LPA. Although ATX/lysoPLD is known to be widely expressed in normal human tissues, the exact distribution of ATX-producing cells has not been fully investigated. In this study, we evaluated ATX/lysoPLD expression by immunohistochemical staining using a rat anti-ATX mAb in the human gastrointestinal tract and found that submucosal mast cells (MC) highly expressed this enzyme. This was confirmed by immunofluorescent double staining using mAbs to tryptase and chymase. Then, we isolated MC from human gastric tissue by an immunomagnetic method using CD117-microbeads and showed that a subpopulation of CD203c-positive MC showed positive staining for intracellular ATX/lysoPLD on flowcytometry. This was confirmed by Western blotting of the isolated cells. Moreover, a significant level of ATX/lysoPLD release could be detected in the culture supernatants of human MC by Western blot analysis. Our data suggest that submucosal MC play significant roles in various aspects of pathophysiology in the gastrointestinal tract by locally providing bioactive LPA through the production of ATX/lysoPLD.