Identification of alpha-gal sensitivity in patients with a diagnosis of idiopathic anaphylaxis.

IgE antibodies (Ab) specific to galactose-α-1,3-galactose (alpha-gal) are responsible for a delayed form of anaphylaxis that occurs 3-6 hours after red meat ingestion. In a unique prospective study of seventy participants referred with a diagnosis of idiopathic anaphylaxis (IA), six (9%) were found to have IgE to alpha-gal. Upon institution of a diet free of red meat, all patients had no further episodes of anaphylaxis. Two of these individuals had indolent systemic mastocytosis (ISM). Those with ISM had more severe clinical reactions but lower specific IgE to alpha-gal and higher serum tryptase levels, reflective of the mast cell burden. The identification of alpha-gal syndrome in patients with IA supports the need for routine screening for this sensitivity as a cause of anaphylaxis, where reactions to alpha-gal are delayed and thus may be overlooked.

Development and validation of the mastocytosis quality of life questionnaire: MC-QoL.

BACKGROUND: Mastocytosis is a heterogeneous disease characterized by a clonal expansion of mast cells in various organs. The vast majority of patients affected suffer from signs and symptoms caused by mediator release from mast cells. Although the disease burden is high, there is currently no specific instrument to measure health-related quality of life (HRQoL) impairment in patients with mastocytosis. OBJECTIVE: The aim of this study was to develop and validate a disease-specific tool to assess HRQoL impairment in patients with cutaneous and indolent systemic mastocytosis, the Mastocytosis Quality of Life Questionnaire (MC-QoL). METHODS: Sixty-two potential MC-QoL items were developed in a combined approach consisting of semi-structured patient interviews, expert input and literature research. Item selection was performed by impact analysis with 76 patients and a final review for face validity. The resulting MC-QoL was tested for validity, reliability and influence factors. In parallel, an US American-English version of the MC-QoL was developed. RESULTS: A total of 158 patients (41 CM, 41 MIS and 76 ISM) took part in the MC-QoL validation study. The final 27-item questionnaire was found to have a four-domain structure ('symptoms', 'emotions', 'social life/functioning' and 'skin'), a valid total score and an excellent test-retest reliability. Multiple regression analysis revealed disease duration, but not age, gender or skin involvement to be a significant determinant of HRQoL impairment in mastocytosis. CONCLUSIONS: The MC-QoL is the first disease-specific HRQoL questionnaire for adult patients with cutaneous and indolent systemic mastocytosis. This short, validated and reliable instrument will serve as a valuable tool in future clinical studies and in routine patient care.

Refined diagnostic criteria and classification of mast cell leukemia (MCL) and myelomastocytic leukemia (MML): a consensus proposal.

Mast cell leukemia (MCL), the leukemic manifestation of systemic mastocytosis (SM), is characterized by leukemic expansion of immature mast cells (MCs) in the bone marrow (BM) and other internal organs; and a poor prognosis. In a subset of patients, circulating MCs are detectable. A major differential diagnosis to MCL is myelomastocytic leukemia (MML). Although criteria for both MCL and MML have been published, several questions remain concerning terminologies and subvariants. To discuss open issues, the EU/US-consensus group and the European Competence Network on Mastocytosis (ECNM) launched a series of meetings and workshops in 2011-2013. Resulting discussions and outcomes are provided in this article. The group recommends that MML be recognized as a distinct condition defined by mastocytic differentiation in advanced myeloid neoplasms without evidence of SM. The group also proposes that MCL be divided into acute MCL and chronic MCL, based on the presence or absence of C-Findings. In addition, a primary (de novo) form of MCL should be separated from secondary MCL that typically develops in the presence of a known antecedent MC neoplasm, usually aggressive SM (ASM) or MC sarcoma. For MCL, an imminent prephase is also proposed. This prephase represents ASM with rapid progression and 5%-19% MCs in BM smears, which is generally accepted to be of prognostic significance. We recommend that this condition be termed ASM in transformation to MCL (ASM-t). The refined classification of MCL fits within and extends the current WHO classification; and should improve prognostication and patient selection in practice as well as in clinical trials.

Proposed diagnostic algorithm for patients with suspected mastocytosis: a proposal of the European Competence Network on Mastocytosis.

Mastocytosis is an emerging differential diagnosis in patients with more or less specific mediator-related symptoms. In some of these patients, typical skin lesions are found and the diagnosis of mastocytosis can be established. In other cases, however, skin lesions are absent, which represents a diagnostic challenge. In the light of this unmet need, we developed a diagnostic algorithm for patients with suspected mastocytosis. In adult patients with typical lesions of mastocytosis in the skin, a bone marrow (BM) biopsy should be considered, regardless of the basal serum tryptase concentration. In adults without skin lesions who suffer from mediator-related or other typical symptoms, the basal tryptase level is an important parameter. In those with a slightly increased tryptase level, additional investigations, including a sensitive KIT mutation analysis of blood leucocytes or measurement of urinary histamine metabolites, may be helpful. In adult patients in whom (i) KIT D816V is detected and/or (ii) the basal serum tryptase level is clearly increased (>25-30 ng/ml) and/or (iii) other clinical or laboratory features suggest the presence of 'occult' mastocytosis or another haematologic neoplasm, a BM investigation is recommended. In the absence of KIT D816V and other signs or symptoms of mastocytosis or another haematopoietic disease, no BM investigation is required, but the clinical course and tryptase levels are monitored in the follow-up. In paediatric patients, a BM investigation is usually not required, even if the tryptase level is increased. Although validation is required, it can be expected that the algorithm proposed herein will facilitate the management of patients with suspected mastocytosis and help avoid unnecessary referrals and investigations.

Bone marrow stromal cells inhibit mast cell function via a COX2-dependent mechanism.

BACKGROUND: Mast cells (MCs) have a central role in the induction of allergic inflammation, such as seen in asthma, and contribute to the severity of certain autoimmune diseases, such as rheumatoid arthritis. The MC thus represents an important inflammatory cell, and one which has resisted therapeutic attempts to alter its role in disease. OBJECTIVE: Because bone marrow-derived stromal cells (BMSC, also known as mesenchymal stem cells or MSCs) have been reported to alter allergic inflammation in vivo, we chose to study the interaction between mouse BMSC and mouse bone marrow-derived MCs. METHODS: MC degranulation, cytokine production and chemotaxis were evaluated in vitro following co-culture with BMSCs either in cell contact or a transwell. In addition, MC degranulation was assessed in vivo following administration of BMSCs in a model of passive cutaneous anaphylaxis and a peritoneal degranulation assay. Mechanisms of MC suppression by BMSCs were determined through use of inhibitors or antibodies to COX1, COX2, nitric oxide, indoleamine 2, 3-dioxygenase, EP1-4 receptors, TGF-ß and IL-10. Lastly, we utilized either BMSCs or MCs deficient in COX1, COX2 or EP1-4 receptors to confirm the mechanisms of inhibition of MC function by BMSCs. RESULTS: We discovered that BMSCs will effectively suppress specific MC functions in vitro as well as in vivo. When MCs are cocultured with BMSCs to allow cell-to-cell contact, BMSCs suppressed MC degranulation, pro-inflammatory cytokine production, chemokinesis and chemotaxis. Similarly, MC degranulation within mouse skin or the peritoneal cavity was suppressed following in vivo administration of BMSCs. Further, we found that these inhibitory effects were dependent on up-regulation of COX2 in BMSCs; and were facilitated through the activation of EP4 receptors on MCs. CONCLUSION AND CLINICAL RELEVANCE: These observations support the concept that BMSCs have the ability to suppress MC activation and therefore could be the basis for a novel cell based therapeutic approach in the treatment of MC driven inflammatory diseases.

The mast cell and allergic diseases: role in pathogenesis and implications for therapy.

Mast cells have long been recognized for their role in the genesis of allergic inflammation; and more recently for their participation in innate and acquired immune responses. Mast cells reside within tissues including the skin and mucosal membranes, which interface with the external environment; as well as being found within vascularized tissues next to nerves, blood vessels and glandular structures. Mast cells have the capability of reacting both within minutes and over hours to specific stimuli, with local and systemic effects. Mast cells express the high affinity IgE receptor (FcepsilonRI) and upon aggregation of FcepsilonRI by allergen-specific IgE, mast cells release and generate biologically active preformed and newly synthesized mediators which are involved in many aspects of allergic inflammation. While mast cells have been well documented to be essential for acute allergic reactions, more recently the importance of mast cells in reacting through pattern recognition receptors in innate immune responses has become recognized. Moreover, as our molecular understanding of the mast cell has evolved, novel targets for modulation have been identified with promising therapeutic potential.

Clinical correlates of blood serotonin levels in patients with mastocytosis.

BACKGROUND: Mastocytosis is a clonal disorder associated with an increased mast cell burden. We have recently demonstrated the ability of human mast cells to express and be activated through multiple serotonin receptors; to synthesize and release serotonin; and that mastocytosis patients may have abnormal serotonin levels. As serotonin has been implicated in the genesis of clinical symptoms found in association with some chronic diseases, we have now determined the whole blood serotonin levels in 29 patients diagnosed with mastocytosis, and correlated these levels with multiple clinical and laboratory parameters. MATERIALS AND METHODS: Patients with mastocytosis were categorized according to disease variant. Blood serotonin values were determined and correlated with values reported for normal subjects; and clinical and laboratory features of the disease. RESULTS: Total blood serotonin levels followed a bimodal distribution in line with our earlier report, unlike the normal distribution reported for normal individuals. Serotonin levels did not correlate with platelet numbers, liver function tests or serum tryptase levels. Patients with lower serotonin values had greater rates of fatigue (P = 0.0001), migraine headaches (P = 0.0028), psychiatric symptoms (P = 0.0001), diarrhoea (P = 0.0407), flushing (0.0085), and abdominal and bone pain (P = 0.0001). CONCLUSIONS: Our study suggests that low blood serotonin levels help define a sub-group of patients with mastocytosis that are more likely to present with neurological and gastrointestinal complaints, and suggests that the use of pharmacologic agents that alter blood serotonin levels could be explored in selected patients.

Practical allergy (PRACTALL) report: risk assessment in anaphylaxis.

Effector mechanisms in anaphylaxis were reviewed. Current approaches to confirmation of the clinical diagnosis were discussed. Improved methods for distinguishing between allergen sensitization (which is common in the general population) and clinical risk of anaphylaxis (which is uncommon) were deliberated. Innovative techniques that will improve risk assessment in anaphylaxis in the future were described.

Glucocorticoids decrease tissue mast cell number by reducing the production of the c-kit ligand, stem cell factor, by resident cells: in vitro and in vivo evidence in murine systems.

The local delivery of glucocorticoids to tissues significantly decreases mast cell number. This pharmacologic effect of glucocorticoids is believed to be one of the mechanisms by which glucocorticoids regulate allergic inflammation. To determine the mechanism by which glucocorticoids are able to exert this effect, we first applied the glucocorticoid fluocinonide to mouse dermis and observed that the decrease in mast cell number was associated with an increase in mast cell apoptosis. This did not appear to be due to a direct effect of the glucocorticoid on mast cells, as the addition of 0.01-1.0 microM of the glucocorticoid dexamethasone into stem cell factor (SCF)-dependent mast cell cultures did not enhance mast cell death. However, addition of dexamethasone to cultured fibroblasts did result in a downregulation of SCF mRNA and a significant decrease in SCF protein production. Similarly, immunohistochemistry performed on fluocinonide-treated mouse dermis revealed a decrease in immunoreactive SCF. Administration of SCF at sites of fluocinonide administration to the dermis abolished the mast cell-depleting effect of this glucocorticoid. Thus, glucocorticoids decrease tissue mast cell number by downregulating tissue SCF production required for the survival of local mast cells. This observation may be applicable to the design of improved strategies to treat mast cell-mediated disorders.

Early expression of high-affinity receptor for immunoglobulin E (Fc epsilon RI) during differentiation of mouse mast cells and human basophils.

Immediate hypersensitivity is due to the release of mediators from mast cells and basophils after the crosslinking of Fc epsilon RI. The appearance of such receptors was examined during differentiation of human and mouse bone marrow cells cultured in the presence of IL-3. As already reported, mouse bone marrow yield cultures of greater than 95% mast cells by 3 wk, whereas human bone marrow develop into cultures comprising 25% basophils by 3 wk. Here we show that transcripts for Fc epsilon RI subunits and membrane-associated receptors are apparent by 1 wk in both human and murine IL-3-dependent bone marrow cells. These cells contain few, if any, granules. The expression of transcripts and the number of receptor-positive cells continue to increase over 3 wk of culture. In parallel, a progressively larger number of cells become increasingly granulated to finally resemble either basophils or mast cells. Mature peripheral human basophils also contain transcripts for Fc epsilon RI and, therefore, may have the potential to synthesize de novo receptors. The early appearance of Fc epsilon FI during cell differentiation may be important for these cells to respond to IgE-mediated stimuli before granulation. The physiologic role of Fc epsilon RI could be to mediate lymphokine production (IL-3, IL-4, IL-6, and granulocyte/macrophage colony-stimulating factor) without inducing cellular degranulation.

Murine mast cells synthesize basement membrane components. A potential role in early fibrosis.

Mast cells are resident in tissues, particularly in association with endothelial and epithelial cell basement membranes, and increase at sites of inflammation, injury, and fibrosis. Although mast cells are known to both release and generate proinflammatory molecules in response to inflammatory stimuli, little is known about their normal biologic function. Here we demonstrate that IL-3-dependent mouse PT18 mast cells, mouse bone marrow-derived mast cells, and rat basophilic leukemia cells express large amounts of mRNA for collagen IV, laminin, and heparan sulfate proteoglycan. Western blot analysis confirmed that mast cells synthesize and secrete significant amounts collagen IV and laminin B1 and B2 chains. These data suggest that mast cells may contribute to normal tissue repair and/or the early overproduction of basement membrane components seen in a variety of fibrotic conditions.

The alpha form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis.

Tryptase, a protease produced by all mast cells, was evaluated as a clinical marker of systemic mastocytosis. Two sandwich immunoassays were evaluated, one which used the mAb G5 for capture, the other which used B12 for capture. The B12 capture assay measured both recombinant alpha- and beta-tryptase, whereas the G5 capture assay measured primarily recombinant beta-tryptase. G5 binds with low affinity to both recombinant alpha-tryptase and tryptase in blood from normal and nonacute mastocytosis subjects, and binds with high affinity to recombinant beta-tryptase, tryptase in serum during anaphylaxis, and tryptase stored in mast cell secretory granules. B12 recognizes all of these forms of tryptase with high affinity. As reported previously, during systemic anaphylaxis in patients without known mastocytosis, the ratio of B12- to G5-measured tryptase was always < 5 and approached unity (Schwartz L.B., T.R. Bradford, C. Rouse, A.-M. Irani, G. Rasp, J.K. Van der Zwan and P.-W.G. Van der Linden, J. Clin. Immunol. 14:190-204). In this report, most mastocytosis patients with systemic disease have B12-measured tryptase levels that are elevated (> 20 ng/ml) and are at least 10-fold greater than the corresponding G5-measured tryptase level. Most of those subjects with B12-measured tryptase levels of < 20 ng/ml had only cutaneous manifestations. The B12 assay for alpha-tryptase and beta-tryptase, particularly when performed in conjunction with the G5 assay for beta-tryptase, provides a more precise measure of mast cell involvement than currently available assessments, a promising potential screening test for systemic mastocytosis and may provide an improved means to follow disease progression and response to therapy.

Isolation and characterization of heparin from human lung.

Heparin as measured by azure A metachromasia and anticoagulant activity has been extracted with 1 M NaCl from (35)S-labeled human lung fragments or dispersed human lung cells enriched for mast cells. The (35)S-labeled metachromatic material in the 3 M NaCl eluate from Dowex-1 chromatography of the extract from lung fragments exhibited an average mol wt of 20,000 by Sepharose 4B gel filtration. The (35)S-labeled metachromatic material with the charge characteristics of commercial porcine heparin on DEAE cellulose chromatography was entirely heparin by the criteria of resistance to degradation by chondroitin ABC lyase and complete degradation by purified heparinase. Antithrombin affinity chromatography of purified heparin with an anticoagulant activity of 137 U/mg, revealed that the one-third that was bound and eluted had a 273 U/mg sp act, whereas the unbound activity was 31 U/mg. Thus, the previously observed heterogeneity of commercial porcine heparin for binding to human antithrombin was also observed with human heparin. The mast cell-enriched human lung cell preparations yielded [(35)S]mucopolysaccharides with an average mol wt of 60,000 by Sepharose 4B gel filtration. Approximately 30% of this fraction was degraded by chondroitin ABC lyase, and the residual 70% was degraded by purified heparinase. When the chondroitin ABC lyase-resistant fraction was subjected to alkali degradation the average mol wt was reduced to 20,000. The calculated human lung mast cell heparin content of 2.4-7.8 mug/10(6) cells gave a ratio to histamine on a weight basis similar to that of intact lung fragments, thereby implying that heparin in the lung fragments was largely restricted to the mast cells.

Structural determinants of the capacity of heparin to inhibit the formation of the human amplification C3 convertase.

The ability of heparin glycosaminoglycan to prevent formation of the properdin-stabilized amplification C3 convertase is independent of antithrombin binding activity and requires substitution of the amino sugar and a degree of oxygen (O)-sulfation which could be on the uronic acid or the amino sugar. Preparations of heparin glycosaminoglycan isolated by different techniques from different species (rat, human, and porcine) exhibited an equivalent capacity to inhibit generation of the amplification C3 convertase. Hyaluronic acid, which is devoid of O-sulfation, had no inhibitory activity; chondroitin 4-sulfate of rat and whale origins, chondroitin 6-sulfate of rat and shark origins, and dermatan sulfate from porcine skin are O-sulfated on the galactosamine and had minimal activity. Porcine heparin glycosaminoglycan, isolated on the basis of affinity for antithrombin III, had no greater anticomplementary activity than porcine glycosaminoglycan, which failed to bind antithrombin III and had essentially no anticoagulant activity. Nitrogen (N)-desulfation of porcine heparin reduced anticomplementary activity to the level of the other sulfated mucopolysaccharides, and both N-resulfation and N-acetylation restored the original activities, thereby indicating a requirement for N-substitution, but not N-sulfation. N-resulfation of N-desulfated and O-desulfated heparin did not restore any activity, thus indicating that O-sulfation and N-substitution represent independent, critical structural requirements for the anticomplementary activity of heparin glycosaminoglycan. Inasmuch as N-desulfated-N-acetylated heparin had no anticoagulant activity, the nature of the N-substitution completely distinguishes the plasma-protein effector pathway that is inhibited.

Food allergy.

Food allergy is now known to encompass a number of distinct clinical entities that follow the ingestion of specific food or food additives. Research continues to shed light on immediate reactions to foods and food protein-induced enterocolitis of newborns and infants. Adverse reactions to food additives remain an area of health concern. Studies of food allergies have entered an era in which improved clinical design is the hallmark of the research and conclusions may now be drawn reliably.

TLR3 activation inhibits human mast cell attachment to fibronectin and vitronectin.

Mast cells are involved in both the genesis of allergic inflammation and in host defense; and reside in tissues where their location and responsiveness is regulated in part by adhesion to extracellular matrix proteins (ECM). We have reported that human mast cells (huMC) express TLR1-7, and 9 and respond to toll-like receptors (TLR) ligands by releasing cytokines and leukotriene C4. To determine if TLR ligation could similarly affect mast cells via an influence on adhesion, we employed huMC; and as substrates, fibronectin (FN) and vitronectin (VN). huMC were thus treated with double-stranded RNA (dsRNA) and adhesion to ECM was quantified. FcvarepsilonRI dependent mast cell degranulation was assessed. Adhesion molecule expression and activation was measured by flow cytometry. Activation of huMC through TLR3 with increasing amounts of polyI:C inhibited mast cell adhesion in a dose-dependent manner. This decrease in adhesion was accompanied by a similar decrease in IgE-mediated mast cell degranulation. Activation of TLR3 on huMC resulted in a change in the conformation of CD29, the receptor for FN, to an inactive form. Thus, TLR3 activation decreases mast cell attachment to VN and FN through an active process and one, which would abrogate mast cell attachment dependent potentiation of IgE-mediated responses.

Proposed diagnostic criteria and classification of basophilic leukemias and related disorders.

Basophils form a distinct cell lineage within the hematopoietic cell family. In various myeloid neoplasms, including chronic myeloid leukemia, basophilia is frequently seen. Acute and chronic basophilic leukemias, albeit rare, have also been described. However, no generally accepted criteria and classification of basophilic leukemias have been presented to date. To address this unmet need, a series of Working Conferences and other meetings were organized between March 2015 and March 2016. The current article provides a summary of consensus statements from these meetings, together with proposed criteria to delineate acute basophilic leukemia (ABL) from chronic basophilic leukemia (CBL) and primary forms of the disease where no preceding myeloid malignancy is detected, from the more common 'secondary' variants. Moreover, the term hyperbasophilia (HB) is proposed for cases with a persistent peripheral basophil count ⩾1000 per µl of blood. This condition, HB, is highly indicative of the presence of an underlying myeloid neoplasm. Therefore, HB is an important checkpoint in the diagnostic algorithm and requires a detailed hematologic investigation. In these patients, an underlying myeloid malignancy is often found and is then labeled with the appendix -baso, whereas primary cases of ABL or CBL are very rare. The criteria and classification proposed in this article should facilitate the diagnosis and management of patients with unexplained basophilia and basophil neoplasms in routine practice, and in clinical studies.

A pilot study of nonmyeloablative allogeneic hematopoietic stem cell transplant for advanced systemic mastocytosis.

Systemic mastocytosis (SM) is a disease characterized by tissue infiltration of neoplastic mast cells originating from hematopoietic stem cells. Patients with advanced SM have a poor prognosis, and there is no mast cell ablative therapy available for most patients who carry an activating point mutation in the c-kit gene. We report results of a prospective study evaluating the safety, engraftment, and possibility of inducing a graft-versus-mast cell (GvMC) effect after allogeneic nonmyeloablative hematopoietic cell transplantation (HCT) from an HLA-identical sibling. Three patients with advanced SM were transplanted. All achieved complete donor T cell chimerism followed by clinical evidence for GvMC effect. However, all patients experienced disease progression with the longest response duration of 39 months. The GvMC effect can be observed after nonmyeloablative HCT with limited efficacy. Effective cytoreductive therapy prior to HCT may be required for long-term disease control and cure.

The isolation, identification and characterization of sulfated glycosaminoglycans synthesized in vitro by human eosinophils.

Human eosinophils were purified to greater than 92% using 16-30% metrizamide gradients, and these cells cultured for up to 72 h in vitro to label sulfated glycosaminoglycans. Over 90% of the sulfated glycosaminoglycan-containing material was extracted in 4 M guanidine HCl and had a hydrodynamic size similar to a glycosaminoglycan marker with an approximate average molecular weight of 60,000. Treatment of this salt-extracted 35S-labeled glycosaminoglycan-containing material with 0.5 M NaOH resulted in a change in mass to approx. 20,000 daltons, suggesting that the larger molecules were proteoglycans with side chains with an approximate molecular weight of 20,000. These salt extracted presumptive 35S-labeled proteoglycans were protease insensitive and behaved in a highly charged fashion on DEAE-cellulose. The composition of 35S-labeled glycosaminoglycans from human eosinophils as identified using selected polysaccharides was 70-81% chondroitin 4-sulfate, 9-12% chondroitin 6-sulfate, and 5-12% dermatan sulfate. The predominance of chondroitin 4-sulfate in human eosinophils is similar to the predominance of chondroitin 4-sulfate in human neutrophils and human platelets.

The mast cell.

The mast cell is the cellular basis for immediate hypersensitivity reactions. The specificity of the immediate hypersensitivity reaction is attributable to IgE molecules fixed to specific membrane receptors which, when stimulated by specific antigen, initiates the process of degranulation of the mast cell. The granules provide three separate sources of biologic activity: performed or primary mediators, newly generated or secondary mediators, and activities associated with the granular matrix. A number of biologic consequences are generated in response to these mediators and these include: increased vascular permeability, vasodilation, smooth muscle spasm, polymorphonuclear leukocyte chemotaxis, stimulation of adenylate and guanylate cyclase, superoxide radical generation, prostaglandin formation, mucous and gastric acid secretion, hypotension, tissue destruction, and mononuclear leukocyte infiltration. This pharmacopia of activities accounts for the clinical aspects of allergic diseases, suggests that the mast cell granule may be involved in the host's defense against parasitic infections, and is compatible with a suggested role of the mast cell as a widely distributed, monocellular endocrine system.