Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation

FcRI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that FcRI also uses a Fyn kinase-dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, FcRI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.

INTRAVASCULAR ANTI-IGE CHALLENGE IN PERFUSED LUNGS - MEDIATOR RELEASE AND VASCULAR PRESSOR-RESPONSE

Intravascular application of goat anti-rabbit immunoglobulin E (IgE) was used to stimulate parenchymal mast cells in situ in perfused rabbit lungs. Sustained pulmonary arterial pressure rise was evoked in the absence of lung vascular permeability increase and lung edema formation. Early prostaglandin (PG) D2 and histamine release into the perfusate was documented, accompanied by more sustained liberation of cysteinyl leukotrienes (LT), LTB4, and PGI2. The quantities of these inflammatory mediators displayed the following order: histamine > cysteinyl-LT > PGI2 > LTB4 > PGD2. Pressor response and inflammatory mediator release revealed corresponding bell-shaped dose dependencies. Cyclooxygenase inhibition (acetylsalicylic acid) suppressed prostanoid generation, increased LT release, and did not substantially affect pressor response and histamine liberation. BW755 C, a cyclo- and lipoxygenase inhibitor, blocked the release of cysteinyl-LT and markedly reduced the liberation of the other inflammatory mediators as well as the pressor response. The H-1-antagonist clemastine caused a moderate reduction of the anti-IgE-provoked pressure rise. We conclude that intravascular anti-IgE challenge in intact lungs provokes the release of an inflammatory mediator profile compatible with in situ lung parenchymal mast cell activation. Pulmonary hypertension represents the predominant vascular response, presumably mediated by cysteinyl-LT and, to a minor extent, histamine liberation.

SOCS3 regulates onset and maintenance of TH2- mediated allergic responses

Members of the suppressor of cytokine signaling (SOCS) family are involved in the pathogenesis of many inflammatory diseases. SOCS-3 is predominantly expressed in T-helper type 2 (TH2) cells, but its role in TH2-related allergic diseases remains to be investigated. In this study we provide a strong correlation between SOCS-3 expression and the pathology of asthma and atopic dermatitis, as well as serum IgE levels in allergic human patients. SOCS-3 transgenic mice showed increased TH2 responses and multiple pathological features characteristic of asthma in an airway hypersensitivity model system. In contrast, dominant-negative mutant SOCS-3 transgenic mice, as well as mice with a heterozygous deletion of Socs3, had decreased TH2 development. These data indicate that SOCS-3 has an important role in regulating the onset and maintenance of TH2-mediated allergic immune disease, and suggest that SOCS-3 may be a new therapeutic target for the development of antiallergic drugs.

Studies on the immunoglobulin E responses to Teladorsagia circumcincta in sheep: purification of a major high molecular weight allergen.

Studies on the immunoglobulin (Ig)E immune responses to the gastric nematode, Teladorsagia circumcincta, have demonstrated a major high molecular weight allergen (HMWTc). Cross reactive allergens of similar MW were demonstrated for Trichostrongylus colubriformis and Cooperia curticei, but not for Haemonchus contortus. Purification of HMWTc was achieved by gel-filtration chromatography, and nonreducing SDS-PAGE and Western blot analysis revealed two closely associated bands with a molecular weight of approximately 140-150?kDa. Reduction showed four IgE reactive bands of 120, 50, 45 and 30?kDa, and deglycosylation abrogated the immunoreactivity of the 120 and 30?kDa bands. Ultrastructural immunolocalization by electron microscopy revealed that the IgE reactivity was confined to the cuticular surface of the infective (L3) larvae. ELISA studies to determine the IgE anti-HMWTc responses in lambs during their first grazing season, demonstrated significantly higher IgE antibody in lambs with low accumulative faecal egg count (FEC) compared to animals with high accumulative FEC. These studies provide evidence for a protective function of IgE antibody in Teladorsagia infections in lambs.

Histamine release from bronchoalveolar lavage cells from asthmatic subjects after allergen challenge and relationship to the late asthmatic response

Background


Metachromatic cells obtained from asthmatic subjects demonstrate increased spontaneous and stimulated histamine release in vitro. Their ability to synthesize and store proinflammatory cytokines has focused renewed interest on their role in asthma.


Objective: The late asthmatic response provides a useful model of clinical asthma. The aim of the study was to examine metachromatic cell derived mediators and histamine releasability in vitro after in vivo allergen exposure in atopic subjects with and without asthma and relate them to the type of physiological response observed.

Methods: Bronchoalveolar lavage (BAL) cells were obtained 4 h after challenge from asthmatics exhibiting a single early response (EAR, n = 5), a dual response (LAR, n = 7), unchallenged (basal, n = 5), atopic non-asthmatic (ANA, n = 6) and non-atopic non-asthmatics (normal, n = 5). BAL histamine and tryptase concentrations and in vitro histamine release (HR) after stimulation with anti-IgE, allergen, A23187, conconavalin A and substance P were compared.

Results:Metachromatic cell numbers were lower in normal controls compared with all asthmatic groups and in LAR compared with EAR. Metachromatic cell derived mediators were higher in asthmatic compared with normal subjects. Spontaneous HR in LAR (20.5 ± 5.0%) was lower than EAR (29.5 ± 3.9%) and ANA (30.2 ± 1.4%) (P < 0.05). No differences were seen in stimulated HR between EAR and LAR. HR in ANA stimulated with anti-IgE was greater than LAR (P < 0.05). HR in ANA stimulated with anti-IgE was greater than LAR (P < 0.05). After stimulation with ionophore A23187 (1 μM), release was greater in LAR compared with basal (P < 0.05) and no different at 5 μM. All subject groups responded to substance P (SP) but was significantly more in the asthmatic subjects compared to normal controls (P < 0.05). Allergen challenge did not modify the response of asthmatic subjects to SP.

Conclusion: Functional differences in metachromatic cell reactivity are present in atopic subjects 4 h after in vivo allergen exposure which relate to the physiological response observed after this time and suggest that there is ongoing metachromatic cell degranulation subjects who subsequently develop LAR.

Langerhans cells are critical in the development of atopic dermatitis-like inflammation and symptoms in mice

Genetic or vitamin D3-induced overexpression of thymic stromal lymphopoietin (TSLP) by keratinocytes results in an atopic dermatitis (AD)-like inflammatory phenotype in mice echoing the discovery of high TSLP expression in epidermis from AD patients. Although skin dendritic cells (DC) are suspected to be involved in AD, direct evidence of a pathogenetic role for skin DC in TSLP-induced skin inflammation has not yet been demonstrated. In a mouse model of AD, i.e. mice treated with the low-calcemic vitamin D3 analogue, MC903, we show that epidermal Langerhans cells (LC)-depleted mice treated with MC903 do neither develop AD-like inflammation nor increased serum IgE as compared to vitamin D3 analogue-treated control mice. Accordingly, we show that, in mice treated with MC903 or in K14-TSLP transgenic mice, expression of maturation markers by LC is increased whereas maturation of dermal DC is not altered. Moreover, only LC are responsible for the polarization of naive CD4+ T cells to a Th2 phenotype, i.e. decrease in interferon-gamma and increase in interleukin (IL)-13 production by CD4+ T cells. This effect of LC on T-lymphocytes does not require OX40-L/CD134 and is mediated by a concomitant down-regulation of IL-12 and CD70. Although it was previously stated that TSLP up-regulates the production of thymus and activation-regulated chemokine (TARC)/chemokine (C-C motif) ligand 17 (CCL17) and macrophage-derived chemokine (MDC)/CCL22 by human LC in vitro, our work shows that production of these Th2- cell attracting chemokines is increased only in keratinocytes in response to TSLP overexpression. These results demonstrate that LC are required for the development of AD in mouse models of AD involving epidermal TSLP overexpression.

Does airways inflammation pre-exist before late onset wheeze in children?

Epidemiological studies show that some children develop wheezing after 3 yr of age which tends to persist. It is unknown how this starts or whether there is a period of asymptomatic inflammation. The aim of this study is to determine whether lower airway allergic inflammation pre-exists in late onset childhood wheeze (LOCW). Follow-up study of children below 5 yr who had a non-bronchoscopic bronchoalveolar lavage (BAL) performed during elective surgery. The children had acted as normal controls. A modified ISAAC questionnaire was sent out at least 7 yr following the initial BAL, and this was used to ascertain whether any children had subsequently developed wheezing or other atopic disease (eczema, allergic rhinitis). Cellular and cytokine data from the original BAL were compared between those who never wheezed (NW) and those who had developed LOCW. Eighty-one normal non-asthmatic children were recruited with a median age of 3.2 . Of the 65 children contactable, 9 (16.7%) had developed wheeze, 11 (18.5%) developed eczema and 14 (22.2%) developed hay fever. In five patients, wheeze symptoms developed mean 3.3- yr (range: 2–5 yr) post-BAL. Serum IgE and blood eosinophils were not different in the LOCW and NW, although the blood white cell count was lower in the LOCW group. The median BAL eosinophil % was significantly increased in the patients with LOCW (1.55%, IQR: 0.33 to 3.92) compared to the children who never wheezed, NW (0.1, IQR: 0.0 to 0.3, p = 0.01). No differences were detected for other cell types. There are no significant differences in BAL cytokine concentrations between children with LOCW and NW children. Before late onset childhood wheezing developed, we found evidence of elevated eosinophils in the airways. These data suggest pre-existent airways inflammation in childhood asthma some years before clinical presentation.

SOCS2 regulates T helper type 2 differentiation and the generation of type 2 allergic responses

The incidence of allergy and asthma in developed countries is on the increase and this trend looks likely to continue. CD4(+) T helper 2 (Th2) cells are major drivers of these diseases and their commitment is controlled by cytokines such as interleukin 4, which are in turn regulated by the suppressor of cytokine signaling (SOCS) proteins. We report that SOCS2(-/-) CD4(+) T cells show markedly enhanced Th2 differentiation. SOCS2(-/-) mice, as well as RAG1(-/-) mice transferred with SOCS2(-/-) CD4(+) T cells, exhibit elevated type 2 responses after helminth antigen challenge. Moreover, in in vivo models of atopic dermatitis and allergen-induced airway inflammation, SOCS2(-/-) mice show significantly elevated IgE, eosinophilia, type 2 responses, and inflammatory pathology relative to wild-type mice. Finally, after T cell activation, markedly enhanced STAT6 and STAT5 phosphorylation is observed in SOCS2(-/-) T cells, whereas STAT3 phosphorylation is blunted. Thus, we provide the first evidence that SOCS2 plays an important role in regulating Th2 cell expansion and development of the type 2 allergic responses.

Basophil models of homeopathy: a sceptical view

This paper examines the activation and inhibition of activation of human basophils. After a brief description of human basophils, different methods to determine basophil activation are discussed with a special emphasis on the use of flow cytometric methods, as these circumvent the potential problems of assays based on the loss of colour by activated basophils. The activation of human basophils by ultra-high dilutions of anti-IgE is discussed. The majority of the paper describes the inhibition of basophil activation by ultra-high dilutions of histamine. The results from published papers are described and discussed.

In vitro effects of the pyrethroid S-bioallethrin on lymphocytes and basophils from atopic and nonatopic subjects

Synthetic pyrethroids are increasingly used as insecticides and marketed as having relatively low human toxicity. The aim of this study was to examine the in vitro effects of the synthetic pyrethroid S-bioallethrin on human blood lymphocytes and basophils in atopic individuals and nonatopic control subjects. S-bioallethrin caused inhibition of lymphocyte proliferation after a 72-h culture period in a concentration-dependent manner. The inhibition of the lymphocyte proliferation by S-bioallethrin at the concentration 6.5 mu M correlated well with the total serum IgE values (r= -0.89, P

EFFECT OF IN-VIVO AND IN-VITRO LOVASTATIN TREATMENT ON MAST-CELL ACTIVATION

The hydroxymethylglutaryl coenzmye A (HMG CoA) reductase inhibitor lovastatin is used to treat hyperlipidaemia. This agent prevents the isoprenylation of some proteins involved in signal transduction processes and inhibits IgE-receptor-linked mediator release from RBL-2H3 cells. In this study the effect of in vivo and in vitro administration of lovastatin on histamine release from rat peritoneal mast cells was examined. Lovastatin (4 mg/kg/day for 2 weeks) inhibited histamine release induced by concanavalin A (con A) from rat peritoneal mast cells of Hooded-Lister rats and both homozygous lean and obese Zucker rats. In contrast, release induced by antirat IgE (anti-IgE) was only significantly inhibited in cells derived from Hooded-Lister rats and that induced by compound 48/ 80 was not altered. Lovastatin (20 mu M, 24 h, in vitro) caused a significant inhibition of the subsequent histamine release to con A, anti-IgE and compound 48/80 but not to the calcium ionophore A 23187. It is important to determine whether such inhibitory effects are also observed after the chronic, clinical administration of lovastatin and other HMG CoA reductase inhibitors.

MODULATORY ACTION OF HELICOBACTER-PYLORI ON HISTAMINE-RELEASE FROM MAST-CELLS AND BASOPHILS IN-VITRO

Helicobacter pylori is important in the aetiology of peptic ulceration. Despite inducing an inflammatory response in the mucosa, the organism persists, suggesting that it has efficient protective mechanisms. Some bacterial and viral products modulate histamine secretion from inflammatory cells. Therefore, this study examined the modulatory effects of H. pylori preparations on histamine release from rat peritoneal mast cells and human basophils. Eleven clinical isolates of H. pylori were prepared in different ways: as whole washed bacteria, washed sonicated bacteria, and formalin-killed bacteria, and as outer-membrane and lipopolysaccharide (LPS) extracts. Histamine release from mast cells or basophils was not elicited by any of these bacterial preparations alone. However, when mixed with various secretory stimulants, the bacterial preparations caused inhibition of histamine release from rat mast cells (calcium ionophore A23187, compound 48/80, concanavalin A, anti-rat IgE) and human basophils (A23187, N-formyl Met-Leu-Phe). The degree of inhibition ranged from 48 % to 97 %. These results indicate that H. pylori exerts an inhibitory effect on cells of the immune system that contributes to its persistence within the gastric mucosa.

Obesity associated severe asthma represents a distinct clinical phenotype - Analysis of the British Thoracic Society Difficult Asthma Registry patient cohort according to body mass index

BACKGROUND: Obesity has emerged as a risk factor for the development of asthma and it may also influence asthma control and airways inflammation. However, the role of obesity in severe asthma remains unclear. OBJECTIVE: To explore the association between obesity (defined by BMI) and severe asthma. METHODS: Data from the National Registry for dedicated UK Difficult Asthma Services were used to compare patient demographics, disease characteristics and healthcare utilisation between three body mass index (BMI) categories (normal weight: 18.5 -24.99, overweight: 25 -29.99, obese: =30) in a well characterised group of severe asthmatic adults. RESULTS: The study population consisted of 666 severe asthmatics with a median BMI of 29.8 (interquartile range 22.5 -34.0). The obese group exhibited greater asthma medication requirements in terms of maintenance corticosteroid therapy (48.9% versus 40.4% and 34.5% in the overweight and normal weight groups, respectively), steroid burst therapy and short-acting ß2-agonist (SABA) use per day. Significant differences were seen with gastro-oesophageal reflux disease (GORD) (53.9% versus 48.1% and 39.7% in the overweight and normal weight groups, respectively) and proton pump inhibitor (PPI) use. Bone density scores were higher in the obese group, whilst pulmonary function testing revealed a reduced FVC and raised Kco. Serum IgE levels decreased with increasing BMI and the obese group were more likely to report eczema, but less likely to have a history of nasal polyps. CONCLUSIONS: Severe asthmatics display particular characteristics according to BMI that support the view that obesity associated severe asthma may represent a distinct clinical phenotype.1Royal Brompton Hospital, London, UK;2Department of Computing, Imperial College, UK3Airways Disease, National Heart & Lung Institute, Imperial College, UK;4Centre for infection and immunity, Queen's University of Belfast, UK;5University of Leicester, UK;6The University of Manchester and University Hospital of South Manchester, UK;7Birmingham Heartlands Hospital, University of Birmingham, UK;8Gartnavel General Hospital, University of Glasgow, UK;9Glasgow Royal Infirmary, Glasgow, UKCorrespondence: Dr Andrew N. Menzies-Gow, Royal Brompton Hospital, Fulham Road, London SW3 6HP.

Characteristics, complications, and gaps in evidence-based interventions in rheumatic heart disease: the Global Rheumatic Heart Disease Registry (the REMEDY study)

AIMS: Rheumatic heart disease (RHD) accounts for over a million premature deaths annually; however, there is little contemporary information on presentation, complications, and treatment.

METHODS AND RESULTS: This prospective registry enrolled 3343 patients (median age 28 years, 66.2% female) presenting with RHD at 25 hospitals in 12 African countries, India, and Yemen between January 2010 and November 2012. The majority (63.9%) had moderate-to-severe multivalvular disease complicated by congestive heart failure (33.4%), pulmonary hypertension (28.8%), atrial fibrillation (AF) (21.8%), stroke (7.1%), infective endocarditis (4%), and major bleeding (2.7%). One-quarter of adults and 5.3% of children had decreased left ventricular (LV) systolic function; 23% of adults and 14.1% of children had dilated LVs. Fifty-five percent (n = 1761) of patients were on secondary antibiotic prophylaxis. Oral anti-coagulants were prescribed in 69.5% (n = 946) of patients with mechanical valves (n = 501), AF (n = 397), and high-risk mitral stenosis in sinus rhythm (n = 48). However, only 28.3% (n = 269) had a therapeutic international normalized ratio. Among 1825 women of childbearing age (12-51 years), only 3.6% (n = 65) were on contraception. The utilization of valvuloplasty and valve surgery was higher in upper-middle compared with lower-income countries.

CONCLUSION: Rheumatic heart disease patients were young, predominantly female, and had high prevalence of major cardiovascular complications. There is suboptimal utilization of secondary antibiotic prophylaxis, oral anti-coagulation, and contraception, and variations in the use of percutaneous and surgical interventions by country income level.