Airway protease/antiprotease imbalance in atopic asthmatics contributes to increased influenza A virus cleavage and replication.

Asthmatics are more susceptible to influenza infections, yet mechanisms mediating this enhanced susceptibility are unknown. Influenza virus hemagglutinin (HA) protein binds to sialic acid residues on the host cells. HA requires cleavage to allow fusion of the viral HA with host cell membrane, which is mediated by host trypsin-like serine protease. We show data here demonstrating that the protease:antiprotease ratio is increased in the nasal mucosa of asthmatics and that these changes were associated with increased proteolytic activation of influenza. These data suggest that disruption of the protease balance in asthmatics enhances activation and infection of influenza virus.

[Diagnosis and venom specific immunotherapy (VIT) in allergic children in Poland--how much the current practice follows the international guidelines?]. / Diagnostyka i immunoterapia u dzieci z alergia na jad owadów blonkoskrzydlych (VIT) w Polsce--jak wyglda w praktyce zgodnosc z miedzynarodowymi wytycznymi?

INTRODUCTION: Insect venom allergy requires a high level approach adequate to allergy intensity. In case of severe IgE-mediated sting reactions, in children older than five years, venom immunotherapy is a treatment of choice. AIM: Identification of current practices applied to venom allergic children in Poland and their adherence to the international guidelines. METHOD: Questionnaire survey concerning diagnostic and treatment rules was carried out in 8 centres of pediatric allergology, based on a similar audit conducted in the United Kingdom [Diwakar L. et al. Clin Exp Allergy 2008, 38: 1651]. RESULTS: In 5 centres both RAST and SPT tests were used as the first line of investigation. Subsequently 6 centres performed IDT. In three centres baseline serum tryptase levels were estimated. In case of sensitization to both bee and wasp venom in a child with the history of severe systemic reaction, but uncertain culprit insect, specific venom immunotherapy with both venoms was practised by 2 centres. In systemic reaction and not-detectable IgE in 6 centres child was followed-up in 6-12 months. Antihistamine premedication concerned all children in 7 centres. Six-week interval between booster doses was applied in half of centres. A target dose equal 100 mcg was used in 7 centres. Similarly all centres practiced 3-5 five year period of VIT. CONCLUSIONS: In Poland current practice with venom allergic children was conducted in congruence with most of the recommendations.

N-acetyltransferases as markers for asthma and allergic/atopic disorders.

An increasing prevalence of asthma noted worldwide has stimulated research on the phenotypic complexity resulting from interaction between the genetic and environmental components. Particularly, an increase in the prevalence of allergic rhinitis and asthma in industrialized countries indicate the importance of pulmonary metabolism of environmental xenobiotics. The arylamine N-acetyltransferases (NATs) are a unique family of enzymes that are involved in the biotransformation and detoxification of hydrazine and arylamine drugs/xenobiotics and could have a major role to play in atopy/asthma pathogenesis. Association studies on NAT1 and NAT2 polymorphisms focused in this review indicate the genetic significance of slow acetylation phenotype in bronchial and occupational asthma as well as in other allergic diseases in different populations worldwide. In contrast, fast acetylators have been found to have higher susceptibility to contact allergic dermatitis. Further in-depth research on the functional role of N- acetylation phenotype in disease pathogenesis is the requisite of the day, so that N-acetylation polymorphisms could serve as a genetic marker. Also, such genetic variations may have important implications in the efficacy of drugs for asthma treatment. The present review also makes a comment on the role of arylalkylamine N-acetyltransferase, an important enzyme involved in the conversion of serotonin to melatonin, in asthma pathogenesis.

Regulatory effects of histamine and histamine receptor expression in human allergic immune responses.

Histamine influences several immune/inflammatory and effector functions in addition to its dominant role in type I hypersensitivity reactions. Histamine can selectively recruit the major effector cells into tissue sites and affect their maturation, activation, polarization, and other functions leading to chronic inflammation. Histamine also regulates monocytes, dendritic cells, T cells and B cells, as well as related antibody isotype responses. The diverse effects of histamine on immune regulation appear to be due to differential expression and regulation of four types of histamine receptors and their distinct intracellular signals. In addition, differences in affinities of these receptors for histamine are highly decisive for the biological effects of histamine and drugs that target histamine receptors.

Association of toxic and essential metals with atopy markers and ventilatory lung function in women and men.

The association of age, smoking, alcohol, superoxide dismutase (SOD), glutathione peroxidase (GPx), blood lead (BPb) and cadmium (BCd) levels, and serum levels of copper (SCu), zinc (SZn) and selenium (SSe) with atopic status and ventilatory function was examined in the groups of 166 women and 50 men with no occupational exposure to metals or other xenobiotics. Markers of atopy included serum total IgE, skin prick test (SPT) to common inhalatory allergens, non-specific nasal reactivity (NNR) and non-specific bronchial reactivity (NBR). Parameters of ventilatory function included forced vital capacity (FVC) and forced expiratory volume in the first second (FEV(1)). Significantly higher BPb, SZn, IgE and prevalence of positive SPT, and lower SCu and NNR was found in men than in women. Fifteen women taking female sex hormones (HT) had significantly higher SCu than women without HT. Regression models showed significant inverse associations between IgE and SCu (P=0.021) and NNR and SCu (P=0.044) in women. When excluding women with HT, the association of SCu and total IgE became of borderline significance (P=0.051), association between SCu and NNR disappeared, and significant positive association between total IgE and BPb emerged (P=0.046). In men, significant inverse association was found between positive SPT and SSe, and between NBR and SSe. A decrease in FVC% and FEV(1)% was associated with an increase in smoking intensity (P<0.001) and a decrease in SZn (P=0.043 and P=0.053, respectively). These results were observed at the levels of the metals comparable to those in general populations worldwide. The observed differences between men and women may partly be explained by different levels of relevant toxic and essential metals, and their combination. The role of female HT in associations of atopy markers and SCu should be further investigated.

Anaphylactic relase of a basophil kallikrein-like activity. II. A mediator of immediate hypersensitivity reactions.

This report describes the immune release of a new mediator from human peripheral leukocytes, a basophil kallikrein-like activity (BK-A). The release process is initiated by the interaction of antigen on anti-IgE with cell-bound IgE, and appears to be similar in mechanism to the relase of histamine and other mediators of the immediate hypersensitivity reaction. The dose-response relationships and kinetics of histamine and BK-A release from antigen-challenged peripheral leukocytes are similar. The relase of the BK-A is calcium and temperature dependent, requires metabolic energy, and is controlled by hormone-receptor interactions that influence the cellular level of cyclic AMP, as has been described for other mediators of immediate hypersensitivity reactions. The data indicate that the interaction of BK-A with human plasma kininogen, generates immunoreactive kinin. We conclude that the antigen-IgE interation leads to the release from human basophils of a new mediator, a basophil kallikrein-like activity which may well be a link between reactions of immediate hypersenstivitity and the plasma and/or tissue kinin-generating systems.

Role of Txk, a member of the Tec family of tyrosine kinases, in immune-inflammatory diseases.

Txk/Rlk, a member of the Tec family of tyrosine kinases, is an important signaling mediator. We previously reported that human Txk is expressed in Th1/Th0 cells, and Txk translocates from cytoplasm into nuclei upon activation. Txk regulates specifically interferon-gamma gene transcription. Txk, poly(ADP-ribose) polymerase 1, and elongation factor 1alpha make a complex to bind to interferon-gamma gene promoter region-53/-39 (Txk responsive element) to exert positive effects on transcription as a Th1 cell-associated transcription factor. Txk expression is enhanced in rheumatoid arthritis and Behçet's disease, where Th1 dominant immunity occurs. In bronchial asthma and atopic dermatitis, typical Th2 diseases, Txk expression is reduced. Modulation of Txk expression by gene transfer or similar modality may lead to the correction of aberrant immunity and, consequently, disease treatment.

Indoleamine 2,3-dioxygenase (IDO) activity is lower in atopic than in non-atopic individuals and is enhanced by environmental factors protecting from atopy.

Tryptophan catabolism activated by the indoleamine 2,3-dioxygenase (EC 1.13.11.42) (IDO) enzyme in antigen presenting cells has a central role in induction of mechanisms suppressing T cell activation or clonal expansion. There is evidence suggesting that IDO activity is mainly upregulated by typical Th1-differentiating signals such as interferon-gamma and bacterial lipopolysaccharide (LPS). Therefore, we hypothesized that IDO activity would be lower in a Th2-associated disease such as atopy and that it would be higher in the presence of environmental factors known to favor Th1 differentiation. Here we show that this was the case. Concentrations of tryptophan (trp) and kynurenine (kyn), the main metabolite, were determined by reverse phase liquid chromatography from serum samples of a cohort of 392 non-asthmatic individual of whom 149 were atopics (one or more positive skin test when tested with a panel of 22 allergens). Kyn/trp ratio, as an indicator of IDO activity, was significantly lower in atopic than in non-atopic individuals. The cohort was stratified according to two known atopy-protecting factors, presence of antibodies against Helicobacter pylori or anamnestic information about childhood on a farm environment. As expected, IDO activity was significantly higher in their presence than absence.

FcepsilonRI cross-linking activates a type II phosphatidylinositol 4-kinase in RBL 2H3 cells.

Crosslinking of FcepsilonRI on rat basophilic leukemia (RBL 2H3) cells leads to an increase in Phosphatidylinositol 4-kinase activity. This increase in Ptdlns 4-kinase activity is strongly correlated with its tyrosyl phosphorylation state. Characterization of the enzyme activity in anti phosphotyrosine immunoprecipitates suggests it as a type II Ptdlns 4-kinase. Membrane cholesterol depletion studies showed a reduction in type II Ptdlns 4-kinase activity suggesting that lipid rafts play an important role in activation of the enzyme. The enzyme activity was inhibited by resveratrol. In situ inhibition of type II Ptdlns 4-kinase activity showed a reduction in beta-hexosaminidase release upon FcepsilonRI cross-linking. These studies suggest that a type II Ptdlns 4-kinase is an integral component of FcepsilonRI mediated signal transduction mechanisms.

Caspase cleavage of the nuclear autoantigen LEDGF/p75 abrogates its pro-survival function: implications for autoimmunity in atopic disorders.

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a nuclear autoantigen in atopic disorders implicated in cellular protection against stress-induced apoptosis. We observed that LEDGF/p75 was cleaved during apoptosis into fragments of 65 and 58 kD generated by caspases-3 and -7 cleaving at three sites: DEVPD30/G, DAQD486/G and WEID85/N. Sequence analysis revealed that the DEVPD30/G and WEID85/N sites lie within the highly conserved HATH (homologous to amino terminus of hepatoma-derived growth factor) region, also known as PWWP domain. Alignment of proteins containing this domain failed to reveal conservation of the DEVPD30/G and WEID85/N sites, suggesting that the HATH/PWWP domain of LEDGF/p75 may be specifically targeted by caspases. Overexpression of LEDGF/p75 protected HepG2 cells from serum starvation-induced cell death, whereas expression of the 65 kD fragment failed to protect. The apoptotic cleavage of LEDGF/p75 may contribute to the pathogenesis of atopic disorders by abrogating its pro-survival function and enhancing its immunogenicity.

Dietary linoleic acid-induced changes in respiratory beta-adrenergic receptor function and the form of arrhenius plots of isoprenaline- and prostaglandin E2-stimulated adenylate cyclase activity in a model for atopy.

Varying dietary linoleic acid altered lung membrane fatty acid composition with linoleic acid content increasing from approximately 6% total in those on 3 en% diet to approximately 14% total fatty acid in those on a 12 en% diet. Accompanying this were two- to three-fold increases in the levels of the elongation products of linoleic acid, namely 20:2 (n-6) and 22:5 (n-6) and a decrease in 18:1 oleic acid from approximately 26% to approximately 19% total. Administration of Haemophilus influenzae, to animals on 6 en% linoleic acid, serving as a model for atopy, effected a small increase in the levels of 22:5 (n-3) and doubled those of 22:6 (n-3). beta-Adrenergic-induced tracheal relaxation and stimulation of lung adenylate cyclase were elevated by increasing dietary linoleic acid from 3 to 6 en%, although such differences were abolished in the atopic model and when dietary linoleic acid was increased to 12 en%. Arrhenius plots of NaF-stimulated lung adenylate cyclase activities exhibited a break (t1) at approximately 26 degrees C in all dietary groups with unchanged activation energies and activity. In contrast, whilst both isoprenaline and PGE2-stimulated adenylate cyclase activities showed similar break-points in their Arrhenius plots, dietary linoleic acid manipulation markedly altered their form. As with NaF-stimulated activities then, irrespective of dietary manipulation and induction of atopy, these plots showed an invariant break occurring at approximately 26 degrees C. But, for animals on 3 and 6 en% diets, a second break was apparent at approximately 15 degrees C, which was slightly decreased to approximately 12 degrees C upon induction of atopy and completely abolished on increasing dietary linoleic acid to 12 en%. Accompanying such changes were marked alterations in activation energies. We suggest that profound changes in lung plasma membrane bilayer properties occur upon both altering dietary linoleic acid levels and in atopy. These selectively perturb adenylate cyclase activity when it is receptor-stimulated but not when it is activated by direct G-protein stimulation with NaF. We suggest that atopy and dietary challenge elicit an asymmetric perturbation of the plasma membrane that predominantly affects the outer half of the lipid bilayer.

Effect of oromucosal administration of IFN-alpha on allergic sensitization and the hypersensitive inflammatory response in animals sensitized to ragweed pollen.

Oromucosal (o.m.) administration of interferon-alpha (IFN-alpha) during either allergic sensitization (days 0-6) or the hypersensitive response (days 11 and 12) or both periods caused a dose-dependent reduction in allergen-specific IgE production and allergen-induced eosinophil recruitment in mice sensitized to ragweed pollen, a common allergen in humans. Treatment during the hypersensitive response period alone appeared to be most effective. Oromucosal treatment was as effective as intraperitoneal (i.p.) treatment, with maximum inhibition of both allergen-specific IgE production and allergen-induced eosinophil recruitment observed at a dose of a 1000 IU IFN-alpha. Treatment of animals with up to 10(5) IU murine IFN-alpha/beta (MuIFN-alpha/beta) by either the om. or i.p. route did not inhibit significantly allergen-specific IgG production, which may even have been increased at certain doses of IFN. Treatment of animals with up to 10(5) IU MuIFN-alpha/beta by either the o.m. or i.p. route did not affect significantly total serum IgE or IgG levels. Oromucosal administration of IFN-alpha reduced allergen-specific IgE production and allergen-induced eosinophil recruitment in the absence of detectable toxicity, the induction of H(2) antigen expression, and 2',5'-oligoadenylate synthetase activity associated with parenteral administration of IFN-alpha and thus may find application for the treatment of asthma and associated viral infections.

Restricted cyclooxygenase-2 expression in the central nervous system following acute and delayed-type hypersensitivity responses to bacillus Calmette-Guérin.

The expression of cyclooxygenase-2, a key enzyme in prostaglandin and thromboxane synthesis in inflammation, was studied immunohistochemically in in vivo models of acute and chronic inflammatory responses in rat central nervous system. In the acute inflammatory response to intracranial injection of heat-killed bacillus Calmette-Guérin as well as in the immune-mediated, delayed-type hypersensitivity response to the same pathogen, cyclooxygenase-2 expression was restricted to major infiltrating haematogenous cell populations such as neutrophils and mononuclear phagocytes, while the expression of the enzyme by brain non-neuronal resident cells (astrocytes, microglia, perivascular cells) appeared to be limited to perivascular cells of the blood vessels in the vicinity of the lesion and in the surrounding area. On the basis of their morphology and location, these perivascular cells were identified as perivascular macrophages, but we could not rule out the possibility that some endothelial cells also expressed cyclooxygenase-2. The constitutive neuronal cyclooxygenase-2 was not affected by the ongoing inflammation. Interestingly, in spite of the extensive astrocyte and microglial reaction occurring over a broad area surrounding the inflammatory lesions, there was no obvious cyclooxygenase-2 staining in these cells. These data indicate that the up-regulation of cyclooxygenase-2 expression in acute and chronic, immune-mediated lesions in the brain parenchyma is remarkably restricted to the lesion site. Since cyclooxygenase metabolites can regulate important functions of resident as well as infiltrating cells, the increased synthesis of prostaglandins and thromboxanes, which is likely to occur as a consequence of the expression of cycloxygenase-2 at the lesion site, might represent an important component of the inflammatory processes within the brain.

Pharmacology of IgE-mediated desensitization of human basophils: effects of protein kinase C and src-family kinase inhibitors.

IgE-mediated down-regulation of secretion from basophils and mast cells is an important component of the overall cellular response that determines the ultimate extent of mediator release. The down-regulatory process that occurs during active secretion has also been associated with the methodological phenomenon called desensitization, but the mechanisms underlying desensitization are not understood. A variety of studies have suggested that activation of protein kinase C (PKC) results in down-regulation of IgE-mediated secretion so we have examined the effect of the PKC inhibitors Ro-31-8220 (3-[1-[3-amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3- yl) maleimide) and bis-indolylmaleimide II on desensitization in human basophils. At concentrations that have been shown previously to inhibit PKC-mediated functions in basophils completely, these two drugs had no effect on IgE-mediated desensitization. We did find, however, that the src-family kinase inhibitors PP1 [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] and PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d]pyrimidine] inhibited desensitization as well as secretion. These data suggest that PKC has little role in down-regulating the IgE-mediated basophil response. However, like the activation signaling cascade, the desensitization process is dependent on the activation of src family kinases.

Der f 2 activates phospholipase D in human T lymphocytes from Dermatophagoides farinae specific allergic individuals: involvement of protein kinase C-alpha.

The major house-dust mite allergen, Der f 2, stimulates the phospholipase D (PLD) in T lymphocytes from Dermatophagoides farinae specific allergic individuals. PLD activity increased more than two-fold in T cells from allergic patients compared with those cells from normal controls with maximal responses within 30 min after exposure of Der f 2. A well-known PLD activator PKC-alpha was found to be translocated to membrane from cytosol in Der f 2-treated T cells from Dermatophagoides farinae specific allergic individuals. Down-regulation of PKC-alpha with phorbol myristate acetate pretreatment for 24 h abolished Der f 2-induced PLD activation. Ro 320432, PKC inhibitor also reduced the effects of Der f 2-induced PLD activation suggesting that PKC-alpha acts as upstream activator of PLD in Der f 2-treated T cells. Taken together, the present data suggest that Der f 2 can stimulate PLD activity through the PKC-alpha activation in T cells from Dermatophagoides farinae allergic individuals.

Serum antitrypsin activity and alpha1 antitrypsin level in atopic bronchial asthma.

The aim of our investigations was to evaluate the behavior of the important proteolitic inhibitor--serum alpha1 antitrypsin in patients with atopic and non-atopic bronchial asthma. The inhibitor concentration was determined by means of the immunodiffusion method, and antitrypsin activity was evaluated using the synthetic substrate BAPNA. The investigations were carried out on 53 patients with bronchial asthma and on 13 healthy persons. It was found that in some patients with atopic asthma alpha1 antitrypsin level and antitrypsin activity were very low but this did not lower significantly the mean value of the whole group. The results presented suggest that a relative alpha1 antitrypsin deficiency, at least in some patients, can be considered as being one of the pathogenetic factors in atopic asthma. In addition, determination of serum antitrypsin activity may be of practical significance in differential diagnosis of bronchial spastic reactions of bacterial and allergic origins.

Eosinophil peroxidase: a new serum marker of atopy and bronchial hyper-responsiveness.

Do markers of eosinophil activation differ in their ability to detect subjects with atopy or bronchial hyper-responsiveness (BHR)? Comparisons of serum levels of eosinophil peroxidase (S-EPO), of eosinophil cationic protein (S-ECP) and the blood eosinophil count (B-Eos) have been made between 154 subjects aged 20-44 years, participating in the European Community Respiratory Health Survey in Uppsala, Sweden. Subjects with atopy had significantly higher levels of S-EPO and S-ECP than those without atopy (P <0 center dot 001). Subjects with BHR had significantly higher levels of S-EPO (P <0 center dot 001) and B-Eos (P <0 center dot 01) than subjects without BHR. Persons reporting asthma-related symptoms had significantly higher levels of S-EPO and B-Eos than subjects without such symptoms (P <0 center dot 001 and P <0 center dot 01, respectively). Asthma symptom score correlated significantly to S-EPO (r = 0 center dot 26, P <0 center dot 01), S-ECP (r = 0 center dot 20, P <0 center dot 05) and B-Eos (r = 0 center dot 18, P <0 center dot 05). Finally, S-EPO was significantly more sensitive than S-ECP for detecting subjects with BHR (P <0 center dot 05) and significantly more sensitive than B-Eos for detecting both subjects with BHR and subjects with a combination of atopy and BHR (P <0 center dot 05). It is concluded that S-EPO is a promising marker with a higher sensitivity for BHR than S-ECP or B-Eos. Further studies are needed to define the value of S-EPO when following disease activity.

Expression of cyclooxygenase in nasal polyps from atopic and nonatopic subjects.

Cyclooxygenase (COX) 1 and 2 are two isoforms of a crucial enzyme in the metabolism of arachidonic acid to prostaglandins, thromboxanes and prostacyclin. It is now clear that COX-1 is expressed in the majority of cells constitutively, and COX-2 appears after stimulation. The role of cyclooxygenase expression in the pathophysiology of the airways has not been elucidated. In this study we aimed to compare cyclooxygenase expression in nasal polyps removed from well-defined atopic and nonatopic subjects. Monoclonal antibodies against COX-total (detecting both COX-1 and COX-2 epitopes) and anti-COX-2 combined with peroxidase-antiperoxidase (PAP) visualizing system were used to assess COX-total and COX-2 expression in cryostat sections of nasal polyps from both groups of patients. Chromotrope R2 or toluidine blue staining were used to detect the presence of eosinophils and mast cells, respectively. We demonstrated that cryostat sections of nasal polyps from both groups of patients revealed COX-total and COX-2 expression with similar intensity. There were no significant differences in distribution of COX-total or COX-2 immunoreactivity in nasal polyps tissue between atopic and nonatopic group. No correlation between density of cells expressing COX-2 and the number of mast cells and eosinophils was found. Our data indicate that COX-2 is expressed in nasal polyps from both atopic and nonatopic patients.

[Serum angiotensin converting enzyme (SACE) in patients with atopic bronchial asthma]. / Surowicza aktywnosc enzymu konwertujacego angiotensyne (SACE) u chorych z atopowa astma oskrzelowa.

The aim of the study was to assess serum ACE (S-ACE) activity in atopic bronchial asthma. S-ACE activity values were determined in 50 subjects (11 blood donors, 39 asthmatics) using the radioenzymatic method with Ventrex Laboratories kits. The authors did not find any differences between asthmatics in clinical remission, not receiving therapy, patients receiving prednisone (mean daily dose 10.0 +/- 2.5 mg) and healthy blood donors. In subjects with exacerbations of bronchospasm (not receiving steroids) S-ACE activity was significantly higher.

Tec family kinases: regulation of FcεRI-mediated mast-cell activation.

Mast cells express the high-affinity receptor for IgE (FcεRI) and are key players in type I hypersensitivity reactions. They are critically involved in the development of allergic rhinitis, allergic asthma and systemic anaphylaxis, however, they also regulate normal physiological processes that link innate and adaptive immune responses. Thus, their activation has to be tightly controlled. One group of signaling molecules that are activated upon FcεRI stimulation is formed by Tec family kinases, and three members of this kinase family (Btk, Itk and Tec) are expressed in mast cells. Many studies have revealed important functions of Tec kinases in signaling pathways downstream of the antigen receptors in lymphocytes. This review summarizes the current knowledge about the function of Tec family kinases in FcεRI-mediated signaling pathways in mast cell.