Effect of Dermatophagoides pteronyssinus extract on the expression of genes involved in inflammation and tissue remodeling by peripheral blood mononuclear cells of allergic asthma patients.

PURPOSE: House dust mite allergy constitutes a risk factor for asthma development and is associated with a faster decline of lung function in allergic asthmatics (AAs). To evaluate the effect of Dermatophagoides pteronyssinus (Dp) allergens on the expression of genes involved in inflammation and tissue remodeling by peripheral blood mononuclear cells (PBMCs) isolated from the blood of AAs. MATERIALS AND METHODS: The cells from AAs, allergic rhinitis without asthma patients (ARs), and healthy controls (HCs) were cultured in the presence of Dp, lipopolysaccharide (LPS), or without any stimulation. The expression of 84 genes was evaluated using a low-density microarray whereas, the quantitative expression analysis of selected genes was performed using a real-time polymerase chain reaction. The concentration of selected proteins in the cell culture supernatants was assessed using ELISA. RESULTS: Stimulation of PBMCs with Dp and LPS resulted in a significant upregulation of 8 and 15 among 84 studied genes, respectively. The greatest upregulation was observed for CCL2 and CCL3 using Dp and LPS, respectively. In comparison with HCs, in AAs, significantly increased upregulation of CCL2 in response to Dp was found. The secretion of CCL2 and CCL3 by PBMCs reflected the pattern of gene expression at the mRNA level. The mean Dp-stimulated secretion of CCL2 by PBMCs of ARs was less than in AAs (p â€‹< â€‹0.01), both being notably greater than in the HCs (p â€‹< â€‹0.01). CONCLUSION: Rapid and potent upregulation of CCL2 expression by PBMCs in response to Dp may constitute an important contribution to the development of allergic asthma.

Sensitization to major Dermatophagoides pteronyssinus allergens in house dust mite allergic patients from North Eastern Poland developing rhinitis or asthma.

PURPOSE: Recognition of individual allergens by IgE is crucial for triggering symptoms in allergic rhinitis (AR) or asthmatic (AA) patients. House dust mite (HDM) allergy is frequent around the world, the sensitization profile to individual HDM allergens varies in individual HDM-allergic patients (APs). The aim of this study was to evaluate the pattern of IgE sensitization to three major Dermatophagoides pteronyssinus (Dp) allergens among patients from North Eastern Poland suffering from HDM-AR and/or AA. PATIENTS AND METHODS: The study was performed on 323 HDM-AR and/or AA patients and 106 controls (CG) including 30 healthy non-atopic subjects, 32 AR patients not sensitized to Dp and 44 non-atopic asthmatics. IgE levels to natural (n)Der p 1, nDer p 2, recombinant (r)Der p 2.0101 and rDer p 23 allergens were measured by ELISA. RESULTS: The majority of HDM-APs were sensitized to nDer p 1 (72.1%), nDer p 2 (81.7%), rDer p 2.0101 (78.3%) and rDer p 23 (70.9%). The frequency of positive results to individual allergens depended on clinical manifestations and the level of IgE to the whole Dp extract. In HDM-AA patients, reactivity to nDer p 1 and rDer p 23 was detected more frequently than in HDM-AR patients. The whole Dp extract completely inhibited IgE binding to nDer p 1 and nDer p 2 but only partially to rDer p 23. CONCLUSIONS: HDM-APs from North-Eastern Poland display sensitization profile to major allergens which is similarly observed in western Europe. HDM-based diagnostic and therapeutic products should include all major allergens.

Serum immunoglobulin E reactivity to cross-reacting panallergen components in north-eastern Poland patients pollen sensitized.

Background: The presence of immunoglobulin E (IgE), which cross-reacts with allergen components, such as profilins, polcalcins, and cross-reacting carbohydrate determinants (CCD), creates a problem when selecting patients for allergen immunotherapy by using conventional methods. The aim of this study was to evaluate the prevalence of sensitization to profilins, polcalcins, and CCDs in patients with seasonal pollen allergic rhinitis. Methods: The study was performed on a group of 112 patients with seasonal pollen allergic rhinitis, ages 14 to 55 years, with sensitization to at least one seasonal allergen (IgE > 0.7 kUA/L). The presence of IgE sensitization to recombinant (r) Bet v 2, rPhl p 12, rBet v 4, rPhl p 7, and CCDs, in addition to rBet v 1, rPhl p 1, rPhl p 5, was evaluated by using a multiparameter immunoblot. Results: Among the studied patients, 64.3, 80.4, and 41.1% were sensitized to birch, timothy grass, and mugwort pollen, respectively. Sensitization to profilins rBet v 2/Phl p 12 was demonstrated in 28.6%, to polcalcins Bet v 4/Phl p 7 in 8.9%, and to CCDs in 25%. In 29.3%, serum IgE reactivity to any of the cross-reactive components could be demonstrated. Serum IgE reactivity to rBet v 2 was always accompanied by IgE reactivity to rPhl p 12, and IgE reactivity to rBet v 4 was always accompanied by IgE reactivity to rPhl p 7. Among the patients with pollinosis co-sensitized to at least two allergen sources according to extract-based diagnosis, possible false-positive results due to sensitization to cross-reactive components were detected in 17.9%. Conclusion: Evaluation of sensitization to cross-reacting components may be useful in evaluation of patients with pollen allergy who are being assessed for allergen immunotherapy to optimize the constitution of their immunotherapy vaccines.

Production and Use of Recombinant Profilins Amb a 8, Art v 4, Bet v 2, and Phl p 12 for Allergenic Sensitization Studies.

Four recombinant (r) allergens (rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101) were successfully produced and used for sensitization studies. The allergens belong to the profilin family which is one of the most numerous allergen families. These four proteins represent allergens originating from pollen of weeds (rAmb a 8.0101 and rArt v 4.0101), tree (rBet v 2.0101) and grass (rPhl p 12.0101). The recombinant allergens were characterized using various biochemical and biophysical methods and tested for their ability to bind patient-derived antibodies. One hundred patients aged 2 to 50 years sensitized to pollen and plant-derived food allergens (IgE > 0.35 kU/L) were included. Sensitization to individual allergen sources and components of birch and timothy pollens was evaluated using multiparameter immunoblots. The presence of IgE to pollen-derived recombinant profilins rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101 in serum was evaluated using ELISA method. The presence of IgE against pollen profilins was detected in 20 out of 100 studied patients. High correlation was seen between IgE ELISA results with individual pollen profilins. In summary, it was shown that the recombinant versions of the four allergenic profilins can be used for sensitization studies and for component-resolved allergy diagnostics.

Predictors of airway hyperreactivity in house dust mite allergic patients.

INTRODUCTION: Airway hyperresponsiveness (AHR) is a cardinal feature of asthma. Asthma is a heterogenous disorder which consists of different phenotypes and endotypes. Mechanisms leading to AHR may differ in different asthma subtypes. Allergy to perennial allergens, including house dust mites (HDM) is a major risk factor for asthma development. The aim of this study was to determine predictors of AHR in a well-characterized population of HDM-allergic patients. MATERIAL AND METHODS: In a retrospective analysis 843 patients with HDM-allergic rhinitis with/without asthma were evaluated. The following parameters were included in the analysis: serum concentration of total (t)- and Dermatophagoides pteronyssinus (Dp)-specific IgE, fractional exhaled nitric oxide concentration (FeNO), lung function tests, bronchial challenge with histamine, age sex, and body mass index (BMI). Linear regression analysis was used to determine predictors of AHR. RESULTS: In a simple linear regression analysis baseline lung function results expressed as either forced expiratory volume in 1 s (FEV1) or maximal expiratory flow at 50% of the forced vital capacity (MEF50), FeNO, tIgE, DpIgE, age and BMI affected AHR. A multiple regression analysis demonstrated that in the whole group of HDM-allergic patients the most important, independent predictors of AHR were MEF50, FeNO and DpIgE. CONCLUSION: Even in a well-characterized asthma phenotype several processes participate in development of AHR. Major, independent predictors of AHR: lung function parameters, FeNO and DpIgE indicate possible targets for therapeutic intervention in a population of HDM-allergic patients.

Synergistic effect of Dermatophagoides pteronyssinus allergens and dexamethasone on the expression of CD163 by peripheral blood mononuclear cells.

BACKGROUND: The CD163 is a marker of monocyte/macrophage anti-inflammatory function. Its soluble form (sCD163) also exert anti-inflammatory activities including inhibition of T cell proliferation. OBJECTIVE: To evaluate the effect of dexamethasone (Dx) and Dermatophagoides pteronyssinus (Dp) on ex vivo production of sCD163 by peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs from 26 allergic asthma patients (AAs) and 12 non-atopic healthy controls (HCs) were cultured with Dp, Dx, Dp + Dx or without any stimulation for up to 144 h (T144). Concentration of sCD163, interleukin (IL) -6 and IL-10 in PMBC culture supernatants was evaluated using ELISA. The mRNA expression of CD163 by PBMCs was estimated using quantitative PCR (qPCR). RESULTS: At T144 the median concentration of CD163 in unstimulated PBMC cultures of AAs was greater than that in HCs (p = 0.008). Concomitant application of Dp and Dx resulted in a synergistic effect reflected by a dramatic increase of sCD163 concentration both in HCs (p = 0.0002) and AAs (p < 0.0001). Also a synergistic effect of Dp and Dx on CD163 mRNA expression was seen at T24 and T48 but not at T6 or T12. Among asthmatic patients the effect of Dx on sCD163 production was attenuated in severe in comparison to mild-to-moderate AAs (p = 0.0007). Moreover, Dp-induced production of IL-6 but not IL-10 was inhibited by Dx (p < 0.0001). Inhibition of IL-10 decreased sCD163 concentration by more than 50%. CONCLUSIONS: Dx-triggered upregulation of anti-inflammatory CD163 expression by monocytes is synergistic with endogenous mechanisms involved in the resolution of Dp-induced inflammation. This effect is impaired in severe asthma patients.

Platelet-leukocyte mixed conjugates in patients with atrial fibrillation.

Although platelets may contribute to the inflammatory component in atrial fibrillation (AF), the impact of platelet-leukocyte mixed conjugates has not yet been determined. Seventeen patients with persistent AF (8/9 m/f; mean age 68.1 +/- 2.5 years), not on anticoagulant therapy, were recruited and compared to 34 healthy controls with normal sinus rhythm (16/18 m/f; mean age 60.8 +/- 1.2 years). Platelet-leukocyte mixed conjugates, platelet P-selectin and leukocyte activation markers (CD11b, myeloperoxidase) were measured by flow-cytometry in whole blood both in basal condition and after in vitro ADP/collagen challenge. Plasma D-dimer and soluble P-selectin were also measured. Statistical analyses were performed by Mann-Whitney or Wilcoxon U test for intergroup differences. In AF patients platelet count, as well as platelet P-selectin expression and percent platelet-leukocyte conjugates were all significantly lower both in basal condition and upon activation with ADP/collagen. In contrast, both soluble P-selectin and D-dimer were significantly higher than in controls; white blood cell count and leukocyte activation markers were unchanged. In conclusion, the formation of platelet-leukocyte mixed conjugates was unexpectedly reduced in AF, possibly due to less reactive platelets as a consequence of previous in vivo activation by ongoing formation of trace amounts of thrombin.

Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome.

Thrombin is an agonist inducing platelet activation. We combined two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MALDI-TOF MS) to analyse differentially expressed proteins secreted from thrombin-stimulated platelets. Human washed platelets, from healthy volunteers, were stimulated with thrombin 0.5 U/ml at 37 degrees C without stirring and the secreted proteins were resolved by 2D-DIGE. By image analysis, 1094 spots were detected in the 2D gel. The spots whose mean intensity showed at least a five-fold change intensity increase or decrease in the thrombin-activated platelet gel in comparison with unstimulated control were digested by trypsin and subjected to MALDI-TOF MS analysis. Peptides from mass spectra of in-gel digest samples were matched against available databases, using the Mascot search engine (Matrix Science) for peptide mass fingerprint. In the activated platelet secretome, transferrin, glutathione-transferase, WD repeat protein, ER-60, thrombospondin-1 precursor and thrombospondin were the most abundant. Also lamin A, a nuclear protein, not previously identified in platelets, appeared to be released. The novel strategy to combine 2D-DIGE with MALDI-TOF MS is a useful approach for a quantitative analysis of the effect of thrombin on the secretome profile of human platelets.

Determinants of platelet conjugate formation with polymorphonuclear leukocytes or monocytes in whole blood.

Following preliminary in-vitro experiments, platelet-leukocyte conjugates and their determinants were evaluated in citrated whole blood from 349 subjects (209 women, age 16-92 years) randomly recruited from the general population. Platelet activation by ADP/collagen but not leukocyte stimulation by fMLP or LTB4 resulted in formation of platelet conjugates with PMN or monocytes. In the population study, mixed cell conjugates, platelet P-selectin and leukocyte CD11b were measured by flow cytometry both at baseline and after in-vitro stimulation with ADP/collagen. The latter significantly increased platelet conjugates with either PMN or monocytes, platelet P-selectin and leukocyte CD11b expression. Platelet count significantly correlated with platelet-PMN, platelet-monocyte conjugates and P-selectin both at baseline and upon stimulation. In all conditions, both conjugate levels correlated with each other, when adjusted for gender, age and platelet count. Age correlated with platelet-PMN conjugate numbers in basal and stimulated conditions and with basal P-selectin. ADP/collagen stimulation resulted in higher P-selectin and conjugates values in women. Among risk factors, a significant correlation was found between conjugate and glucose levels. In conclusion, the presence and formation in whole blood from a large population of platelet-leukocyte conjugates reflects primary platelet - but not leukocyte - activation and varies with gender, age, platelet count and blood glucose.

Plasminogen activator inhibitor-1 plasma concentration in allergic asthma patients during allergen challenge.

BACKGROUND: The -675 4G/5G plasminogen activator inhibitor-1 (PAI-1) polymorphism is linked with asthma and bronchial hyperreactivity. The aim of this study was to evaluate the effect of allergen challenge on plasma PAI-1 concentration in relation to the -675 4G/5G PAI-1 gene polymorphism in house dust mite-allergic asthma patients (HDM-AAs). MATERIALS AND METHODS: The study was performed in 54 HDM-AAs and 54 healthy nonatopic controls (HCs). Plasma samples were collected in HDM-AAs before, as well as 30 min, 6 h and 24 h after allergen challenge and at corresponding time points in sham-challenged HCs. RESULTS: In subjects carrying the individual PAI-1 genotype, the mean baseline plasma PAI-1 concentration was greater in HDM-AAs than in HCs. At 30 min the mean increase in plasma PAI-1 concentration was significantly greater in HDM-AAs (14.4 +/- 12.9 ng/ml) than in HCs (3.4 +/- 3.2 ng/ml; p < 0.001). At 6 h, plasma PAI-1 concentration greater than before challenge was found in only 4 HCs (7.4%) but in 48 HDM-AAs (88.9%; p < 0.0001). An increase in plasma PAI-1 concentration at 6 h was found in all HDM-AAs carrying the 4G allele but only in 33.3% of the 5G homozygotes (p < 0.0001). The strongest correlation was found between log PC20 and PAI-1 plasma concentration over the period of 24 h (r = -0.507; p = 0.0001). CONCLUSION: Changes in plasma PAI-1 concentration associated with allergen-induced bronchoconstriction are modulated by the -675 4G/5G polymorphism of the PAI-1 gene. Allergen-induced upregulation of PAI-1 synthesis may participate in the development of bronchial hyperreactivity in HDM-AAs.

Comparison of VASP-phosphorylation assay to light-transmission aggregometry in assessing inhibition of the platelet ADP P2Y12 receptor.

There is need to improve platelet function testing to monitor the response to antiplatelet drugs. We compared flow-cytometric analysis of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) to light-transmission aggregometry for the detection of drug-induced in-vitro inhibition of the platelet P2Y12 ADP receptor on 22 healthy subjects (10 males, 12 females, 28.5 +/- 6.6 years). The platelet reactivity index (PRI) of VASP was calculated both from mean fluorescence intensity (MFI) and percent of fluorescence-positive platelets in the presence of PGE1 with or without ADP (10 microM). Platelet aggregation was induced by ADP (1.25, 2.5 and 5 microM). Cangrelor, a competitive inhibitor of the P2Y12 receptor, preincubated 5 minutes, induced a concentration-dependent inhibition of platelet ADP-receptor function in both tests. Indeed PRI (%) based on either MFI or percent platelets gated were highly correlated with each other (r = 0.97, p %lt; 0.0001) and with aggregation induced by ADP. The IC50 of cangrelor against each of the three ADP concentrations used in aggregometry increased from 5.8 +/- 3.4 nM to 23.1 +/- 4.0 nM and to 98 +/- 25 nM, respectively. The IC50 of cangrelor based on VASP-P was within the same range (25.5 +/- 7.7 nM). No correlation was observed between IC50 values of cangrelor and ADP concentrations giving 50% effect (EC50) in the absence of the drug. However, at 10 nM cangrelor seven subjects could be identified by the VASP-P assay as " low responders " to the drug (PRI > 50%), and six of them also had an aggregation response to 5 micro M ADP > 50%. These six subjects showed the lowest ADP EC50 values in the absence of the drug, possibly reflecting high sensitivity of their platelet P2Y12 receptors to ADP. In conclusion, both the VASP-P assay and light-transmission aggregometry detect in a comparable way in-vitro pharmacological inhibition of the platelet P2Y12 ADP receptor and its individual variability.

[Thrombin activatable fibrinolysis inhibitor (TAFI) in allergic asthma patients]. / Aktywowany trombina inhibitor fibrynolizy (TAFI) w astmie alergicznej.

Increased thrombin generation occurs in the airways of asthmatic patients. Thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme which after activation by a thrombin-thrombomodulin complex inhibits fibrinolysis. The aim of this study was to evaluate TAFI concentration and activity in plasma of bronchial asthma patients challenged with Dermatophagoides pteronyssinus (Dp) extracts. The study was performed on 23 asthma patients mean age 28.7 +/- 9.8 years with a typical history and positive skin prick test with Dp allergens. Seventeen patients developed both early asthmatic reaction (EAR) and late asthmatic reaction (LAR), 6 patients developed only EAR. Blood was collected before allergen challenge (AO), 1 (A1), 8 (A2) and 24 hours (A3) after allergen challenge. Five healthy persons, mean age 24.5 +/- 3.6 years with negative skin prick tests to common aeroallergens served as negative controls. Healthy controls underwent sham bronchial provocation with 0.9% solution of NaCl. TAFI antigen and activity, levels of thrombin-antithrombin III complexes (TAT) and prothrombin fragments F 1+2 were measured in plasma by ELISA method. At A0 in dual responders mean TAFI concentration (101.4 +/- 23.82%) and activity (19.6 +/- 5.92 microg/ml) were higher than in healthy controls (69.35 +/- 21.49%; p < 0.05 and 10.49 +/- 3.53 microg/ml; p = 0.01; respectively). Significant fall in TAFI plasma concentration was detected at A1 and the decreased concentration of TAFI persisted until A3 (85.15 +/- 26.36%; p < 0.05). No significant change in plasma TAFI concentration was observed in healthy controls or in patients who responded with an EAR only. Allergen induced inflammation is associated with significant changes in plasma TAFI concentration.

[The role of platelets in allergic inflammation]. / Rola plytek krwi w alergicznej reakcji zapalnej.

Platelets are the smallest blood cells, which perform various functions. They are able to activate and cooperate with leukocytes and endothelial cells by biologically active substances released from their granules. They play a key role in the inflammatory process, and are important contributors to allergic diseases. This article presents a short review of actual knowledge about platelets and their role in allergic inflammation.