22 research outputs found
Fine characterization of immunological mechanisms mediated by the major allergens of Parietaria judaica and hypoallergenic hybrid, rPjEDcys
Purpose: Allergy is a hypersensitivity disease IgE-mediated, affecting more than 25% of the population. The
symptoms of IgE-mediated allergies reactions can be transiently ameliorated pharmacologically, but the only
curative treatment of allergies is Allergen-Specific Immunotherapy (SIT). Recombinant hypoallergenic
allergen derivatives with reduced allergenic activity have been engineered to reduce side effects during SIT.
Parietaria judaica (Pj) pollen contains two major allergens belonging to the family of Lipid Tranfer Proteins
(Par j 1 and Par j 2). By means of DNA recombinant technology, a hybrid hypoallergenic (PjEDcys),
expressing disulphide bond variants of Par j 1 and Par j 2, was generated. The aim of this research project is
to study the immunological mechanisms activated by the major allergens of Parietaria judaica, Par j 1 and
Par j 2, and hypoallergenic hybrid rPjEDcys. Moreover, the project I am involved is trying to address the
question whether this engineered hypoallergenic derivative can be a potential products for safer Allergen
Specific Immunotherapy (SIT).
Methods: Par j 1, Par j 2 and PjEDcys were produced as recombinant proteins. Human Peripheral Blood
Mononuclear Cell (PBMC) from P. judaica allergic patients were stimulated in vitro with wild-type
recombinant allergens and hybrid. PBMC proliferation assay, cytokine secretion assay, magnetic cell sorting
of different subset of regulatory T cells, multiparametric flow cytometric analysis and molecular
characterization using Real Time-PCR on sorted cells allow to study the biological properties of wild-type
recombinant allergens and hybrid hypoallergenic derivate.
Results: In vitro analysis suggested that PjEDcys have a reduced allergenity and maintained T cells
reactivity. PBMC of P. judaica allergic patients stimulated in vitro with the hybrid and the wild-type
recombinant allergens scored a percentage of proliferating CD4+ and CD56+ cell higher than unstimulated
sample. Consistent with these data, cytokine secretion assay on CD4+ cells demonstrated that PBMC
stimulation with rPjEDcys showed a percentage of IL-5 and IL-13 secreting T CD4+ cells lower than the
wild-type allergens. Both rPjEDcys and wild-type stimulation promote the secretion of IFN- \u3b3 and IL-10 by
T CD4+ cells. Finally whit the aim to study which subset of regulatory cells respond to wild-tipe allergens
and hypoallergenic hybrid new experiment are setting.
Discussion: In this experimental setting, the use of the major allergens of Pj and the hybrid polypeptides,
rPjEDcys allows me to study the immunological mechanisms activated by the two different antigen
stimulation and to investigate differences between the wild-type allergen and the hypoallergenic mutant
rPjEDcys. Our data showed that CD4+ cells are clearly the predominant cell population proliferating in
response to mixture of Par j 1 and Par j 2 allergens. The hypoallergenic derivate rPjEDcys retain the ability
to stimulate CD4+ cells proliferation like the mixture of allergens (rPar j 1 and rPar j 2). Moreover these
results highlighted a particular interesting datum; the mixture of allergens and the rPjEDcys hybrid showed
the ability to stimulate an innate immune response, inducing CD56+ cells proliferative response. Cytokine
secretion assay demonstrate that rPjEDcys reduce the secretion of IL-5 and IL-13, Th2 cytokines with a
critical role in the development of allergy, compared to wild-type allergens. This may reflect the different
biological function exerted by rPjEDcys.
Conclusion: Collectivelly, our findings demonstrate that PjEDcys show a reduced allergenicity but
maintained its immunogenicity and maybe it is also capable to regulate and redirect the immune response.
These results suggest that PjEDcys represent a useful approach for immunotherapy of allergic disease
Proportional Venn diagram and determinants of allergic respiratory diseases in Italian adolescents
Large variations in prevalence of atopy and allergic diseases are reported worldwide in children, but in epidemiological studies the use of skin prick tests (SPT) and spirometry along with questionnaires is not common in the Mediterranean Area. The present work was aimed at evaluating the prevalence of current asthma (CA), rhinoconjunctivitis (RC), and eczema (E), with atopy and respiratory function, and the role of risk factors for allergic respiratory diseases. A total of 2150 Italian schoolchildren were cross-sectionally investigated through respiratory questionnaire, SPT, and spirometry. A proportional Venn diagram quantified the distribution of CA, RC, and E, stratifying for allergic sensitization to show differences in prevalence of allergic diseases among subjects with and without positive SPT. CA prevalence was 4.2%, RC 17.9%, and E 5.3%. CA and RC increased, while E decreased, with respect to previous local studies. Allergic sensitization prevalence (evaluated as positive response to at least one SPT) was 39.2%. A double Venn diagram identified 15 categories. Atopic CA was threefold more frequent than non-atopic CA. Atopic vs non-atopic RC and E were 9.6% vs 10.3% and 2.0% vs 3.3%, respectively. Atopic vs non-atopic RC associated with CA were 1.6% vs 0.5%; the same figures for RC associated with E were 0.8% vs 1.3%. Asymptomatic atopic subjects were 27.0%. Atopy, RC, parental asthma, and environmental risk factors were associated with CA. Atopy and environmental factors were risk factors also for RC. Asthma and traffic exposure were linked to reduced lung function. Respiratory allergic diseases are still increasing and largely concomitant in Italian adolescents. Atopy is more important for CA than RC. Avoiding exposures to measured environmental risk factors would prevent 41% of current asthma and 34% of rhinoconjunctivitis
Multiple in vitro and in vivo regulatory effects of budesonide in CD4+ T lymphocyte subpopulations of allergic asthmatics.
Abstract
BACKGROUND:
Increased activation and increased survival of T lymphocytes characterise bronchial asthma.
OBJECTIVES:
In this study the effect of budesonide on T cell survival, on inducible co-stimulator T cells (ICOS), on Foxp3 and on IL-10 molecules in T lymphocyte sub-populations was assessed.
METHODS:
Cell survival (by annexin V binding) and ICOS in total lymphocytes, in CD4+/CD25+ and in CD4+/CD25- and Foxp3 and IL-10 in CD4+/CD25+ and in CD4+/CD25-cells was evaluated, by cytofluorimetric analysis, in mild intermittent asthmatics (n = 19) and in controls (n = 15). Allergen induced T lymphocyte proliferation and the in vivo effects of budesonide in mild persistent asthmatics (n = 6) were also explored.
RESULTS:
Foxp3 was reduced in CD4+/CD25- and in CD4+/CD25+ cells and ICOS was reduced in CD4+/CD25+ cells but it was increased in CD4+CD25-in asthmatics when compared to controls. In asthmatics, in vitro, budesonide was able to: 1) increase annexin V binding and to reduce ICOS in total lymphocytes; 2) increase annexin V binding and Foxp3 and to reduce ICOS in CD4+/CD25- cells; 3) reduce annexin V binding and to increase IL-10 and ICOS in CD4+/CD25+ cells; 4) reduce cell allergen induced proliferation. In vivo, budesonide increased ICOS in CD4+/CD25+ while it increased Foxp3 and IL-10 in CD4+/CD25+ and in CD4+/CD25- cells.
CONCLUSIONS:
Budesonide modulates T cell survival, ICOS, Foxp3 and IL-10 molecules differently in T lymphocyte sub-populations. The findings provided shed light on new mechanisms by which corticosteroids, drugs widely used for the clinical management of bronchial asthma, control T lymphocyte activation
Use of a comprehensive diagnostic algorithm for Anisakis allergy in a high seroprevalence Mediterranean setting
Background. Diagnosis of anisakis allergy (AA) is based on the skin prick test (SPT) and specific IgE (sIgE) determination. Anyway, false positivity cases are due to cross reactivity with numerous allergens. The aim of the study was to evaluate the reliability of a comprehensive diagnostic algorithm for the AA. Methods. An observational study was conducted on a sample of consecutive subjects accessing the allergology outpatient ambulatories of two hospitals located in Western Sicily. All the recruited outpatients were tested by Skin Prick Test performed using anisakis extracts by ALK-Abello (Madrid, Spain). Specific IgE dosage for anisakis extracts was then performed by using ImmunoCAP250 (Immunodiagnostics Uppsala, Sweden). Consequently, outpatients who tested positive to first line tests underwent sIgE testing for ascaris and tropomyosin. Lastly, outpatients positive to the first line were invited to be further tested by basophil activation test (BAT) by using Flow CAST kit and anisakis commercial extract (Buhlmann Laboratories AG, Schonenbuch, Switzerland), as confirmatory analysis. Results. One hundred and eleven outpatients with an anamnesis suggestive of sensitization to anisakis (AS) and 466 subjects with chronic urticaria (CU) were recruited in the study. Of these, 22 with AS and 41 with CU showed a sensitization to anisakis allergens. The diagnostic algorithm revealed that 8.8% of outpatients who tested positive to sIgE determination were affected by CU, while 82.5% of all the sIgE positivity was related to cross-reactivity. Overall, a genuine anisakis seroprevalence of 2.3% was documented. Within a sub-sample of 15 subjects with clinical symptoms related to AA, n. 8 showed a real positivity after BAT. A greater response to A. pegreffii allergens as compared to A. simplex was reported. Conclusions. Our preliminary findings support the high clinical specificity of BAT for AA diagnosis, suggesting implementing this method in a comprehensive diagnostic algorithm
Heme oxygenase-1 and carbon monoxide in pulmonary medicine
Heme oxygenase-1 (HO-1), an inducible stress protein, confers cytoprotection against oxidative stress in vitro and in vivo. In addition to its physiological role in heme degradation, HO-1 may influence a number of cellular processes, including growth, inflammation, and apoptosis. By virtue of anti-inflammatory effects, HO-1 limits tissue damage in response to proinflammatory stimuli and prevents allograft rejection after transplantation. The transcriptional upregulation of HO-1 responds to many agents, such as hypoxia, bacterial lipopolysaccharide, and reactive oxygen/nitrogen species. HO-1 and its constitutively expressed isozyme, heme oxygenase-2, catalyze the rate-limiting step in the conversion of heme to its metabolites, bilirubin IXα, ferrous iron, and carbon monoxide (CO). The mechanisms by which HO-1 provides protection most likely involve its enzymatic reaction products. Remarkably, administration of CO at low concentrations can substitute for HO-1 with respect to anti-inflammatory and anti-apoptotic effects, suggesting a role for CO as a key mediator of HO-1 function. Chronic, low-level, exogenous exposure to CO from cigarette smoking contributes to the importance of CO in pulmonary medicine. The implications of the HO-1/CO system in pulmonary diseases will be discussed in this review, with an emphasis on inflammatory states
Tropomyosin: A panallergen that causes a worldwide allergic problem
Background: Panallergens are proteins that take part in key processes of organisms and, therefore, are ubiquitously distributed with highly conserved sequences and structures. One class of these panallergens is composed of the tropomyosins. The highly heatstable tropomyosins comprise the major allergens in crustaceans and mollusks, which make them important food allergens in exposed populations. Tropomyosins are responsible for a widespread immunoglobulin E cross-reactivity among allergens from different sources. Allergic tropomyosins are expressed in many species, including parasites and insects. Methods: This panallergen class is divided, according to it capacity of induced allergic symptoms, into allergenic or nonallergenic tropomyosin. Although vertebrate tropomyosins share ~55% of sequence homology with invertebrate tropomyosins, it has been thought that the invertebrate tropomyosins would not have allergic properties. Nevertheless, in recent years, this opinion has been changed. In particular, tropomyosin has been recognized as a major allergen in many insects. Results: A high grade of homology has been shown among tropomyosins from different species, such as crustaceans and insects, which supports the hypothesis of cross-reactivity among tropomyosins from divergent species. Moreover, the emerging habit of consuming edible insects has drawn the attention of allergists to invertebrate tropomyosin protein due to its potential allergenic risk. Nevertheless, evidence about tropomyosin involvement in clinical allergic response is still scarce and deserves more investigation. Conclusion: This review intended to report allergic reactions associated with different tropomyosins when considering house dust mites, parasites, seafood, and insects, and to summarize our current knowledge about its cross-reactivity because this could help physicians to accurately diagnose patients with food allergy
MODULATING ALLERGIC RESPONSE BY UNLOCKING THE STRUCTURE OF THE MAJOR PARIETARIA ALLERGENS
Fine characterization of immunological mechanisms mediated by the major allergens of Parietaria judaica and by a hypoallergenic hybrid, rPjEDcys.
Allergy is a hypersensitivity disease IgE-mediated, affecting more than 25% of the population. Actually the only curative treatment of allergies is Allergen-Specific Immunotherapy (SIT). Recombinant hypoallergenic allergen derivatives with reduced allergenic activity have been engineered to reduce side effects during SIT.
Parietaria judaica (Pj) pollen contains two major allergens, Par j 1 and expressing disulphide bond variants of Par j 1 and Par j 2, was generated. The aim of this research project is to compare the immunological mechanisms activated by the major allergens of Pj and by rPjEDcys.
In vitro analysis suggested that rPjEDcys has a reduced allergenity and maintains T cells reactivity. In particular we showed that PBMC of Pj allergic patients stimulated in vitro with the hybrid and the wild-type recombinant allergens scored a percentage of proliferating CD4+ and CD56+ cells higher than unstimulated samples. Furthermore, cytokine secretion assays on CD4+ cells demonstrated that rPjEDcys reduces the secretion of two Th2 cytokines that are critical in the development of allergy such as IL-5 and IL-13. Furthermore we observed the selection of a putative pTreg cell subset (defined as CD4+ CD25++ CD127-) in both the w.t. mixture and the rPjEDcys. We characterized these cells at molecular level by REAL-TIME PCR. Moreover, we addressed the kinetic of functional surface marker expression, such as GARP (Glycoprotein A Repetitions Predominant), LAP (Latency-Associated Peptide) and CD39 on CD4+ cells. Our analyses demonstrated that rPjEDcys induces a number of GARP-LAP-CD39 co-expressing cells higher than wild-type recombinant allergens. These results suggest that rPjEDcys represents a useful approach for immunotherapy of allergic disease
Cilomilast counteracts the effects of cigarette smoke in airway epithelial cells
Cigarette smoke extracts (CSE) alter TLR4 expression and activation in bronchial epithelial cells. Cilomilast, a phosphodiesterase-4 inhibitor, inhibits cigarette smoke-induced neutrophilia. This study was aimed to explore whether cilomilast, in a human bronchial epithelial cell line (16-HBE), counteracted CSE effects. In particular, TLR4 expression, IP-10 and IL-8 release, lymphocyte and neutrophil chemotactic activity and ERK and IkBa phosphorylation in CSE and LPS-stimulated 16-HBE were assessed. CSE increased TLR4 expression, reduced IP-10 release and lymphocyte chemotactic activity and increased IL-8 release and neutrophil chemotactic activity. Cilomilast reduced TLR4 expression, IL-8 release and neutrophil chemotactic activity as well as it increased IP-10 release and lymphocyte chemotactic activity. All these cilomilast mediated effects were associated with a reduced ERK1/2 and with an increased IkBa phosphorylation. In conclusion, the present study provides compelling evidences that cilomilast may be considered a possible valid therapeutic option in controlling inflammatory processes present in smokers
IL-33/ST2 axis controls Th2/IL-31 and Th17 immune response in allergic airway diseases
IL-33 targeting ST2 receptor (T1/ST2), expressed on Th2 cell surface, regulates the production of cytokines like IL-17A and IL-31.We studied the role of IL-33/ST2 axis in IL-31 and IL-17A production in patients with allergic rhinitis (AR) and with concomitant allergic asthma and rhinitis (AAR).20 healthy control subjects (HC), 14 AR and 17 AAR subjects were recruited and blood samples collected. IL-33, soluble ST2 (sST2), IL-17A and IL-31 plasma concentrations were measured by ELISA method. T1/ST2, IL-31 and IL-17A cellular expression were studied in peripheral blood mononuclear cells (PBMC) from HC, AR and AAR (n =6 for each group) by flow-cytometry. In vitro, we also evaluated the effect of beclomethasone dipropionate (BDP) on T1/ST2, IL-31 and IL-17A expression in CD3+T-cells from PBMC of AAR (n =6).Plasma levels of IL-33, IL-31 and IL-17A were significantly higher and sST2 was lower in patients with AR and AAR than in HC. IL-31 and IL-17A intracellular levels significantly increased, whereas T1/ST2 expression was significantly lower, in CD3+T-cells from AR and AAR compared to HC. Positive correlations were observed between plasmatic components of IL-33/ST2 axis and IL-31 in both AR and AAR and IL-17A in AAR. In vitro IL-31 and IL-17A intracellular levels decreased after BDP treatment, whereas T1/ST2 expression increased in cultured CD3+T-cells obtained from AAR.IL-33/ST2 axis is involved in Th2/IL-31 and Th17 immune response during the progression of allergic airway disease. In vitro BDP is able to control Th2/IL-31 and Th17 immune response in PBMC from allergic patients