Alternaria alternata acts on human Monocyte-derived Dendritic cells to mediate Th2/Th17 polarisation

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Two examples demonstrate the flow cytometric analysis of peripheral blood samples from one control (top panels) and on the patient (down panels). CD19+ B-cells (R2) are gated within the lymphocyte scatter region (R1). Naive B-cells (R3), marginal zone-like B-cells (R4), switched memory B-cells (R5), and IgM-only memory B-cells (R6) are defined within the IgM lymphoma based on expression of CD27 and IgD. Also, transitional B-cells (R7), plasmablasts (R8) and CD21low B-cells (R9) are defined within the CD21 lymphogate based on expression of CD38 and IgM." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "R. Yazdani, R. Seify, M. Ganjalikhani-Hakemi, H. Abolhassani, N. Eskandari, F. Golsaz-Shirazi, B. Ansaripour, E. Salehi, G. Azizi, N. Rezaei, A. Aghamohammadi" "autores" => array:11 [ 0 => array:2 [ "nombre" => "R." "apellidos" => "Yazdani" ] 1 => array:2 [ "nombre" => "R." "apellidos" => "Seify" ] 2 => array:2 [ "nombre" => "M." 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"apellidos" => "Aghamohammadi" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S030105461630101X?idApp=UINPBA00004N" "url" => "/03010546/0000004500000002/v1_201702200044/S030105461630101X/v1_201702200044/en/main.assets" ] "itemAnterior" => array:18 [ "pii" => "S0301054616301033" "issn" => "03010546" "doi" => "10.1016/j.aller.2016.05.006" "estado" => "S300" "fechaPublicacion" => "2017-03-01" "aid" => "787" "copyright" => "SEICAP" "documento" => "article" "crossmark" => 1 "subdocumento" => "fla" "cita" => "Allergol Immunopathol (Madr). 2017;45:169-74" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:2 [ "total" => 81 "formatos" => array:3 [ "EPUB" => 1 "HTML" => 26 "PDF" => 54 ] ] "en" => array:12 [ "idiomaDefecto" => true "cabecera" => "Original Article" "titulo" => "Airway tone dysfunction among pre-schoolers with positive asthma predictive index: A case–control study" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => "en" "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "169" "paginaFinal" => "174" ] ] "contieneResumen" => array:1 [ "en" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1362 "Ancho" => 1452 "Tamanyo" => 80360 ] ] "descripcion" => array:1 [ "en" => "Comparison in basal central airway resistance (R20) by IOS, and post BD changes in FEV0.5 and FEF25–75 by spirometry, between positive API with and without ICS. Footnote: API, asthma predictive index; BD, bronchodilator; FEF, mid forced expiratory force; FEV0.5, forced expiratory volume in 0.5s; ICS, inhaled corticosteroids; IOS, impulse oscillometry." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "V. Lezana, A. Gajardo, L. Bofill, M. Gutierrez, S. Mora, J.A. Castro-Rodriguez" "autores" => array:6 [ 0 => array:2 [ "nombre" => "V." "apellidos" => "Lezana" ] 1 => array:2 [ "nombre" => "A." "apellidos" => "Gajardo" ] 2 => array:2 [ "nombre" => "L." "apellidos" => "Bofill" ] 3 => array:2 [ "nombre" => "M." "apellidos" => "Gutierrez" ] 4 => array:2 [ "nombre" => "S." "apellidos" => "Mora" ] 5 => array:2 [ "nombre" => "J.A." "apellidos" => "Castro-Rodriguez" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0301054616301033?idApp=UINPBA00004N" "url" => "/03010546/0000004500000002/v1_201702200044/S0301054616301033/v1_201702200044/en/main.assets" ] "en" => array:19 [ "idiomaDefecto" => true "cabecera" => "Original Article" "titulo" => "Alternaria alternata acts on human Monocyte-derived Dendritic cells to mediate Th2/Th17 polarisation" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "175" "paginaFinal" => "182" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "A. Loghmani, R. Raoofi, A. Ownagh, N. Delirezh" "autores" => array:4 [ 0 => array:4 [ "nombre" => "A." "apellidos" => "Loghmani" "email" => array:1 [ 0 => "Alireza_trtr@yahoo.com" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0010" ] 1 => array:2 [ "etiqueta" => "*" "identificador" => "cor0005" ] ] ] 1 => array:3 [ "nombre" => "R." "apellidos" => "Raoofi" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0005" ] ] ] 2 => array:3 [ "nombre" => "A." "apellidos" => "Ownagh" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0005" ] ] ] 3 => array:3 [ "nombre" => "N." "apellidos" => "Delirezh" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0005" ] ] ] ] "afiliaciones" => array:2 [ 0 => array:3 [ "entidad" => "Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran" "etiqueta" => "a" "identificador" => "aff0010" ] 1 => array:3 [ "entidad" => "Department of Infectious Diseases, University of Jahrom Medical Science, Jahrom, Iran" "etiqueta" => "b" "identificador" => "aff0005" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0030" "etiqueta" => "Figure 6" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr6.jpeg" "Alto" => 2064 "Ancho" => 2310 "Tamanyo" => 192974 ] ] "descripcion" => array:1 [ "en" => "The representative flow cytometric histograms obtained from phagocytic analysis of MCM-DC and Induced-DC. Both types of DCs were incubated with FITC-conjugated latex beads for 48h also DCs without beads were used as a negative control then washed with quenching buffer and subjected to flow cytometric analysis." ] ] ] "textoCompleto" => "IntroductionAsthma exacerbation is a major agent of disease for the patients involved with moderate asthma. Sensitisation and exposure to the fungal allergen Alternaria alternata (A. alternata) are a risk factor for the start of severe asthma symptoms, including threatening and mortal responses.<a class="elsevierStyleCrossRefs" href="#bib0175">1–3</a> The exclusive relation between A. alternata and exacerbation of asthma is clear; but the aetiology of the exclusive pathogenesis of A. alternata has not been precisely understood. These responses are mediated by CD4-derived T-cells that are polarised to Th2 or Th17cells phenotypes.<a class="elsevierStyleCrossRef" href="#bib0190">4</a> Dendritic cells (DCs) are one of the main cellular members regulating immune tolerance and response.<a class="elsevierStyleCrossRefs" href="#bib0195">5–7</a> DCs by producing various secretory substance or membrane ligand determine the eventuality of T cell responses, such as T-helper1 (Th1), T-helper2 (Th2), or T-regulatory (Treg) responses.<a class="elsevierStyleCrossRef" href="#bib0210">8</a> Currently, limited data exist concerning how asthma patients develop such disturbance Th2 immune responses to environmental allergens. Overall, dendritic cell pulse for an innocuous antigen or medium is considered a tolerogenic occurrence.<a class="elsevierStyleCrossRefs" href="#bib0195">5,9</a> On the other hand, the mechanism of Th2 immune responses may reflect Th2 immune responses to parasite infection. In specific terms, asthma may offer overexpression of immune responses to chitin-containing organisms, specially mites, and fungi.<a class="elsevierStyleCrossRefs" href="#bib0220">10,11</a> However, the association of immunological mechanisms with impulsive due to chitin-containing organisms and development of Th2 immune responses are not fully understood. The aetiology of human asthma is complicated and multifactorial; probably it involves the interference between genetic factors and environmental motives. As a major environmental motive, the relationship between fungal exposure and asthma has been recognised pathologically and epidemiologically.<a class="elsevierStyleCrossRef" href="#bib0175">1</a> In particular, there is a great deal of evidence suggesting a relationship between a ubiquitous environmental fungus A. alternata and asthma.<a class="elsevierStyleCrossRefs" href="#bib0180">2,12</a>A. alternata is ubiquitous and unique both in outdoor and indoor,<a class="elsevierStyleCrossRef" href="#bib0235">13</a> and for the high rates of spore germination and antigen propagation.<a class="elsevierStyleCrossRef" href="#bib0240">14</a> Exposure to A. alternata is a major risk factor for asthma and allergic.<a class="elsevierStyleCrossRef" href="#bib0245">15</a> Severe asthma and acute exacerbations of asthma have also been associated with increased airborne exposure to A. alternata spores and consequently its pollen pollution.<a class="elsevierStyleCrossRef" href="#bib0250">16</a> In mice, the relationship between asthma and stimulation by fungal antigen, such as aspergillus and A. alternata, has been clinically and pathologically explained.<a class="elsevierStyleCrossRefs" href="#bib0175">1,2</a> The mouse models of asthma offer a wide range of experimental possibilities, but due to their limitations such as their different physiology, we decided to use other models.<a class="elsevierStyleCrossRef" href="#bib0255">17</a> To survey the immunological mechanisms involved in Th2 responses, we used A. alternata to model environmental exposure correlated to asthma.Materials and methodsThe objective of this study was to perceive the mechanisms engaged in asthma. The study was conducted in the cleaning room of the research centre of biotechnology at Urmia University and zoonosis researches central of Jahrom Universty of medical sciences from September 2013 to February 2014. Various tests have been used to evaluate Autologous T cell responses by co-culturing with DCs. In the meantime, the phagocytic activity of pulsed DCs by A. alternata was examined. These experiments were replicated five times.Preparation of fungus extractA. alternata spore purchased from Iranian industrial and scientific research organisation (PTCC 5248) was cultured on Sabaroud dextrose agar at 25°C for five days. Mature fungi subculture on Czapek's agar was used to produce a large number of spores. In addition, the amount of 1×107 spores was collected by Hanks solution and passed through sterile Tampon, subsequently transported on liquid culture of Yeast nitrogen base (37°C, 5% CO2, 5% humidity) for 48h in order to improve the growth of mycelium. Then grown-up centrifuged myceliums (2000×g) were added to PBS buffer containing 2×10−3M protease inhibitor (Sigma-USA), 50×10−3M EDTA (Sigma-USA), 50×10−3 Tris–HCl and sonicated on a sonicator (20,000 AMP), 10s interval (apparatus 10s was off and 10s was on) and totally 5min duration. After sonication, the homogenised fungi were centrifuged (7000×g at 4°C), and the supernatant were dialysed by dialysing tube (cut off: 14,000) full off. The extract was dehydrated by freeze dryer (CHRIST ALPHA 1.4, UK) to reduce the volume of water.<a class="elsevierStyleCrossRef" href="#bib0260">18</a> The resulted extract was filtered through fine pores (22μm in diameter) and protein of solution was measured by BRADFORD method.<a class="elsevierStyleCrossRef" href="#bib0265">19</a> The final extract was considered 1mg/ml and it was stored at −70°C until use.Preparation of Peripheral Blood Mononuclear Cell (PBMC)Heparinised blood was obtained from volunteer donors (200U/ml) in sterile conditions and mixed with the equal volume of culture medium RPMI-1640 (Gibco, UK). Diluents blood was transported gently on Ficoll-Hypaque (Sigma, USA) and centrifuged for 15min in 800×g. PBMC located between Ficoll-Hypaque and diluents blood were collected, and washed by RPMI-1640 then it was centrifuged for 10min in 480×g in order to be deleted from platelets. Cellular pellet was washed again by RPMI-1640 for 10min in 200×g. The number and viability of cells were assigned by Trypan blue.<a class="elsevierStyleCrossRef" href="#bib0270">20</a>Production of control DCs in the presence of Monocyte-conditioned Medium (MCM) and its Inductive with A. alternata extractsMCM was prepared as described elsewhere. Briefly,<a class="elsevierStyleCrossRef" href="#bib0270">20</a> PBMC was plated onto the human Ig-coated Petri dish for one hour, non-adherent cells were washed away and Ig-adherent cells were incubated in fresh complete medium with 1% autologous plasma at 37°C for 24h. The medium was collected and saved at −20°C until time of use. The isolation of T cells was performed using anti-CD3 magnetic bead cell sorting technique (Miltenyi Biotec, Germany). 4×106 PBMC in RPMI-1640 (6ml) were transferred to T-25 flask and after 2h the adhered cells to the T-25 flask were washed three times with RPMI-1640 and finally the adhered cells cultured with RPMI-1640 (5ml) supplemented 2% serum AB+. The preparation of control DC has been occurred at five stages; at the first stage and day 0, the adherent cells were converted to immature dendritic cells (ImDC) by using granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 (500U/ml). At the second stage (day 3), the equal amount of GM-CSF and IL-4 (Sigma, USA) was added to the culture for the maturation of DC. At the third stage (4 day) MCM was added to the DC culture. Additionally, 100μg/ml of A. alternata extracts and MCM (20%, v/v) were added to the induced group (Induced-group), but in the control group, only MCM (MCM-DC) was used. All of these stages occurred in sterile conditions and incubation performed at 37°C; 5% CO2. On day 7, DCs were harvested, and morphological changes of DC; stimulation of T cell phagocytic capability were assigned.<a class="elsevierStyleCrossRef" href="#bib0270">20</a>Estimation of DC yield and viability from plated PBMCSeparated DCs from cell culture flasks in day 7 were submitted to the count and assessment of their viability by trypan blue exclusion test. The percentage of yield was estimated by the following formula<a class="elsevierStyleCrossRef" href="#bib0275">21</a>:<elsevierMultimedia ident="eq0005"></elsevierMultimedia>Microscopic analysisThe bottoms of the culturing flasks were observed daily by inverted microscope. The shape and size of cells as well as their composition were compared in both groups. Extended cells without projections were considered as macrophages<a class="elsevierStyleCrossRef" href="#bib0270">20</a> and the round and unchanged cells were accounted as lymphocytes. Platelets were considered as the smallest cells with irregular surfaces.Phenotypic analysisFor phenotypic analysis, direct immunofluorescence was used for the cell surface staining of DCs, which were stained in FACS buffer (0.2 BSA, 0.02 sodium azaid in PBS) contain 1×105 cells per staining. Staining was performed by incubation DC with FITC-conjugated mouse antibodies against CD14, CD83, anti-human leukocyte antigen-DR (HLA-DR) and the appropriate isotype-matched controls at 4°C for twenty minutes (DAKO, Denmark). Samples were analysed on FACS DAKO (Partec, Germany) using FlowMax Software.<a class="elsevierStyleCrossRef" href="#bib0270">20</a>Cytokine production assayAfter DCs were stimulated with or without A. alternata extract, they were analysed for cytokine profile then were incubated with T cells and cytokine production of T cell was analysed the same as DCs by ELISA method. The non-adherent DCs were stimulated with or without 100μg/ml A. alternata extract for 24h. After that DCs were washed three times with Foetal Bovine Serum (FBS). 100μl of the suspended 2×105 cell/ml in RPMI 1640 supplemented 2% serum AB+ were seeded in round-bottom 96-well microplates. T cells, which were isolated from autologous human, were added to the wells at a 1:5 of DC:T cell ratio and co-cultured for 24. The concentrations of IL-12, IL-10 and IL-23 were measured from DC supernatant capture as well as IL-17, Il-4 and Interferon-γ (IFN-γ) were measured as DC-T cell co-culture profile (R&D Systems); sensitivities for IL-4, IFN-γ and IL-17 were 2, 3 and 0.5pg/ml, respectively.Evaluation of phagocytic activityThe phagocytic activity of DCs was computed by evaluating the uptake of FITC-conjugated latex beads (Sigma, Munich, Germany). 20μl latex bead florescent (FITC-conjugated) at concentration 2.5×105 bead per ml was incubated on human serum AB+ for seven minutes. Then, the amount of 25×105 mature dendritic cell (mDC) along with 60μl phagocytic buffer reached to the total volume 100μl in 96-well plate. DCs without beads were used as a negative control. After 48h of incubation, the cells were harvested and the extracellular fluorescence was quenched in quenching buffer. Finally, the number of engulfed latex beads in both MCM-DC and Induced-DC were assigned. Phagocytic activity was analysed in terms of percentage and mean fluorescence intensity (MFI) of phagocytic cell on DAKO flow cytometry system (Partec, Germany) and FlowMax Software by flow cytometry (DAKO, Germany). The percentage of phagocytosis was identified according to the following equation<a class="elsevierStyleCrossRef" href="#bib0275">21</a>:<elsevierMultimedia ident="eq0010"></elsevierMultimedia>All of the statistical analyses were calculated by SPSS-17 Software, and Tukey test was used for data analysis. p<0.05 was considered significant. Microsoft Excel was employed to draw diagrams. The data were presented as Mean±SEM.ResultsYield and viabilityOur results indicated that by using MCM or A. alternata extract, either 5.56±1.45 or 6.69±1.85 percent of plated PBMCs was differentiated into DCs, respectively. The viability of MCM-DC and A. alternata extract DC (Induced-DC) were 70.66±9.8 and 91.65±7.08 percent, respectively. The differences in both yield and viability of the resultant DCs by the two methods were significant (p<0.05).Microscopic analysisMorphological studies were conducted by inverted microscope at different magnifications and the results indicated that after five days, the adherent cells (at the present of GM-CSF, IL-4) lost their adhesion and caused the induced group of floating cells after adding A. alternata extract. These cells looked larger than monocytes containing large intracellular granules (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>).<elsevierMultimedia ident="fig0005"></elsevierMultimedia>Immunophenotyping generated DCsWhen DCs were incubated for 24h with A. alternata extract, their expression of MHC-II and costimulatory molecule including CD83, increased dramatically to the DCs which were cultured with only MCM. After 48h, A. alternata extract down-regulated the expression of CD14 that was implicating in DC maturation development. The flow cytometric analysis of DCs showed the significant increase of CD83 expressing DCs through A. alternata extract-DCs compared with MCM-DCs (19.7±3.9 vs. 50.4±4.5) (p<0.01). In addition, a higher percentage of HLA-DR were expressed by A. alternata extract-DCs (77.6±7 vs. 45.5±1.01) and lower percentage of CD14 (15.8±1.2 vs. 29.9.±4.1) were observed by MCM-DCs, with their differences being significant (p<0.05) (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>).<elsevierMultimedia ident="fig0010"></elsevierMultimedia>DCs stimulated with A. alternata extract promote T cell production of Th2-type cytokinesThese in vitro observations suggested that DCs are activated by A. alternata extract and promoted CD4_T cell differentiation towards a Th2 type. Monocyte-derived DCs (MoDc) were stimulated with A. alternata extract or medium control, and washed, and then incubated with isogenic T cells. In this study, IL-10, IL-12 and IL-23 were evaluated as DC cytokine productions. Therefore, the DCs which had been stimulated by A. alternata were shown their IL-10 secretion dominated to IL-12 and IL-23 significantly (p<0.01). Isogenic T cells incubated with control DCs (stimulated with medium control) made IL-4, IL-17, and IFN-γ. Importantly, isogenic T cells incubated with A. alternata-stimulated DCs produced more IL-4 and IL-17 but they produced less IFN-γ. The results confirmed that the frequency of IL-4 and IL-17 producing T cells increased significantly (p<0.01), on the other hand IFN-γ producing T cells significantly decreased when those were incubated with A. alternata-stimulated DCs (p<0.05) (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>).<elsevierMultimedia ident="fig0015"></elsevierMultimedia>Phagocytic activityThe results of phagocytic evolution have been shown in two forms: (A) the phagocytosis of thorough DCs and the percentage of phagocytosis in each DC (MFI), shown in <a class="elsevierStyleCrossRefs" href="#fig0020">Figs. 4 and 5</a>, respectively. Furthermore, the data are shown graphically in <a class="elsevierStyleCrossRef" href="#fig0030">Fig. 6</a>. The results of florescence intensity and percentage of phagocytosis (shown in <a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>) indicated that the rate of phagocytosis in MoDc stimulated with A. alternata extract significantly decreased (p<0.01), but this percentage significantly increased in MFI (p<0.05).<elsevierMultimedia ident="fig0020"></elsevierMultimedia><elsevierMultimedia ident="fig0025"></elsevierMultimedia><elsevierMultimedia ident="fig0030"></elsevierMultimedia>DiscussionAlthough frequent studies have been conducted on the properties of dendritic activity in mice, their importance in the development of human allergic responses remains largely unknown. In this study, we have used an A. alternata-related asthma model to evaluate the importance of dendritic activities in the presence an A. alternata extract for triggering Th2 responses in vitro. MCM can perform DC maturation without specific antigen; such soluble non-pathogenic proteins, when delivered through DC, do not excite potent immune responses but instead induce a state of hypo responsive specific to medium.<a class="elsevierStyleCrossRef" href="#bib0195">5</a> Consistent with previous reports,<a class="elsevierStyleCrossRef" href="#bib0195">5</a> we found minimal sensitisation to MCM when DCs were exposed to only MCM. In contrast, when MCM was administered in the presence of A. alternata extracts, DC maturation and CD4-drived Th2 cytokine responses developed sturdily.<a class="elsevierStyleCrossRefs" href="#bib0280">22,23</a> Herein, when DCs were stimulated with A. alternata in vitro, we observed the following: they up-regulated their expression of MHC-II and costimulatory molecules, including CD83. Generally, when DCs show low levels of MHC-II and costimulatory molecules, they induce T cell tolerance or anergy.<a class="elsevierStyleCrossRef" href="#bib0215">9</a> However, the A. alternata-mediated Th2 response was independent of TLR4 and TLR2.<a class="elsevierStyleCrossRef" href="#bib0285">23</a> Now what products of A. alternata possess such strong Th2 adjuvant activity? Oliver, regarding this issue, mentions that protease from A. alternata is able to activate TLR4 and Myd88 signalling and the outcome development Th2 potent response.<a class="elsevierStyleCrossRefs" href="#bib0280">22,24</a> Whereas the IL-12 family cytokines are strong stimulator of the Th1 response thus DCs exposed to A. alternata can polarise T cell response to the Th2 type,<a class="elsevierStyleCrossRefs" href="#bib0290">24,25</a> one important condition for DCs to induce Th2 responses is likely the down-regulation of these cytokines. Several studies suggested that Th2 responses are likely resulted from the absence of IL-12 or over-existence of IL-10.<a class="elsevierStyleCrossRefs" href="#bib0275">21,23,26</a> This conclusion was conspicuous in our study. In contrast, other studies show that Th2 cell development requires ligands expression or soluble compound secretory with the mediation of DCs.<a class="elsevierStyleCrossRefs" href="#bib0285">23,26,27</a> For example, the interaction between OX40L on Antigen Presenting Cell and OX40 on T cells is a major key in the proliferation of CD4-T cell and participation of Th2 responses. One of our findings that was analogical with asthma subject showed the increase of IL-10 level in Induced-DC group, which is consistent with a previous report that there was an increased IL-10 mRNA expression in allergy and atopic asthma.<a class="elsevierStyleCrossRef" href="#bib0310">28</a> On the other hand, it is possible that the discovery of IL-17 producing T cells has added an additional layer of complexity to the regulation of allergic inflammation. Although IL-10 was dominant in the presence of A. alternata extract, IL-17 promoted parallel one.<a class="elsevierStyleCrossRefs" href="#bib0175">1,29</a> Concerning this issue, Wang explained that a novel subset of Th2 memory/effector cells exists that co-expresses the transcription factors GATA3 and RORγt and co-produces Th17 and Th2 cytokines. This subset that is termed Classical Th2 memory/effector cells had the potential to produce IL-17 after stimulation with pro-inflammatory cytokines IL-1β, IL-6, and IL-2.<a class="elsevierStyleCrossRef" href="#bib0320">30</a> It was also shown that chitin led to the production of IL-17A with involving TLR2 signalling through the MyD88- independent pathway.<a class="elsevierStyleCrossRef" href="#bib0320">30</a> Recent studies have disclosed that IL-23 is an arch axis to maintain Th17 cells. Despite families and structural similarity between IL-12 and IL-23,<a class="elsevierStyleCrossRef" href="#bib0325">31</a> it seems that IL-23, more than IL-12, plays pro-inflammatory roles in this study. On the contrary, in the previous report, we observed DCs co-pulsed with A. alternata and Myelin Basic Protein decreased IL-17.<a class="elsevierStyleCrossRef" href="#bib0330">32</a> It is likely that DCs appear in the form of down regulatory when faced with multi-epitopes of IL-17 inducer. In our finding, observed T cells were significantly differentiated. Another notable characterisation of DC is phagocytic capabilities, so that it can be expected that turning ImDC into mDC can decrease the ability of DC to phagocytosis as well as all needs to engulf antigen such as surface receptor. In contrast, it increases the presentation of antigen, which leads to escalating T cell stimulation ability in DC.<a class="elsevierStyleCrossRef" href="#bib0205">7</a> Furthermore, the IL-4 that abolished the release of cytokines in DC and macrophages had a minimal inhibitory effect on DC and macrophages from patients with asthma.<a class="elsevierStyleCrossRef" href="#bib0315">29</a> According to the results, it was indicated that the rate of phagocytosis in Monocyte-derived DCs, which were stimulated with A. alternata extract was significantly decreased. DC exposure to A. alternata probably prevents tolerance to innocuous placebo as MCM and facilitates towards Th2 polarisation. Therefore, the potent biological activities of A. alternata facilitate both the sensitisation and effectors phases of immunologic Th2 responses. These activities and responses may offer the mechanism of triggering asthma and the well-perceived correlation between fungi, development and exacerbation of asthma and allergic airway diseases in humans.<a class="elsevierStyleCrossRefs" href="#bib0285">23,33,34</a> Our findings suggest that the DCs when accompanied with A. alternata antigen through interfering in cytokines or presentation and shift to the aberrant secretion of T cells pro-inflammatory cytokines such as IL-17 subsequent Th2/Th17 potent polarisation may be engaged in or exacerbate asthma. In the end, one question remained: If A. alternata is a strong Th2 response contrast with innocuous medium, why only some, but not all, individuals or animals promote allergic airway diseases? Further study of genetic analysis, individual specific immune and identification of antigens of A. alternata that interfere in the potent Th2 effects of A. alternata will show the mechanisms involved in the development of Th2 immune responses better.Conflict of interestThe authors have no conflict of interest to declare.FundingJahrom university of medical sciences and Urmia university.Authors’ contributionsAll authors had equal role in design, work, statistical analysis and manuscript writing.Ethical disclosuresConfidentiality of dataThe authors declare that no patient data appears in this article.Right to privacy and informed consentThe authors declare that no patient data appears in this article.Protection of human subjects and animals in researchThe authors declare that no experiments were performed on humans or animals for this investigation." "textoCompletoSecciones" => array:1 [ "secciones" => array:13 [ 0 => array:3 [ "identificador" => "xres802996" "titulo" => "Abstract" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Introduction" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Materials and methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusion" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec801326" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "xpalclavsec801327" "titulo" => "Abbreviations" ] 3 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 4 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:8 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Preparation of fungus extract" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Preparation of Peripheral Blood Mononuclear Cell (PBMC)" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Production of control DCs in the presence of Monocyte-conditioned Medium (MCM) and its Inductive with A. alternata extracts" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Estimation of DC yield and viability from plated PBMC" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "Microscopic analysis" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Phenotypic analysis" ] 6 => array:2 [ "identificador" => "sec0045" "titulo" => "Cytokine production assay" ] 7 => array:2 [ "identificador" => "sec0050" "titulo" => "Evaluation of phagocytic activity" ] ] ] 5 => array:3 [ "identificador" => "sec0055" "titulo" => "Results" "secciones" => array:5 [ 0 => array:2 [ "identificador" => "sec0060" "titulo" => "Yield and viability" ] 1 => array:2 [ "identificador" => "sec0065" "titulo" => "Microscopic analysis" ] 2 => array:2 [ "identificador" => "sec0070" "titulo" => "Immunophenotyping generated DCs" ] 3 => array:2 [ "identificador" => "sec0075" "titulo" => "DCs stimulated with A. alternata extract promote T cell production of Th2-type cytokines" ] 4 => array:2 [ "identificador" => "sec0080" "titulo" => "Phagocytic activity" ] ] ] 6 => array:2 [ "identificador" => "sec0085" "titulo" => "Discussion" ] 7 => array:2 [ "identificador" => "sec0090" "titulo" => "Conflict of interest" ] 8 => array:2 [ "identificador" => "sec0095" "titulo" => "Funding" ] 9 => array:2 [ "identificador" => "sec0100" "titulo" => "Authors’ contributions" ] 10 => array:3 [ "identificador" => "sec0105" "titulo" => "Ethical disclosures" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0110" "titulo" => "Confidentiality of data" ] 1 => array:2 [ "identificador" => "sec0115" "titulo" => "Right to privacy and informed consent" ] 2 => array:2 [ "identificador" => "sec0120" "titulo" => "Protection of human subjects and animals in research" ] ] ] 11 => array:2 [ "identificador" => "xack269025" "titulo" => "Acknowledgments" ] 12 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2016-04-29" "fechaAceptado" => "2016-07-08" "PalabrasClave" => array:1 [ "en" => array:2 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec801326" "palabras" => array:4 [ 0 => "Alternaria alternata" 1 => "Dendritic cell" 2 => "Environmental fungus" 3 => "Th2/Th17" ] ] 1 => array:4 [ "clase" => "abr" "titulo" => "Abbreviations" "identificador" => "xpalclavsec801327" "palabras" => array:17 [ 0 => "A. alternata" 1 => "CD" 2 => "DCs" 3 => "FACS" 4 => "FBS" 5 => "GM-CSF" 6 => "HLA-DR" 7 => "IFN-γ" 8 => "IL" 9 => "ImDC" 10 => "MCM" 11 => "mDC" 12 => "MFI" 13 => "MoDc" 14 => "PBMC" 15 => "Th" 16 => "Treg" ] ] ] ] "tieneResumen" => true "resumen" => array:1 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "IntroductionAlthough the mechanism of asthma is not precisely understood in humans, clinical and epidemiological studies have offered a potential relationship between exposure to environmental fungi, such as Alternaria alternata (A. alternata) and the development and exacerbation of asthma. The aim of this project is to investigate the mechanisms of Th2 responses by A. alternata as a clinically relevant model for the environmental exposure. Materials and methodsPlastic adherent monocytes were cultured with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to convert these cells into Monocyte-derived Dendritic cells (MoDc) and then matured in the presence of Monocyte-Conditioned Medium (MCM) as the control group and MCM+ A. alternata extract as the inductive groups. ResultsThe results indicated that the expression of CD14 decreased and CD83 and anti-human leukocyte antigen-DR (HLA-DR) increased in the inductive groups in comparison with the control group. More importantly, A. alternata inhibited IL-12 production by activated dendritic cells (DCs), and the DCs exposed to A. alternata enhanced the Th2 polarisation of CD4+ T cells. The production amount of IL-10 overcame IL-12 as well as Il-23 increased significantly, and hand in T cells the production of cytokines Interferon-γ (IFN-γ) decreased. However, both IL-17 and IL-4 increased (p<0.05). Phagocytic activity in the inductive groups decreased significantly compared with the control group. ConclusionThe asthma-related environmental fungus A. alternata, with an effect on dendritic cells profile mediates TH2/TH17. Such immunodysregulation properties of causative environmental fungi may explain their strong relationship with human asthma and allergic diseases." "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Introduction" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Materials and methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusion" ] ] ] ] "multimedia" => array:8 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 697 "Ancho" => 1400 "Tamanyo" => 196343 ] ] "descripcion" => array:1 [ "en" => "Morphological appearance of MCM-DCs (A) and Induced-DCs (B). Analysis by inverted microscope revealed that in comparison, Induced-DC were longer and had more frills than MCM-DC. These cells (both groups) looked larger than monocytes containing large intracellular granules also were more non-adherent." ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 2731 "Ancho" => 2698 "Tamanyo" => 311710 ] ] "descripcion" => array:1 [ "en" => "Immunophenotyping of DCs. (A) Representative flow cytometric histograms, obtained from MCM-DCs and Induced-DCs, stained with FITC conjugated mAb against CD14, CD83, and HLA-DR. As shown in histograms, the Induced-DCs (down) produced a higher fluorescent intensity relative to MCM-DCs (up) using the three stained markers. (B) Flow cytometric analysis showing increased CD83, HLA-DR and decreasing expression among Induced-DCs compared with MCM-DCs. Considering the data p<0.001 into CD83 and HLA-DR, as well as p<0.01 into CD14 significant differences existed between the two groups." ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 2089 "Ancho" => 1396 "Tamanyo" => 86111 ] ] "descripcion" => array:1 [ "en" => "Both A and B show data using MCM-DCs and Induced-DCs, A show Concentrations of IL-10, IL-12 and IL-23 in the supernatant of DCs and B show IL-4, IFN-γ and IL-17 in the supernatant from the DC-T cell co-culture. The measurement was performed using commercially available ELISA kits. * and ** represent significant difference between these three tested groups p<0.05 and p<0.01 respectively" ] ] 3 => array:7 [ "identificador" => "fig0020" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 1170 "Ancho" => 1329 "Tamanyo" => 36871 ] ] "descripcion" => array:1 [ "en" => "Flow cytometric analysis of phagocytic cells revealed significant increase in number of phagocyting cells accompany with mature among MCM-DCs to Induced-DCs (p<0.01). * and ** represent significant difference between these three tested groups p<0.05 and p<0.01 respectively." ] ] 4 => array:7 [ "identificador" => "fig0025" "etiqueta" => "Figure 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 1015 "Ancho" => 1288 "Tamanyo" => 32640 ] ] "descripcion" => array:1 [ "en" => "Flow cytometric analysis of phagocytic cells revealed significant increased MFI as phagocytosis power per DC accompany with maturation among MCM-DCs to Induced-DCs (p<0.05)." ] ] 5 => array:7 [ "identificador" => "fig0030" "etiqueta" => "Figure 6" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr6.jpeg" "Alto" => 2064 "Ancho" => 2310 "Tamanyo" => 192974 ] ] "descripcion" => array:1 [ "en" => "The representative flow cytometric histograms obtained from phagocytic analysis of MCM-DC and Induced-DC. 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"vista" => "all" ] ] ] "idiomaDefecto" => "en" "url" => "/03010546/0000004500000002/v1_201702200044/S0301054616301112/v1_201702200044/en/main.assets" "Apartado" => array:4 [ "identificador" => "5554" "tipo" => "SECCION" "en" => array:2 [ "titulo" => "Original articles" "idiomaDefecto" => true ] "idiomaDefecto" => "en" ] "PDF" => "https://static.elsevier.es/multimedia/03010546/0000004500000002/v1_201702200044/S0301054616301112/v1_201702200044/en/main.pdf?idApp=UINPBA00004N&text.app=https://www.elsevier.es/" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0301054616301112?idApp=UINPBA00004N"]

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A. Loghmania,

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Alireza_trtr@yahoo.com

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, R. Raoofib, A. Ownaghb, N. Delirezhb

a Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

b Department of Infectious Diseases, University of Jahrom Medical Science, Jahrom, Iran

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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Asthma exacerbation is a major agent of disease for the patients involved with moderate asthma&#46; Sensitisation and exposure to the fungal allergen <span class="elsevierStyleItalic">Alternaria alternata</span> &#40;<span class="elsevierStyleItalic">A&#46; alternata</span>&#41; are a risk factor for the start of severe asthma symptoms&#44; including threatening and mortal responses&#46;<a class="elsevierStyleCrossRefs" href="#bib0175"><span class="elsevierStyleSup">1&#8211;3</span></a> The exclusive relation between <span class="elsevierStyleItalic">A&#46; alternata</span> and exacerbation of asthma is clear&#59; but the aetiology of the exclusive pathogenesis of <span class="elsevierStyleItalic">A&#46; alternata</span> has not been precisely understood&#46; These responses are mediated by CD4-derived T-cells that are polarised to Th2 or Th17cells phenotypes&#46;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">4</span></a> Dendritic cells &#40;DCs&#41; are one of the main cellular members regulating immune tolerance and response&#46;<a class="elsevierStyleCrossRefs" href="#bib0195"><span class="elsevierStyleSup">5&#8211;7</span></a> DCs by producing various secretory substance or membrane ligand determine the eventuality of T cell responses&#44; such as T-helper1 &#40;Th1&#41;&#44; T-helper2 &#40;Th2&#41;&#44; or T-regulatory &#40;Treg&#41; responses&#46;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">8</span></a> Currently&#44; limited data exist concerning how asthma patients develop such disturbance Th2 immune responses to environmental allergens&#46; Overall&#44; dendritic cell pulse for an innocuous antigen or medium is considered a tolerogenic occurrence&#46;<a class="elsevierStyleCrossRefs" href="#bib0195"><span class="elsevierStyleSup">5&#44;9</span></a> On the other hand&#44; the mechanism of Th2 immune responses may reflect Th2 immune responses to parasite infection&#46; In specific terms&#44; asthma may offer overexpression of immune responses to chitin-containing organisms&#44; specially mites&#44; and fungi&#46;<a class="elsevierStyleCrossRefs" href="#bib0220"><span class="elsevierStyleSup">10&#44;11</span></a> However&#44; the association of immunological mechanisms with impulsive due to chitin-containing organisms and development of Th2 immune responses are not fully understood&#46; The aetiology of human asthma is complicated and multifactorial&#59; probably it involves the interference between genetic factors and environmental motives&#46; As a major environmental motive&#44; the relationship between fungal exposure and asthma has been recognised pathologically and epidemiologically&#46;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">1</span></a> In particular&#44; there is a great deal of evidence suggesting a relationship between a ubiquitous environmental fungus <span class="elsevierStyleItalic">A&#46; alternata</span> and asthma&#46;<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">2&#44;12</span></a><span class="elsevierStyleItalic">A&#46; alternata</span> is ubiquitous and unique both in outdoor and indoor&#44;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">13</span></a> and for the high rates of spore germination and antigen propagation&#46;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">14</span></a> Exposure to <span class="elsevierStyleItalic">A&#46; alternata</span> is a major risk factor for asthma and allergic&#46;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">15</span></a> Severe asthma and acute exacerbations of asthma have also been associated with increased airborne exposure to <span class="elsevierStyleItalic">A&#46; alternata</span> spores and consequently its pollen pollution&#46;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">16</span></a> In mice&#44; 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The study was conducted in the cleaning room of the research centre of biotechnology at Urmia University and zoonosis researches central of Jahrom Universty of medical sciences from September 2013 to February 2014&#46; Various tests have been used to evaluate Autologous T cell responses by co-culturing with DCs&#46; In the meantime&#44; the phagocytic activity of pulsed DCs by <span class="elsevierStyleItalic">A&#46; alternata</span> was examined&#46; These experiments were replicated five times&#46;</p><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Preparation of fungus extract</span><p id="par0015" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">A&#46; alternata</span> spore purchased from Iranian industrial and scientific research organisation &#40;PTCC 5248&#41; was cultured on Sabaroud dextrose agar at 25<span class="elsevierStyleHsp" style=""></span>&#176;C for five days&#46; Mature fungi subculture on Czapek&#39;s agar was used to produce a large number of spores&#46; 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The isolation of T cells was performed using anti-CD3 magnetic bead cell sorting technique &#40;Miltenyi Biotec&#44; Germany&#41;&#46; 4<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span> PBMC in RPMI-1640 &#40;6<span class="elsevierStyleHsp" style=""></span>ml&#41; were transferred to T-25 flask and after 2<span class="elsevierStyleHsp" style=""></span>h the adhered cells to the T-25 flask were washed three times with RPMI-1640 and finally the adhered cells cultured with RPMI-1640 &#40;5<span class="elsevierStyleHsp" style=""></span>ml&#41; supplemented 2&#37; serum AB<span class="elsevierStyleSup">&#43;</span>&#46; The preparation of control DC has been occurred at five stages&#59; at the first stage and day 0&#44; the adherent cells were converted to immature dendritic cells &#40;ImDC&#41; by using granulocyte macrophage colony stimulating factor &#40;GM-CSF&#41; and IL-4 &#40;500<span class="elsevierStyleHsp" style=""></span>U&#47;ml&#41;&#46; At the second stage &#40;day 3&#41;&#44; the equal amount of GM-CSF and IL-4 &#40;Sigma&#44; USA&#41; was added to the culture for the maturation of DC&#46; At the third stage &#40;4 day&#41; MCM was added to the DC culture&#46; Additionally&#44; 100<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;ml of <span class="elsevierStyleItalic">A&#46; alternata</span> extracts and MCM &#40;20&#37;&#44; v&#47;v&#41; were added to the induced group &#40;Induced-group&#41;&#44; but in the control group&#44; only MCM &#40;MCM-DC&#41; was used&#46; All of these stages occurred in sterile conditions and incubation performed at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#59; 5&#37; CO<span class="elsevierStyleInf">2</span>&#46; On day 7&#44; DCs were harvested&#44; and morphological changes of DC&#59; stimulation of T cell phagocytic capability were assigned&#46;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">20</span></a></p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Estimation of DC yield and viability from plated PBMC</span><p id="par0030" class="elsevierStylePara elsevierViewall">Separated DCs from cell culture flasks in day 7 were submitted to the count and assessment of their viability by trypan blue exclusion test&#46; The percentage of yield was estimated by the following formula<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">21</span></a>&#58;<elsevierMultimedia ident="eq0005"></elsevierMultimedia></p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Microscopic analysis</span><p id="par0035" class="elsevierStylePara elsevierViewall">The bottoms of the culturing flasks were observed daily by inverted microscope&#46; The shape and size of cells as well as their composition were compared in both groups&#46; Extended cells without projections were considered as macrophages<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">20</span></a> and the round and unchanged cells were accounted as lymphocytes&#46; Platelets were considered as the smallest cells with irregular surfaces&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Phenotypic analysis</span><p id="par0040" class="elsevierStylePara elsevierViewall">For phenotypic analysis&#44; direct immunofluorescence was used for the cell surface staining of DCs&#44; which were stained in FACS buffer &#40;0&#46;2 BSA&#44; 0&#46;02 sodium azaid in PBS&#41; contain 1<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">5</span> cells per staining&#46; Staining was performed by incubation DC with FITC-conjugated mouse antibodies against CD14&#44; CD83&#44; anti-human leukocyte antigen-DR &#40;HLA-DR&#41; and the appropriate isotype-matched controls at 4<span class="elsevierStyleHsp" style=""></span>&#176;C for twenty minutes &#40;DAKO&#44; Denmark&#41;&#46; Samples were analysed on FACS DAKO &#40;Partec&#44; Germany&#41; using FlowMax Software&#46;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">20</span></a></p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Cytokine production assay</span><p id="par0045" class="elsevierStylePara elsevierViewall">After DCs were stimulated with or without <span class="elsevierStyleItalic">A&#46; alternata</span> extract&#44; they were analysed for cytokine profile then were incubated with T cells and cytokine production of T cell was analysed the same as DCs by ELISA method&#46; The non-adherent DCs were stimulated with or without 100<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;ml <span class="elsevierStyleItalic">A&#46; alternata</span> extract for 24<span class="elsevierStyleHsp" style=""></span>h&#46; After that DCs were washed three times with Foetal Bovine Serum &#40;FBS&#41;&#46; 100<span class="elsevierStyleHsp" style=""></span>&#956;l of the suspended 2<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">5</span> cell&#47;ml in RPMI 1640 supplemented 2&#37; serum AB<span class="elsevierStyleSup">&#43;</span> were seeded in round-bottom 96-well microplates&#46; T cells&#44; which were isolated from autologous human&#44; were added to the wells at a 1&#58;5 of DC&#58;T cell ratio and co-cultured for 24&#46; The concentrations of IL-12&#44; IL-10 and IL-23 were measured from DC supernatant capture as well as IL-17&#44; Il-4 and Interferon-&#947; &#40;IFN-&#947;&#41; were measured as DC-T cell co-culture profile &#40;R&#38;D Systems&#41;&#59; sensitivities for IL-4&#44; IFN-&#947; and IL-17 were 2&#44; 3 and 0&#46;5<span class="elsevierStyleHsp" style=""></span>pg&#47;ml&#44; respectively&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Evaluation of phagocytic activity</span><p id="par0050" class="elsevierStylePara elsevierViewall">The phagocytic activity of DCs was computed by evaluating the uptake of FITC-conjugated latex beads &#40;Sigma&#44; Munich&#44; Germany&#41;&#46; 20<span class="elsevierStyleHsp" style=""></span>&#956;l latex bead florescent &#40;FITC-conjugated&#41; at concentration 2&#46;5<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">5</span> bead per ml was incubated on human serum AB<span class="elsevierStyleSup">&#43;</span> for seven minutes&#46; Then&#44; the amount of 25<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">5</span> mature dendritic cell &#40;mDC&#41; along with 60<span class="elsevierStyleHsp" style=""></span>&#956;l phagocytic buffer reached to the total volume 100<span class="elsevierStyleHsp" style=""></span>&#956;l in 96-well plate&#46; DCs without beads were used as a negative control&#46; After 48<span class="elsevierStyleHsp" style=""></span>h of incubation&#44; the cells were harvested and the extracellular fluorescence was quenched in quenching buffer&#46; Finally&#44; the number of engulfed latex beads in both MCM-DC and Induced-DC were assigned&#46; Phagocytic activity was analysed in terms of percentage and mean fluorescence intensity &#40;MFI&#41; of phagocytic cell on DAKO flow cytometry system &#40;Partec&#44; Germany&#41; and FlowMax Software by flow cytometry &#40;DAKO&#44; Germany&#41;&#46; The percentage of phagocytosis was identified according to the following equation<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">21</span></a>&#58;<elsevierMultimedia ident="eq0010"></elsevierMultimedia></p><p id="par0055" class="elsevierStylePara elsevierViewall">All of the statistical analyses were calculated by SPSS-17 Software&#44; and Tukey test was used for data analysis&#46; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 was considered significant&#46; Microsoft Excel was employed to draw diagrams&#46; The data were presented as Mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SEM&#46;</p></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Results</span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Yield and viability</span><p id="par0060" class="elsevierStylePara elsevierViewall">Our results indicated that by using MCM or <span class="elsevierStyleItalic">A&#46; alternata</span> extract&#44; either 5&#46;56<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;45 or 6&#46;69<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;85 percent of plated PBMCs was differentiated into DCs&#44; respectively&#46; The viability of MCM-DC and <span class="elsevierStyleItalic">A&#46; alternata</span> extract DC &#40;Induced-DC&#41; were 70&#46;66<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>9&#46;8 and 91&#46;65<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>7&#46;08 percent&#44; respectively&#46; The differences in both yield and viability of the resultant DCs by the two methods were significant &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Microscopic analysis</span><p id="par0065" class="elsevierStylePara elsevierViewall">Morphological studies were conducted by inverted microscope at different magnifications and the results indicated that after five days&#44; the adherent cells &#40;at the present of GM-CSF&#44; IL-4&#41; lost their adhesion and caused the induced group of floating cells after adding <span class="elsevierStyleItalic">A&#46; alternata</span> extract&#46; These cells looked larger than monocytes containing large intracellular granules &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Immunophenotyping generated DCs</span><p id="par0070" class="elsevierStylePara elsevierViewall">When DCs were incubated for 24<span class="elsevierStyleHsp" style=""></span>h with <span class="elsevierStyleItalic">A&#46; alternata</span> extract&#44; their expression of MHC-II and costimulatory molecule including CD83&#44; increased dramatically to the DCs which were cultured with only MCM&#46; After 48<span class="elsevierStyleHsp" style=""></span>h&#44; <span class="elsevierStyleItalic">A&#46; alternata</span> extract down-regulated the expression of CD14 that was implicating in DC maturation development&#46; The flow cytometric analysis of DCs showed the significant increase of CD83 expressing DCs through <span class="elsevierStyleItalic">A&#46; alternata</span> extract-DCs compared with MCM-DCs &#40;19&#46;7<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;9 vs&#46; 50&#46;4<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>4&#46;5&#41; &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;01&#41;&#46; In addition&#44; a higher percentage of HLA-DR were expressed by <span class="elsevierStyleItalic">A&#46; alternata</span> extract-DCs &#40;77&#46;6<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>7 vs&#46; 45&#46;5<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;01&#41; and lower percentage of CD14 &#40;15&#46;8<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;2 vs&#46; 29&#46;9&#46;<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>4&#46;1&#41; were observed by MCM-DCs&#44; with their differences being significant &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">DCs stimulated with <span class="elsevierStyleItalic">A&#46; alternata</span> extract promote T cell production of Th2-type cytokines</span><p id="par0075" class="elsevierStylePara elsevierViewall">These in vitro observations suggested that DCs are activated by <span class="elsevierStyleItalic">A&#46; alternata</span> extract and promoted CD4&#95;T cell differentiation towards a Th2 type&#46; Monocyte-derived DCs &#40;MoDc&#41; were stimulated with <span class="elsevierStyleItalic">A&#46; alternata</span> extract or medium control&#44; and washed&#44; and then incubated with isogenic T cells&#46; In this study&#44; IL-10&#44; IL-12 and IL-23 were evaluated as DC cytokine productions&#46; Therefore&#44; the DCs which had been stimulated by <span class="elsevierStyleItalic">A&#46; alternata</span> were shown their IL-10 secretion dominated to IL-12 and IL-23 significantly &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;01&#41;&#46; Isogenic T cells incubated with control DCs &#40;stimulated with medium control&#41; made IL-4&#44; IL-17&#44; and IFN-&#947;&#46; Importantly&#44; isogenic T cells incubated with <span class="elsevierStyleItalic">A&#46; alternata</span>-stimulated DCs produced more IL-4 and IL-17 but they produced less IFN-&#947;&#46; The results confirmed that the frequency of IL-4 and IL-17 producing T cells increased significantly &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;01&#41;&#44; on the other hand IFN-&#947; producing T cells significantly decreased when those were incubated with <span class="elsevierStyleItalic">A&#46; alternata</span>-stimulated DCs &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Phagocytic activity</span><p id="par0080" class="elsevierStylePara elsevierViewall">The results of phagocytic evolution have been shown in two forms&#58; &#40;A&#41; the phagocytosis of thorough DCs and the percentage of phagocytosis in each DC &#40;MFI&#41;&#44; shown in <a class="elsevierStyleCrossRefs" href="#fig0020">Figs&#46; 4 and 5</a>&#44; respectively&#46; Furthermore&#44; the data are shown graphically in <a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>&#46; The results of florescence intensity and percentage of phagocytosis &#40;shown in <a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>&#41; indicated that the rate of phagocytosis in MoDc stimulated with <span class="elsevierStyleItalic">A&#46; alternata</span> extract significantly decreased &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;01&#41;&#44; but this percentage significantly increased in MFI &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia><elsevierMultimedia ident="fig0025"></elsevierMultimedia><elsevierMultimedia ident="fig0030"></elsevierMultimedia></span></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Discussion</span><p id="par0085" class="elsevierStylePara elsevierViewall">Although frequent studies have been conducted on the properties of dendritic activity in mice&#44; their importance in the development of human allergic responses remains largely unknown&#46; In this study&#44; we have used an <span class="elsevierStyleItalic">A&#46; alternata</span>-related asthma model to evaluate the importance of dendritic activities in the presence an <span class="elsevierStyleItalic">A&#46; alternata</span> extract for triggering Th2 responses in vitro&#46; MCM can perform DC maturation without specific antigen&#59; such soluble non-pathogenic proteins&#44; when delivered through DC&#44; do not excite potent immune responses but instead induce a state of hypo responsive specific to medium&#46;<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">5</span></a> Consistent with previous reports&#44;<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">5</span></a> we found minimal sensitisation to MCM when DCs were exposed to only MCM&#46; In contrast&#44; when MCM was administered in the presence of <span class="elsevierStyleItalic">A&#46; alternata</span> extracts&#44; DC maturation and CD4-drived Th2 cytokine responses developed sturdily&#46;<a class="elsevierStyleCrossRefs" href="#bib0280"><span class="elsevierStyleSup">22&#44;23</span></a> Herein&#44; when DCs were stimulated with <span class="elsevierStyleItalic">A&#46; alternata</span> in vitro&#44; we observed the following&#58; they up-regulated their expression of MHC-II and costimulatory molecules&#44; including CD83&#46; Generally&#44; when DCs show low levels of MHC-II and costimulatory molecules&#44; they induce T cell tolerance or anergy&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">9</span></a> However&#44; the <span class="elsevierStyleItalic">A&#46; alternata</span>-mediated Th2 response was independent of TLR4 and TLR2&#46;<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">23</span></a> Now what products of <span class="elsevierStyleItalic">A&#46; alternata</span> possess such strong Th2 adjuvant activity&#63; Oliver&#44; regarding this issue&#44; mentions that protease from <span class="elsevierStyleItalic">A&#46; alternata</span> is able to activate TLR4 and Myd88 signalling and the outcome development Th2 potent response&#46;<a class="elsevierStyleCrossRefs" href="#bib0280"><span class="elsevierStyleSup">22&#44;24</span></a> Whereas the IL-12 family cytokines are strong stimulator of the Th1 response thus DCs exposed to <span class="elsevierStyleItalic">A&#46; alternata</span> can polarise T cell response to the Th2 type&#44;<a class="elsevierStyleCrossRefs" href="#bib0290"><span class="elsevierStyleSup">24&#44;25</span></a> one important condition for DCs to induce Th2 responses is likely the down-regulation of these cytokines&#46; Several studies suggested that Th2 responses are likely resulted from the absence of IL-12 or over-existence of IL-10&#46;<a class="elsevierStyleCrossRefs" href="#bib0275"><span class="elsevierStyleSup">21&#44;23&#44;26</span></a> This conclusion was conspicuous in our study&#46; In contrast&#44; other studies show that Th2 cell development requires ligands expression or soluble compound secretory with the mediation of DCs&#46;<a class="elsevierStyleCrossRefs" href="#bib0285"><span class="elsevierStyleSup">23&#44;26&#44;27</span></a> For example&#44; the interaction between OX40L on Antigen Presenting Cell and OX40 on T cells is a major key in the proliferation of CD4-T cell and participation of Th2 responses&#46; One of our findings that was analogical with asthma subject showed the increase of IL-10 level in Induced-DC group&#44; which is consistent with a previous report that there was an increased IL-10 mRNA expression in allergy and atopic asthma&#46;<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">28</span></a> On the other hand&#44; it is possible that the discovery of IL-17 producing T cells has added an additional layer of complexity to the regulation of allergic inflammation&#46; Although IL-10 was dominant in the presence of <span class="elsevierStyleItalic">A&#46; alternata</span> extract&#44; IL-17 promoted parallel one&#46;<a class="elsevierStyleCrossRefs" href="#bib0175"><span class="elsevierStyleSup">1&#44;29</span></a> Concerning this issue&#44; Wang explained that a novel subset of Th2 memory&#47;effector cells exists that co-expresses the transcription factors GATA3 and ROR&#947;t and co-produces Th17 and Th2 cytokines&#46; This subset that is termed Classical Th2 memory&#47;effector cells had the potential to produce IL-17 after stimulation with pro-inflammatory cytokines IL-1&#946;&#44; IL-6&#44; and IL-2&#46;<a class="elsevierStyleCrossRef" href="#bib0320"><span class="elsevierStyleSup">30</span></a> It was also shown that chitin led to the production of IL-17A with involving TLR2 signalling through the MyD88- independent pathway&#46;<a class="elsevierStyleCrossRef" href="#bib0320"><span class="elsevierStyleSup">30</span></a> Recent studies have disclosed that IL-23 is an arch axis to maintain Th17 cells&#46; Despite families and structural similarity between IL-12 and IL-23&#44;<a class="elsevierStyleCrossRef" href="#bib0325"><span class="elsevierStyleSup">31</span></a> it seems that IL-23&#44; more than IL-12&#44; plays pro-inflammatory roles in this study&#46; On the contrary&#44; in the previous report&#44; we observed DCs co-pulsed with <span class="elsevierStyleItalic">A&#46; alternata</span> and Myelin Basic Protein decreased IL-17&#46;<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">32</span></a> It is likely that DCs appear in the form of down regulatory when faced with multi-epitopes of IL-17 inducer&#46; In our finding&#44; observed T cells were significantly differentiated&#46; Another notable characterisation of DC is phagocytic capabilities&#44; so that it can be expected that turning ImDC into mDC can decrease the ability of DC to phagocytosis as well as all needs to engulf antigen such as surface receptor&#46; In contrast&#44; it increases the presentation of antigen&#44; which leads to escalating T cell stimulation ability in DC&#46;<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">7</span></a> Furthermore&#44; the IL-4 that abolished the release of cytokines in DC and macrophages had a minimal inhibitory effect on DC and macrophages from patients with asthma&#46;<a class="elsevierStyleCrossRef" href="#bib0315"><span class="elsevierStyleSup">29</span></a> According to the results&#44; it was indicated that the rate of phagocytosis in Monocyte-derived DCs&#44; which were stimulated with <span class="elsevierStyleItalic">A&#46; alternata</span> extract was significantly decreased&#46; DC exposure to <span class="elsevierStyleItalic">A&#46; alternata</span> probably prevents tolerance to innocuous placebo as MCM and facilitates towards Th2 polarisation&#46; Therefore&#44; the potent biological activities of <span class="elsevierStyleItalic">A&#46; alternata</span> facilitate both the sensitisation and effectors phases of immunologic Th2 responses&#46; These activities and responses may offer the mechanism of triggering asthma and the well-perceived correlation between fungi&#44; development and exacerbation of asthma and allergic airway diseases in humans&#46;<a class="elsevierStyleCrossRefs" href="#bib0285"><span class="elsevierStyleSup">23&#44;33&#44;34</span></a> Our findings suggest that the DCs when accompanied with <span class="elsevierStyleItalic">A&#46; alternata</span> antigen through interfering in cytokines or presentation and shift to the aberrant secretion of T cells pro-inflammatory cytokines such as IL-17 subsequent Th2&#47;Th17 potent polarisation may be engaged in or exacerbate asthma&#46; In the end&#44; one question remained&#58; If <span class="elsevierStyleItalic">A&#46; alternata</span> is a strong Th2 response contrast with innocuous medium&#44; why only some&#44; but not all&#44; individuals or animals promote allergic airway diseases&#63; Further study of genetic analysis&#44; individual specific immune and identification of antigens of <span class="elsevierStyleItalic">A&#46; alternata</span> that interfere in the potent Th2 effects of <span class="elsevierStyleItalic">A&#46; alternata</span> will show the mechanisms involved in the development of Th2 immune responses better&#46;</p></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Conflict of interest</span><p id="par0090" class="elsevierStylePara elsevierViewall">The authors have no conflict of interest to declare&#46;</p></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Funding</span><p id="par0095" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleGrantSponsor" id="gs1">Jahrom university of medical sciences and Urmia university</span>&#46;</p></span><span id="sec0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Authors&#8217; contributions</span><p id="par0100" class="elsevierStylePara elsevierViewall">All authors had equal role in design&#44; work&#44; statistical analysis and manuscript writing&#46;</p></span><span id="sec0105" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Ethical disclosures</span><span id="sec0110" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0145">Confidentiality of data</span><p id="par0105" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appears in this article&#46;</p></span><span id="sec0115" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0150">Right to privacy and informed consent</span><p id="par0110" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appears in this article&#46;</p></span><span id="sec0120" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0155">Protection of human subjects and animals in research</span><p id="par0115" class="elsevierStylePara elsevierViewall">The authors declare that no experiments were performed on humans or animals for this investigation&#46;</p></span></span></span>"
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          "titulo" => "Abstract"
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              "identificador" => "abst0005"
              "titulo" => "Introduction"
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              "titulo" => "Materials and methods"
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              "identificador" => "abst0015"
              "titulo" => "Results"
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              "titulo" => "Conclusion"
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          "titulo" => "Keywords"
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          "titulo" => "Abbreviations"
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          "titulo" => "Introduction"
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          "titulo" => "Materials and methods"
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              "titulo" => "Preparation of fungus extract"
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            1 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "Preparation of Peripheral Blood Mononuclear Cell &#40;PBMC&#41;"
            ]
            2 => array:2 [
              "identificador" => "sec0025"
              "titulo" => "Production of control DCs in the presence of Monocyte-conditioned Medium &#40;MCM&#41; and its Inductive with A&#46; alternata extracts"
            ]
            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "Estimation of DC yield and viability from plated PBMC"
            ]
            4 => array:2 [
              "identificador" => "sec0035"
              "titulo" => "Microscopic analysis"
            ]
            5 => array:2 [
              "identificador" => "sec0040"
              "titulo" => "Phenotypic analysis"
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              "identificador" => "sec0045"
              "titulo" => "Cytokine production assay"
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            7 => array:2 [
              "identificador" => "sec0050"
              "titulo" => "Evaluation of phagocytic activity"
            ]
          ]
        ]
        5 => array:3 [
          "identificador" => "sec0055"
          "titulo" => "Results"
          "secciones" => array:5 [
            0 => array:2 [
              "identificador" => "sec0060"
              "titulo" => "Yield and viability"
            ]
            1 => array:2 [
              "identificador" => "sec0065"
              "titulo" => "Microscopic analysis"
            ]
            2 => array:2 [
              "identificador" => "sec0070"
              "titulo" => "Immunophenotyping generated DCs"
            ]
            3 => array:2 [
              "identificador" => "sec0075"
              "titulo" => "DCs stimulated with A&#46; alternata extract promote T cell production of Th2-type cytokines"
            ]
            4 => array:2 [
              "identificador" => "sec0080"
              "titulo" => "Phagocytic activity"
            ]
          ]
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        6 => array:2 [
          "identificador" => "sec0085"
          "titulo" => "Discussion"
        ]
        7 => array:2 [
          "identificador" => "sec0090"
          "titulo" => "Conflict of interest"
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        8 => array:2 [
          "identificador" => "sec0095"
          "titulo" => "Funding"
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        9 => array:2 [
          "identificador" => "sec0100"
          "titulo" => "Authors&#8217; contributions"
        ]
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          "identificador" => "sec0105"
          "titulo" => "Ethical disclosures"
          "secciones" => array:3 [
            0 => array:2 [
              "identificador" => "sec0110"
              "titulo" => "Confidentiality of data"
            ]
            1 => array:2 [
              "identificador" => "sec0115"
              "titulo" => "Right to privacy and informed consent"
            ]
            2 => array:2 [
              "identificador" => "sec0120"
              "titulo" => "Protection of human subjects and animals in research"
            ]
          ]
        ]
        11 => array:2 [
          "identificador" => "xack269025"
          "titulo" => "Acknowledgments"
        ]
        12 => array:1 [
          "titulo" => "References"
        ]
      ]
    ]
    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2016-04-29"
    "fechaAceptado" => "2016-07-08"
    "PalabrasClave" => array:1 [
      "en" => array:2 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec801326"
          "palabras" => array:4 [
            0 => "<span class="elsevierStyleItalic">Alternaria alternata</span>"
            1 => "Dendritic cell"
            2 => "Environmental fungus"
            3 => "Th2&#47;Th17"
          ]
        ]
        1 => array:4 [
          "clase" => "abr"
          "titulo" => "Abbreviations"
          "identificador" => "xpalclavsec801327"
          "palabras" => array:17 [
            0 => "<span class="elsevierStyleItalic">A&#46; alternata</span>"
            1 => "CD"
            2 => "DCs"
            3 => "FACS"
            4 => "FBS"
            5 => "GM-CSF"
            6 => "HLA-DR"
            7 => "IFN-&#947;"
            8 => "IL"
            9 => "ImDC"
            10 => "MCM"
            11 => "mDC"
            12 => "MFI"
            13 => "MoDc"
            14 => "PBMC"
            15 => "Th"
            16 => "Treg"
          ]
        ]
      ]
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    "resumen" => array:1 [
      "en" => array:3 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Introduction</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Although the mechanism of asthma is not precisely understood in humans&#44; clinical and epidemiological studies have offered a potential relationship between exposure to environmental fungi&#44; such as <span class="elsevierStyleItalic">Alternaria alternata</span> &#40;<span class="elsevierStyleItalic">A&#46; alternata</span>&#41; and the development and exacerbation of asthma&#46; The aim of this project is to investigate the mechanisms of Th2 responses by <span class="elsevierStyleItalic">A&#46; alternata</span> as a clinically relevant model for the environmental exposure&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Materials and methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Plastic adherent monocytes were cultured with granulocyte macrophage colony stimulating factor &#40;GM-CSF&#41; and interleukin-4 &#40;IL-4&#41; to convert these cells into Monocyte-derived Dendritic cells &#40;MoDc&#41; and then matured in the presence of Monocyte-Conditioned Medium &#40;MCM&#41; as the control group and MCM&#43; <span class="elsevierStyleItalic">A&#46; alternata</span> extract as the inductive groups&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">The results indicated that the expression of CD14 decreased and CD83 and anti-human leukocyte antigen-DR &#40;HLA-DR&#41; increased in the inductive groups in comparison with the control group&#46; More importantly&#44; <span class="elsevierStyleItalic">A&#46; alternata</span> inhibited IL-12 production by activated dendritic cells &#40;DCs&#41;&#44; and the DCs exposed to <span class="elsevierStyleItalic">A&#46; alternata</span> enhanced the Th2 polarisation of CD4<span class="elsevierStyleSup">&#43;</span> T cells&#46; The production amount of IL-10 overcame IL-12 as well as Il-23 increased significantly&#44; and hand in T cells the production of cytokines Interferon-&#947; &#40;IFN-&#947;&#41; decreased&#46; However&#44; both IL-17 and IL-4 increased &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; Phagocytic activity in the inductive groups decreased significantly compared with the control group&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusion</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">The asthma-related environmental fungus <span class="elsevierStyleItalic">A&#46; alternata</span>&#44; with an effect on dendritic cells profile mediates TH2&#47;TH17&#46; Such immunodysregulation properties of causative environmental fungi may explain their strong relationship with human asthma and allergic diseases&#46;</p></span>"
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          2 => array:2 [
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            "titulo" => "Results"
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          "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Morphological appearance of MCM-DCs &#40;A&#41; and Induced-DCs &#40;B&#41;&#46; Analysis by inverted microscope revealed that in comparison&#44; Induced-DC were longer and had more frills than MCM-DC&#46; These cells &#40;both groups&#41; looked larger than monocytes containing large intracellular granules also were more non-adherent&#46;</p>"
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          "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Immunophenotyping of DCs&#46; &#40;A&#41; Representative flow cytometric histograms&#44; obtained from MCM-DCs and Induced-DCs&#44; stained with FITC conjugated mAb against CD14&#44; CD83&#44; and HLA-DR&#46; As shown in histograms&#44; the Induced-DCs &#40;down&#41; produced a higher fluorescent intensity relative to MCM-DCs &#40;up&#41; using the three stained markers&#46; &#40;B&#41; Flow cytometric analysis showing increased CD83&#44; HLA-DR and decreasing expression among Induced-DCs compared with MCM-DCs&#46; Considering the data <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001 into CD83 and HLA-DR&#44; as well as <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;01 into CD14 significant differences existed between the two groups&#46;</p>"
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          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Flow cytometric analysis of phagocytic cells revealed significant increased MFI as phagocytosis power per DC accompany with maturation among MCM-DCs to Induced-DCs &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p>"
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          "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">The representative flow cytometric histograms obtained from phagocytic analysis of MCM-DC and Induced-DC&#46; Both types of DCs were incubated with FITC-conjugated latex beads for 48<span class="elsevierStyleHsp" style=""></span>h also DCs without beads were used as a negative control then washed with quenching buffer and subjected to flow cytometric analysis&#46;</p>"
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Article information

ISSN: 03010546
Original language: English

DOI:10.1016/j.aller.2016.07.005

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 November	2	0	2
2024 October	31	9	40
2024 September	38	11	49
2024 August	31	11	42
2024 July	32	13	45
2024 June	28	6	34
2024 May	36	6	42
2024 April	31	14	45
2024 March	53	17	70
2024 February	62	9	71
2024 January	72	8	80
2023 December	68	8	76
2023 November	85	7	92
2023 October	103	27	130
2023 September	62	13	75
2023 August	65	7	72
2023 July	113	30	143
2023 June	100	4	104
2023 May	167	2	169
2023 April	131	3	134
2023 March	137	4	141
2023 February	58	3	61
2023 January	91	10	101
2022 December	57	5	62
2022 November	69	9	78
2022 October	52	22	74
2022 September	52	8	60
2022 August	30	12	42
2022 July	44	12	56
2022 June	35	5	40
2022 May	62	18	80
2022 April	77	8	85
2022 March	42	14	56
2022 February	29	9	38
2022 January	61	11	72
2021 December	43	11	54
2021 November	40	10	50
2021 October	50	25	75
2021 September	65	13	78
2021 August	42	6	48
2021 July	39	10	49
2021 June	19	11	30
2021 May	17	8	25
2021 April	52	29	81
2021 March	25	7	32
2021 February	18	13	31
2021 January	23	11	34
2020 November	0	1	1
2020 October	1	1	2
2020 August	0	2	2
2020 July	0	4	4
2020 June	0	1	1
2020 May	0	4	4
2020 February	0	3	3
2020 January	0	6	6
2019 December	0	4	4
2019 November	0	4	4
2019 October	0	5	5
2019 September	0	3	3
2019 August	1	8	9
2019 July	0	3	3
2019 June	0	11	11
2019 May	0	23	23
2018 February	7	1	8
2018 January	9	6	15
2017 March	1	0	1
2017 February	1	1	2
2016 December	0	1	1
2016 November	0	1	1

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Allergologia et Immunopathologia is no longer published on Elsevier since the 2021 year.Transferred to Codon Publications

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Allergologia et Immunopathologia is no longer published on Elsevier since the 2021 year.Transferred to Codon Publications

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