http://ijaai.tums.ac.ir/index.php/ijaai/issue/feed Iranian Journal of Allergy, Asthma and Immunology 2019-09-21T10:58:25+0430 Shahnaz Rafiei Tehrani, MD, Ph.D. ijaai@tums.ac.ir Open Journal Systems http://ijaai.tums.ac.ir/index.php/ijaai/article/view/2095 Factors Influencing Atopic Dermatitis Incidence in Offspring 2019-09-21T10:53:56+0430 Siqi Ye 776402535@qq.com Xiumei Mo cdc@gzucm.edu.cn Junfeng Liu cdc@gzucm.edu.cn Fenggen Yan cdc@gzucm.edu.cn Dacan Chen szyadzj@126.com <p>Atopic dermatitis (AD) is a chronic, recurrent skin condition resulting from both genetic and environmental factors. In recent decades, the prevalence of AD has increased considerably in some countries. However, given that the role of genetics is unlikely to have changed over this short period, the increased prevalence is more likely to be explained by changes in environmental and maternal factors. The aim of this review is to comprehensively summarize the various factors impacting AD incidence in offspring and provide guidance for primary prevention. Recent research has demonstrated that environmental and climate factors, maternal history of allergies, gestational diabetes, and stress play essential roles in increasing the risk of AD in infants. Some factors have protective effects against the incidence of AD, including probiotic supplementation, fish intake, and moisturizers. This review also considers fundamental research into AD prevalence and factors that in the past were mistakenly thought to affect that prevalence, such as caesarean section and antigen avoidance. The potential influence of these factors on infant AD incidence remains inconclusive and needs further study. Furthermore, infants with a family history of atopic disease may benefit from early weaning or reduced breastfeeding duration.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/1926 In Vitro Analysis of Nine MicroRNAs in CD8+ T Cells of Asthmatic Patients and the Effects of Two FDA-approved Drugs 2019-09-21T10:54:41+0430 Mohsen Badalzadeh mbadalzadeh@ut.ac.ir Marzieh Mazinani marzieh.mazinani@yahoo.com Zahra Pourpak pourpakz@tums.ac.ir Hassan Heidarnazhad heidarnazhad@ams.ac.ir Esmaeil Mortaz emortaz@gmail.com Mostafa Moin mmoin@tums.ac.ir Ali Farazmand afarazmand@ut.ac.ir <p>In this study, we first tried to determine whether the expression level of 9 miRNAs in the peripheral blood CD8+ T cells of asthmatic patients varies from that of controls, and secondly, we investigated the effects of fluticasone furoate and vilanterol on the expression level of these miRNAs. Fifteen subjects including 8 healthy individuals and 7 asthmatic patients were included in this study. CD8+T cells were isolated from participants' peripheral blood by a negative selection method using magnetic-activated cell sorting (MACS). The expression of 9 miRNAs was examined between the healthy individuals and asthmatic patients. Then the expression level of 9 miRNAs before and after treatment with the drugs was examined by quantitative real-time PCR. No significant changes in the expression level of 9 miRNAs were observed in asthmatic patients compared to the healthy controls. Fluticasone and vilanterol,&nbsp;in combination, had the greatest effect on miRNA expression. MiR-150 and miR-106a were the most and the least miRNAs, respectively, present in CD8+ T cells of patients and controls. MiR-106a and miR-126 had a positive correlation in CD8+ cells of asthmatic patients. Although no significant difference in the expression level of studies miRNAs was observed, the correlations among miRNAs were significant. Therefore, we suggest that the correlation between miRNAs would be a very important factor in physiological and pathological conditions in healthy individuals and asthmatic patients. Such a miRNA-miRNA correlation network can be even more critical than any changes in the variation of their expression in the CD8+ T cells.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/2064 CD4+CD25+ Regulatory T Cells Decreased CD8+IL-4+Cells in a Mouse Model of Allergic Asthma 2019-09-21T10:55:50+0430 Ping Li ping_li19@163.com Ze Li 1714169628@qq.com Guqin Zhang 542175963@qq.com Jiong Yang yangjiongwhu@126.com Junwen Chen 547785476@qq.com <p>Interleukin (IL)-4-producing-CD8 (cytotoxic T cells, Tc) contribute to lung eosinophilia and airway hyper-responsiveness (AHR) to an antigen. CD4<sup>+</sup>CD25<sup>+ </sup>regulatory T cells (Tregs) attenuate airway inflammation and AHR. This study investigated whether Tregs decrease Tc2frequencies in ovalbumin (OVA)-induced asthma model of mice.&nbsp;Female C57BL/6 mice were sensitized with OVA intraperitoneally and challenged with OVA intranasally to induce allergic asthma model. Tregs were sorted by fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS) microbeads. OVA-sensitized mice were injected with Tregs or phosphate buffer saline (PBS) by tail vein ahead of the first challenge. Airway inflammation and airway hyper-responsiveness (AHR)were evaluated by histological analysis and invasive method, respectively. OVA-specific IgE and cytokine levels were detected by ELISA. Flow cytometry was used to detect the percentages of Tc1 and Tc2. Gata3 and T-bet mRNA was determined by quantitative PCR (qPCR).&nbsp;OVA-sensitized and challenged mice displayed typical asthma features, which included eosinophilic airway inflammation, higher levels of Th2 cytokines and AHR. Gata3 mRNA, Tc2 frequencies and OVA-specific IgE levels were significantly increased in OVA-sensitized and challenged mice. Compared to PBS treatment, Tregs decreased Tc2 frequencies, airway inflammation, Th2 cytokine levels and AHR in OVA-sensitized and challenged mice. IL-13 levels were negatively correlated with Tc1 frequencies and with IFNg levels in experimental mice. Our results demonstrated that Tregs could prevent airway inflammation and AHR by decreasing Tc2 frequencies and cytokine levels in OVA-induced asthma model of mice, supporting Tregmight be as a potent therapeutic target for alleviating airway inflammation and AHR.</p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/2291 In Vitro Evaluation of CMV Specific CD8+T Cells Function in CMV+ Colorectal Cancer Patients Compared to Healthy Controls 2019-09-21T10:56:36+0430 Mona Shaker Ardakani mona_shakeri69@yahoo.com Fatemeh Pak parisa_amin@yahoo.com Parviz Kokhaei p_kokha@yahoo.com Mohammad Sadegh Fazeli mhfazeli@yahoo.com Yadollah Shakiba yshakiba@gmail.com Seyed Morteza Tabatabaei Yazdi tabatabaei-y@yahoo.com Ali Abbasian abbasian1343@yahoo.com Maryam Nourizadeh nourizadeh.maryam@gmail.com <p>The oncogenic role of human cytomegalovirus (HCMV) has been recently shown in different cancers like colorectal cancer (CRC). According to the recent immunotherapy approach to target the CMV-expressing tumor cells, we investigated the CMV peptide-stimulated CD8+T cells functions in CRC patients compared to healthy individuals.&nbsp;All sixteen patients and seven controls were CMV seropositive. Blood samples were obtained from patients without chemotherapy and radiotherapy before surgery. Cytotoxic CD8+ T cells were generated using 14-day culture of PBMCs in the presences of CMV peptide epitopes and rhIL-2. In addition to the supernatant evaluations for TNF-α and IFN-γ, the functionality of CD8+ T cells was examined by detecting CD107a and intracellular IFN-γ using flow cytometry. CMV DNA was detected in tissues by Real Time PCR. CMV DNA was found in 31% of tumor tissues, while it was not seen in the adjacent non-tumor tissues. There was a close association between CMV in tumor tissue and tumor grade. Surface expression of CD107a and intracellular IFN-γ in CMV-stimulated CD8+T cells and the level of IFN-γ production in patient and control groups increased significantly after culture. The number of functions increased in patients (<em>p</em>&lt;0.05) and healthy individuals after culture. Followingstimulation, expressions of CD107a and intracellular IFN-γ were elevated in tumor CMV positive patients while the TNF-α secretion was decreased. In vitro stimulation of PBMC in the presence of CMV peptide epitopes and IL-2 can be an applicable method to generate cytotoxic CD8+ T cells in CRC patients for future T cell therapy.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/2222 Genetic Polymorphisms of CXCL8 (−251) Are Associated with the Susceptibility of Helicobacter pylori Infection Increased the Risk of Inflammation and Gastric Cancer in Thai Gastroduodenal Patients 2019-09-21T10:58:25+0430 Wongwarut Boonyanugomol wongwarut.boo@mahidol.ac.th Kamolchanok Rukseree Kamolchanok.ruk@mahidol.edu Worrarat Kongkasame Worrarat-eab@hotmail.com Prasit Palittapongarnpim Prasit.pal@mahidol.ac.th Seung-Chul Baik scbaik@gnu.ac.kr Mereerat Manwong Mereerat_s@hotmail.com <p>CXC Chemokine Ligand 8 (CXCL8) plays an important role in gastric inflammation and in the progression of gastric cancer induced by <em>Helicobacter pylori </em>(<em>H. pylori</em>) infection. The association of <em>CXCL8</em>, CXC Chemokine Receptor 1 (<em>CXCR1</em>), and CXC Chemokine Receptor 2 (<em>CXCR2</em>) polymorphisms with <em>H. pylori </em>infection and gastric cancer progression needs to be investigated in a population within an enigma area consisting of multiple ethnicities, such as Thailand. To analyze the relative risk of <em>H. pylori</em> infection and gastric cancer among Thai gastroduodenal patients, gene polymorphisms in <em>CXCL8</em> (promoter region -251) and in <em>CXCR1</em> and <em>CXCR2</em> (receptors for CXCL8) were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele specific-PCR (AS-PCR). We also determined the presence of cytotoxin-associated gene A (<em>cag</em><em>A</em>) in Thai patients with <em>H. pylori</em> infection. Correlation between the <em>CXCL8</em> (-251) polymorphism and <em>CXCL8</em> gene expression was evaluated by quantitative reverse transcriptase-PCR (qRT-PCR). We found a significant association between the T/A and A/A genotypes of <em>CXCL8</em> (-251) with <em>H.&nbsp;pylori</em> infection. However, no significant correlation was found between the <em>CXCR1</em> (+2607) and <em>CXCR2</em> (+1208) gene polymorphisms with <em>H. pylori</em> infection among Thai gastroduodenal subjects. Within the <em>H. pylori</em>-infected group of Thai gastroduodenal patients, no significant differences in <em>cag</em><em>A</em> were observed. In addition, the A/A genotype of <em>CXCL8</em> (-251) significantly correlated with the risk of gastric cancer and correlated with higher <em>CXCL8</em> gene expression levels in Thai gastroduodenal patients. These results suggest that <em>CXCL8 </em>(-251) polymorphisms are associated with <em>H. pylori</em> infection, an increased risk of stronger inflammatory responses, and gastric cancer in Thai gastroduodenal patients.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/2163 Histologic, Metabolic, and Inflammatory Changes in the Liver of High-fat Diet-induced Obese Rats before and after Vitamin D Administration 2019-09-18T13:45:20+0430 Mahdi Yaghchiyan abbasalizad_m@yahoo.com Leila Roshangar abbasalizad_m@yahoo.com Mahdieh Abbasalizad Farhangi abbasalizad_m@yahoo.com Mehran Mesgari-Abbasi abbasalizad_m@yahoo.com Leila Rafiei abbasalizad_m@yahoo.com Parviz Shahabi abbasalizad_m@yahoo.com <p>The current study aimed to investigate the effects of vitamin D administration on the markers of inflammation and metabolic damages in the liver of high-fat diet-induced obese rats. Forty male Wistar rats were divided into two groups of control receiving a normal diet (ND) and intervention receiving a high-fat diet (HFD). After 16 weeks, each group was divided into two groups including ND, ND + vitamin D, HFD, and HFD + vitamin D. Vitamin D was administered by oral gavage for five weeks at the dose of 500 IU/kg. Hepatic MCP-1, TGF-β, and NF-κB levels, serum liver enzymes, and serum lipids, and histological and structural changes in the liver were determined. Vitamin D administration significantly reduced the monocyte chemoattractant protein (MCP)-1 concentrations in the HFD + vitamin D group compared with the HFD group and reduced liver Transforming growth factor beta (TGF-β) levels in both vitamin D-treated groups (<em>p</em>&lt;0.05). Moreover, a significant reduction in the serum levels of aspartate amino transferase (AST) and alanine amino transferase (ALT) in vitamin D treated groups was identified (<em>p</em>&lt;0.05). A significant improvement in lipids and a pronounced improvement in the markers of liver histology damage including fat accumulation, aggregation of inflammatory cells, pre-apoptotic changes, hepatic sinusoidal dilatation, and necrotic pyknosis in the Kupffer cells were also identified. Our results demonstrated that vitamin D has potential effects in ameliorating the inflammatory, metabolic, and histologic changes in the liver of these animals.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/1850 The Study of Relationship between Serum Levels of Soluble fms-like Tyrosine Kinase-1 and Soluble Fibrinogen-like Protein 2 with Delayed Graft Function after Kidney Transplantation 2019-09-18T13:53:19+0430 Hossein Akbari akbari1350_h@yahoo.com Zahra Hooshyar hooshyar1993@yahoo.com Saeede Shabanitabar saiedeh1992@gmail.com Ali Salmani ali.salmani70@yahoo.com Hassan Nikoueinejad hnikuinejad@yahoo.com Behzad Einollahi behzad.einollahi@gmail.com <p>Delayed graft function (DGF) is a transplant complication which means a need to dialysis throughout the first week after transplantation. This study aimed to ascertain the relationship between the two immunomodulatory factors of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble fibrinogen-like protein 2 (sFGL-2) with DGF after transplantation. This case-control study was done in 2 groups of 58 kidney transplant patients with and without DGF. The control group included the patients who didn’t show DGF symptoms. Then, serum levels of sFlt-1and sFGL-2 in all blood samples were measured by ELISA. Serum sFlt-1 and sFGL-2 levels were significantly higher in the DGF group compared to those in the control group (<em>p</em>≤0.001). sFlt-1 and sFGL-2 serum levels significantly affect DGF (<em>p</em>&lt;0.001) in such a way that they may be diagnostic factors of DGF. This study showed a significant relationship between sFlt-1 as well as sFGL-2 and DGF. Therefore, plasma levels of sFlt-1 and sFGL-2 may be used as diagnostic tools to determine the risk of DGF.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/2240 Molecular Cloning, Expression and Purification of G-CSF Isoform D, an Alternative Splice Variant of Human G-CSF 2019-09-18T14:07:23+0430 Fatemeh Sadat Toghraie ftoghraie@gmail.com Mahsa Yazdanpanah-Samani hosseiniahmad59@gmail.com Elham Mahmoudi Maymand emmahmoodi@yahoo.com Ahmad Hosseini hosseiniah@sums.ac.ir Amir Asgari asgari.a72@yahoo.com Amin Ramezani aramezani@sums.ac.ir Abbas Ghaderi ghaderia@gmail.com <p>Granulocyte colony-stimulating factor (G-CSF) is the major regulator of hemopoiesis and granulopoiesis. However, overexpression of G-CSF has been implicated in several important processes in tumor biology such as tumor growth, angiogenesis, and metastasis. Four different mRNA isoforms resulting from alternative splicing have been reported for G-CSF (transcript variants 1, 2, 3 and 4). The mRNAs and protein products of splice variants 1 and 2 have been isolated for the first time, from tumor cell lines. In the present study for the first time we isolated the G-CSF transcript variant 4 encoding G-CSF isoform D from a highly malignant tumor cell line (Mehr80) with overexpression of G-CSF. Both the full-length G-CSF isoform B and G-CSF isoform D were cloned from Mehr80 cell line, overexpressed in <em>Escherichia coli </em>as N-terminal glutathione-S-transferase fusion proteins in the form of inclusion bodies and affinity purified by the batch method using glutathione-Sepharose 4B resin. Both fusion proteins were successfully cloned and expressed. Folded recombinant proteins were solubilized from inclusion bodies using sarkosyl, Triton X-114 and CHAPS and purified. The purity of G-CSF isoforms was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and they were clearly detected in western blot analysis using anti-G-CSF polyclonal antibody. The G-CSF plays various roles in physiological and pathological conditions, however to date, the differential function of G-CSF isoforms remains unknown. Considering the fact that G-CSF isoform D was isolated from a highly malignant tumor cell line with overexpression of G-CSF, the role of this splice variant in tumorigenesis requires further investigation.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/2192 Functional Deimmunization of Interferon Beta-1b by Identifying and Silencing Human T Cells Epitopes 2019-09-18T14:17:34+0430 Amin Moradi Hasan-Abad amin.moradi63@yahoo.com Elham Adabi Elham.a3188@yahoo.com Esmaeil Sadroddiny sadroddiny@sina.tums.ac.ir Mohammad Reza Khorramizadeh khoramza@tums.ac.ir Mohammad Ali Mazlomi m-mazlomi@tums.ac.ir Saeed Mehravar saeed.mehravar@gmail.com Gholam Ali Kardar gakardar@tums.ac.ir <p>Interferonbeta-1b (IFNβ-1b) developed as therapeutic protein for the treatment of multiple sclerosis (MS). Studies have been shown that Long-term usage of this protein can lead to the development of anti-drug antibodies (ADAs) and this phenomenon cause total loss or reduced efficacy of IFNβ-1b. The aim of this study was to predict and silence IFNβ-1b T-cells epitopes by in silico methods and genetic engineering. Based on bioinformatics studies we identified optimal sets of conservative point mutations for eliminating T-cells epitopes in IFNβ-1b protein. Four synthetic genes with desirable mutation constructed and PET26b+ was used as an expression vector in E. coli. The expression of this proteins confirmed by SDS-PAGE and Western blotting, consequently, IFNβ-1b proteins was purified by His-tag chromatography. To determined activity of mutants’ variants anti-proliferative and anti-viral activity compared to wild form was evaluated using MTT assay in A549 and Vero cells lines respectively. Also the immunogenicity of mutant proteins compared with Betaseron measured in BALB/c mice. The in vitro bioactivity analysis demonstrated that functional activities of all mutant proteins were maintained and is the same as biological activity of Betaseron. Pharmacokinetic studies suggest that, in engineered proteins that contain substitution of Histidine to Glutamic Acid at position 131 (mut 2 and mut 1+2) antibodies response reduced by about 50%, as compared to that for Betaseron. Computational analysis expedites identification and prediction of epitopes in therapeutic protein, therefore, we used immunoinformatic tools for modification of dominant T-cell epitope in IFNβ-1b protein, and this strategy has capacity to create proteins which have naturally reduced immunogenicity.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/2261 Identification of a Novel C16orf57 Mutation in Iranian Patient with Clericuzio-type Poikiloderma with Neutropenia (CPN): A Case Report 2019-09-18T14:20:55+0430 Farhad Abolnezhadian abolnezhadian@yahoo.com Sara Iranparast sara.iranparast@yahoo.com <p>Poikiloderma is a hereditary pathologic situation in which the appearance of skin rash is associated with epidermal atrophy, telangiectasia, and reticular dyspigmentation skin symptoms of poikiloderma are usually caused by sun damage. The main reason forpoikiloderma is unknown. We introduce a 14- month-old boy who referred to our center with a complaint of fever and cough. Furthermore, hepatosplenomegaly symptoms had been presented at the time of birth and were continuously observed at age one. He had transient thrombocytopenia when he was born due to his prematurity condition, which was resolved during Intravenous Immunoglobin (IVIG) treatment. Therefore, the presence of various mutation scan lead to distinct clinical symptoms. Immunohematologic abnormalities such as increased level of IgM and IgE antibodies, as well as increased C-reactive protein (CRP) and Erythrocyte sedimentation rate (ESR), have been reported. However, mutation of the C16orf57 gene was identified in this patient. We also introduced a new genetic mutation in a particular part of DNA sequence (NM_001195302: exon6: c.T703C) that leads to new clinical finding in PN.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/2275 Skewed X-inactivation in a Female Carrier with X-linked Chronic Granulomatous Disease 2019-09-18T14:29:24+0430 Itzel López-Hernández itzelh.lh.31@gmail.com Caroline Deswarte caroline.deswarte@inserm.fr Miguel Ángel Alcantara-Ortigoza malcantaraortigoza@gmail.com María del Mar Saez-de-Ocariz mariadelmar71@prodigy.net.mx Marco Antonio Yamazaki-Nakashimada yzki71@yahoo.com.mx Sara Elva Espinosa-Padilla saraelvaespino@gmail.com Jacinta Bustamante jacinta.bustamante@inserm.fr Lizbeth Blancas-Galicia blancas.lizbeth@gmail.com <p>Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defective phagocytic NADPH oxidase, causing a complete lack or significant decrease in the production of microbicidal reactive oxygen metabolites. It mainly affects male children; however, there are scarce reports of adult females diagnosed with X-linked-CGD attributed to an extremely skewed X-chromosome inactivation. This condition is characterized by severe and recurrent infections that usually develop after childhood. In clinical practice, physicians who usually confront these patients should suspect this entity and differentiate it from a secondary immunodeficiency. Here, we report a 38-year-old Mexican female with juvenile-onset X linked-CGD, caused by a <em>de novo</em> mutation and extremely skewed X-inactivation, whose clinical features were similar to those in patients with classic X-linked-CDG.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement## http://ijaai.tums.ac.ir/index.php/ijaai/article/view/1671 Lupus Erythematosus and Chronic Granulomatous Disease: Report of Four Iranian Patients with AR-CGD and One XL-CGD 2019-09-18T14:36:09+0430 Marzieh Maddah marzieh.maddah@gmail.com Mohammad Reza Fazlollahi fazlollahi@tums.ac.ir Reza Shiari shiareza@yahoo.com Farhad Shahram shahramf@tums.ac.ir Setareh Mamishi smamishi@sina.tums.ac.ir Delara Babaie delara77@yahoo.com Maryam Monajemzadeh maryam.monajemzadeh@gmail.com Soheila Sotudeh ssotudeh@tums.ac.ir Amir Ali Hamidieh aahamidieh@sina.tums.ac.ir Mohsen Badalzadeh m.badalzadeh@gmail.com Shaghayegh Tajik sh-tajik@farabi.tums.ac.ir Leila Sedighipour lsedighipour@yahoo.com Zahra Pourpak pourpakz@tums.ac.ir <p>Chronic granulomatous disease (CGD) is a rare genetic disorder of neutrophil activity, resulting in increased rate of recurrent infections with catalase–positive bacteria and fungi, as well as various autoimmune diseases such as sarcoidosis, rheumatoid arthritis, and discoid and/or systemic lupus erythematosus. Few reports have reported lupus erythematosus (LE) in patients with X–linked CGD (XL-CGD) and carriers, and very few in autosomal recessive CGD (AR-CGD). Here, we present 5 patients with CGD developing LE at different ages to emphasize on the importance of appropriate follow–up and treatment in patients with CGD with clinical signs and symptoms of autoimmune diseases and even in those with negative serologic results.</p> 2019-08-17T00:00:00+0430 ##submission.copyrightStatement##