Madridge Journal of Immunology

ISSN: 2638-2024

International Conference on Immunology and Immunotechnology

November 1-3, 2017, Barcelona, Spain
Accepted Abstracts
DOI: 10.18689/2638-2024.a1.004

Genome Wide Transcriptome Analysis of IL-17 and STAT3 Loop Activation Psoriasiform Murine Model

Xinran Xie1,2*, Ping Li1, Lei Zhang1, Ning Wang1, Xin Liu1, Xue Li2 and Lu Zhang1

1Beijing Institute of Traditional Chinese Medicine, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, China
2Beijing University of Chinese Medicine, China

Psoriasis is an autoimmune skin disease characterized by T lymphocytes activation and keratinocytes excessive proliferation and abnormal differentiation. Recently, T-helper 17 (Th17) cells and its related cytokines have been important influence factors in the pathology of psoriasis. Signal transducers and activators of transcription 3 (STAT3) over expression mice representing for keatinocytes proliferation. We tried to apply IL-17 (2μg / mouse) intradermal injection on STAT3 mice for 24 hours forming psoriasis-like lesions. The experiment was divided into three groups: wild-type (C57BL/6) mice, STAT3 mice and IL-17-treated STAT3. Illumumia sequence technique was applied to the dorsal skin of three groups (n=3). The results showed that 203 up regulated and 75 down regulated mRNAs were identified in IL-17A treated STAT3 mice compared to WT mice. The up-regulated included the members of S100A protein family such as S100A8 and S100A9, small proline-rich protein 2 (Sprr2) such as Sprr2e, Sprr2g and Sprr2d, late cornified envelope-3 (LCE) genes such as LCE3d, LCE3e and LCE3f. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis dispicted the De-genes annotated pathway including innate immune response, arachidonic acid metabolism, cytokine-cytokine receptor interaction, et al. Those were in accordance with the pathological characteristics of psoriatic patients. In conclusion, our study verified that IL-17 and STAT3 loop activation play a key role in the pathogenesis of psoriasis, and IL-17-treated STAT3 mice model could be used as an animal model for the study of psoriasis.

Xinran Xie is on-job doctorate, assistant researcher. The main research direction is the study of pathogenesis in psoriasis.

The Inhibition of Acetyl-11-Keto-β-Boswellic Acid on Activated Dendritic Cells in an Imiquimod-Induced Psoriasis Mouse Model and in vitro

Ping Li*, Mingxing Wang*, Yan Wang, Jingxia Zhao, Tingting Di, Xiaolong Xu, Yujiao Meng, Xiangjiang Xie, Zhitong Ruan, Lu Zhang, Yan Lin, Xin Liu and Ning Wang

Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing Institute of Traditional Chinese Medicine, China

Psoriasis vulgar is an inflammatory skin disease that is mediated by T cells and dendritic cells which is the most accessible human disease. In this study we aimed to investigate the effects of Acetyl-11-keto-b-boswellic acid (AKBA) on activated dendritic cells using imiquimod (IMQ)-induced psoriasis-like mouse model. The mice treated with IMQ and intragastrically administered 25~100 mg/kg/day of AKBA, 1 mg/kg/day of methotrexate (MTX), or normal saline. The inflammation of IMQ mice skin lesions were evaluated by psoriasis area and severity index (PASI) and pathological staining. The related proteins of TLR7/8 pathways were assessed by western blotting and the quantity of interleukin IL-23, IL-12p40 mRNA were measured by RT-PCR. The levels of DCs were assessed by flow cytometry and cytokines by enzyme linked immunosorbent assay (ELISA). In this study we found that the AKBA and MTX obviously improve the psoriasis-like skin lesions of IMQ mice. Also obviously decreased each score of PASI, and reduced the thickness of epidermis, ameliorated the infiltration of CD3+, CD11c+ cells in skin lesions, decreased the percentage of CD11c+ cells in spleen, suppressed the expression of TLR8 and MYD88, reduced the transcription of IL-23, IL-12p40 mRNA and the secretion of related cytokines. This study inferred that AKBA might be ameliorate psoriasis-like skin lesions of IMQ-induced psoriasis mice by suppressing the function of activated DCs and inhibiting the activation of TLR8 signal pathway.

Li Ping was born in 1965, China. She received D.S.A from Beijing University of Traditional Chinese Medicine in 1998. Now she is the deputy director of Beijing Institute of Traditional Chinese Medicine. She is also the director of Beijing Key Laboratory of Clinic and Basic Research with Traditional Chinese Medicine on Psoriasis. Her main research interest is TCM on autoimmune dermatosis and wounds healing.

Punicalagin, a PTP1B Inhibitor, Induces M2c Phenotype Polarization via Up-Regulation of HO-1 in Murine Macrophages

Xiaolong Xu1,2,3*, Jingxia Zhao1,2,3, Di Tingting1,2, Wang Yan1,2, Ping Li1,2 and Qingquan Liu1,2,3

1Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, China
2Beijing Institute of Traditional Chinese Medicine, China
3Beijing Key Laboratory of Basic Research with Traditional Chinese Medicine on Infectious Diseases, China

Punicalagin (PUN) is the most abundant ellagitannin in pomegranate, a well-documented traditional Chinese medicine used for inflammatory diseases. We reported that PUN possesses considerable anti-inflammatory and anti-oxidant properties; However, its direct targets have not yet been reported. Here, we demonstrated that Protein Tyrosine Phosphatase-1B (PTP1B) serves as a direct target of PUN, with IC50 value of 1.04 μM. Results from NPOI further showed that the Kon and Koff of PUN-PTP1B complex were 3.38e2 M-1s-1 and 4.13e-3s-1, respectively. The active site Arg24 of PTP1B was identified as a key binding site of PUN by computation simulation and point mutation. Moreover, inhibition of PTP1B by PUN promoted an M2c-like macrophage polarization and enhanced anti-inflammatory expression, including IL-10 and M-CSF. Based on gene expression profile, we elucidated that PUN treatment significantly up-regulated 275 genes and down-regulated 1059 genes. M1 like macrophage marker genes, such as Tlr4, Irf1/2, Hmgb1, and Stat1 were down-regulated, while M2 marker genes, including Tmem171, Gpr35, Csf1, Il1rn, Cebpb, Fos, Vegfα, Slc11a1, and Bhlhe40 were up-regulated in PUN-treated macrophages. Hmox-1, a gene encoding HO-1 protein, was preferentially expressed with 16-fold change. Inhibition of HO-1 obviously restored PUN-induced M2 polarization and IL-10 secretion. In addition, phosphorylation of both Akt and STAT3 contributed to PUN-induced HO-1 expression. This study provided new insights into the mechanisms of PUN-mediated anti-inflammatory and anti-oxidant activities and provided new therapeutic strategies for inflammatory diseases.

Xu Xiaolong was born in 1988, China. He received M.S.A and D.S.A from China Agriculture University in 2012 and 2015, respectively. Now he is a research associate in Beijing Hospital of Traditional Chinese Medicine and Beijing Institute of Traditional Chinese Medicine. He is also a research fellow in Beijing Key Laboratory of Basic Research with Traditional Chinese Medicine on Infectious Diseases. His main research interest is natural plant extract on inflammatory diseases including Sepsis, IBD, Shock and so forth.

Berberine Regulates IL-17+Gamma Delta T Cells Mediated Immune Response Inimiquimod-Induced Mice through Toll-like Receptor

Ting-Ting Di*, Jingxia Zhao, Yan Wang, Xiaolong Xu and Ping Li

Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing Institute of Traditional Chinese Medicine, China

Berberine (BBR) is an isoquinoline alkaloid extracted from numerous plants of the general Berberis and Coptis. BBR has been shown to exhibit antimicrobial, ant oxidation, antitumor, antidiabetic, and even neuroprotective effects. Upon stimulation, dermal γδT cells are quickly activated to release IL-17 and other proinflammatory cytokines. Meanwhile, they also expand more to enhance acute immune response. This study aimed to demonstrate the immunoregulation effect of BBR in inflammatory process of gamma delta T cells and explore the mechanism further. Imiquimod (IMQ) and R848 both were Toll-like receptor7, 8 agonist. We employed an IMQ-induced psoriasis-like mouse model to investigate the effect of BBR in γδT cell mediated inflammation. Keratinocytes proliferation and differentiation of psoriasis-like lesions was assessed by histology, circulating levels of T cells were assessed by flowcytometry and cytokines by bead-based immunoassay. The percentage of CD3+ T cells were elevated in the spleen and skin. However, the percentage of CCR6+gamma delta T cells was reduced. Jak/Stat3 pathway was assessed by Western blotting. Administration of BBR ameliorated IMQ-induced keratinocyte proliferation and inhibited IL-17A related inflammation. In vitro, BBR directly inhibited IL-17 secretion in a dose-dependent manner. It has synergy with toll-like receptors to promote γδT cells to transform IFN-γ producing cells and inhibited Il-17 producing phenotypic transformation.

Ting-Ting Di was born in January 1987, China. She received PhD from Beijing University of Chinese Medicine, Beijing, China. Now she mainly engaged in studies of the effect of traditional Chinese medicine on psoriasis, working at Beijing Hospital of Traditional Chinese Medicine, affiliated with Capital Medical University, Beijing Institute of Traditional Chinese Medicine, Beijing Key Laboratory of Clinic and Basic Research with Traditional Chinese Medicine on psoriasis, Beijing, China.

Optical Immunosensor Based on Photoluminescence of ZnO Thin Films for the Detection of GVA-Antigen Proteins

Alla Tereshchenko1, Viktoriia Fedorenko1,2, Valentyn Smyntyna1, Igor Konup3, Anastasiya Konup4, Martin Eriksson5, Rositsa Yakimova5, Sebastien Balme2, Mikhael Bechelany2 and Arunas Ramanavicius6

1Experimental Physics Department, Odessa National I. I. Mechnikov University, Ukraine
2Institut Européen des Membranes IEMM, France
3Department of Microbiology, Virology and Biotechnology, Odessa National I. I. Mechnikov University, Ukraine
4National Scientific Centre “Institute of Viticulture and Wine Making Named After V. Ye. Tairov”, Ukraine
5Department of Physics, Chemistry and Biology, Linköping University, Sweden
6Department of Physical Chemistry, Vilnius University, Sweden

Photoluminescence based immunosensor for the determination of Grapevine virus A-type (GVA) proteins (GVA-antigens) has been developed [1]. The immunosensor was based on thin films of ZnO (100 nm thickness) formed on the Silicon substrates by atomic layer deposition (ALD). The ZnO-based films have demonstrated favorable surface-structural properties for the direct immobilization of antibodies against GVA-antigens in order to form a biosensitive layer sensitive to the GVA antigens. The absorption of anti-GVA antibodies resulted in a new photoluminescence band appearance in the region of 400-550 nm (Fig. 1) that can be caused by the formation of some chemical bounds during the anti-GVA adsorption on ZnO surface. The possibility to detect GVA-antigens without additional labels (e.g enzymes or fluorescent dyes) has been demonstrated. The GVA-antigen detection was performed by the evaluation of changes and behavior of the photoluminescence band, related to protein adsorption. The sensitivity of as-formed label-free biosensor towards the GVA-antigens was determined in the range from 1 pg/ml to 10 ng/ml.


[1] A. Tereshchenko, V. Fedorenko, V. Smyntyna, I. Konup, A. Konup, M. Eriksson, R. Yakimova, A. Ramanavicius, S. Balme, M. Bechelany. ZnO films formed by atomic layer deposition as an optical biosensor platform for the detection of Grapevine virus A-type proteins, Biosensors and Bioelectronics, 2017, 92, 763–769.

TCR Cross linking Promotes Crk Adaptor Protein Binding to Tyrosine-Phosphorylated CD3ζ Chain

Noah Isakov*, Guangyu Dong, Rachel Kalifa, Pulak Ranjan Nath and Sigal Gelkop

Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, Israel

Tcell antigen receptor (TCR) binding of a peptide antigen presented by antigen-presenting cells (APCs) in the context of surface MHC molecules initiates signaling events that regulate T cell activation, proliferation and differentiation. A key event in the activation process is the phosphorylation of the conserved tyrosine residues within the CD3 chain immune receptor tyrosine-based activation motifs (ITAMs), which operate as docking sites for SH2 domain-containing effecter proteins. Phosphorylation of the CD3ζ ITAMs renders the CD3 chain capable of binding the ζ-chain associated protein 70 kDa (ZAP70), a protein tyrosine kinase that is essential for T cell activation.

We found that TCR/CD3 cross linking in Jurkat T cells promotes the association of Crk adaptor proteins with the transiently phosphorylated CD3ζ chain. Pull down assays using bead-immobilized GST fusion proteins revealed that the Crk-SH2 domain mediates binding of phospho-CD3ζ. Phospho-CD3ζ binding is selective and is mediated by the three types of Crk, including CrkI, CrkII, and CrkL, but not by other SH2 domain-containing adaptor proteins, such as Grb2, GRAP and Nck.

Crk interaction with phospho-CD3ζ is rapid and transient, peaking one minute post TCR/CD3 cross linking. The results suggest the involvement of Crk adaptor proteins in the early stages of T cell activation in which Crk might help recruiting effector proteins to the vicinity of the phospho-CD3ζ and contribute to the fine-tuning of the TCR/CD3-coupled signal transduction pathways.

Genetics, Genomics and Epigenomics Approaches to Identify Key Players in SLE

Shaheen Khan

Department of Immunology, UT Southwestern Medical Center, USA

Systemic lupus erythematosus (SLE) is a complex chronic multisystem autoimmune disease with extensive clinical heterogeneity. Identification of novel genes in SLE has been challenging in the field due to complex genetics and epigenetics underlying disease pathogenesis. One of my research interests focuses on understanding genetic basis of SLE and identifying novel genes using state of the art next generation sequencing technologies. I have extensively used NZM2410 spontaneous mouse models of SLE to identify novel candidate genes using congenic dissection strategy combined with genomics and epigenomics approaches. This will focus on identification of KLF13 as a novel transcription factor that drives SLE pathogenesis by modifying the chromatin landscape of immune cells. In support of plethora of emerging studies, this study sheds light on the importance of non coding variations associated with epigenetics marks as regulators of inflammation and autoimmunity.

Shaheen Khan is an Instructor in Department of Immunology at UT Southwestern Medical Center. She received her Ph.D in Genetics from Texas A & M University where she studied molecular mechanisms underlying estrogen-mediated breast cancer. She then did her post-doctoral training at UT Southwestern Medical Center in Department of Immunology. During this time she received extensive training in the field of immunology, mouse genetics and next generation sequencing technologies. She utilizes these approaches to address key questions of her own research interests. Her current research focuses on understanding genetic Basis of autoimmunity in mice as well as SLE/Pediatric lupus patient cohorts. She also has a deep interest in translational studies in the field of cancer immunotherapy and collaborates with clinicians at Simmons Cancer Center at UT Southwestern Medical center. This study is specifically focused to understand roles of patientʼs genetic and immune status in development of toxicity in patients undergoing immune checkpoint blockade therapy.

TLR3 Elicits Constitutive HSV-1 Resistance in Human Cortical Neurons and Inducible Resistance in Trigeminal Neurons

Osefame Ewaleifoh1, Shen-Ying Zhang4,6,7, Bastian Zimmer2, Jean-Laurent Casanova4,6,7,8,9, Lorenz Studer2,3, Luigi D. Notarangelo5 and Gregory A. Smith1

1Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, USA
2The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, USA
3Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, USA
4St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, USA
5Division of Immunology, Boston Childrenʼs Hospital, USA
6Laboratory of Human Genetics of Infectious Diseases, Necker Hospital for Sick Children, France
7Paris Descartes Université, Imagine Institute, Paris, France
8Howard Hughes Medical Institute, USA
9Pediatric Hematology-Immunology Unit, Necker Hospital for Sick Children, France

Background: HSV-1 infections of the central nervous system are associated with life-threatening Herpes Simplex Encephalitis (HSE). Inborn errors in Toll like receptor 3 (TLR3) increase susceptibility to HSE. This study investigates the role of TLR3 in restricting HSV-1 infection using human iPSC-derived neuronal culture models.

Methods: We examined the mechanisms by which TLR3 protects human neurons from HSV-1 infection. Induced pluripotent stem cell derived trigeminal and cortical neurons obtained from TLR3 and STAT-1 deficient patients, and healthy controls, were infected with recombinant strains of HSV-1. Viral entry, retrograde axonal transport, and gene expression were assessed.

Results: Our studies indicate that control cortical neurons exhibit TLR3-dependent constitutive resistance to HSV-1 that manifested from blockades in viral entry and retrograde axonal transport. STAT-1 was dispensable for constitutive resistance to HSV-1. TLR3-deficient cortical neurons revealed that HSV-1 entry occurred by transient endocytosis followed by bafilomycin-sensitive membrane fusion to release capsids into the cytosol. In contrast to cortical neurons, TLR3-dependent resistance in trigeminal neurons required advanced stimulation to establish an antiviral state prior to HSV-1 challenge.

Conclusions: Human iPSC-derived cortical and trigeminal neurons accurately model the selective neurotropic properties of HSV-1 exhibited in vivo. Absence of functional TLR3 results in cortical neuron infection in culture that is consistent with the presentation of HSE in TLR3-deficient patients. We propose that the rapid and constitutive TLR3-based resistance to HSV-1 exhibited in iPSC-derived cortical neurons underlies HSV-1ʼs selective neurotropism for the human peripheral nervous system.

Immunohistochemical Study of Cytokine Profile Present In Local Immune Response against Hydatid Cyst

M. Jiménez, F. Corrêa, C. Stoore, C. Hidalgo, C. González and R. Paredes

Laboratory of Veterinary Medicine, Faculty of Ecology and Natural Resources, University of Andres Bello, Chile

Introduction: Cystic Echinococcosis (CE) is a zoonotic infection with high prevalence in part of Eurasia, Africa, Australia, and South America. Previous reports show during CE, the distinguishing feature of the host-parasite relationship is that chronic infection coexists with detectable humoral and cellular responses against the parasite, but this response regarding the hydatid cyst fertility is scarcely studied. The objective of the present work is to evaluate locally response of lymphocytes T and B with the cytokine profiles Th1 / Th2 present in adventitial layer (AL) of hydatid cysts.

Materials and Methods: Samples from animals with either fertile or infertile hydatid cysts were included in this study, using 5 samples per condition. Histological samples were evaluated by immunohistochemistry with a panel of antibodies for CD3, CD79a, INFγ, TNFα, IL4 and IL10 molecular markers. Digital images were obtained using an Olympus FSX100 Microscope and analyzed with software for morphometric analysis (Image J).

Results: Immunohistochemical analysis of lymphocyte populations in AL of bovine hydatid cysts showed a predominance of CD3+ T cells compared to CD79+ B cells. With respect to fertility, infertile cysts had a statistical significant increase of CD3+ T cells and CD79+ B cells (p <0.01) in AL. The expression of cytokines showed a statistically significant increase of INFγ and TNFα (p < 0.02) in infertile cysts, unlike the fertile cysts that present an increase of IL4 (p=0.02) and IL10 (p=0.2).

Conclusions: These findings suggest a predominant Th1 polarized local immune response, and a high CD3+ T cell population could contribute to infertility condition of bovine hydatid cysts.

Role of ‘Serum Amyloid A’ as a Marker of Disease Activity in Juvenile Idiopathic Arthritis

Anup Singh* and Sharad Dev

Dept. of General Medicine, Institute of Medical Sciences, Banaras Hindu University, India

Background/Purpose: Although several studies have validated the use of serum amyloid A (SAA) protein as a biomarker for the disease activity in different rheumatoid conditions, it has not been extensively used in clinical practice. Moreover only one such study is available in relation with JIA.

Methods: A total of 50 newly diagnosed cases of JIA (modified ILAR criteria 2001), covering all subtypes and 40 healthy controls of same age and sex, were included in this study. Serum amyloid A (SAA), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were measured in both patients and healthy controls. Quantitative measurement of SAA level was done using standard human SAA ELISA kit. Disease activity was assessed by number of large joints affected, ie. USG score (Out of total 8; viz bilateral elbow, wrist, knee, ankle) as detected by the USG study as well as clinical examination in all patients.

Results: SAA levels were significantly higher in JIA patients versus healthy controls (p<0.001). Significant positive correlations were found between SAA and the presence of active joints (r=0.64, p<0.001), ESR(r=0.39, p<0.05) and CRP(r=0.36, p<0.05). Similar but less significant correlations between ESR and the presence of active joints (r=0.39, p<0.05) and between CRP and the presence of active joints (r=0.36, p<0.05) were demonstrated in JIA patients.

The correlation between CRP and ESR was also significant (r=0.57, p=<0.001). The mean USG score of patients with increased SAA level was significantly higher than that of patients with normal SAA level (p<0.05).

Conclusion: We discovered a significant increase in SAA levels in JIA patients and strong positive correlation between SAA level and JIA disease activity. We also discerned SAA to be a more sensitive laboratory marker than ESR and CRP for evaluating the presence of active joints.

Consensus S1 Glycoprotein Induces Partial Protection against Avian Infectious Bronchitis in Chicken

Saravanan Ramakrishnan1, Sohini Dey2 and Madhan Mohan2

1Avian Immunology laboratory, Immunology Section, India
2Recombinant DNA laboratory, Division of Veterinary Biotechnology, India

Avian Infectious Bronchitis (IB) is an economically important OIE listed acute contagious viral disease of the poultry. We studied the protection potential of consensus S1 glycoprotein against IB. A consensus sequence was derived out of 67 S1 glycoprotein gene sequences of IB virus and synthesized commercially. The gene was cloned in yeast expression vector, transformed into Saccharomyces cereviciae cells and induced to express the recombinant S1 protein (rS1) which was confirmed by Western blot. Specific pathogen free (SPF) chicks (n=50) were randomly divided into five groups (n=10/group) namely, rS1 (100μg/bird), rS1 plus adjuvant (rS1 glycoprotein 100μg plus Montanide ISA 71 R VG), inactivated vaccine, live IBV vaccine and unvaccinated control. At two weeks of age, the birds were vaccinated through intramuscular route except live IBV vaccine, which was administered intranasally. A booster was given two weeks later. The yeast expressed rS1 induced significantly higher antibody response (P<0.01) in ELISA as well as stimulation index in lymphocyte transformation test (P<0.05) than that of the unvaccinated control group. All the vaccinated groups showed significantly higher (P<0.01) CD4+ as well as CD8+ T cells than that of the unvaccinated control group. The adjuvant also enhanced the antigen specific humoral as well as cellular immune responses. The experimental birds were challenged with Massachusetts standard challenge virus (104 ELD50 per bird through ocular instillation) two weeks post booster immunization and representative birds (n=6/group) were sacrificed to collect the trachea on day 5 post-challenge for assessing the protection by ciliostasis test. The live IBV vaccine afforded 71.7% protection, while it was only 40 and 45% in the rS1 alone or with adjuvant, respectively. In conclusion, the rS1 glycoprotein conferred partial protection against IB, though it induced antigen specific immune responses.

Immunomodulatory Therapeutics from Phytochemicals and Novel Format Antibody in Ameliorating Ovalbumin-Induced Lung Inflammation

Ena Ray Banerjee

Immunobiology and Regenerative Medicine Research laboratory, Dept. of Zoology, University of Calcutta, India

Background: Asthma is a chronic allergic airway disease characterized by persistent inflammation and airway hyper-responsiveness. T cells, especially Th2 cells secreting IL-4, IL-5 and IL-13 are pivotal in orchestrating the disease process. Under the influence of chemo attractants and IL-5, eosinophils proliferate, migrate in to the lung, and are activated to secrete histamine and other mediators. IgE plays a major role in allergic disease by causing the release of histamine and other inflammatory mediator from mast cells. Presently, the most effective treatments are down regulation of the inflammatory process with corticosteroids. A more direct approach to stop the systemic reaction is to block IgE, via an anti-IgE. The serendipitous discovery of naturally occurring single chain antibodies in animals revived interest in these molecules. Our aim was to generate a single heavy chain antibody from Indian Camels (Camelus dromedarius) to ameliorate the disease of Ova specific asthma. Further we evaluated the potential activity of natural compounds like fisetin and curcumin mediated anti-asthma activity.

Methods: We used molecular biology techniques where single heavy chains were expressed in bacterial expression systems. At first, we immunized male Camel and B lymphocyte blood was isolated to isolate RNA. RT-PCR was carried out and cDNA was synthesized. The Variable domain of camelid heavy chain antibody fragments were isolated by nested PCR. Phage display technology was performed to select best clone for antigen specificity. We further developed ovalbumin induced chronic asthma in pre-clinical model (C57Bl/6) to test efficacy of anti-IgE. Mice were sensitized and challenged with OVA with adjuvant up to 55 days. We investigated Bronchoalveolar Lavage fluid (BALF), Histological analysis, measurement of gene expression by RT-PCR and Flow cytometry analysis. For drug treated groups, 5 MPK Fisetin and Curcumin was given intratracheally one hour before each intra tracheal challenge.

Results: Fisetin and Curcumin significantly reduced the numbers of infiltrating leukocytes in the airways of OVA-challenged mice, especially lymphocytes. Fisetin and Curcumin treated group Eosinophil significantly reduce 2.71 and 1.44 fold in blood whereas in Balf number of Eosinophils reduced 2.52 and 1.52 fold. Ova induced inflammatory cells and subepithelial collagen content around the bronchiloles and small vessels was detected by H&E, Massonʼs Trichrome stained paraffin-embedded lung sections. Further gene expression of IL-5, IL-13 and TGF-β significantly reduce 1.02, 0, 1.14 fold in case of Curcumin treated group whereas, Fisetin treated group reduced 1.27, 2.67 and 1.06 fold. More interestingly, CD45 cells become reduced in lung 1.84 and 2.72 fold in Curcumin and Fisetin treated group by Flow cytometry analysis.

Furthermore, we are developing anti-IgE antibody of Camelid format. We obtained VHH single chain antibody of 15 kDa protein in bacterial expression system. The small size allows for good tissue penetrability making them molecules of choice to combat lung diseases.

Conclusion: Both Fisetin and Curcumin reduce airway eosinophil trafficking and mucus hypersecretion as well as collagen deposition in a mouse model of Ovalbumin induce asthma. Expected administered with anti IgE single chain antibody will have the ability to drastically and rapidly reduce the local and systemic lung inflammation. The camelidantibodies will be potential and successful drugs against lung diseases (Asthma, COPD etc.) in the market as their high selectivity and affinity to their targets reduces the chances of side effects and the production will be also cost-effective.

Dr. Ena Ray Banerjee is Professor of Zoology in University of Calcutta, India and heads the Immunobiology and Regenerative Research Unit of the department. Her interests are manifold but centered around translational outcomes research in life sciences by exploring biodiversity through bioprospecting and converting them into bioresources currency. Her work on target identification and validation in asthma and other related lung diseases helped her transition from a completely academic pursuit to a more applied one. She began with immunological studies defining key molecules in inflammation and eventually super-specialized into lung inflammation particularly allergy and made a natural transition onto regenerative medicine of the lung, having worked with some renowned names in the field. Her work validated several key targets in hematopoiesis and inflammation and also pioneered tissue engineering of lung lineage specific cells of the non-ciliated variety from Human embryonic stem cells and identified stem cell niches in mouse lung.

The Role of Dynamic Genome-Wide Changes of Histone H3K4Me3 Methylation in Regulating the Immune Response of Macrophages to Mycobacterium Tuberculosis Infection

Arijita Subuddhi*, Manish Kumar, Manikuntala Kundu and Joyoti Basu

Bose Institute, India

Objectives: The role of epigenetics in dictating the triggering of an appropriate immune response from the macrophage, and the ability of virulent Mycobacterium tuberculosis (Mtb) to dampen this reponse, has been scantily explored. Our objective was to analyze the role of genome-wide histone H3K4Me3 in the immune response to virulent (H37Rv) as opposed to the avirulent (H37Ra) strain of Mtb.

Methods: We infected THP-1 macrophages with Mtb followed by chromatin immunoprecipitation using H3K4me3 antibody and deep sequencing to map the global occupancy of H3K4Me3. A select set of genes were chosen to validate the occupancy of H3K4Me3 in their upstream regions by ChIP-qPCR. The expression levels of selected genes were analyzed. The levels of selected cytokines were measured after infection.

Results: Differences were observed in the temporal occupancy profiles in H37Rv vs. H37Ra-infected, differentiated THP-1 macrophages. We focused on a set of genes for which H3K4Me3 occupancy mapped close to the TSS, and which were of importance to the innate immune response, namely SATB1, DUSP4, a MAP kinase phosphatase and ATF2, a transcription factor. We observed enhanced H3K4Me3 occupancy near the TSSs of all three genes, in infected macrophages. Occupancy levels were higher in H37Rv than H37Ra in the time scales analyzed. This correlated with higher gene and protein expression levels of all three of the above in H37Rv vs. H37Ra. ASH1L, an H3K4 methylating enzyme occupied the promoters of all three of the above genes. Knockdown of ASH1L attenuated their expression. SATB1 and DUSP4 were also linked to attenuation of the expression of a set of inflammatory cytokines.

Conclusions: We hypothesize that differential H3K4Me3 modification triggered in macrophages by virulent vs. avirulent Mtb, is linked to differential regulation of the expression of immune-regulatory genes in macrophages. Virulent Mtb manipulates the host epigenetic machinery as a possible host subversion strategy.

Arijita Subuddhi is currently working on two projects as a senior research fellow (Phd scholar) in Bose Institute, India. She has one paper as a co author named “MicroRNA let-7 Modulates the Immune Response to Mycobacterium tuberculosis Infection via Control of A20, an Inhibitor of the NF-kB Pathway” published in Cell Host & Microbe 17, pages 1-12, March 11, 2015.

Effect of Chronic Physical Stress on Immune System

Ahmed S. Hamada*, Mohamed S. Alshorbagy and Mohamed Khadr

Department of Clinical Pathology, Faculty of Medicine, Al Azhar University, Egypt

Background: Physical stress affects most people in some way, but prolonged exposure to physical stress can negatively affect all body systems including immune system. Chronic stress suppresses the immune system and its functions, which can lead to an increase in the susceptibility to infections and cancer.

Objective: Investigating the effect of chronic physical stress on some parameters of immune system.

Patients and Methods: This study has been carried out on 50 patients subjected to chronic physical stress, and 20 healthy persons of matched age and sex as a control group. Serum cortisol level (fasting, morning), IgG, IgM, IgA, C3 and C4 were measured, in addition to CBC and leucocyte differential count.

Results: There was a positive correlation between cortisol level and chronic physical stress, and a negative correlation between chronic stress and IgG and IgA levels. Results also revealed a statistical significant increase in WBCs count in the case group.

Conclusions: Chronic stress has a deleterious effect on immune system.

Keywords: Chronic stress, immune system, immunoglobulins, complement.

The Role of T Regulatory Cells and Pro-and Anti-Inflammatory Cytokines in Viral Persistence and Clinical Outcome in HCV-Infected Patients

Lobna Metwally1, Anwar Ahmed Heiba1, Omar Fathy Dessouki2, Nader Nemr3, Nahed Ibrahim Gomaa and GehanSedik Elhadidy

1Microbiology and Immunology Department, Faculty of Medicine, Suez Canal University, Egypt
2Clinical Pathology Department, Faculty of Medicine, Suez Canal University, Egypt
3Gastroenterology and Hepatology Department, Faculty of Medicine, Suez Canal University, Egypt

The immuno pathogenesis of chronic HCV infection is a matter of great controversy. We aimed to investigate the distributional profiles of T-regulatory cells (Tregs) and the balance between pro-inflammatory (interferon [IFN] and interleukin-[IL] 2) and anti-inflammatory (transforming growth factor [TGF] 1 and IL-10) cytokines among chronic HCV-infected patients in comparison to asymptomatic HCV patients and normal healthy controls. Ninety individuals (50 viremic HCV with elevated liver enzymes, 20 asymptomatic HCV patients with normal liver enzymes and 20 healthy blood donors as control) were investigated. Levels of Tregs subpopulation (CD4 + CD25 +, CD4 + FoxP3 + and CD4 + CD25 + FoxP3 +) were analyzed using 4-colour flow cytometry. In addition, IL-10, TGF- 1, IFN-, and IL-2 were measured in serum using ELISA. A significantly higher proportion of CD4 + CD25 + cells were found in those with chronic HCV infection compared to asymptomatic and normal controls. Similar results were obtained when comparing CD4 + FoxP3 + Treg subset among the chronic HCV group and the other two groups with p values equaling 0.009 and 0.04 respectively, similarly, with CD4 +CD25 +FoxP3 + significant differences were obtained between chronic and asymptomatic (p <0.004) and between chronic and control group (p <0.008). A positive correlation was found between CD4 +CD25 +FoxP3 +and HCV RNA titer (R=0.601, P>0.005), meanwhile, no relation between them and degree of fibrosis. Comparison of the cytokines profile of chronic HCV group with the asymptomatic one revealed significantly higher serum levels of IL-2, IL-10, TGF- and IFN-. Patients with chronic viral hepatitis display increased numbers of Tregs compared to other groups which highlight the importance of Tregs in immunopathogenesis of chronic HCV infection.

New Insights in Chaperone Mediated Extracellular Vesicle Communications and Regulations: Roles in Neuroinflammation-Mediated Neurodegeneration

Joy I. Irobi

Hasselt University, Biomedical research institute, Belgium

Immune responses to small heat-shock proteins (HSPBs) are observed in almost all chronic inflammation. HSPBs provide protection from cellular and environmental stress factors as molecular chaperones to maintain protein homeostasis. In Multiple Sclerosis (MS), there is an up regulation of HSPBs which are secreted out of the cells into the blood. Extracellular HSBPs have a protective role in mediating immunological functions and immunomodulatory activity. MS is a chronic autoimmune disease of the central nervous system, featured by immune cell mediated destruction of the insulating myelin around neuronal processes. Previously we showed that HSPB1 and HSPB8 have critical neuroprotective functions in the peripheral nervous system where mutations in these chaperones cause peripheral neuropathy and axonal death. We showed that expression of mutant HSPB1 decreased acetylated α-tubulin abundance and induced severe axonal transport deficits. Intracellular HSPB are released out and their potential extracellular functions during neuroinflammation have not been studied extensively. Neural cells secrete exosomes or extracellular vesicles expressing HSPBs. Exosomes are nanovesicles which are of great importance for their biomarker potential in disease diagnosis and therapy. We hypothesize that HSPB8 loaded extracellular vesicles restores the disrupted protective mechanisms of extracellular HSPB8 induced by neuroinflammation. Understanding the mechanism of EV loaded-HSPBs chaperoning activities in neuroinflammation will provide new insights to advance the development of therapeutic strategies in neuro degeneration.

Dr. Joy Irobi earned her doctorate in the field of molecular biology, and spent 17 years as the lead researcher for the internationally renowned Institute VIB and UAntwerp Center for Molecular Neurology. Currently, she is a professor of medicine and life sciences in the Biomedical Research Institute (BIOMED) at Hasselt University in Belgium. Her Neuro-Functional Genomics group at Hasselt University focuses on elucidating chaperone mediated extracellular vesicle communications and regulations in neuroinflammation-mediated neuro degeneration. She has produced more than 30 international peer-reviewed publications and has won several awards for her contributions to the field of biomedical sciences.

Impact of HCMV on HLA-I and Consequences on Gamma Delta T cell and Alpha Beta T cell Activation

Layal Massara

University of Bordeaux, College of Health Sciences, France

Cytomegalovirus (CMV), a Beta Herpes virus, is considered as a paradigm for viral evasion. It is an important opportunistic pathogen in immunocompromised patients and a major cause of congenital birth defects when acquired in utero. CMV encodes molecules to prevent antigen presentation to αβ T cells through inhibition of MHC Class I expression and to suppress NK cell functions by mimicking or down-regulating ligands of NK receptors (NKR). These evasion mechanisms are not expected to affect γδ T cells and, as a matter of fact, their response to CMV has been widely reported in many different physio-pathological contexts as well as in CMV-seropositive healthy donors (Dechanet et al, 1999)(Scheper, 2013).

Our aim was to understand how CMV induces γδ T cell response. We used recombinant adenovir uses expressing each of the four US genes, and a mutant HCMV deleted for these 4 genes (CMV-DUS). We observed an induction of HLA-I expression by the control adenovirus, and an inhibition by US2, US3 and US11. When using CMV-DUS, infected cells expressed much more native HLA-I than CMV-WT infected cells. Interestingly and in sharp contrast to αβ Tcells, γδ T cell were activated to produce IFNg when cultured with fibroblasts infected with CMV-WT, but not when fibroblasts were infected with CMV-DUS. These results indicate that HLA-I molecules regulate γδ T cells through mechanisms that are under investigation in our team. The immune escape processes developed by CMV could thus promote γδ over αβ T cell response and explain the important response of γδ T cells to the virus in immune suppressed individuals.

Pathological Manifestations in Lymphatic Filariasis Correlate with Lack of Inhibitory Properties of IgG4 Antibodies on IgE-Activated Granulocytes

Tomabu Adjobimey1,3*, Ulrich F. Prodjinotho1, Charlotte von Horn1 and Achim Hoerauf1,2,3

1Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Germany
2German Center for Infectious Disease Research (DZIF), Germany
3University Abomey -Calavi, Faculty of Health Sciences (FSS), Germany

Helminthes parasites are known to be potent modulators of their hostʼs immune system. To guarantee their survival, they induce alongside the classical Th2 a strong regulatory response with high levels of anti-inflammatory cytokines and elevated plasma levels of IgG4. This particular antibody was shown in different models to exhibit immunosuppressive properties. How IgG4 affects the etiopathology of lymphatic filariasis (LF) is however not well characterised. Here we investigated the impact of affinity-purified IgG4 positive and negative fractions from endemic normals (EN), LF infected pathology patients (CP), asymptomatic microfilaraemic (Mf+) and amicrofilaraemic (Mf-) individuals on IgE/IL3 activated granulocytes. The activation and degranulation states of the granulocytes were monitored using the expression of CD63/HLADR and the release of granule contents (neutrophil elastase (NE), eosinophil cationic protein (ECP) and histamine) by flow cytometry and ELISA respectively. Our results indicate that affinitypurified IgG4 antibodies from EN, Mf+ and Mf- individuals bound granulocytes and inhibited activation and the release of ECP, NE and histamine. In contrast, IgG4 from CP could not bind granulocytes and presented no suppressive capacity. More interestingly, a significant reduction of the anti-inflammatory effect on granulocytes was reached only when FcγRI and FcγRII were blocked simultaneously. These data indicate that IgG4 antibodies from Mf+, Mf- and EN, in contrast to those of CP exhibit FcγRI/IIdependent suppressive properties on granulocytes. Our findings suggest that both quantitative and qualitative alterations in IgG4 molecules are associated with the different clinical phenotypes observed in LF endemic regions.

Dr. Tomabu Adjobimey completed his PhD Studies on the modulation of antibodies secretion by regulatory T cells at the University of Bonn, Germany. He did his Postdoc at the Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn, Germany. Since 2011, he was working as a Group leader, with focus on the mechanisms of parasite-induced immune tolerance in human at Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn, Germany.

Intra-Tumoral Injection of SS1P Immunotoxin Combined with Anti-CTLA-4 Induce Massive Local Inflammation that Eradicates Resistant Multi-Centric Murine Cancer

Yasmin Leshem*, James OʼBrien, Yoram Reiter and Ira Pastan

Laboratory of Molecular Biology, National Cancer Institute, USA

Background: SS1P is an immunotoxin against mesothelin. Here we evaluated if SS1P injected directly into tumorscan transform tumors into living source of antigens that boost the effect of anti-CTLA-4.

Method: A BALB/c breast cancer cell line was transfected with human mesothelin (66C14-M) and injected in two different locations. SS1P was injected directly into one tumor loci and anti-CTLA-4 administered IP.

Result: In mice treated with anti-CTLA-4 and SS1P, we observed complete tumor regressions in 26 out of 30 SS1P injected tumors (87%) and in 16 out of 30 distant un-injected tumors (53%). No tumor regressions were observed in mice treated with the drugs separately supporting the case for synergic anti-tumor effect. Pathological evaluation showed a central abscess forming in injected tumors that was more prominent in combination treated mice. Surrounding the abscess, a collar of inflammation emerged only in combination treated mice containing a mixture of lymphocytes and mononuclears. Additionally, injected tumors were harvested in few time points demonstrating a time dependent increase in CD8+ cells located mainly in the inflammation collar. Furthermore, analyzing combination treated tumors showed that out of 768 immune related gene transcripts tested, 385 were elevated by twofold or more including CD8a, PDL2 and IL1 alfa (by a factor of 13, 21 and 27 respectively). Importantly, tumors from combination treated mice were tested negative for bacteria indicating that the massive inflammation found was targeting cancer cells.

Conclusion: Altogether, combining local SS1P with anti-CTLA-4 promotes massive inflammation that results in cure of multicentered cancer disease providing a strong rational to explore this combination therapy in patients.

Yasmin Leshem is a PhD candidate of Graduate Partnerships Program of the NIH (Bathesda, MD, USA) and The Technion University (Haifa, Israel). She is a Research Fellow/Post-Doc at Laboratory of Molecular Biology, National Institutes of Health (NIH), Bethesda, MD, USA.

Jinyangjo-Jangdan of DaegeumSanjo Musichas Positive Effects on Depression

Hyun-Ja Jeong1*, Hee-Yun Kim2, Kyung-Ja Ko2 and Hyung-Min Kim2

1Department of Food Science & Technology, Hoseo University, Republic of Korea
2Department of Pharmacology, College of Korean Medicine, Kyung Hee University, Republic of Korea

Music therapy is a commonly used adjuvant therapeutic method in depression. The purpose of music therapy is to improve, maintain, remediate, or prevent clinical issues, as determined by need for habilitation and rehabilitation. Daegeumsanjo is a Korean traditional folk music played with a Daegeum (a traditional Korean transverse flute). Jinyangjo-jangdan provides catharsis as very slow rhythm in pansori or sanjo. However, the effects of the jinyangjo-jangdan of Daegeumsanjo (JJDS) on depression have not been investigated. In this study, we investigated the antidepressant-like effects of JJDS. Mice were exposed to JJDS music daily for 21 days. Immobility times in the forced swimming test (FST) and distance moved in open field test (OFT) were measured, and after the FST, levels of depression biomarkers were analyzed. JJDS music significantly decreased immobility times as compared with control, and increased levels of serotonin, brain-derived neurotrophic factor, phosphorylated extracellular signal-regulated kinase, and estrogen receptor-β in brains. Levels of inflammatory cytokine were significantly lower in mice exposed to JJDS music. Furthermore, JJDS music did not affect locomotor activity in OFT as compared control. These findings indicate that JJDS music therapy be considered a potential therapeutic intervention for the treatment of depression.

Hyun-Ja Jeong was born in 1974 and received Ph.D. degree in College of Natural Science, Jeonbuk National University in 2003. She has been published about 200 articles about allergy, inflammation, cancer, and immune responses. She won Women scientist fellowship prize award from Korea Loreal-UNESCO and Womenʼs Bioscience Forum. In 2014 and 2017, she won the Best Research Teacher Award from Hoseo University. Currently, she is working in Hoseo University as an associate professor.

Indirubin Ameliorates Imiquimod-Induced Psoriasis-like Skin Lesions in Mice by Inhibiting IL-17+ γδ T cell Mediated Inflammatory Responses

Jing-Xia Zhao*, Ting-Ting Di, Yan Wang, Xiaolong Xu and Ping Li

Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing Institute of Traditional Chinese Medicine, China

Objectives: Indirubin is extracted from the leaves of Indigo naturalist, a traditional Chinese herbal medicine that has been used in China to treat inflammatory diseases for years. Psoriasis is a chronic immune-mediated inflammatory skin disease in which γδ T cells play an important role. This study aimed to demonstrate the immunoregulation effect of indirubin in inflammatory process of psoriasis and explore the mechanism further.

Methods: A mouse model of psoriasis was induced by administration of imiquimod. Mice were administered with indirubin, methotrexate, or normal saline intragastrically. Keratinocyte proliferation, inflammatory cell infiltration, cytokines mRNA levels, and JAK/STAT signaling pathway related proteins were examined. Moreover, mouse spleen cells were incubated under γδ T cell polarizing conditions with indirubin and cell viability was tested by CCK-8, secretion of IL-17 was measured by ELISA, activation of JAK3/STAT3 signaling pathway was detected by Western blotting.

Results: Indirubin ameliorated keratinocyte proliferation, reduced infiltration of CD3+T cells, γδ T cells in dermis to psoriatic lesions, altered γδ T+ cell amounts in spleen and lymph nodes, inhibited IL-1mRNA, IL-6mRNA, IL-23 P40 mRNA, IL-17AmRNA and IL-22mRNA levels expression and JAK3/STAT3 signaling in psoriatic lesions. In vitro, indirubin inhibited the IL-17A expression and secretion of isolated γδ T cells, suppressed the JAK3/STAT3 signaling activation.

Conclusion: Indirubin alleviates IMQ-induced psoriasis-like inflammation by reducing the infiltration of inflammatory cell and cytokine expression, especially inhibiting IL-17+ γδ T cell mediated inflammatory responses via JAK3/STAT3 mediated signaling pathway. Our results indicate that indirubin is a promising drug in the treatment of psoriasis.

Jingxia Zhao was born in 1981, China. She received PhD from Beijing University of Chinese Medicine in 2013. Now she is an associate researcher in Beijing Hospital of Traditional Chinese Medicine and Beijing Institute of Traditional Chinese Medicine. Her research focus on the potential activities of Chinese herbal extracts on anti-inflammation and immunoregulation effect. As principal investigator and co-investigator, she has completed 10 research programs totally, and has more than 20 peer-reviewed publications in journals in the field of immunology as first and/or correspondence author.

Serum Interleukin-8 and Insulin like Growth Factor-1 in Egyptian Bladder Cancer Patients

Gehan S. El-Hadidy*, Moushira A. Mahmouda, Mohamed H. Alib and Howayda M. Hassoba

Background: Bladder cancer is a major health problem in Egypt as it represents the most common malignancy.

Aim: Evaluation of the potential usefulness of serum IL-8 and IGF-1 in Egyptian bladder cancer patients.

Methods: Serum levels of IL-8 and IGF-1 were determined in 51 bladder cancer patients and 45 controls using a chemiluminescence enzyme immunometric assay.

Results: Serum IL-8 was significantly higher in cancer patients than in controls (P < 0.0001). It was significantly higher in patients with invasive cancer than those with superficial cancer (P < 0.01). Also, IL-8 showed a significant elevation in relation to schistosomal infection (P = 0.02) however, it did not differ in relation to either pathological type of tumor or its grade (P > 0.05). Receiver operating characteristic (ROC) curve indicated that IL-8 cut-off value of 35 pg/mL yielded the best combination of sensitivity (71%) and specificity (98%) for differentiating patients with bladder cancer from control subjects. Serum IGF-1 levels showed no significant difference between bladder cancer patients and controls (P > 0.05). There was no relationship between IGF-1 levels and clinicopathological parameters.

Conclusions: In Egyptian patients with bladder cancer, serum IL-8 is significantly elevated and its level is related to tumor invasion and associated schistosomal infection. Moreover, serum IGF-1 level does not help as a serum tumor marker in these patients.

Keywords: Interleukin-8, insulin like growth factor-1, bladder cancer.

Role of Cys-LT1 and NMDA Receptors in Protective Effect of Montelukast on an Animal Model of Post-Inflammatory Irritable Bowel Syndrome

Pariya Khodabakhsh1*, Hamed Shafaroodi2 and Pooneh Khodabakhsh3

1Department of Pharmacology, ShahidBeheshti University of Medical Sciences, Iran
2Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Iran
3Department of Computer and IT, Islamic Azad University South Tehran Branch, Iran

Irritable bowel syndrome (IBS) is a functional gut disorder with multi-factorial pathophysiology. Although the roles of many inflammatory mediators, involved in the progression of this disorder, have been thoroughly researched and studied, the part played by cysteinyl-leukotrienes (Cys-LTs) remains widely neglected. The aims in the present study were to evaluate the chronic effects of a cysteinyl leukotriene-1 (CysLT1) receptor antagonist, montelukast (ML), on acetic acid induced diarrhea predominant IBS (D-IBS) in rats and identify the involvement of CysLT1 and N-methyl-D-aspartate (NMDA) receptors and CysLTs in mediating the effects. After 4% acetic acid-induction of colitis, separate groups of animals had been treated with vehicle, ML (10mg/kg) alone and in combination with NMDA for 7 days. Visceral hypersensitivity was assessed after subsidence of inflammation on the seventh experimental day. Also, we analyzed pathomorphism of distal colon, Cys-LTs levels, and mRNA expression of CysLT1 and NMDA receptors to investigate the mechanisms of protective effects of ML. Behavioral pain responses to noxious mechanical stimulation were abolished by chronic treatment of ML. The effect of ML was significantly inhibited by NMDA pretreatment. Surprisingly, Cys-LT levels and CysLT1R mRNA expression showed no remarkable differences among groups. However, there was a significant increase in expression of NMDA receptor NR1 subunit in D-IBS control group, while ML treatment was associated with significantly decreased NR1 mRNA level in IBS rats. It is possible that the mechanism of such beneficial effects of ML in IBS at least to some extent is through modulating NMDA receptors.

Pariya Khodabakhsh was born in Tehran, Iran, in 1989. She received her doctorate in pharmacy from Islamic Azad University of Pharmaceutical Sciences, Tehran, Iran, in 2014. She is a second-year Ph.D student of pharmacology at ShahidBeheshti University of Medical Sciences, Tehran, Iran. She has authored numerous papers in national and international journals and conferences. Her current areas of research interest include Molecular and Structural Immunology, Immunopharmacology, Allergology and Neuroinflammation.

Evaluation and Comparison of IFN-γ Molecular Expression at Protein Level with T Lymphocytes Proliferation via Recombinant Fusion Peptide Gag-Tat-Env-Pol-Nef (HIV-1) in Balb/c Mice

Fatemeh Rouhollah1*, Roya Lahimgarzadeh2 and Kimia Hariri2

1Department of cellular and Molecular biology, Tehran Medical Science Branch, Islamic Azad University, Iran
2Tehran Medical Science Branch, Islamic Azad University, Iran

Introduction: Interleukins are among the group of cellular combinations and from the cytokines family that have an important role in the fortification and maintenance of immune system. IFN-γ is among the group of molecules wherein its expression causes stimulation of cellular immunity system. In this research we have studied the role and importance of Interferon gamma molecular expression as a cellular immunity index and its comparison with the cellular immune proliferation and its comparison with cellular proliferation in the control and the case groups that received 20 mM Hepes and gag-tat-env-pol-nef immunogen sequence respectively.

Material and Methods: The 6 to 8 weeksʼ male mice subcutaneously received protein at the rate 20 μg/mouse in the form of Prime/ Booster. At the end of second and fourth weeks, the rats were subjected to spinal cord injury; their spleen was extracted in a completely sterile environment for 72 hours cellular growth. The second week groups received only an injection and the fourth week groups received two injections. The cellular suspension with 20x106/cell was prepared, the growth medium included RPM1640 at 10% FCS. The cellular suspension at the rate of 100 μL/well was introduced in ELISA plates for Brdu and LTT assay.

Results: The results based on the statistical analysis showed that in the Booster and Prime recipient groups separately in each group, in the Case a significant difference was not observed in T lymphocytes proliferation rate and IFN-γ expression in relation to the control group, whereas a significant difference at P<0.05 was observed in the booster recipient groups in relation to the prime and this significance relates to increase of IFN-γ gene expression and T cells proliferation( with two methods) in the booster recipient groups in relation to the Prime recipient groups. This recombinant fusion Peptide be introduced as a powerful immunogen candidate in the design of HIV poly-epitope vaccines.

Discussion: The findings shows that the acquired immunogen sequences from the HIV-1 virus have an ability to stimulate the immune system at the protein expression level such as IFN-γ and T lymphocytes proliferation and resultantly fortification of the cellular immune system and in the future with the new design of these sequences the novel therapeutic routes can be used to design medicines for HIV treatment. Today, Interferons have diverse applications in the treatment of diseases typeʼs viz. types of cancers.

Keywords: IFN-γ, LTT, Brdu, Gag-Tat-Env-Pol-Nef

Fatemeh Rouhollah is Doctor of Philosophy in the field of Cellular and Molecular Biology, Assistant Professor and Faculty Member of Islamic Azad University, Tehran, Iran. She is the head of the Department of Cellular and Molecular Biology, former deputy at medical college of modern science, research attitudes in the field of recombinant vaccines for HIV and Evaluation of immune responses and T-cell vaccination and proinflammatory cytokines in infectious and autoimmune diseases.

Preparation Indirect ELISA Test for Detection of Antibodies against Serotype A13 of Foot and Mouth Disease (FMD) Virus in Cattle

Fatemeh Malekdar

RAZI Vaccine and Serum Researcher Institute, Iran

Foot and Mouth Disease (FMD) Virus is a contagious animal disease that causes irreparable damage to the economy of a country, including Iran where the disease is native in that. Among the ways to combat against FMD is vaccination and slaughter. Because of the specific situation of Iran, it is not possible to kill infected animals. Therefore, the most important way to fight the disease is to vaccinate. The methods used to evaluate the safety and determine the titer of antibody in a serum are mainly SNT and ELISA.

In this research, designing an indirect ELISA test based on coating of complete particle of viral 140S particle makes it possible to determine antibody and following that determining serotype and viral type without need for time-consuming and complex molecular tasks, including gene expression.

In addition, in the event of a new epidemic, a new epidemic condition can be detected by using serum antibody method. However, coating complete viral particle leads us to need virus purification as well as the anti-immunoglobulin conjugate antibody testing of the same animal.

In this study, SNT test was used as a Gold Test to determine the serum antibody level and comparing its results with indirect ELISA method to determine the sensitivity and specificity of the indirect ELISA test for measuring the anti-virus antibody rate of type (A13) FMD through rock analysis with 100% sensitivity and the specificity of 90% sensitivity using routine formulas with 100% sensitivity and specificity of 82%.

The results indicate that in our review, approximately 100% of the items which were examined by the SNT test having protective antibody, have been confirmed with the evidence that was tested by SNT with protective antibodies was also confirmed by indirect ELISA test as well. Besides, 82% of the serums that in terms of SNT test were lack of anti-body titer at a protective level were confirmed by an indirect ELISA test.

In this study, considering Cut off OD=0.3, there was a significant difference between the vaccinated animals and the unvaccinated animals in terms of antibody level against the A13 type. This indicates the correctness of the test and the accurate and proportional antibody detection against the under study viral types of FMD.

Keywords: ELISA, serum neutralization test, Food and mouth disease

Fluorescent Isothiocyanate Dextran Evaluates the Permeability of Blood-Brain Barrier in Rabies Infected Mice Model

Waqas Ahmad1,2, Liu Xinyue1, Li Yingying1, Xinyu Wang1, Ming Duan1, Zhenhong Guan1 and Zhang Maolin1

1Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University, Peopleʼs Republic of China
2Section of Epidemiology and Public Health, College of Veterinary and Animal Sciences, Pakistan

Rabies is primarily a horrifying viral zoonosis that annually accounts for 55,000 deaths worldwide. Acute encephalitis develops as the rabies virus (RABV) enters to the central nervous system by crossing the blood brain barrier (BBB) which is a tight junction of endothelial cells. In this study, three different molecular weights (70 kDa, 150 kDa and 200 kDa) of fluorescent isothiocyanate dextrans (FITC-Dextrans) were used to measure the extent of BBB damage and subsequent leakage patterns in brain tissues of rabies infected mice which were post-immunized with neutralizing antibodies to observe whether it has positive effect on infected mice by decreasing the death ratio. The brains were processed for immunofluorescence to observe the neutralizing antibodies and its relevant compatibility with the leakage of FITC-Dextrans.

Results showed that 70 kDa and 150 kDa FITC-Dextrans efficiently crossed BBB, and produced fluorescent illumination mainly in the cerebral cortex of brain. The enhancement of BBB permeability was significant at 5th day of post-immunization, while the neutralizing antibody neutralized some rabies virus particles by crossing BBB, but it did not present enough treatment effect to the dying mice. These findings suggest that FITC-Dextran is an important fluorescent marker to investigate the integrity of BBB permeability.

Keywords: Rabies virus, blood-brain barrier, Evans blue, FITC-Dextrans, neutralizing antibody.

Immunotherapy of Glioblastoma Spheroids Tumor Cultured in Fibrin Gel by Atorvastatin: In 3D in vitro Model

Armin Ai

School of Dentistry, Tehran University of Medical Sciences, Iran

Glioblastoma multiform (GBM) is the most aggressive glial neoplasm. Absolutely, the survival, growth, and invasion of GBM cells are promoted by various inflammatory cytokines. Statins, such as atorvastatin, are known to exert anti-inflammatory effects. Chronic inflammation is a pathological feature of cancer. Growth of solid tumors results in most cases in a hypoxic microenvironment and the release of various cytokines and growth factors, which together increase inflammation, angiogenesis in tumor stroma, and triggering signaling cascades that activate NFkappa B and STAT3 that produces predominantly by a specific subset of T helper cells (Th cells), namely Th17 cells. Interleukin-17 (IL-17) has emerged as a central player in the mammalian immune system. IL-17RA is expressed in most tissues examined to activate many of the same signaling cascades as innate cytokines such as TNFα and IL-1β. Furthermore, emerging knowledge regarding IL-17A/IL-17RA signaling in numerous tissues suggests an important role in health and disease beyond the immune system. This increasing evidence suggests that IL-17A and Th17 play a main role in autoimmune inflammation. A VEGF independent pathway was also found via NF-κB, which leads to suppression of the immune response targeting cancer cell. In this study, we investigated the anti-inflammatory and anti-angiogenesis activity of atorvastatin on engineered three-dimensional (3D) human tumor models using glioma spheroids and Human Umbilical Vein Endothelial cells (HUVECs) in fibrin gel as tumor models in different concentrations of atorvastatin (1, 5, 10μM). After 48 hours exposing with different concentrations of atorvastatin, cell migration of HUVECs were investigated. After 24 and 48 hours exposing with atorvastatin VEGF, CD31, IL-17R genes expression by real time PCR were assayed. In the current study, results have demonstrated a potential impact of IL-17R in glioma growth and progression. The results showed that atorvastatin has potent anti-inflammatory and anti-angiogenic effect against glioma spheroids by downregulates IL-17RA and VEGF expression especially at 10 μM concentration. The most likely mechanisms are the inhibition of inflammation by IL-17RA interaction with NFKB signaling pathway. Finally, these results suggest that this biomimetic model with fibrin may provide a vastly applicable 3D culture system to study the effect of anti-cancer drugs such as atorvastatin on tumor malignancy in vitro and in vivo and atorvastatin could be used as agent for glioblastoma treatment.

Keywords: IL-17RA. NF-κB, Inflammation, Angiogenesis, Glioblastoma, VEGF, Atorvastatin.

Human Herpesvirus Type 8 in Mild Cirrhotics

Cheng-Chuan Su1,2,3, Kuo-Chih Tseng1,4, Ming-Nan Lin1,5, Tang-Yuan Chu6,7 and Jen-Pi Tsai1,4,8

1School of Medicine, Tzu Chi University, Taiwan;
2Departments of Clinical Pathology,
3Anatomic Pathology,
4Internal Medicine,
5Family Medicine, Buddhist Dalin Tzu Chi Hospital, Taiwan
6Institute of Medical Sciences, Tzu Chi University, Taiwan
7Department of Obstetrics and Gynecology, Buddhist Tzu Chi Medical Center, Taiwan
8Institute of Medicine, Chung Shan Medical University, Taiwan

The high seroprevalence of human herpesvirus type 8 (HHV-8) in moderate or severe cirrhotics appears to be associated with male sex, hepatitis B virus (HBV) infection, alcoholism, and disease severity. The status of HHV-8 infection in mild cirrhotics remains unclear. Plasma samples collected from 93 mild cirrhotics and 93 age and sex-matched healthy controls were analyzed for HHV-8 antibodies and HHV-8 DNA. Mild cirrhotics had higher seropositivity for HHV-8 antibodies than healthy controls (P=0.0001). Univariate logistic regression analysis revealed that an age ≥55 years (odds ratio [OR] 2.88, P=0.02), hepatitis C virus (HCV) infection (OR 3.42, P=0.01), and hepatitis activity (OR 4.10, P=0.004) were associated with KSHV seropositivity in mild cirrhotics. Stepwise multivariate logistic regression analysis confirmed that age ≥55 years (adjusted OR [aOR] 1.92, P=0.04) and hepatitis activity (aOR 3.55, P=0.005) were independent factors. The rate of hepatitis activity was higher in HCV-infected than in HBV-infected patients (P<0.0001) and in women than in men (P=0.0001). Mild cirrhotics who were seropositive for HHV-8 or HCV or had hepatitis activity were significantly older (P=0.02, <0.0001, and <0.0001, respectively). Plasma samples from all participants were negative for HHV-8 DNA. HHV-8 antibody titers in mild cirrhotics also markedly exceeded those in controls (P<0.0001), as did those in patients ≥55 years old vs. younger patients (P=0.01), those in patients with vs. without HCV-infection (P=0.0008), and those in patients with vs. without hepatitis activity (P=0.0005). Mild cirrhotics had high HHV-8 seroprevalence and HCV infection, and, in particular, old age and hepatitis activity were predictors.

Dr. Cheng-Chuan Su completed residency training in Anatomic Pathology at the age of 31 years and in Clinical Pathology two years later; and obtained the Master degree from the Institute of Biomedical Engineering, National Cheng Kung University, Taiwan when he aged 32 years. At present, he is the Medical Director of Department of Clinical Pathology, Attending Physician of Department of Anatomic Pathology, Buddhist Dalin Tzu Chi Hospital, and Professor of Departments of Laboratory Medicine and Pathology, Tzu Chi University, Taiwan. He has published more than 40 papers in reputed journals and has been serving as an editorial board member of repute.

Spatholobus Suberectus Column Extract Suppresses Dendritic cell Maturation and has Therapeutic Potential for Psoriasis

Wang Yan*, Jingxia Zhao, Tingting Di, Xiaolong Xu, Mingxing Wang, Yujiao Meng, Xiangjiang Xie, Zhitong Ruan, Lu Zhang, Yan Lin, Xin Liu, Ning Wang and Ping Li

Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing Institute of Traditional Chinese Medicine, China

Spatholobus suberectus column extract (SSCE) is a natural product from the plant Spatholobus suberectus Dunn, a kind of traditional Chinese medicine, which is widely used to invigorate the circulation of blood and replenish blood, has antitumor and anticoagulant properties, and improves hematopoiesis. This study evaluated the clinical effects of Spatholobus suberectus column extract (SSCE) in an imiquimod (IMQ)-induced psoriasis mouse model and investigated its role in regulating the differentiation and maturation of dendritic cells (DCs). BALB/c mice were used to establish the animal model for psoriasis-like skin lesion; SSCE at 12 mg/kg (high), 6 mg/kg (medium), 3 mg/kg (low), respectively, were intragastrically administered. Psoriasis area and severity index (PASI) was used to evaluate the skin lesions. Histological changes, the thickness of epidermis, and the quantity of CD11c+ DCs in skin lesion and spleens were measured. In vitro experiments, bone marrow cells of mice were obtained, and CD11c+ cells were isolated. DCs with a mature state in differentiation and function were identified by flow cytometry. The influence of DCs on proliferation of allogenic lymphocytes was analyzed with CCK-8. SSCE treatment alleviated psoriasis-like skin with the decreased Psoriasis Area and Severity Index (PASI) score and obviously reduced the vertical thickness of epidermis. Besides, SSCE treatment decreased the quantity of CD11c+ DCs in skin lesions and spleens. Furthermore, SSCE reduced R848-induced murine bone marrow–derived DC maturation, characterized by reduced expression of CD80/86, and inhibited the alloproliferation of T cells. SSCE inhibited DC function and had potential as a therapeutic agent for psoriasis.

Wang Yan was born in 1982, China. She received D.S.A from Beijing University of Traditional Chinese Medicine in 2017. Now she is a research associate in Beijing Hospital of Traditional Chinese Medicine and Beijing Institute of Traditional Chinese Medicine. She is also a research fellow in Beijing Key Laboratory of Clinic and Basic Research with Traditional Chinese Medicine on Psoriasis. Her main research interest is TCM on autoimmune dermatosis.

Reduced Central Serotonin Up-Regulates Prostaglandin E2 Production in the Anteroventral Preoptic Region during Systemic Inflammation

Luiz G. S. Branco*, Clarissa M. D. Mota and Evelin C. Carnio

Department of Physiology, Medical School of Ribeirao Preto, University of Sao Paulo, Brazil Department of Morphology, Physiology and Basic Pathology, Dental School of Ribeirao Preto, University of Sao Paulo, Brazil

Serotonin (5-HT) is a neuromodulator involved in several central-mediated mechanisms, such as endocrine processes, behavior and sleep. Dysfunction of the serotonergic system is mainly linked to psychiatric disorders, but emerging evidence suggests that immune system activation may also alter brain 5-HT signaling. However, whether central 5-HT modulates systemic inflammation (SI) remains unknown. Thus, we measured 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the anteroventral preoptic region [AVPO - the hierarchically most important region for body temperature (Tb) control] during lipopolysaccharide (LPS)-induced SI. We also combined LPS treatment with intracerebroventricular (icv) injection of 5-HT and measured Tb (“hallmark” of SI), AVPO PGE2 (an essential mediator of fever) and PGD2 (a cryogenic mediator), plasma corticosterone (CORT - a stress marker with an endogenous anti-inflammatory effect) and IL-6 (an immune mediator) levels. We also assessed tail skin temperature [used to calculate heat loss index (HLI)] to assess a key thermo effector mechanism.

As expected we observed LPS-induced increases in Tb, AVPO PGE2 (whereas PGD2 remained unchanged), plasma CORT and IL-6 levels, as well as a decrease in HLI. These changes were accompanied by reduced levels of AVPO 5-HT and 5-HIAA. Besides we also observed negative correlations between 5-HT and 5-HIAA with plasma CORT levels and Tb, respectively. Moreover, icv 5-HT microinjection caused a U-shaped dose-response curve in LPS fever, in which the intermediate dose reduced the febrile response. Icv 5-HT microinjection prevented the LPS-induced increases in AVPO PGE2 (whereas did not alter PGD2), plasma CORT and IL-6 levels; besides preventing the reduced HLI. Our data are consistent with the notion that the AVPO 5-HT synthesis is downregulated during SI favoring AVPO PGE2 synthesis and consequently potentiating the immune response.

These results reveal a novel effect of central 5-HT as an anti-inflammatory neuromodulator that may take place during psychiatric disorders treatment with 5-HT reuptake inhibitors besides suggesting that 5-HT modulation per se is a potential therapeutic approach for inflammatory diseases.

Type 1 Diabetes and Coeliac Disease in Algeria

Meriche Hacene* and Gadiri S

Department of immunology, clinic st Therese, UHC Annaba, Algeria

Introduction: The co-occurrence of celiac disease and type 1 diabetes has been reported as 5-7 times more prevalent than celiac disease alone. The clinical presentation of celiac disease in patients with type 1 diabetes may vary considerably. Less than 10% of patients with type 1 diabetes and celiac disease show gastrointestinal symptoms. Celiac disease is more prevalent in type 1 diabetic patients than in the general population in Algeria country. As follows the ESPGHAN guidelines, diagnosis of celiac disease is based on the presence of villous atrophy and crypt hyperplasia by intestinal biopsy and the presence of antibodies against tissue transglutaminase.

Material and Methods: In a total of 420 diabetic adults were screened for coeliac disease by simultaneous detection of human IgA isotype antibodies directed against tissue transglutaminase, gliadin and deaminated peptide of gliadin by FIDIS Celiac DPG kits (Theradiag).

Resultants: Forty diabetic adults were positive for IgA class transglutaminase and a Deaminated Peptide of Gliadin antibody, all underwent biopsy of the small intestine. Twenty two cases of coeliac disease were found; all of these adults had characteristic biopsy establishing partial or total villousatrophy.

Conclusion: It was concluded that IgA class transglutaminase and deaminated peptide of gliadin antibody were a good marker of coeliac disease for screening tests of high risk populations. The prevalence of coeliac disease in Algerian diabetic population was 5.2% and we suggest that diabetic adults be screened routinely for antibody for coeliac disease at diagnosis of type 1 diabetes, every year in the first five years of follow-up.

Bcl-2 Inhibitor ABT-737 Alleviated in Vivo and in Vitro Allergic Rhinitis Reactions

Hee-Yun Kim1*, Hyun-Ja Jeong2 and Hyung-Min Kim1

1Department of Pharmacology, College of Korean Medicine, Kyung Hee University, Republic of Korea
2Department of Food Science & Technology, Hoseo University, Republic of Korea

ABT-737 is an inhibitor of Bcl-2 and has an anti-cancer property. Recent studies reported that tumor growth, invasion, angiogenesis, and metastasis were accelerated by inflammatory reactions. Here the aim of this study is to assess the anti-allergic inflammatory effect of an anticancer agent, ABT-737 on human mast cell line HMC-1 and allergic rhinitis (AR) animal model. ABT-737 significantly diminished production and mRNA expression of pro-inflammatory cytokines on activated human mast cell line, HMC-1. In an AR animal model, ABT-737 decreased rub scoring and IgE, histamine, thymic stromal lymphopoietin, pro-inflammatory cytokines, and vascular endothelial growth factor levels from the serum of ovalbumin-challenged mice. ABT-737 reduced numbers of infiltrated mast cells and eosinophils in nasal mucosa tissues of AR mice. In addition, levels of Th2 cytokines and chemokines were significantly reduced by ABT-737 in nasal mucosa tissues of AR mice. In conclusion, the results suggest that ABT-737 is potential candidate for treatment of AR.

Hee-Yun Kim was born in 1985 and received M.S. degree in Biological Engineering from Inha University, R.Korea. Currently, he is a Ph.D. candidate at Kyung Hee University, R. Korea. He has been published many articles about allergic inflammation. He won an Excellent Research Paper Award from Kyung Hee University in 2017.