Clinical \u0026 Cellular Immunology Immunology Summit-2014 Accepted Abstracts

$Page 1: Clinical \u0026 Cellular Immunology Immunology Summit-2014 Accepted Abstracts$
3rd International Conference and Exhibition on

Clinical & Cellular ImmunologySeptember 29-October 01, 2014 DoubleTree by Hilton Baltimore-BWI Airport, USA

216th OMICS Group Conference

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Immunology Summit-2014

Accepted Abstracts(Oral)

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Volume 5, Issue 5J Clin Cell Immunol 2014

ISSN: 2155-9899, JCCI an open access journalImmunology Summit-2014

September 29-October 01, 2014

September 29-October 01, 2014 DoubleTree by Hilton Baltimore-BWI Airport, USA


Clinical & Cellular ImmunologyInhibitor of differentiation 3 blocks oxidized phospholipid induction of monocyte chemotactic protein-1 in vascular smooth muscle cellsColeen McNamara, Jack Hensien, Aidan Toms and James GarmeyUniversity of Virginia Health System, USA

Background: Atherosclerosis, the most common cause of death worldwide, is a chronic inflammatory disease initiated by accumulation of oxidized phospholipids (oxPL) in the artery wall. OxPL in arteries induce vascular smooth muscle cells (VSMCs) to produce chemokines such as monocyte chemotactic protein-1 (MCP-1), which attracts monocytes to the artery. Inhibitor of differentiation 3 (Id3) is a transcription factor that has been shown to limit artery plaque buildup in mice. Humans with a specific single nucleotide polymorphism (SNP) in Id3 have greater artery plaque buildup, underscoring the relevance of identifying pathways whereby Id3 may regulate artery plaque buildup.

Hypothesis: OxPL induces expression of more chemokines, such as MCP-1, in VSMCs lacking Id3 compared to control.

Methods and Results: Cultured VSMCs with the Id3 gene deleted and control VSMCs were stimulated with oxPL, harvested 24 hours later and mRNA was isolated. The mRNA was reverse transcribed and then specific mRNA was quantitated using polymerase chain reaction (PCR). Loss of Id3 increased MCP-1 expression in VSMCs treated with oxPL. OxPL-induced expression of another chemokine unrelated to macrophage chemotaxis, CCL11, was not Id3-dependent.

Conclusions: Id3 may be an important factor that limits MCP-1 expression and monocyte infiltration in the artery wall and humans with the Id3 SNP may have increased plaque due to loss of Id3 inhibition of MCP-1 expression in VSMCs. The Id3 SNP may be able to identify humans that could benefit from local delivery of inhibitors of MCP-1.

BiographyInhibitor of Differentiation 3 Blocks Oxidized Phospolipid Induction of Monocyte Chemotactic Protein-1 in Vascular Smooth Muscle Cells. Jack Hensien, Aidan

Toms, James Garmey and Coleen McNamara Cardiovascular Research Center, University of Virginia Health System.

Jack Hensien and Aidan Toms are students in the Math, Engineering and Science Academy at Albemarle High School. They performed this work as part of a research assignment in the laboratory of Dr. Coleen McNamara. Mr. Garmey is the laboratory manager in the McNamara lab. Dr. McNamara is the Edward W. and Betty Knight Scripps Professor of Medicine/Cardiovascular Division at the University of Virginia. Her laboratory studies the role of inhibitor of differentiation 3 and vascular and immune cells in the development of atherosclerosis using murine models and through translational human studies.

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Coleen McNamara et al., J Clin Cell Immunol 2014, 5:5http://dx.doi.org/10.4172/2155-9899.S1.019

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Clinical & Cellular ImmunologyErythrocyte is the first line of blood antibacterial defenseHayk MinasyanPrivate Laboratory, Armenia

Background: After discovery of phagocytosis, it has become an axiom that blood cellular immunity is provided by leukocytes and in blood plasma phagocytes engulf, kill and digest germs. As to erythrocytes, it is generally accepted that they have no role in immune response and their main function is respiration. Our research provides objective video and photo evidence regarding erythrocyte bactericidal function.

Methods: Phase-contrast immersion vital microscopy of the blood of patients with bacteremia was performed and the process of bacteria entrapping and killing by erythrocytes was shot by means of video camera.

Results: Phase contrast microscopy reveals that erythrocytes take active part in blood bactericidal action entrapping and killing microorganisms. Video evidence demonstrates that human erythrocytes can repeatedly engulf and kill bacteria of different species and size.

Conclusion: The research demonstrates that erythrocytes are extremely important integral part of human blood cellular immunity. Compared with phagocytic leukocytes the erythrocytes (as cellular bactericidal agent) have the following advantages: (a) they are more numerous; (b) they are able to entrap and kill microorganisms again and again without being injured; (c) they are more resistant to infection and better withstand the attacks of pathogens; (d) they have longer life span and are produced faster; (e) their inner space filled with hemoglobin is inauspicious media for survival and proliferation of microbes and does not support replication of smaller intracellular parasitic organisms (chlamidiae, mycoplasmas, rickettsiae, viruses, etc.); (f) they are more effective and uncompromised bacterial killers. Blood cellular immunity theory and traditional view regarding the function of erythrocytes in human blood should be revised.

BiographyPlace and date of birth: Yerevan, Armenia, April 29, 1953 Education: MD, Yerevan State Medical Institute, Armenia, 1976.Scientific degrees: Candidate of Medical sciences (PhD): Leningrad Military-Medical Academy, 1984. Doctor of Medical Sciences (DSci): Leningrad Military-Medical Academy, 1988. Research interests: Gastroenterology, Immunology, Bacteriology. Publications: over 80 peer-reviewed journal papers, 1 monography (book) “The Functional Blocks And The Problems Of Clinical Medicine”, Moscow-Leningrad, 1990, 360 pp. 4 Patents regarding new methods of treatment. Memberships in professional societies: Academician of the Academy of Natural Sciences of Russia Academician of the Academy of Ecology (Russia) Member of the editorial board of “Russian Journal of Gastroenterology, Hepatology, Coloproctology” (Moscow).

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Hayk Minasyan, J Clin Cell Immunol 2014, 5:5http://dx.doi.org/10.4172/2155-9899.S1.019

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Clinical & Cellular ImmunologyThe immunoregulatory axis of tBregs in cancer escape and metastasis Arya BiragynNational Institute on Aging, USA

Recently we found a new subset of regulatory B cells, designated tumor-evoked Bregs (tBregs), which is generated in response to cancer-produced 5-lipoxygenase metabolites by targeting PPARα in resting B cells. The role of tBregs is to

convert CD4+ T cells into FoxP3+ Treg cells and thereby protect metastasizing cancer cells through the inactivation of effector NK cells and CD8+ T cells. tBregs also activate immunoregulatory functions of MDSCs needed for suppression of antitumor effector cells and metastasis. In the absence of tBregs, metastasis cannot be established despite expansion of MDSCs. Thus, inactivation of tBregs or the blockage of any step of its immunoregulatory axis will provide anti-metastatic benefits. We show that the depletion of Tregs and tBregs by targeting their surface-expressed CD25 abrogates breast cancer lung metastasis in mice, although some strategies, such as the use of B-cell depleting anti-CD20 antibody, can instead be harmful and greatly enhance metastasis due to depletion of beneficial B cells and enrichment of CD20Low tBregs. The regulatory axis can also be blocked using modified chemokines to deliver siRNA or immunostimulatory CpG-ODN to tBregs and Tregs. For example, transient inactivation of FoxP3 or IL-10 in Tregs via siRNA delivered by CCL17 and, alternatively, inactivation of tBregs with CpG-ODN bound CXCL13 can efficiently abrogate metastasis. Overall, our data underscores the importance of tBregs as key initiators of immunoregulatory events needed for successful breast cancer metastasis.

BiographyArya Biragyn received his PhD from the Institute of Molecular Biology at Engelgardt, Academy of Sciences of Russia, Moscow, in 1991. Since 2011, he is a tenured senior investigator and section chief at the National Institute on Aging. His research focus is to understand the “immunological paradox of aging”; that is, why cancer incidence is enhanced but cancers often grow more slowly in older people, and why autoimmunity is more prevalent in the elderly but older people have poor vaccine responses. He studies cancer-mediated immunoregulation to gain insight on the role of regulatory immune cells and to develop potent immunotherapeutics tailored for elderly. He has published more than 60 papers in peer-reviewed journals. He is an editorial board member or ad hoc reviewer of various journals and grant funding organizations.

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J Clin Cell Immunol 2014, 5:5http://dx.doi.org/10.4172/2155-9899.S1.019

Leveraging bacterial and human genomic datasets at NCBI for immunological researchBen BusbyNational Institute of Health, USA

In the last 15 years, we have gone from sequencing a single human genome to sequencing material, including exomes, transcriptomes and/or whole genomes from millions of individuals. This presentation will describe how NCBI displays

this genomic information, sometimes in conjunction with phenotypic information and how some of its public databases can be queried for computational biology and clinical questions. We are in the process of indexing the exons of over 1 million samples from over 800,000 individuals housed in the dbGaP database at every position in the human genome, as well as having expanded access to the raw data in several ways, and made variant data in these and our medical genomics databases widely accessible. Examples of how investigators can leverage bacterial and human genomic datasets to further immunological research will be described.

BiographyBen Busby is the Genomics Outreach Coordinator for NCBI, and the Chair of the Bioinformatics Department at the Foundation for the Advanced Education in the Sciences. He is very interested in the integration of large genomic datasets, specifically in the metadata harmonization and statistical normalization necessary to do so.

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Clinical & Cellular ImmunologyIL-7R: A target in ALL and autoimmunityScott K Durum1, W Q Li1, P Zenatti2, D Ribeiro3, L Zuurbier 4, M C Silva3, M Paganin5, J Tritapoe1, J A Hixon1, A B Silveira2, B A Cardoso3, L M Sarmento3, N Correia3, M L Toribio6, J Kobarg7, M Horstmann8,9, R Pieters4, S R Brandalise2,10, A A Ferrando5, J P Meijerink4, J A Yunes2,10 and J T Barata3

1National Cancer Institute, USA2Centro Infantil Boldrini, Brazil 3Universidade de Lisboa, Portugal 4Erasmus Medical Center, The Netherlands 5Columbia University, USA 6Universidad Autónoma de Madrid, Spain7Centro Nacional de Pesquisa em Energia e Materiais, Brazil 8German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia, Germany 9University Medical Center Hamburg-Eppendorf, Germany 10Universidade Estadual de Campinas, Brazil

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from leukemic transformation of T-cell progenitors in the thymus. It accounts for approximately 15% of ALL cases in childhood and

20-25% in adults and is a leading cause of death in children. IL-7 and its receptor (IL-7R) play a critical role in normal T-cell development and homeostasis. Mutations in IL-7R were identified in 9% of pediatric T-ALL patients. These mutations usually involved insertions of three amino acids including cysteine and proline in the extracellular juxtamembrane region. WT or mutant forms of the human IL-7R (hIL-7R) from patients were retrovirally transfected into an IL-7-dependent murine thymic cell line D1. Mutant hIL-7Rs induced ligand-independent activation of the Jak-Stat and PI3K pathways, cell survival and proliferation. Notably, mutant hIL-7R-expressing D1 cells formed subcutaneous tumors in Rag1-/- mice, with substantial infiltration into various organs that are normally affected in advanced stages of T-ALL, such as bone marrow, liver, lymph nodes and spleen. Further functional assays revealed that mutant hIL-7Rs constitutive signaling required homodimerization via cysteines in the inserted sequences and downstream Jak1 activation and were IL-7, γc and Jak3-independent. Jak inhibitors were effective in blocking proliferation and survival of transformed cells. The hotspot for insertions lies in exon 6 in precisely the same region as a coding polymorphism regulating risk for MS and other autoimmune diseases, and we observe that this polymorphism affects strength of signaling. Our findings indicate that IL-7R mutations drive T-ALL, whereas polymorphisms that increase signaling promote autoimmunity, implicating IL-7R and Jak1 as therapeutic targets in these diseases.

BiographyScott K Durum, Head of Immunological Cytokine Group, Laboratory of Molecular Immunoregulation and Deputy Laboratory Chief. After receiving his PhD in 1978, Dr. Durum received his postdoctoral training in immunology at the National Jewish Hospital in Denver and at Yale Medical School. Dr. Durum joined the Laboratory of Molecular Immunoregulation in 1984.

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Immunoregulation of E-FABP in breast cancer developmentBing LiUniversity of Minnesota, USA

Fatty acid binding proteins (FABPs) are known as central regulators of both metabolic and inflammatory pathways, but their role in tumor development remains largely unexplored. Here, it is reported that host expression of epidermal FABP

(E-FABP) protects against mammary tumor growth. It was found that E-FABP is highly expressed in macrophages, particularly in a specific subset, promoting their antitumor activity. In the tumor stroma E-FABP-expressing tumor associated macrophages (TAMs) produce high levels of interferon β (IFNβ) through upregulation of lipid droplet (LD) formation in response to tumors. E-FABP-mediated IFNβ signaling can further enhance recruitment of tumoricidal effector cells, in particular NK cells, to the tumor stroma for antitumor activity. These findings identify E-FABP as a new protective factor to strengthen IFNβ responses against tumor growth.

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Clinical & Cellular ImmunologyReversing immunological ignorance in a mouse model of human prostate cancer Gerardo Rivera SilvaUniversity of Monterrey, Mexico

Cytotoxic T cells have the competence to attack cancer cells and eradicate a complete tumor. However, this mechanism has showed challenging for two motives. First, T cells and other elements of the immune system ignore self-molecules

and cells. Second, tumor microenvironment has immunosuppressive elements capable of producing a mechanism called immunological tolerance. CD4+ T cells generate signaling molecules and activate immune cells that deliver efficient CD8+ T cell response. CD70 is essential for dendritic cells-facilitated delay of T cell tolerance initiation. CD80 and CD86 cells are implicated in the refunctionalizing the tolerized T cell. Here, these cells (CD4+, CD8+, CD70, CD80 and CD86) were obtained from a tumor prostate tissue treated with androgen ablation and were applied in a mouse model of human prostate cancer. Count cell number and purity for the particular cell population was realized by flow cytometric analysis. Preliminary results report that the activity of the CD8+T cells persisted for up to 45 days after treatment with CD4, CD70, CD80 and CD86 cells, and resulted in a significant diminution of tumor size. The stimulation of T cell infiltration and other immune cells in cancer tissues could have effects for the immunotherapeutic treatment of other hormone-related malignant tumors.

BiographyGerardo Rivera Silva has completed his PhD from University of Salamanca, Spain, Diploma in Molecular Biology, Pasteur Institute, Paris, and Postdoctoral studies from Laval University, Quebec and Northwestern University, Chicago. He is the Director of Laboratory for Tissue Engineering and Regenerative Medicine, University of Monterrey. He has published more than 25 papers in reputed journals and has been serving as a technical reviewer - Spanish translation- of Immunology books.

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Irrelevancy of a currently popularized concept accompanied by a novel cure method for immune diseasesKimihiko OkazakiThe Japan Medical Association, Japan

Although it is well established that an equilibrium state exists among antibody molecules in the vicinity of their receptors on the surface of immune cells, namely, cytolytic T lymphocytes and mast cells, it has long been taken for granted that

molecular substitutions of antibodies on their receptors never occur. Obviously, these two concepts disagree with each other because existence of equilibrium state itself indicates that every molecule of antibody keeps changing its status being attached to or detached from its receptor. In addition, a certain molecule of antibody does not necessarily keep attaching to one certain receptor. In other words, every receptor of antibody keeps changing antibodies. The alternative concept, namely, molecular substitutions of antibodies on their receptors do occur all the time, is extremely useful. Reason is that the concept can be applied to complete cure of immune diseases; allergic and autoimmune diseases. Still reason, of course, is that pathogenic specific antibodies could be replaced by harmless non-specific antibodies if the latter are accumulated in patients’ bodies. The necessary and sufficient condition for the accumulation is to repeat injecting the patient with non-specific antigens.

BiographyKimihiko Okazaki graduated from Kyoto University, Faculty of Medicine in 1959. He was then engaged in medical chemical research till 1981 and started working as an internist as of July 16, 1981. His main achievements are discoveries of (1) A novel coenzyme in Baker’s yeast, (2) Initiator of rat liver regeneration, and (3) A radical cure method of immune diseases.

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Clinical & Cellular ImmunologyLeishmania donovani negatively regulates TLR4 mediated host response by preventing ubiquitination dependent degradation of TRAF3Purnima GuptaUniversity of Calcutta, India

Leishmania donovani, causative agent of fatal visceral leishmaniasis, is an intramacrophage pathogen. It escapes host immune response by subverting Toll like receptor (TLR) signaling, which is critically regulated by protein ubiquitination.

Our work identified tumour necrosis associated factor (TRAF) 3 as a target used by Leishmania to deactivate LPS-mediated TLR4 signaling. TRAF3 is an E3 ubiquitin ligase of the membrane associated signaling complex that regulates TLR pathway through distinct protein ubiquitination at specific residues. We observed that TRAF3 which is ubiquitinated at lys 48 position and subsequently degraded following LPS treatment, persisted in L. donovani as well as L. donovani + LPS co-stimulated cells due to defective lys 48 ubiquitination. Unlike lys 48, lys 63-linked ubiquitinated proteins has been implicated in the signaling mediated activation of the molecules. Our results revealed lys 63-linked ubiquitination of upstream proteins in the cascade (cIAP1/2 and TRAF6), mandatory for lys 48 linked TRAF3 ubiquitination and subsequent degradation was significantly reduced during infection. The reason may be reduced association between ubiquitin conjugating enzyme Ubc13 and TRAF6 during infection. Persistence of TRAF3 resulted in stabilization of signalosome complex at the membrane resulting in inhibition of TAK-1 mediated pro-inflammatory responses. Inhibition of TRAF3 prior to infection by small hairpin RNA in Balb/c mice also showed enhanced production of IL-12 and TNF-α and significantly decreased spleen and liver parasite burden. Our findings identified TRAF3 as a novel molecular regulator exploited by Leishmania for successful infection.

BiographyPurnima Gupta is pursuing her PhD in Biochemistry from Calcutta University. She has received the prestigious CSIR-fellowship for Doctoral studies. Her research interest includes understanding how intramacrophage pathogens evade host cell defenses utilizing host signaling molecules to aid their survival within macrophage hostile environment.

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Clinical & Cellular ImmunologyCross talk between apoptosis, autophagy and necrosis-pathways involved in treatment of neuroblastoma, an in-vitro studyShailasree SekharUniversity of Mysore, India

Immunology ranks as one of the most rapidly expanding areas of biomedical science, covering topics from cancer and vaccine research to immune deficiencies and autoimmunity. Neuroblastoma and solid extracranial cancer of childhood

with an annual incidence of ~ 9.1 cases per million children is reported. Standard treatments include radiation along with chemotherapy, however results in side effects to children due its acute toxicity. Advances in technology has allowed for search of new nontoxic drugs to be used as a single or multidrug therapy in natural products with almost 60% of drugs approved for cancer treatment being from natural origin. Apoptosis, defined as a controlled suicide program to remove defective cells, without damage to neighboring cells, acts via death receptor (extrinsic) and the mitochondrial (intrinsic) pathway. The mitochondrial pathway, involving loss of mitochondrial membrane potential (Δψm), is regulated through a highly intricate cross-talk, includes caspases 8, 9 and 3 situated at pivotal points of the signaling process. Caspase-8 studies represent a yet unknown pro-apoptotic pathway activated by cytotoxic drugs, and further, it was identified as an important p53 target gene in drug-induced death of cancerous cells. Autophagy, type-II cell death, a catabolic process, is phenotypically characterized by formation of large intracellular vacuoles, termed autophagosomes. It includes sequestration of cellular proteins and organelles in a specific sequence prior to the destruction of the nucleus and has been recognized in various cancer cell types as a response to anticancer therapies. Induction of autophagy, results in post-translational processing of cytosolic-associated microtubule-associated protein-1A/1B light chain 3 (LC3-I; ~17kDa), to autophagosome membrane-bound LC3-phosphatidylethanolamine conjugate (LC3-II; ~19 kDa), leading to formation of autophagosomes. p-coumaric acid (p-CA), a ubiquitous plant phenolic acid, has been proven to render protection against pathological conditions. Studies on p-CA as evaluated for its capacity to induce cytotoxic effect to neuroblastoma, N2a, cells and possible mechanism of its action will be discussed.

BiographyShailasree Sekhar received her PhD from the CSIR Institute, Central Food Technological Research Institute at Mysore, Karnataka, India, in 2000. The thrust area under the Institution of Excellence was identified the biodiversity of Western Ghats medicinal plants (MP), with immunological affections and cancer prevention properties, due to location advantage of this hot spot to the University. She has been actively involved in compilation of their scientific data as reviews. Recently, she has brought out a database on medicinal plants of Western Ghats in an efficient way. Screening of MP with immunological affections used by tribes has resulted in identification of several of them with inflammation/cancer inhibiting property. Fingerprinting their metabolites has been her priority. She has published more than 30 papers in peer-reviewed journals, has 2 patents and is an adhoc reviewer of various journals. She has to her credit grants from National scientific agencies under Government of India.

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Clinical & Cellular ImmunologyBinding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist maravirocOlga Latinovic, Kate Schneider, Henryk Szmacinski, Joseph Lakowicz, Alonso Heredia and Robert R RedfieldUniversity of Maryland, USA

The CCR5 chemokine receptor is crucial for human immunodeficiency virus type 1 (HIV-1) infection, acting as the principal coreceptor for HIV-1 entry and transmission and is thus an attractive target for antiviral therapy. Studies have

suggested that CCR5 surface density and its conformational changes subsequent to virion engagement are rate limiting for entry, and consequently, infection. Not all CCR5 antibodies reduce HIV-1 binding, entry and infection, suggesting a need for more potent reagents. Here full length single chain (FLSC) IgG1, a novel CD4-gp120BAL fusion protein with several characteristics was evaluated that makes it an attractive candidate for treatment of HIV-1 infections, including bivalency and a potentially increased serum half-life over FLSC itself. FLSC IgG1 binds two domains on CCR5, the N-terminus and the second extracellular loop, lowering the levels of available CCR5 viral attachment sites. Furthermore, FLSC IgG1 is synergistic with Maraviroc, the only one licensed CCR5 antagonist. In this study, both, microscopy and functional assays were used to address the mechanistic aspects of the interactions of FLSC IgG1 and Maraviroc in the context of CCR5 conformational changes and viral infection. A novel Stochastic Optical Reconstruction Microscopy (STORM) was used based on high-accuracy of photoswitchable dyes localization with high resolution to visualize direct contacts between FLSC IgG1 and CCR. Viral entry inhibition by FLSC IgG1 with that of other CCR5 blockers was compared and showed FLSC IgG1 to be the most potent. Also it was also shown that lower CCR5 surface densities in HIV-1 infected primary cells result in reduced FLSC IgG1 EC50 values. In addition, CCR5 binding by the FLSC IgG1, but not by 2D7, was significantly increased when cells were treated with MVC, suggesting MVC increases exposure of the relevant epitope. Together, this data may have implications on future anti-viral therapy efforts.

BiographyOlga Latinovic received her M. Sc (2001) and PhD (2006) from Lehigh University, USA, where she was awarded the Sherman Fairchild scholarship for outstanding academic performance. As of 2010, she is an assistant professor working at the Institute of Human Virology led by Robert C. Gallo, MD at the University of Maryland, School of Medicine in Baltimore, USA. She heads the Laboratory for Imaging Studies of Pathogens and Host Cells Interactions. Dr. Latinovic’s research focus is on HIV-1 entry and its inhibition into host target cells, particularly focusing on the CCR5 coreceptor which plays a major role in HIV-1 infection and is consequently an attractive target for anti-viral therapy. Dr. Latinovic wrote the book Micromechanics and Structure of Soft and Biological Materials, as a sole author and published by Verlag Dr. Muller in 2010, and co-authored the book Handbook of Photonics for Biomedical Engineering published bySpringer-Verlag in 2013. Dr. Latinovic lectures Virus Entry course for the grad students at the Department of Microbiology and Immunology at the University of Maryland and she has lectured at various national/international conferences, and is on the Editorial Board of three scientific journals. She is a member of several national and international scientific societies.

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Acute radiation-combined syndrome: Immunological profile and response to therapeutic interventionsRisaku FukumotoUniformed Services University of the Health Sciences, USA

Victims of nuclear disasters often suffer from a radiation injury (RI) combined with other types of injury; radiation-combined injury (CI). Medical countermeasures proposed for RI are not necessarily effective for CI, and vice versa. There is a strong

need to identify pathology specific to CI and plan for effective treatments. The talk will summarize current CI research with respect to: An animal model, a unique pathology including immunological aspects, and medical countermeasures that have been tested. These together may direct us to prospective CI management.

BiographyRisaku Fukumoto has completed his PhD at the age of 28 years from the University of Tokyo and Postdoctoral studies from the National Institutes of Health. He is the lead scientist of Armed Forces Radiobiology Research Institute in the University. He has published more than 10 papers in a few years of current affiliation and is serving as an international editor of the Journal Progress in Health Sciences.

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Clinical & Cellular ImmunologyProtracted protection against malaria is maintained by memory T cellsUrszula KrzychWalter Reed Army Institute of Research, USA

Immunologic memory is one of the cardinal features of adoptive immune responses and is inextricably linked to lasting protection against infections. While many re-infections are prevented by memory T and B cells, malaria re-infections occur

frequently and the reasons for the absence of lasting protection in endemic areas remain poorly understood. By contrast, exposures of humans and laboratory rodents to radiation-attenuated Plasmodium sporozoites (γ-spz) induce sterile and durable protection against experimental sporozoite challenge. The presence of memory CD4 T cells has been shown to be associated with lasting protection in humans exposed P. falciparum (Pf) γ-spz. Studies focusing on durable protection induced in mice with P. yoelii and P. berghei γ-spz have, instead, implicated memory CD8 T cells as crucial features of lasting protection. We observed that in the Pbγ-spz model, MHC class I-dependent liver-stage (LS) Ag-specific memory CD8 T cells are indispensable for the maintenance of protection. We hypothesize that long-term protection to malaria, whether mediated by CD8 or CD4 T cells, can be induced and maintained. As such, it requires the formation and persistence of memory T cells with a reservoir of central memory cells maintained in part by LSAg-depot, IL-15 and possibly IL-4 and other cytokines that promote conscription of IFN-γ producing effector/effector memory CD8 T cells during re-infections.

BiographyUrszula Krzych received her PhD degree from Rutgers University and subsequently as a Postdoctoral fellow studied with the late Eli Sercarz at UCLA. She currently heads the Department of Cellular Immunology at WRAIR, where she has been conducting research on the various aspects of malaria, most notably investigating immune mechanisms of protection induced by malaria vaccines in humans and mice. She is also affiliated with the George Washington University, Washington, DC.

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Autophagy supports influenza A replication in the distal respiratory epitheliumDavid R Hahn and Timothy E WeaverUniversity of Cincinnati College of Medicine, USA

Autophagy (macroautophagy) is a highly conserved catabolic pathway in eukaryotes which supports cellular homeostasis through recycling of metabolites and clearance of cytotoxic agents and damaged organelles from the intracellular space.

Impaired autophagy has been linked to disease pathogenesis in multiple organ systems,with recent studies hinting at the existence of cell or tissue-specific requirements for autophagic function. To date, the requirement for autophagy in lung epithelial homeostasis has been poorly defined. Similarly, while a growing body of evidence suggests a role for autophagy in influenza A pathogenesis, a relative lack of in vivo study obfuscates the clinical relevance of this connection. The present study aimed to address these knowledge gaps through the generation and analyses of transgenic mouse models for targeted deletion of the Atg5 in the distal lung epithelium. Partial inhibition of autophagy, by recombination in up to 50% of targeted lung epithelial cells, was associated with significantly decreased morbidity and mortality, preservation of lung structure/function, and reduced levels of cellular apoptosis and viral replication following infection with influenza A/H3N2 virus. Importantly, a 50% reduction of autophagy in the lung epithelium was both stable and well tolerated throughout the lifespan of the mouse. In contrast, a model with >90% loss of Atg5 in the lung epithelium developed progressive lung pathology beyond one year of age and fared poorly with viral challenge, suggesting that at least some autophagy is needed. Taken together, these findings support the concept of targeting autophagy inhibition to lung epithelia as an adjunct to current therapies.

BiographyDavid R Hahn, PhD completed his Doctoral studies in Molecular and Developmental Biology at the Cincinnati Children’s Hospital Medical Center and the University of Cincinnati College of Medicine. Presently, he is pursuing his Postdoctoral studies in the Department of Clinical Pharmacology at Cincinnati Children’s Hospital with research interests in pharmacogenomic studies and pharmacological targeting of pathogen-host molecular interactions.

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Clinical & Cellular ImmunologyTolerance of graftsA P MalyshkinOrenburg State Medical Academy, Russia

Studies on computer simulation of the genome leads to understanding of not only the characteristics of body elements (structure, weight, color, etc.), but also all interactions between them are directly determined at the genomic level. Tolerance

of self-antigens should also be directly determined by the genome, through genomic or "smart" recognition, rather than through negative selection against self-reactive lymphocytes. The concept of linked functions holds that the very presence of the genes of class I MHC self-antigens in the genome "automatically" precludes immune response to these antigens. Therefore, integration of certain class I MHC genes of the donor into the genome of the recipient's hematopoietic stem cells in the course of preoperative treatment should result in tolerance of the donor's MHC antigens. Conceivably, this approach to the formation of tolerance should also work for xenogenic grafts, which would considerably enhance the possibilities of tissue and organ transplantation. The technique for integrating foreign class I MHC genes into the genome of hematopoietic stem cells has not been developed thus far. Solution of the numerous problems involved requires experimental research.

BiographyAlexander P. Malyshkin, male, microbiologist, graduated from Orenburg State Medical Academy in 1979 and worked for this academy as a researcher. After defending his Candidate of Science (Med.) dissertation (PhD thesis), he headed the Division of Laboratory Diagnosis of Orenburg Regional Tuberculosis Dispensary for some time. Dr. Malyshkin's field of research includes microbiology, immunology, and issues of infectious diseases and their prevention. He is the author of the active susceptibility hypothesis and a fundamentally new approach to the prevention of infectious diseases of plants, animals, and humans (including the HIV infection) based on it. The main recent work (now in press) is the chapter on the prevention of infectious diseases in the book Aquatic Plants and Plant Diseases (to be published by Nova Science). Dr. Malyshkin is exploring the possibility of collaboration in further developing and implementing his novel approach to disease prevention, which could be used, in particular, for breeding infection-resistant animal and plant varieties.

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Does vitamin D modulate the effect of acute or chronic stress on helper T cell subsets in peripheral bloodBilge Pehlivanoglu Hacettepe University, Turkey

Stress is an inevitable fact of life and affects all body systems. Maladaptive stress response is involved in pathophysiology of various diseases in a wide range of severity. The common point of all stress induced diseases is the dys/malfunction

of immune system. On the other hand acute or chronic exposure to stressor and individual differences varies the immune response where helper T (Th) lymphocytes have a central role. The balance between subtypes of Th cells (Th1, Th2, Th17) is the key for the survival and health of the organism. Vitamin D besides being an essential component of metabolism has critical modulatory role in immune response. Its deficiency is reported to increase the duration and severity of diseases, among which autoimmune conditions predominate. Our studies concerning the effect of acute and chronic stress pointed out the reversal effect of Vitamin D on stress induced changes in Th cells. Acute stress augmented Th1 response, in contrary chronic stress shifted the immune response to Th2 as presented by increased IFN-γ and IL-4 respectively. These changes in Th1/Th2 balance reversed back to control levels with Vit D application. Similar changes with vitamin D exposure were observed in the cytokine IL-17 indicating Th17 cell activity. These results support the role of vitamin D as an immunomodulatory agent especially in stress related conditions, a role that should further be studied.

BiographyBilge Pehlivanoglu, MD, after graduating from Hacettepe University Faculty of Medicine received the degree of physiologist from same University Department of Physiology in 2000. She continued her studies mainly on stress and immune system in Hacettepe University. Since 2001, she has been a member of the faculty at Hacettepe University Faculty of Medicine. Her publications are primarily related to stressful conditions and modulation of various body functions. She is on the editorial board of various medical journals.

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Clinical & Cellular ImmunologyIdentification of EhTIFIA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IAAnkita Srivastava and Sudha BhattacharyaJawaharlal Nehru University, India

Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at rDNA promoter containing the RNA Polymerase-I (Pol-I) with many auxiliary factors. RNA Pol-I forms transcriptionally active enzyme, in association

with a factor known as Rrn3P in yeast and Transcription Initiation Factor IA (TIFIA, Rrn3P homologue) in mammals. TIF-IA interacts with RPA43, a unique subunit of Pol I, and with two Pol I-specific TAF (TATA binding protein-associated factors), thereby serving as a bridge between Pol I and the pre-initiation complex at the rDNA promoter. TIFIA is phosphorylated at multiple sites, and signals in response to that affect cell proliferation and metabolism. In E. histolytica, the rRNA genes are present exclusively on 24.5 kb circular extra chromosomal plasmid named as EhR1. EhR1 contains two rDNA repeats (I & II), which are arranged palindromically, and separated by upstream and downstream spacers. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified and characterized the E. histolytica equivalent of the transcription initiation factor TIF-1A (EhTIFIA) which is known in other systems to control the initiation of rDNA transcription. EhTIFIA was identified by sequence analysis and have cloned and expressed the gene in E. coli. Immuno-localization studies showed, EhTIFIA co-localizes partially with Ehfibrillarin and the RNA Pol I specific subunit (EhRPA12) both of them are in the nucleolus (in eukaryotic cells), which in E. histolytica is located at the nuclear periphery. EhTIFIA was extensively phosphorylated in presence of E. histolytica nuclear extract and one phosphorylation site at S461 was identified through mass spectroscopy. EhTIFIA was shown to interact with EhRPA12 both in vivo and in vitro. Mass spectroscopy data showed the major interacting partners of EhTIFIA to be RNA Pol-I specific subunits as well as some nucleolar proteins Further, we are trying to characterize EhTIFIA in relation to RNA Polymerase-I transcription regulation. Our study demonstrates presence of similar transcriptional machinery in primitive E. histolytica as in higher eukaryotes with certain structural and functional reform.

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B16 Mouse melanosomes can serve as a chemoattractant for macrophagesAmeera Bukhari, Kyle Di Vito, Cynthia Simbulan-Rosentha and Dean RosenthalGeorgetown University, USA

Previous research in the Rosenthal lab has shown that the inhibitor of differentiation-4 (Id4) stimulates pigment production, in vivo, resulting in histiocyte recruitment and tumor necrosis. Histiocytes are tissue macrophages derived from the monocyte

lineage. However, the chemotactic factor that attracts macrophages to the melanoma has not been described. Two mouse macrophage cell lines (RAW264.7 and IC-21) were therefore incubated with different concentrations of conditioned medium (CM) containing melanosomes obtained from B16F0 mouse melanoma cells. HaCaT immortalized human keratinocytes were used as a negative control. After 3 hours of incubation with CM, macrophages were collected and melanosome uptake was measured by absorbance at 340 nm. Significant melanosome uptake was observed in both macrophage cell lines when compared to negative control, indicating that macrophages readily phagocytose melanosomes present in CM. A trans-well migration assay was performed to further examine the interaction between RAW264.7 and IC-21 macrophages and pigment-producing B16F0 melanoma cells. Our initial observation suggests a trend toward increased migration of macrophages toward pigment-producing melanoma cells and thus merits further investigation. Since Id4 expression was required for melanin synthesis in vivo, we investigated the effect of ectopic Id4 on the production of tyrosinase, the rate-limiting enzyme in controlling the production of melanin. However, no association was detected after 48 hours. Further time points, may be required to determine if Id4 alters tyrosinase production in B16 melanoma cells.

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Clinical & Cellular ImmunologyInnate and adoptive immune response to Chlamydia trachomatis infectionAruna MittalDelhi University, India

The role of intracellular recognition molecules like NODs expressed by cells of the female reproductive tract (e.g. Fallopian tube epithelium) in the response to chlamydial infection, or any STI, remains poorly understood. A suboptimal innate

immune response may result in a permissive environment for pathogen colonization, whereas an over-exuberant response will cause excessive inflammation and tissue damage. Modulation of the host response to infection is an attractive alternative or adjuvant approach to antibiotic therapies in treatment of genital tract infections. Genome sequence analysis has revealed that Chlamydia possesses numerous novel genes that might be involved in the manipulation of the host cells. The infected cells often display altered metabolic, immunological and cell biological characteristics, however, at the same time the microbes have to maintain the integrity and viability of host cells before completing their own intracellular replication. To achieve this goal chlamydiae have evolved the ability to both prevent the infected cells from undergoing apoptosis induced by intracellular stress and to protect these cells from recognition and attack by lymphocytes. Analysis of C. trachomatis genome had identified more than two dozens of open reading frames encoding proteins with potential proteolytic activity. Some of these proteases may be used to target host cell proteins because some proteins are cleaved and/or degraded in infected cells. The attacked host proteins include transcriptional factors, pro and anti apoptoytic proteins, DNA repairing enzymes, cyclins and cytoskeletal protein. However, survival strategies of Chlamydia at the tissue level and the relevance of these findings in disease pathogenesis have yet to be determined. Since till date a Chlamydia vaccine is unavailable, a better definition of human immune response along with Chlamydial survival strategies needs to remain an important research priority if we are to develop a vaccine against C. trachomatis infection that has protective and not deleterious effects.

BiographyAruna Mittal has been working in the field of Immunology for more than thirty years after obtaining Doctorate degree from Delhi University in 1977. She was awarded ‘Shakuntala Amir Chand’ Prize for Young Scientists for miniaturizing radiometric assay for M. leprae viability and drug resistance by ICMR in 1984. She worked at Rockefeller University, USA on purification of dendritic cells from peripheral blood and study of antigen presentation in leprosy. She initiated research in female genital chlamydial infections in 1990, subsequent to her DBT overseas Research Associateship at the Center for Disease Control, Atlanta, USA, where she was awarded the title of ‘Chlamydia Farmer’. In addition, she is the first one in India who has made considerable efforts to understand mucosal host immune responses locally in persistent and recurrent C. trachomatis infection in order to have a mechanistic understanding of immunopathogenesis of C. trachomatis infection which will help in vaccine development against Chlamydia. She is the only one in India carrying out extensive research on chlamydial pathogenesis and the first ones worldwide to publish reports on responses of mucosal (cervical) immune responses to chlamydial infection.

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Emerging functions for the Staphylococcus aureus RNome: Its relationships with antibiotic resistance, immune evasion and toxic peptide secretionBrice FeldenRennes University, France

Staphylococcus aureus is a serious pathogen for animals and humans, being one of the most frequently isolated bacteria in hospital-associated infections and also causing diseases in the community. To coordinate the expression of its numerous

virulence genes for growth, survival and adaptation, S. aureus uses various signalling pathways that include two-component regulatory systems, transcription factors, and hundreds of regulatory RNAs (sRNas). Biological roles have only been determined for a handful of these sRNAs, including cis, trans, and cis-trans acting RNAs, some internally encoding small, functional peptides and others possessing dual or multiple functions. Recent investigations from the author’s lab have identified as RNA that influence antibiotic resistance in S. aureus, a novel sophisticated translational control of an mRNA by two differentially expressed sRNAs that ensures supervision of host immune escape by S. aureus, and a novel toxin-antitoxin system producing membrane and secreted toxic peptides destroying host cells and competing bacteria with dissimilar strengths.

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Clinical & Cellular ImmunologyAnti-proliferative and anti-angiogenic activities of thalidomide dithiocarbamate analoguesBishoy Y A El-AaragMenoufia University, Egypt

Inhibition of angiogenesis is currently perceived as one of the promising strategies in the treatment of cancer. The anti-angiogenetic property of thalidomide has inspired a second wave of research on this teratogenic drug. The aim of the current

study is to investigate the anti-proliferative and the anti-angiogenic activities of two thalidomide dithiocarbamate analogues by determining their anti-proliferative abilities towards human umbilical vein endothelial cells (HUVECs) and MDA-MB-231 human breast cancer cell lines. Furthermore, their effects on the expression level of TNF-α and VEGF165 mRNA in MDA-MB-231 had been assessed. Moreover, we evaluated their effects on angiogenesis using HUVECs through tube formation assay and wound healing assay. Results illustrated that, MDA-MB-231 and HUVECs proliferations were not significantly affected by thalidomide at 6.25-100 µM. Besides, thalidomide failed to block angiogenesis at similar concentrations. However, thalidomide diminished the expression level of TNF-α and VEGF165 by 27% and 34.4%, respectively. Conversely, thalidomide dithiocarbamate analogues 1 and 2 were significantly established anti-proliferative action in MDA-MB-231 and HUVECs without causing cytotoxicity. Two analogues showing powerful anti-angiogenicity in the tube formation assay and wound healing assay as well as analogue 1 demonstrated more potent inhibition ability to suppress TNF-α (79%) and VEGF165 (52%) than analogue 2, TNF-α (56%) and VEGF165 (41%). Analogue 1 consistently showed the highest potency and efficacy in all assays. Taken together, our results support the further development and evaluation of novel thalidomide dithiocarbamate analogues as anti-tumor and anti-angiogenic agents.

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Vitamin A and immunity in HIV+ adults from BotswanaBoitumelo Stokie MotswagoleNational Food Technology Research Centre, Botswana

Diets ensuring the availability of micronutrients can provide the foundation for improving the quality of life for HIV positive individuals. The aim of this study was to investigate the efficacy of vitamin A fortified sorghum in improving

the immune status of HIV positive adults in Botswana. This was a double-blind randomised placebo controlled trial. 132 HIV infected adults were randomised to receive either sorghum fortified with vitamin A (N=67) or control (N=65) micronutrients. Serum retinol, CD4 cell count and HIV viral load were assessed at baseline and every 3 months for one year. The mean serum retinol level was 1.6µmol/L at baseline in both groups and did not change at the end of the intervention (experiment 1.6(0.7)µmol/L and control 1.5(6.8)µmol/L). Similarly there was no significant improvement in the CD4 cell counts (experiment 338(228,426) cells/mm3 and control 348(220,451)cells/mm3). The fortified sorghum did not improve the immune status of the study participants. Future research is needed to investigate the contribution of micronutrient supplementation in improving immune function of HIV infected people.

BiographyBoitumelo Stokie Motswagole received her PhD in Human Nutrition from the North West University in South Africa in 2010. She is employed at National Food Technology Research Centre as a Principal Research Scientist and Head on Nutrition & Dietetics Department. She started the Nutrition & Dietetics Department at the centre and through her hard work the department has grown to what it is today. Her main job is to conduct research aimed at improving the nutritional status of individuals, households, communities and the entire Botswana population and also conduct studies that provide evidence for the solutions nutritional problems that do exist in Botswana. She is considered an expert in body composition analysis. She has worked on numerous projects assessing body composition of individuals and has presented on the topic of fat distribution patterns in several fora. Her expertise is recognized by the International Atomic Energy Agency hence has been engaged in providing expert services in this area to several research groups in Africa. In addition, she has delivered nutritional education talks to various institutions around the country.

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Clinical & Cellular ImmunologyIn-vitro analysis of cytokines responses of visceral leishmaniasis and pulmonary tuberculosis patients to homologous and heterologous antigen stimulationHadeel Faisal Gad and Maowia M MukhtarUniversity of Khartoum, Sudan

Background: Leishmaniasis-tuberculosis co-infection has been reported many times mainly in the east region of Africa, but little is known about the immunological interactions of the co-infection. A case control study was carried out to analyze in-vitro cytokines responses in visceral leishmaniasis (VL) patients and pulmonary tuberculosis (TB) patients.

Method: The cytokine profiles of 30 leishmaniasis patients, 30 tuberculosis patients and 10 healthy individuals were compared after stimulation with live Leishmania Promastigotes and BCG. Th-1 (IFN-γ and TNF-α), Th-2 (IL-10) and inflammatory cytokine IL-15 were measured in the supernatants of stimulated whole blood samples whole blood using ELISA.

Results: The concentration of Th-1 cytokines (IFN-γ and TNF-α) were significantly higher in the supernatants of stimulated whole blood of VL patients compared with TB patients mainly when stimulated by L.donovani antigen. Th-2 cytokine IL-10 was significantly produced by whole blood of TB patients particularly stimulated with BCG. A significant concentration was detected in stimulated whole blood of VL and TB patients compared by healthy controls.

Conclusion: The Th-1 cytokines expressions to the homologous antigen stimulation in visceral leishmaniasis patients were higher compared to the non-stimulated which suggests a strong adaptive response. Meanwhile, the Th-2 cytokine IL-10 expression to the homologous antigen stimulation in TB patients was higher than the non-stimulated which led to strong suppression the protective Th-1 cytokines expression. This finding suggests that a re-occurring TB infection may generate a weak protective immune response which could lead to a more persistent infection.

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Investigating existing drug targets for autoimmune disease treatmentDeepti BistaAsian University for Women, Bangladesh

Autoimmune diseases are an emerging global health problem. The increasing incidence of these diseases worldwide, in addition to the economic and psychological burdens, calls for a sense of urgency for the development of efficient and robust

therapeutic treatments. There has been abundant research on the discovery of drug for autoimmune diseases but researchers still have not been able to restore the pathogenic immune response back to the normal one; instead, the success has been only to curtail the symptoms and related inflammation. The major challenge in the repertoire of treatments for autoimmune diseases that subsists is resetting the immune system back to normal functions. The complex and multifactorial pathogenesis of autoimmune diseases has made the development of robust therapeutics sluggish. Even so, the ongoing studies in parallel with the development of cutting-edge technology have shown promising results in the realm of autoimmunity treatment. This talk aims to study the available drug targets that have shown some promising results along with the challenges they are currently facing, which include anti-TNF therapies and activation of the TNF-TNFR2 pathway, stem-cell transplantation, altering the balance between pathogenic and regulatory T-cells, and genetic modulation. For the effective, feasible and affordable treatment, tilting the pro-inflammatory response to anti-inflammatory and increasing the number of regulatory T-cell or Th2 -either by targeting cytokine IL-6 or construction of APL are favored treatments for autoimmune diseases. Since there is cross-talk among different cytokines, it is important to investigate more on the primary roles of individual cytokines in order to get more insight into the specific drug targets.

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Clinical & Cellular ImmunologyEmerging paradigms in immunonutritionEbenezer SatyarajNestle Research Center, USA

Nutritional immunology is an emerging discipline that evolved with the study of the detrimental effect of malnutrition on the immune system. While malnutrition still remains a worldwide problem, life-stage [neonate or old age] and

natural stress are increasingly becoming the major causes of lowered immune status in both humans and animals. Unlike immunodeficiency caused by malnutrition, life-stage and natural stress need a more comprehensive strategy and cannot be addressed simply by correcting nutritional problems. Lowered immune status because of life-stage or natural stress is characterized by a reduced of antigen presenting cells [APC] function, resulting in a less efficient or altered immune response, leading to increased susceptibility to infections disease, increase in autoimmunity and cancers. Beyond providing essential nutrients, diet can actively influence the immune system. Over 65% of the immune cells in the body are located in the gut, technically making the gut the ‘largest immune organ’. The immune receptors of the innate immune system present in the gut are the primary targets of strategies for immunomodulation via diet. Diet interacts with the immune system at multiple levels, starting with providing basic nutrients, moving on to providing higher levels of key nutrients such as protein, vitamins & minerals, leading to a more focused modulation of the immune system. A framework elaborating diet - immune system interaction, with relevant examples will be discussed along with specific examples of how an immune-enhancing ingredient is evaluated, tested and formulated into diets.

BiographyEbenezer Satyaraj graduated with a Masters degree in Medical Microbiology from University of Madras, Madras, India in 1990. He completed his formal education by earning a Doctor of Philosophy degree in Immunology from the National Institute of Immunology, New Delhi, India in 1996. He continued his training at the University of Chicago, Illinois, as a Postdoctoral Fellow in Department of Molecular Genetics & Cell Biology doing research in Molecular Immunology. He subsequently accepted an Instructor’s position at the Department of Medicine, Northwestern University Medical School, Chicago, where he taught Immunology and conducted research in the area of autoimmunity. He joined Nestle Purina in 2003 where he currently serves as Group Manager, Nutritional Immunology at the Nestlé Research Center in St Louis, MO, leading a team that focuses on research in the area of nutritional immunology & cytokine biology. He has authored numerous scientific papers in the areas of cellular/molecular immunology and cytokine biology, including a recent publication in the journal Science that explains size variations in dogs and lectures internationally in the area of Nutritional Immunology. He is a member of the American Association of Immunologist and the American Veterinary Immunology Association, serves on the Editorial Board of ‘International Journal of Immunological Studies’ and is a reviewer for several journals including British Journal of Nutrition, Arthritis & Rheumatism and Journal of Gerontology.

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Anti-inflammatory effects of neurotropic drugs at pneumofibrosis are associated with their inhibitory impact on regional hematopoietic stem cells and progenitor cellsEvgenii Skurikhin, Ekaterina Khmelevskaya, Natalia Ermakova, Olga Pershina, Vyacheslav Krupin, Alena Reztsova, Inna Stepanova and Alexander DygaiRussian Academy of Medical Sciences, Russia

Pharmacological influence on the regional adult hematopoietic stem cells (HSC) and hematopoietic progenitor cells by neural-pharmacological agents may be a promising approach in the treatment of alveolar epithelium inflammation at

the fibrotic diseases. We investigated neural-pharmacological agents (reserpine, ketanserine, spiperone) influence on the inflammatory reaction development in the lungs of mice C57BL/6 during bleomycin-induced pulmonary fibrosis. We conducted cytometric analysis of HSCs (Lin-, Sca-1+, c-Kit+, CD34-), progenitor hematopoietic cells (Lin-, Sca-1+, c-Kit+), pan-hematopoietic cells (CD45+) from bone marrow, blood and lung. We evaluated ability of stem cells and progenitor cells to self-maintenance, their clonal activity and differentiation. It was shown neural-pharmacological agents decreased bleomycin-induced alveolar interstitial infiltration by lymphocytes, neutrophils, monocytes and plasma cells. At the same time with the improvement of histopathological measures in lung reserpine, spiperone and ketanserine reduced not only by the number of HSCs and hematopoietic progenitor cells in bone marrow, blood and lung, but also their proliferation and differentiation decreased. We assume the anti-inflammatory effects of neurotropic agents may be associated with their inhibitory impact on HSCs and progenitor cells.

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Clinical & Cellular ImmunologyAssessing the sustainability of the expansion of the expanded programme on immunisation in the GambiaFrancis SarrUniversity of the Gambia, The Gambia

The aim was to assess the sustainability of the expansion of the Expanded Programme on Immunisation (EPI) in the Gambia. This is a rare policy approach to assess immunization programmes in countries where the sustainability of the introduction of new

vaccines is particularly important. The review of the literature in this study has demonstrated that while some tools may prove useful for measuring the component of funding sustainability of immunization programmes, they were insufficient with regard to questions on other aspects of sustainability. To address these issues of sustainability sufficiently in the study, a stakeholder analysis questionnaire was identified from the literature for adoption. Selected indicators of financial sustainability in the literature, in addition to country-specific indicators, were incorporated in the stakeholder analysis questionnaire. Also, a resource map questionnaire was developed, based on the National Health Accounts methodology, to complement the stakeholder analysis tool. This approach provided qualitative information about stakeholders’ opinion concerning the issue of whether to use additional resources for improving efficiency in the supply chain rather than introducing new vaccines to determine whose interests should be taken into account, but more importantly to assess where stakeholders think the system works and where it doesn’t work, and if different parties have contradictory incentives. It also provided detailed information on the flow and uses of resources within the immunisation system, in addition to data that focus on questions of power, relationships, processes and accountability in the EPI. Thus, this combination of methods can be useful for systematic assessment of the sustainability of EPI systems and the efficiency of having multiple organisations running programmes.

BiographyFrancis Sarr is Head of the Department of Nursing & Reproductive Health, University of The Gambia. In addition to his professional preparation, he was educated at the University of Wales, Cardiff (MEd, Curriculum Development & Educational Administration), London University School of Hygiene and Tropical Medicine (MSc & Post-Graduate Diploma, Public Health), London University Institute of Child Health (Nutrition & Child Health Cert.) and London South Bank University (PhD, Public Health concentration). He is a Fellow of the West African College of Nursing. He has published 5 articles in reputed journals and a book on community health education for health professionals.

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Standardization of interferon-gamma assay for diagnosis of bovine tuberculosis in EgyptAbdellrazeq G S1, El-Naggar M M1, Mahmoud A H1, Davis W C2 and Singh M3

1Alexandria University, Egypt2College of Veterinary Medicine, USA3Helmholtz Centre for Infection Research, Germany

Bovine tuberculosis (bTB) is a chronic disease of animals caused primarily by Mycobacterium bovis (M. bovis), a member of the M. tuberculosis complex. In Egypt, bTB continues to cause significant losses in the cattle industry and a major public health

problem. There are critical gaps in the current Egyptian national bTB surveillance program (NbTBS) as it based only on the use of single cervical tuberculin test (SCT) with a mammalian type of PPD. Despite of its world wide application, the IFN-γ assay has not been applied in Egypt until today. This assay is a fully validated test by the OIE, EU and USDA as an ancillary test to the tuberculin skin tests. The objective of this study was to determine appropriate cut-off values for protein purified derivatives (PPD) and ESAT6-CFP10 antigens to obtain optimal results of IFN-γ assay in terms of sensitivity and specificity to complement official skin-test screening in Egypt. The sensitivity and specificity (Ser and Spr) of PPD and antigen cocktail-based IFN-γ assays (IFN-γ-BA and IFN-γ-EC) were analyzed by different cut-off points, relative to gold standards bTB positive confirmation (culture and PCR), retrospectively using blood samples collected from 34 SCT reactors from recognized bTB-infected zones in five high bTB prevalence governorates during the 2011, 2012 and 2013. The absolute specificity (Sp) was studied using blood samples collected from 14 cattle from one bTB-free herd from a bTB-free zone. Results revealed the use of IFN-γ-EC provided high sensitivity but equal specificity, comparable to the estimates obtained for IFN-γ-BA. Data analysis suggested the use of (PPDbOD>0.1, PPDbOD-NILOD>0.05 and PPDbOD>PPDaOD) strategy to get optimal IFN-γ-BA results {PPD-Ser (90%) and PPD-Spr (100.0%)}, and (ECOD-NILOD≥0.1) strategy to get optimal IFN-γ-EC results {EC-Ser (97%) and EC-Spr (100.0%)}. In conclusion, this is the first report determine the appropriate cut-off points to optimize use of the of IFN-γ assay as a serial test for diagnosis of bTB in Egypt.

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Clinical & Cellular ImmunologyNon-invasive fetal RHD genotyping: Validation of the method with 200 patientsGuinchard EmmanuelleEFS Rhône-Alpes, France

Background: Non invasive fetal RHD genotyping is an important tool to assess the risk of fetuse’s hemolytic disease of anti-D allo-immunized pregnant women by non invasive method

Method: A method of genotyping has been developed in the laboratory of Lyon-GHE according to Minon’s team: Exon 4, 5, and 10 are amplified by real time PCR. At first, genotyping results of 200 pregnant women have been compared with RH1 phenotype at birth.

Results: The most important parameters of validation have been tested: The sensibility and the specificity; the negative predictive value; the correlation study permitted to define criteria of biological interpretation. The validation of this method permitted to determine critical points and the limits of the method due to the minor amount of fetal DNA in the maternal plasma and existence of many variant forms of the RHD gene.

Conclusion: Fetal RHD genotyping can optimize the obstetrical management of women RH -1 allo-immunized by a simple, reliable and minimally invasive method

BiographyI am an M.D/PhD in Biology. I work currently in a laboratory of a Health Clinical Center, CHU of Lyon. I specialize in immuno-haematology as well as in NIPT: fetal RHD genotyping on maternal plasma. I was invited to present this at several international conferences (ESHG 2012) and I am the author of a recently published article (Non-invasive fetal RHD genotyping: Validation of the method with 200 patients, TCB, 2014 ).

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Air pollution, innate immunity and inflammationGangamma SNational Institute of Technology Karnataka, India

Epidemiological studies have established the relationship between air pollution exposure and adverse health effects. Studies have also shown that, significant part of the air pollution induced adverse health effects is inflammatory in nature. However,

the biological pathways of air pollution induced inflammation and different facets of inflammation are poorly understood. A new approach to understand air pollution was attempted which induced inflammation in which, air pollution associated inflammation is compared with inflammation induced by a model compound, endotoxin.The reason to select endotoxin as a model will be explained. But, this simplification may not take account of possible complex interactions of air pollution components with host, but could be enough to shed light on the mechanisms of inflammation due to air pollution. The role of innate immune receptors of endotoxin, signalling pathways and phenomena such as endotoxin tolerance in air pollution induced inflammation were explored. The study shows that endotoxin and associated signalling molecules play an important role in air pollution induced inflammation. The results also show that, the small amount of endotoxin present in air pollutant samples is important component in air pollution induced inflammation. Low level endotoxemia and inflammation among a group of workers with elevated levels of exposure is reported. The average plasma endotoxin concentration in the group of workers was an order of magnitude higher than that observed in the controls. However, the study results show that, despite of presence of endotoxin in the circulatory system, tolerance or priming is not observed in ex-vivo stimulation.

BiographyGangamma S has completed her PhD from Indian Institute of Technology, Bombay. She is an Assistant Professor in the Department of Chemical Engineering, NITK Suratkal. She is working in the area of environmental immunology and the focus is to understand the air pollution induced inflammation. She is actively involved in teaching environmental immunology at Post-graduate level. She has significant experience in aerosol measurement methods, development of lung deposition models, aerosol instrumentation development and in environmental health studies.

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Clinical & Cellular Immunology


Recent insight into pathogenesis of inflammatory bowel disease for novel therapeutic strategiesFatemeh Zare ShahnehJohannes Gutenberg University, Germany

The activation of intestinal epithelial, innate and adaptive immune cells through Pattern Recognition Receptors (PRRs) are generally lead to defense mechanism or chronic inflammatory disorders and respond through secretion of pro-

inflammatory cytokines and chemokine in host immune responses. The gastrointestinal tract is constantly exposed to wide range of antigens including enteric bacteria and foods but gut homeostasis is maintained and controlled by suppressing excessive immune responses to foreign antigens. In both innate and adaptive immunity, the disruption of regulatory mechanisms may result in inflammatory immune responses to enteric antigens and cause chronic intestinal inflammation such as Inflammatory Bowel Disease (IBD). Dysfunction of Toll-like receptors as a PRRs prototype is associated with infectious and inflammatory diseases, for which therapeutic management with PRPs antagonists demonstrate a promising drug strategy. IBD patients are mostly treated with anti-inflammatory and immunosuppressive drugs, antibiotics, and biological therapies and/or surgery. Oral medicinal plants-derived compound can be a future promising to the prevention and treatment of IBD. Better understanding of the etiopathology basis of IBD lead to the development of novel targeted therapeutics. Experimental animal models can be representative of human IBD and provide new insights into the role of the pathways, cells and molecules which is essential for intestinal homeostasis. Animal models of IBD (genetic, chemical or induced by immune cell transfer) are particularly useful to study the contribution of innate immune mechanisms of IBD and reveal the mechanisms of intestinal pathologies. Experimental animal models (gene targeting approaches and/or chemical models of colitis) of intestinal inflammation have studied the role of the epithelial barrier in intestinal homeostasis to demonstrate epithelial mechanisms deregulated in IBD. Association between IBD and genes that control immune pathways such as TLR, RAGE (Receptor of advance glycation end product), nucleotide oligomerization domain (NOD)-like receptor (NLRs) genes that regulate inflammatory response (NF-KB, AP1 and IRFs) and genes in the T helper cells pathway indicate the significant roles of host-microbe interactions in regulating intestinal immune hemostasis. Inflammatory cell infiltrates were dominated by abundant lymphoid follicles, large numbers of DC and granulocytes, CD4+ T cells and granulocytes and increased expression of inflammatory cytokines and chemokines (IL-6, IL-11, IL-12, IL-18, IFN-γ, COX-2) in colitis mice. Active compounds derived from natural compounds were also found with unique features as PRPs antagonists. Recent evidences encourage further research works on characterization for these compounds, which will become promising drug candidates in PRPs-based therapy in the future.

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Clinical & Cellular ImmunologyTrypanosoma cruzi strains cause different myocarditis patterns in infected miceHéctor O Rodríguez AInstituto Venezolano de Investigaciones Científicas, Venezuela

Aims: Chagas disease pathology is dependent on the infecting T. cruzi strain. However, the relationship between the extent and type of myocarditis caused by different T. cruzi strains in the acute and chronic phases of infection has not been studied in detail. To address this, we infected mice with three genetically distant T. cruzi strains as well as infected in vitro different cell types.

Methods and Results: Parasitemia was detected in mice infected with the Y and VFRA strains, but not with the Sc43 strain; however, only the Y strain was lethal. When infected with VFRA, mice showed higher inflammation and parasitism in the heart than with Sc43 strain. Y and VFRA caused homogeneous pancarditis with inflammatory infiltrates along the epicardium, whereas Sc43 caused inflammation preferentially in the auricles in association with intracellular parasite localization. We observed intramyocardic perivasculitis in mice infected with the VFRA and Y strains, but not with Sc43, during the acute phase, which suggests that endothelial cells may be involved in heart colonization by these more virulent strains. In in vitro infection assays, the Y strain had the highest parasite-cell ratio in epithelial, macrophage and endothelial cell lines, but Y and VFRA strains were higher than Sc43 in cardiomyocytes.

Conclusions: This study supports parasite variability as a cause for the diverse cardiac outcomes observed in Chagas disease, and suggests that endothelial cells could be involved in heart infection during the acute phase.

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Gut and integrated pathophysiology of immune response: Observations from experiments in oral tolerance in mice and the response associated with a model of metabolic syndrome surgeryGiovani Marino FaveroState University of Ponta Grossa, Brazil

The gut mucosa is the place that most contact with foreign antigenic proteins occurs and forms with the immune system an integrated, dynamic and adaptive complex that has evolved to provide effective digestion and defense. The whole

intestinal area is 100-fold larger than the skin, presents the largest amount of lymphoid tissue of the body and the more number of activated lymphocytes. The Peyer´s patches and the lamina propria of the gut present a very large number of T cells. Immunoglobulin production, especially IgA, is the only antibody secreted by mucosal, that offers the first protection to neonates. For the experiment with oral tolerance, a protocol on adult Swiss mice by oral administration of a recombinant dermonecrotic toxin of brown spider Loxosceles intermedia (LiRecDT1) and its mutated form (LiRecDT1H12A) for three weeks was proposed. Our results demonstrated evidences of tolerance induction through decrease in IgG anti-dermonecrotic toxin levels, paw edema reduction and increased survey in 24 h after challenge. All statistical analysis was performed using ANOVA following Bonferroni’s pos hoc test. Related to bowel surgery readjustment we observed that the removal of the greater omentum decreases the secretion of cytokines, particularly IL-6, regressing other diseases associated with obesity such as bronchitis. In conclusion, the intestine can be considered the main immune organ of the body and this association between immunity and digestion begin prior to birth and mediate allergic responses and/or tolerance throughout the life of the individual.

BiographyGiovani Marino Favero is an Adjunct Professor at the State University of Ponta Grossa. He guides Master’s and Doctoral students of Pharmaceutical Sciences and Evolutionary Biology programs. He is currently Head of the Department of General Biology, Coordinator of the Ethics Committee on Animal Use, Editor-in-Chief of the magazine Publication UEPG Biological and Health Sciences. He works with associated gut immunology, drug development and cancer.

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Clinical & Cellular ImmunologyNovel expression of Schistosoma mansoni cathepsin L1 suggesting potential role as a diagnostic marker for human schistosomiasisIbrahim R B Aly1, Eman E L-Ahwany1, Enas Nweir2, Shreif Edris2 and Suher Zad2 1Theodor Bilharz Research Institute, Egypt 2American University in Cairo, Egypt

Schistosomes utilize proteinases to accomplish several activities such as tissue penetration, tissue digestion and evasion of host immune responses. Cathepsin L which indicates that this enzyme contributes to the proteolysis of ingested hemoglobin is a

cysteine proteinase of the papain superfamily detected in their gut lumen. Due to the roles played in the schistosome biology, proteolytic enzymes are considered potential targets for developing and guiding antischistosomal therapies. In the present work, the cathepsin L cDNA coding of Schistosoma mansoni was cloned providing high-level expression of heterologous proteins in Escherichia coli to produce recombinant schistosome cathepsin L with 597 bp. Our product had 99% similarity to a Schistosoma mansoni Puerto Rican preprocathepsin L (SmCL1) mRNA. The recombinant fusion protein was then analyzed by SDS-PAGE and Western blotting resulting in a molecular weight of approximately 31 kDa. The recombinant protein was expressed as inclusion bodies, purified under denaturing conditions. This recombinant protein could be recognized specifically by rabbit antiserum against Schistosoma adult worm antigen preparation (SWAP), showing that the expressed product possessed good antigenicity. The specificity and sensitivity of the purified recombinant cathepsin L against S. mansoni infected patients was assessed using ELISA. Sera from 97 S. mansoni infected patients, 30 patients infected with other helminthic infection and 30 healthy controls were collected. Anti-Cathepsin-L S. mansoni antibodies were detected with the sensitivity reached to 99% and specificity was 99.3 %. These results suggest that our product of S. mansoni cathepsin L might be considered as a suitable candidate for diagnosis of human schistosomiasis.

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AID expression in CD4+ T cells is associated with a IL-10-producing subset that increases with ageHongyan QinFourth Military Medical University, China

Activation-induced cytidinedeaminase (AID) is essential for immunoglobulin gene alterations to form immune memory. In addition, AID has the genotoxicactivity that is involved in tumorigenesis even in non-B cells. To examine the feature

of AID-experienced lymphocytes, a genetic system was used, in which both past and current Aicda (a gene encoding AID) expression can be detected with high sensitivity. With this system, fractions of AID-expressed (exAID) T and B cells accumulated by aging were observed. Most of the accumulated exAID B cells remained IgM positive, but many of them bore mutated Ig. The Aicda expression were also detected in a substantial fraction of CD4+ T cells that increased by aging, and can reach to almost 25% of total CD4+ T cells at the age around 18 months. The expression was rather augmented in the absence of B cells using μMT mouse, thus T-B interaction may not be critical for AID expression in T cells. Because the exAID T cells showed a unique feature that produces IFN-γ and IL-10, but few IL-4 and IL-17, raising a possibility that AID is involved in a function of particular subset of CD4+ T cells. Further, it was found that almost exAID CD4+ T cells in aged animals were PD1+ cells, also indicating exAID expression accumulation with increasing age. These results imply the involvement of AID in multiple biological processes in T and B lymphocytes development.

BiographyHongyan Qin has completed her PhD from Fourth Military Medical University and Postdoctoral studies from Department of Immunology and Genomic Medicine in Graduate School of Medicine of Kyoto University. She is the Vice-Director of Department of Medical Genetics and Developmental Biology of Fourth Military Medical University. Until now she has published more than 20 papers in reputed international journals.

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Clinical & Cellular ImmunologyFirst description of auto-antibodies to vascular endothelial cadherin in humans: Association with autoimmune diseasesIsabelle Y VilgrainGrenoble Alpes University, France

Patients with autoimmune diseases may present high risk of developing vascular cell dysfunction. To identify them, early biomarkers are required. VE-cadherin is an endothelial specific protein in charge of vascular integrity. This study

was designed to detect and characterize circulating autoantibodies to VE-cadherin (AAVEs) in patients with early-stage autoimmune diseases. An enzyme-linked immunosorbent assay (ELISA) was developed to capture autoantibodies, using a recombinant human VE-cadherin fragment covering the extracellular domains as a target antigen. Basal AAVEs levels were determined for healthy donors (HD, n=75) and for patients (n=100) with various autoimmune diseases, including rheumatoid arthritis (n=23), systemic lupus erythematosus (SLE, n=31), systemic sclerosis (n=30), and Behçet's disease (BD, n=16) were also tested. Levels of AAVEs were significantly higher in rheumatoid arthritis (P<0.0001), SLE (P<0.05), and BD (P<0.05) patients as compared to HD. Epitope mapping of AAVEs from a BD patient, showed a specificity for the EC3 and EC4 domains of VE-cadherin, whereas SLE patients preferentially recognized the EC1 fragment. Purified IgG from BD patients was found to induce endothelial cell retraction, redistribution of VE-cadherin, and cause the formation of numerous intercellular gaps. Altogether, these data demonstrate a potential pathogenic effect of AAVEs isolated from patients. In addition, distinct epitopes of human VE-cadherin might be specific for distinct immune diseases. Because regions EC1 and EC3-4 have been shown to be involved in homophilic VE-cadherin interactions, AAVEs produced in the course of dysimmune diseases might be specific biomarkers for endothelial injury, which is a part of the early pathogenicity of these diseases.

BiographyIsabelle Y Vilgrain completed her PhD in 1985 (France) and is a full-time Research Scientist at the National Scientific Research Council of France (CNRS) since 1985. From 1981 to 1989, she was a pioneer in protein kinase C in endocrine tissues. She has been awarded by the Foundation pour la Recherche Médicale in 1987. In 1989, she moved to the Whittier Institute in San Diego (CA, USA) for two years. From 1992, she gradually turned from cell signalling and kinases toward in vivo models and vascular biology in oncology with applications in clinic. In 2006, she developed means for the detection of biomarkers in blood and has 3 patents in the field of in vitro diagnosis. Her current research is focused on the study of knock in mice and the identification of biomarkers in cancer, inflammatory and autoimmune diseases.

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Virulence gene markers and biofilm formation of Aeromonas species recovered from cow Isoken H Igbinosa1,2, Etinosa O Igbinosa1,3 and Anthony I Okoh1

1University of Fort Hare, South Africa2University of Pretoria, South Africa3University of Benin, Nigeria

This study was carried out to assess the antibiotic susceptibility profiles of Aeromonas strains recovered from cow feces, and the virulence potentials of the isolates. Aeromonas isolates obtained in the study were found to show total resistance to

novobiocin, oxacillin, clindamycin, ticarcillin and trimethoprim. Also Aeromonas strains demonstrated susceptibility against oxytetracycline, tobramycin and cefotaxime. Aeromonas strains from Lovedale farm were found to harbor virulence genes in the following proportion:- ast 35.7%, aer 71.4%, fla 60.7%, hlyA.25%, lip 35.7%. While isolates from Fort Cox farms were found to possess virulence markers in the following fraction:- ast 55.2%, fla 78.9%, alt 10.5%, aer 63.1%, hlyA 35.9%, lip 21%. Class 1 integron was present in 27.2% of isolates; blaTEM gene was detected in 34.8% of isolates while blaP1 class A β-lactamase gene was present in 12.1% of isolates. About 86% of the isolates formed biofilm on microtitre plates. The possession of multidrug-resistant determinants and virulence genes presence in Aeromonas isolates from cow feces reveals the pathogenic and infectious significance of these isolates and is of great significant to public health. The biofilm forming potential of the isolates may pose challenge during treatment of infection associated with Aeromonas species.

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Clinical & Cellular ImmunologyThe role of miR-155 in the apoptosis of human lymphoma cell induced by CIK cells Jiang Yi Wuhan University, China

Objective: To observe the effect of cytokine-induced killer cells (CIK) on the apoptosis of human lymphoma cells (Raji and BJAB), and explore the role of micro RNA-155(miR-155) in this process.

Methods: MiR-155 was determined by real time quantitative PCR and the apoptosis was detected by flow cytometry in Raji and BJAB cells. Psi CHECK2-CEBPB 3’-UTR containing the binding site of miR-155 was constructed, and then transfected into Raji and BJAB cells. Luciferase activity of CEBPB (CCAAT/enhancer binding protein beta) was determined with the assistance of dual luciferase report system.

Results: CIK cells could promote Raji and BJAB cells apoptosis (t=3.634, P<0.05; t=3.976, P<0.05), and increase the expression of miR-155 in Raji by (6.87±0.19) fold (t=2.787, P<0.05), meanwhile, in BJAB cells by (5.06±0.25) fold (t=3.513, P<0.05). Moreover, miR-155 inhibitor might block this process (t=3.842, P<0.05; t=4.016, P<0.05). Furthermore, miR-155 mimics induced Raji and BJAB cells apoptosis (t=4.239, P<0.05; t=3.477, P<0.05). MiR-155 targeted at the site of CEBPB 3’-UTR, and CIK cells could decrease the luciferase activity of Raji cells by (42.89±2.06)% (t=3.281, P<0.05); meanwhile, in BJAB cells by (37.02±1.98)% (t=4.933, P<0.05).

Conclusion: CIK cells could enhance human lymphoma Raji and BJAB cells apoptosis by upregulating miR-155, which may provide a new database to elucidate lymphoma cell therapy using CIK cells.

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Critical role of all-trans retinoic acid in stabilizing human nature regulatory T cells under inflammatory conditionsLing Lu1,2, Qin Lan2,3, Zhiyuan Li4, Xiaohui Zhou2,3, Jian Gu1,2, Qiang Li2, Julie Wang2, Maogen Chen2, Ya Liu2, Yi Shen3, David Brand5, Bernhard Ryffel6, David A Horwitz2, Francisco P Quismorio Jr2, Zhongmin Liu3, Bin Li4, Nancy Olsen7 and Song Guo Zheng2,3,7

1First Affiliated Hospital of Nanjing Medical University, China2University of South California, USA3Shanghai East Hospital at Tongji University, China4Chinese Academy of Sciences, China5Veterans Affairs Medical Center, USA6University of Orléans and Molecular Immunology and Embryology, France7Penn State University Hershey College of Medicine, USA

Recent studies have demonstrated that thymus-derived naturally-occurring CD4+Foxp3+ regulatory T cells (nTregs) in both human and mouse are unstable and dysfunctional in the presence of pro-inflammatory cytokines. All-trans Retinoic Acid

(atRA), the active derivative of vitamin A, has been shown to regulate Treg and T effector cell differentiation. It is hypothesized atRA stabilizes human nTregs under inflammatory conditions. atRA prevents human nTregs from converting to Th1 and/or Th17 cells and sustains their Foxp3 expression and suppressive function in vitro or in vivo following encountering with IL-1 and IL-6. Interestingly, adoptive transfer of human nTregs pre-treated with atRA significantly enhanced their suppressive effects on xeno-graft versus host diseases (xGVHD) and atRA- but not rapamycin-pretreated nTregs sustained the functional activity against xGVHD after stimulating with IL-1/IL-6. atRA suppresses IL-1R upregulation, accelerates IL-6R downregulation, and diminishes their signaling events as well as prevents the upregulation of Stub1 on Foxp3+ cells following IL-1/IL-6 stimulation. atRA also increases histone acetylation on Foxp3 gene promoter and CpG demethylation in the region of Foxp3 locus (TSDR). These results strongly implicate that nTregs primed with atRA may represent a novel treatment strategy to control established chronic immune-mediated autoimmune and inflammatory diseases.

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Clinical & Cellular ImmunologyFLT3L-Primed in situ vaccine for patients with low-grade lymphoma: Tumor regression at untreated sites (NCT01976585)Joshua Brody, Seunghee Kim-Schulze, Nina Bhardwaj and Miriam MeradMount Sinai School of Medicine, USA

Objectives: Though low-grade lymphoma is generally incurable, T-cell based therapy such as allogeneic transplant can induce prolonged remissions, possibly cures, even for chemo-resistant disease. If anti-tumor T cell immunity could be mobilized without the morbidity of transplant, it could change the paradigm of lymphoma therapy. Previously, we completed three trials of an 'in situ vaccine' combining low-dose XRT with intratumoral administration of a TLR agonist demonstrating partial and complete remissions of patients’ non-irradiated sites of disease, lasting as long as 4+ years. One obstacle to the induction of potent anti-tumor immunity with the in situ vaccine approach is the paucity of professional antigen presenting cells, e.g. dendritic cells (DC) at the tumor site.

Methods: To address this problem, we have adopted the approach by adding a priming step of intratumoral administration of the predominant DC differentiation factor - Flt3L. The aim of the adopted approach is to use:

• intratumoral Flt3L administration to recruit DC to the tumor,• low-dose XRT to induce immunogenic cell death and release tumor-associated antigens, and• intratumoral poly-ICLC administration to activate tumor antigen-loaded DC, inducing anti-tumor T cells and anti-tumor

immunity.

Results: Preliminary immune correlative data for the first two patients receiving the in situ vaccine demonstrate a significant (up to 200-fold) increase in the proportion of intratumoral DC subsets (BDCA-1 and BDCA-3) at the treated tumor site. We also observe a marked increase in the intratumoral proportion of CD80 (high) PD-L1 (low) activated DCs after XRT and poly-ICLC administration. This latter finding is in contrast to the activation status of DC in the peripheral blood.

Conclusions: Flt3L-primed in situ vaccination demonstrates immunologic and clinical proof-of-principle and warrants continued investigation. We will present additional immunologic and clinical results as well as ongoing studies to quantify anti-tumor T cells and to determine potential tumor-associated antigens.

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Manipulation of lymphocytes by sphingolipid analogue for therapeutic purposesJing Song, Osnat Almogi-Hazan, Shimon Gatt, Arie Dagan and Reuven OrHadassah-Hebrew University Medical Center, Israel

Immune cells have been revealed to be beneficial for immune therapy in various pathological conditions. However, the inability to control the cells once administrated is still a major obstacle to the development of immunotherapeutic treatments.

Recent studies demonstrate that sphingolipid analogues influence some crucial processes during immune response. In immune cells, sphingolipid metabolism is involved in a common signaling pathway which controls the main stages of immune cell development and function. The use of sphingolipid analogues, such as FTY-720, is currently under investigation as a therapy for different immune disorders. We have synthesized a group of sphingolipid analogues. The effects of the analogue AD-2900, on human lymphocytes activation was investigated and compared with sphinogsine-1-phosphate (S1P) and the commercial sphingolipid analogues FTY-720and SEW-2871. We found out AD-2900 can activate sphinogsine-1-phosphate S1P receptor 1 and may be also work through other S1P receptors. AD2900 affects different signal transduction pathways comparing to the other sphingolipid analogues tested and induces different extracellular signal-regulated kinase ERK phosphorylation pattern. In addition, AD-2900 also inhibits T cell proliferation, inflammatory cytokine secretion and induces apoptosis dose-dependently, but in a compromised way comparing with FTY-720, a commercialized sphingolipid analogue. To further illustrate the mechanism of AD-2900, the effect of AD-2900 on different immune cells such as dendritic cells and macrophages will be tested. Hopefully, sphingolipid analogue, AD-2900 may play a key role as immunomodulator for immunotherapy and other therapeutic purposes in the future.

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Clinical & Cellular ImmunologyDrug resistance patterns of Mycobacterium tuberculosis complex and associated factors among retreatment cases at Jimma University Specialized Hospital, South West EthiopiaKedir-Abdella, Gemeda-Abebe and Ketema AbdissaJimma University, Ethiopia

Background: The global burden of tuberculosis (TB) has been accompanied with the emergence of multidrug-resistant tuberculosis (MDR-TB). The rate of MDR-TB is five times higher among previously treated tuberculosis cases than new cases.

Objective: The objective of this study was to determine drug resistance patterns of Mycobacterium tuberculosis complex (MTBC) isolates and associated factors among retreatment cases.

Methods: A facility-based cross-sectional study was conducted in from March 2012 to April 2013. A total of 79 sputum smear positive retreatment cases were enrolled in the study. Socio-demographic characteristics and clinical data of patients were collected using questionnaires. Sputum specimens were collected, cultured and drug susceptibility testing (DST) was done for four first line drugs of streptomycin, isoniazid, rifampicin and ethambutol system using indirect proportion method. Statistical analysis of the data (Chi-square and logistic regression) was done using SPSS V-20.

Results: DST was conducted for 70 MTBC isolates. Any drug resistance was detected in 58.6% of isolates. The overall prevalence of MDR-TB was 31.4%. Place of residence (p=0.032, 95% CI =1.11, 10.60), duration of illness (p=0.035, 95%CI= 1.10, 10.62) and multiple treatment (p=0.048, 95%CI=1.01, 8.86) were associated with any drug resistance. The history of treatment failures (p=0.028, 95% CI: =1.14, 10.28) was found to be predictor for MDR-TB.

Conclusion: The prevalence of MDR-TB was higher than the world health organization estimation for Ethiopia. The rate of MDR-TB was higher in patients with the history treatment failures. Hence, patients with the history of treatment failures should timely be identified and referred for culture and drug susceptibility testing.

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Strain improvement of Brevibacillus borostelensis R1 for optimization of α-amylase production by mutagensK Suribabu1, T Lalitha Govardhan1 and K P J Hemalatha2

1Dr.Lankapalli Bullayya Post-Graduate College, India2Andhra University, India

Physical and chemical mutagens are promising and are used for screening of high yielding strains. The exponential increase in the application of amylases in various fields has placed stress and demand in both qualitative improvement and quantitative

enhancement through strain improvement. Ultraviolet light exerts its mutagenic effect by exciting electrons in molecules. The potent UV mutants which showed more than 20 mm zone of starch hydrolysis were screened and selected at 42% of survival time at 80minutes of exposure. The wild strain with fixed parameters yielded (3000 U/ml). The major findings of the strain improvement were out of ten mutants isolated, two (UV-3 and UV-10) showed 3000-4000 U/ml of amylase activity. The % of survival of Brevibacillus borstelensis R1 in Pikovskaya’s medium was 25.75% at 120 minutes of exposure. Ten mutants (HNO2-10, HNO2-30, EMS-4, EtBr-40, EtBr-50, Acr-1, Acr-20, Acr-30, Acr-4 and 5'-FU-50) out of fifty mutants isolated showed 3000-4300 U/ml of amylase activity which was higher than the wild strain. The potent Bacillus species screened from marine water was Brevibacillus borstelensis R1. The α-amylase was found to be useful in bakery, food, fodder for poultry, automation dishwashing and laundry industries.

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Clinical & Cellular ImmunologyAutoimmune hepatitis: Evolution/development and treatmentLaura IliescuFundeni Clinical Institute, Romania

Introduction: Autoimmune hepatitis and overlap syndromes are poorly defined and require individualized therapies.

Purpose: Comparative analysis of the types of autoimmune hepatitis in order to determine prognosis and optimal therapy selection.

Material and Methods: The study was performed on 33 cases using revised diagnostic criteria of the International Group Study (2005, 2007).

Results: Distribution was: 58% autoimmune hepatitis, 42% overlap syndromes. One case was acute, severe onset. Associated diseases were represented by: autoimmune hemolytic anemia, autoimmune thyroiditis, renal impairment and diabetes mellitus. A positive HCV-RNA associated with anti-LKM antibodies was noted in 4 cases. Immunophenotyping revealed a ratio Th/Ts=4.8. HLA profile was A1, A3, B35, BW4, BW6, DR4, DR10, DR53, DQ1, DP3. The overlap syndromes of autoimmune hepatitis/primary biliary cirrhosis were treated with ursodeoxycholic acid in combination with immunosuppressant medication (Imuran, Cellcept), with very encouraging results.

Conclusions: Autoimmune hepatitis type I was predominant and the overlap syndrome of AIH/HCV. Overlap syndromes respond favorably to immunosuppressive therapy in combination with ursodeoxycholic acid. Extrahepatic manifestations are more important and more severe for atypical forms with renal impairment that may lead to CKD.

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Autoimmune co-morbidities of HCV infection: From case reports to the experience of a single center and literature review Laura IliescuFundeni Clinical Institute, Romania

HCV infection is associated with several autoimmune co-morbidities such as cryoglobulinaemia, or even malignant pathologies, for example lymphomas. Many HCV patients have circulating autoantibodies. For example, 70% of patients

with hepatitis C have circulating rheumatoid factor. Approximately one third have cryoglobulins, and anywhere from 13% to 21% have low-titer or high-titer antinuclear antibodies (ANA). A slightly lower percentage have smooth muscle antibodies—about 5% have antibodies to liver or kidney microsomes—and about 7% have antithyroid antibodies. A direct relationship between systemic lupus (SLE) and HCV infection has been described. There may be a possible causal link between HCV and SLE: there is a high prevalence of HCV infection in SLE patients and a higher prevalence of liver involvement in HCV-positive patients with SLE. Constant immune proliferation and clonal expansion of B cells driven by hepatitis C can at times transfer or progress to B-cell lymphoma. In HCV patients, B cell non-Hodgkin lymphoma may evolve either related to mixed cryoglobulinaemia type II or in patients without cryoglobulinaemia. This talk aims to draw the attention towards the importance of correct and complete evaluation of HCV patients in respect to such co-morbidities. The author will discuss three cases of HCV infection associated with different pathologies (cryoglobulinaemia, SLE, B cell lymphoma), study their diagnosis, evolution and prognosis from the clinical and paraclinical point of views and review the literature data.

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Clinical & Cellular ImmunologyImmunology and sleepLourdes DelRossoThe Children’s Hospital of Philadelphia, USA

The relationship between sleep and immunity has been well established. Cytokines have been demonstrated to both promote and inhibit REM sleep, hence viral and bacterial infections have different effects on our sleep patterns through

immune related mechanisms. Sleep deprivation has been shown to affect immunity through altering the ratio of interleukin-6 and C-reactive protein (CRP); sleep excess has also been shown to elevate levels of CRP with concomitant increased risks in cardiovascular disease and diabetes. Interleukin-4 and tumor necrosis factor alpha have been found to be elevated in obstructive sleep apnea (OSA), while interleukin-10 is decreased. These changes have been hypothesized to play an intrinsic role in the pathophysiology of excessive sleepiness found in patients with OSA. Narcolepsy is another sleep disorder with significant immune pathophysiology. There is a strong association between narcolepsy and HLA DQB1*0602. Molecular mimicry, an immune response reaction, has been implicated in the cases of H1N1 vaccine -related narcolepsy. Other sleep disorders like insomnia and restless leg syndrome have also been linked to immune conditions. This presentation is intended to review the immune changes in sleep deprivation and the immunology related findings in the most common sleep disorders: OSA, narcolepsy, restless leg syndrome and insomnia.

BiographyLourdes DelRosso completed her MD degree from University of Miami. She moved to California where she completed her Family Medicine Residency at Kaiser Permanente, a program affiliated to University of California Irvine. After 7 years in practice, she moved to Louisiana to train in sleep medicine under the direction of Dr. Andrew L. Chesson Jr. She joined the faculty at Louisiana State University School of Medicine. Since then she has published about 20 publications in reputed journals. She joined the Sleep Center at The Children’s Hospital Of Philadelphia in November 2013.

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Incidence and predictors of tuberculosis among adult people living with human immunodeficiency virus at the University of Gondar Referral Hospital, Northwest EthiopiaKefyalew Addis Alene, Ansha Nega and Belay New Wasie TayeUniversity of Gondar, Ethiopia

Background: Tuberculosis (TB) is the leading killer of people living with HIV (PLHIV). Many of these deaths occur in developing countries. This study aimed at determining the incidence and predictors of tuberculosis among PLHIV.

Methods: A five year retrospective follow up study was conducted among adult PLHIV. The Cox proportional hazards model was used to identify predictors.

Results: A total of 470 patients were followed and produced 1724.13 Person-Years (PY) of observation, and 136 new TB cases occurred during the follow up period. The overall incidence density of TB was 7.88 per 100 PY. It was high (95.9/100PY) in the first year of enrolment. The cumulative proportion of TB- free survivals was 79% and 67% at the end of the first and fifth years, respectively. Baseline WHO clinical stage III (AHR =2.88, 95% CI =1.53-5.43), WHO clinical stage IV (AHR =3.82, 95% CI =1.86-7.85), CD4 count <50 cell/ul (AHR =2.13, 95% CI =1.28-3.53) and ambulatory or bed ridden functional status (AHR =1.64, 95%CI =1.13-2.38) were predictors of time to TB occurrence.

Conclusions: TB incidence rate among PLHIV, especially in the first year of enrollment was high. Advanced WHO clinical stage, limited functional status, and low CD4 count (<50 cell cell/ul) were found to be the independent predictors of TB occurrence. Early care seeking and initiation of HAART to improve the CD4 count and functional status are important to reduce the risk of TB infection.

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Clinical & Cellular ImmunologyPotential impact of metal oxide nanoparticles on the immune system: The role of integrins, L-selectin and the chemokine receptor CXCR4Mercedes ReyHospital Donostia, Spain

The impact of metal oxide Nanoparticles (NPs) on the immune system has been studied in vitro using human peripheral blood lymphocytes (PBLs). Metal oxide NPs (ZnO, CeO2, TiO2 and Al2O3) induced changes in the expression levels of

adhesion molecules and the C-X-C chemokine receptor type 4 (CXCR4) in these cells. Proliferation studies were carried out with CFSE in response to PHA, finding an increase in T cell proliferation upon cell exposure to TiO2 and Al2O3 NPs. For ZnO NPs, a decrease in the chemotactic response to SDF-1α was observed. No changes were found in basophil activation and leukocyte oxidative burst after phagocytosis. Despite the absence of cytotoxicity, metal oxide NPs are not inert; they alter the expression levels of adhesion molecules and chemokine receptors, key actors in the immune response, and affect important cell functions such as T cell proliferative response to mitogens and chemotaxis.

BiographyMercedes Rey is qualified as a specialist in Immunology in 2002 and gained her PhD from the Universidad Autónoma de Madrid (Madrid, Spain) in 2007, for her work on the chemokine receptors in T lymphocytes and their interaction with cytoskeletal proteins. In 2008, she moved to Institut Curie (Paris, France), where she worked on the molecular mechanisms underlying the extracellular matrix degradation associated with metastasis of breast cancer cells. From 2010 she has been working as an Immunologist at the Hospital Universitario Donostia (San Sebastián, Spain), where she combines her assistential duties (dignosing leukaemias and lymphomas by flow cytometry, among others) with research work, especially focusing on the potential adverse effects of the interactions between metallic nanoparticles and the immune cells.

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Antitumor and antioxidant activities of Escherichia coli are accompanied by changes in the metabolic pathways of L-arginine and creatine in Ehrlich ascites carcinomaM R Hovhannisyan, N H Movsesyan, N Kh. Alchujyan and G A KevorkianNational Academy of Sciences, Republic of Armenia

Avirulent strains of Escherichia coli isolated from healthy humans, exert antitumor and antioxidant activities and affect the subcellular metabolic pathways of L-arginine and creatine in peritoneal leucocyte following Ehrlich ascites carcinoma

(EAC). Two days after EAC transplantation, a single non-invasive treatment the eyes and mouth of mice with alive cells of E. coli increases life span by 75 %, and on 11-th day of EAC development decreases thrice the volume of ascites fluid, and inhibits EAC-induced lipid peroxidation (LPO) processes in the peritoneal leucocyte. The level of malondialdehyde (MDA) and its in vitro formation are decreased by 3.2 and 1.8 times in cytoplasm and mitochondria respectively, thus ameliorating deleterious effects of oxidative stress and immunosuppression. EAC caused alterations in the L-arginine metabolism are corrected by bacterial treatment. E. coli-treatment reduces twice the content of L-arginine in the cytoplasm of peritoneal leucocyte which was elevated by 4.4 times in EAC, as well as causes a decrease of arginase and NOS activities in the leucocyte cytoplasm and mitochondria that were increased during EAC. The correlated changes were observed in the peritoneal leucocyte basal levels of ornithine (produced partially by arginase), and generated mainly by NOS reactive nitrogen species and L-citrulline. E. coli-treatment completely suppresses the arginase activity in the EAC-cells and could diminish their proliferation due to inhibition of the synthesis of polyamines. At the same time E. coli-treatment up-regulates the NOS in tumor cells there through stimulates both nitrosation stress and suppression of intracellular antioxidant system. Moreover, E. coli-treatment cancels completely the EAC-induced stimulation of the cytoplasmic creatine kinase (CK) and restores the mitochondrial CK activity in the peritoneal leucocyte (dropped in EAC) stabilizing their structure. Simultaneously, the level of creatine significantly decreased in EAC-cells, disrupting the energy metabolism. In conclusion, it was demonstrated for the first time that E. coli antitumor effects are accompanied by changes in the L-arginine and creatine pathways and oxidative stress processes in the peritoneal leucocyte and tumor cells and should be taken into account in E. coli application in adjuvant cancer therapy.

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Clinical & Cellular ImmunologyHigh levels of IgG3 against N-terminal Pv-MSP1 and a broad panel of proteins of polymorphic block 2 region in symptomless Plasmodium vivax-infected individualsPaulo A Nogueira1,2, Maria Edilene M Almeida1, Fernanda Versiani2, Leidiane A Soares1, Patrícia P Orlandi1, Luciana P Sousa1, Antonio A Balieiro1, Juliane Correa1, Lucas Barbosa1, Yury Chaves1, Marcus V G Lacerda3, Fabio T M Costa4 and Luis Andre Mariuba1

1Instituto Leônidas e Maria Deane, Brazil2Universidade Federal do Amazonas, Brazil3Tropical Heitor Vieira Dourado, Brazil4Universidade de Campinas (UNICAMP), Brazil

Plasmodium vivax has the potential to infect 2.85 billion individuals worldwide. Nevertheless, the limited number of studies investigating the immune status of individuals living in malaria-endemic areas, as well as the lack of reports investigating

serological markers associated with clinical protection, has hampered development of vaccines for P. vivax. The humoral immune response against the N-terminus of P. vivax merozoite surface protein 1 (Pv-MSP1) of symptomless Plasmodium vivax-infected individuals from two different endemic areas was associated with reduced risk of malarial infection. The last study, a cross-sectional and longitudinal follow up was performed with 313 residents of the Rio Pardo rural settlement (Amazonas State, Brazil). Symptomless Plasmodium vivax-infected individuals were identified after follow up over two months using thick blood smear and rRNA gene-based nested real-time PCR to diagnose malaria infection. The majority of P. vivax-infected individuals (52-67%) showed immune recognition of the N-terminus of Pv-MSP1. Interesting data on infected individuals who have not developed symptoms, total IgG levels against the N-terminus Pv-MSP1 were age-dependent and the IgG3 levels were significantly higher than levels of subjects had acute malaria or those uninfected ones. The total IgG anti ICB2-5 was detected to be an important factor of protection against new malaria vivax attacks in survival analysis in a prospective survey (p=0.029). Our preliminary data has illustrated the importance of IgG3 associated to symptomless in P. vivax infected individuals and open perspectives for the rationale of malaria vaccine designs capable to sustain high levels of IgG3 against polymorphic malaria antigens. Nonetheless one of the major difficulties in developing a malaria vaccine is the genetic diversity of highly polymorphic surface antigens of Plasmodium sp. and this problem tends to be more important for blood stage targets of naturally-acquired immunity. A panel of proteins of major polymorphic domain of Pv-MSP1 (Block-2) was produced to assess the influence of genetic diversity in modulating the prevalence of repertoire of IgG responses. Individuals with clinical immunity to malaria had higher levels of IgG3 antibodies to panel proteins than symptomatic individuals and the frequency of IgG3 responders was increased when the panel of proteins was joined. The profit with malaria protective antibodies observed here using a panel of allelic types of Pv-MSP1 should contribute to reduce effect of polymorphism in the humoral response and encourages further development of malaria vaccine designs.

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Immunomodulant activity of medicinal plants: A reviewMuhammad AkramThe University of Poonch, Pakistan

Since ancient times, plants have been an exemplary source of medicine. Researchers have discovered some important compounds from plants. The present work constitutes a review of the medicinal plants whose immunomodulant activity

has been proven. PUBMED, EMBASE, Google scholars and CENTRAL searches for research papers of medicinal plants having immunomodulant activity were performed. Medicinal plants used by traditional physician or reported as having immunomodulant activity includes; Acacia concocinna, Camellia sinensis, Lawsonia inermis Linn, Piper longum Linn, Gelidium amansii, Larrea divaricata, Petroselinum crispum, Plantago major and Allium sativum. Present review indicates that various plants have immunomodulant activity. Medicinal plants documented have immunomodulant activity and will be further investigated via clinical trial

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Clinical & Cellular ImmunologyCereblon RING finger E3-ubiquitin ligase receptor is a novel regulator of T-cell activation and homeostasisPearlie Epling-BurnetteMoffitt Cancer Center, USA

Cereblon (CBBN) is a RING-domain containing protein that likely functions as a substrate receptor for the DDB1/Cul4/CRBN E3 ubiquitin ligase (UbL) complex. Recently, CRBN was shown to be a target of the immunomodulatory (IMiD)

drug thalidomide. Although known for inducing severe teratogenicity in the 1950s, this drug class is now used extensively for anticancer immunotherapy. CRBN is expressed in the hematopoietic compartment, but currently has no known function in immune regulation. Ubiquitin ligases including C-Cbl, cbl-b, GRAIL, and ITCH underlie T-cell homeostatic regulation and protect against autoimmunity. To understand the role of CRBN in T-cell function, we studied a mouse with a germline deletion of exon 3 and 4 of this gene. First, crbn-/- mice are viable, fertile and normal in appearance without limb malformations. In the T-cell compartment, splenic and peripheral blood lymphocytes are increased at 3 months of age. The expanded population of splenic T-cells was also evident by immunohistochemical staining. Mature differentiated cell lineages such as CD4 and CD8 single positive (SP) thymocytes, SP peripheral T-cells and naive and memory T-cells are similar to wild-type littermates with no apparent spontaneous autoimmune features at 3 months. Similar to IMiD responses, the mature T-cells from crbn-/- mice showed superior activation after T-cell receptor (TCR) stimulation. Proximal TCR signaling events including pZAP70 and pLck, cytokine production and survival are increased in knockout mice relative to wild-type littermates. In summary, our findings demonstrate a novel role for CRBN, the molecular target of thalidomide and other members of the IMiD-family, in T-cell activation.

BiographyPearlie Epling-Burnette completed PharmD training at the University of Florida and PhD training at the University of South Florida in 1994. Her Postdoctoral studies were conducted at the H. Lee Moffitt Cancer Center & Research Institute in Tampa, FL. She is now a Senior Member and Professor with Tenure in the Immunology Department at Moffitt Cancer Center and is the author of 74 papers in prestigious medical journals in the field of malignant hematology and immunology.

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A combined ex vivo local lymph node assay-BrdU ELISA and irritancy assay to investigate the sensitization and irritancy potential of the chemicalsOzge Cemiloglu Ulker1, Seren Arancioglu2 and Asuman Karakaya1

1Ankara University, Turkey2Kulu State Hospital, Turkey

In our study to discriminate the sensitization and irritancy potential of the chemicals, the parameters of ex vivo local lymph node assay-BrdU ELISA and irritancy assay were combined. Different concentration of sensitizer and irritant chemicals

and a negative control (PABA) were administered on the ears of the Balb/c mice. As parameters of lymph node related assays, the auricular lymph node weights, lymph node cell counts and % change in ear swelling and stimulation index values were calculated. Cytokine releases (IL-2, IFNγ, IL-4, IL-5, IL-1 and TNF-α) from lymph node cell culture were also investigated as endpoints. According to our lymph node related results, the auricular lymph node weights, lymph node cell counts were found to be increased both sensitizers and irritants in high concentrations. On the other hand, according to lymph node cell proliferation results, it was found that there was no 3 fold increase in proliferation of lymph node cells (stimulation index) for irritant chemicals and negative control whereas there was 3 fold increase for sensitizer chemicals in the applied concentrations. Cytokine analyze results indicated that IL-2, IFNγ, IL-4, IL-5 were among the most differentially released cytokines discriminating between irritant contact dermatitisand allergic contact dermatitis.

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Clinical & Cellular ImmunologyImmunomdulatory effects of probiotics and prebiotics on immune system during (DSS)-induced experimental colitisRyma Toumi, Katia Abdelouhab, Hayet Rafa, Imene Soufli and Chafia Touil-BoukoffaUniversity of Science and Technology Houari Boumediene, Algeria

Introduction: The inflammatory bowel diseases (IBDs), represented mainly by ulcerative colitis and Crohn disease refer to chronic and relapsing disorders that affect the gastrointestinal tract. Studies have indicated that these diseases result from a lack of tolerance to resident intestinal bacteria in genetically susceptible hosts. It seems that in IBD the mucus barrier is broken. The production of high levels of the nitric oxide (NO) by the inflammatory cells plays a part in tissue injury; in the current study, we investigate the potential preventive effects of inulin on dextran sulphate sodium (DSS)-induced experimental colitis in Swiss albino mice.

Methods: The colitis was induced in mice via administration of 2.5% DSS in drinking water for 7 days. During this period the Swiss albino mice were given 1% (w/v) inulin in their drinking water ad libitum for 7 days. The production of NO was evaluated in the supernatants of peritoneal macrophages (pMϕ) cultures. The mucus production by the goblet cells in the colon was determined.

Results: A significant shortening of colon length was observed and high levels of NO in pMϕ cultures were observed in DSS group compared to control. Oral administration of inulin decreased the severity of DSS-induced colitis. NO production in pMϕ supernatants was lower in inulin group and correlated with a reduction of colonic lesions.

Conclusion: The prebiotic inulin improved DSS-induced colitis symptoms by down regulation of NO production and induction of mucus secretion. Prebiotics seem to be promising for development of preventive strategies approach for IBD using dietary supplementation.

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Gene frequencies of ABO and Rh antigens among six populations of Jammu and Kashmir, IndiaMohd. Fareed and Mohammad AfzalAligarh Muslim University, India

Background: Identification of blood group antigen frequencies in a population has various benefits in transfusion medicine, transplantation and disease risk. ABO and Rhesus (Rh) protein in human populations are used as the immunogenetic markers for anthropological, basic and applied medical studies.

Objective: The aim of the study was to record gene frequencies of ABO blood groups, their subtypes and Rh antigen for six different endogamous groups.

Methods: Nine hundred and ninety five (995) individuals irrespective of the age and sex were selected from six populations of J&K viz., Gujjar and Bakarwal (n=201), Mughal (n=155), Khan (n=151), Malik (n=155), Mir (n=160) and Syed (n=173). ABO blood groups and Rh antigens were tested by simple agglutination reactions on a clean slide by using specific antisera (Tulip Diagnostics; Goa, India).Genotypes and allele frequencies for each population were calculated by Hardy-Weinberg method and heterozygosity was determined.

Results: The ABO phenotypic frequency varies among six different populations showing significant difference (p<0.0005). Gujjar and Bakarwal (a tribal population) shows highest (42.29%) of B blood phenotypes. A1 is the highest among Syeds (39.31%), O blood group frequency highest among Mughals (43.23%) and A1B and A2B are rare phenotypes showing very low frequency among all populations. The pattern of allele frequencies (p<0.025) is in order of IO> IB> IA1> IA2, except Syeds (IO>IA1>IB>IA2). The rhesus protein (Rh) phenotypic frequency (p<0.01) shows significant increase in Rh(D) positive (87.86% in Syed to 96.03% in Khan) among all populations. The Rh allele (p<0.05) and genotype (p<0.02) frequencies shows a significant difference. Heterozygosity for Rh protein is less than homozygosity among six populations. The result from this study provides information on the genetic variation in blood antigens and rhesus protein among human populations inhabiting Jammu and Kashmir. Bringing health awareness through information, education, and communication activities about blood and Rh related disorders can help in preventing many of the immunogenetic, hematological and transfusion problems.

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Clinical & Cellular ImmunologyKIR Genotyping in the selected population in Andhra Pradesh, India S Krupanidhi1, G Vaishnav2 and C B Sanjeevi31Vignan University, India2SRM Medical College, India3Karolinska Institute of Medical Sciences, Sweden

Background: The population is not always homogeneous in relation to the representation and functioning of genes. Therefore, the presence of allogenicity is a universal phenomenon. The profound variability is noticed among the members of the human population with reference to the resistance against infections and late-on-set of diseases. In this line, a few sets of alleles which come under the domain of immune function namely KIRs (Killer Cell immunoglobulin-like receptor genes) and HLA-I have been chosen to report in the population of Puttaparthi (India).

Objectives: The genotyping of the population is the current ongoing focus of our team wherein the distribution of the following alleles has been taken up in the mixed ethnic groups of Puttaparthi as a prelude to earmark them as genotypic markers in future studies relating to susceptible diseases.

Methods: The PCR protocols for the identified immune related genes viz., KIR-2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 3DL1, 3DL2, 3DL3, 3DS1, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 2DP1, 3DP1; HLA- C1 and HLA- C2 have been standardized.

Results & Interpretation: In the present study, except KIR 2DL2, the other non-framework inhibitory KIR genes were represented at higher percentage and ranged from 57% to 80%in the chosen population which would suggest its higher survival adaptation. Interestingly, majority of activating KIR genes were least represented and varied between 5% to 32.5% which is also in compliance with the survival adaptation of the chosen population. The carrier gene frequencies of KIRs were compared with the other populations’ viz., Chinese Mongolian, Chinese Han, Greek and Brazilian data. The expected heterozygosity of KIR alleles and their rank in gene diversity among the population of Puttaparthi were also discussed.

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Expression of Epstein-Barr virus (EBV)-encoded latent membrane protein 1(LMP-1) in patients with nasopharyngeal carcinoma (NPC) is associated with a worse clinical prognosisSamuel Rosales-PMexican Social Security Institute, Mexico

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a key effector of EBV-mediated B cell transformation. LMP1 displays oncogenic properties in fibroblasts, and induces a wide range of effects in B cells and a

variety of growth-promoting effects in human epithelial cells. The constitutive activation of these signaling cascades explains LMP1's ability to induce morphological and phenotypic effects in cells. Taken as a whole, these findings demonstrate that LMP1 can induce profound effects in epithelial cells, many of which may account for its oncogenic properties. There is now strong evidence supporting a role of EBV encoded LMP-1 protein in the pathogenesis of nasopharyngeal carcinoma (NPC), but also, there are other molecular pathology variables which can predict clinical outcome in these patients. Accordingly, we aimed to systematically analyze LMP1 immunoexpression in patients with non-endemic NPC and do a correlation with clinicopathological features and patients survivals. Our data suggest that LMP1 expression could be correlated with a poorer clinical outcome and prognosis in patients with NPC. To our knowledge, this series is the first one published in non-endemic/non-white population of NPC; and supports the importance to explore the molecular signaling pathways to provide a substantial opportunity for identification of novel diagnostic and prognostic biomarkers that could improve individual treatment in patients with NPC.

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Clinical & Cellular ImmunologyContribution of muscle cells and BM-derived APCs to CD8 T cells priming upon SAM® vaccinationSandra Lazzaro1, Cinzia Giovani1, Simona Mangiavacchi1, Barbara Baudner1, Domenico Maione1, Andrew J Geall2, Ennio De Gregorio1, Ugo D’Oro1 and Cecilia Buonsanti11Novartis Vaccines, Italy2Novartis Vaccines, USA

SAM® vaccines are self-amplifying mRNA derived from the positive-strand alphavirus genome. They contain genes encoding non structural proteins which drive the RNA replication, but lack the viral structural proteins which are replaced by vaccine

antigens.In this way; RNA amplification within the cytoplasm of transfected cells allows an increase of antigen expression. In addition, dsRNA intermediates exert an intrinsic adjuvant effect resulting in the induction of enhanced immune responses. It has been shown that SAM® vaccines are effective at eliciting both humoral and cellular antigen-specific immune responses in animal models of infectious and non-infectious diseases. However their mechanism of action has not been fully elucidated. To date, no evidence of in vivo transduction of APCs by the SAM® vectors has been produced, while the antigen expression has been shown to occur mostly in the muscle fibers. Bone marrow chimeric mice were used to assess the respective contribution of muscle cells and bone marrow derived antigen presenting cells (APCs) to CD8 T cells priming following SAM®vaccination. Our results show that bone marrow derived APCs rather than muscle cells are responsible for direct induction of Class-I restricted CD8 T cells. Nevertheless, direct transfection of APCs by SAM® vectors is it not required for CD8 T cells priming, suggesting that the antigen is expressed within muscle cells and then presentedby professional bone marrow derived APC to CD8 T cells, most likely through a cross-priming.

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Immune checkpoint inhibitor therapy for lymphomasSattva S Neelapu The University of Texas MD Anderson Cancer Center, USA

Non-Hodgkin lymphomas of B-cell origin are highly immune-responsive. However, immune checkpoints in the tumor microenvironment may impair optimal antitumor immune responses. Analysis of tumor-infiltrating lymphocytes from

lymphoma patients shows expression of many coinhibitory molecules, with programmed death (PD)-1 being the most highly expressed. PD-1 expression is associated with impaired T-cell function and blocking PD-1 enhances their tumor-reactivity. In a phase II clinical trial, administration of pidilizumab, an anti-PD-1 monoclonal antibody and rituximab, an anti-CD20 monoclonal antibody in patients with relapsed follicular lymphoma induced an overall response rate of 66% and a complete response rate of 52%, both of which are markedly superior to results previously reported with rituximab monotherapy. Analysis of peripheral blood and tumor samples before and after therapy with pidilizumab showed activation of both T and NK cells. In addition, predictors of clinical outcome based on the molecular features of tumor-infiltrating immune cells at baseline were identified. These results suggest that anti-PD-1 monoclonal antibody therapy is effective in follicular lymphomas and suggest that immune checkpoint inhibitor therapy is worthy of further exploration.

BiographySattva S Neelapu is currently a tenured Associate Professor in the Department of Lymphoma and Myeloma at The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. He completed his medical school at Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India and Clinical Fellowship in Medical Oncology and Postdoctoral Fellowship in Tumor Immunology and Immunotherapy at the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. His research is focused on characterization of immunoregulatory mechanisms in the tumor microenvironment in patients with lymphoma and developing strategies to target them.

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Clinical & Cellular ImmunologyImage assessment in immunotherapeutic trials: Use of novel combined RECIST 1.1 and immune-related response criteria (irRC)S Agarwal1, P Chokron2, A Thabet1, R S Arellano1, X Ma2, H Le2, I Kazam2, M Harisinghani1 and R Walovitch2

1Massachusetts General Hospital, USA2World Care Clinical, LLC, USA

The objective of this study was to validate an image assessment method that combines RECIST 1.1 and irRC criteria. Regulatory agencies often request that immunotherapeutic tumor burden be assessed using validated methods (i.e.,

RECIST) and newer irRC paradigm. Two sets of image datasets (10 RECIST and 10 irRC/RECIST), consisting of pre/post-treatment CT images from melanoma subjects enrolled in a PD-1 immunotherapeutic trial, were read twice by five independent radiologists (IR). Inter- and intra- reader variability of the combined irRC/RECIST and RECIST methods were measured. In a separate analysis, overall read time for the combined method was compared to irRC and RECIST alone. The combined irRC review had the best inter-reader agreement (90-98%; kappa 0.74-0.94) and intra-reader agreement (80 -100%; kappa 0.38-1.0) with excellent intra-reader homogeneity for number of lesions assessed. No significant differences were observed between RECIST IR variability using the combined vs. independent method. Inter-reader agreement ranged from 85 to 95% with kappa 0.55-0.84. Combining the RECIST and irRC assessments resulted in an approximate 20% decrease in assessment time compared to individual RECIST and irRC assessments. In conclusion, in immunotherapeutic trials RECIST may lead to errors in tumor burden as lesions initially grow despite favorable treatment response. This study indicates that the Combined irRC/RECIST review method is robust, as it is reproducible and simplifies the process of simultaneously assessing the cytotoxic and immunotherapeutic responses of drugs, and may be an efficient time saving analytical tool.

BiographyS Agarwal is a practicing radiologist in the division of Abdominal Imaging and Intervention at Massachusetts General Hospital (MGH). She is Associate Director of Radiology Clinical Trials at MGH and an instructor in radiology at Harvard Medical School. She is board certified in diagnostic radiology.

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Understanding and defining food allergy, intolerance, and sensitivityStephen WangenIBS Treatment Center, USA

Inflammation is probably the single most important health issue of our time. Food allergy and intolerance is one of the most underestimated and controllable causes of inflammation in modern day humans, with celiac disease being just the

tip of the ice berg. Yet we continue to underestimate the impact of this profound problem, which affects millions of people. Why? Because we lack a common language to properly discuss it. The words allergy, intolerance, and sensitivity are used by healthcare practitioners and the public without any consistency in the science or the mechanism behind the reaction. Reactions are frequently labeled based on symptoms, standard of practice, or personal preference. Incorrect assumptions are commonly made and promoted as fact, and there is no consistency or foundation on which to build. This must change in order for us to move forward in this exciting and important field of immunology. A logical process for doing that is the focus of this presentation.

BiographyStephen Wangen is one of the world's leading experts on IBS, food allergies and intolerances, and the ecosystem of the digestive tract. He is the Co-founder and Medical Director of the IBS Treatment Center, and the author of two popular books, “The Irritable Bowel Syndrome Solution” and the award winning “Healthier without Wheat: A New Understanding of Wheat Allergies, Celiac Disease, and Non-Celiac Gluten Intolerance.” He is a licensed and board certified physician with a Doctoral degree in naturopathic medicine from the internationally renowned Bastyr University in Seattle, WA, where he also is a faculty member.

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Clinical & Cellular ImmunologyAssessment of peripheral blood immunoglobulins (IgG, IgM & IgA), lymphocyte subsets (T & B cells) and natural killer cells in children with Down syndromeSheetal ShardaPost Graduate Institute of Medical Education and Research, India

Down syndrome (DS) is the most frequent cause of mental retardation with an incidence of approximately 1 in 800 live births. Individuals with DS are cognitively impaired, although severity is highly variable. Characteristic facial features

and hypotonia are present in almost all patients. Abnormalities of the cardiovascular system, gastrointestinal tract, eye and ear abnormalities occur with increased frequency compared with non-Down syndrome patients (NDS). It has been known that subjects with DS have an increased susceptibility to bacterial and viral infections, a high risk of malignancies, and autoimmune disorders in comparison to healthy population. Autoimmune phenomenon such as acquired hypothyroidism, celiac disease and diabetes mellitus also occur at higher frequency compared with NDS. Leukemia and pneumonia are still the major causes of mortality and morbidity in these children. The increased susceptibility to infection, malignancies and autoimmune disease, suggest that immunodeficiency is an integral part of DS. However, a majority of them do not show clear features of immunological disease. Multiple immunologic disturbances are commonly observed in individuals with DS including abnormal proportions of peripheral lymphoid subsets, cellular dysfunction and autoimmune phenomena. The abnormalities of the immune system associated with DS include: Mild to moderate T and B cell lymphopenia, with marked decrease of naive lymphocytes, impaired mitogen-induced T cell proliferation, reduced specific antibody responses to immunizations and defects of neutrophil chemotaxis. The clinical relevance of these immunological disturbances is also unclear. Although a high incidence of infectious diseases, particularly viral hepatitis and upper-tract respiratory infections in DS, is currently reported in the literature, the existence of an immunodeficiency associated to DS is not firmly established. There is no clear evidence of enhanced susceptibility to infection, suggestive of serious immunodeficiency, in both adults and children with DS, in spite of the existence of compelling evidence of immune derangement. Life expectancy in DS is significantly poorer than that for the general population, but over half of DS individuals can be expected to survive into their 50’s. Deaths from infection have shown the largest decline because of improvements in the health care provision. There is lack of data regarding the immunologic status of children with DS in India. At present there are no recommendations or guidelines to routinely investigate these children for immunological abnormalities, thus increasing the morbidity due to recurrent infection and also autoimmune disorders. The study is designed with an aim to determine the relationship between abnormalities in immune system with relation to peripheral blood lymphocytes and their subsets and immunoglobulin levels in children with DS, thus giving us an opportunity to identify those groups of patients with severe abnormalities and instituting improved care.

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An insight review on immunopathogenesis of bovine and human Mycobacteria infectionsSimenew Keskes MelakuDilla University, Ethiopia

Mycobacterium is one of the first infectious agents to spring to mind in connection with chronic or persistent infections. The causative organism of bovine tuberculosis is Mycobacterium bovis (M. bovis), a member of the Mycobacterium

tuberculosis complex (MTBC), which includes Mycobacterium tuberculosis (M. tuberculosis), M. bovis, Mycobacterium africanum (M. africanum), Mycobacterium microti (M. microti), Mycobacterium canetti (M. canetti), Mycobacterium caprae (M. caprae) and Mycobacterium pinnipedii (M. pinnipedii), and many of the species and subspecies of MTBC show specific host association. Immunity against mycobacteria is multifactorial and it is believed that the host innate immunity provides initial resistance to mycobacteria before the adaptive cell-mediated immunity fully develops. There are still many unsolved problems associated with the pathogenesis and immune response to tuberculosis. Therefore multi-disciplinary approach to develop more complete understanding of the pathogenic strategies is mandatory. Special consideration to bovine tuberculosis might help scientists to devise proper mechanisms to prevent human tuberculosis as they are closely related.

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Clinical & Cellular ImmunologyAlpha-enolase is up regulated on the cell surface and responds to plasminogen activation in mice expressing a delta133p53 alpha mimicTania SlatterUniversity of Otago, New Zealand

The p53 tumor suppressor is an intrinsic part of the cellular stress response. To add to the complexity of understanding p53 function 12 p53 isoforms are produced, many of which have tumor promoting properties. The delta133p53alphaisoform

lacks the N-terminal 133 amino acids due to an alternative promoter in intron four and it is aberrantly expressed in multiple tumors including breast and colon. Previous work attributed pro-inflammatory and proliferative properties todelta133p53alphausing a mouse model expressing a delta133p53alpha mimic (delta122p53). To identify the mechanism by which delta122p53 triggers inflammation the current study used a proteomic-based approach. The bone marrow, thymus, and lung proteome from Δ122p53, wild-type mice (p53+), and p53 null mice (p53-) were compared using two dimensional fluorescence difference gel electrophoresis and western blotting. In the bone marrow alpha-enolase was increased in delta122p53 cells. Further analysis showed alpha-enolase was increased in the cytosol and cell surface of delta122p53 bone marrow and peripheral blood mononuclear cells. Alpha-enolase on the delta122p53 peripheral blood mononuclear cell surface acted as a plasminogen receptor, with tumor necrosis factor alpha induced upon plasminogen stimulation. Taken together, these data identified new proteins associated with p53 function. One of which, alpha-enolase, is regulated differently by delta122p53.Increased cell surface alpha-enolase function with delta122p53 provides a possible explanation for the model’s pro-inflammatory features and suggests the delta133p53 alpha isoform may direct an inflammatory response by increasing the amount of alpha-enolase on the cell surface.

BiographyTania Slatter completed her Doctorate in Biochemistry at the University of Otago, New Zealand in 2007. Her current position is as a Senior Research Fellow in the field of molecular pathologyuses animal models and human clinical samples to investigate how inflammation contributes to different pathologies including cancer. She has published 18 papers in reputed journals.

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Protective anti-inflammatory responses mediated by a commensal microbial molecule require both innate and cognate interactionsSuryasarathi Dasgupta and Dennis L KasperHarvard Medical School, USA

The microbiota is critical in shaping the mammalian host’s immune system. Polysaccharide A, the archetypical immunomodulatory microbial molecule of the gut commensal Bacteroides fragilis, induces regulatory T cells to secrete

the anti-inflammatory cytokine interleukin 10. We show, in a model of colitis, that PSA requires both innate and adaptive immunity to generate protection. Dendritic cells mediate PSA’s effect on IL-10 production. Unlike conventional DCs, plasmacytoid DCs exposed to PSA do not produce the proinflammatory cytokines tumor necrosis factor-α and IL-12 but PDCs do specifically stimulate IL-10 secretion by CD4+ T cells and efficiently mediate PSA-mediated immunoprotection. PSA induces and preferentially ligates Toll-like receptor 2 on PDCs but not on CDCs. Compared with other TLR2 ligands, PSA better enhances PDC expression of co-stimulatory molecules required for protection against colitis. PDCs orchestrate beneficial immunoregulatory interaction of commensal microbial molecules with CD4+ T cells through both innate and adaptive immunity.

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Clinical & Cellular ImmunologyMolecular mechanisms in the pathogenesis of sepsisValentina Pop-Began, D Pop-Began, V Grigorean and C PopescuClinical Emergency Bagdasar Arseni Hospital, Romania

Innate immune system is an universal form of host defense against infections. The recognition of the innate immunity is based on a limited number of encoded receptors that have evolved to recognize microbial metabolism products. Recognition

of these molecular structures allows the immune system to distinguish own infectious components from non-communicable structures. Immune suppression is a hallmark of sepsis. The complement system is activated in the early stages of sepsis, generating large amounts of anaphylatoxin C5a. Complement and TLR family are two major upstream sensors and effectors systems of innate immunity. It was found that TLR4 and complement system are involved in initiating the inflammatory response in sepsis. Clinical studies in which TLR4 was blocked have not shown beneficial effects (41). Toll-like receptors (TLRs) that are a subfamily of PRRS have emerged as the crucial receptors for the recognition of DAMPs. Recently, in the complex cascade of sepsis was highlighted a special form of non-coding genetic material called microRNA. The individual role of every microRNA and the exact role of microRNA network is under investigation. Currently, studies are performed in order to find micro RNA to be used as biomarkers of sepsis. Researches are performed to determine microRNA, small fragments of non-coding RNA, in order to distinguish patients with sepsis and healthy patients, and if the plasma levels of microRNA correlate with the severity of the disease. Recent researches reports that the regulation of gene expression through microRNA plays a very important role in the following cellular processes, for example: Apoptosis, the differentiation process, and the cell cycle.

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Presence of partial RH1 by fetal RHD genotyping in maternal plasma: Clinical and biological expression at birth. About a caseGuinchard Emmanuelle EFS Rhône-Alpes, France

Introduction: Mrs. B. Marie, caucasian, 5th pregnancy, presents a poly- alloimmunization already known during a previous pregnancy: A non evolutive anti-RH1 + an anti-RH2 + an anti-JK1. RHD genotyping in maternal plasma fetal performed at 23 weeks and 33 weeks shows an amplification of exon 10, but no amplification of exons 4 and 5. The study of the maternal buffy coat showed no amplification. It may be a partial D inherited from his African father whose RHD gene could not be analyzed. The birth took place at 37 weeks: at birth, the child had no anemia, moderate hyperbilirubinemia quickly contained by phototherapy, no transfusion was necessary.

Results and hypothesis: At birth, the direct antiglobulin test was positive. The elution test found the presence of anti-RH2, anti-JK1 and anti-RH1 while the newborn was Rh: -1.2 and JK: 1. The sequencing of the RHD gene showed an aspect of partial D associated with a deletion of some exons: How is the expression profile? It may be a non-functional gene and anti-RH1 found in the eluate was present in the free state in the close environment of the erythrocyte (Matuhasi-Ogata phenomenon). Conversely, in case of a gene with partial antigen expression, the result of RH1 phenotype at birth may be due to saturation of antigenic sites by corresponding antibodies.

Conclusion: A new sample a few months after birth would confirm its RH1 phenotype.

BiographyI am an M.D/PhD in Biology. I work currently in a laboratory of a Health Clinical Center, CHU of Lyon. I specialize in immuno-haematology as well as in NIPT: fetal RHD genotyping on maternal plasma. I was invited to present this at several international conferences (ESHG 2012) and I am the author of a recently published article (Non-invasive fetal RHD genotyping: Validation of the method with 200 patients, TCB, 2014 ).

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Clinical & Cellular ImmunologyReactive oxygen species involved in the inflammation and wound healingYu-Sheng Wu and Shiu-Nan ChenNational Taiwan University, Taiwan

Immune cells are involved in virtually every aspect of the physiology process that may participate in hemostasis and work to prevent infection while scar formation or pathogen infected. Evidence supporting a central role for T lymphocytes

in the regulation of immune system is provided by studies which examine the in vivo effects of alternate forms of T cell manipulation on various parameters and neutrophils also as important to the infection process as they help to prevent the pathogen, however they also release harmful enzymes which damage healthy tissue surrounding the wound site. While in the wound or infected process, the one of the most important materials released is the ROS and recent researches also indicated that the reactive oxygen species (ROS) are essential messengers that may act as inflammation activator. ROS consist of many molecular species, including H2O2, O2−, and OH− that act as signaling molecules. It was reported that ROS may affect the cell migration for hepatic pro-fibrogenic cells and peripheral blood monocytes. In this inflammation process, cooperation among cells and mediators occurs, and a wide range of factors are involved in the classical immune response. Macrophage is critical for the uptake and degradation of infectious agents and senescent cells, as it also plays important roles in tissue growth, tissue remodeling, and inflammation by producing oxidants, proteinases, and antimicrobial peptide. Activated inflammatory cells serve as sources of ROS and reactive nitrogen intermediates (RNI) that are capable of initiating changes in cell function to include cell signaling pathways, transcription factor activation, mediator release, apoptosis, and compensatory cell proliferation but it is not clear whether ROS and RNI produced and released by neutrophils or macrophages are sufficient to diffuse through the extracellular matrix, enter epithelial cells, cross their cytoplasm even the physiological role of ROS and RNI involved in the cellular response. In our recent research, we major in the function of ROS role in the macrophage or other physiology responses in the microenvironment full of ROS production. We proposed that immune cells interacts with oxidative stress, which promotes cellular messenger to affect permeability of the calcium ion channel to increase intracellular Ca2+ concentration to the cellular migration mechanism.

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Multidrug-resistant Acinetobacter baumannii infection in intensive care unit patients in a hospital with building construction: Is there an association?Haleh Talaie, Arezou Mahdavinejad, Sepideh Kamalbeik, Abdollah Karimi and Alireza SalimiShahid Beheshti University of Medical Sciences, Iran

Background: Acinetobacter baumannii (A. baumannii) has emerged globally as a significant pathogen in hospitals. It is also present in soil and water. In a previous study, it was discovered that the A. baumannii class 2 integron occurred most frequently. Here, whether the A. baumannii class 2 integron is in the soil around the hospital, and if the soil is the cause for increasing numbers of A. baumannii infections in the intensive care unit (ICU) patients were determined.

Methods: This cross-sectional prospective study was conducted in two ICUs at Loghman-Hakim Hospital, Tehran, Iran, from November 2012 to March 2013. Patient, soil, and hospital environment samples were collected. All isolates were identified using standard bacteriologic and biochemical methods. The phenotypes and genotypes were characterized. The standard disc diffusion method was utilized to test antimicrobial susceptibility. Integron identification was performed by multiplex polymerase chain reaction.

Results: A total of 42 A. baumannii clinical strains were isolated, all from patient samples; 65% of the isolated species were classified as class 2 integrons. The strains were 100% resistant to piperacillin, piperacillin-tazobactam, ceftazidime, ceftriaxone, cotrimoxazole, cefepime, ceropenem, and cefotaxime. However, all of the strains were sensitive to polymyxin B. A. baumannii was detected around the lip of one patient.

Conclusions: Further research is necessary to establish a relationship between A. baumannii and soil, (especially in regards to its bioremediation), as well as to determine its importance in nosocomial infections and outbreaks in the ICU.

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Clinical & Cellular ImmunologyNeurocognitive rehabilitation in improvement of immune systemZarghi A, Zali A, Ashrafi F and Moazzezi SShahid Beheshti University, Iran

Background: Neurocognitive rehabilitations are complex set of techniques that are designed to enhance cognitive domains among individuals who are ill or disabled.

Method: Neurocognitive rehabilitation therapy is science and art for restoring mental process and remediation strategies training and it improves cellular and molecular processing with integrating behavioral and cognitive changes for increasing of immune system. This method is achieved to the ability of cognitive and neurological function improvement with successful development of cell transplantation, nanotechnology and appropriate expertise in rehabilitation environments.

Results: Advancement of this science is with effectiveness interventions that it has become a priority and it has been achieve to desire objectives of theoretical and empirical chain transfer made of neuroscience, cognitive neuroscience, psychology, physiology, pharmacology, medical imaging and other medical disciplines with behavioral interventions and achieved success in compensatory strategies. Cognitive impairment is a health challenge much more than common disorders related illnesses and immune disorders. Sub-systems affect different aspects of a person's life, such as emotions, diet, health, stress, and social performance and interference in the passive skills can lead to neurocognitive rehabilitation which includes a tailored experience based on neural structure and brain function.

Conclusion: These methods can improve immune system and abnormal brain processing based on the principles of neuroplasticity and damaged cortical reorganization by the nerve regeneration morphological and physiological reactions.

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Acute generalized exanthematous pustulosis in patient with hepatitis C infection during treatment with pegylated interferon/ribavirin + telaprevir: A case reportTugba Sari Buldan Chest Diseases Hospital, Turkey

HCV infection is associated to several autoimmune comorbidities such as cryoglobulinaemia, porphyria cutanea tarda, lichen planus, cutaneous polyarteris nodosa and for a variety of other dermatological conditions including psoriasis,

urticaria, and erythema multiforme (EM). Chronic hepatitis C and peg-interferon alpha/ribavirin treatment dermotological side effects are well known. New direct-acting antivirals have led to significant improvements in sustained virologic response rates, but several have led to an increase in dermatological side affects versus peginterferon (peg-İFN)/ribavirin (RBV) alone. A 55-year old man was administered peg-İFN alpha-2a (180 mcg, once a week )/RBV (1200 mg daily) and telaprevir (2250 mg daily) with the diagnosis of chronic hepatitis C. At the 8th week of the treatment erythematous rash developed. On the 12th administration of the combination therapy, the patient admitted to our hospital with, fever, elevated neutrophils, facial edema with excoriations and rash initially involved, rapidly spread to the body. The patient had been diagnosed as acute generalized exanthematous pustulosis and he was hospitalised. AGEP is generally characterized by an acute, widespread edematous erythema with the presence of small non-follicular pustulosis mostly in the folds and the face, and is associated with elevated neutrophils and high fever. As a result, with the advent of the new direct-acting antivirals, dermatological manifestations will be seen more frequently so patients should be monitored closely in terms of dermatological side effects.

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Clinical & Cellular ImmunologyClinical evaluation of rheumatologic manifestations in hepatitis B & C A Ramouz1, I Nazari Haghighi2 and M T Afkhami21Tabriz University of Medical Sciences, Iran2Tabriz University, Iran

Introduction: Although most of patients with chronic hepatitis B or C are asymptomatic, an appreciable number will experience symptoms that are due to the liver disease or rheumatologic manifestations of HBV and HCV infection. Recognition of these symptoms will lead to early diagnosis and treatment of hepatitis B and C.

Material and Methods: 100 patients, 95 infected with HBV and 5 with HCV were recruited from the hepatitis clinic. All patients were questioned for their age, marriage, job, education and rheumatological symptoms and were examined carefully for oral and cutaneous manifestations, tenosynovitis, enthesopathy, arthritis, and joint effusion.

Results: Out of 100 patients, 66 were male, 34 were female, and mean age was 38.7 years (45±29). In 36% of patients their hepatitis has been discovered occasionally at routine checkup. CRP in 65% of patients and RF in 77% and ANA in 90% was negative. 69% of patients had fatigue and 41% myalgia. LBP was been presented in 50% and morning stiffness in 32% and numbness in 28% of patients. On examination, 24% had dry mouth oral apheta in 26%, rush 4%, raynaud's 6%, CTS 19%, palmar tenosynovitis 11%, enthesopathy 21% and arthritis in 32% of patients. Arthralgias and arthritis most commonly involved the medium and large size joints. The most of arthralgias and arthritis were mono or oligo arthrcular and all of them were symmetrically.

Conclusion: Hepatitis B and C can present with rheumatologic manifestations. Risk factors or a history of jaundice should be included in the history of present illness of any patient with arthritis or unexplained other rheumatic problems. The predominant clinical findings include icterus, dry mouth, oral apheta, CTS, clubbing, enthesopathy and arthritis. Although symmetrically mono-oligo arthicular involvements in medium and large size joints are the most common pattern, there is not a single clinical picture of arthritis in patients with HBV or HCV infection.

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Cyclosporin a combined with gentamicin suppressed human Wa rotavirus replication in vitro and in vivo Yu-Zhang Wu, Zi-Gang Shen and Hai-Yang He and Jin-Tao LiThird Military Medical University, PR China

Rotavirus infection is the most common cause of acute diarrheal diseases among infants and young children worldwide. This work investigated whether cyclosporin A (CsA) combined with gentacimin inhibits human Wa rotavirus replication

in HT-29 cells and in neonate BALB/c mice model. CsA combined with Gentacimin after Wa rotavirus infection significantly suppressed virus replication/infection, which is evidenced by reduced rotavirus antigen, decreased expression of rotavirus RNA, protein and infectious viruses in intracellular and extracellular. The inhibition of Wa rotavirus replication/infection by CsA combined with gentacimin can restore the expression of IFN-β in HT-29 cells. And CsA combined with gentacimin treatment of Wa rotavirus-infected HT-29 cells upregulated the expression of IFN regulatory factor-5, 7 and β-transducin repeat containing protein and inhibited the expression of suppressor of cytokine signaling-1, protein inhibitor of activated signal transducers and activators of transcription-1 and y, the primary negative regulators of IFN signaling pathway. CsA combined with gentamicin treatment of Wa rotavirus-infected-neonate mice shortened the recovered time against Wa rotavirus infection and speeded up the elimination of rotavirus antigen. These findings indicate that further evaluation and characterization of the CsA combined with gentacimin on Wa rotavirus-infected diarrhea are warranted.

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Clinical & Cellular ImmunologyFrequency of autoimmune diseases in those suffering from vitiligo in comparison with normal population Ali Ramouz, Shahla Babaee Nejad and Leila NazemanTabriz University of Medical Sciences, Iran

Objectives: Vitiligo is more common in people with certain autoimmune diseases. Here we studied the association between vitiligo and autoimmune diseases.

Materials and Methods: In this case control study, 86 patients with vitiligo were questioned about the location of vitiligo, family history, treatment and therapeutic response. All patients were examined both clinically and with laboratory tests to detect the presence of autoimmune disorders including autoimmune thyroid disease, pernicious anemia, insulin dependent diabetes, systemic lupus erythematic (SLE) and Addison disease. The prevalence of autoimmune disorder in vitiligo patients with that in a group of age- and gender-matched normal population was compared.

Results: Average age of disease onset was 21.8+11 years; 61% of patients were females and 39% were males. The most common locations of vitiligo were hands (33.7%) and face (32.1%). The most common pattern of onset was vulgaris type (40%). Nearly one-fourth of patients had a positive family history of vitiligo. Prevalence of thyroid disorders in vitiligo patients and control group was 21.1% and 7%, respectively. The difference was statistically significant (P=0.008).

Conclusion: The most common autoimmune disorder in patient with vitiligo was hypothyroidism. Family history had a poor prognostic effect on response to therapy.

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Evaluation of IgA kit versus the gold standard blood culture method to detect Yersinia enterocolitica contamination of blood products Badlou B A1, Timori N H2, Dabirmoghadam A2 and Razjou F2

1BBAdvies and Research, The Netherlands2High Institute for Research and Education in Transfusion Medicine, Iran

Background: Current detection materials and methods of bacterial detection tests of blood products are rather controversial and lethargic. The gold standard screening test to detect is blood culture method. A rapid serological YOP IgA kit was introduced by the Company Microgen, which was developed to detect Yersinia in the whole blood sub-products to our blood bank (IBTO). The aim of this study is to compare whether IgA kit detect Yersinia contamination in the whole blood product stored at 4-6°C in the IBTO.

Materials and Methods: The sera from 492 healthy blood donors in the IBTO were selected for study with Yersinia anti-Yop IgA antibodies kit (MICROGEN, Germany) by using two different techniques, enzyme immunoassay (EIA) and recomLine Yersinia Western blot to confirm the prevalence of Yersinia antibody.

Results: We carried out ELISA tests as manufacturer instruction has described. The recom well Yersinia EIA measurements revealed that the prevalence rates of Yesinia Yop-specific IgA antibodies were 12.5% (n=62) positive, and 87.5% (n=430) negative. The confirmation Western blot test on 60 ELISA-positive samples showed 71% positive and 29% negative, respectively.The blood culture results of 62 seropositive donors’ samples were all negative (100%), which were stored at 4-6 °C, for 35 days.

Discussions: Based on observed data IgA ELISA kit, become clear that it is indirect in detection of Yersinia, has high false positivity, shows controversial results versus the gold standard blood culture, increases blood donors deferral, affects negatively blood supply, and is not cost effective as well.

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Clinical & Cellular ImmunologyCD21 positive B cell: A novel target for treatment of multiple sclerosisMojtaba FarjamFasa University of Medical Sciences, Iran

Etiologic-based therapy is an ideal pharmacological option to treat or prevent diseases. There is no known etiology for multiple sclerosis (MS); however, environmental risk factors have been suggested to predispose genetically susceptible

people to be affected by the disease. One of these risk factors is infection with Epstein-Barr virus (EBV). Eradication of this virus has not been effective in modulation of MS, probably due to being inhabitant in the CD21 (EBV receptor) positive B cells. To eradicate this virus, targeting CD21 on these EBV-infected B cells is hypothesized here. A sequential study plan to test this hypothesis has been suggested too. This study might eventually suggest an effective immunopharmacological strategy to treat MS. Moreover, testing this strategy will help in better clarification of the role of EBV in MS disease triggering and predisposition.

BiographyMojtaba Farjam graduated in medicine in 2001 and received a PhD in Pharmacology from Shiraz University of Medical Sciences, Iran. He studied neuroimmune pharmacology of multiple sclerosis as a Research Scholar in Thomas Jefferson Medical College PA, USA in 2012. Currently, he is the Scientific Executive of Fasa Cohort Study, Fasa, Iran and Assistant Professor of Medical Pharmacology in Fasa University of Medical Sciences.

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BMI1 and TWIST1 downregulated mRNA expression in skin cancerFatemeh Vand Rajabpour, Narges Sadeghipour, Reza Raoofian, Hamidreza Fathi, Pedram Noormohammadpour, Kambiz Kamyab Hesari and Mina TabriziTehran University of Medical Sciences, Iran

Bmi-1 is a proto-oncogene with a role in self-renewal. Twist1 can regulate Bmi-1 expression, and Twist1 also regulates Snai2 which has been proven to be essential for Twist1 function. These molecules play a role in aggressive behavior of

cancerous cells. BMI1 expression could identify subtypes of Merkel cell carcinoma (MCC). However, BMI1, TWIST1 and SNAI2 expression levels in basal cell carcinomas (BCCs) has not been elucidated. It was hypothesized spectrum of skin cancers from BCC to Melanoma could be good model systems to shed some light on mechanisms that drive tumor metastasis. The aim of this study was to examine the mRNA expression level of BMI1, TWIST1, and SNAI2 in BCCs, SCCs and melanomas. Eighty fresh non-metastatic BCC tissue samples, eight SCC's, three melanomas and fifty fresh normal skin tissues were collected. As a pilot study, thirty-five fresh BCC tissue samples and seven fresh normal skin tissue samples were evaluated by real-time RT-PCR. BMI1 and TWIST1 demonstrated marked down-regulation (p<0.00l, p=0.00l respectively), but SNAI2 showed no significant change (p=0.12). Previous literature has clearly demonstrated a positive association between BMI1 and TWIST1 expression and metastatic BCC, aggressive SCC and melanoma. This pilot study demonstrates Bmi-1 and Twist1 dichotomy of action in locally invasive BCC in contrast to aggressive skin cancers. Local invasion of BCC shows no correlation with Snai2 expression level. It would be interesting to compare these results with SCC and melanoma findings.

BiographyFatemeh Vand Rajabpour is a PhD student of Medical Genetics at Tehran University of Medical Sciences (TUMS), Tehran, Iran. She completed her MSc jointly with Shahrekord University of Medical Sciences and the Medical Genetics Dept at TUMS. She has two review articles published on Basal Cell Carcinoma and miRNAs.

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Clinical & Cellular ImmunologyThe effect of NR2B-containing NMDA receptor antagonist on neurological damage and calpain and caspase3 activities in experimental model of multiple sclerosisMojtaba FarjamFasa University of Medical Sciences, Iran

Neurodegeneration is the pathophysiological basis for permanent neurological disabilities in multiple sclerosis (MS); thus neuroprotection is emerging as a therapeutic approach in MS research. Modulation of excitotoxicity by inhibition of

NMDARs has been suggested for neuroprotection. Decrease in Calcium-dependent protease activity secondary to NMDAR inhibition, has been postulated as a mechanism for this approach. However, this probable mechanism remains to be proven yet. Moreover, selective antagonisation of NR2B subtype of these receptors, an NMDAR subtype believed to play a more pivotal role in neurodegeneration, has not been studied too. In this study, the effect of inhibition of NR2B-containing NMDAR was evaluated on the animal model of MS, experimental autoimmune encephalomyelitis (EAE). EAE induction was done using MOG in C57/Bl6 mice. Therapeutic administration of different doses of highly selective NR2B-containing NMDAR inhibitor (RO25-6981) was compared with memantine (non-selective NMDAR antagonist) and vehicle in different experimental groups. Behavioral, histological and western blot analysis of Calpain and Caspase 3-dependent α-spectrin break down were studied comparatively among groups. Neurological deficits in EAE animals were more efficiently decreased by selective inhibition of NR2B-containing NMDARs. Histological studies of the spinal cords also showed decreased inflammation, myelin degradation, neuro-axonal degeneration and Calpain activity when RO25-6981was administered with higher doses. Regarding the role of NR2B-containing NMDARs in excitotoxicity, selective inhibition of these receptor subtypes appears to modulate the neurological disabilities and pathological changes in EAE. Decreasing the activity of Calcium-dependent protease, Calpain, by blockade of NR2B-containing NMDAR using high dose of RO25-6981 can be seen simultaneously. More experimental studies could be performed to suggest NR2B-containing NMDAR inhibition as a potentially effective treatment strategy for slowing down the clinical deterioration of disability in MS.

BiographyMojtaba Farjam graduated in Medicine in 2001 and received a PhD degree in Medical Pharmacology from Shiraz University of Medical Sciences, Iran in 2012. He studied neuroimmune pharmacology of multiple sclerosis as a Research Scholar in Thomas Jefferson Medical College PA, USA in 2012. Currently, he is the Scientific Executive of Fasa Cohort Study Center, Fasa, Iran and Assistant Professor of Medical Pharmacology in Fasa University of Medical Sciences. He has been working in Fars MS Aid Charity as advisor and supervisor of the data base in this organization since 2010.

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Oral and dental health status in preschool children with asthma Sara EhsaniTehran University of Medical Sciences, Iran

Asthma is a chronic inflammatory disorder of the airways, which is diagnosed by periodic symptoms of inflammation, bronchial spasm, and increased mucosal secretions. It has higher incidence among the preschool children. There are many

contradictory reports based on the effect of asthma on oral health, however it has been hypothesized that asthma could lead to poor oral health. The objective of the present study was to investigate oral health indices in 44 preschool children of three to six years old with mild to moderate asthma and 46 matched healthy children in Tehran Children's Respiratory Center. Dental plaque, gingival inflammation, mouth breathing, and dental caries were evaluated by one trained examiner according to World Health Organization [WHO] criteria. Culture and colony counting of streptococcus mutants and Lactobacillus species were carried out in saliva specimens of the patients. The effects of different factors on the colony counts were statistically analyzed using linear regression analysis. The level of mother’s education and preexisting asthma disease in children had significant effect on the colony counts of streptococcus species whereas no factor was found to influence the number of Lactobacillus counts significantly. The results indicated no significant difference between the children with asthma and those without asthma regarding dmft (decayed, missing, filled, teeth) index (mean of 3.34 in asthmatic children and 3.0 in the control group). Therefore, it can be deduced that the presence of asthma disease did not increase the probability of tooth decay.

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Clinical & Cellular ImmunologyEffect of oral herbal oil supplement (Cannabis sativa L, Oenothera biennis L) and advised hot-nature diet on clinical signs and symptoms and inflammatory factors in patients with multiple sclerosisSoheila Rezapour-FirouziUniversity of Medical Sciences, Iran

Background: Multiple sclerosis (MS), ensuring demyelization results in physical disability, is the most chronic and inflammatory disorder. Because of limited efficacy and adverse side effects of the current treatments, identifying novel therapeutic and protective agents is important. For many years, it has suspected that the risk of developing MS might be associated with increased dietary intake of saturated fatty acids and consumption of Cold nature foods. This study aimed to assess the potential therapeutic and protective effects of hemp seed and evening primrose oils as well as Hot-nature dietary intervention on mild MS patients.

Methods and materials: In this double blind, randomized trial, 100 RRMS patients (Expanded Disability Status Scale<6) allocated into three groups: “Group A” who received co-supplemented oils with advising Hot nature diet, “Group B” who received olive oil as placebo, “Group C” who received co-supplemented oils. Mizadj, Clinically EDSS, relapse rate and functional score as well as immunological factors (plasma cytokines of IL-4, IFN-γ and IL-17), biochemical parameters (GGT, AST, ALT, red blood cells PUFA and erythrocyte membrane fatty acids composition, D-6-desaturase (FADS2), secretory PLA2 (sPLA2) assessed at baseline and after 6 months.

Results: Mean follow-up was 180±2.9SD days (N=65, 23 male and 42 female aged 34.25±8.07 years with disease duration 6.80±4.33 years). There was no significant difference in studies parameters at baseline. After 6 months, significant improvements in Mizadj, EDSS score and relapse rate found in the groups A and C while the group B only showed a border significant decrease in relapse rate. Immunological and biochemical parameters showed improvement in the groups A and C whereas there was worsening condition for the group B after the intervention, and immunological parameters correlated with the EDSS score in-group A. After 6 months, the erythrocyte cell membrane with regard to specific fatty acids, showed improvement in the group A and C whereas there was worsening condition for the group B after the intervention.

Conclusion: This study suggests that co-supplemented herbal oils with hot nature diet can have beneficial effects in reversing the signs and improve clinical outcome in RRMS patients which were confirmed by immunological and biochemical findings. This intervention causes an increase PUFAs in MS patients and improvement in the erythrocyte membrane fatty acids composition and it could be an indication of restored plasma stores, and a reflection of disease severity reduction.

BiographySoheila Rezapour-Firouziis completed PhD from Department of Immunology, Microbiology and Genetics; Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran. In 2013, she published her papers on "Effect of Oral Herbal Oil Supplement (Cannabis sativa L, Oenothera biennis L) and Advised Diet with Hot-Nature on Clinical Signs and Symptoms and Inflammatory Factors in Multiple Sclerosis Patients". This supplement oil was named MS-NUT; it means Nutrition for MS Patients. Her research is going to be published with the name of the peace, justice and freedom for all around the world, presents for the treatment of suffers of Multiple Sclerosis.

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Clinical & Cellular ImmunologyTumor suppressor gene alteration as early markers in ulcerative colitis associated carcinoma and dysplasia Zainab Mohammed Taher JaafarMinistry of Science and Technology, Iraq

Ulcerative colitis (UC), involves the colon and rectum. Inflammation and ulcers typically affect only the innermost lining in these areas. The etiology of UC is not fully elucidated the causes are likely based on combination of heredity and

environmental factors. People who had UC for at least eight years have a higher risk of developing colon cancer. The risk is even greater when inflammation affects the entire colon, approximately 10% of UC develop colorectal neoplastic. In long standing UC, a dysplasia carcinoma sequences is widely accepted, the tumor genesis being considered associated with long term inflammation and oxidative stress causing DNA damage.The early marker of neoplastic progression is mutation in P53 gene is reported to be an early event in UC -associated carcinogenesis, as analyzed with reference to genetic instability, the other marker chromosomal alteration such as chromosome aberration, sister chromatid breaks or exchange and also detected by comparative genomic hybridization and flow cytometry. In this study, the colonic biopsies which collected from patients of UC and colon cancer were examined for gene expression of P53 gene and Bcl-2 gene on the levels of in situ hybridization. The results indicated accumulated mutations in P53 gene in the inflamed colonic biopsies, this give an indication for progression of carcinogenesis. While on the level on Bcl-2 gene, the results showed increasing in the activity of the gene, resulting in increase in gene expressing which is similar in gene expression in tissues of colon cancer biopsies. The extent of p53 gene was 33.3 in high grade, 63.93 in moderate grade and 2.77 in low grade. The extent of Bcl-2 was 75.03 in high grade, 24.97 in moderate grade in tissue of colon cancer while the intensity of P53 was 36.1in low grade, 55.6 in moderate grade and 8.3 in high grade, and the intensity of Bcl-2 was 41.63 in high grade, 55.53 in moderate grade and 2.76 in low grade in colon cancer tissues. The extent of P53 was 63.97 in high grade, 27.73 in moderate grade, 8.3 in low grade in UC tissues, while in Bcl-2 was 66.3 in high grade, 25 in moderate grade and 8.37 in low grade in UC tissues. The intensity of P53 was 18.47 in high grade, 47.2 in low grade and 33.3 in high grade in UC tissues and the intensity of Bcl-2 was 7.86 in high grade, 50.87 in moderate grade and 21.27 in low grade of UC tissues. In addition to some immunological tests to insight into the disturbance associated with clinical expression of UC. It was noticed that the immunoglobulins that include IgG, IgA, IgM, as well as some complements such as C3, C4 were increased in both UC and Colon cancer in the sera of the patients.

Conclusion: The detection of P53 gene and Bcl-2 gene in chronic UC and colon cancer give early detection for diagnostic and therapeutic and monitoring purposes.

BiographyZainab Mohammed Taher Jaafar did his PhD in Biology and works in Ministry of Science and Technology, Head Department in the Biotechnology Center. He is interested in immunology field, especially in autoimmune diseases. He has more than 35 published articles in biological fields.

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Clinical & Cellular ImmunologyAdvances and challenges in the diagnostic accuracy for surgical management of inflammatory bowel disease: Potential role of molecular biometricsAmosy E M'KomaMeharry Medical College School of Medicine, USA

In the 20th century, it has become possible to plant a new organ instead of diseased or lost organ. The ways to replace organs has been implemented in three different approaches: i) use of artificial organ (extra-corporeal or intra-corporeal), ii)

transplantation of organs from other humans and iii) construction of new organ by use of sources from the same patient’s own body. One of the most extensive procedures following the third option, is the “restorative proctocolectomy (RPC) and ileal pouch-anal anastomosis (IPAA)” for surgical treatment of ulcerative colitis (UC) and familial adenomatous polyposis (FAP). RPC replaces the colon and rectum by a pouch formed from the ileum and sutured to the anal canal preserving the anal sphincters. The reconstruction restore gastrointestinal continuity, defecation, deferral and discrimination. Successes of RPC and IPAA depend on the correct diagnosis, which is currently not offered accurately in up to 30% of inflammatory bowel disease (IBD) patients. In trying to find a solution to diagnosis inaccuracy dilemma in IBD, proteomic evaluation of surgical pathology specimens of colonic mucosal and submucosal layers, individually; of IBD, encompassing Crohn’s colitis (CC) (a colonic Crohn’s disease (CD)) and UC has been the focus to the advancement of diagnostic medicine to improve diagnostic accuracy in IBD. Mass spectrometry (MS) is unique among analytical technologies in its advantage and ability to directly measure individual molecular species in complex clinical specimens, allowing it to make significant contribution to our understanding of biological molecules in health and in disease. It has become an essential tool for qualitative and quantitative analysis of cellular systems. The biochemical complexity and functional diversity of the biometric system are well suited to proteomic studies. The direct analysis of cells and tissues by matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) has developed significant momentum for applications that have diagnostic potential. MALDI IMS methodology permits histology-directed target highly specific areas in colonic tissue for analysis without the need for extensive sample preparation. MALDI IMS provides molecular information from specific cell types within tissue sections. This laser-based approach significantly reduces the analysis time for each location sampled. This presentation discusses a perspective on the application of sophisticated MALDI IMS and bioinformatics technologies, to detect unique molecular biometrics to trying to improving diagnostic accuracy in IBD, elucidation of advances involving the identification of differentiating proteomic patterns, while offering a perspective on the challenges faced in the transfer.

BiographyAmosy E M'Koma is Assistant Professor of Surgery, Biochemistry and Cancer Biology in the Department of Biochemistry and Cancer Biology at Meharry Medical College and Vanderbilt University Medical Center, Division of General Surgery, Colon and Rectal Surgery. Dr. M'Koma received medical training initiated and completed his MD degree at Kharkov Medical Institute in Kharkov, Ukraine in 1984. He did his postgraduate and surgical residency program at the Karolinska University Hospital, Huddinge in Stockholm, Sweden. He was Board Certified in General Surgery (The Swedish Board of Health and Welfare in 1990). Dr. M'Koma pursued a Licentiate of Medical Science degree (1999) and a PhD (2001) in Surgery from the Karolinska Nobel Institute, Sweden. Dr. M'Koma came to the United States to further pursue academic endeavors. He was able to work at the Mayo Clinic in Rochester, Minnesota as a Research Trainee where he interacted with some of the leaders in the field of Colon and Rectal Surgery. The interactions with investigators at the Mayo clinic provided additional insight into the development of his research career in inflammatory bowel disease (IBD). He interacted with some of the leading physicians in the areas of IBD and received guidance and advice on the research activities that had the potential to significantly impact the understanding and surgical treatment of the disease. His research efforts during this time related to an in depth study of the scientific and clinical background of inflammatory colitis and it maladies. Dr. M'Koma research interest is focused on the pathophysiology of IBD more focused on diagnostic methodologies and surgical management of ulcerative colitis (UC) and familial adenomatous polyposis (FAP). The research is gravitated to gastrointestinal disease, specifically relating to the restorative physiology of intestinal continuity after operations for IBD, specifically, restorative proctocolectomy (RPC). The research efforts are directed towards developing strategic methodologies based on MALDI-mass spectrometry, proteomics and recombinant single-chain antibody to identify molecular biometrics that can provide discriminatory diagnostic tool for the colitides.

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Clinical & Cellular ImmunologyCharacterization of two Moroccan scorpion venoms: Proteomic analysis, neurotoxicity, myotoxicity and sensitivity to neutralization by experimental polyclonal anti-venom Oukkache NaoualPasteur Institute of Morocco, Morocco

Buthus occitanus and Androctonus mauretanicus are two medically important scorpions implicated in the pathogenesis of scorpion stings in Morocco. In this study, we first report on the neurotoxicity and myotoxicity of Androctonus

mauretanicus (Am) and Buthus occitanus (Bo) venoms, as well as on the neutralizing capacity of experimental antivenom towards the neuromuscular and myotoxic activities of these venoms. Second, we examined the efficacy of commercially-available polyvalent antivenom regarding its ability to prevent or reduce mortality (in vivo testing in mice), myotoxicity and neurotoxicity of venoms. The protein contents of Am and Bo venoms were investigated using an original scorpion venomic approach. The pharmacological effects of Am and Bo venoms were investigated in vitro using chick biventer cervicis nerve-muscle preparations. The protective potency of commercial polyclonal anti-venom was also investigated. This anti-venom was made from serum of horse that was hyperimmunized with a mixture of three scorpion venoms: Two venoms from the most venomous species of North Africa, Bo and Androctonus australis hector (Aah) and one venom from the most venomous species living in Middle East (Leiurus quinquestriatus). The results obtained were finally compared to the values of ED50 in assays of lethal activity neutralization in mice. Our findings: 1) The sequential analysis of venom fractions revealed a highly complex venom proteome which counted a total of 80 and 140 proteins for Bo and Am venoms, respectively. In Am venom, we report a content of 21% enzymes, 43% sodium channel-specific toxins (89% are alpha-toxins and 11% are beta-toxins), 20% potassium channel-specific toxins, 4% chloride channel-specific toxins, 3% unknown peptides and 9% other activities. Among Am toxins, 17% are insect toxins whereas 83% are mammal toxins. In Bo venom, we found 18% enzymes, 58% sodium channel-specific toxins (90% are alpha-toxins and 10% are beta-toxins), 15% potassium channel-specific toxins, 5% chloride channel-specific toxins, 1% unknown peptides and 8% other activities. Among Bo toxins, 19% are insect toxins and 81% are mammal toxins. From data analyses, it appears that Am and Bo venoms contain a number of compounds sharing high sequence identities with alpha-toxins from structural and immunological groups 1 to 3. 2) It was found that Am and Bo venoms contain myotoxins and postsynaptic neurotoxins. In agreement with lethal potencies of these venoms in mice, Am venom exhibited greater neurotoxicity and myotoxicity than those observed with Bo venom.

Significance: 1. Interestingly, our results show that Am and Bo venoms are complex mixtures of molecules (especially Am venom). When

Am venom is concerned, our results agreed well with previous reports highlighting a high frequency of compounds sharing a high structural identity with toxins (suggesting a particular danger of Am venom to humans

2. These findings strongly suggest that Am and Bo venoms contain distinct toxin components that are responsible for myotoxic and/or neurotoxic effects. The data also indicate it would be appropriate to add the Am venom, together with Bo and Aah venoms, to potentially produce more efficient polyvalent antivenom.

BiographyOukkache Naoual has completed his PhD in Biochemistry. She has an expertise in the field of study of venoms and the improvement of antivenoms. She did internships on the study of venoms and antivenoms production from Butantan Institute in Brazil, The Institute of Biotechnology of Mexico and Monash University in Malaysia. She works now as Researcher at Pasteur Institute in Morocco.

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Clinical & Cellular ImmunologyEffect of flaxseed supplementation on immune response of peripheral blood mononuclear cellsHanan Al KhalaifaKuwait Institute for Scientific Research, Kuwait

Introduction: The phagocytosis assay allows quantitative measurement of the percentage of phagocytes and the enzymatic activity of each phagocyte (i.e. the number of ingested antigens per phagocytic cell). Bacteria such as Escherichia coli are commonly used as substrates for the phagocytosis assay. Immunomodulation of fatty acids in flaxseed may alter phagocytosis activity.

Aims and objective: The objective of the current work was to compare the effect of feeding normal broiler chickens 15% of dietary flaxseed on phagocytic activity of monocytes and heterophils in the peripheral blood.

Methodology:Diet and sample collection

One-day-old male Cobb-500 broiler chicks were used. Water and feed were provided ad libitum. Upon hatching, all chicks were given the same basal diet for 13 d. Following this, dietary supplementation of flaxseed started at 14 d of ages until the end of the cycle at 35 d of age. At slaughter, samples of blood were collected from each bird.

Phagocytic activity of blood The quantitative analysis of the phagocytic activity of peripheral blood mononuclear phagocytes in whole blood was

performed using phagotest commercial kits. Results were expressed as percentage of fluorescent cells (% phagocytosing cells) and mean fluorescence intensity (MFI). Data were analysed by CellQuestTM software. Discrete populations of polymorphonuclear heterophils and monocytes were gated in the software programme based on identification by forward and side scatter and its green fluorescence histogram (FL1) was analysed. The control sample was used to set a marker for fluorescence-1 (FL1) so that the events above this marker position were considered to be positive. The overall differences between the dietary treatments were analysed using one-way analysis of variance (ANOVA) and the general linear model procedure of Minitab was applied in all the tests. Differences between the treatment groups were considered statistically different at P≤0.05. When significant differences occurred (P≤0.05), treatment mean differences were identified by pairwise comparison using Tukey tests.

Results: Feeding flaxseed at 15% did not affect either the percentage of cells participating in phagocytosis, or the Mean fluorescence intensity (MFI). However, there was a trend towards a decrease in the % of monocytes involved in phagocytosis in birds fed diets containing 15% flaxseed. Also, there was a trend towards a decreased MFI (p=0.056) for monocytes.

Overall, studies in mice showed that n-3 PUFA decreased phagocytosis. n-3 PUFA either increased or had no effect on phagocytosis activity in rats and human studies demonstrated little evidence of an effect of n-3 PUFA on phagocytosis of blood cells. Studies in the literature which investigated the effect of n-3 PUFA on phagocytosis in chickens are quite limited. Results of the current study showed no effect of flaxseed on phagocytosis of peripheral blood cells.

BiographyHanan Al Khalaifa has a PhD in Immunonutrition from the University of Reading- United Kingdom and MSc in Medical Immunology from the University of Manchester. She has been working in Kuwait Institute for Scientific Research since 1996. She participated in many research projects and activities. She gained skills and experience in immunological techniques, Immunonutrition approaches, biodiversity monitoring and assessment techniques, poultry production research, production of added-value products, analysis techniques including gas chromatography, proximate analysis, flow cytometry etc. She published more than 20 scientific papers in refereed journals and conferences.

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Clinical & Cellular ImmunologyThe promise of sonodynamic therapy: Using ultrasonic irradiation and chemotherapeutic agents as a treatment modalityMatthew TrendowskiSyracuse University, USA

Sonodynamic therapy (SDT) is showing promise as a potentially vital alternative to traditional cancer treatment modalities. SDT is a form of ultrasound therapy in which chemotherapeutic agents known as sonosensitizers are administered to

increase the efficacy of ultrasound’s preferential damage on neoplastic cells (Trendowski., 2014). The attractive features of SDT emerges from the ability to focus ultrasound energy on malignancy sites buried deep in tissues and to locally activate a preloaded sonosensitizer (Rosenthal, Sostaric, Riesz., 2004; Kuroki et al., 2007). Furthermore, SDT has shown to induce cell damage in many cancer types and appears to be a versatile treatment method (Bai W, Shen E, Hu B., 2012; Kuroki et al., 2007). It has even been shown that SDT can illicit an immune response as observed in a substantial variety of in vivo studies. Evidence of this remarkable mechanism include transient increases in infiltration of non-T regulatory (non-Treg) tumor infiltrating lymphocytes (TILs), continual infiltration of CD8+ cytotoxic T-lymphocytes (CTL) and an observable macrophage response due to cell lysis (Hu et al., 2007; Liu et al., 2012; Wu., 2013; Wu et al. 2007).

Perhaps one of the most intriguing capabilities of ultrasound is its ability to preferentially lyse cells based on size (Miller, Luque, Battaglia, Mazza, Everbach., 2003). This known fact invariably gives rise to the idea of grossly enlarging tumor cells to increase their already noticeable size difference with normal cells. Cytochalasin B is a known pharmacological agent that disrupts the actin cytoskeleton and inhibits cytokinesis by interfering with formation of the contractile ring as well as the development of the cleavage furrow (Trendowski., 2014). Consequently, the cell does not divide and an immature actin cytoskeleton remains. However, the cell continues to form nuclei and eventually becomes grossly enlarged and multinucleated. Such cells invariably have more DNA targets, increasing the likelihood of apoptosis. Furthermore, the multinucleated cells have a large cell volume, making them more susceptible for direct cell destruction. Preferential damage of malignant cells is actually easily attainable as normal cells exposed to cytochalasin B exit the cell cycle and enter a resting state until sufficient actin levels are restored. Therefore, only malignant cells that have lost the ability to enter the rest phase will become grossly enlarged and multinucleated, providing an ideal target for ultrasonic irradiation.

Work from our lab has indicated that cytochalasin B does indeed only damage leukemia cells, leaving normal blood cells, unaffected (Trendowski., 2014). The designated cell line has been promyleocytic leukemia U937 cells as they are a frequent choice for in vitro studies. The U937 cells have routinely become grossly enlarged and multinucleated, providing an ideal target based on size. The typical erythrocyte is 6-8µm, while leukocytes fair slightly better with a range of 10-15µm and an average of 12µm. By contrast, work from our lab has shown that cytochalasin B treated leukemia cells easily grow in excess of 20µm with some reaching 40µm in diameter after enough exposure(Trendowski et al., 2014). Such cells have reduced cytoskeletal integrity and are easy targets for ultrasonic irradiation. Furthermore, cytochalasin B treated leukemia cells are substantially multinucleated as cytokinesis is inhibited. This provides plenty of targets for a nucleic acid directed agent such as cisplatin or doxorubicin to attack.

The proposed therapeutic approach could provide for a potent clinical method to treat leukemias and other hematological malignancies. Ultrasound can be directed towards any area of the body, allowing for search and destroy methods to be created in which there is no place for malignant cells to hide. Preferential damage should be substantially increased when a cytochalasin B-nucleic acid agent drug cocktail is applied to ultrasonic irradiation as leukemia cells will be most affected by the treatment. SDT has even been shown to reverse drug resistance in K562/A02 leukemia cells when doxorubicin, another nucleic acid agent is combined with ultrasound (Meng, Chen, Wu, Shao, Gao, Zhao., 2008). Such results are promising and further substantiate SDT as a viable treatment modality. By using cytochalasin B and a nucleic acid agent as sonosensitizers, I propose that ultrasonic irradiation will significantly cripple leukemia cell populations, creating a basis to promote the therapeutic approach for in vivo and eventual clinical studies.

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Clinical & Cellular ImmunologySerum levels of TNF-α but not CRP predict retinopathy prior to diabetic diagnosisMohammad J Alemzadeh-Ansari, Mohammad Omidi, Afsaneh Mortaza and Manuchehr NakhjavaniTehran University of Medical Sciences, Iran

Objective: It is shown that chronic inflammation is the leading cause of many diseases including coronary heart disease, type 2 diabetes and even some cancers. Increase C reactive protein (CRP) and tumor necrosis factor α (TNF-α) levels are associated with the incidence of diabetes and its major complication. It was hypothesized that the active immune system predicts the long term diabetes complications. Here we aimed to study two important inflammatory markers, TNF-α and CRP in patients with and without diabetic retinopathy as well as newly diagnosed diabetes, and also evaluate the effects of metformin therapy on these markers.

Methods: A cross sectional study on patients with long standing diabetes and patients with newly diagnosed diabetes were performed. The patients with long standing diabetes were classified into three groups: Proliferative diabetic retinopathy (PDR), non-proliferative diabetic retinopathy (NPDR), and diabetes without retinopathy (NDR). Patients with newly diagnosed diabetes were diagnosed within recent 6 months who not on any type of treatment. Another follow up study for patients with newly diagnosed diabetes before and after 3 months of therapy with metformin. Serum level of TNF-α and CRP were measured in patients with long standing diabetes and also, in patients with newly diagnosed diabetes before and after of therapy. The local ethics review committee of Tehran University of Medical Sciences approved the study protocol. The statistical package SPSS 17 for windows (Chicago, Illinois, USA), was used for analysis.

Results: Of the 113 patients enrolled including 83 patients had long standing diabetes (29 with PDR, 27 with NPDR, and 27 with NDR) and 30 patients had newly diagnosed diabetes. There were no significant differences in the serum TNF-α level between patients with retinopathy compared to those with newly diagnosed diabetes. Although, serum TNF-α levels in patients with retinopathy (either PDR or NPDR) were significantly higher than those without retinopathy (p value <0.01). Whereas, there was no significant difference found between groups in serum levels of CRP. Serum levels of CRP were significantly lower in patients with newly diagnosed diabetes compared to other groups (p value <0.01). After therapy with metfomin in groups with newly diagnosed diabetes, no significant change was observed in serum levels of TNF-α or CRP.

Conclusions: Our findings show TNF-α was not affected by diabetes duration or therapy by metformin, but the incidence of retinopathy is predictable by the primary level of TNF-α. Serum level of CRP was lowest in patients with newly diagnosed diabetes, although presence of retinopathy or therapy by metformin does not influence the serum level of CRP.

BiographyMohammad Javad Alemzadeh-Ansari graduated as a general physician with a first degree from Jondishapour University of Medical Sciences, Ahwaz, Iran. In the same year, he was accepted as resident of cardiology in the Tehran University of Medical Sciences, Iran. He has participated in various international conferences, such as 4th, 5th, and 6th International Online Medical Conferences (IOMC), 17th congress of Iranian Heart Association, and World Congress of Cardiology 2012. Also in 4th and 6th International Online Medical Conferences participated as a member of Conference Students’ Board. He has published 26 articles in various international journals, and acts as a reviewer for some international journal including BMJ Journal, Anatolian Journal of Cardiology, African Journal of Biotechnology, and Saudi Journal of Kidney Disease and Transplantation.

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Clinical & Cellular ImmunologyThe association between vitamin D status and recurrent wheezing in rural Bangladeshi childrenMd. Atiar RahmanBangabandhu Sheikh Mujib Medical University, Bangladesh

Background: The exact mechanism between the vitamin D level and recurrent wheezing is not obvious. There are a few studies investigating the relationship between recurrent wheezing and vitamin D level. Vitamin D appears to play a role in immune system and lung development in the fetus. Vitamin D receptors (VDR) have been found in fetal type II alveolar pneumocytes of rats, and may play a role in lung development, pneumocyte differentiation and surfactant secretion. A study in a large cohort of children with asthma in Costa Rica showed an association between low vitamin D levels (<30 ng/ml) and asthma severity in terms of hospitalizations, medication use and airway responsiveness. The rising prevalence of asthma may be linked to vitamin D deficiency but asthma is a multifactorial chronic disease. It may be useful to measure the levels of 25 (OH) vitamin D of the pre-school children with recurrent wheezing and give them vitamin D supplementation until the level is increased up to 30 IU/L by controlling the calcium levels in order to prevent asthma.

Objective: To observe association between vitamin D status and recurrent wheezing in preschool children.

Methods: Twenty five preschool children with recurrent wheezing and twenty five healthy, similar aged children without any history of acute or chronic illness were included in the study. The clinical features of children were recorded and serum 25-hydroxyvitamin D [25(OH) D] levels were measured. Data analysis was performed using SPSS 16 package program.

Results: The mean value of 25 (OH) D vitamin levels were 32.1±8.9 IU/L and 28.8±11 IU/L for the control and recurrent attack group respectively. Thirteen percent of subjects with recurrent wheezing had vitamin D levels in the deficient range (<20 ng/ml) and 7.9% had vitamin D levels under <20 ng/ml in the control group. The percentage of insufficient vitamin D levels (<30 ng/ml) were 8 and 7 for the patient and control group respectively. There was no statistical significance between the groups in terms of the distribution of 25 (OH) D levels.

Conclusions: The present study showed that vitamin D status did not influence recurrent wheezing in the pre-school children because there may be sufficient ultraviolet B (UVB) intensity for cutaneous synthesis of 25(OH) D in rural Bangladeshi children and or small sample size may not reflect the actual scenario.

BiographyMd. Atiar Rahman has completed his MD in Pediatrics from Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh and Fellowship in Pediatrics from Bangladesh College of Physician and Surgeon. He had also an opportunity to receive Fellowship training in Pediatric Respiratory Medicine from Great Ormond Street Hospital for Children, London, UK and “Anna Meyer” Children Hospital, Florence Italy. He is the Associate Professor of Pediatric Pulmonology, Department of Pediatrics of BSMMU. He has published more than 16 papers in reputed journals and serving as a reviewer of reputed international journals.

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Clinical & Cellular Immunology3D Chromatin structure of the immunoglobulin heavy chain geneTatiana GerasimovaNational Institute of Aging, USA

Immunoglobulin heavy chain (IgH) genes are assembled by rearrangement of variable (VH), diversity (DH) and joining (JH) gene segments spread over more than 2.5 Mb of the genome. The order of recombination is precisely determined,

with DH to JH recombination occurring first, followed by VH recombination to newly-created DJH junctions. One key mechanism involves folding of the IgH locus into conformations that minimize hazardous translocation events during V(D)J recombination. In a previous study we proposed a two-step model for generating IgH locus conformation. The first step, which is Eμ-independent, permits pro-B-specific CTCF binding to the IgH locus and generates multiple 250-400 kb sub-domains. The second step involves Eμ-dependent interactions with distant sites in the VH region that juxtapose sub-domains in the VH part of the locus to the 3’ end of the IgH locus, thereby leading to locus compaction. Here we verify that Eμ-dependent interactions with the VH regions are involved in locus compaction and demonstrate that integrity of Eμ-dependent loops requires YY1 protein. We also provide evidence that YY1-dependent locus compaction is mediated by the condensin components Smc 2 and 4. In contrast, sub-domains in the VH are CTCF dependent but YY1- independent. Furthermore, these CTCF-dependent sub-domains are B lineage-specific and Pax5-independent. These observations strongly substantiate the hypothesis that IgH locus compaction occurs by two independently-regulated steps that first fold the entire locus into sub-domains followed by juxtaposition of sub-domains to accomplish full contraction. Using novel multi-probe FISH technique we also demonstrated a great variability and diversity of chromatin conformation even inside one genotype using IgH mouse locus. We suggest that these folding principles may apply more generally in folding megabase-sized chunks of the genome.

BiographyTatiana Gerasimova received her education, PhD and Doctorate degree in Moscow, Russia. She worked in the Biology Department of Johns Hopkins University, USA, where she made significant contributions in molecular genetics of Drosophila melanogaster, in characterization of Insulators elements. During 15 years she was an Associate Editor of journal “Genetica” ((The International Journal of Genetics, Springer, Netherlands). She has published more than 100 papers in peer-reviewed journals, including Nature, Cell, Molecular Cell, Genes and Development, etc. Since 2007, she is a Staff Scientist of the National Institute of Aging, Laboratory of Molecular Biology and Immunology. During these years she dedicated herself to investigate chromatin structure and nuclear architecture of the murine immunoglobulin heavy chain (IgH) gene using 3D-multicolor FISH technique.

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Clinical & Cellular ImmunologyQualitative study of knowledge and attitudes to bio banking among lay persons in NigeriaMichael A Igbe and Clement A AdebamowoFederal Ministry of Health, Nigeria

Background: Interest in biobanking for collection of specimens for non-communicable diseases research has grown in recent times. This paper explores the perspectives of Nigerians on donation of specimen for the biobanking research.

Methods: We conducted 16 Focus Group Discussions (FGD) with individuals from different ethnic, age and socio-economic groups in Kano (North), Enugu (Southeast), Oyo States (Southwest) and Abuja, the Federal Capital Territory (Central) of Nigeria. We used topic guides and prompt statements to explore the knowledge and understanding of interviewees to general issues about biobanking of biospecimens, their use and specifically about role of biobanking in non-communicable diseases research.

Results: A total of 123 individuals participated in 16 focus group discussions in 2011. Our participants had limited knowledge of the concept of biobanking but accepted it once they were educated about it and saw it as a worthwhile venture. Half of our study participants supported use of broad consent, a quarter supported restricted consent while the remaining quarter were in favour of tiered consent. Most discussants support shipment of their samples to other countries for further research, but they prefer those collaborations to be done only with competent, ethical researchers and they would like to receive feedback about such projects. The majority preferred health care as a benefit from participation, particularly for any unexpected condition that may be discovered during the course of the research instead of financial compensation. Participants emphasized the need to ensure that donated samples were not used for research that contradicts their religious beliefs.

Conclusions: Our study demonstrates that our participants accepted biobanking once they understand it but there were different attitudes to elements of biobanking such as type of consent. Our study highlights the need to carefully document population attitudes to elements of modern scientific research and the consenting process.

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Clinical & Cellular ImmunologyAssociation of interleukin 1 β polymorphism with mRNA expression and risk of non small cell lung cancer Imtiyaz A Bhat1, Niyaz A Naykoo1, Iqbal Qasim1, Aasif Ahmad Mir1, Qaiser Yousuf1, Farooq A Ganie1, Roohi Rasool1, S A Aziz1, M A Siddiqi2 and Zafar A Shah1

1Sher-I-Kashmir Institute of Medical Sciences, India2Transworld Muslim University Srinagar Jammu & Kashmir, India

Introduction: Interleukin-1beta (IL- 1β), a key proinflammatory cytokine encoded by the interleukin 1 beta gene, has been associated with chronic inflammation and plays an important role in lung inflammatory diseases including lung cancer. Elevated levels of Interleukin 1 proteins, in particular interleukin 1 beta greatly enhance the intensity of the inflammatory response.

Aim: To study the role of interleukin 1 beta -31 C>T and -511 T>C polymorphism in the pathogenesis of non small cell lung cancer (NSCLC).

Materials and Methods: 190 non small cell lung cancer patients and 200 healthy age, sex, smoking and dwelling matched controls were used for polymorphic analysis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by sequencing. Normal tissues of 48 histopathologically confirmed non small cell lung cancer patients were taken for mRNA expression analysis. Quantitation of interleukin 1 beta was carried out by quantitative real time PCR.

Result: The T/T genotype of Interleukin 1 beta-31 gene was significantly associated with increased risk of NSCLC [(P=0.001, OR-2.8 (95%CI 1.52-5.26)]. The interleukin І beta −511 T>C does not show any difference between the non small cell lung cancer and control group (P=0.3, OR-0.72 (95%CI 0.41-1.28). Quantitative analysis of mRNA showed significant association with interleukin 1 beta T allele as compared to the interleukin 1 beta -31C allele (p=0.006).

Conclusion: We conclude that lung cancer risk genotype interleukin 1 beta -31TT results in increased expression of Interleukin 1beta mRNA in lung cancer patients. Our data suggest that this genotype (IL1β -31TT) in the interleukin 1 beta regulatory region provide a microenvironment with elevated inflammatory stimuli and thus increasing the risk for lung cancer.

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Clinical & Cellular ImmunologyGut-liver cross talk: Role of toll-like receptors in liver fibrogenesis and development of hepatocellular carcinoma Mostafa El AwadyNational Research Center, Egypt

The increase in intestinal barrier permeability due to bacterial over growth, viral infection or heavy fat or alcohol intake is associated with excessive release of several pathogen associated molecular patterns into the portal circulation such as LPS,

FFA or viral CpG containing nucleic acids. The subsequent binding of these factors to toll like receptors in the liver activates downstream intracellular signaling pathways resulting in innate immunological, molecular and pathological responses in liver cells ending with fibrogenesis, and hepatocarcinogenesis. We examined the expression profiling of 168 genes related to fibrogenesis and IFN induced genes in a hundred patients chronically infected with HCV type 4. Those patients suffer different stages of liver fibrosis (F0-F4) as well as HCC. The transcriptome data were compared with twenty healthy subjects proven to have normal liver functions and undetectable viral hepatitis markers. The studied array included TLR, IFN stimulated genes, inflammatory cytokines, chemokines and their receptors, adhesion molecules, extracellular matrix proteins, Metalloproteinases, growth factors and signal transduction mediators. Based on the initial explorative study few genes were selected for validation based on fold changes, statistical significance, their relation to chronic liver disease and novelty. The validation included analyses of expression profiles at both RNA and protein levels. Members of innate immune responses and their transcription factors such as TLR and NFkB were clearly up regulated in fibrosis. Chemokines and their receptors involved in hepatic stellate cell activation were also up regulated in fibrosis. The role of SMAD genes in directing the dialogue between TGFB and TLR signaling has been confirmed. The roles of matrix Metalloproteinases and their tissue inhibitors in fibrogenesis as well as the roles of inflammatory cytokines IL1 A & B and TNFα in inflammation of Kupffer cells were confirmed in the current profiling. Besides, 11 genes whose roles are not directly related to TLR signaling were found differentially regulated in fibrosis. THBS1, TGIF1, STAT1, STAT6, SERPINH1 were up regulated. While IL5, ITGB3, ITGB1, ITGA2, ITGAV and SERPINA1 were down regulated. The current study provides gene signatures associated with liver fibrosis and HCC as well as transcriptome data on mechanisms of HCV related liver disease.

BiographyMostafa El Awady is a Professor of Molecular Genetics and Biochemistry at the National Research Center, Cairo, Egypt. He obtained his PhD in Biochemistry in 1981 and received several Postdoctoral fellowships in South Western Med. School Dallas TX, Liver Research Center, Albert Einstein College of Medicine NY and University of North Carolina Chapel Hill (1981-1993). He was the Dean of Genetic Engineering Research Institute Mubarak Science Park Alexandria, President of the Division of Genetic Engineering at NRC. He has been granted several state awards for his accomplishments in science. He has published over 100 articles in peer reviewed journals in molecular biology of genetics and viral diseases. His current interests include development of prophylactic vaccine against HCV and exploration of novel therapeutic targets and prognostics of chronic liver disease.

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Clinical & Cellular ImmunologyCytokines profile and the phenotyping of lymphocyte subsets in chronic pain patients Durval Campos Kraychete, Marcia de Miguel and Roberto José Meyer NascimentoBahia Federal University, Brazil

The relationship between chronic pain and immune system is not yet understood. Besides that, the studies conducted in humans are controversial and bring limited data.

Objective: This research aims to study the immunological profile of patients with chronic pain at Pain Clinic of the Bahia Federal University who had a poor response to medical treatment and waiting for another therapeutic possibility.

Methods: An observational study of the epidemiological survey type was conducted comparing the immunological profile of 20 patients with chronic pain (neuropathic and fibromyalgia) and 10 healthy individuals. Social-demographic characteristics of the groups were evaluated, and the phenotyping of lymphocyte subsets and serum levels of inflammatory cytokines IL-1β, IL-4, IL-6, IL-10, IFN-γ and TNF-α in peripheral blood.

Results: Patients with chronic pain had higher mean age, predominantly women and individuals away from work. Regarding cytokines, high levels of IFN-γ, IL- 1β, IL-4 and TNF-α besides reduced levels of IL-6 and IL-10 were found in the group with chronic pain. As for the phenotyping of lymphocytes, were found reduced CD8 and elevated NK cells and B lymphocytes in the group with chronic pain. There was a slight elevation in CD4 count and elevated CD4/CD8 ratio in the group with chronic pain.

Conclusion: Despite the small groups and the non-randomic selected size of the study, it was possible to observe differences between the immunological profiles of patients with chronic pain compared to subjects without pain, as described in the literature.

BiographyDurval Campos Kraychete Graduated in Medicine from Bahia Federal University (1986), did his Master’s in Anesthesiology Department, São Paulo Federal University (1993) and PhD at Clinical Medicine Bahia Federal University (2004). He has been coordinating the Pain Clinic since 1996, focusing on clinical research. He has published papers in national journals and has been serving as an Editorial Board Member of Brazilian Journal of Anesthesiology; Brazilian Journal of Rheumatology, and Revista Dor-Pesquisaclínica e terapêutica (Official Journal of the Brazilian Pain Society). Currently, he is the Vice-President of Brazilian Pain Society.

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Clinical & Cellular ImmunologyFluorescent studies of lymphocytes subpopulations in colorectal cancer patientsInta Kalnina, Elena Kirilova, Tija Zvagule, Georgiy Kirilov and Galyna GorbenkoDaugavpils University, Latvia

The novel fluorescent probe ABM (derivative of benzanthrone) was used to characterize lymphocyte membranes from colorectal cancer patients at stages II-IV and advanced cancer. The aim of studies was to evaluate the potential utility

of measures of ABM fluorescence parameters as a standard tool in the analysis of organism immune status and for a clinical interpretation of lymphocytes functional activity in patients. ABM spectral parameters in lymphocytes reflect interrelated properties of the cells: 1) outer membrane physicochemical state, 2) membrane microviscosity, 3) proliferative activity, 4) lipid metabolism, and/or 5) phenotypical profile. Specific relatioship was obtained between ABM fluorescence in lymphocytes and characteristics of cells: Anisotropy index, binding constant, binding sites number etc. The lymphocyte distribution among the subsets also differed. Interestingly, the ABM fluorescence intensity in the cell suspension correlate with select immunological parameters (CD4+, CD8+, ratioCD4+/CD8+, level of immunoglobulines IgA, IgG, IgM etc.). Decrease in the CD4+/CD8+ ratio mainly depend on an increase in the T-suppressor cells in patients without metastases, whereas it is due to a decrease in the T-helper cell in most patients with metastatic disease. Surgical treatment affects immunological parameters and apperead to elevate lymphocytes functional activity. Immunosuppression increased gradually with progress of of cancer. In advanced cancer, in contrast to other groups, the absolute number of lymphocytes had direct (not inverse) correlation with ABM fluorescence intensity. The higher level of lymphocytes number, T-cell proliferative activity have a beneficial effect on overall survival. Significant note: Excellent agreement is obtained between changes in spectral characteristics and both clinical and pathological estimates of disease severity. Measures of ABM fluorescence intensity values in lymphocytes (as reflection of their functional activity might be a useful tool in the evoluation of the immune status of patients in clinics. including prognosis, prediction of therapeutic efficacy, treatment outcomes. The fluorescence-based method is less expensive, not very time-consuming, technically simple, 100 times more sensitive than standart ones.

BiographyInta Kalnina, biochemist, Dr. Med. graduated from Latvia State University 1972. His interests include fluorescent based methods for development of structural and functional properties of membranes and plasma albumin in different pathologies, immune status of patients. He is publications Board member of J. India Research Publications. He has ~220 publications, participated in ~100 conferences and congresses. He is coauthor of 5 patents.

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Clinical & Cellular ImmunologyVitamin D antibodies in systemic sclerosis patients: Antibodies presence and clinical and laboratory correlationsNarin-Nard Carmel1,2, Alexey Lavrov2, Pnina Rotman2 and Yair Levy2

1Tel Aviv University, Israel2Meir Medical Center, Israel

Background: Vitamin D is a pivotal factor not only in disorders that involve calcium metabolism, such as osteoporosis and osteomalacia as well as an immunomodulatory effect as noted in several autoimmune conditions in diseases. Very low levels of vitamin D were also demonstrated in systemic sclerosis (SSC) patients. Patients with vitamin D deficiency showed longer and more severe disease. Furthermore, an inverse relationship was found between skin involvement and vitamin D serum concentrations. Associations were found Between Systemic Sclerosis pattern of disease and Scleroderma-Specific Autoantibodies. Novel research demonstrated the presence and importance of anti-vitamin D antibodies in SLE; this motivated our research team to seek for similar antibodies among scleroderma patients.

Materials and Methods: Our study population comprised of 55 scleroderma patients and 41 donors from our hospital staff served as the control group. Levels of IgG & IgM autoantibodies against Vitamin D2 and D3 were compared between scleroderma patients & controls. Furthermore, scleroderma patients were assessed for disease severity and auto-antibodies profile was taken.

Results: We found significant differences in the levels of Anti vit D2 antibodies between scleroderma & controls. IgM hydroxyl vitamin D levels were higher among scleroderma group as compared to controls (0.48±0.22 vs. 0.39±0.33, p=0.013, Mann Whitney test). However, we found no significant differences in the levels of Anti DiHydroxyl vitamin D antibodies between scleroderma & control. Furthermore, no correlation was found to other auto-antibodies or disease severity or sub-organ damage.

Conclusions: To our knowledge, this is the first time these novel anti-vitamin D antibodies are studied in scleroderma patients. Furthermore, it is the first time a correlation to hydroxy vitamin subgroup is identified. Further research and evaluation regarding the role, pathophysiological significance and therapeutic potential is required.

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Clinical & Cellular ImmunologyTherapeutic potential of ixmyelocel-T, an expanded autologous multicellular therapy, derived from bone marrow, for treatment of ischemic cardiovascular diseasesRoss Tubo and Kelly LedfordAastrom Biosciences Inc., USA

A variety of bone marrow derived cell therapies have been utilized to treat ischemic tissue damage and promote more balanced tissue repair under inflammatory conditions. Ixmyelocel-T, is an autologous, culture expanded, bone marrow-

derived multicellular therapy which contains MSCs and M2-like macrophages, as well as many of the CD45+ cells found in the bone marrow. MSC and M2-macrophages have been shown to exhibit properties useful for angiogenesis and modulation of tissue repair in response to ischemia. In this study, it was used a rat model of hind limb ischemia to determine the effects of ixmyelocel-T on restoration of blood flow to the ischemic limb. Further, the mechanism(s) for action of ixmyelocel-T in angiogenesis on endothelial cells, by co-culturing with human umbilical vein endothelial cells (HUVEC) in non-contacting Transwell® inserts, in vitro was explored. Co-culture of HUVECs with ixmyelocel-T resulted secretion of a variety of pro-angiogenic factors, eNOS expression and nitric oxide (NO) production, and enhanced migration, proliferation, and branch formation. In TNFα-stimulated HUVECs, ixmyelocel-T co-culture decreased apoptosis and reactive oxygen species generation, increased super oxide dismutase activity, and decreased NFκB activation. Treatment with ixmyelocel-T in a rat model of hind limb ischemia resulted in significantly increased blood flow perfusion and capillary density, gene expression and plasma levels of the anti-inflammatory cytokine IL-10, plasma nitrates, plasma PDGF-BB, VEGF expression, and significantly decreased plasma TBARS. Fluorescently labeled ixmyelocel-T was detected in the ischemic limb up to six weeks after transplantation. Transplanted ixmyelocel-T did not co-localized with CD31+ endothelial cells, but did co-localize with the M2 macrophage marker CD206 in the ischemic tissue suggesting that ixmyelocel-T promotes angiogenesis in a paracrine manner. This data suggests that ixmyelocel-T could be useful for promoting of angiogenesis and tissue repair in ischemic tissue injury and disease.

BiographyRoss Tubo, PhD, is Chief Scientific Officer, Aastrom Biosciences. He has more than twenty years of experience in cell therapy, regenerative medicine, and stem cell biology. He was a pioneer in the research, development, and commercialization of the first autologous cell therapy for articular cartilage repair, known as Carticel. As Vice President of Stem Cell and Chemokine Biology for Genzyme Corporation, he developed a world-class research organization designed to understand the underlying cell and molecular mechanism(s) of action of mesenchymal stem cells (MSCs) in autoimmune disease and cancer. These efforts led to the identification of specific therapeutic targets for treatment of these diseases. He holds a PhD in Cell and Molecular Biology from State University of New York at Buffalo.

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Clinical & Cellular ImmunologyEnhanced functional maturation of dendritic cells by exponential wave electric pulse application Sunil K JoshiOld Dominion University, USA

The development of specific adaptive immunity requires pathogen/tumor-specific antigen presentation to T cells by professional antigen-presenting cells (APCs). The most specialized APCs, known as Dendritic Cells (DCs), are critical for

priming antigen-specific T cell responses. The antigen-presentation function of DCs strictly depends on functional maturation characterized by (a) high expression of MHC I/II and co-stimulatory molecules (CD40, CD80, and CD86) and (b) efficient antigen processing and presentation. The pathogens that cause Malaria, Tuberculosis, Flu, AIDS, cancer, and many other diseases suppress immune responses by inhibiting DC maturation leading to inefficient antigen processing, presentation, and dysfunctional CD8+ T cell priming. In the past several decades, many strategies have been developed to functionally mature and activate DCs in vivo. These strategies have included the use of adjuvants (substances that enhance immune response to antigens) such as Toll-like receptor (TLR) ligands (Lipid A, Poly [I:C], CpG etc.), cytokines, and immune-modulators. However, these approaches have had limited efficacy and have posed a risk of adverse immune responses. In this study we tested an innovative approach to enhance DC maturation and antigen-presenting function without the use of cytokines, LPS or TLR ligands. The central hypothesis of this study is that “Defined Exponential Pulse Electric Fields” (pEFs) delivered to skin and superficial lymph nodes can significantly enhance DC maturation and antigen-presenting function without inducing cell death, thus, triggering antigen-specific T cell priming and activation". The experimental data supporting this hypothesis will be discussed.

BiographySunil Joshi is an outstanding young scientist, with an excellent education and background, and includes the unusual albeit valuable combination of a veterinary degree as well as a PhD degree. He is an established cellular immunologist and employing novel applications of bioelectrics in modulating innate immunity. Recently, he has established his own laboratory at the Frank Reidy Research Center for Bioelectrics which is newly created multidisciplinary advanced biomedical engineering center at Old Dominion University. His lab is also working to define molecular changes in the plasma membrane in response to various electrical functional waves. He employs multi-dimensional approaches to address key issues in vaccinology particularly vaccination against Malaria and Tuberculosis. He has been awarded Bill & Melinda Gates Foundation Grand Challenge Exploration award to work on use of Bioelectrics in vaccination.

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Clinical & Cellular ImmunologySeptember 29-October 01, 2014 DoubleTree by Hilton Baltimore-BWI Airport, USA

216th OMICS Group Conference

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Immunology Summit-2014

Accepted Abstracts(Posters)

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Clinical & Cellular ImmunologyA national survey of ankylosing spondylitis in IranAhmadreza JamshidiShariati Hospital, Iran

Objective: Ankylosing spondylitis (AS) is a chronic systemic inflammatory disease with variable clinical expression. Ethnic, racial, geographical and gender-related factors have been associated with disease incidence and clinical manifestations. The study intended to describe clinical characteristics and assess disease severity, gender influences, and treatment status in the Iranian population.

Subjects and methods: 320 patients diagnosed with primary AS throughout Iran were evaluated for baseline characteristics, clinical manifestations, HLA-B27 status, disease severity, functional indices, quality of life, and treatment status.

Results: A gender ratio of 3.8:1, average age onset of 27±7.3, and a mean diagnostic delay of 8 years was observed. The incidence of juvenile-onset cases was 11%. Positive family history was higher than that observed in most other countries. Both gender groups were similar in terms of ethnicity, positive family history, juvenile-onset AS, and diagnostic delay. Enthesitis was present in more than two-thirds of patients with females demonstrating a higher rate (p<0.05). Uveitis was the leading extra-articular manifestation. Overall HLA-B27 positivity was 73% and four HLA-B27 subtypes were found. HLA-B27 positivity was higher among males (78.3% vs. 55.2%; p<0.001). Disease activity was high and the functional status was poor as indicated by mean Bath AS disease activity, functional, and metrology indices. Female disease was at least as severe as male disease and more severe impairment was present in some aspects. Extra-articular manifestations and treatments modalities presented similar frequencies among genders. Biological medication was utilized less frequently than disease modifying anti-rheumatic drugs and corticosteroids. Quality of life was considerably impaired.

Conclusion: The study exhibits a broad characterization of Iranian AS patients providing better understanding of the disease status and allowing for healthcare development and advancement of earlier diagnostic and treatment strategies. Early detection and specialized care would be of great practical importance.

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Silencing of microRNA-21 confers silica-induced cytotoxicity through inhibition of the PI3K/AKT pathway and enhancing autophagy in RAW264.7 cellsChunhui NiNanjing Medical University, China

Silica is a core part of the cause of Coal Workers’ Pneumoconiosis (CWP), which is a chronic occupational lung disease characterized by irreversible pulmonary fibrosis. However, the underlying immunologic mechanisms of CWP are poorly

understood. Macrophages constitute the first line of cellular defense against pathogens. Autophagy is a fundamental cellular homeostasis pathway that operates with the intracellular degradation/recycling system. In response to invading pathogens, induction of the autophagic process in macrophages constitutes a crucial mechanism in innate immunity. Here, it is demonstrated that microRNA 21 (miR-21), an elevated miRNA in silica-induced pulmonary fibrosis, is one of the main players in silica- cytotoxicity. Silica-cytotoxicity in RAW264.7 cells measured by cytotoxic cell survival assays was closely associated with the expression of miR-21. P62, indicator for autophagic degradation, increased in a dose/time-dependent way in cultured RAW264.7 cells due to silica exposure. Blocking miR-21 with anti-miR-21 led to a decreased expression of p62 in 200 µg/ml silica-treated RAW264.7 cells for 12 hours and the suppression of phosphor-Akt (ser473) was as well observed. In a cell cycle analysis, a significant increased arrest in the G1/G0 phase by anti-miR-21 was found at 48 hours after silica-treatment. Notably, obtained results demonstrated that anti-miR-21 raised factors involved in autophagosome formation. Moreover, augmented autophagy by anti-miR-21 resulted in an increase in the apoptotic population after silica exposure. The findings show that miR-21 is a crucial molecule for alleviating silica-induced cell death in RAW264.7 cells by inhibiting the PI3K/AKT pathway and enhancing autophagy, providing a new insight into the mechanisms of CWP.

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Clinical & Cellular ImmunologyImmunodiagnosis of Haemonchus contortus infection in sheep by indirect enzyme linked immunosorbent assay (ELISA)Bashir A Lone, M Z Chishti, Fayaz Ahmad, Suhaib A Bandh, Javaid A Parray and Abida KhanUniversity of Kashmir, India

Indirect plate enzyme-linked immunosorbent assay was standardized and evaluated for its effectiveness in immunodiagnosis of haemonchosis in experimental and clinical cases in sheep by using somatic whole adult antigen of H. contortus. Plate

ELISA was standardized using 5 μg/well antigen concentration with 1:100 and 1:1000 of sera and conjugate dilution. Indirect plate ELISA was able to demonstrate the antibody titer at different weeks post infection in experimental sheep. A comparison of plate ELISA on suspected field sera and faecal sample examination by floatation method revealed that 74 samples were found to be positive by ELISA but only ten by faecal examination. Sensitivity of plate ELISA was found to be 80.0%, whereas specificity was 21.42% indicating that this test is quite sensitive for clinical cases; an early diagnosis, however, lacks specificity.

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Listeria moncytogenes induces inflammasome activation of macrophages regulating trophoblast invasion and immune functionFei Zhong1, Wenyan Li1, 5, Yumei Chang2, Zhenyu Zhong3, Xiujin Li4, Liyue Wang1, Hongyu Lin4, Jiexia Wen1, Jianlou Zhang1, Yonghong Zhang1 and Heling Huang2 1Agricultural University of Hebei, China2252 Hospital of Chinese PLA, China3University of California San Diego, USA4Yanshan University, China5Hebei University, China

To investigate the molecular mechanism of inflammasome activation in Listeria monocytogenes (LM)-infected macrophages and the regulatory effects of secretory products of LM-infected macrophage on trophoblast invasion and

immune functions, the mouse B6 macrophages and its derived NLRP3-/- B6 macrophages were infected with wild-type LM and Listeriolysin O-/- LM (LLO-/- LM), respectively. IL-1β concentration in the medium, caspase 1 and pro-IL-1β and TNFα mRNA in the B6 macrophages were detected by ELISA, western blot and RT-PCR, respectively. Apoptosis was detected by Annexin V/PI double staining. Then the mouse SM9-1 trophoblasts were treated by the secretory products of LM-infected macrophages. The trophoblast invasion and immune functions were detected in vitro by cell invasion assay and measurements of matrix Metalloproteinases (MMPs), monocyte chemotactic protein-1 (MCP-1) and TNFα and IL-6 by RT-PCR. Results showed that the wild-type, not LLO-deficient LM, could significantly induce the IL-1β release, caspase 1 maturation and pyroptosis of the B6 cells, but not NLRP3-/- B6 cells, indicating that LM, not LLO-/- LM, could activate NLRP3 inflammasome of mouse macrophages by its LLO. Regarding the regulation of LM-infected macrophage on trophoblasts, our results showed that LM-infected macrophage products, as well as recombinant IL-1β, could significantly facilitate trophoblast invasion, increase MMP-2, MMP-9 MMP-26 expressions, and up-regulate pro-inflammatory cytokine TNFα and IL-6 expressions and down-regulate MCP-1 expression of mouse trophoblasts in vitro, which indicate that the macrophage secretory products, mainly IL-1β can facilitate trophoblast invasion by increasing MMP expressions, and regulate trophoblast immune functions, including modulating monocyte/macrophage recruitments by inhibiting MCP-1 expression and shaping the inflammatory reaction by improving pro-inflammatory cytokine production. Above results indicate that LM can activate macrophage NLRP3 inflammasome and promote cell apoptosis by its LLO, the inflammasome activation pathway-mediated IL-1β release from LM-infected macrophages plays a crucial role in regulating trophoblast invasion and immune functions during LM infection.

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Clinical & Cellular ImmunologyAssessment of factors which leads to irrational use of drugs among outpatients in Gondar town, North-Western EthiopiaBefikadu Taye TadesseUniversity of Gondar, Ethiopia

Background: It is now evident that both developed and developing countries are experiencing many aspects of inappropriate use of drug in their health care facilities. There is also concern regarding the irrational use of drugs in Gondar town. These studies aimed to describe factors responsible for irrational drug use by outpatients, which consists of all categories of ages.

Objectives: To assess factors that leads to irrational use of drugs among outpatients in Gondar town.

Methods: A study was done in Gondar town, by administering a questionnaire for all volunteer outpatients to participate in the study. The questionnaire was structured based on the pretest, which was done. This study was patients based cross-sectional study. The study populations were outpatients who were selected by simple random sampling methods. Total samples of 385 outpatients were involved.

Result and discussion: Concerning the factors which lead to irrational use of drug among the respondents (outpatients), 216 (56.10%) respondents have given multiple responses. The major reasons for irrational drug use in majority of the society, which were responded by respondents includes; 287 (51.25%) rapid relief from illness, 162 (28.93%) unusual route of administration of drug, 108 (19.28%) unfavorable work time and work status to take the drug.

Conclusion: Irrational drug use practice by outpatient is not only risky on patient but also the state itself, so a great emphasis should be given on awareness creation to the community on RDU by improving the knowledge and attitudes about drugs in general which could be done by mass media or drug information program for the community.

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Protective effect of Eucalyptus oil in COPD-like rat model induced by LPS and Klebsilla pneumoniaChunzhen Zhao1, Lin Wang1, Wanzhong Li1 and Fadi Tang2

1Weifang Medical University, China2Zhejiang University, China

Eucalyptus oil, an essential oil isolated from Eucalyptus leaves, was examined for its possible influence on LPS and Klebsilla pneumonia induced COPD in rats. The COPD model was induced by instilling intratracheally with LPS and Klebsiella

pneumonia (K.P, density, ≥6×108 CFU/mL). The test compound, Eucalyptus oil (30, 100 and 300 mg/kg), Prednisone Acetate (10 mg/kg) or vehicle was instilled intragastrically after three weeks exposure of LPS and K.P and lasted for 4 weeks. Eucalyptus oil significantly reduced amounts of inflammatory cells in bronchoalveolar lavage fluid (BALF) and blood, and significantly decreased bronchiolitis, alveolar destruction, emphysematous changes and thickness of bronchioles. It also significantly reduced the increased AB-PAS-positive goblet cells in bronchioles. Prednisone acetate also significantly attenuated pulmonary inflammation and airway mucus hypersecretion, but no significant difference was found in emphysema. Eucalyptus oil exerts its antioxidant activities and decreased the elevated MDA levels and increased the level of SOD in lung tissues. These findings suggest Eucalyptus oil as an active controller of airway inflammatory responses and oxidant stress in COPD rats. Further studies are in progress in order to elucidate the mechanisms. Our results provide evidence that Eucalyptus oil might have its potential to be a proper candidate drug in the treatment of COPD.

BiographyChunzhen Zhao, PhD is a Lecturer, Department of Pharmacology, 7166 Baotong Street, Weifang Medical University, Weifang 261053, China. He graduated from Zhejiang University, China and earned a Doctor’s Degree in Pharmacology from college of Medicine in 2011. He is member of Chinese Pharmacological Society, Member of Pharmacological Society of Shandong Province.

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Clinical & Cellular ImmunologyThe preparation of recombinant chicken interleukin-7 and its antiviral activity against chicken IBDV infectionFei Zhong1, Liyue Wang1, Hongyu Lin2, Wenyan Li1, Jiexia Wen , Yonghong Zhang1 and Xiujin Li21Agricultural University of Hebei, China2Yanshan University, China

Interleukin-7 (IL-7), produced mainly by thymic stromal cells, is an important cytokine playing a critical role in host immune system, especially in B cell growth and pre-B cell survival, differentiation and proliferation. IL-7 also stimulates pre-bone

marrow cell differentiation into dendritic cells (DCs) and promotes DC maturation. Meanwhile, Il-7 triggers CD4+/CD8+T cell proliferation and maintains normal immune function. As IL-7 possesses the character of promoting the immune functionit was extensively used as biological adjuvant to enhance the antigenicity of many vaccines including DNA vaccines. Il-7 has also been used to stimulate CD4+/CD8+T cell proliferation for the cancer patients after chemotherapy and the AIDS patients. Chicken infectious bursal disease (IBD), caused by IBD virus (IBDV), is an immunosuppressive viral disease, causing huge economic loss in poultry industry. Since IBDV attacked chicken bursal, which is a central immune organ for chicken B cell development and maturation, the B cells and antibodies against IBDV in the peripheral blood of IBDV-infected chicken were severely depleted during the late period of infection, leading to B cell-associated humoral immune suppression. As mentioned above, IL-7 has the ability to stimulate B cell differentiation and proliferation and restore humoral immune activity, whether IL-7 can revive the suppressed immune system of the IBDV-infected chicken, especially B cell-associated humoral immune system is still unknown. In this study, therefore, we prepared recombinant chicken IL-7 and investigated its therapeutic effects on IBD prevention and treatment. First, we amplify IL-7 cDNA by RT-PCR from chicken spleen, constructed His-tagged IL-7 eukaryotic expression vector using pcDNA3.1A plasmid. The recombinant chicken IL-7 was expressed in HEK293T cells, isolated by Ni-NTA Agarose affinity chromatography and identified for its biological activity by cell proliferation assay using 2E8 mouse B cell line. For therapeutic evaluation on IL-7 in chicken IBD, 4-week old chickens were treated with the recombinant IL-7 before or after IBDV challenge, the B cell number and IBDV antibody titers in the blood were analyzed by flow cytometry and ELISA, respectively. The histopathologic changes of IBDV-infected chicken tissues were observed as well. The results showed that the amplified chicken IL-7 gene sequence and its expression vector structure were correct based on the sequencing result, the IL-7 could stimulate 2E8 mouse B cell proliferation and maturation, suggesting the prepared IL-7 possesses the biological activity. After purification with Ni-NTA Agarose affinity chromatography, its purity reached more than 95%. The chicken experiment showed that the B cell numbers in the bursal significantly increased in IL-7-treated IBDV-infected chickens compared with control group (IBDV-infected chickens untreated with IL-7). The titers of anti-IBDV antibodies in the IL-7-treated IBDV-infected chicken were also significantly higher than that of IL-7-untreated chicken, indicating that chicken IL-7 can significantly stimulate humoral immune response to IBDV in IBDV-infected chicken, therefore the recombinant chicken IL-7 might have the potential therapeutic value to be further developed for IBD prevention and treatment.

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Antigen conjugated PMIDA coated cobalt oxide nanoparticles highly accelerate anticancer immune responseSomenath RoyVidyasagar University, India

The viability of using N-phosphonomethyliminodiacetic acid (PMIDA) modified cobalt oxide nanoparticles (CoO NPs) as an antigen carrier was studied. The whole Daltons lymphoma (DL) cell lysate conjugated PMIDA-CoO NPs (CPCNs) were

well characterized by DLS, TEM and SEM study. The CPCNs successfully activated antigen presenting cells (APCs) which was evident by the increasing levels of serum IFN-γ and TNF-α. Immunization of mice with the CPCNs induced IgG responses against the peptide, and increased the antibody dependent cellular cytotoxicity (ADCC). CPCNs also enhance the anti tumor CD4+T cell response in mouse. This result demonstrates that CPCNs are able to serve as antigen carriers to induce humoral immune responses against tumor antigens.

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Clinical & Cellular ImmunologyChromoendoscopy associated with endoscopic laryngeal surgery for treatment of recurrent respiratory papillomatosis (phase II)Daniel G Rey Caro, Enrique P Rey Caro and Enrique A Rey CaroChutro Clinic in Cordoba, Argentina

Introduction: Chromoendoscopy is an endoscopic technique which uses a contrast stain to paint the aerodigestive tract mucosal lining followed by an optical assessment to highlighting any epithelial abnormalities. Detailed and high-definition magnified views achieved with the aid of rigid endoscopes can often allow for identification of the tissue type or pathology based upon the pattern uncovered. According to the literature we reviewed, we may have been the first ones to use indigo carminein the field of otolaryngology. Tiny lesions that usually go overlooked with conventional microlaryngoscopy become visible upon the instillation of indigo carmine and further decreasing the chances of an early lesion postoperative recurrence. Chromoendoscopy, in recurrent respiratory papillomatosis (RRP), helps identify unsuspected intraoperative lesions by clearly enhancing the view of their boundaries and surface type. It is also suitable to assess the presence of residual lesions, if any, after their surgical removal.

Objectives: To demonstrate the usefulness of chromoendoscopy in RRP in laryngotracheal surgery.

Material and Methods: Indigo carmine associated with endoscopic laryngeal surgery was used. Before staining, the mucosa may need to be treated with a mucolytic agent to get rid of excess mucus to boost staining. Rigid suspension laryngoscopes of different proximal and distal diameters were used with chromoendoscopy. Patients underwent chromoendoscopy associated with endoscopic laryngeal surgery under general anesthesia in the O.R.

Results: In the second phase of research work, diagnostic technique was applied to eighteen patients with recurrent laryngeal papillomatosis and two patients with suspected carcinoma of the larynx. It was able to optimize the intraoperative diagnosis and reduce the likelihood of the relapse risk in all patients.

Conclusion: Chromoendoscopy associated with endoscopic laryngeal surgery is an excellent low-cost intraoperative diagnostic method for the treatment of invasive diseases of the larynx such as laryngeal papillomatosis.

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Mucosal IL-6 mRNA expression level and Helicobacter pylori infection in Iranian adult patients with chronic gastritisFatemeh Azadegan-Dehkordi1, Nader Bagheri2, Abbas Mirshafiey2, Mahmoud Rafieian-Kopaei1 and Hedayatollah Shirzad1

1Shahrekord University of Medical Sciences, Iran2Tehran University of Medical Sciences, Iran

Objective: Helicobacter pylori (H. pylori) infection is associated with gastritis and marked infiltration of the gastric mucosa by several cytokines secreting inflammatory cells that contribute to sustain and expand the local inflammation. In this study, the author sought to examine IL-6 expression in the H. pylori-infected and uninfected gastric mucosa and to elucidate the implication in the pathogenesis of H. pylori-associated gastritis in human.

Methods: Using endoscopic biopsies taken from the gastric antrum of 58 subjects infected with H. pylori and 44 uninfected subjects, mucosal IL-6 mRNA levels was measured by real-time PCR. Presence of vacA and cagA virulence factors was evaluated using PCR.

Results: The IL-6 mRNA expression levels were significantly more elevated in H. pylori-positive patients than uninfected individuals. There was a correlation between IL-6 expression level and the degree of chronic gastritis.

Conclusion: The enhanced induction of IL-6 may be involved in the pathogenesis of H. pylori-associated [email protected]

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Clinical & Cellular ImmunologyImmune mechanism as the mechanism maintenance stability of both internal energy an organism as well as internal energy of cells of an organismMichail PonizovskiyKiev Regional p/n Hospital, Germany

Regulative mechanism of an organism was divided into three levels: highest level regulation, high level regulation and low level regulation which realize mechanism regulation of maintenance stability Internal Energy (stable temperature

36.6°C-36.9°C by which all enzymes operate etc.) and Internal Medium (stable concentration of substances in blood and neurolymph) as an organism as well as cells of an organism. Biochemical and biophysical Equilibrium Constants in three levels regulation support balance catabolic exoergonic processes and anabolic endoergonic processes, which determine intracellular and extracellular chemical potentials. The influences of cellular potentials on cellular walls form different cellular capacitors. Also the interactions between nuclear processes, due to nuclear capacitors, and mitochondrial processes, due to mitochondrion capacitors, determine stabile basophilic chemical potential in cytoplasm, i.e. stability cellular internal energy. Relative interactions between cellular capacitors of cells maintain common stability of internal energy both in cells and in an organism. The mechanism of mutual interactions between cellular capacitors of all cells and an organism promote remote defensive reactions across distance for immune responses on strange objects. Biophysical mechanism of immune cells remote reactions transit into contact biochemical immune reactions for decomposition of the strange object for maintenance stability internal energy and internal medium of an organism and as well as cells of an organism.

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Influence of bFGF, VEGF, PlGF, TGFβ on endothelial cells tube formation in presence of THP-1 cells Okorokova L S, Lvova T Yu, Stepanova O I, Beljakova K L, Selkov S A and Sokolov D ID.O.Otta Research Institute of Obstetrics and Gynecology, Russia

During angiogenesis, interaction of endothelial cells (EC) with cells of microenvironment, including monocytes/macrophages, and the extracellular matrix are controlled by cytokines. The aim of the research was investigation of

cytokine influence on capillary-like tube formation of endothelial cells EA.Hy926 in presence of THP-1 cells. For cell activation the following recombinant cytokines were used: bFGF - (BD, США) (1 ng/ml, 10 ng/ml, 20 ng/ml); VEGF - (BD, США) (1 ng/ml, 10 ng/ml, 100 ng/ml); PlGF - (BD, США) (1 ng/ml, 5 ng/ml, 20 ng/ml); TGFβ - (BD, США) (1 ng/ml, 5 ng/ml, 10 ng/ml). Endothelial cells were seeded on Matrigel-coated 24-well plates (BD, USA) at a density of 150000 cells/well, also were added cytokines and 2,5% fetal bovine serum. To part of wells THP-1 cells were added (250000 cells/well), to part of wells - the cultural medium without THP-1 cells. Capillary tube formation was assessed 24 hours later using microscope AxioObserver Z1. Stimulating effect of bFGF and VEGF on capillary-like tube formation in monoculture and co-culture system was shown, while the effects of cytokines were more pronounced in co-culture system. PlGF had no effect on EC tube formation in monoculture, but at high concentrations proangiogenic activity of PlGF in co-culture system was shown. TGFβ inhibited EC tube formation, while its anti-angiogenic potential was more pronounced in the co-culture system. Thus, cytokines of microenvironment could make a definite contribution to the interaction of EC and monocytes/macrophages during angiogenesis.

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Clinical & Cellular ImmunologyMechanism of cellular immune response to strange objectsPonizovskiy M RKiev Regional p/n Hospital, Germany

The interactions of cellular transport across cellular wall promote stability internal energy of a cell via creating extracellular chemical potential and intracellular chemical potential which induce different electrical charges on external and internal

cellular membranes of a cellular wall. Thus the formed different cellular capacitors into cellular wall are functioned. Just the mechanism of mutual interactions between cellular capacitors of all cells and an organism promote remote reactions across distance for immune responses on strange objects. Interactions between all cells of an organism occur due to remote reactions across distance as the results of cellular capacitors operations via production of resonance waves. Interactions between cellular capacitors of cells maintain common stability of internal energy both in cells and in an organism. Penetration of strange high-molecular substance into an organism creates local change of chemical potential and promotes remote reactions across distance of cellular capacitors via cellular waves on common molecular wave of strange high-molecular substance, due to the wave function of any molecule which is determined as the total wave functions of the nuclear orbitals, according to Schrödinger (wave) equation of linear combination of atomic orbitals (MO LCAO). The forming resonance waves cause attraction the immune cells to strange high-molecular substance and create the contact reaction of decomposing the high-molecular substance of the strange object, ruining it. Biophysical mechanism of immune cell’s remote reactions transit into contact biochemical immune reactions like, Phagocytosis, Autophagy etc.

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IFN-γ, IL-8, IL-10 Regulate surface molecules expression in natural killer cell lineLarisa Viazmina, O M Ovchinnikova, K N Furaeva, D I Sokolov, S A SelkovD. O. Ott Research Institute of Obstetrics and Gynecology, Russia

Natural Killer (NK) cells are dominant effectors of early host defense. Their activity depends on balance of signals from activation and inhibition receptors. Various soluble factors, including cytokines have an influence on phenotype of NK

cells.To investigate this effect, we used pro-inflammatory and anti-inflammatory cytokines. IFN-γ induces Class II major histocompatibility complex (MHC) molecule expression. IL-8 basic functions include chemotaxis, activation and the induction of neutrophil infiltration. IL-10 is an anti-inflammatory cytokine with multidirectional effects in immunoregulation and inflammation. Assay was performed using NK-92MI natural killer cell line. Cells were treated with 3 different doses of cytokines (IFN-γ, IL-8, IL-10, separately) for 24 hours. Surface molecules expression was assessed by flow cytometric analysis using fluorochrome-conjugated mABs and FACSC. IFN-γ significantly (p<0.05) upregulated expressions of KIR2DL1, KIR3DL1, CD11a, CD11b, CD18, CD29, CD44 (dose-dependent), CD47 (dose-dependent), CD184 (dose-dependent), CD49d and CD54. Cells stimulated byIL-8 showed significally higher expression level ofCD29. However, IL-8 downregulated KIR2DL3, KIR2DL4, KIR3DL1 (dose-dependent), KIR2DS4, CD11b (dose-dependent), CD18, CD49d (dose-dependent), CD44 and CD58. Significantly increased expression of CD47, CD58 (dose-dependent) and CD49 was discovered in cells treated with IL-10, but expression of KIR2DL1, KIR2DL3, CD29, CD44, and CD184 was significally decreased.

BiographyLarisa Viazmina is a five-year student of biotechnology faculty of Saint Petersburg Chemical Pharmaceutical Academy. She is laboratory research assistant in immunology department of D. O. Ott Research Institute of Obstetrics and Gynecology. Her research interests are related to reproductive immunology, mainly preeclampsia and NK cells.

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Clinical & Cellular ImmunologyNatural regulatory T cells in some parasitic diseasesNashwa K Abousamra1, Raida S Yahya1, Soha I Awad1, Hanan Azzam1, Gehan Atia1 and Hatim A El-Baz2

1Mansoura University, Egypt2King Abdulaziz University, KSA

Parasitic infection in human alimentary tract causes a significant change in immune system through its continuous antigens secretion. The aim of this study was to estimate the change in natural regulatory T cell population in peripheral blood of

patients infected with different types of alimentary tract parasites. Regulatory T cells (CD4+CD25+Foxp3+) were detected in eighty patients infected with intestinal parasites and forty healthy volunteers using flow cytometry technique. Statistical analysis showed a significant increase in regulatory T cell percentages in infected patients compared to healthy group (P<0.001). Patients infested with Giardia showed significantly higher CD4+CD25+Foxp3+ cell percentages than those infested with other parasites (P<0.001). Also, mixed infestation showed significantly higher CD4+CD25+Foxp3+ cell percentages than single infestation. In conclusion, natural regulatory T cell frequencies (CD4+CD25+Foxp3+) increase significantly in patients with parasitic diseases compared to healthy controls. The higher levels were associated with mixed infection compared to single infection and in older than younger patients.

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PD-L1 regulation by PspA antigen during S. pneumoniae infectionMohit Vashishta1, Naeem Khan2, Subhash Mehto1, Devinder Sehgal2 and K Natarajan1

1University of Delhi, India 2National Institute of Immunology, India

Pneumonia is a major cause of mortality in children under the age of five worldwide resulting in close to 20 percent of all deaths in this age group. Consequently, investigations into the host-pathogen interactions during S. pneumoniae

infection are keys to devising strategies towards the development of better vaccines and drugs. To that end in this study it was investigated the role of S. pneumoniae and its antigen pneumococcal surface protein A (PspA) in modulating the expression of the co-inhibitory molecule Programmed Death Ligand -1 (PD-L1) on bone marrow derived dendritic cells (DCs) and the subsequent effects of increased PD-L1 levels on immune responses. PD-L1 is a co-stimulatory molecule that largely induces suppressor responses and causes T cell inactivation and anergy. Obtained data indicates that stimulation of DCs with PspA increases the surface expression of PDL1 in a time and dose dependent manner. Characterization of intracellular signalling molecules indicate that PspA induced expression of PDL1 was dependent on MAPK pathway and routes of calcium influx. While calcium release from intracellular stores positively regulated PDL1 expression, calcium influx from external milieu negatively regulated PDL1 expression. Knockdown of intermediates in the TLR pathway showed that the expression of PDL1 was dependent on MyD88. Knockdown of PD-L1 promoted apoptosis of DCs and increased autophagic responses together with increase in the levels of pro-inflammatory cytokines like IL-12 and IL-6. These results indicate that increased expression of PD-L1 mediated by PspA could be an immune evasion strategy adopted by S. pneumoniae to establish a long term infection. Further characterization of the responses is underway.

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Clinical & Cellular ImmunologyTMP1 significantly suppresses mammary tumor metastasis via inhibition of granulocytic myeloid-derived suppressor cell differentiationNing-Sun Yang1, Sheng-Yen Lin1,2, Ying-Chu Wang1, Chun-Wen Lan1, Wen-Chin Yang1, Pei-Wen Hsiao1 and Wen-Chi Wei11Agricultural Biotechnology Research Center, Taiwan2National Defense Medical Center, Taiwan

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells including immature granulocytes, macrophages, and dendritic cells that expand extensively during cancer, inflammation and infection.

MDSCs are increasingly recognized as having a crucial role in protecting tumor cells from immune surveillance by suppressing anti-tumor immunity. This study aimed to investigate the immune-regulatory and antitumor activities of TMP1 on MDSC expansion and tumor metastasis. The results showed that TMP1 effectively suppressed mammary 4T1 tumor metastasis and increased mouse survival in a mammary tumor resection mouse model. In addition, TMP1 significantly decreased tumor-induced splenomegaly and the number of granulocytic MDSCs in test mice. In ex vivo cell culture assays, TMP1 did not decrease the level of granulocyte colony stimulating factor (G-CSF, a key cytokine for granulocytic MDSC differentiation) produced by tumor tissue, tumor associated stromal cells and 4T1 cells. However, TMP1 significantly inhibited granulocyte-macrophage colony stimulating factor (GM-CSF) or G-CSF-induced MDSC differentiation from bone marrow cells. TMP1 also strongly inhibited GM-CSF-induced expression level of G-CSF receptor in bone marrow cells. Taken together, our results suggest that TMP1 significantly suppresses mammary tumor metastasis via inhibition of granulocytic MDSC differentiation.

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Soluble CD83 inhibits human peripheral blood mononuclear cell differentiation into dendritic cells in vitroXiujin Li1, Shuang Liang3, Zhenyu Zhong3, Wenyan Li2, Jian Xu1, Fei Zhong2 and Hongyu Lin1,2

1Yanshan University, China2Agricultural University of Hebei, China3University of California San Diego, USA

Human CD83 is type I transmembrane glycoprotein, mainly expressed on mature dendritic cells (DCs). The CD83 is not only a molecular marker for mature DCs, but a regulatory molecule, especially its extracellular domain released from

DCs, called soluble CD83 (sCD83), possessing many functions in immune regulations. However, whether the sCD83 has the regulation on human peripheral blood mononuclear cell (PBMC) differentiation into DCs is unknown. To investigate its regulatory function on human PBMC differentiation into DCs in vitro, we prepared the glycosylated and deglycosylated sCD83 in HEK293T cells and used the sCD83 to treat human PBMCs being differentiated into DCs. The results showed that both glycosylated and deglycosylated sCD83 could bind the PBMCs and significantly up-regulated CD14 expressions (P<0.01) and down-regulated CD1a and CD80 expressions (P<0.01), indicating that the sCD83 can inhibit PBMC differentiation into DCs, suggesting that sCD83 possesses the negative feedback regulation on human PBMC differentiation into DCs in vitro.

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Clinical & Cellular ImmunologyPrevalence of class 2 integrons in multidrug-resistant Acinetobacter baumannii in toxicological ICU patients in TehranSepideh Kamalbeik1, Mehran Kouchek2, Majid Baseri Salehi1, Fatemeh Fallah2, Mohammad Ali Malekan2 and Haleh Talaie2

1Islamic Azad University Fars Science and Research Branch, Iran2Shahid Beheshti University of Medical Sciences, Iran

Background: Acinetobacter baumannii is an important opportunistic pathogen which causes complications in hospitalized patients, especially those in ICU. The aim of thisstudy was to determine the frequency of class 1 and 2 integrons in multi-drug resistant A. baumannii and to investigate the association between the presence of integrons and antibiotic resistance patterns.

Methods: A total of 40 A. baumannii strains were isolated from 372 ICU patients from June to Oct 2012. A. baumannii was detected in 50% of tracheal cultures, 15% in blood, 15% in urine samples and 22.5% in other locations. In accordance with CLSI 2011, 12 antibiotics were used through disc diffusion method. Existence of integron classes was investigated by PCR assay with the amplification of integrase genes.

Results: The most effective antibiotic against Acinetobacter baumannii was polymyxin B with 100% susceptibility, followed by meropenem, piperacillin, cotrimoxazole, ceftazidime with 100% resistance; this was followed by ciprofloxacin 97.5%, tetracycline, 92.5%, imipenem 62.5%, and gentamicin 60% resistance. The presence of integrin class 1 was 7.5%, class 2 was 67.5%, and non-integron was 20%.

Conclusion: The association between multidrug resistance and class 2 integron was not statistically significant. Other factors accounting for the lack of significance of thefindings may be the impact of other resistance determinants such as transposons orplasmids, not investigated in the current study. Considering the increasing trend of MDRinfections among ICU patients with critical problems in follow up, the use of appropriate infection control strategy and a regular surveillance system is necessary in our hospital.

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Phenotypic characteristic of THP-1 cell-derived microvesiclesOvchinnikova O M, Korenkov D A, Onohin K V, Benken K A, Urodkova A A, Viknyaschuk A N, Selkov S A and Sokolov D ID.O.Ott Research Institute of Obstetrics and Gynecology, Russia

Microvesicles are small cell-derived membrane vesicles shed from cells during activation or apoptosis. In peripheral blood are found leucocyte-, thrombocyte-, and endothelial-derived microvesicles. Peripheral blood microvesicles are

known to participate in coagulation, inflammation and immune response. In the present study phenotype and size distribution of monocyte-like cell line THP-1 microvesicles were evaluated. Microvesicles from intact, TNFα or IFNγ activatedTHP-1 were fractionated from cell conditioned media. Microvesicle fraction was analyzed by atomic force microscopy and also was stained forCD11a, CD11b, CD11c, CD18, CD31, CD29, CD49d, HLA-DR, CD47, CD54, VEGFR1, CD181, TRAIL, CD120a, CD120b and analyzed by flow cytometry. Atomic force microscopy showed a population of spherical objects characterized by the height distribution within 50-400 nm with a peak at 200 nm. Particle size analysis of a 2×2 nm field detected large 700 nm microvesicles in addition to that having the 230 nm mean size. Microvesicles derived from intact THP-1 bore CD11a, CD31, CD29, CD49d, CD47, VEGFR1, CD181, TRAIL, CD120a, CD120b. After TNFαactivation, THP-1 cellsproduced more CD11а+, CD18+, CD54+, VEGF-R1+, CD120b+ microvesicles than intact cells. Activation of THP-1 by TNFα or IFNγ led to the increase in CD54 fluorescence intensity on THP-1-derived microvesicles. Thus, intact or activated THP-1 cells produce microvesicles 50-700 nm in diameter, with the peak at 200 nm. Intact THP-1-derived microvesicles bear adhesion molecules, cytokine and growth factor receptors. Activation of THP-1 by TNFα or IFNγ leads to the increase in production of CD11а+, CD18+, VEGF-R1+, CD120b+, CD54+ microvesicles.

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Clinical & Cellular ImmunologyInfluence of THP-1 cells on endothelial cells tube formation in the presence of soluble factors derived from the placentas on early and late stage of pregnancyLvova T Yu, Stepanova O I, Okorokova L S, Beljakova K L, Selkov S A and Sokolov D ID.O.Ott Research Institute of Obstetrics and Gynecology, Russia

Macrophages play an essential role in angiogenesis regulation. The aim of the research was investigation of THP-1 cells influence on capillary-like tube formation of endothelial cells EA.Hy926 (EC) at presence of soluble placental products

obtained from healthy pregnant women on 9-11 weeks of gestation (n=20, group 1) and from healthy pregnant women on 38-39 weeks of gestation (n=20, group 2). EC were seeded on Matrigel-coated 24-well plates (BD, USA) at a density of 150000 cells/well, also were added placental tissue supernatants and 2,5% fetal bovine serum. To part of wells THP-1 cells were added (250000 cells/well), to part of wells - the cultural medium without THP-1 cells. Tube formation was assessed 24 hours later using microscope AxioObserver Z1. Addition of THP-1 cells to the EC cultivated in the presence of soluble placental products of group 1 resulted in increasing of tube length and reducing of tube number, while in the presence of soluble placental products of group 2 addition of THP-1 cells did not lead to changes in length and number of tubes. In monoculture system and in co-culture system were established increasing of tube length in the presence of soluble placental products of group 2 compared to group 1. Our data suggest that THP-1 cells can modulate tube formation of EC in the presence of soluble placental products. Obtained data may reflect the influence of monocytes/macrophages on EC tube formation in placenta at different stages of physiological pregnancy.

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Fine-tuning of host immune responses: A tale of adeno-vector expressing hepatitis C virus antigen NS4Shakti Singh, Satish Vedi, Wen Li, Subodh Kumar Samrat, Rakesh Kumar and Babita AgrawalUniversity of Alberta, Canada

Non-replicative adenoviral vectors expressing antigens induce strong cellular and humoral immune responses, and are the basis of several vaccines being developed. The critical role of both route and doses of immunization is still unknown.

Hepatitis C virus (HCV) leads to chronic infection in the majority of infected patients due to failure or inefficiency of the immune responses generated, but can be resolved in those who mount strong immune responses directed against various structural and non-structural antigens of HCV. In this study, we sought to analyze the influence of both route and dose of immunization by non-replicative recombinant adenovirus expressing HCV NS4 protein (rAd-NS4) on antigen specific cellular and humoral immune responses, and their role in viral clearance. Female C57BL/6 mice were immunized with various doses of rAd-NS4 via intramuscular (i.m.) or intraperitoneal (i.p.) route. The induction of antigen specific antibodies, T cell proliferation and pro-inflammatory cytokine responses in both spleen and lymph nodes and their role in the clearance of Vaccinia-HCV chimeric virus in a mouse challenge model were evaluated. Our results show that an optimum dose of adenovirus vector (2x107 PFU/Mouse) administered by i.m. route induces high T cell proliferation, granzyme B-expressing CD8+ T cells, pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-2 and IL-6, and antibody responses that can significantly reduce the Vaccinia-HCV viral load in the ovaries of female C57BL/6 mice. Interestingly, immunization by i.m. route induces lower adeno vector antigens specific neutralizing antibodies compared to i.p. route. Our results clearly demonstrate that recombinant adenovirus vector can induce strong humoral and cellular protective immunity against HCV-NS4 antigen, and that immunity is intricately controlled by route and dose of immunizing vector.

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Clinical & Cellular ImmunologyMetformin inhibitory function on tumor cell induce by hypoxiaZ Safari1,2, M H Seyedzadeh2, R Safaralizadeh1, Ayla Valinezhad Orang1 and G A Kardar2 1University of Tabriz, Iran 2Tehran University of Medical Sciences, Iran

Background: The formation of the hypoxic acidic in tumor is an important factor of invasion and metastasis. Although it has been reported that metformin acts as a proliferation inhibitor and apoptotic elevator in cancer cells, the role of metformin in low oxygen supplies has not been yet identified. In line with the differences in oxygen supplies in cancer cells compared to normal cells, in this current study we provided both hypoxia and normoxia condition in order to evaluate the actual anticancer effects of metformin on cancer cells.

Material and method: Normal cells (HEK239) and cancer cells (MCF-7) in both hypoxia and normoxia condition were cultured and treated with different concentrations of metformin. Their proliferation, apoptosis, and necrosis rate assessed using MTT test and annexin V assay. The phosphorylation rate of S6K1 assessed using western blotting. Zymography and western blot were used to measure the expression levels of MMP-9.

Results: Metformin inhibits proliferation more effectively in hypoxia condition compared to normoxia in cancer cells, while it has no significant difference between normoxia and hypoxia conditions in normal cells. Statistical analysis indicates that metformin causes an increase of apoptosis rate in cancer cells with hypoxia condition (p value <0.05), while there is no acceptable increase in normal cells. In addition metformin cause a significant decrease in S6K1 phosphorylation and activation in cancer cells under hypoxia condition compared to normoxia. But there was no significant difference between normoxia and hypoxia condition in normal cells. It also leads to a significant decrease in MMP-9 expression levels more effectively in hypoxia condition compared to normoxia.

Conclusion: Our results indicate that in hypoxia condition metformin exerts its anti-cancerous function by inhibiting proliferation and tumor progression and inducing cell apoptosis more effectively than normoxia condition.

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5-Lipoxygenase and phospholipase A2 inhibitory piperine as anti-inflammatory and anticancer agentNagendra Sastry Yarla, Umadevi Parimi, Venugopal, Durvasula V R Venugopal, Kaladhar DSVGK and Govinda Rao DuddukuriGitam University, India

Piperine is an alkaloid responsible for the pungency of black pepper and long pepper. It has also been used in some forms of traditional medicine. Piperine was discovered in 1819 by Hans Christian, who isolated it from the fruits of Piper nigrum,

the source plant of both the black and white pepper grains. Piperine is alkaloid, have been reported for various medicinal properties including antimicrobial, anti-inflammatory and anticancer activities. In present study, we have screened isolated piperine for 5-LOX and PLA2, which have important role in inflammatory related diseases and cancer. Piperine showed significant 5-LOX inhibition and moderate PLA2 inhibition in cell free system. Thus, further investigations are needed to develop piperine or its analogs as anti-inflammatory and anticancer agent(s). Acknowledgement: Govinda Rao Duddukuri, Y.Nagendra sastry, Kaladhar DSVGK are thankful to UGC-MRP (F.No. 42-643/2013) for financial support in antiPLA2 studies.

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Clinical & Cellular ImmunologyThe anti proliferative and cytotoxic activity of sodium butyrate (SB) as a microbial product on human colorectal carcinoma cell lines Kazemi Sefat N A1, Mohammadi M M1, Hadjati J2, Talebi S2, Ajami M2,3 and Daneshvar H1

1Kerman University of Medical Sciences, Iran2Tehran University of Medical Sciences, Iran3Shahid Sadoughi University of Medical Science, Iran

Background: Sodium butyrate (SB), a short chain fatty acid, is a histone deacetylase inhibitor (HDI) and produced in the colonic lumen as a consequence of anaerobic bacterial fermentation of dietary fiber, undigested starch and proteins. SB serves as an energy source of colonic epithelium and plays a role in the maintenance of colonic homeostasis; in addition HDIs induce growth arrest and apoptosis in a variety of human cancer cells. Colorectal cancer metastases result in a significant number of cancer related deaths.

Methods: The viability of cells was measured by MTT assay. The adherent SW480 and HCT116 human colorectal carcinoma cells were seeded in 96-well plates. Different concentrations of SB (0, 1.0, 2.5, 5.0, 10, 20 μmol/L) were added and then incubated for 12, 24, 48, and 72 hours at 37°C with 5% CO2. MTT solution was added to each well. Dimethylsulfoxide was added after 4 hours and the absorbance measured at 550 nm in a spectrophotometer. The 50% inhibiting concentration IC50 were determined graphically.

Results: After 24 hours, a dose dependent cytotoxicity and morphological changes were observed which were accentuated after 48 and 72 hours. MTT assay results confirmed the morphologic observations. IC50 for SW480 and HCT116 cells was 10 and 20 μmol/L respectively. Results confirmed the anti-proliferative effect of Sodium butyrate on two mentioned cell lines.

Conclusion: Although many risk factors involved in engender colorectal cancer but it has not been possible to harness a rational approach to its prevention or treatment until now. Our results may suggest that the gene expression which is contributed in cell proliferation and apoptosis may be changed under pressure of HD inhibition due to SB which is a byproduct of bacterial fermentation in colon and this suggested mechanism may play a role for prevention of colon cancer; whereas more future investigation is necessary for confirmation.

BiographyI was born in Tehran, in 29 May 1978, Where I Attended Aeen Roshan high school and graduated in 1996. After that I studied Medical Laboratory Sciences at the Uniersity of Tehran and attained My Bachelor of Science (B.Sc.) degree in 2006. In 2011, I was Accepted in Master of Sciences Immunology Examination and Now I have MSc in Medical Immunology. I am interested in clinical research in the field of cancer therapy. I have experience of working in several Medical Laboratories having about 11 years.

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Clinical & Cellular ImmunologyAnemia and risk factors in HAART naive and HAART experienced HIV positive participants in South West Ethiopia: A comparative studyLealem Gedefaw, Tilahun Yemane, Zewdineh Sahlemariam and Daniel YilmaJimma University, Ethiopia

Background: Human immunodeficiency virus (HIV) infection and its treatment cause a range of hematological abnormalities. Anemia is commonly observed and multifactorial in origin in HIV positive people and has been associated with increased mortality rates and disease progression.

Objective: We aimed to determine the prevalence and risk factors of anemia in highly active antiretroviral therapy (HAART) naive and HAART experienced HIV positive people.

Methods: A facility-based comparative cross sectional study was conducted in Jimma University Specialized Hospital from February 1 to March 30, 2012. A total of 234 HIV positive persons, 117 HAART naive and 117 HAART experienced, were enrolled in this study. Blood and stool specimens were collected from each participant for hematological, immunological and parasitological investigations. Socio-demographic characteristics and clinical data of patients were collected using pre-tested questionnaire. Statistical analysis of the data (Chi-square, student’s t-test, logistic regression) was done using SPSS V-16.

Results: The overall prevalence of anemia was 23.1%. The prevalence of anemia in HAART naive and HAART experienced people was 29.9% and 16.2% respectively (P=0.014). Presence of opportunistic infections (P=0.004, 95%CI=1.69-15.46), CD4+<200 cells/μl (P=0.001, 95%CI=2.57-36.89) and rural residence (P=0.03, 95%CI=1.12-10.39) were found to be predictors of anemia for HAART naive participants. On the other hand, HAART regime (ZDV/3TC/NVP) and the duration of HAART were found to be predictors of anemia for HAART experienced groups.

Conclusion: The prevalence of anemia in HAART naive patients was higher than HAART experienced patients. Risk factors for anemia in HAART naive and HAART experienced HIV positive persons were different. Hence, there is a need for a large scale and longitudinal study for further characterization of the type of HIV associated anemia.

BiographyLealem Gedefaw acquired his first degree from Hawassa University in medical laboratory science and an MSc in Clinical Laboratory Science Speciality Haematology and Immunohematology from Jimma University in 2012. Currently he is working at Jimma University, one of the top leading universities in Ethiopia, as lecturer. He is involved in lecturing and advising postgraduate and undergraduate students in their research project in the Department of Medical Laboratory science and pathology, Jimma University. He has participated in different local and international workshops prepared by different organizations. He is also doing different researches in the university in the area of HIV, Immunology, parasitology and nutrition.

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Clinical & Cellular ImmunologyDynamic association of cytokines (IL-6, IL-8, IL-10, IL-12, TNF-α, IL-1 β) in trauma hemorrhagic shock patientsManoj Kumar1, D N Rao2, Sujata Mohanty3, Arul Selvi4 and Sanjeev K Bhoi11JPN Apex Trauma Center, India2All India Institute of Medical Sciences, India

Objective: This study investigates the serum cytokines levels in patients with trauma hemorrhagic shock and the relationship of these cytokines with clinical outcome.

Background: Trauma remains a significant public health issue and is the leading cause of death in persons younger than 40 years. Up to 50% of early deaths are due to massive hemorrhage. Severe injury and hemorrhagic shock may result is an excessive production of pro-inflammatory cytokine and mediators, which play a significant role in the development of multiple organ dysfunctions.

Design: Prospective cohort study

Patients: Seventy patients with trauma hemorrhagic shock, thirty with normal healthy control.

Intervention: Peripheral blood samples were collected in each patient for determination of serum cytokines concentration. Samples were obtained within 8 h of post injury with T/HS patients. Standard resuscitation techniques as per Advance Trauma Life Support were used in each patient. Clinical and laboratory data were prospectively collected.

Methodology: High concentrations of circulating IL-6, IL-10, IL-8, IL-12, (p<0.05) but not TNF-α, IL-1 β) were detected in a trauma hemorrhagic shock as compared with healthy control group. At study entry, IL-8 concentrations were higher in non-survivors as compared with survivors T/HS patients but not TNF-α, IL-1 β, IL6, IL10. Increased IL-6 value was an indicator of the development of an infection in patients of trauma hemorrhagic shock.

Conclusions: In trauma hemorrhagic shock, increased IL-6, IL-10, IL-8, IL-12 are detected while compared to normal healthy control. In these patients, increased IL-8 value in non-survivors as compared to survivors and TNF-α, IL-1 β, IL6, IL10 do not correlate with clinical outcome. This study suggests a much higher degree of activation of immune-inflammatory in T/HS than in normal healthy control. Increased IL-8 values were found to be reliable markers of mortality following T/HS.

BiographyManoj Kumar is Member of Indian immunology Society (ISS), Indian Society of Trauma and Acute (ISTAC) and International Society of Cellular Therapy (ISCT). He has done M.Phil, Microbiology, Ch. Charan Singh University, Meerut, India. Now, he is doing PhD, Department of Emergency Medicine, JPN Apex Trauma Center at All India institute of Medical Sciences, New Delhi, India. His expertise includes, Hematopoietic Stem cells, Immunology (cytokine analysis) patients with Trauma hemorrhagic shock.

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Clinical & Cellular ImmunologyDefining the early in vivo immune response after pulmonary Aspergillus challenge under different immune suppressive regimensNatarajaswamy Kalleda1,2, Jorge Amich1, Katharina Mattenheimer1, Katja Ottmüller1,2, Ana-Laura Jordán-Garrote1,Christian Brede1, Marcel Münstermann1, Andreas Brandl1, Hermann Einsele1 and Andreas Beilhack1,2

1Würzburg University Hospital, Germany2University of Würzburg, Germany

Background: The number of patients with immune suppression is steadily increasing due to various clinical therapies that require administration of immunosuppressive drugs as it is the case for patients undergoing allogenic hematopoietic stem cell or solid organ transplantation and patients with autoimmune conditions. Other disease conditions such as cancer, retroviral infections and genetic immune disorders also compromise the immune system. Fungal infections with the invasive mold Aspergillus fumigatus cause high morbidity and mortality in these clinical situations. The degree of immune suppression influences the severity of A. fumigatus infections. Furthermore, how virulence factors impact the immune response and consequently the outcome of infection remains elusive. Therefore, a better understanding of immune cell responses in various degrees of immune suppressive states following infection with virulent and attenuated A. fumigatus strains is required.

Methods: To investigate the immune cell response under different immune suppressive regimens, we employed corticosteroid treated and cyclophosphamide treated leukopenic mouse models of invasive aspergillosis. We challenged immune competent, corticosteroid treated and leucopenic mice with 106 A. fumigatus virulent wild type or avirulent melanin layer deficient pksP mutant conidia. The immune cell recruitment in post-infection early and late time points (4h, 16h and 40h) was quantified using FACS analysis and histology. We utilized dynamic in situ multi-photon microscopy to corroborate our findings and to visualize the dynamic interactions between eGFP labeled immune cells and growing td tomato transgenic A. fumigatus in the lung under ex vivo conditions.

Results and conclusion: Innate and adaptive immune cell populations significantly decreased after corticosteroid treatment. Infection of these mice with A. fumigatus wild type conidia resulted in a significant early (4 h) recruitment of neutrophils, alveolar macrophages, conventional macrophages, monocytes and eosinophils. However, at 16 h and 40 h after infection, immune cells decreased below steady state levels. In the leukopenic model, most of the immune cells disappeared from the lungs. However, at 4h after A. fumigatus infection reduced, but significant numbers of neutrophils, macrophages and monocytes were recruited to the lungs. In contrast to the corticosteroid model, the neutrophil recruitment persisted even at 16 h and 40 h after infection in leukopenic mice, even if immune cells decreased below the steady state levels. There was no significant recruitment of DCs, NK- and T cell subpopulations in both models. Surprisingly, the immune cell recruitment did not differ after infection with WT or the avirulent pksP conidia after 4h and 16h.This data suggest that the lack of melanin in pksP A. fumigatus mutants does not impact immune cell recruitment, but rather acts via suppression of reactive oxygen intermediates produced by immune cells. Taken together, these results demonstrated that myeloid cells are crucial for defense against A. fumigatus infections under immune suppression conditions.

BiographyNatarajaswamy Kalleda completed his Masters in biotechnology from Osmania University, Hyderabad, India. He worked as an ICMR-Senior Research Fellow in University of Delhi for couple of years and at present he is working in Wuerzburg University Hospital, Wuerzburg, Germany. He has background in molecular biology and immunology of fungal infections.

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Clinical & Cellular ImmunologyThe elimination of cytomegalovirus in patients with ulcerative colitis without antiviral therapyO V Knyazev1, K N Shakhpazian2, A I Parfenov1, A A Churikova1 and A G Konopliannikov3

1Moscow Clinical Research Center, Russia2Russian Oncological Scientific Center, Russia3Medical Radiological Scientific Center, Russia

Introduction: It is known that immune-suppressive therapy increases the risk of infectious complications and activation of an opportunistic infection in patients with inflammatory bowel diseases. The accumulated data for today shows that, despite the pronounced immune-suppressive effect, mesenchymal stromal cells (MSCs) do not inhibit the functional activity of immunocompetent cells against infectious agents.

Objective: To identify a link between the presence of cytomegalovirus and hormone-dependence\hormone-resistance of patients with ulcerative colitis and to assess the impact of MSCs on to the level of cytomegalovirus (CMV) in patients with ulcerative colitis (UC).

Material and methods: 31 Patients with chronic UC were examined, 13 of them with hormone-dependent and/or hormone-resistant forms (group 1), 18-without hormone dependence and hormone-resistance (group 2). The identification of DNA viruses, Epstein-Barr, cytomegalovirus, and herpes of type 6 in biological samples was conducted by multiplex real-time PCR. For DNA extraction from biological samples and PCR sets of production of FBSI, Central Research Institute of epidemiology of Rospotrebnadzor, DNA-Sorb-B and АmpliSense EBV/CMV/HHV6-screen-FL" were used respectively. PCR with the detection of amplification products in real time was held in the thermocycler "RotorGen 3000" (Corbet, Australia). The conditions of amplification (the number of cycles, temperature mode and parameters of detection of the products of amplification) and the interpretation of data were carried out according to the instructions and methodical recommendations to sets of reagents.

Results: It was established that CMV was detected much more frequently in patients with hormone-dependent and/or hormone-resistant forms: in 9 patients (69.2%) in group 1 vs. 5 patients (27.8%) in group 2 (RR - 2.49; 95% Cl 1.09-5.71, x2-3.7; p=0.05). The average level of copies of DNA of CMV in patients of the first group was 0.86±0.12 Lg/105 cells, in the second group-0.35±0.15 Lg/105 cells (p<0.05). 150 million MSCs were introduced in 10 patients of the first group. In 4 weeks after the introduction of MSCs in 6 patients without antiviral therapy, CMV by PCR method was not determined. The average level of viral load in patients of the first group after the introduction of MSCs significantly decreased and amounted to 0.24±0.1 Lg/105 cells (p<0.05). Out of the 18 patients of the second group, 12 patients were introduced with MSCs. In 4 weeks after the introduction of MSCs in 3 patients without antiviral therapy, CMV by PCR method was not determined. The elimination of CMV did not depend on the presence or absence in hormone-dependent and/or hormone-resistant patients with UC (RR - 0.83; 95% Cl 0.2- 3.44, x2-0.11; p=0.74).

Conclusions: Cytomegalovirus is the most common in patients with hormone-dependent and/or hormone-resistant forms of ulcerative colitis. The introduction of MSCs culture contributed to the elimination of CMV without holding antiviral therapy in patients both with hormone-dependent and/or hormone-resistant forms of ulcerative colitis, or without hormone- dependence/hormone-resistance.

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Clinical & Cellular ImmunologySerum of patients with Behçet's disease induces activation of human lymphocytes and monocytes in vitroDjamel Messaoudene1,2, Houda Belguendouz1, Mohamed Laid Ahmedi1, Malika Terahi3, Pierre Youinou4 and Chafia Touil-boukoffa1

1Université de Bab-Ezzouar, Algeria2Universite de Boumerdes, Algeria3CHU Bab El Oued, Algeria4Centre Hospitalier Universitaire, France

Background: Although several immunological abnormalities have been demonstrated in Behçet's disease (BD), the exact mechanism of the inflammatory changes occurring is still unknown. Monocytes and lymphocytes are now recognized as key effector cells in BD.

Objective: To assess effects of Behçet disease serum on blood peripheral mononuclear cells from healthy donors and to evaluate if complement activation and complex immune were involved.

Methods: Monocytes and lymphocytes from a healthy donor were incubated with 33 sera from BD patients in both active and inactive disease. After incubation cells were analyzed by flow cytometry. The apoptosis-inducing effect (AIE) of these sera was monitored with flow cytometry using annexin V and propidium iodide (PI) binding and proliferation cells was assessed by CFSE labeling.

Results: Proliferation in monocytes and lymphocytes was significantly higher in sera of patient’s compared to the cells stimulated by serum of healthy donor and staurosporine (P<0.01), the CD25/CD69 and CD64 expression was statistically significant compared to the positive and negative stimulation, this activation correlate with disease activity. Levels of all cytokines [interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), interleukin (IL)-6, interleukin (IL)-10 and IL-4] were highest in Behçet patients. Level of complement, circulating immune complexes and AECA were higher in serum of patients of Behçet disease. Heat inactivation and complexes immunes’ depletion of sera affect significantly CD69 and CD64 expression.

Conclusion: Taken together, our results indicate that serum of patients with BD induces activation of lymphocyte and monocytes in vitro. Thus, BD serum factor(s) have a strong and apparently disease-specific activation inducing capacity, which could contribute to the physiopathology of the disease.

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Clinical & Cellular ImmunologyDo immune organ weights indicate immunomodulation of polyunsaturated fatty acids?Hanan Al KhalaifaKuwait Institute for Scientific Research, Kuwait

Introduction: The main immune organs in poultry are the thymus, spleen and bursa of Fabricius. During an immune response, mature lymphocytes and other immune cells interact with antigens in these tissues. Consequently, the mass of these organs can in some cases indicate immune status. Wang et al. (2000) observed that feeding laying chickens on diets rich in PUFA, especially n-3 PUFA, promoted the growth of the thymus, spleen and bursa up to 4 weeks of age. From the age of 4 weeks onwards, the immune tissue weight started to be suppressed and the bursa was degenerated in the course of 4 to 8 weeks of age. However, the authors observed that these changes in the immune tissues did not correlate with the immune function of thymus and spleen cells. Interestingly, the same phenomenon was observed in mice. For example, subcutaneous injection of 3.6 mg/day of pure linoleic, linolenic, or arachidonic acid into male C57Bl/6 mice daily for 10 days resulted in up to a two- to threefold increase in spleen weight. In another study, the authors fed Listeria monocytogenes infected or non-infected mice on 20% fatdiet rich in either n-3 PUFA, linoleic oleic, or saturated fatty acids for 6 weeks. Results showed that spleens were 12-22% heavier in the infected mice fed the n-3 PUFA diets than in those fed the saturated diets.

Results: Results showed that dietary supplementation with flaxseed did not affect the weights of the spleens of broiler chickens. However, it significantly lowered bursa weights (p<0.01), compared to the control diet. In addition, the bursae were thinner in appearance compared with bursii from chickens fed the control diets

Discussion & conclusion: Dietary supplementation with flaxseed did not affect the growth of the spleen of the chickens. Conversely, it was reported in some studies that feeding PUFA to chickens and mice results in increased spleen weights. However, chickens fed a diet containing flaxseedhad smaller and thinner bursii than chickens fed the control diets (P=0.001). This modulation in the weight of immune organs may indicate immunomodulation effect of fatty acids in flaxseed. More investigation studies need to be applied to shed light on the mechanism behind this immunomodulation.

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Clinical \u0026 Cellular Immunology Immunology Summit-2014 Accepted Abstracts

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