The Institute for Medical Research Israel-Canada, the Hebrew University, Medical School Ein Kerem, Israel
Recent thymic emigrants (RTEs) represent an immature T cell subset characterized by reduced propensity to proliferate following stimuli. Here we describe the discovery of a metabolic checkpoint modulating T cells propensity to initiate a response upon priming. We demonstrate that RTEs are phenol typically distinct from their more mature naïve CD8+ T cells by reduced OXPHOS, increased glycolysis, and substantially elevated mitochondrial membrane polarization. We define mitochondrial complex V restriction as the mechanism governing these metabolic differences. Following these findings we show that mitochondrial hyper polarization, driven by ATP synthase restriction, limits naive CD8+ T cells propensity to respond to diverse stimuli independent of ATP production. Tracing mitochondrial biogenesis in vivo, we reveal that mitochondrial polarization modulates proliferation capabilities of naive CD8+ T cells upon priming by regulating the acquisition of mitochondrial biomass. Our study defines mitochondrial hyper polarization induced by complex V restriction as a critical checkpoint directly controlling RTEs propensity to proliferate upon stimuli allowing intact T cell population and diversity.
Biography:
Dr. Michael Berger is a Senior Lecturer at the Lautenberg Center for Immunology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University Medical School, The Hebrew University, Jerusalem. He completed his PhD in the Faculty of Medicine, The Hebrew University of Jerusalem, Israel. He did his Postdoc at laboratory of Prof. Bruce Beutler, Department of Genetics, The Scripps Research Institute, TSRI, CA, USA. He honored as an Excellence Teaching at Faculty of Medicine, The Hebrew University of Jerusalem, Israel.
1Department of Immunobiology, Kingʼs College London, UK
2Wellcome Trust Sanger Institute, UK
3The Francis Crick Institute, UK
Epidermal tissue is the first line of animal defense against external factors. Mouse epidermis is populated by gδ T cells, also known as dendritic epidermal T cells (DETC), most of which belong to Vγ5Vd1 subset. These cells form a dense network in parallel to the network of Langerhans cells. However, it is still not clear which genes define the tissue homeostasis and functionality of these cells. As part of the Infection, Immunity and Immunophenotyping (3i) Consortium we conducted epidermal immunophenotyping of over 500 mouse gene knockouts generated by the Welcome Trust Sanger Institute. The epidermal sheets were isolated from 16 week old C57BL/6N mice and analysed by confocal microscopy and automated 3D image quantification in order to establish the effects of gene knockouts on the number and morphology of DETC and Langerhans cells. During the course of the project over 3000 mouse samples were processed. The multiparametric analysis showed a clear sexual dimorphism in wild type mice for most data readouts. Novel phenotypes were revealed for knockout lines with an overall hit rate ~4%. As a result we identified a number of new genes which were not previously associated with immune functions opening new avenues for further in depth investigation. Particularly Hbs1l knockout mice displayed severe defects in DETC morphology. Further study using structured illumination microscopy showed that although Hbs1l KO DETC are still able to migrate and form the immunological synapse they do not form actin-reach protrusions which may affect function of these cells in skin stress surveillance.
Biography:
Dmitry S. Ushakov is a research fellow at Kingʼs College London. His research is centered on understanding the fundamental principles governing immune cell motility, maintenance and interactions, which he investigates using advanced fluorescence microscopy technologies. Recently he established a novel automated 3D image processing method enabling multiparametric analysis of cells residing in tissues. This approach was utilized for the Infection, Immunity and Immunophenotyping (3i) consortium where hundreds of mouse lines were screened revealing new genes involved in immune cell homeostasis. He now takes these findings forward to further investigate the role of the identified genes in the intraepithelial leukocyte biology.
Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University, South Korea
Rationale: We studied whether the nasal mucosa in allergic rhinitis (AR) would be more susceptible to influenza A virus (IAV) infection due to lower induction of interferon (IFN)-related immune responses.
Objectives: To determine whether IFN induction would be impaired in allergic nasal mucosa and to identify which IFN was correlated with higher viral loads in IAV-infected allergic nasal mucosa.
Methods: IAV mRNA, viral titers and IFN expression were compared in IAV-infected normal human nasal epithelial (NHNE, N=10) and allergic rhinitis nasal epithelial (ARNE, N=10) cells. We used in vivo model of AR (BALB/C mouse, N=10) and human nasal mucosa from healthy volunteers (N=72) and AR patients (N=29) to assess the induction of IFNs after IAV infection.
Results: IAV mRNA levels and viral titers were significantly higher in ARNE compared with NHNE cells. IFN-β and -λs were induced in NHNE and ARNE cells up to 3 days after IAV infection. Interestingly, induction of IFN-λs mRNA levels and the amount of secreted proteins were considerably lower in ARNE cells. The mean IFN-λs mRNA level was also significantly lower in the nasal mucosa of AR patients. We found that recombinant IFN-λ treatment attenuated IAV mRNA levels and viral titers in IAV-infected ARNE cells and completely controlled IAV infection in an in vivo AR model.
Conclusion: Higher susceptibility of the allergic nasal mucosa to IAV may depend on impairment of type III IFN induction, and type III IFN is a key mechanistic link between higher viral loads and control of IAV infection in allergic nasal mucosa.
Keywords: Influenza A virus; type III interferon; allergic rhinitis; nasal mucosa.
Biography:
Yung Jin Jeon is a Clinical Research Fellow of Department of Otorhinolaryngology at Seoul National University Hospital in South Korea. She graduated from Seoul National University College of Medicine and has completed her resident training at Seoul National University Hospital. She was honored as Excellent Resident. She has done International Medical Education and Research program of University of Minnesota in 2011 and visited the Department of Otorhinolaryngology at University of Harvard Medical School as a visiting physician in 2016. She is a Ph.D. candidate in Immunology of Seoul National University and currently doing basic research focusing on respiratory allergic diseases.
1Department of Microbiology, National Institute of Respiratory Diseases, Mexico
2Department of Environmental and Occupational Health, Rutgers School of Public Health, USA
3Office for Global Public Health Affairs, Rutgers School of Public Health, USA
Introduction: Alveolar macrophages play a central role in the protective immune response to respiratory infections and are the first line cellular responders to inhaled particulate matter (PM) and microbial pathogens. Monocytes are recruited from blood during inflammation and then mature into alveolar macrophages. Mechanisms by which PM modulate innate responses of macrophages are not understood.
Objectives: To examine the effects of PM exposure on human monocytes during in vitro differentiation into macrophages and assessphagocytosis of PM, expression of cell-surface molecules and TNFα production.
Methods: Peripheral blood was obtained from healthy adult volunteers (n=10). Monocytes were isolated from peripheral blood mononuclear cells by plastic adherence and positive immunomagnetic selection and exposed to PM during in vitro differentiation into macrophages for seven days. Cell morphology, proportions of cells containing PM and TNF-α production were assessed by microscopy, flow cytometry for cell-surface expression of CD14, CD16, CD33, CD36, CD163, CD206 and CD209 and ELISA, respectively. Ambient PM2.5(aerodynamic diameters <2.5μm) for in vitro exposure studies, were collected with high-volume PM2.5 samplers (GMW Model 1200, VFC HVPM10, airflow rate 1.13m3/min) at the National Center for Environmental Research and Training (CENICA, Mexico City).
Results: Monocytes exposed to PM during their in vitro differentiation exhibited a round morphology, while unexposed monocytes showed a non-rounded morphology and were elongated. The proportion of cells containing PM increased according to the concentration of PM with the highest proportion of cells containing PM being 22% (15-32, 10mg/mL). Exposure to 1, 5 and 10mg /ml of PM did not show significant changes to the cell-surface expression of CD14, CD16, CD163 and CD33. Although exposure to 1, 5 and 10mg/ml of PM did not significantly alter the expression of CD36 a trend to a decrease in proportion of cells expressing CD36 was observed when PM concentrations were increased. The expression of CD206 was significantly increased in cells exposed to 10mg /mL of PM (p<0.005). In addition, exposure to 10mg /mL of PM induced significant production of TNF-α in comparison to non-exposed cells (p<0.005).
Conclusions: In vitro exposure to PM enhanced the expression of CD206 and production of TNFα during macrophage differentiation. Our data support the hypothesis that PM affects the differentiation of monocytes to macrophages and modify macrophage function.
1,2University of the Western Cape, South Africa
Silver nanoparticles (AgNPs) are commonly found in consumer products due to their antimicrobial properties. However, very little is known about the effects of AgNPs on the immune system. This study evaluates the effects of AgNPs on the murine macrophage cell line RAW 264.7 and human whole blood cell cultures (WBCs). The effects of AgNPs were assessed in the presence or absence of a mitogen, lipopolysaccharide (LPS). The effects of AgNPs on WBCs were monitored under basal conditions, LPS or phytohaemmagglutinin (PHA). A number of parameters were evaluated for both cultures, which included cytotoxity, biomarkers of inflammation, cytokines of the acquired immune system and a proteome profile analysis. AgNP concentrations tested had no effect on RAW cell viability. However, cytotoxicity of WBCs was evident at 250 μg/ml AgNP. Under basal conditions, AgNPs concentrations ≥ 62.5 μg/ml and > 25 μg/ml induced inflammation in RAW cells and WBCs respectively. Under a simulated inflammatory response (+ LPS), 250 μg/ml AgNP inhibited the inflammatory response for both RAW and WBCs. The acquired immune cytokines IL-10 and IFNγ were both induced by 250 μg/ml AgNP in the absence of PHA. While IL-10 was partially inhibited by 250 μg/ml AgNP in a simulated (+ PHA) acquired immune response. Proteome profiles of RAW cell supernatants show that AgNPs do in fact modulate specific protein synthesis. Upregulated RAW cell proteins due to AgNP exposure indicate induction of proteins associated with inflammation and wound and tissue healing. The mitogen activated WBCs proteome analysis indicates the partial inhibition of inflammatory proteins. Monitoring those proteins in future experiments will give a better indication of the effects of AgNPs.
Biography:
Currently concluding her PhD research project in the Medical Bioscience Department at the University of the Western Cape. She is also currently the lab manager for our Ecotoxicology/Immunotoxicology Laboratory. Her work includes investigating the in vitro effects of heavy metals and nanoparticles on the immune system. She has previously presented work at various international conferences. These include the 16th International Symposium on Toxicity Assessment (ISTA), 7th Society of Environmental Toxicology and Chemistry (SETAC) Africa conference and the ChinaAfrica Water Forum.
1Department of Infection, Immunity and Cardiovascular Disease, the University of Sheffield, UK
2Department of Biomedical Science, the University of Sheffield, UK
Cystic fibrosis (CF) is a disease that affects respiratory function and in the UK it affects about 151 young persons per 100,000 people. The disease arises due to dysfunction in cystic fibrosis transmembrane conductance regulator (CFTR) protein, a protein that has been shown recently to influence calcineurin activities in cell secretion. CFTR dysfunction causing mucus lodging and bacteria colonisation of the airways and intestinal linings leading to functional alterations of immune cells. In airways, CFTR has been shown to form a functional complex with S100A10 and AnxA2 in a cyclic adenosine monophosphate (cAMP)/ protein kinase A (PKA) dependent pathway. The multiprotein complex of CFTR, S100A10 and AnxA2 is also regulated by protein phosphatase 2B (PP2B). The objective of this study was to investigate whether chloride ion (Cl-) channels are activated by lipopolysaccharide (LPS) from Pseudomonas aeruginosa (PA), and whether this activation requires cAMP/PKA/PP2B pathway.
Human monocytes and macrophages were used in the study. Whole cell patch records showed that LPS from PA can activate Cl- channels, and this activation appears to require an intact PKA/PP2B signalling pathway. The Gout in the presence of LPS was 2185.97± 226 μS/cm2 (n=27). Gout was significantly inhibited by diisothiocyanatostilbene-disulfonic acid (DIDS), an outwardly rectifying Cl- channel (ORCC) blocker, 1204.40±132 μS/cm2 in the presence of DIDS. CFTR channels were inhibited using CFTRinh172 and this reduced Gout to 838.68±101 μS/cm2. Data from cells stimulated with LPS from PA that were pre-incubated with PKA inhibitor or PP2B inhibitor showed no DIDS and CFTRinh172 sensitive currents. Activation of both CFTR and ORCC is therefore observed in response to exposure of monocytes and macrophages to LPS. Ongoing work is investigating whether this activation plays a subsequent role in the release of pro-inflammatory molecules.
1Neurosciences and Cell Biology of the Federal University of Para, Brazil
2Evandro Chagas Institute/Health Ministry, Federal Fluminense University, Brazil
3Postgraduate Program in Biology of Infectious and Parasitic Agents of the Federal University of Para, Brazil
4Institutional Scholarship Program/Evandro Chagas Institute/Health Ministry, Brazil
Background: P. vivax is the most prevalent species in the Brazilian Amazon region. A growing body evidences indicates that the immunity is important in the outcome of P. vivax infection. Single-nucleotide polymorphisms (SNPs) in cytokine genes can alter the production of these proteins and consequently affect the parasite density, which has been recognized as an important factor in the outcome of malaria infection.
Materials and Methods: We investigated whether SNPs in cytokine genes are associated with parasite density in 50 malaria patients and 79 healthy individuals from Itaituba, municipality situated on southwest of Para state. Three SNPs, TNFA -308G/A (rs1800629), INFB +874T/A (rs2430561), IL6 -174G/C (rs1800795) and the haplotype IL10 -1082G/A (rs1800896), -819C/T (rs1800871) and -592C/A (rs1800872), were analyzed by PCR-SSP. The parasite density was determined by counting the number of parasite in 100 separate fields and converted to the number of parasites per microliter of blood assuming 8,000 leucocytes/ μL. The existence of association was determined by Mann-Whitney test, with level of significance of 0.05, using Graph-Pad Prism software, version 6.0.
Results: Parasite density levels ranged from 30 to 60,000 parasites/ mm3 (10910.4 ± 14406.25) and all SNPs tested were in Hardy-Weinberg equilibrium. No significant association was found between the SNPs tested and parasite density.
Conclusions: Due to the obvious importance of cytokine effects on malaria, studies that elucidate the complex host-parasite interaction may be useful for understood pathophysiology. In the present study, the SNPs in cytokine gene appear to have no effect on the parasite density, since no association was found. The inclusion of a larger number of samples may confirm this assertion and therefore may have been a limitation of the study.
Keywords: Polymorphisms, Cytokine, Parasite Density, P. vivax.
1Molecular, Biochemical and Immunobiology Lab, Zacatecas Autonomous University of Mexico, Mexico
2Department of Hematology, Oncology and Molecular Medicine Superior Health Institute, Italy
Th1 type cellular activity plays a key role for the control of M. tuberculosis (MTb) as well as the induction of long-term memory conferring protective immunity. In order to better understand Mtb-specific immune mechanisms, both cellular and innate immune responses should be taken into account and the role of pivotal cytokines orchestrating their complex interactions should be elucidated. Type I interferonʼs (IFN-I)(α/β) are an important family of infection-induced cytokines that among other multiples functions, promote differentiation/activation of DCs in both human, and participate in the regulation of the Th1 responses. However, contrasting and controversial results have been provided against microbial infections either in vivo as well as in vitro. At this point, our group has reported that IFN-alpha associated to BCG protects against M. lepraemurium, a mice pathogen elicited a similar skin lesion than M. leprae in adults persons with a concomitant increase in NO synthase. In the present work, we are reporting that under a specific experimental protocol of prime-boost settings, IFN-alpha boosting of BCG vaccine in mice and/or in human cell lines (A549, macrophages derived from PBMC), elicited IFN-γ, TNF-α, as well as IL-17, correlated with the induced protection against M. tuberculosis. Altogether, the data strengthened IFN-alpha as a potential candidate boosting of BCG immunity.
Biography:
Dr. Guerrero obtained his PhD from the National Autonomous University of Mexico. Thereafter, he did two post doctorates. One of them in the Institute of Biology of Institute Pasteur in Lille, France under the supervision of Dr Camille Locht. The second postdoc was in the lab of Jean Laureant Casanova, working with the human tuberculosis. Right after that he spent almost two years in the Institute Superiore di Sanita in Rome, Italy in which he started to work with interferon alpha and the role in mycobacterial infections. He is the head of the Molecular Biochemistry and Immunobiology lab in the Unit of Biological Science of the University Autonomous of Zacatecas in Zacatecas State Mexico. His principle interest in the study of the molecular mechanism of tuberculosis in the three models, bovine, human and using the murine model, with the aim to identify biomarkers that can be used potentially in therapy and candidate vaccines.
1Research Center for Cell Dynamics (IICBA), Autonomous University State of Morelos, Mexico
2Inserm, Tagc, France
Human neonates have a poor response to intracellular pathogens, despite a high inflammatory response. This leads to a high morbidity and mortality rates, reaching 37% of deaths of children under 5 years of age. A major cause of death is infections and inflammatory syndromes, like sepsis. To better understand this phenomenon, we evaluated the transcriptome and epigenomic landscape of naive CD8+- and CD4+- T cells from neonate and adult blood. We show that neonatal T cells have a specific genetic program established by epigenetic mechanisms, biased towards innate immunity. Functional studies corroborated that CD8+ T cells are less cytotoxic and transcribe antimicrobial peptides. CD4+ T cells have a high expression of negative TCR signalling molecules and a low expression of positive signalling molecules, explaining the high threshold of activation of these cells. Altogether, these properties could explain the high susceptibility of neonates to infections and inflammation and will contribute to a better diagnosis and management of the neonatal immune response.
Biography:
Dr. Santana studied her B.Sc. at the Autonomous University of Metropolitan in Mexico City. She obtained her PhD degree from University Louis Pasteur in Strasbourg, France and did post doctorate work at the Medical School of The University of Manchester and at the Institute of Biotechnology, National Autonomous University of Mexico. She now directs the Laboratory of Cellular Immunology at the Centro de Investigation in Cellular Dynamics, Autonomous University, State of Morelos in Mexico. She has over 30 publications and has been responsible for national and international projects.
Laboratory of Molecular Immunology, Institute of Molecular Medicine, National Cheng Kung University, College of Medicine, Taiwan
Forced maturation of leukemia T cells requires up regulation of Ser14 phosphorylation of WWOX and the WWOX/ERK/IκBα signaling. WWOX is generally regarded as a tumor suppressor. Here, we showed that synthetic pSer14-WWOX7-21 peptide significantly induced the expansion of CD3+, CD4+, CD8a+, and CD8b+ T and CD19+ B cells in the spleens of immune-competent BALB/c mice bearing with melanoma. Despite immune cell expansion, no cancer growth suppression was observed. Notably, restoration of immune cells occurred in naive immunodeficient NOD-SCID mice during prolonged treatment with pSer14-WWOX7-21. In stark contrast, non-phosphorylated WWOX7-21 and WWOX7-11 peptides caused spleen Hyal-2+ CD3- CD19- or Z cells to undergo clonal expansion for blocking melanoma growth and metastasis and breast cancer growth. Both peptides induced the expression of spleen Z cells with up regulation of Hyal-2, Zfra, Noxa, Fam46A and Ademdec1 expression. Stimulation of mice with Hyal-2 antibody or sonicatedhyaluronan (HAson) activated Z cells to suppress cancer growth in vivo. In contrast, native hyaluronan (HAn) had no inhibitory effect. Time-lapse microscopy showed that activated Z cells aggressively bound and caused explosion of breast 4T1 cancer cells in vitro. In addition, WWOX7-21 enhanced ceritinib or U0126/protease inhibitor cocktail-induced 4T1 stem cell sphere explosion, whereas pSer14-WWOX7-21 protected the cells from death. Together, our observations suggest that pSer14-WWOX supports T and B cell expansion, whereas de-phosphorylated WWOX blocks cancer growth via induction of Z cells or direct assistance of therapeutic chemicals. Ser14 phosphorylation probably switches WWOX from a tumor suppressor to promoter.
Biography:
Dr. Nan-Shan Chang is currently the Distinguished Professor of the Molecular Medicine Institute, National Cheng Kung University (NCKU) in Taiwan, and the Adjunct Professor with the SUNY Upstate Medical University and the NYS Institute for Basic Research in Developmental Disabilities, New York. He is most noted for his discovery of tumor suppressor WWOX in 2000. Recent Awards: Breast cancer and neurofibromatosis research awards from the Department of Defense, USA, in 2008 and 2010; Distinguished Professor Award 2010, 2013, 2016 from NCKU; Distinguished Scientist Award 2011 from the Society of Experimental Biology & Medicine, USA.
University of Illinois, Department of Animal Sciences, USA
The consequence of prenatal stress exposure of gestating sows on the immune responsiveness of her progeny is not well understood. The objective was to assess the effects of social rank, and diet and feeding stall treatments of gestating sows on immune status of her progeny. Pigs (n =40) born to dominant (DOM) and submissive (SUB) sows that were housed in group-pens with different feeding stalls [0.6 m (SHT) or 1.8 m (LNG)] and fed modified gestation diets [wheat middlings and soybean hulls (MID-SOY) or distillers dried grains and corn germ-meal (DDG-GM)] were used in this study. Blood samples were collected. Total IgM was less (P = 0.01) in pigs from SUB fed DDG-GM, but cortisol was greater (P = 0.05) in pigs from SUB fed MID-SOY (social × diet). Total IgG (P < 0. 01) and IgG1:IgG2 (P < 0.01) were greater in pigs from sows in LNG and fed DDG-GM, but ConA (P = 0.05) and IgM (P = 0.02) were greater in pigs from sows fed MID-SOY (diet × stall). LPS-induced (P <0.01) and cortisol (P < 0.01) were greater in pigs from sows fed DDG-GM. Total IgG (P < 0.01) and IgM (P <0.01) were greater in pigs from sows in pens with LNG. Pigs from SUB had higher IgG2 (P <0.01), but pigs from DOM had higher (P = 0.10)IgG1:IgG2. These data imply that sow social rank and dietary treatment and environment during gestation may affect the immune responsiveness of her progeny.
Biography:
Dr. Janeen Salak-Johnson obtained her BS, MS, and PhD degrees in Animal Science from Texas Tech University. After earning her PhD in 1994, she was awarded a NIH Postdoctoral Training Fellowship in Psychoneuroimmunology and a NIH-NSRA Pain Fellowship in Pain at the University of Minnesota. She specialized in the areas of immunology, virology, and pain. In late 1999, she joined the faculty at the University of Illinois in Animal Sciences. She has authored or co-authored over 150 refereed publications, proceedings, and abstracts and given over 80 presentations in areas of Stress Physiology and Animal Well-being and Animal Behavior and Care.
Moffitt Cancer Center & Research Institute, USA
Checkpoint blocking antibodies have dramatically changed the landscape of therapy in melanoma and are rapidly improving the outcome of many cancer patients. There is still an unmet clinical need to develop novel therapeutic combinations that target mechanism of tumor immune suppression to enhance the duration of response or the number of responders after checkpoint blockade. We found that cereblon (Crbn), an E3-ubiquitin ligase substrate receptor for the DDB1/Cul4A/Roc1 complex, genetic deficiency markedly improves effector T-cell function in the B16 melanoma mouse model. T-cells present in this hostile tumor microenvironment are exposed to hypoxia, high lactic acid, and poor nutrient availability that limit their anti-tumor cytotoxic potential. While similar total intratumoral T-cells and splenic populations are present in tumor-bearing Crbn-/-mice, the proportion of CD44+ melanoma reactive (TRP2+) tumor infiltrating lymphocytes (TIL) was increased significantly relative to Crbn+/+ mice. To explore this mechanistically, differentially-regulated genes and metabolic profiling demonstrated an increase in the uptake and metabolism of intermediates in the Arginine/Proline pathway. This pathway was also modulated by immunomodulatory drugs pomalidomide and CC-122 by suppressing CRBN function. Therefore, treatment with immunomodulatory compounds that block CRBN may act to overcome a metabolic checkpoint induced through nutrient restrictions in the tumor microenvironment.
Biography:
P.K. Epling-Burnette, PharmD, PhD is a researcher at the H. Lee Moffitt Cancer Center & Research Institute and a Professor at the University of South Florida (USF) in Tampa, Florida, USA. Her laboratory program focuses on novel therapies that overcome basic mechanisms of T lymphocyte and NK cell immune suppression. Dr. Epling-Burnette has an established track record of research in hematological malignancies including myelodysplastic syndromes (MDS) which has contributed to the discovery of cereblon and its role in metabolite regulation in T cells. Her research has identified several mechanisms that contribute fundamentally to cancer-associated immune suppression through translational studies.
Infectious Diseases Research Lab, Department of Zoology, Kakatiya University, India
Background: The current treatment modality for HIV/AIDS is HAART (Highly active anti-retroviral therapy) but this treatment is not an ultimate cure to HIV/AIDS. Therefore there is need to develop inexpensive alternative anti-HIV/AIDS therapy. Different parts of Ecliptaalba crude extracts are used traditionally for the treatment of several diseases of liver, skin, stomach and sexually transmitted infections.
Objective: The objective of this study is to isolate alkaloid from E. Alba leaves for their activities against HIV-1 reverse transcriptase.
Methods: Collected leaves of E. Alba were extracted with different solvents and the purity of isolated alkaloids was checked by TLC and qualitative phytochemical analysis and total alkaloids were quantified. Peripheral Blood Mononuclear Cells (PBMCs) isolated from healthy donors by ficoll–hypaque density gradient centrifugation method. Cell viability test was performed on all crude extracts by MTT assay against PBMC and HIV-1 RT inhibition activity was determined by HIV-1 Reverse Transcriptase (p66) Capture ELISA.
Results: In the HIV-1 reverse transcriptase assay, the isolated alkaloid showed 89% of HIV-1 reverse transcription with IC50 of 5 μg/ ml. MTT assay revealed that, the alkaloid isolated from E. Alba had no cytotoxic activity (IC50 values higher than 100 μg/ml). Characterization of important biologically-active alkaloid from E. Alba plant will certainly be helpful in protecting and treating various viral diseases in human beings.
Conclusion: The results of the present study support the medicinal usage of the alkaloid isolated from the leaves of E. Alba can be used as antiviral agents and can be subjected to characterize the therapeutic drugs and undergo further pharmacological screening that can be used as sources for new drugs.
Keywords: Ecliptaalba, alkaloid, HIV-1 reverse transcriptase, MTT assay, PBMCs.
Biography:
Dr. Estari Mamidala completed his B.Sc in Kakatiya University and did post graduation in Zoology in Department of Zoology, Kakatiya University, India. He did his Ph.D in the same University on ‘HIV prevalence in rural areas and understanding of its pathogenesisʼ. He attended 32 national and international conferences and presented papers and he published 28 research publications in reputed journals and doing his post-doctoral research. He received Rapid Grant Young Investigator Award and also received DST-Young Scientist project from DST, India at the time his post doctoral research. He did vaccine trial on SIV at Department of Microbiology, Emory University, Atlanta, USA during his post-doctoral research.
1Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand
2Center of Research excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand
3Faculty of Allied Health Sciences, Thammasat University, Thailand
4School of parasitology, Institute of Medicine, Suranaree University, Thailand
Hepatitis C virus (HCV) infects ~3% of world population causing chronic hepatitis which frequently leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. NS5A is a multifunctional and pivotal protein which has an important role in the viral replication, the virus morphogenesis, and pathogenesis. Thus, the NS5A protein is an attractive novel anti-HCV target. In this study, human single chain (HuscFv) that inhibited the bioactivity of HCV was generated using antibody phage display library as the antibody producing tools for the ultimate purpose of developing further as a safe, side effect free and viral mutation tolerable novel anti-HCV remedy. Recombinant (r) NS5A was produced, purified, and used as bait for fishing out phage clones that displayed the NS5A-HuscFv from the established HuscFv phage display library. Based on indirect ELISA and Western blot analysis, 5 phage transfected E. coli clones produced rNS5A-bound-HuscFvs. The HuscFvs of these E. coli clones were linked to a cell penetrating peptide (Nonaarginine; R9) to make cell penetrating format. The R9-HuscFvs did not cause cytotoxicity, entered the huh7 cells (transbodies) and inhibited HCV replication (significantly reduced HCV RNA). The HCV-infected cells treated with the transbodies had up-regulation of the innate immune response genes. Computerized homology modeling and intermolecular docking (simulation) indicated that the HuscFvs formed interface contact with several critical residues of NS5A leading to interference with the protein functions and hence HCV replication inhibition. The transbodies produced in this study are worthwhile developing and testing further for future use as safe, interferon-free, broadly effective and robust DAAs.
Biography:
KittiratGlab-ampai is a last-year Ph.D. student in Immunology program, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand. His research focuses on the production of antibodies to infectious diseases, especially Hepatitis C. He received the Royal Golden Jubilee Ph.D. scholarship, from Thailand research fund. His interests in the field of immunology and virology. His Ph.D. work was published in Biochemical and Biophysical Research Communications Journal, 2016. He also briefly had a research over Zika virus while working in Australia which is a part of the recent publication in Scientific Reports journal, 2017.
Comparative Medicine Department and Organ Transplant Centre, King Faisal Specialist Hospital and Research Centre, Saudi Arabia
Introduction: Microvascular injuries due to excessive inflammatory responses has been associated with transplant malfunctions, which play a major role in the development of chronic rejection in all solid organ transplants, and as reported in both clinical and preclinical studies that there are no ongoing immunosuppressive regimens sufficiently affect the restoration of functional microvascular flow during the transplantation. Microvascular reestablishment, and repair during rejection is a promising new avenue to prevent acute and chronic rejection with Tregulatory cell (Treg) mediated immunosuppression but the direct molecular correlations between Treg and microvascular reestablishment has never been examined before. The present study was designed to analyze the molecular mechanisms of Treg-mediated restoration of microvascular flow during the state of immunotolerance and, in particular, the allograft rejection microenvironment.
Methods: We depleted IL-10 in BALB/cJ→C57Bl/6J allografts, and serially monitored peripheral and graft infiltrating Tregs, key proinflammatory and regulatory cytokines, donor-recipient microvascular connections, microvascular leakiness, tissue oxygenation and microvascular blood flow during four week in orthotopic mouse airway transplants.
Results: We demonstrated that depletion of IL-10 in BALB/cJ→C57Bl/6J allografts significantly suppressed CD4+CD25+FOXP3+ Tregs and regulatory IL-10 while upregulated major proinflammatory cytokines, IL-1β, IFN-γ and IL-15, and thereby facilitated donor-recipient microvascular associated injuries followed by a steep drop in tissue oxygenation, microvascular blood flow levels, which proceeds subepithelial injuries at d8, d10 and d28 post transplantation.
Conclusion: Altogether, these findings demonstrate that the targeted blocking of IL-10 severely affected the establishment of microvascular connections between donor-recipients graft, which are regulated through IL-10 and T regulatory cells.
Biography:
Mohammad Afzal Khan earned his PhD in Biotechnology from Aligarh Muslim University, India, and spent eight years as a post-doctoral fellow at Stanford University, USA. His primary research interests are in the field of lung transplant rejection, cell and complement-based Immunotherapies. Lung transplantation is the only effective therapy for many end-staged lung diseases. Unfortunately, lung transplantation has the worst survival of any major organ transplant because the patientʼs own immune system attack the transplanted lung and can cause significant irreversible damage, which leads to chronic rejection. Chronic rejection is the major obstacle to lung transplant success, and there are no effective therapies to avoid this failure. To investigate the clinical benefits of cell and complement-based immunotherapies, he established mouse model of orthotopic airway transplantation and used it together with assessment of microvascular flow after transplantation in MHC mismatched transplants. They use wide array of microsurgical, translational, immunological, confocal imaging, cell and molecular biology techniques to dissect potential benefits of cell based therapies. In global drug discovery program, developing targeted cell-mediated immunotherapies which prevent the transplant rejection without drug induced toxic immunosuppression could, therefore, be of singular importance in the prevention of organ failure following transplantation. Mohammad Afzal Khan et al.,
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