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1
Taylor, Kristen Rea. "Activation of cutaneous innate defense by glycosaminoglycans." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3211370.
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Thesis (Ph. D.)--University of California, San Diego, 2006.Title from first page of PDF file (viewed June 13, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 142-162).
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Krynak, Katherine L. "ENVIRONMENTAL INFLUENCES ONAMPHIBIAN INNATE IMMUNE DEFENSE TRAITS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1435590530.
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Hill, David Richard. "THE ROLE OF HYALURONAN IN INNATE INTESTINAL DEFENSE." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1364999901.
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Subramanian, Vignesh Kavitha. "Zinc: An Immunomodulator of Innate Defense against Pathogenic Infection." University of Cincinnati / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1383909171.
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Rose-Martel, Megan. "Innate Mechanisms of Antimicrobial Defense Associated with the Avian Eggshell." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32299.
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During the course of evolution, the avian egg has developed multiple physical and chemical barriers in order to resist microbial challenges. These barriers are essential for the successful reproduction of avian species as well as to maintain safe and nutritious food for human consumption of the table egg. The calcified eggshell is a biomineralized barrier with an integrated organic matrix containing antimicrobial proteins, a hallmark of sophisticated biological structures. Calcium carbonate is deposited onto the outer shell membranes to form the calcified mammillary, palisade and vertical crystal layers; the final layer to be deposited is the outer eggshell cuticle. In this thesis, mass spectrometry-based technology was used to investigate the proteome of the outer cuticle, the mammillary cones and the shell membranes in order to gain insight into biomineralization and antimicrobial functions of the avian eggshell. Proteomics analysis of the eggshell cuticle revealed multiple antimicrobial proteins, supporting the hypothesis that the outermost cuticle layer is the first barrier against invading pathogens. The two most abundant cuticle proteins identified are similar to Kunitz-like protease inhibitor (ovocalyxin-25) and ovocalyxin-32. Multiple antimicrobial proteins were also revealed to be associated with the shell membrane fibres. Among the most abundant proteins were lysozyme C, avian β-defensin-11, ovotransferrin, ovocalyxin-36 and gallin. The biomineralized shell is also an important physical barrier against invading pathogens. Proteomics analysis of the mammillary cones, the initiation sites for shell calcification, revealed several candidate proteins involved in calcitic biomineralization. Promising candidates include nucleobindin-2 and SPARC, two calcium binding proteins previously shown to modulate mineralization. In-depth analysis of the comprehensive proteomes generated by this study revealed the presence of histones in the shell membranes, shell and cuticle compartments. Histones are cationic antimicrobial peptides, which are key molecules of the innate immune defense system of many species. This thesis reports the minimal inhibitory concentrations and minimal bactericidal concentrations of histones extracted from avian erythrocytes against Gram-positive, Gram-negative and antibiotic-resistant bacteria. Results suggest that the underlying antimicrobial mechanism is based on the interaction between histones and lipopolysaccharides / lipoteichoic acids, which are negatively charged components of bacterial cell membranes. Histones also inhibit the growth of Gram-positive biofilms; the minimal biofilm eradication concentrations were determined for S. aureus and methicillin-resistant S. aureus (MRSA). Sensitive proteomics analyses have provided great insight into the protein constituents of the eggshell matrix, with two primary roles in the innate immune defense of the egg: regulation of calcitic biomineralization and antimicrobial protection. Further research on these proteins and their functions can provide a new focus for selective breeding programs looking to enhance the egg’s natural defenses, or provide inspiration for alternatives to conventional antibiotics, such as the histones.
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Madera, Laurence. "Mechanisms of immune response regulation by innate defense regulator peptides." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43066.
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The growing threat of antibiotic-resistant bacteria necessitates the development of new anti-infective therapeutics. Innate defense regulator (IDR) peptides are a novel class of immunomodulatory agents shown to combat bacterial pathogens in murine models of infection via the augmentation of host immune functions, including the stimulation of chemokine production and enhancement of leukocyte recruitment, while suppressing bacterial-induced inflammation. Although IDR-peptides present the potential for future broad-range anti-infective agents, our limited understanding of how they modulate host immunity remains an obstacle in their development as clinical therapeutics. I hypothesized that IDR-peptides impact host immunity by modulating the immune responses of monocytes, a cell population necessary for IDR-mediated protection against infection. In this study, IDR-1002 was found to be a multi-faceted regulator of monocyte migration. IDR-1002 induced the production of monocyte-specific chemokines MCP-1 and MCP-3, as well as neutrophil-specific chemokines, IL-8 and GRO-α in human peripheral blood mononuclear cells (PBMCs), correlating with the activation of the mitogen-activated protein kinases (MAPK), p38 and extracellular-regulated kinase (ERK)-1/2, in monocytes. IDR-1002 was also found to enhance human monocyte migration towards chemokines through the enhancement of β1-integrin-mediated adhesion to fibronectin via regulation of the phosphatidylinositol-3-kinase (PI3K)-Akt signalling pathway. In addition, IDR-1002 increased monocyte responsiveness to the chemokines MIP-1α and RANTES via modulation of CCR5 expression. These results demonstrate an overall promotion of monocyte motility by IDR-1002. In contrast to the immune-strengthening effects of IDR-1002, the production of pro-inflammatory cytokines in human PBMCs stimulated with bacterial lipopolysaccharide (LPS) was suppressed by the peptide, and correlated with a suppression of LPS-induced NFκB and p38 MAPK signalling and activation of PI3K-Akt signalling in monocytes. These results demonstrate that IDR-peptides are potent modulators of human monocyte function via their extensive regulation of monocyte signalling networks, potentially accounting for their multifunctional effects on host immunity in murine models of bacterial infection.
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Kohatsu, Luciana. "Novel roles for human lectins in innate defense against pathogens." Diss., Restricted to subscribing institutions, 2006. http://proquest.umi.com/pqdweb?did=1155572661&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Sang, Yongming. "Porcine innate antiviral immunity : host defense peptides and toll-like receptors." Diss., Manhattan, Kan. : Kansas State University, 2008. http://hdl.handle.net/2097/960.
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Kim, Yeojung. "The Role of Hyaluronan in Innate Host Defense against Bacterial Infection." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1499340461710323.
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Ribeiro, Andrea. "Activation of innate immune defense mechanisms contributes to polyomavirus BK-associated nephropathy." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-165813.
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Sherwood, Cara. "ARSENIC ALTERS KEY COMPONENTS OF INNATE IMMUNE DEFENSE IN AIRWAY EPITHELIAL CELLS." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/203448.
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Chronic exposure to arsenic-contaminated drinking water is correlated with obstructive lung disease (i.e. chronic obstructive pulmonary disease (COPD), bronchiectasis), reduced lung function and other respiratory effects (e.g. chronic cough, chest sounds). Researchers have associated arsenic exposure with reduced airway immunity. The airway epithelial innate immune system protects underlying tissue from inhaled particulates and pathogens through a variety of mechanisms. Such defects in innate immunity are associated with chronic bacterial infections and development of obstructive airway diseases, including COPD and bronchiectasis. We hypothesize that arsenic exposure may lead to recurrent lung infection and eventual obstructive lung disease by compromising mechanisms essential in airway innate immunity. In the work presented herein we evaluated the effects of arsenic on airway epithelial barrier properties, wound repair capacity, and signaling pathways essential in innate immunity. We previously published that acute (24 hr) arsenic (0.4-3.9 μM as Naarsenite) slowed wound repair in a human bronchial epithelial cell line (16HBE14o-). In the first study we hypothesized arsenic may be affecting wound repair by altering Ca²⁺ signaling that is important in multiple aspects of wound repair, including cell migration. We found wound-induced Ca²⁺ signaling was largely mediated by paracrine ATP in 16HBE14o- cells, and acute (24 hr) arsenic (0.8, 3.9 μM) exposure reduced ATPmediated Ca²⁺ signaling. We identified functional reductions in the ATP receptors P2Y₂ and P2X₄ following arsenic exposure. Both of these receptors are essential in airway innate immunity (e.g. mucociliary clearance). In the second study we found similar reductions in wound repair capacity and ATP-mediated Ca²⁺ signaling in 16HBE14o cells using a chronic (4-5 week) low-dose (0.13, 0.33 μM) arsenic exposure representative of U.S. drinking water standards. Further, wound-induced Ca²⁺ signaling was reduced in primary cultured tracheal cells derived from mice fed arsenic-free or arsenic-supplemented (50 ppb; 1μM=75 ppb) water for four weeks prior to experimentation. In the last study we demonstrated that the structure and function of the airway epithelial barrier was altered by a five-day exposure of arsenic (0.8, 3.9 μM). We conclude that arsenic at environmentally relevant levels compromises key functions in airway epithelial innate immunity that may underlie development of lung disease.
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Möllerherm, Helene Alwina [Verfasser]. "Innate immune defense against zoonotic bacterial infections at physiological oxygen conditions / Helene Alwina Möllerherm." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2017. http://d-nb.info/1150337141/34.
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Jia, Ting. "Analysis of CC-chemokines and monocyte trafficking : in the innate immune defense against listeria monocytogenes /." Access full-text from WCMC, 2009. http://proquest.umi.com/pqdweb?did=1692648201&sid=1&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Tao, Yufeng. "Studies on the membrane receptors sensing heat shock protein 70 in an innate defense system." Kyoto University, 2006. http://hdl.handle.net/2433/136502.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第12664号
農博第1587号
新制||農||934(附属図書館)
学位論文||H18||N4190(農学部図書室)
UT51-2006-U369
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 北畠 直文, 教授 吉川 正明, 助教授 谷 史人
学位規則第4条第1項該当
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Guedes, Mariana da Rocha Soares. "Mitochondria and peroxisomes : role within cellular antiviral defense." Master's thesis, Universidade de Aveiro, 2014. http://hdl.handle.net/10773/12960.
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Mestrado em Biomedicina MolecularThe present paper presents a review and compilation of all the scientifically relevant bibliography to date, regarding the antiviral signalling pathways implicated in the cellular innate immune system in humans. Emphasizing the mitochondrial antiviral signalling adaptor (MAVS), this paper explores the special features of the signal transduction pathways and their components in two specific organelles: mitochondria and peroxisomes. These pathways, ultimately, result in the expression of interferon-stimulated genes (ISGs), which are primarily responsible for fighting against viral replication, viral particle assembly and virion release within the cell. In this paper, several proposals for further investigation are also presented, since there is still a lot to learn about the role of peroxisomes in the antiviral innate immune responses.
O presente trabalho propõe-se a rever e compilar toda a bibliografia cientificamente relevante até à data, no que respeita as vias de sinalização antivirais implicadas na imunidade celular inata em células humanas. Com ênfase na proteína adaptadora MAVS, este trabalho explora as particularidades das vias de transdução de sinal e respetivos intervenientes em dois organelos celulares específicos: mitocôndrias e peroxissomas. Estas vias, em última instância, resultam na expressão de genes estimulados por interferões (ISGs), principais responsáveis pelo combate celular eficaz contra a replicação viral, montagem de partículas virais e libertação de viriões na célula infetada. Neste trabalho são ainda apresentadas propostas para investigações futuras, uma vez que ainda muito pouco se sabe sobre o papel dos peroxissomas nas respostas imunitárias inatas contra infeções virais.
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Bergsson, Gudmundur. "Antimicrobial polypeptides and lipids as a part of innate defense mechanism of fish and human fetus /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-546-1/.
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Long, Matthew Eugene. "Manipulation of the innate immune response and evasion of macrophage host defense mechanisms by Francisella tularensis." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/1683.
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Tularemia is a potentially fatally illness caused by the facultative intracellular Gram-negative bacterium Francisella tularensis. Virulent strains of F. tularensis can cause a fatal disease after inhalation of a few as ten organisms. Due to the highly pathogenic features of Francisella, it has been designated as a Tier 1 select agent, meaning that its possession and handling is highly restricted. Macrophages are phagocytes that play a central role in the innate immune response to infection that can be used by certain pathogens, including Francisella, as a niche for bacterial replication and dissemination during infection. After infection of macrophages Francisella escapes from the phagosome and replicates in the cytosol, however the bacterial factors required for these aspects of virulence are incompletely defined.
Here we describe the isolation and characterization of F. tularensis subspecies tularensis strain Schu S4 mutants in iglI, iglJ, and pdpC, three genes located in the Francisella Pathogenicity Island. Our data demonstrate that these mutants were unable to replicate in macrophages due to a defect in phagosome escape. However, a small percentage of pdpC mutants were able to reach the cytosol and replicate moderately. Both iglJ and pdpC mutants were highly attenuated for virulence in a mouse intranasal infection model, however pdpC but not iglJ mutants, were able to disseminate from the lung before eventual clearance. These data demonstrated that the FPI genes tested were essential for F. tularensis Schu S4 virulence, but suggest that they may have different functions due to the unique phenotype observed for pdpC mutants.
Our studies also characterized the role of F. tularensis O-antigen and capsule to facilitate interactions with components of the serum complement system; demonstrating that the O-antigen is required for binding of IgM to the bacteria in order to initiate complement opsonization. IgM dependent complement opsonization of both F. tularensis Schu S4 and LVS strains facilitated enhanced phagocytosis of the bacteria by complement receptors 3 and 4 of human macrophages. In addition, we examined the mechanisms of macrophage cytotoxicity and proinflammatory cytokine secretion that was induced after infection with a Schu S4 LPS O-antigen and capsule mutant. The response to the mutant was dependent on phagosome escapes, suggesting a cytosolic pattern recognition receptor was involved in recognition of the bacteria. We found that the cytotoxic and proinflammatory responses had both similar and distinct requirements between human and murine macrophages. Infection with the O-antigen mutant induced robust proinflammatory cytokine secretion that was dependent on caspase-1, cathepsin B, and ASC while cytotoxicity was partially dependent on these molecules. Importantly, we demonstrated that wild-type Schu S4 predominately activated apoptotic caspases, and not inflammatory caspases, during infection and had a blunted cytotoxic response. This was in contrast to the robust cytotoxicity and activation of inflammatory caspases after infection with the non-virulent strain LVS. Together, these studies demonstrated that the Schu S4 LPS O-antigen and capsule are required for evasion of macrophage cytosolic host defense mechanisms.
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Baurhoo, Bushansingh. "Reduction of salmonella-induced enteric and systemic inflammation by mannan-oligosaccharide prebiotic through improvement of innate defense mechanism." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=106385.
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Salmonella Enteritidis has become a major burden in human health due to emergence of multiple-antibiotic resistant strains that has made antibiotic treatment difficult. Today, there is urgency to develop efficacious alternatives to the utilization of sub-therapeutic antibiotics in poultry. We, therefore, hypothesized that, in contrast to sub-therapeutic antibiotics, mannose-rich oligosaccharides (MOS), a natural prebiotic, would improve intestinal innate defense mechanisms in healthy and Salmonella-infected chickens, and abrogate S. Enteritidis invasion of intestinal epithelium, thus mitigating Salmonella-induced intestinal and systemic inflammation. In contrast to virginiamycin (VIRG) and bacitracin (BACT) antibiotics, dietary MOS (0.2%) significantly improved intestinal innate defensive mechanisms in chickens raised under good sanitary conditions, as demonstrated by increased development of mucin-secreting goblet cells that ultimately secretes higher amounts of mucins, and microflora enrichment with beneficial bacteria, especially bifidobacteria. Moreover, MOS was equally effective as VIRG and BACT in the control of intestinal E. coli. But, at higher (0.5%) than the recommended (0.2%) dosage, MOS conferred no additional intestinal health benefits. To examine whether MOS may control S. Enteritidis through improvement of host's innate defense mechanisms, young chicks were deliberately infected with S. Enteritidis. Interestingly, whereas control (CTL) chicks suffered from drowsiness, diarrhea, starvation, exfoliation of epithelial cells and damaged villi integrity, such classic signs of Salmonella-induced enteric inflammation were abrogated by MOS and VIRG. However, IL 12 down-regulation by MOS revealed that MOS terminated S. Enteritidis-induced enteric inflammation earlier than VIRG. Whereas VIRG relied mostly on its bactericidal properties, MOS significantly improved host's mucins-mediated defense mechanism against S. Enteritidis. MOS increased secretions of neutral and acidic mucins through increased numbers of neutral and acidic goblet cells, rather than increased mucins-secreting capacity of goblet cells, as indicated by non-differential MUC 2 expressions between infected chicks fed VIRG and MOS. MUC 1 down-regulation by MOS indicated that MOS more significantly reduced S. Enteritidis damage of epithelial cells than VIRG. Evidently, intestinal villi were longer and healthier in S. Enteritidis-infected chicks fed MOS than VIRG. S. Enteritidis invasion of the intestinal epithelium triggers extra-intestinal infections and systemic inflammation. A Salmonella LPS-induced systemic inflammation chicken model and microarray analysis approach was employed to determine whether MOS, in contrast to VIRG, may potentially mitigate systemic inflammation, and reduce glucose mobilization during late systemic inflammation. MOS inherently induced IL 3 expression in non-challenged control hosts. However, consequent to LPS challenge, IL 3 was induced in VIRG hosts but not differentially expressed in MOS hosts, therefore revealing that MOS counteracted LPS's detrimental inflammatory effects. Indeed, the lower energy demands of LPS-challenged birds fed MOS were sufficiently met through TCA citrate-derived energy, as indicated by ATP citrate synthase (CS) up-regulation. Contrastingly, VIRG host's elevated energy requirements increased gene expressions for intestinal gluconeogenesis (PEPCK) and liver glycolysis (ENO2). In conclusion, this study revealed the mechanisms by which MOS, in contrast to VIRG, i) enhanced host's intestinal innate defense mechanisms against Salmonella; ii) terminated Salmonella-induced intestinal and systemic inflammation earlier; iii) and modulated innate immunity to markedly reduce glucose mobilization during late systemic inflammation. Therefore, MOS represents a biological strategy that can prevent or treat S. Enteritidis infections in poultry and humans, without posing the risk of developing antibiotic-resistance.Le développement des souches de S. Enteritidis résistantes aux antibiotiques compromet le traitement de la salmonellose avec des antibiotiques thérapeutiques dans la santé humaine. Donc, il y a urgence de développer des remplaçants efficaces aux antibiotiques de croissance chez la volaille. Notre hypothèse était que contrairement aux antibiotiques sous-thérapeutiques, l'utilisation d'oligosaccharides riches en mannose (MOS), un prébiotique naturel, améliorerait les mécanismes de défense intestinale innés chez les poulets sains et infectés par la Salmonelle, prévenant ainsi l'invasion de l'épithélium intestinal par S. Enteritidis et atténuant de ce fait l'inflammation intestinale et systémique induite par la Salmonelle. Contrairement aux antibiotiques tels que la virginiamycine (VIRG) et la bacitracine (BACT), le MOS (0.2%) a amélioré les mécanismes de défense intestinale innés chez les poulets, tel que démontré par la prolifération des cellules à gobelet sécrétant ainsi plus de mucines, et l'enrichissement de la flore microbienne avec des bactéries bénéfiques, particulièrement bifidobacteria. Le MOS était aussi efficace que la VIRG et BACT dans le contrôle intestinal d'E. coli. Mais, à une dose plus élevée (0.5%) que le dosage recommandé (0.2%), le MOS n'a démontré aucun bénéfice additionnel dans la santé intestinale. Afin d'examiner si le MOS pourrait mieux contrôler le S. Enteritidis intestinale tout en améliorant les mécanismes de défense innés, des poussins ont été infectés avec S. Enteritidis. Les poussins infectés alimentés avec la diète témoin ont souffert de somnolence, diarrhée, perte d'appétit, exfoliation des cellules épithéliales et endommagement des villosités, tandis que ces symptômes ont été abrogés par le MOS et la VIRG. Cependant, une réduction dans l'expression de IL 12 par le MOS relatif à la VIRG démontra que le MOS termina l'inflammation induite par S. Enteritidis plus tôt. Le MOS augmenta la sécrétion des mucines neutres et acides en augmentant les nombres de cellules à gobelet neutres et acides plutôt que par une sécrétion accrue de mucines par chaque cellule à gobelet; l'expression de MUC 2 était inchangé entre les poussins infectés et alimentés avec la VIRG ou MOS. En revanche une diminution dans l'expression du MUC 1 induite par le MOS indiqua que le MOS a réduit les dommages des cellules épithéliales causés par S. Enteritidis. Les villosités intestinales étaient plus longues et saines chez les poussins infectés et alimentés avec le MOS que la VIRG. L'invasion de l'épithélium intestinal par S. Enteritidis cause l'inflammation systémique. Pour déterminer si le MOS, contrairement à la VIRG, pourrait atténuer l'inflammation systémique et réduire la mobilisation de glucose pendant la phase avancée de l'inflammation, nous avons utilisé un modèle d'inflammation systémique et des analyses de microréseaux d'ADN. Le MOS induit l'expression d'IL 3 chez les poulets non soumis au LPS. Mais, suite à l'injection de LPS, l'expression d'IL 3 a été augmentée chez les poulets alimentés avec la VIRG que le MOS, indiquant que le MOS a aboli les effets inflammatoires du LPS. En effet, une augmentation dans l'expression de l'ATP citrate de synthase (CS) indiqua que les demandes énergétiques inférieures des poulets injectés avec du LPS et alimentés avec le MOS ont été suffisamment satisfaites par l'énergie dérivée par le cycle de l'acide citrique. Par contre, les besoins énergétiques plus élevés chez les poulets alimentés avec la VIRG ont entrainé une augmentation de l'expression des gènes de la néoglucogenèse intestinale (PEPCK) et de la glycolyse du foie (ENO2). En conclusion, cette étude a démontré les mécanismes par lesquels le MOS, contrairement à la VIRG, i) a augmenté les mécanismes de défense intestinaux innés contre la Salmonelle; ii) a mis fin à l'inflammation intestinale et systémique causée par la salmonelle plus tôt; iii) et a réduit la mobilisation du glucose pendant une phase avancée dans l'inflammation.
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Ribeiro, Andrea [Verfasser], and Franz-Xaver [Akademischer Betreuer] Beck. "Activation of innate immune defense mechanisms contributes to polyomavirus BK-associated nephropathy / Andrea Ribeiro. Betreuer: Franz-Xaver Beck." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1048361497/34.
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Roberts, Sean Anthony. "A GENE THERAPY APPROACH TO THE INHIBITION OF HIV-1 REPLICATION BY RESTORATION OF INNATE ANTIVIRAL DEFENSE PATHWAYS." Diss., Temple University Libraries, 2010. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/99935.
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Microbiology and ImmunologyPh.D.
Since it emerged as an infectious agent in 1981, the human immunodeficiency virus type 1 (HIV-1) is continually disseminated and remain fatal to the majority of those infected. Strategies including highly active retroviral therapies (HAART) with nucleoside analogues and protease inhibitors have shown limited success in therapy due to the virus' ability to evolve rapidly at every replication cycle as a consequence of it's highly error prone reverse transcriptase, generating resistant retroviral strains and in addition to latent HIV-1 reservoirs. Thirty years of research efforts to find a cure or to generate a vaccine has been met with failure. It is, therefore, of necessity to broaden our paradigm of therapy for the treatment and eventual cure of HIV-1 infection. In this study, I look beyond the current anti-retroviral strategies and instead rely on the mammalian host immune system to inhibit HIV-1 replication through molecular genetic manipulation. Here, we approach the inhibition of HIV-1 replication by up-regulation of the innate antiviral pathway that is natural to mammalian cells. HIV-1 derived self-inactivating lentiviral (SIN) vectors were designed and constructed to deliver the antiviral payloads of two antiviral enzymes, p68 kinase (PKR) and 2'-5' oligoadenlyate synthetase (2-5OAS), to target cell, SupT1 lymphoblastoid cells and CD4+ T lymphocytes under the control of a constitutive cytomegalovirus (CMV) promoter. These data here demonstrates a significant inhibition of HIV-1 replication in cells transduced with the anti HIV-1 transgenes PKR and 2-5OAS as determined by HIV-1 induced syncytia formation and HIV-1 p24 antigen capture assay. Furthermore, here demonstrated is an increase up-regulation of PKR and 2-5OAS 96 hr post cell transduction in all the clones when compared to pHIV empty vector control. These results demonstrate that the over-expression of PKR and 2-5OAS can inhibit HIV-1 replication and also confirm the involvement of PKR and 2-5OAS in the IFN-associated antiviral pathway against HIV-1 infection.
Temple University--Theses
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Hamad, Shaimaa Kamal. "Developmental gene expression of host defense peptides in immune organs and the small intestine of turkey poults (Meleagris gallopavo)." Thesis, Virginia Tech, 2016. http://hdl.handle.net/10919/73056.
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Host defense peptides (HDPs) are a large group of small positively charged peptides that play an important role in innate immunity. Their role is more critical at early ages when other components of the immune system have not fully developed. There are three classes of avian HDPs: avian beta defensins (AvBDs), cathelicidins (Cath) and liver-expressed antimicrobial peptide 2 (LEAP-2). The objective was to compare expression of HDPs in male turkey poults at day of hatch (D0), D7, D14, D21 and D28 from the thymus, spleen, bursa, duodenum, jejunum and ileum. The expression of AvBD1, AvBD2, AvBD8, AvBD9, AvBD10, AvBD13, Cath2, Cath3 and LEAP-2 was measured using qPCR (n=6 birds/tissue/age). Data were analyzed by one-way ANOVA and Tukey's test, and significance considered at P ≤ 0.05. AvBDs and Caths exhibited greater expression in immune organs than intestinal tissues, with the greatest expression of AvBDs observed in the spleen. The intestinal tissues showed very low expression of AvBDs except for AvBD10 at D0. Similar to AvBDs, Caths expression in the immune organs was greater than the intestinal tissues with the spleen having the greatest expression among immune organs. Conversely, LEAP-2 showed greater expression in the intestinal tissues than in the immune tissues, which showed very low LEAP-2 expression unlike other HDPs. Understanding the differential expression of HDPs could reveal the innate immune status of poults, and may subsequently allow improvement of their health through appropriate mitigation strategies.Master of Science
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Shekhar, Sudhanshu. "A study on the role of lung dendritic cells and their interaction with innate lymphocytes in host defense against a bacterial lung infection." Karger, 2015. http://hdl.handle.net/1993/30622.
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Chlamydia is an obligate intracellular bacterial pathogen that causes a wide spectrum of diseases worldwide. At present, there are no vaccines to prevent chlamydial infections due to poor understanding of how anti-chlamydial immunity ensues. In this study, we employed a variety of in vitro and in vivo systems, including knockout (KO) mice and adoptive transfer, to investigate the role of lung dendritic cells (LDCs) and their relationship with innate lymphocytes, natural killer (NK) and invariant NKT (iNKT) cells, in host defense against chlamydial lung infections in mice. We found that iNKT cells altered the phenotype and cytokine production pattern of LDCs following C. pneumoniae infection. Adoptive transfer of LDCs from infected Jα18-KO mice, which lack iNKT cells, into naïve wild-type (WT) mice promoted Th2 (IL-4) immunity following infection challenge, whereas the transfer of LDCs from the infected WT mice induced protective Th1/Tc1 (IFN-γ) immunity. On the other hand, upon adoptive transfer, LDCs from C. muridarum-infected NK-cell-depleted mice (NK-LDCs) conferred reduced protection after chlamydial challenge than the recipients of LDCs from infected sham-treated mice (NK+LDCs). NK+LDC recipients exhibited an enhanced Th1/Th17, in contrast to Th2, response compared to the NK-LDC recipients. In coculture experiments, NK cells isolated from the infected mice promoted IL-12p70, IL-6, and IL-23 production by LDCs through NKG2D receptor signaling. These findings indicate that iNKT and NK cells condition LDCs to confer protective Th1/Tc1/Th17 immunity against chlamydial lung infection.
We also analyzed the contribution of major LDC subsets, CD103+ and CD11bhi LDCs, in host defense against C. muridarum infection. We found that CD103+ and CD11bhi LDC subsets expanded following chlamydial infection. CD103+ LDCs showed higher expression of costimulatory molecules and greater production of Th1- and Th17-inducing cytokines (IL-12, IL-6 and IL-23) than CD11bhi LDCs. Coculture of Chlamydia-specific CD4+ T cells with LDC subsets revealed that the T cells cultured with CD103+ LDCs produced larger amounts of IFN-γ and IL-17 compared to those with CD11bhi LDCs. To test their function in vivo, we isolated CD103+ and CD11bhi LDC subsets from infected mice and transferred them into naïve syngeneic mice that received chlamydial challenge. CD103+ LDC-recipients showed better protection, as evidenced by their reduced body weight loss, bacterial burden and lung pathology, than CD11bhi LDC recipients. Mice that received CD103+, compared to CD11bhi, LDCs produced enhanced Th1/Th17 cytokines (IFN-γ and IL-17) in the lung and the MLNs. In conclusion, these findings demonstrate that CD103+ LDCs are more efficient in inducing Th1/Th17 immunity to chlamydial infection than CD11bhi LDCs.
Taken together, our findings have provided direct in vivo evidence on the role of LDCs and their conditioning by iNKT and NK cells in generating mucosal T-cell immunity against a bacterial lung infection. The findings have added new knowledge to the field of lung immunology, which have implications for developing prophylactic and/or therapeutic strategies against respiratory diseases.October 2015
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Pyle, Charlie Jacob. "Impact of macrophage zinc metabolism on host defense against Mycobacterium tuberculosis." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469135576.
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Winkle, Sean M., Andrea L. Throop, and Melissa M. Herbst-Kralovetz. "IL-36γ Augments Host Defense and Immune Responses in Human Female Reproductive Tract Epithelial Cells." FRONTIERS MEDIA SA, 2016. http://hdl.handle.net/10150/617371.
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IL-36 gamma is a proinflamatory cytokine which belongs to the IL-1 family of cytokines. It is expressed in the skin and by epithelial cells (ECs) lining lung and gut tissue. We used human 3-D organotypic cells, that recapitulate either in vivo human vaginal or cervical tissue, to explore the possible role of IL-36 gamma in host defense against pathogens in the human female reproductive tract (FRT). EC were exposed to compounds derived from virus or bacterial sources and induction and regulation of IL-36 gamma and its receptor was determined. Polyinosinic-polycytidylic acid (poly I:C), flagellin, and synthetic lipoprotein (FSL-1) significantly induced expression of IL-36 gamma in a dose-dependent manner, and appeared to be TLR-dependent. Recombinant IL-36 gamma treatment resulted in self amplification of IL-36 gamma and its receptor (IL-36R) via increased gene expression, and promoted other inflammatory signaling pathways. This is the first report to demonstrate that the IL-36 receptor and IL-36 gamma are present in the human FRT EC and that they are differentially induced by microbial products at this site. We conclude that IL-36 gamma is a driver for epithelial and immune activation following microbial insult and, as such, may play a critical role in host defense in the FRT.
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Lindenberg, Valesca [Verfasser], Klaus [Gutachter] Pfeffer, and Lutz [Gutachter] Schmitt. "Functional characterization of guanylate binding proteins (GBPs) in the innate and adaptive host defense against pathogens / Valesca Lindenberg ; Gutachter: Klaus Pfeffer, Lutz Schmitt." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2017. http://d-nb.info/1138436828/34.
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Ulvila, J. (Johanna). "Functional genomic analysis of the Drosophila immune response:identification of genes essential for phagocytosis, viral defense and NF-κB signaling." Doctoral thesis, University of Oulu, 2008. http://urn.fi/urn:isbn:9789514289941.
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Abstract Innate immunity provides the first line of defense against invading, pathogenic microorganisms in all multicellular organisms. The fruit fly Drosophila melanogaster has turned out to be an excellent model organism to elucidate mechanisms of innate immune responses because of the highly conserved intracellular signaling cascades mediating these ancient immune functions in flies and mammals. In the present study, RNA interference (RNAi) -based functional genomics were utilized to identify novel components of Drosophila’s immune reactions. Mediators of bacterial phagocytosis, nuclear factor kappa B (NF-κB) signaling and the antiviral RNAi pathway were screened in hemocyte-like S2 cells. Follow-up studies were executed in mammalian cells as well as in Drosophila larvae and adult flies to gain broader significance for the results. Seven novel components essential for efficient phagocytosis of bacteria were identified. Eater was defined as Drosophila’s most important phagocytic receptor showing novel epidermal growth factor (EGF)-repeat -based microbial recognition properties. Additionally, Abelson interacting protein (Abi), capping protein alpha (cpa), 14-3-3ζ, tousled-like kinase (tlk), CG2765 and CG15609 were determined as intracellular effectors of phagocytosis, the three former ones executing their evolutionarily conserved functions through remodeling of the actin cytoskeleton. Eater, together with Scavenger receptor class C, type I (Sr-CI), was demonstrated to be responsible for double-stranded RNA (dsRNA) uptake into S2 cells and, when ectopically expressed, into mammalian cells via clathrin-mediated endocytosis. Proteasome component Pros45 and RNA helicase Belle were established as mediators of the intracellular RNAi pathway, whereas essential roles in antimicrobial signaling via the immune deficiency (Imd) pathway were addressed for Inhibitor of apoptosis 2 (Iap2) and Tak1-associated binding protein (TAB). Iap2 and TAB were shown to affect nuclear translocation of NF-κB -like transcription factor Relish. The present study identifies several novel mediators of the Drosophila immune response and provides insight into mechanisms of fly host defense. As insects serve as vectors of human diseases (e.g. malaria), knowledge about Drosophila immune mechanisms may help to better understand the transmission and pathogenesis of these diseases and develop treatments to fight these infections. Additionally, knowledge gained from model organisms serves as valuable background information, often conducting human research into new tracksTiivistelmä Synnynnäinen immuniteetti on elintärkeä puolustusjärjestelmä taudinaiheuttajia vastaan. Kodeissakin yleinen banaanikärpänen, Drosophila melanogaster, on osoittautunut erinomaiseksi synnynnäisen immuniteetin tutkimusmalliksi, erityisesti teknisesti yksinkertaisen ja eettisesti ongelmattoman geneettisen muunneltavuutensa ansiosta. On myös havaittu, että solunsisäiset, immunologisia signaaleja välittävät mekanismit ovat evoluutiossa hyvin säilyneitä. Hyvin usein samankaltaiset geenituotteet toimivat signaalinsiirtäjinä sekä kärpäsen että ihmisen soluissa. Tämän työn tarkoituksena oli RNA-häirintää (RNAi) sekä muita nykyaikaisia solu- ja molekyylibiologisia tutkimusmenetelmiä hyödyntäen tunnistaa uusia kärpäsen synnynnäiselle immuunipuolustukselle välttämättömiä geenituotteita. Bakteerien fagosytoosille, viruspuolustukselle ja tumatekijä nuclear factor kappa B:n (NF-κB) välittämälle signaloinnille välttämättömiä signalointimolekyylejä pyrittiin identifioimaan laajan mittakaavan RNA-häirintään perustuvilla seuloilla kärpäsen soluissa. Saatujen tulosten merkitystä nisäkkäiden immuunipuolustukselle tutkittiin myös hiiren soluissa. Seitsemän geenituotteen osoitettiin olevan bakteerien fagosytoosille tärkeitä kärpäsen soluissa. Aiemmin tuntematon geenituote, joka nimettiin Eateriksi, osoitettiin kärpäsen tärkeimmäksi bakteereja fagosytoivaksi reseptoriksi. Eaterin solun ulkoisen osan osoitettiin tunnistavan taudinaiheuttajia uudella epidermaalisen kasvutekijän (epidermal growth factor, EGF) kaltaisella toistosekvenssillä. Myös useiden solun tukirankaan, sytoskeletoniin, liittyvien proteiinien (Abi, cpa, 14-3-3ζ) sekä aiemmin vähemmän tunnettujen geenituotteiden (CG2765, CG15609, tlk) osoitettiin osallistuvan bakteerien fagosytoosiin. Näistä kolmen ensinmainitun immunologinen tehtävä havaittiin evoluutiossa säilyneeksi, kärpäsestä hiireen. Eaterin, yhdessä kärpäsen toisen scavenger reseptorin (Sr-CI) kanssa, havaittiin myös toimivan kaksijuosteisen RNA:n (dsRNA) reseptoreina kärpäsen soluissa, mahdollistaen helpon ja tehokkaan RNA-häirinnän. RNA-häirinnän, ja siten mahdollisesti myös viruspuolustuksen, välittäjiksi identifioitiin proteasomin alayksikkö Pros45 ja RNA-helikaasi Belle. Lisäksi Inhibitor of apoptosis 2 (Iap2) ja Tak1-associated binding protein (TAB) todettiin kärpäsen immune deficiency (Imd) signalointireitin komponenteiksi, jotka osallistuvat antimikrobisten peptidien tuotantoon välittämällä NF-κB:n kaltaisen kärpäsen transkriptiotekijän (Relish) siirtymisen tumaan aktivoimaan immuunipuolustusta välittävien geenien ilmentymistä. Tämän tutkimuksen tulokset valottavat banaanikärpäsen immuunipuolustuksen mekanismeja. Koska hyönteiset toimivat monien ihmisten infektiotautien välittäjinä, kärpäsen immuniteetin tuntemus luo mahdollisuuksia kehittää hoitoja näitä tauteja vastaan. Lisäksi malliorganismeista saatu tieto luo uusia teorioita ja näkökulmia, johtaen usein myös lääketieteellistä tutkimusta uusille raiteille
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Arranz, Trullén Javier. "Unveiling the multifaceted antimicrobial mechanism of action of human host defence RNases." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/400666.
Full textAbstract:
La presente tesis doctoral se encuentra integrada dentro del estudio a gran escala de la
estructura-función de las ribonucleasas antimicrobianas humanas. Estas proteínas catiónicas
y de bajo peso molecular son secretadas por la mayoría de los organismos vertebrados
agrupándose dentro la superfamilia de la ribonucleasa A, una de las enzimas mejor
caracterizadas del siglo XX. De interés remarcable podríamos considerar su amplio abanico
de propiedades biológicas, teniendo en cuenta su diverso historial de propiedades
biológicas no catalíticas, convirtiéndolas en un buen modelo de proteínas multifunción.
Junto a su principal característica como enzima catalizador de ácidos ribonucleicos, es
importante destacar también otro tipo de propiedades biológicas no menos esenciales,
como su actividad antimicrobiana, que comparten miembros distantes de la familia
sugiriendo una función ancestral en el sistema inmune. Además, se ha visto que la
expresión de algunas RNasas humanas puede ser inducida en procesos infecciosos. En
particular, las RNasas estudiadas en este trabajo, las RNasas humanas 3, 6 y 7, se expresan
principalmente en eosinófilos, monocitos y células epiteliales, respectivamente. Estas
proteínas muestran una alta cationicidad debido a su alta proporción de residuos básicos y
una notable actividad antimicrobiana frente a una amplia gama de patógenos humanos.
Nuestro grupo de investigación posee una larga trayectoria en el estudio del mecanismo de
acción de las ribonucleasas humanas y el trabajo teórico-experimental que se presenta en
esta tesis ha contribuido a consolidar el actual proyecto de investigación. Los principales
avances llevados a cabo por la presente tesis doctoral se enumeran a continuación:
- La caracterización del mecanismo antimicrobiano de la ribonucleasa 6, evaluando
sus propiedades microbicidas frente a patógenos y modelos de membrana.
Concretamente se ha revelado su actividad aglutinadora además de demostrarse
que su actividad antimicrobiana está localizada básicamente en su extremo Nterminal.
- La resolución de la primera estructura tridimensional de la ribonucleasa 6, obtenida
a 1.72 Å, que ha permitido asentar las bases estructurales para futuros estudios
funcionales. Análisis complementarios sobre su caracterización cinética y predicción
de complejos con diferentes ligandos han revelado sitios de unión y de catálisis que
posteriormente han sido confirmados mediante mutagénesis dirigida.
- El estudio de la efectiva actividad antipatogena a nivel intracelular que presentan las
ribonucleasas 3,6 y 7 asi como sus péptidos derivados N-terminales frente a
micobacterias en un modelo de macrófagos infectados.
- La expansión del conocimiento sobre las bases antipatogenas de diferentes péptidos
y proteínas antimicrobianas que participan en la erradicación de las infecciones por
micobacterias, asi como las terapias derivadas.
- La caracterización del mecanismo antimicrobiano de los 8 peptidos N-terminales
derivados de las ribonucleasas frente a Candida albicans, como modelo de patógeno
eucariota
Como conclusión, los resultados presentados en esta tesis contribuyen a profundizar en la
comprensión de las bases moleculares del papel que desempeñan algunas ribonucleasas en
el sistema inmune y expandir el proyecto al diseño de agentes terapéuticos basados en
péptidos antimicrobianos con el objetivo de erradicar enfermedades infecciosas causadas
por patógenos resistentes.The present doctoral thesis is integrated into the large-scale study of the structure and function of human antimicrobial ribonucleases. These cationic and low molecular weight proteins are grouped into the ribonuclease A superfamily, considered one of the best characterized enzymes of the twentieth century. The RNase A superfamily is specific for vertebrates and has attracted remarkable interest due to the diversity of displayed biological properties; and represents an excellent example of a multifunctional protein´s family. Together with the main enzymatic activity we must highlight other biological properties such as the angiogenic, immunomodulatory and antimicrobial activities. The reported antimicrobial activity of distantly related family members in early vertebrates suggests that the family arouse with an ancestral function in host defence. Besides, the expression of several human RNases has been reported to be induced by infection. In particular, the RNases studied in this work, the human RNases 3, 6 and 7, are mainly expressed in eosinophils, monocytes and epithelial cells respectively. These proteins show a high cationicity due to the high proportion of basic residues and a remarkable antimicrobial activity against a wide range of human pathogens. Our research group has a consolidated experience in the study of the mechanism of action of human ribonucleases and the experimental work presented in this thesis is contributing to this overall research project. The main results achieved by the present PhD study are outlined below: - The characterization of the antimicrobial mechanism of RNase 6, both in bacteria cell cultures and using membrane models. Results highlight that the antimicrobial and cell agglutinating activities are mainly located at the N-terminus. - The resolution of the first three-dimensional structure of ribonuclease 6, obtained at 1.72 Å, which has set the structural basis for future functional studies. The kinetic characterization of RNase 6 mutant variants and the prediction of protein- substrate complexes have identified the enzyme nucleotide binding sites. - The study of the intracellular activity of ribonucleases 3, 6 and 7 and their derived Nterminal peptides against intracellular resident mycobacteria using a macrophage infected model. - The analysis of the anti-pathogenic mechanism of action of human antimicrobial proteins and peptides in mycobacterial infections and their applied therapies. - The comparative characterization of the antimicrobial mechanism of action of human RNases and their N-terminal derived peptides towards Candida albicans, as an eukaryote pathogen model. The results presented in this thesis will contribute to the understanding of the role of human RNases in the immune system and provide the structure- function basis to expand the initial project into the design of novel peptide mimetic therapeutics agents towards the eradication of resistant infectious diseases.
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Zilbauer, Matthias. "Innate immune defence to Campylobacter jejuni." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1445170/.
Full textAbstract:
Campylobacter jejuni is the most prevalent cause of bacterial diarrhoea worldwide and is frequently associated with severe post-infectious complications such as the Guillain-Barre syndrome. Despite the serious health burden caused by the bacterium disease pathogenesis remains ill defined. Human (3-defensins (hBDs)), a family of epithelial antimicrobial peptides, are a major component of host innate defence at mucosal surfaces. In the present study we investigated the effect of C. jejuni on intestinal epithelial innate responses. Up-regulation of IL-8, hBD-2 and hBD-3 gene and peptide expression was observed in Caco-2 and HT-29 cell-lines in response to C. jejuni strains 11168H and 81-176. Furthermore, recombinant hBDs were found to exhibit potent bactericidal activity against C. jejuni suggesting a major role for these peptides in disease pathogenesis. Secondly, we aimed to identify host receptor(s) involved in sensing of C. jejuni and initiating innate defence. Given the invasive nature of infection, we investigated the potential role of cytoplasmic nucleotide-binding oligomerisation domain (NOD) proteins. Using small interfering (si) RNA targeting NODI and transfection of NOD2 overexpression plasmids, we identified NODI as a major pattern recognition receptor involved in mediating innate host defence to C. jejuni while NOD2 was found to play a minor role. Additionally, reduced NODI expression resulted in an increased number of intracellular C. jejuni thus highlighting a critical role for NODI mediated antimicrobial defence in limiting infection. In the final part of the study an ex-vivo model of C. jejuni infection using human intestinal biopsies was developed. Additionally, a vertical diffusion chamber system was utilised to improve culture conditions in C. jejuni infection models. In conclusion, this study highlights the important role of intestinal innate host defence to C. jejuni. The development of new and improved models of infections has the potential to provide previously unavailable opportunities to study C. jejuni disease pathogenesis.
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Boughan, P. K. "Innate immune defence to Helicobacter pylori." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1444551/.
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Helicobacter pylori exhibits tropism for the human stomach causing a spectrum of complications ranging from gastritis to gastric cancer in susceptible individuals. The mechanism(s) that allow the bacteria to persist and cause disease are unfolding. p-defensins are a family of endogenous, epithelial anti-microbial peptides that engage in host defense most prominently at mucosal surfaces. We and others have previously shown that human p-defensin (hBD)-2 and -3 are potent bactericidal agents against H. pylori. At present the identity of signalling pathways involved in host-bacterial cross talk leading to modulation of host antimicrobial immunity are unknown. The present study firstly investigated the potential role of bacterial virulence factors in mediating human p-defensin gene expression during H. pylori infection. AGS gastric epithelial cells were infected with cytotoxic H. pylori strains (60190, 84-183) and isogenic mutant strains (cagA-, cagE-, vacA- and CagPAl-). Human p-defensin (hBD2 & -3) gene expression quantified by RT-PCR and p-defensin transcriptional regulation was followed by transient transfection studies utilising hBD2 and -3 promoter luciferase constructs. We found hBD2 induction was dependent upon an intact cagPAI and minimal involvement was observed for the bacterial virulence factors CagA and VacA in modulating P-defensin expression. We sought to investigate the bacterial component responsible for instigating epithelial innate immune responses. Through the use of siRNA for NODI we determined a role for NODI-dependent NF-kB activation in mediating hBD2 but not hBD3 expression. Experiments utilising specific inhibitors of the MAP Kinase pathways directed us to delineate the role of each pathway in modulating p-defensin expression by the activation of stably transfected conditional MAP Kinase mutants. These studies revealed critical involvement of ERK pathway in the regulation of hBD3 but not hBD2 gene expression. Signalling upstream of ERK was explored and revealed EGFR as the host receptor responsible for detection and initiation of hBD3 gene and peptide production. Our studies demonstrated a crucial role for NODI in H. /Ty/ort-mediated hBD2 but not hBD3 expression and implicate EGFR transactivation in mediating hBD3 but not hBD2 expression, thus indicating two distinct regulatory mechanisms at play during innate immune host response to H. pylori infection.
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Rose, Felicity. "The elucidation of antimicrobial activity in the human gastrointestinal tract." Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285756.
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Lakkis, Sara. "Résistance systémique induite par Pseudomonas fluorescens et Bacillus subtilis chez des génotypes de vigne de différentes sensibilités au mildiou et à la pourriture grise Strengthening Grapevine Resistance by Pseudomonas fluorescens PTA-CT2 Relies on Distinct Defense Pathways in Susceptible and Partially Resistant Genotypes to Downy Mildew and Gray Mold Diseases." Thesis, Reims, 2019. http://www.theses.fr/2019REIMS025.
Full textAbstract:
Le mildiou causé par l'oomycète Plasmopara viticola et la pourriture grise causée par le champignon Botrytis cinerea font partie des maladies les plus menaçantes dans les vignobles. La stratégie actuelle de lutte contre ces maladies repose entièrement sur l'utilisation de fongicides chimiques. L'utilisation de bactéries bénéfiques apparaît comme une stratégie durable permettant de contrôler diverses maladies. Cette méthode est principalement basée sur l'activation de l’immunitaire innée des plantes, se traduisant par une résistance systémique induite (ISR). Une telle stratégie est rendue possible pour lutter contre la pourriture grise grise, mais son efficacité contre le mildiou est encore inconnue et les mécanismes impliqués dans l’état de résistance restent à explorer. Dans cette étude, nous nous sommes focalisés sur la capacité de Pseudomonas fluorescens PTA-CT2 à induire une ISR chez la vigne contre P. viticola et B. cinerea, en utilisant deux cultivars greffés avec des sensibilités différentes au mildiou ; le Pinot noir comme sensible et le Solaris comme partiellement résistant. Nous avons ensuite examiné les différences et les similitudes entre l'ISR et la potentialisation induite par Bacillus subtilis et P. fluorescens après infection par P. viticola. Sur la base de la différence de sensibilité phénotypique des cultivars, nous avons exploré les mécanismes associés à l’ISR avant et après infection par l’agent pathogène. Nos résultats ont montré que P. fluorescens induit une ISR contre P. viticola et B. cinerea en potentialisant des voies de défense communes et distinctes en fonction de l'immunité basale du génotype. Bien que P. fluorescens élicite une synthèse hormonale et une stimulation de la photosynthèse avant l'infection chez les deux génotypes, nos résultats soulignent le rôle important des voies de défense dépendantes de l'acide salicylique (SA) et de la synthèse de phytoalexines, mais pas celles dépendantes de JA et ET dans l’ISR contre P. viticola chez les deux cultivars. L'état de potentialisation repose en outre sur la forte expression du gène HSR impliqué dans la mort cellulaire de type HR chez Solaris, alors que chez le cultivar sensible, il implique une accumulation d'ABA, plutôt qu'une mort cellulaire. L’induction de l’ISR contre B. cinerea est plutôt liée à l’activation des voies de signalisation JA et ET, mais aussi à l’accumulation de stilbènes et la réduction de la mort cellulaire chez les deux cultivars.L’approche comparative entre l’ISR induite par Bacillus subtilis et P. fluorescens a permis de révéler des différences significatives en terme de potentialisation de défenses contre P. viticola. Bien que les deux bactéries induisent une ISR de niveau comparable en potentialisant principalement la voie de SA et l’accumulation de stilbènes chez les deux cépages, les fortes réponses sont procurées par B. subtilis qui induit aussi une expression élevée du gène HSR et l’accumulation de SA surtout chez le cépage hybride résistant. P. fluorescens quant à elle, elle potentialise dans une moindre mesure l’expression d’autres gènes liés au SA et l'accumulation d'ABA dans les deux variétés. Ces résultats suggèrent que l’ISR induite par B. subtilis et P. fluorescens contre P. viticola pourrait opérer selon des mécanismes communs et distincts en fonction de la résistance basale du génotype de la vigne.Des analyses transcriptomiques supplémentaires ont été effectuées sur du Pinot noir traité avec P. fluorescens après inoculation de B. cinerea et P. viticola, afin de caractériser l'expression de gènes associés à l’ISR contre des agents pathogènes de modes de vie différents. Un grand nombre de gènes exprimés de manière différentielle (DEG) identifiés à partir des données d'ARN-seq suggère que l’ISR induite par P. fluorescens pourrait être associé à la potentialisation de plusieurs mécanismes en fonction du mode de vie de l'agent pathogèneDowny mildew caused by the oomycete Plasmopara viticola and grey mold caused by the fungus Botrytis cinerea are among the highly threatening diseases in vineyards. The current strategy to control these diseases relies totally on the use of chemical fungicides. The use of beneficial bacteria is arising as a sustainable strategy in controlling various diseases. This can be achieved through the activation of the plants own innate immune system, known as induced systemic resistance (ISR). Such a strategy is effective in controlling grey mold disease, but the efficiency against downy mildew is still in unknown, and the mechanisms underpinning the resistance state remain to be explored. In this study we focused on the capacity of Pseudomonas fluorescens PTA-CT2 to induce ISR in grapevine against P. viticola and B. cinerea by using two grafted cultivars differing in their susceptibility to downy mildew, Pinot noir as susceptible and Solaris as partially resistant. We then examined the differences and similarities of Bacillus subtilis- and P. fluorescens-mediated ISR and priming state after P. viticola challenge. On the basis of the contrasting phenotypic susceptibility of cultivars, we explored mechanisms underlying ISR before and upon pathogen infection. We provide evidence that P. fluorescens induces ISR against P. viticola and B. cinerea by priming common and distinct defensive pathways depending on the basal immunity of genotype. Although P. fluorescens elcits hormonal synthesis and photosynthetis efficiency before infection in both genotypes, our results highlight an important role for salicylic acid (SA)-dependent defense and phytoalexin synthesis, but not JA and ET signaling in ISR against P. viticola in both cultivars. The priming state relies further on the strong expression of HSR gene involved in HR-like cell death in Solaris, whereas in the susceptible cultivar it involves accumulation of ABA, rather than HR-like cell death. The induced ISR against B. cinerea is rather related to the activation of JA/ET signaling, but also to stilbene accumulation and reduction of cell death in both cultivars.The comparative approach between Bacillus subtilis- and P. fluorescens-ISR revealed significant differences in terms of primed defenses against P. viticola. Although both bacteria induce comparable level of ISR by mainly priming SA pathway and stilbene accumulation in both grape varieties, the strong responses are provided by B. subtilis which also induces high expression of the HR-related gene and the accumulation of SA, especially in the resistant hybrid variety. P. fluorescens potentiates to a lesser extent the expression of other SA-related genes and accumulation of ABA in both varieties. These results suggest that B. subtilis- and P. fluorescens-induced ISR against P. viticola could operate through common and distinct mechanisms depending on the basal resistance of grapevine genotype.Further transcriptomic analyses were performed on mock and P. fluorescens-treated Pinot noir after inoculation with B. cinerea and P. viticola, in order to characterize the expression of genes associated to ISR against pathogen with different lifestyle. A large number of differentially expressed genes (DEGs) identified from the RNA-seq data suggests that P. fluorescens-ISR could be associated with priming of several mechanisms depending on the pathogen lifestyle
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El, Asmi Faten. "Rôle différentiel des isoformes de PML en réponse au trioxyde d’arsenic et dans la défense antivirale." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T102.
Full textAbstract:
Les interférons (IFN) constituent une famille de cytokines aux propriétés antiprolifératives et antivirales. Ils activent, via la voie Jak/STAT, des gènes spécifiques dont les produits sont les médiateurs des effets biologiques des IFN. C’est le cas de PML (Promyelocytic leukemia), appelée aussi TRIM19, qui joue un rôle central dans la défense antivirale. PML appartenant à la famille des protéines Tripartite Motif (TRIM), caractérisée par la présence en N-terminal d’un motif RBCC, constitué d’un domaine RING, d’une ou de deux boites B et d’un domaine coiled-coil. PML a été identifiée dans la leucémie aiguë promyélocytaire, une pathologie causée par la translocation chromosomique t(15 ;17) qui fusionne les gènes PML et RARA, aboutissant à la synthèse d'une protéine chimère PML-RARA. Le trioxyde d'arsenic (As2O3) cible la portion PML de la protéine oncogénique, entraînant sa dégradation et la rémission complète des patients. Dans les cellules saines, les transcrits PML issus d’un gène unique génèrent par épissage alternatif 7 isoformes principales de PML, dont six sont nucléaires (PMLI à PMLVI) et une cytoplasmique (PMLVIIb). Toutes possèdent la même extrémité N-terminale mais diffèrent au niveau de leur extrémité C-terminale, conférant à chaque isoforme des fonctions spécifiques.PML est l’organisatrice d’une structure multi-protéique appelée corps nucléaires (CN), impliquée dans divers processus cellulaires tels que l’apoptose, la dégradation des protéines ou encore la défense antivirale.PML est modifiée par SUMO de façon covalente au niveau de trois sites lysines (K65, K160, K490) et de façon non covalente, via son domaine SIM (pour « SUMO Interacting Motif »). Ces modifications sont requises pour la formation de CN fonctionnels et le recrutement de protéines partenaires au sein de ceux-ci. Le but de ma thèse a été d’étudier le rôle différentiel des différentes isoformes de PML en réponse à l’As2O3 et suite à l’infection virale. Nous avons montré que le SIM de PML est nécessaire à sa dégradation en réponse à l'As2O3. Ce motif est présent dans toutes les isoformes de PML, hormis l’isoforme nucléaire PMLVI et l’isoforme cytoplasmique PMLVIIb. Le SIM de PML n’est pas requis pour sa SUMOylation et son interaction avec RNF4 (une E3 ubiquitine ligase responsable de la dégradation de PML via le protéasome). En revanche, ce motif est requis pour l’ubiquitination de PML, le recrutement des composants du protéasome et sa dégradation en réponse à l’As2O3. Concernant les propriétés antivirales de PML, l’étude que nous avons menée avec toutes les isoformes de PML a permis de montrer que seules PMLIII et PMLIV confèrent une résistance au Virus de la Stomatite Vésiculaire (VSV). L’effet antiviral de PMLIII n'est observé qu'à faible multiplicité d’infection (MOI) et est indépendant de la production d’IFN. Par contre, PMLIV exerce une puissante activité anti-VSV, y compris à forte MOI et s'exerce selon deux mécanismes distincts : (i) PMLIV inhibe la réplication du VSV par un mécanisme précoce indépendant de l’IFN, (ii) PMLIV augmente tardivement la production d’IFN-β via une plus forte activation d’IRF3 qui est due à la séquestration spécifique de Pin1 au sein des CN par PMLIV. Ces deux processus nécessitent la SUMOylation de PMLIV. Ces résultats montrent que PMLIV exerce une activité antivirale intrinsèque et est impliquée dans l’immunité innée en régulant positivement la voie de transduction conduisant à la synthèse d’IFN-βInterferons (IFNs) are a family of cytokines with antiproliferative and antiviral properties.They activate, via the Jak/Stat pathway, specific genes whose products are the mediators of the biological effects of IFNs. This is the case of PML (Promyelocytic leukemia), also known as TRIM19, which plays a central role in antiviral defense.PML belongs to the Tripartite Motif (TRIM) protein family, characterized by the presence of an N- terminal RBCC pattern, consisting of a RING domain, one or two B-boxes and a coiled-coil domain. PML was identified in acute promyelocytic leukemia, a disease caused by the chromosomal translocation t(15 ;17), which fuses the PML and RARA genes, leading to the synthesis of a chimeric protein PML-RARA . Arsenic trioxide (As2O3) targets the PML moiety of the oncogenic protein, resulting in its degradation and in the complete remission of patients.In healthy cells, PML transcripts derived from a single gene generate seven major isoforms of PML by alternative splicing, including six nuclear (PMLI to PMLVI) and one cytoplasmic (PMLVIIb). All share the same N-terminus but differ at their C-terminus, giving each isoform specific functions.PML is the organizer of a multi-protein structure called nuclear bodies (NBs) that are involved in various cellular processes such as apoptosis, protein degradation or antiviral defense.PML is covalently modified by SUMO at three lysine residues (K65, K160, K490) but also non-covalently via its SIM domain (for « SUMO Interacting Motif »). These modifications are required for the formation of functional NBs and the recruitment of partner proteins within them.The aim of my thesis was to study the differential role of the different PML isoforms in response to As2O3 and during viral infection.We have shown that the SIM PML SIM is necessary for its degradation in response to As2O3. This motif is present in all PML isoforms, except the nuclear PMLVI and the cytoplasmic PMLVIIb isoforms. The SIM of PML is not required for its SUMOylation and its interaction with RNF4 (the E3 ubiquitin ligase responsible for PML proteasome-dependent degradation). However, this motif is required for the ubiquitination of PML, the recruitment of proteasome components and the degradation of PML in response to As2O3.Concerning the antiviral properties of PML, the study that we conducted with all PML isoforms allowed us to show that only PMLIII and PMLIV confer resistance to Vesicular Stomatitis Virus (VSV). Whereas the antiviral activity of PMLIII is only observed at low multiplicity of infection (MOI) and is independent of IFN production, PMLIV has a potent anti-VSV activity, including at high MOI, which is mediated through two distinct mechanisms: (i) PMLIV inhibits the replication of VSV by an early and IFN-independent mechanism, (ii) PMLIV later increases the production of IFN-β via a stronger activation of IRF3, which is due to the specific sequestration of Pin1 by PMLIV within NBs. Both processes require the PMLIV SUMOylation. These results show that PMLIV has an intrinsic antiviral activity and is also involved in innate immunity by positively regulating the transduction pathway leading to IFN-β synthesis
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Afacan, Nicole. "Linking cellular metabolism and Innate Defence Regulator peptide function." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/57024.
Full textAbstract:
Appropriate cellular metabolism is essential to immune cells to survival and their ability to mount effective and appropriate responses to the pathogen and host derived insults they encounter. Malnutrition, caused by nutrient deficiency or excess, can result in significant dysregulation of immune cell activity. Immune cells undergo metabolic reprogramming in response to pathogen and host-derived signals found in their environment. These changes modulate the type of response they will mount. Innate defence regulatory (IDR) peptides, synthetic derivatives of host defence peptides, were developed as anti-infectives that modulate host immune system responses however, much of the mechanisms behind their activity are unknown. In this study, IDR-1018 was shown to modulate glycolytic activity in macrophages, which appeared to be important to its immunomodulatory activity. Activation of the ERK signalling pathway, a major regulator of metabolism and inflammatory responses, by IDR-1018 was found to be a possible mechanism by which IDR-1018 induced both glycolysis and chemokine production. Inhibition of glycolysis using 2-deoxy-d-glucose (2DG) suppressed IDR-1018 induced chemokine production. However, 2DG also suppressed IDR-1018 activity through induction of endoplasmic reticulum stress and the unfolded protein response (UPR), specifically the anti-inflammatory PERK arm of the UPR. The anti-endotoxin activity of IDR-1018 was also found to be associated with modulation of glycolysis. IDR-1018 suppressed lipopolysaccharide (LPS)-induced chemokine and cytokine production, possibly through inhibition of LPS-induced glycolysis. Interestingly, dysregulation of both glycolysis and the UPR by 2DG enhanced the anti-endotoxin activity of IDR-1018, suppressing LPS-induced chemokine and cytokine production. Finally, this study identified a potential new activity for IDRs, the modulation of metabolic pathways dysregulated in response to nutrient excess. Specifically, this study showed that IDR-1018 enhanced HDL-mediated cholesterol efflux from macrophages and smooth muscle cells, two important cellular mediators of atherosclerosis. This may have been a result of IDR-1018 interacting with HDL particles found in serum, facilitating their binding to the plasma membrane of cells. The results presented in this study demonstrated that IDR peptides are potent modulators of both immune cell function and cellular metabolism as well as identified a novel mechanism by which IDR peptides exert their immunomodulatory activity.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Rousso, Christopher. "Characterizing the Role of α-Synuclein in Innate Defenses." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/40011.
Full textAbstract:
Typical Parkinson’s disease (PD) is thought to be caused by a combination of genetic and environmental factors. α-Synuclein (SNCA) is central to PD pathogenesis; however, functions of SNCA outside the brain remain largely unknown. We, and others, have found that wild-type Snca expression confers anti-microbial effects in mice by reducing the severity of viral infections. Our aim is to further characterize a role of SNCA in systemic and brain health of the host during infection. We hypothesize that SNCA plays a role in innate defenses and that SNCA gene dosage will modulate outcomes of infection in the brain following pathogen exposure. Intranasal delivery of reovirus in mouse pups causes systemic illness, leading to encephalitis. In this study, intracranial inoculations of reovirus are used to differentiate the relative contribution of Snca-mediated protection in the brain versus the periphery. Two outcomes are monitored: survival and viral titres in select organs. When comparing wild-type Snca, heterozygous, and knock-out mice, I found that Snca expression did not confer any protection with respect to survival or regarding viral brain titres. These results are paralleled by cellular overexpression models. Unexpectedly, the anti-viral property of Snca, which was previously observed systemically with three distinct dsRNA viruses, did not extend to a paradigm where neural cells were directly exposed to reovirus. These results suggest a complex, anti-viral role for Snca in host defenses that may be mediated, in part, outside the central nervous system. Future studies will address whether this occurs in peripheral neurons or cells of hematopoietic lineages.
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Ivory, Catherine P. "The Gal-lectin and innate host defenses against Entamoeba histolytica /." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111883.
Full textAbstract:
Entamoeba histolytica, etiological agent of amebiasis, continues to be a significant threat to human health worldwide. The disease affects 10% of the world's population and leads to an estimated 100, 000 deaths a year. The parasite's surface Gal-lectin is an immunodominant protein that also mediates colonization and pathogenicity. The Gal-lectin is the most promising vaccine candidate against amebiasis. However, the immune mechanisms involved in protection against disease remain unclear. The objective of this study was to characterize the immunological basis of the host defense mechanisms using a Gal-lectin based vaccine. Exposure of the Gal-lectin with immature dendritic cells increased cell maturation and activation and upregulated co-stimulatory molecules and pro-inflammatory cytokines production. Dendritic cell activation was dependent on NF-kappaB and MAPK activation. In vaccination studies, the adjuvant effect of CpG-ODN, a synthetic oligodeoxynucleotide capable of stimulating Th1 immune responses enhanced the immune response to the Gal-lectin when administered systemically or mucosally. Protected animals had elevated anti-Gal-lectin serum and stool IgA antibodies capable of blocking parasite adherence in vitro. Analysis of cytokine responses in vaccinated and protected animals revealed increased IFN-gamma production compared to controls. Finally, E. histolytica DNA was shown to activate macrophages in a TLR9 and MYD88-dependent manner. Immunized gerbils with Gal-lectin and E. histolytica DNA induced protective immunity against a challenge infection. Taken together, these findings underscore the importance of multivalent subunit vaccines in Th1 mediated immune responses in host defense against amebiasis.
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Franzoi, Marco. "Welcome to the jungle: insights into the innate defence peptides." Doctoral thesis, Università degli studi di Padova, 2017. http://hdl.handle.net/11577/3422412.
Full textAbstract:
Understanding innate defence mechanisms is not only an exercise of pure science, but can give the scientific community new tools in fighting pathogens. In particular, the emergence of strains multi-resistant to common antibiotics gave a rush to the identifi-cation of new bioactive molecules. Possible candidates are a variety of endogenous gene-encoded antibiotics, evolved together with other humoral effectors to protect hosts from infective agents, beyond adaptive immunity.
The main target of the thesis is to get further insights in the actual “jungle” of AMPs, in particular on their mechanism of action, with the final goal of improving their perfor-mance against so-called ESKAPE pathogens. Thus, I’ve developed new specific methods, starting from in silico predictions to high-throughput characterisation of mutants, and characterized new molecules, in particular peptides derived from bivalves, as lead com-pounds for anti-infective drugs. To achieve these goals, I worked with different labora-tories expert in peptide synthesis, structural bioinformatics or recombinant protein pro-duction.
The first chapter gives an overview on antimicrobial peptides, with particular attention to CSαβ peptides. Such peptides display features useful for the development of new antibiotics, such as high stability and immunomodulatory properties.
The second chapter presents the work done in collaboration with the Chemical sci-ences Department of the University of Padua on the structural characterization of CSαβ peptides. Considering the abundance of putative AMP sequences and the scarcity of ef-fectively produced and market-oriented peptides, we developed a cysteine-guided mo-lecular dynamics protocol for CSαβ structure refinement. This could support the research of active fragments before the recombinant production or chemical synthesis of the whole peptide. The proposed protocol is now published in the Journal of Biomolecular Structure and Dynamics, issue of Sep 2016.
As proof of application of chapter 2 protocol, the third chapter illustrates the exper-imental work on myticin C, a polymorphic AMP from M. galloprovincialis. Such molecule displays interesting immuno-related properties. Starting from a structural model, we were able to design MytC-derived active fragments showing properties similar to the whole peptide. Part of results presented in this thesis are now published in the Journal of Agricultural and Food Chemistry, issue of Oct. 2015.
Aiming to increase the repertoire of available bioactive AMPs, we selected five putative antimicrobial peptide sequences from M. galloprovincialis transcriptomic data and, after preliminary trials in E. coli, the yeast Pichia pastoris was selected for a more advantageous recombinant production. This work, described in the fourth chapter, was done in collaboration with ETHZ (Swiss), namely in the laboratory of Prof. Markus Aebi, where I spent six months under the supervision of Doctor Andreas Essig. Actually two proteins have been produced, one of them showing an interesting range of antimicrobial activity.
During my staying at ETHZ, I’ve worked on copsin, a defensin-like peptide previously identified in the fungus Coprinopsis cinerea and the fifth chapter describes the high throughput production of site-directed copsin mutants and the in silico characterization of membrane interaction of copsin and of one copsin mutant with improved features, named k-copsin.
In the appendix are reported experimental inputs supporting other research lines carried on in the Dott. Paola Venier laboratory.Capire come funziona l’immunità innata non significa solo sviluppare nuove cono-scenze di base, ma può fornire alla comunità scientifica nuovi strumenti per combattere organismi patogeni. In particolare, la comparsa di ceppi multi-resistenti ai comuni anti-biotici ha spinto la ricerca di nuove molecole bioattive. Tra i possibili candidati vi sono antibiotici endogeni codificati da geni ed evoluti insieme ad altre molecole effettrici per proteggere ospite da agenti infettivi indipendentemente dalla immunità di tipo adattativo. L’obiettivo principale della tesi è stato quello di cercare nuove evidenze riguardo questa giungla di AMPs, in particolare sul loro meccanismo d’azione, con l’obiettivo di migliorare la loro performance contro il gruppo di patogeni cosiddetti ESKAPE. Per fare ciò ho sviluppato nuovi metodi di indagine specifici, a partire dalla predizione in silico fino alla caratterizzazione di mutanti, e caratterizzato nuove molecole, in particolare derivate da bivalvi. Per raggiungere questi risultati ho collaborato con diversi laboratori esperti nella sintesi di peptidi, in bioinformatica strutturale e nella produzione ricombinante di proteine. I peptidi antimicrobici naturali sono strutturalmente diversi, ma presentano alcune caratteristiche comuni. Il primo capitolo offre una panoramica sui peptidi antimicrobici, con particolare attenzione ai cosiddetti peptidi CSαβ che posseggono alcune caratteristiche interessanti, come elevata stabilità e proprietà immunomodulatorie. Il secondo capitolo presenta il lavoro fatto in collaborazione con il Dipartimento di Scienze chimiche dell’Università di Padova e riguardante la caratterizzazione strutturale di peptidi CSαβ. Considerando l’abbondanza di sequenze di possibili peptidi antimicrobici e la scarsità di peptidi efficacemente prodotti e orientati al mercato, abbiamo sviluppato un protocollo bioinformatico basato sui ponti disolfuro ed in grado di migliorare predizioni di struttura. Questo potrebbe essere utile nella ricerca di frammenti attivi senza la necessità di produrre l’intero peptide. Il protocollo è stato pubblicato nel Settembre del 2016 in Journal of Biomolecular Structure and Dynamics. Come prova dell’utilità di tale protocollo, il terzo capitolo riporta il lavoro speri-mentale effettuato sulla miticina C, un peptide antimicrobico polimorfico di M. gallopro-vincialis con interessanti proprietà legate all’immunità. Partendo da predizioni strutturali, siamo stati in grado di disegnare dei frammenti attivi derivanti dalla miticina C aventi proprietà simili a quelle del peptide intero. Parte di questo lavoro è già stato pubblicato nell’Ottobre del 2015 nel Journal of Agricultural and Food Chemistry. Con l’obiettivo di aumentare il repertorio di AMP bioattivi disponibili, abbiamo sele-zionate cinque sequenze di possibili peptidi antimicrobici dai dati trascrittomici di M. galloprovincialis puntando su Pichia pastoris, dopo i primi tentativi in E. coli, per una più vantaggiosa produzione ricombinante di tali peptidi. Questa parte di lavoro, descritta nel quarto capitolo, è stata svolta in collaborazione con l’ETH di Zurigo, in particolare nel laboratorio del Prof. Markus Aebi, sotto la supervisione del Dott. Andreas Essig. Per il momento siamo stati in grado di produrre due peptidi, uno dei quali ha un interessante spettro di attività antimicrobica. Durante il periodo all’ETHZ, ho lavorato sulla copsina, una defensina identificata nel fungo Coprinopsis cinerea. Nel quinto capitolo è descritta la produzione di mutanti della copsina e la caratterizzazione computazionale dell’interazione a livello di membrana di due versioni della copsina, la prima wild-tipe ed una seconda con caratteristiche migliorative. L’articolo relativo ai dati presentati sta per essere sottoposto a peer review. In appendice sono presentati i contributi sperimentali a supporto di altre linee di la-voro presenti nel laboratorio della Dott.ssa Paola Venier.
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Cadwell, Kevin. "Investigations of the gut innate defences of commercial broilers." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/3067.
Full textAbstract:
The E.U. ban on the use of anti-microbial growth promoters in poultry feed, introduced to counter global problems of bacterial antibiotic resistance, has increased the risk of enteric disease in commercially reared broiler chickens. Development of strategies to prevent such diseases requires further knowledge and understanding of avian gut defences and particularly the innate immune defences. In collaboration with Aviagen Ltd., the objective of this study was to investigate, through two farm trials, the effects of bacterial exposure on host avian β-defensin (AvBD) expression profiles and gut health of two commercial broiler lines (X and Y). Furthermore, two host defense peptides, avian beta defensin 1 (AvBD1) and 10 (AvBD10) were analysed in vitro for their anti-microbial efficacies. In Trial 1, Lines X and Y, differing in their gut health, were exposed to one of three bacterial challenges on the day of hatch, namely a combination of Bacteroides dorei and Barnesiella viscericola (B/BV), Lactobacillus johnsonii (LJ) or a mixture of the two challenges (B/BV + LJ). At days 4, 7, 14, 21, 28 and 35, birds were scored for gut health using an industry approved system and digesta were sampled and analysed for microbiotae (pyrosequencing). The data revealed that, relative to control and LJ challenged birds, the B/BV challenge was associated with gut health deterioration. Furthermore, relative to Line X, there was a trend for the gut health of Line Y birds to be superior for all challenged groups. Although microbiome analyses did not reveal any clear differences between Lines X and Y, the data did suggest that birds with better gut health outcomes were associated with higher ileal Lactobacillus spp. levels at Day 4 and higher caecal levels of Bacteroides spp. at Day 21. Despite less optimal gut health, Line X is important to the Aviagen Ltd. breeding programme. To understand the roles, if any, of the AvBDs in bird gut health, a second trial was performed in which gut AvBD1 and 10 gene expression were assessed in Line X birds following B/BV challenge. Relative to control birds, the B/BV challenge suppressed gene expression of AvBD1 in the duodenum/jejunum (P < 0.05) and AvBD10 in the duodenum/caecum (P < 0.05) and AvBD1 down-regulation was confirmed at the cellular level by data from an in vitro challenge model (P < 0.001). Interestingly, within the B/BV challenged group, birds with higher AvBD1 expression were associated with better gut health assessments. The AvBD1 gene contains single nucleotide polymorphisms (SNP) within the region encoding region the mature peptide. Three AvBD1 variants were synthesised that were typical to Line X (NYH), Y (SSY) and another commercial Line, Z (NYY), and were assessed, together with AvBD10, for in vitro anti-microbial activities (AMAs) against a variety of gut bacterial isolates. Despite Line X displaying the least optimal gut health, the ‘NYH’ variant exhibited the greatest potency against all bacterial species. The data for AvBD10 revealed that, although bacterial growth was inhibited, this peptide had lower AMA than AvBD1, indicative of additional physiological functions. An in vitro examination of wound healing capacity using a scratch assay was inconclusive. The in vivo data indicated that gut AvBD expression is susceptible to gene down-regulation by bacteria and that this, in turn, may have an adverse outcome on gut health. However, selectively breeding for birds able to maintain high AvBD expression presents a strategy to protect flocks against the threat of endemic gut health problems.
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Nicholls, Erin Frances. "Immunomodulatory and wound-healing effects of the host defence peptide LL-37 and related innate defence regulators." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/41389.
Full textAbstract:
LL-37, the only known human cathelicidin peptide, possesses a variety of immunomodulatory properties that extend its role in host defence far beyond its original classification as an antimicrobial peptide. Recently, work has been underway to elucidate signalling pathways initiated by LL-37, with the aim of further understanding this peptide’s role in the immune system. The aim of this study was to further uncover the role of transcription factors during the responses of immune cells to LL-37 and related innate defence regulator peptides. Secondary aims were to investigate potential wound-healing properties of these peptides and to compare host defence peptides with chemokines in terms of immunomodulatory function. Here, I demonstrated involvement of AP-1 in LL-37-induced wound healing. I also showed a functional overlap between chemokine CXCL9/MIG and host defence peptide LL-37 and demonstrated similarities between LL-37 and the antibiotic azithromycin.
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Crumlish, Margaret. "A study on the innate defence mechanisms in farmed tropical Rana spp." Thesis, University of Stirling, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321973.
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Funderburg, Nicholas Thomas. "Human beta defensin 3 linking innate and adaptive immune responses /." Connect to text online, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1189084245.
Full textAbstract:
Thesis (Ph. D.)--Case Western Reserve University, 2007.[School of Medicine] Department of Molecular Biology and Microbiology. Includes bibliographical references. Available online via OhioLINK's ETD Center.
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Lanz, Marcelo. "Investigations of the innate immune defences in the urogenital tract." Thesis, University of Newcastle upon Tyne, 2014. http://hdl.handle.net/10443/2433.
Full textAbstract:
The urinary tract (UT) is a sterile environment despite being constantly challenged by pathogens from its surrounding environment. Protection of the uroepithelium from microbial assault involves the innate immune system, but still, to date, little is known of the actual mechanisms that function in such defences. The focus of the research presented in this thesis, which used RT4 and VK-2 E6/E7 in vitro cell systems to model the bladder and vaginal epithelia respectively, was to interrogate the mechanisms by which these epithelia recognise and respond to microbial assault. The RNA of bladder RT4 cells challenged with motile and non-motile Escherichia coli strains was analysed via a TLR related gene array. The data supported upregulation of the NF-κB dependent cytokine IL-8 and thus NF-κB signalling was used to measure the uroepithelial response to microbial infection. Flagellin, recognised by TLR-5, and responsible for bacterial motility, induced a significant (p<0.001), 44.9±1.7 fold increase, in RT4 NF-κB signalling within 4h of challenge and with use of TLR-5 blocking antibody was identified as the key activating PAMP in the UT. Other bacterial cell wall components, namely LPS and peptidoglycan, failed to induce comparable NF-κB activation (maximum 10.7± 0.8 and 6.3±0.6 fold increase at 16 hours post challenge). These data led to the hypothesis that strains associated with urinary tract infection (UTI) have reduced or no motility. However, 12/24 clinical strains associated with UTI were motile and induced a NF-κB signalling response. Variability in the responses (3.4 0.3 to 40.3 1.8 fold at 8 hours post challenge), could not be explained by flagellin, suggesting that other factors, host and bacterial, may also contribute to the intensity of the response. Analysing UPEC strains isolated from the urine of patients carrying a C1174T SNP encoding a truncated TLR-5 revealed such strains to induce normal NF-κB signalling (39.5 0.3 fold) in RT4 cells. These data emphasised the importance of TLR-5 and bacterial motility in the innate response of the host to a UTI. Human (h) βD-2, a member of the Defensin family with microbial killing properties, is important in the innate defences of the uroepithelium. To identify agents that could be used therapeutically to enhance the innate defences of the UT, a reporter construct (phβD-2-Luc), containing 2 kbp of hβD- 2 5’UTR sequence, was transfected into RT4 and VK-2 E6/E7 vaginal cells. Reporter activity (fold increase) was significantly increased with calcitriol (VitD, 10 nM), or 17β-oestradiol (17β-oes, 4 nM), treatments when challenging VK-2 E6/E7 with either flagellin (68.1±8.5 vs. 196.3±28.3 for VitD, p<0.001, or vs. 199.5 37.7 for 17β-oes, p<0.01) or E. coli NCTC 10418(19.9±3.8 vs. 40.1±4.3 for VitD, p<0.01) or vs. 40.2±6.6 for 17β-oes, p<0.05). The hβD-2 reporter data also revealed Zymosan (50 μg/ml) enhancing reporter activity in both cell lines (RT4 34.8±5.0 and VK-2 E6/E7 27.2±2.9, both p<0.001) and had a synergistic effect to flagellin increasing reporter activity by more than 60 % (p<0.05). These data suggested that Zymosan, vitamin D as well as oestrogen regulate the hβD-2 gene. The expression of hβD-3, another member of the Defensin family with microbial killing properties, was identified in human bladder biopsies so its expression was further investigated in vitro. While no hβD-3 expression was measured in the RT4 cells, constitutive and inducible expression was identified in VK-2 E6/E7 cells. Human βD-3 expression was enhanced at 8 hours following challenge of the cells (PBS 70.5±13.4) with E. coli NCTC 10418 (1094.5±293.8, p<0.001), flagellin (518±139.2, p<0.01) and Zymosan (280.8±65.7, p<0.01). Induction was also observed with LPS but at 16 hours (27.0±8.1 (PBS), 612.2±260.8, p<0.001) but not with peptidoglycan (32.2±12.3, p>0.05). These data suggested that hβD-3 functions in the defence of urogenital tract and as its expression was inducible, a potential target for therapeutic strategies. The fungal cell wall β1,3-glucan, Zymosan, induced NF-κB signalling as well as hβD-2 in the RT4 and VK-2 E6/E7 cells, and hβD-3 expression and secretion in VK-2 E6/E7 cells. Molecular analysis of the cell RNA identified the expression of three isoforms (417, 330, 211 bp) of the Dectin-1 (CLEC7A) receptor and immunocytochemistry identified the receptor protein localised to the cell membrane. Following Zymosan challenge of VK-2 E6/E7 cells, receptor clustering and the phosphorylation of a Dectin-1 signalling protein, SYK was observed supporting the presence of functional receptors. Induction of NF-κB signalling by Zymosan was inhibited by blocking TLR-5 (4.2±0.4 vs 2.8±0.1, p<0.01) suggesting functional interactions between Dectin-1 and TLR-5. These data provide support for the presence of Dectin-1 receptor in the urogenital tract. In conclusion, the data presented in this thesis confirms the importance of TLR-5 in the defence of the urogenital tissues. Investigations of hβD-2 and hβD-3 expression and their enhancement in the UT have led to the discovery of a role for the Dectin-1 receptor. This receptor represents a target for future therapeutic strategies to enhance the innate response.
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Stanton, Anna Maria. "Estrogen and the innate epithelial defences of the urogenital tract." Thesis, University of Newcastle upon Tyne, 2016. http://hdl.handle.net/10443/3560.
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Approximately 40% of women will experience at least one urinary tract infection (UTI) in their lifetime and 25% of these women will go on to suffer recurrent UTIs. After the menopause, the risk of developing a recurrent UTI doubles, which is putatively linked to reduced estrogen levels. Topical vaginal estrogen treatment in postmenopausal women has been shown to reduce the incidence of UTIs. However, the mechanism by which vaginal estrogen helps to protect against UTIs is not well understood. Antimicrobial peptides (AMPs), secreted in response to infection and functioning via bacterial cell lysis, are an important component of the host innate immune response to infection. The aim of this project was to investigate the effects of estrogen treatment on vaginal epithelial AMP expression and synthesis. Immortalised vaginal epithelial cells (VK2 E6/E7), used to model the vaginal epithelium, were treated with 4nM 17β-estradiol for seven days and then challenged with 50ng/ml flagellin (isolated from Escherichia coli clinical UTI isolate) for 24 hours. Combined estrogen pretreatment plus flagellin resulted in a 2.3- and 2.1-fold increase in expression of the AMPs human β-defensin 2 (hBD2) and hBD3, respectively, above that observed with flagellin challenge alone (p-values < 0.001). Microarray analyses identified upregulation of the AMPs LCN2, RNase 7, S100A7, S100A12, SLPI, by estrogen pretreatment plus flagellin in addition to hBD2 and hBD3. Furthermore, several genes relating to keratinisation (for example keratin and SPRR genes) and inflammation (for example SERPINB4, S100A8 and S100A9) were also upregulated suggesting that estrogen pretreatment stimulated multiple protective responses in VK2 cells. A hBD2 luciferase reporter vector containing a 2032bp hBD2 promoter region was used to measure hBD2 expression in response to estrogen and flagellin treatments. Reporter activity in VK2 cells significantly increased 2.1-fold following seven day estrogen pretreatment (4nM) plus flagellin (50ng/ml) challenge compared to flagellin challenge without estrogen pretreatment (p=0.0367). Six estrogen response elements (EREs) were identified in the hBD2 promoter and were mutated by site-directed mutagenesis. Mutation of each of the EREs abolished hBD2 gene expression potentiation by estrogen pretreatment plus flagellin, suggesting that hBD2 is regulated through ER-α or ER-β binding to EREs in the hBD2 promoter. Pathway analysis of the microarray data identified IL-17A as a potential regulator of AMP expression. Thus, VK2 cells were challenged with exogenous IL-17A in concentrations ranging from 0.1ng/ml to 100ng/ml for 24 hours. The expression of hBD2, LCN2, RNase 7, and S100A7 was significantly increased between 2- and 21- fold in an IL-17A dose dependent manner (p-values < 0.001). In addition, estrogen pretreatment of VK2 cells prior to challenge with IL-17A (100ng/ml) plus flagellin (50ng/ml) significantly increased the expression of hBD2, hBD3 and S100A7 by between 1.4- and 1.6-fold compared to IL-17A plus flagellin without estrogen (p-values < 0.05). These data indicated an important role for estrogen in AMP expression even in the presence of proinflammatory cytokines, such as IL-17A. Altogether, these data indicated that estrogen is an important regulator of innate epithelial defences of the urogenital tract. These results suggest that estrogen protects against UTIs by augmenting the vaginal antimicrobial response to infection and by strengthening the epithelial barrier to infections. Hence, loss of estrogen following the menopause leaves postmenopausal women susceptible to repeated UTIs.
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Funderburg, Nicholas Thomas. "Human Beta Defensin 3: Linking Innate and Adaptive Immune Responses." Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1189084245.
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Al-Hababi, Fadhil H. "Inhibition of HCV replication by host cell innate antiviral defences." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/6828.
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RIG-I and MDA5 are the major intracellular sensors for the induction of IFN-β, by dsRNA, in Huh7 cells. It was found that the expression of RIG-I, MDA5 and the signalling mediators TRIF, MAVS and MyD88 were upregulated in Huh7 HCV RNA replicon cells. Similar findings were obtained in Huh7 cells infected with JFH-1 HCV. When replicon and HCV infected cells were further stimulated by dsRNA the induction of these genes were reduced compare to control cells. The expression of NS3 and NS3/4A proteins, using adenovirus vectors, reduced RIG-I, MDA5, MAVS and MyD88 expression levels in response to dsRNA stimulation. Ablation of MDA5, MAVS, MyD88 and TRIF in HCV infected Huh7.5 cells, using siRNAs, increased virus replication demonstrating their importance in inhibiting HCV infection. These results show that HCV inhibits the induction of interferon in response to dsRNA and that NS3 plays an important role in this inhibition. Analysis of gene expression during HCV infection showed significant changes in the expression of genes involved in the innate immune response, interferon, apoptosis and cell cycle regulation pathways. These include SOCS1, 2 and 3, OAS3, MXA1, IRF9, IRF1, IFNAR2 and STAT1. SOCS3 knockdown by siRNAs reduced HCV replication indicating an additional mechanism of IFN inhibition by HCV. Infection with HCV also caused cell cycle arrest leading to DNA fragmentation and apoptosis. A decrease in cyclin B1 and an increase in GADD45-β expression during HCV infection indicates that they may play an important role in HCV induced alterations to the cell cycle.
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Wroblewska, Natalia. "Role of the ventromedial hypothalamus in control of innate defensive behaviours." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/276036.
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Our senses are constantly bombarded with information. How does the brain integrate such a variety of inputs to generate appropriate behaviours? Innate defensive behaviours are a good model to address this question. They are essential for animal survival and the brain circuits that control them are highly conserved across species. Moreover, the sensory inputs and behavioural outputs can be well defined and reliably reproduced in the lab. This allows us to study function of the individual components of the circuit controlling these behaviours. Ventromedial hypothalamus (VMH) is a key brain region for controlling responses to predators; it has been shown that inactivating the VMH can reduce defensive behaviours. Interestingly, activating the VMH output neurons (SF1+ cells) can produce a variety of different behaviours, from immobility to escape, depending on the intensity of activation. During my PhD I used a variety of approaches to address the question of the function of the VMH in control of defensive behaviours. At first I hypothesised that the VMH might act as a centre responsible for choosing an appropriate behavioural response according to the stimulus. I set to investigate how different activation levels of SF1+ neurons can produce such different behavioural outputs, and how this activity is modulated in vivo in response to predator stimuli. I began the project by quantifying mouse defensive behaviours in response to olfactory and auditory predator cues, as well as to the optogenetic activation of SF1+ neurons. I then questioned whether there was heterogeneity within the population of SF1+ neurons, which could explain their ability to trigger different behaviours. I performed patch clamp recordings from acute brain slices and conducted a study of the electrophysiological properties of SF1+ neurons. I next investigated how SF1+ neurons integrate excitatory inputs from the medial amygdala, a region which receives olfactory inputs from the accessory olfactory bulb. By combining optogenetics with slice electrophysiology and behavioural assessment, I described the physiology and relevance of this connection. Finally, I investigated in vivo activity in the VMH in response to predator cues by performing calcium imaging of the VMH neurons in freely moving mice. By presenting different sensory stimuli, I addressed the question of heterogeneity of the input pattern to the VMH neurons and the relationship between the VMH activity and the behavioural output. Taken all together, the results of this project have led to a hypothesis whereby the function of the VMH is to facilitate rather than directly control the choice of an appropriate behavioural response.
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McGlasson, Sarah Louise. "Regulation of the innate immune system." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17911.
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The innate immune system is the first line of defence against pathogen invasion. The range of diseases that are caused by deficiencies in or deregulation of the innate immune system illustrates the importance of maintaining an effective balance between clearance of infectious agents and minimisation of inflammatory mediated tissue damage. This thesis explores the role of two proteins in the regulation of the innate immune system. Primarily, this work investigates the effect of human β-defensin 3 (hBD3) on the response to self-DNA and pathogenic DNA. HBD3 is an antimicrobial peptide (AMP), which has been shown to have a role in regulating the immune response; increased copy number of the region containing the gene for hBD3, DEFB103, is linked to an increased risk of psoriasis. Additionally, a similar cationic AMP, LL37, has been shown to exacerbate the pathogenesis of psoriasis by forming an immunogenic complex with self-DNA. This lead to the hypothesis that hBD3 may also affect the innate immune response to DNA. Therefore this project investigates what effect hBD3 has on the response of the innate immune system to self and pathogenic DNA. Flt-3 dendritic cells were used to show that whilst hBD3 increased cellular uptake of self-DNA, it did not convert self-DNA into an immune stimulus. However, hBD3 significantly exacerbated the response to bacterial DNA in a TLR9-dependent manner, also by increasing cellular uptake into FLDCs. The finding that hBD3 increased cellular uptake of both self- and pathogenic DNA suggests that at sites of infection or increased cell death, where DNA would be found in the extracellular environment, hBD3 may increase uptake into immune cells and could induce an increased immune response. Since increased hBD3 expression is induced by inflammatory stimuli, this process would cause a positive feedback loop of inflammation during bacterial infections. In conclusion, hBD3’s role in regulating the innate immune response to DNA is at the ligand-receptor level rather than affecting signalling pathways. Furthermore, hBD3 promotes the innate immune response to bacterial DNA by increasing the efficiency of cellular uptake possibly by inducing DNA aggregation. These results implicate a possible role for hBD3 in the earliest stages of psoriatic plaque development, which is often initiated or exacerbated by an infection, and this could be investigated further. Secondly, I investigated the innate immune function of an E3 ubiquitin ligase (E3L) not previously associated with human disease. Mutations in E3L have been identified in three microcephalic primordial dwarfism families; these patients also presented with recurrent respiratory illnesses. E3L has been implicated in the regulation of the innate immune system via interactions with signalling pathways downstream of the receptor, though its role is not clear. We hypothesised that E3L had a dual role both in regulating growth and cell division and in regulating the immune system. Primary patient fibroblasts did not demonstrate an altered cytokine response to bacterial or viral ligands, implying that E3L may have a specific function in immune cells. To investigate this further, and to provide a system to study E3L in vivo, two transgenic mouse lines were designed and engineered, firstly a conditional ‘knock-out’ designed to replicate some of the alternative isoforms of E3L seen in RT-PCRs, and secondly a ‘knock-in’ line to recapitulate the human mutation in exon 7 of E3L, R185X. These mouse lines should offer an insight into the developmental role for E3L, and contribute to establishing a potential role for E3L in the innate immune system. This thesis exemplifies the complexity of the innate immune system and the regulatory pathways that interact to maintain a delicate homeostasis preventing pathogenic inflammation. Understanding these regulatory mechanisms may shed light on the pathogenicity of diseases and identification of potential targets for therapeutics.
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Garcia-Garbi, Natalio. "Interaction of immunostimulants and stress on innate defence mechanisms of rainbow trout, Oncorhynchus mykiss." Thesis, University of Stirling, 1998. http://hdl.handle.net/1893/26672.
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This study investigated the use of non-specific immunostimulants to alleviate stress-mediated suppression of defence mechanisms and subsequent susceptibility to bacterial pathogens in rainbow trout (Oncorhynchus mykiss). One yeast (1-3),)1-6)-β-glucan and a bacterial peptidoglycan were selected as immunostimulants from a panel of test substances on the basis of enhanced intracellular superoxide generation by kidney macrophages stimulated in vitro. Kidney macrophage effector activity was not affected after 1, 2, 3 or 4 weeks of in-feed treatment with 0.05% or 5% of glucan or peptidoglycan. However, production of bactericidal superoxide by inflammatory peritoneal macrophages did increase significantly after four weeks of oral treatment with 0.05% peptidoglycan. Although a single confinement of fish (93% reduction of water volume for five minutes) caused a physiological stress response, as indicated by hyperglycaemia in plasma, kidney and inflammatory macrophage activities were only affected after six daily confinements. Phagocytosis, intracellular superoxide production and killing of Aeromonas salmonicida in vitro by kidney macrophages were significantly reduced. Conversely, production of extracellular superoxide, which may be associated with damage to self, was enhanced. Peritoneal macrophages displayed a similar but less marked respiratory burst response after repeated confinement. Some of the alterations in macrophage function caused by daily confinement were prevented by feeding 0.05% peptidoglycan four weeks before the first confinement. The increase in kidney macrophage extracellular superoxide production caused by repeated confinement was significantly alleviated by in-feed peptidoglycan. Similarly, the decrease in intracellular production by peritoneal macrophages caused by repeated confinement was prevented by in-feed treatment with peptidoglycan. Neither peptidoglycan nor repetitive confinement had any effect on complement lytic activity. These results indicate that dietary peptidoglycan was able to reduce, by regulating macrophage function, the impact of stress on certain bactericidal defences and potential damage to self. However, there was no significant difference in the persistence of viable A. salmonicida in the spleen or blood of infected fish in any of the experimental treatments.
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Fuhrman, Christopher. "RETROCYCLIN RC-101 OVERCOMES CATIONIC MUTATIONS ON THE HEPTAD REPEAT 2 OF HIV-1 GP41." Master's thesis, University of Central Florida, 2007. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4338.
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Retrocyclin RC-101, a θ-defensin with lectin-like properties, potently inhibits infection by many HIV-1 subtypes by binding to the heptad repeat (HR)-2 region of gp41 and preventing six-helix bundle formation. In the present study, we used in silico computational exploration to identify residues of HR2 that interacted with RC-101 and then analyzed the HIV-1 Sequence Database at LANL for residue variations in the HR1 and HR2 segments that could plausibly impart in vivo resistance. Docking RC-101 to gp41 peptides in silico confirmed its strong preference for HR2 over HR1, and implicated residues crucial for its ability to bind HR2. We mutagenized these residues in pseudotyped HIV-1 JR.FL reporter viruses, and subjected them to single round replication assays in the presence of 1.25-10ug/ml RC-101. Except for one mutant that was partially resistant to RC-101, the other pseudotyped viruses with single-site cationic mutations in HR2 manifested absent or impaired infectivity or retained wild-type susceptibility to RC-101. Overall, these data suggest that most mutations capable of rendering HIV-1 resistant to RC-101 will also exert deleterious effects on the ability of HIV-1 to initiate infections - an interesting and novel property for a potential topical microbicide.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology MS
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Staal, Jens. "Genes and mechanisms in Arabidopsis innate immunity against Leptosphaeria maculans /." Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/200669.pdf.
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Wilson, Kirsty. "Modelling the airway epithelium in vitro as a tool for understanding pulmonary innate defence mechanisms." Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/13496/.
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The airway epithelium forms a continuous barrier from the nose to the alveoli and serves a variety of functions. Multiple functionally distinct cell types are involved in these processes. The innate defence functions require a patent airway epithelium, with infections often associated with epithelial defects and phenotypic alterations that are themselves associated with multiple lung diseases. Non-typeable Haemophilus influenzae (NTHi) and respiratory syncytial virus (RSV) are frequently identified in the airways in a range of respiratory diseases These pathogens often trigger exacerbations and worsening symptoms that often result in hospitalisation. This is particularly true in paediatric populations. Although mortality for NTHi and RSV infections alone are themselves low it remains unclear what role these infections play in mortality rates in complex chronic respiratory infections. These studies aimed to establish NTHi and RSV infections within airway epithelium models, and use them as tools to study pulmonary innate defence mechanisms in order to understand the role of these infections in respiratory disease. In vitro airway models were established using lung derived cell lines, undifferentiated primary human bronchial epithelial (uHBE) cells and air-liquid interface (ALI) differentiated uHBE cell cultures. Following establishment of differentiation we validated ALI cultures using a number of markers, including for the putative innate defence PLUNC family proteins, gel-forming mucins and tubulin. These markers are representative of different epithelial cell types within the cultures. Cultures were infected with NTHi or RSV for periods of time ranging from 1 hour to 7 days with a view to establishing chronic infections and allowing biofilm formation. Monolayer cultures showed an enhanced susceptibility to both infections. Cytokine array profiling showed enhanced pro-inflammatory cytokine profiles in response to NTHi and RSV infections in ALI cells resulting in an ability to manage infections compared to monolayer cultures. Expression analysis indicated that both infections altered the transcription of a number of pro-inflammatory genes. Neutrophil products and trypsin were shown to degrade PLUNC proteins in ALI cell secretions. NTHi also appeared to cause degradation of PLUNC proteins suggesting that infection may impair the innate defence shield of the airway epithelium. Our data showed that differential ALI cultures of human airway cells are a useful model for the study of respiratory pathogens.