Dissertations / Theses: 'LL-37'

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Filewod, Niall Christopher Jack. "Immunomodulatory effects of LL-37 in the epithelia." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/927.

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The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics.

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El, Abbouni Sarah. "Microencapsulation of LL-37 Antimicrobial Peptide in PLGA." Digital WPI, 2016. https://digitalcommons.wpi.edu/etd-theses/235.

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Antimicrobial peptides are key actors in organisms€™ immune systems. They play an important role in phagocytosis, breaking bacteria membranes. They destroy the microbes, keeping them from repairing themselves, and therefore do not promote antimicrobial resistance. LL37 is a peptide produced by the human body. It is a short amino acid chain that is particularly active on the skin and mucous membranes. It has antimicrobial and fungal activity as well as wound healing properties, which makes it a very interesting active substance in wound treatment. However, its fragile and sensitive structure is a challenge to its use. Nowadays, encapsulation in a biocompatible polymer system is a promising technique in drug delivery, and presents a solution to LL37 administration and delivery. LL37 is a hydrophilic active substance, it will be trapped in PLGA (poly (lactic-co-glycolic acid)) by double emulsion and the microspheres will be shaped and stabilized by solvent evaporation. The capsules will be characterized by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy. Their main features, drug loading, encapsulation efficiency and release profile, are determined using the Bradford assay. Since the peptide is expensive and delicate, it is important to optimize its encapsulation. For that reason, we will adapt the process to have the best drug loading as possible using water in oil in oil emulsions. For an external use, the capsules would be used over a few days, so having a fast release is very relevant. The larger the specific surface area, the faster the diffusion. For that reason, we will also study the impact of porosity on the release profile. As a result, different types of capsules will be synthesized, with higher porosity and by two processes: aqueous double emulsion and oil double emulsion. Their characteristic features and impact on bacterial pathogens will be determined and compared in order to determine their optimal synthesis process and formulation in given conditions of use.

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Currie, Silke Maria. "Antiviral function of LL-37 on respiratory syncytial virus." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25954.

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Recurrent infection with human respiratory syncytial virus (RSV) is one of the most common causes for lower respiratory tract illness (LRI) in infants, the elderly, and immunocompromised individuals. Due to lack of vaccines and therapeutic interventions, medical care of acute RSV bronchiolitis is mostly limited to supportive measures. Thus, novel treatment options to control RSV infection are desperately required. The cationic host defence peptide human cathelicidin LL-37 possesses both microbicidal and immunomodulatory properties. This essential effector of the innate immune system holds potent antiviral activity against a variety of viruses, including influenza virus, and has been proposed as a promising candidate for antiviral drug development. Previous studies revealed that lower cathelicidin levels put RSV infected infants at risk for more severe RSV disease, while infection of lung epithelial cells induced cathelicidin up-regulation. These findings suggest that LL-37 might possess antiviral activity against RSV. However, its potential antiviral function on RSV remains to be elucidated. This thesis therefore aimed to evaluate the antiviral activity of cathelicidins against RSV, by assessing its relevance in vitro and in vivo and elucidating the underlying antiviral mechanism. Firstly, the antiviral effects of human cathelicidin LL-37 against RSV were addressed in vitro. Presence of LL-37 during infection potently reduced viral titres and protected cells against virus-associated cytopathic effects. Experiments revealed that only the core region of LL-37 holds antiviral activity against RSV. Antiviral effects were also observed for the murine LL-37 orthologue mCRAMP. Administration of LL-37 at different stages in the infection cycle provided evidence that LL-37 can be used preventatively, protecting against RSV infection by directly acting on both cells and viral particles. When given therapeutically, once an infection was established, LL-37 also limited viral spread. Next, the molecular mechanism mediating the peptide’s antiviral activity was investigated. It was demonstrated that LL-37 does not affect the interferon-mediated cellular antiviral immune response to RSV. Experiments established that LL-37 does not contribute to viral clearance by inducing epithelial cell death. Further mechanistic studies revealed that the peptide directly binds to RSV particles, destabilises the integrity of the viral envelope, and prevents adsorption of RSV to epithelial cells during the entry stage of infection. Finally, the in vivo relevance of LL-37 treatment and endogenous cathelicidin expression was examined, employing both murine and human model systems. It was established that LL-37 has protective antiviral effects against RSV in vivo. In contrast to the cell culture model, only co-administration of LL-37 and RSV, but not treatment prior or post infection, protects mice from clinical signs of infection. Levels of the murine LL-37 orthologue mCRAMP were increased in RSV infected lungs, pointing towards its importance in antiviral defence. In keeping with this, mCRAMP-deficient mice were more susceptible to RSV induced disease. Equally, individuals with low nasal LL-37 baseline levels that were experimentally challenged with RSV, were more susceptible to infection. This highlights the importance of endogenous cathelicidin expression to fight and control RSV infection. Overall, these results identify LL-37 as an important antiviral agent against RSV in vitro and in vivo, and emphasise the role of endogenous cathelicidins in the defence against this pathogen. Moreover, unravelling the underlying antiviral mechanism of LL-37 against RSV adds to our understanding of how CHDP act on enveloped viruses, thus supporting the development of new antiviral treatment options.

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Frew, Lorraine. "The production and function of cervical hCAP18/LL-37 in pregnancy." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/18000.

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Antimicrobial peptides (AMPs) are small proteins produced by epithelial surfaces, which have broad-spectrum antimicrobial and immunomodulatory activities. In the lung, skin and alimentary tract AMPs are known to be important in infectious and inflammatory conditions. Far less is known regarding the role of AMPs within the female reproductive tract, but as infection and inflammation are causes of preterm labour, AMPs may have a key function in maintain and protecting pregnancy. The major groups of human AMPs include the human beta defensins (HBDs), two antileukoproteinases (secretory leukocyte protease inhibitor (SLPI) and Trappin-2/Elafin), and the human cathelicidin hCAP18/LL-37, with several studies identifying their presence at sites throughout the reproductive tract. The cervix in pregnancy is positioned between the upper genital tract containing the developing fetus and the lower tract where infections usually arise. I hypothesise that AMPs are fundamental to mucosal immune defence of the cervix in pregnancy, preventing ascending infection and excessive inflammation that can cause preterm labour. This thesis focused on the human cathelicidin hCAP18/LL-37 and its role within the cervix and vagina. The aims of this thesis were to; investigate the inflammatory effects of LL-37 from cervical and vaginal derived epithelial cells and determine the pathways and receptors in which LL-37 may elicit its effects and how production may be regulated; investigate the role of CRAMP in a mouse model of preterm birth; and determine the production of AMPs by the pregnant cervix whilst investigating the relationship between AMP concentrations in cervicovaginal secretions and preterm labour. The inflammatory effect of LL-37 was investigated using cell lines derived from endocervical, ectocervical and vaginal epithelium. The study of these cell lines suggests divergent responses of cervical and vaginal epithelial cells. LL-37 mediated induction of IL-8 and IL-6 production from endocervical epithelial cells was observed in a dose-dependent and time-dependent manner, whilst ectocervical and vaginal cells also respond to treatment with LL-37 through IL-8 and IL-6 production. To determine a possible mechanism of action of LL-37 on IL-8 and IL-6 in the three cell lines, inhibitors against MAPK cascades, ERK, p38 MAPK and JNK, and known LL-37 receptors were investigated. In endocervical cells LL-37 mediated IL-8 occurs via activation of unidentified GPCRs, whilst in ectocervical cells this effect on IL‐8 and IL-6 is via the activation of ERK and p38 MAPK cascades. The mechanism by which LL-37 induces IL-8 secretion in vaginal epithelial cells remains unknown. Expression of LL-37 was shown to be mediated by vitamin D3 in vitro in cervical and vaginal epithelial cells. However when this relationship was investigated in vivo, using matched serum and cervicovaginal secretions from woman at early pregnancy, no correlation was observed between circulating vitamin D and cervicovaginal or circulating hCAP18/LL-37. However, the majority of women in this study reported with insufficient levels of vitamin D, which may effect the relationship observed with hCAP18/LL-37. Using a mouse model of LPS-induced preterm labour, to mimic the presence of intrauterine infection bacterial infection, I aimed to characterise the role of CRAMP, the mouse orthologue of hCAP18/LL-37, in the lower inflammatory and immune response that results in preterm labour. Wild type C57Bl/6J mice receiving an intrauterine injection of LPS deliver prematurely, within 24 hours of injection. However mice deficient in CRAMP (Camp -/-) receiving an intrauterine injection of LPS deliver significantly later and have a non-significant increase in pup survival compared to wild type C57Bl/6J mice. Cervical tissue collected post partum showed no difference in inflammatory markers between wild type C57Bl/6J and Camp -/- mice, however there was increased expression of the neutrophil chemoattractant marker, Cxcl5, and the neutrophil marker, Ngp in Camp -/- mice. In the lower genital tract, levels of antimicrobial peptides were determined in samples of cervicovaginal secretions collected from pregnant women. AMPs, hCAP18/LL-37, HBD-2 and SLPI were found in cervicovaginal secretions, and levels of hCAP18/LL-37 were increased in women with the common vaginal infection bacterial vaginosis. However no relationship was identified between the concentration of AMPs and preterm birth in this study. This work has shown that the lower genital tract, where infections that are associated with preterm labour originate, expresses the human cathelicidin hCAP18/LL-37. It may play an important role in modulating the immune response to invading infection associated with preterm labour. Further investigation of these responses may increase understanding of the physiology and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.

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Oliveira, Junior Luiz Roberto de. "Vitamina D e LL-37 em Pacientes com Doença de Chagas." Botucatu, 2018. http://hdl.handle.net/11449/153022.

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Orientador: Cilmery Suemi Kurokawa
Resumo: O texto apresentado faz parte dos estudos sobre doença de Chagas (DC), suas formas clínicas e correlação com morbidades associadas à obesidade e envelhecimento e confeccionado para obtenção do título de Mestre em Doenças Tropicais. Ele é dividido em dois capítulos, no qual o primeiro texto compreende uma revisão bibliográfica sobre a doença de Chagas, vitamina D3 e catelicidina LL-37; e, no segundo capítulo, um artigo com dados descritivos da população de estudo intitulado de “FATORES DE RISCO CARDIOVASCULAR ASSOCIADOS À DOENÇA DE CHAGAS CRÔNICA” e um artigo intitulado de “VITAMINA D3, CATELICIDINA LL-37 E POLIMORFISMOS DO GENE VDR EM PACIENTES COM A FORMA INDETERMINADA E CARDÍACA DA DOENÇA DE CHAGAS”. Atualmente a DC é uma doença tropical negligenciada em todo o mundo e endêmica em 21 países latino-americanos, com mais de 25 milhões de pessoas em área de risco de transmissão. Afeta de 8 a 10 milhões de pessoas, sendo que no Brasil estima-se que existam 3 milhões de infectados, com 6 mil mortes por ano. Em sua fase crônica, tem a forma cardíaca como a manifestações mais importante da doença, tanto por sua frequência quanto por sua gravidade, com impacto social e financeiro, já que afetam os indivíduos na fase mais produtiva da vida (entre 30 e 50 anos), com altos índices de morbimortalidade. Além dos distúrbios no sistema de condução cardíaco, pode evoluir para manifestações mais graves como cardiomegalia, falência cardíaca e morte súbita. A inexistência de drogas eficazes pa... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The text presented is part of the studies on Chagas disease (CD), its clinical forms and correlation with morbidities associated with obesity and aging and made for the title of Masters in Tropical Diseases. It is divided into two chapters, in which the first text comprises a literature review on Chagas disease, vitamin D3 and catatelicidin LL-37; and in the second chapter an article with descriptive data from the study population entitled "CARDIOVASCULAR RISK FACTORS ASSOCIATED WITH CHRONIC CHAGAS DISEASE" and an article entitled "VITAMIN D3, CATELICIDINE LL-37 AND VDR GENE POLYMORPHOSMS IN PATIENTS WITH THE UNDETERMINED AND HEART CHAGAS DISEASE FORM". Currently, CD is a neglected tropical disease worldwide and endemic in 21 Latin American countries, with more than 25 million people at risk of transmission. It affects 8 to 10 million people, and in Brazil it is estimated that there are 3 million infected, with 6 thousand deaths a year. In its chronic phase, it has the cardiac form as the most important manifestations of the disease, as much by its frequency as by its gravity, with social and financial impact, since they affect the individuals in the most productive phase of the life (between 30 and 50 years), with high morbidity and mortality rates. In addition to disturbances in the cardiac conduction system, it can progress to more serious manifestations such as cardiomegaly, heart failure and sudden death. The lack of effective drugs to treat the disease in its chronic ph... (Complete abstract click electronic access below)
Mestre

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Carlsson, Martin, and Johan Humlén. "Effekter av den antimikrobiella peptiden LL-37 på humana osteoblasters viabilitet." Thesis, Malmö högskola, Odontologiska fakulteten (OD), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-19902.

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Den antimikrobiella peptiden LL-37 finns uttryckt i alla kroppens slemhinnor och lagras i stora mängder i sekundära granula hos neutrofiler och monocyter. Förutom dess antimikrobiella effekt uppvisar LL-37 pro- alternativt anti-apoptotisk effekt på eukaryota celler beroende på celltyp. I denna studie visar vi för första gången den pro-apoptotiska effekten av LL-37 på den humana osteoblastcellinjen MG63. Vid stimulering med 4μM iakttogs en reduktion av cellantalet med 40% och en utbredd celldöd kunde fastställas genom Trypan Blue-infärgning. Genom flödescytometri sågs en förhöjd andel annexin V-positiva celler och en ökning aktivt kaspas-3 kunde påvisas genom ELISA efter stimulering med 4μM LL-37. Tillsammans med morfologiska tecken såsom skrumpning kan den pro-apoptotiska effekten av LL-37 fastställas. En kraftig och ihållande ökning av intracellulära Ca2+-koncentrationer kunde ses i Fluo 4-AM-infärgade celler i konfokalmikroskop efter stimulering med 4μM LL-37 men inte då Ca2+ exkluderades från mediet. Blockering av den dominerande spänningsberoende Ca2+- kanalen av L-typ med nifedipin hade ingen effekt på de tilltagande intracellulära Ca2+-koncentrationerna vilket påvisar en mekanism som ej involverar den kanalen. I efterföljande försök sågs dessutom att LL-37 reducerade cellantalet oberoende om Ca2+ fanns närvarande i mediet eller ej. Sammanfattningsvis visar data från denna studie att LL-37 reducerar antalet MG63-celler genom pro-apoptotisk effekt i koncentrationer som associerats med kronisk parodontit. Vi föreslår att verkningsmekanismen för apoptos sker via en permeabilisering av plasmamembranet, där tilltagande Ca2+-koncentrationer snarare får ses som en bieffekt av den förlorade membranintegriteten än en central faktor vid den apoptotiska signaleringen i detta system.

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Scott, Aaron. "Functional studies of the human antimicrobial proteins LL-37 and Eppin." Thesis, Queen's University Belfast, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546424.

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Cassin, Margaret Emily. "The Design of Antimicrobial Detachable Thin Films for the Study of Hepatic Infections." Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/77426.

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Microbial infections are a global problem. Due to the over and misuse of antibiotics, drug-resistant pathogens are becoming more common. It is imperative to explore broad spectrum antimicrobial approaches. In this work, we modified collagen/hyaluronic acid polyelectrolyte multilayers (PEMs) with the natural antimicrobial peptide, LL-37 to study hepatic infections. LL-37 was physisorbed and covalently linked to the surface of the PEMs. Escherichia coli DH10B were cultured in the presence of LL-37modified PEMs in bacterial adhesion and contact killing models. Physisorbed LL-37 PEMs prevented bacterial adhesion and could also kill pathogens in the surrounding environment due to the release of LL-37 from the film. Immobilized LL-37 PEMs resulted in less bacterial adhesion on the surface due to the presence of the peptide. Films were then placed in contact with primary rat hepatocytes as well as in hepatocyte/bacteria co-cultures. LL-37 input concentrations up to of 16μM did not exhibit cytotoxic effects on hepatocytes. The LL-37 modified PEMs exhibited a hepatoprotective effect on albumin and urea secretion functions in co-cultures. The hepatoprotective effects were dependent on the ratio of hepatocytes and bacteria as well as the concentration of LL-37. These findings are encouraging and demonstrate that LL-37 modified PEMs can be used to investigate hepatic infections caused by bacteria.
Master of Science

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Dannehl, Claudia. "Fragments of the human antimicrobial LL-37 and their interaction with model membranes." Phd thesis, Universität Potsdam, 2013. http://opus.kobv.de/ubp/volltexte/2013/6814/.

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A detailed description of the characteristics of antimicrobial peptides (AMPs) is highly demanded, since the resistance against traditional antibiotics is an emerging problem in medicine. They are part of the innate immune system in every organism, and they are very efficient in the protection against bacteria, viruses, fungi and even cancer cells. Their advantage is that their target is the cell membrane, in contrast to antibiotics which disturb the metabolism of the respective cell type. This allows AMPs to be more active and faster. The lack of an efficient therapy for some cancer types and the evolvement of resistance against existing antitumor agents make AMPs promising in cancer therapy besides being an alternative to traditional antibiotics. The aim of this work was the physical-chemical characterization of two fragments of LL-37, a human antimicrobial peptide from the cathelicidin family. The fragments LL-32 and LL-20 exhibited contrary behavior in biological experiments concerning their activity against bacterial cells, human cells and human cancer cells. LL-32 had even a higher activity than LL-37, while LL-20 had almost no effect. The interaction of the two fragments with model membranes was systematically studied in this work to understand their mode of action. Planar lipid films were mainly applied as model systems in combination with IR-spectroscopy and X-ray scattering methods. Circular Dichroism spectroscopy in bulk systems completed the results. In the first approach, the structure of the peptides was determined in aqueous solution and compared to the structure of the peptides at the air/water interface. In bulk, both peptides are in an unstructured conformation. Adsorbed and confined to at the air-water interface, the peptides differ drastically in their surface activity as well as in the secondary structure. While LL-32 transforms into an α-helix lying flat at the water surface, LL-20 stays partly unstructured. This is in good agreement with the high antimicrobial activity of LL-32. In the second approach, experiments with lipid monolayers as biomimetic models for the cell membrane were performed. It could be shown that the peptides fluidize condensed monolayers of negatively charged DPPG which can be related to the thinning of a bacterial cell membrane. An interaction of the peptides with zwitterionic PCs, as models for mammalian cells, was not clearly observed, even though LL-32 is haemolytic. In the third approach, the lipid monolayers were more adapted to the composition of human erythrocyte membranes by incorporating sphingomyelin (SM) into the PC monolayers. Physical-chemical properties of the lipid films were determined and the influence of the peptides on them was studied. It could be shown that the interaction of the more active LL-32 is strongly increased for heterogeneous lipid films containing both gel and fluid phases, while the interaction of LL-20 with the monolayers was unaffected. The results indicate an interaction of LL-32 with the membrane in a detergent-like way. Additionally, the modelling of the peptide interaction with cancer cells was performed by incorporating some negatively charged lipids into the PC/SM monolayers, but the increased charge had no effect on the interaction of LL-32. It was concluded, that the high anti-cancer activity of the peptide originates from the changed fluidity of cell membrane rather than from the increased surface charge. Furthermore, similarities to the physical-chemical properties of melittin, an AMP from the bee venom, were demonstrated.
Aufgrund der steigenden Resistenzen von Zellstämmen gegen traditionelle Therapeutika sind alternative medizinische Behandlungsmöglichkeiten für bakterielle Infektionen und Krebs stark gefragt. Antimikrobielle Peptide (AMPs) sind Bestandteil der unspezifischen Immunabwehr und kommen in jedem Organismus vor. AMPs lagern sich von außen an die Zellmembran an und zerstören ihre Integrität. Das macht sie effizient und vor allem schnell in der Wirkung gegen Bakterien, Viren, Pilzen und sogar Krebszellen. Das Ziel dieser Arbeit lag in der physikalisch-chemischen Charakterisierung zweier Peptidfragmente die unterschiedliche biologische Aktivität aufweisen. Die Peptide LL-32 und LL-20 waren Teile des humanen LL-37 aus der Kathelizidin-Familie. LL-32 wies eine stärke Aktivität als das Mutterpeptid auf, während LL-20 kaum aktiv gegen die verschiedenen Zelltypen war. In dieser Arbeit wurde die Wechselwirkung der Peptide mit Zellmembranen systematisch anhand von zweidimensionalen Modellmembranen in dieser Arbeit untersucht. Dafür wurden Filmwaagenmessungen mit IR-spektroskopischen und Röntgenstreumethoden gekoppelt. Circulardichroismus-Spektroskopie im Volumen komplementierte die Ergebnisse. In der ersten Näherung wurde die Struktur der Peptide in Lösung mit der Struktur an der Wasser/Luft-Grenzfläche verglichen. In wässriger Lösung sind beide Peptidfragmente unstrukturiert, nehmen jedoch eine α-helikale Sekundärstruktur an, wenn sie an die Wasser/Luft-Grenzfläche adsorbiert sind. Das biologisch unwirksamere LL-20 bleibt dabei teilweise ungeordnet. Das steht im Zusammenhang mit einer geringeren Grenzflächenaktivität des Peptids. In der Zweiten Näherung wurden Versuche mit Lipidmonoschichten als biomimetisches Modell für die Wechselwirkung mit der Zellmembran durchgeführt. Es konnte gezeigt werden, dass sich die Peptide fluidisierend auf negativ geladene Dipalmitylphosphatidylglycerol (DPPG) Monoschichten auswirken, was einer Membranverdünnung an Bakterienzellen entspricht. Eine Interaktion der Peptide mit zwitterionischem Phosphatidylcholin (PC), das als Modell für Säugetierzellen verwendet wurde, konnte nicht klar beobachtet werden, obwohl biologische Experimente das hämolytische Verhalten zumindest von LL-32 zeigten. In der dritten Näherung wurde das Membranmodell näher an die Membran von humanen Erythrozyten angepasst, indem gemischte Monoschichten aus Sphingomyelin (SM) und PC hergestellt wurden. Die physikalisch-chemischen Eigenschaften der Lipidfilme wurden zunächst ausgearbeitet und anschließend der Einfluss der Peptide untersucht. Es konnte anhand verschiedener Versuche gezeigt werden, dass die Wechselwirkung von LL-32 mit der Modellmembran verstärkt ist, wenn eine Koexistenz von fluiden und Gelphasen auftritt. Zusätzlich wurde die Wechselwirkung der Peptide mit der Membran von Krebszellen imitiert, indem ein geringer Anteil negativ geladener Lipide in die Monoschicht eingebaut wurde. Das hatte allerdings keinen nachweislichen Effekt, so dass geschlussfolgert werden konnte, dass die hohe Aktivität von LL-32 gegen Krebszellen ihren Grund in der veränderten Fluidität der Membran hat und nicht in der veränderten Oberflächenladung. Darüber hinaus wurden Ähnlichkeiten zu Melittin, einem AMP aus dem Bienengift, dargelegt. Die Ergebnisse dieser Arbeit sprechen für einen Detergenzien-artigen Wirkmechanismus des Peptids LL-32 an der Zellmembran.

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Neto, Guilherme Tude Coelho. "Peptídeo antimicrobiano LL-37 e seus efeitos em stemness de diferentes células tumorais." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/5/5164/tde-06032017-104147/.

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Os peptídeos antimicrobianos desempenham papéis protetores críticos em uma gama de doenças humanas, incluindo o câncer. Vários estudos demonstraram funções - tais como proliferação, angiogênese, apoptose e imunomodulação - desses peptídeos em vias cancerígenas cruciais. Investigamos o papel do Peptídeo antimicrobiano LL-37 sobre stemness em câncer de mama (SKBR3) e células de melanoma (A375). Análise por PCR array da expressão diferencial de genes em SKBR3 e A375 com knockdown por siRNA para o mRNA de LL-37 revelou uma regulação negativa de genes relacionados com stemness, incluindo transcriptase reversa da telomerase, forkhead box D3 e para o fator indiferenciado de transcrição de células embrionárias 1, notavelmente em células de câncer de mama.Além disso, as células SKBR3 com knockdown para a expressão de LL-37 mostraram uma diminuição da produção de oncosferas em comparação com controles negativos, enquanto as células A375 exibiram uma produção aumentada. Tomados em conjunto, nossos achados indicam um papel para LL- 37 em stemness, dependendo do tipo de celular analisado
Antimicrobial peptides play critical protective roles in a range of human diseases, including cancer. Multiple studies have demonstrated functions -- such as proliferation, angiogenesis, apoptosis and immunomodulation -- of these peptides in crucial cancer pathways. We investigated the role of the antimicrobial peptide LL-37 on stemness in breast cancer (SKBR3) and melanoma cells (A375). PCR array analysis of differential gene expression in SKBR3 and A375 cancer cell lines downregulated for LL-37 expression by siRNA revealed downregulation of genes related to stemness, including telomerase reverse transcriptase, forkhead box D3 and undifferentiated embryonic cell transcription factor 1, remarkably in breast cancer cells. Furthermore, SKBR3 cells knocked down for LL-37 expression showed a decreased production of oncospheres in comparison with negative controls, while A375 cells exhibited increased production. Taken collectively, our findings indicate a role for LL-37 in cancer cell stemness depending on the cell type

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Milhan, Noala Vicensoto Moreira [UNESP]. "Avaliação do peptídeo LL-37 em contato com células-tronco da polpa dentária." Universidade Estadual Paulista (UNESP), 2017. http://hdl.handle.net/11449/149791.

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O peptídeo LL-37 (catelicidina derivada de humano), é liberado por algumas células humanas e capaz de neutralizar os tecidos com lipopolissacarídeo (LPS), além de atrair células da polpa, e induzir a angiogênese, características que o tornam um possível adjunto para a regeneração do complexo dentino-pulpar. O objetivo desse trabalho foi avaliar in vitro a biocompatibilidade do peptídeo LL-37 nas concentrações de 5 e 10 μg/mL, e sua possível atuação na diferenciação de células-tronco da polpa dentária (DPSC) para odontoblastos- like. Com esse propósito, foram avaliados: (a) a citotoxicidade, pelo teste MTT; (b) a genotoxicidade, através do ensaio do micronúcleo; (c) a produção e quantificação de óxido nítrico; (d) as fases do ciclo celular, por citometria; (e) a expressão de alguns genes associados à formação de tecido mineralizado, através do teste qRT-PCR; (f) o conteúdo de proteína total; (g) a atividade de fosfatase alcalina (ALP); e (h) a produção de sialofosfoproteína dentinária (DSPP), pelo ensaio imunoenzimático ELISA. Foi observado que as concentrações de 5 e 10 μg/mL de LL-37 não foram citotóxicas e ainda aumentaram, em geral, a viabilidade celular (p<0,05), sendo que os maiores valores de absorbância foram observados no 3° dia de contato. As concentrações testadas também não induziram genotoxicidade, após 7 dias de contato, tendo sido genotóxico apenas o grupo controle positivo (EMS) (p<0,05). Ainda, não foi observado diferença estatisticamente significativa na produção de nitrito, pelas células expostas ao LL-37 após 7 dias, em ambas as concentrações. A análise do ciclo celular, evidenciou maior porcentual de células na fase G0/G1, em todos os grupos (p<0,05). Quando estes foram comparados, foi observado maior quantidade de células na fase G0/G1 na concentração de 10 μg/mL de LL- 37 comparada ao grupo controle (p<0,05). Por outro lado, o grupo controle exibiu mais células na fase G2 e em mitose (M) que os grupos tratados com 5 e 10 μg/mL de LL-37 (p<0,05), e mais células na interfase (S) que o grupo tratado com 10 μg/mL de LL-37 (p<0,05). A análise da expressão gênica demonstrou que não houve aumento de expressão dos genes fosfatase alcalina, osteocalcina, osteopontina e Runx2 após tratamento com ambas as concentrações do peptídeo, no 3° dia. Além disso, não foi observado diferença estatisticamente significativa na ALP nos grupos tratados e controle, após 3 e 14 dias, enquanto o conteúdo de proteína total foi maior aos 14 dias nos grupos tratados com LL-37 (p<0,05). Ainda, aos 3 dias, a produção da proteína DSPP foi maior no grupo tratado com 10 μg/mL de LL-37 (p<0,05). Com base nesses resultados, pode-se concluir que o LL-37 é biocompatível nas concentrações testadas nesse trabalho, e ainda aumenta o número de células viáveis, principalmente em período inicial. Além disso, aos 3 dias, na concentração de 10 μg/mL, ele retarda o ciclo celular e aumenta a expressão da proteína DSPP, além de aumentar a síntese proteica aos 14 dias, o que indica que esse peptídeo pode desempenhar algum tipo de função na diferenciação odontoblástica.
The LL-37 peptide (human derived cathelicidin) is released by some human cells and able of neutralizing the tissues that present lipopolysaccharide (LPS), as well as, attracts pulp cells and induces angiogenesis; characteristics that makes it a possible adjunct for regeneration of the dentin-pulp complex. The aim of this study was evaluate in vitro the biocompatibility of LL-37 in the concentrations of 5 and 10 μg/mL, and its possible performance in the differentiation of dental pulp stem cells (DPSC) into odontoblasts-like cells. For this purpose, it was evaluated: (a) the cytotoxicity by MTT assay; (b) the genotoxicity by the micronucleus test; (c) the production and quantification of nitric oxide; (d) the cell cycle, by flow cytometry; (e) the expression of genes associated with the mineralization by qRT-PCR; (f) the total protein content; (g) the alkaline phosphatase activity (ALP); and (h) the production of dentine sialofosfoprotein (DSPP) by indirect enzyme-linked immunosorbent assay (ELISA). It was observed that the concentrations of 5 and 10 μg/ml of LL-37 were not cytotoxic, in addition to they increased, in general, the cell viability (p<0,05). Moreover, higher absorbance values were observed on 3rd day of contact. After 7 days, the tested concentrations also did not induce genotoxicity, (p<0,05); only the positive control group (EMS) was genotoxic (p<0.05). Furthermore, there was not statistical significance in the nitrite production by the cells exposed to LL-37 for 7 days, in both concentrations. The cell cycle test showed higher percentage of cells in the phase G0/G1 in all groups (p<0.05). When they were compared, it was noticied that concentration of 10 ug/ml of LL-37 arrested the cells in G0/G1 compared to the control group (p<0.05). On the other hand, the control group, exhibited higher amount of cells in G2 and mitosis (M) than the others (p<0.05) and also higher number of cells in interfase (S) than the group treated with 10 μg/mL of LL-37 (p<0.05). On the 3rd day, the analysis of gene expression demonstrated no increase in the expression of the genes alkaline phosphatase, osteocalcin, osteopontin and Runx2, after treatment with both peptide concentrations. Furthermore, it was not observed statistical significance in the ALP in the treated and control groups after 3 and 14 days, while total protein content was higher in the groups treated with LL-37, at 14 days (p<0.05). On the 3rd day, the production of DSPP protein was higher in the group treated with 10 μg/mL of LL-37 (p<0.05). Based on these results, it can be concluded that LL-37 is biocompatible at these concentrations and increases the number of viable cells, especially in the initial period. Moreover, on the 3rd day, the concentration of 10 μg/mL arrests the cell cycle, and increases the expression of DSPP protein, in addition to raising the protein content at 14 days, which indicates that this peptide may present some kind of function in the odontoblastic differentiation.

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12

Milhan, Noala Vicensoto Moreira. "Avaliação do peptideo LL-37 em contato com células-tronco da polpa dentária /." São José dos Campos, 2017. http://hdl.handle.net/11449/149791.

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Orientador: Samira Esteves Afonso Camargo
Banca: Luana Marotta Reis de Vasconcellos
Banca: Mônica Ghislaine Oliveira Alves
Banca: Cristina Pacheco Soares
Banca: Cacio de Moura Netto
Resumo: O peptídeoLL-37 (catelicidina derivada de humano), é liberado por algumas células humanas e capaz de neutralizar os tecidos com lipopolissacarídeo (LPS), além de atrair células da polpa, e induzir a angiogênese, características que o tornam um possível adjunto para a regeneração do complexo dentino-pulpar. O objetivo desse trabalho foi avaliar in vitro a biocompatibilidade do peptídeo LL-37 nas concentrações de 5 e 10 µg/mL, e sua possível atuação na diferenciação de células-tronco da polpa dentária (DPSC) para odontoblastoslike. Com esse propósito, foram avaliados: (a) a citotoxicidade, pelo teste MTT; (b) a genotoxicidade, através do ensaio do micronúcleo; (c) a produção e quantificação de óxido nítrico; (d) as fases do ciclo celular, por citometria; (e) a expressão de alguns genes associados à formação de tecido mineralizado, através do teste qRT-PCR; (f) o conteúdo de proteína total; (g) a atividade de fosfatase alcalina (ALP); e (h) a produção de sialofosfoproteína dentinária (DSPP), pelo ensaio imunoenzimático ELISA. Foi observado que as concentrações de 5 e 10 µg/mL de LL-37 não foram citotóxicas e ainda aumentaram, em geral, a viabilidade celular (p<0,05), sendo que os maiores valores de absorbância foram observados no 3° dia de contato. As concentrações testadas também não induziram genotoxicidade, após 7 dias de contato, tendo sido genotóxico apenas o grupo controle positivo (EMS) (p<0,05). Ainda, não foi observado diferença estatisticamente significativa na produção de nitrito, pelas células expostas ao LL-37 após 7 dias, em ambas as concentrações. A análise do ciclo celular, evidenciou maior porcentual de células na fase G0/G1, em todos os grupos (p<0,05). Quando estes foram comparados, foi observado maior quantidade de células na fase G0/G1 na concentração de... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract : The LL 37 peptide (human derived cathelicidin) is released by some human cells and able of neutralizing the tissues that present lipopolysaccharide (LPS), as well as, attracts pulp cells and induces angiogenesis; characteristics that makes it a possible adjunct for regeneration of the dentin-pulp complex. The aim of this study was evaluate in vitro the biocompatibility of LL-37 in the concentrations of 5 and 10 µg/mL, and its possible performance in the differentiation of dental pulp stem cells (DPSC) into odontoblasts-like cells. For this purpose, it was evaluated: (a) the cytotoxicity by MTT assay; (b) the genotoxicity by the micronucleus test; (c) the production and quantification of nitric oxide; (d) the cell cycle, by flow cytometry; (e) the expression of genes associated with the mineralization by qRT-PCR; (f) the total protein content; (g) the alkaline phosphatase activity (ALP); and (h) the production of dentine sialofosfoprotein (DSPP) by indirect enzyme-linked immunosorbent assay (ELISA). It was observed that the concentrations of 5 and 10 µg/ml of LL-37 were not cytotoxic, in addition to they increased, in general, the cell viability (p<0,05). Moreover, higher absorbance values were observed on 3rd day of contact. After 7 days, the tested concentrations also did not induce genotoxicity, (p<0,05); only the positive control group (EMS) was genotoxic (p<0.05). Furthermore, there was not statistical significance in the nitrite production by the cells exposed to LL-37 for 7 days, in both concentrations. The cell cycle test showed higher percentage of cells in the phase G0/G1 in all groups (p<0.05). When they were compared, it was noticied that concentration of 10 ug/ml of LL-37 arrested the cells in G0/G1 compared to the control group (p<0.05). On the other hand, the control group, exhibited higher amount of cells in G2 and mitosis...(Resumo completo, clicar acesso eletrônico abaixo)
Doutor

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13

Li, Yue Xin. "The human cationic host defense peptide LL-37 modulates neutrophil apoptosis and chemokine responses." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/31726.

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LL-37 is a human cationic peptide expressed primarily by neutrophils and epithelial cells. It is a 37 amino acid peptide that belongs to the cathelicidin family of the cationic host defense peptides. Accumulating evidence has demonstrated that LL-37 has multiple immunomodulatory properties. The modulatory effects of LL-37 on neutrophils were investigated here, and LL-37 was shown to be a potent inhibitor of spontaneous apoptosis in human neutrophils, signalling through P2X₇ receptors and G-protein-coupled receptors other than the formyl peptide receptor-like- 1 molecule. Inhibition of neutrophil apoptosis involved modulation of Mcl-1 expression, inhibition of BID and procaspase-3 cleavage, and the activation of phosphatidylinositol-3 kinase and protein kinase C but not the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2) pathway. In addition, LL-37 modified neutrophil cytokine/chemokine responses to pro-inflammatory stimuli in a stimulus-specific manner. Specifically, LL-37 abrogated LPS-induced TNF-a cytokine production while enhancing IL-1β elicited release of TNF-a as well as a number of chemokines including IL-8, Gro-a, CCL-22 and Mip-la . The increased release of chemokines induced synergistically by LL-37 and IL-1β resulted from de novo protein synthesis and was found to be associated with the signalling through the ERK1/2 and p38 MAP kinases and nuclear factor κΒ pathways. These novel immunomodulatory properties of LL-37 may contribute to peptide-mediated enhancement of innate host defenses against acute infection and are of considerable significance in the development of such peptides and their synthetic analogs as potential therapeutics for use against multiple antibiotic-resistant infectious diseases.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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14

Zhang, P. "Identification of staphylococcal genes involved in resistance to the human antimicrobial peptide LL-37." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1380282/.

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Staphylococcus aureus is well-known for its ability to acquire resistance to a broad range of antimicrobial agents and a limited number of commercially available antibiotics exist that are active against multidrug resistant strains. Antimicrobial peptides have been suggested as promising alternatives to current antimicrobials due to their potent antimicrobial activity against a broad range of microorganisms including multidrug resistant bacteria, and a membrane-lytic mode of action that is thought to have low possibility of inducing bacterial resistance. This study describes the identification of S. aureus genes involved in resistance to the human cationic antimicrobial peptide LL-37, with a particular interest in the effects of a physiological concentration of bicarbonate on the resistance mechanism. Transposon mutagenesis and recombinase-based in vivo expression technology systems were designed to enable genome-wide screening. A S. aureus transposon mutant library was screened for increased resistance to LL-37 in the presence of bicarbonate. Mutants with insertions in yycH and yycI, demonstrated bicarbonate-dependent resistance to LL-37. Both yycH and yycI form part of a predicted operon yycFGHI in S. aureus, and have been shown to be suppressors of an essential two component system YycFG in B. subtilis that regulates cell wall metabolism. The resistance of S. aureus small colony variants (SCVs) to LL-37 was also investigated. SCVs defective in hemB, menD or aroD, demonstrated bicarbonate-dependent resistance to LL-37. Furthermore, SigB (a global regulator) and TcaR (an activator of protein A) were found to exert opposite effects on resistance to LL-37 in the presence of bicarbonate. Strains defective in TcaR showed bicarbonate-dependent resistance to LL-37, interestingly, this resistance was abolished by either deleting sigB or repairing tcaR in these strains. These data suggest that YycFG, SigB, TcaR and the SCV phenotype may play important roles in resistance to LL-37 under in vivo conditions where bicarbonate is present.

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Bravo, Martha de Oliveira. "Efeito do peptídeo catelicidina LL-37 sobre a propriedade imunossupressora das células-tronco mesenquimais." reponame:Repositório Institucional da UnB, 2016. http://repositorio.unb.br/handle/10482/21629.

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Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2016.
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As células-tronco mesenquimais (CTMs) desempenham importante função imunorregulatória, por escapar do reconhecimento imune e suprimir a resposta imunológica. A infusão de CTMs é útil para o tratamento de situações em que o sistema imunológico apresenta resposta exacerbada, como na doença do enxerto contra o hospedeiro (DECH). Entretanto, o elevado número de células necessário para a obtenção do efeito terapêutico desejado constitui um grande limitante para o uso clínico dessas células. Para realçar o potencial supressivo das CTMs, duas estratégias têm sido realizadas: o condicionamento (licenciamento) e a terapia combinada de CTMs com imunossupressores. A catelicidina (LL- 37) é um peptídeo antimicrobiano que exerce diversas funções modulatórias sobre a resposta imune, estimulando, em alguns contextos a produção de fatores anti-inflamatórios, como IL-10 e, em outros, estimulando mediadores pró-inflamatórios. Com base nesse contexto, avaliamos, in vitro, se o condicionamento de CTMs pelo peptídeo LL-37 realça o potencial supressivo dessas células e se esse peptídeo poderia ser utilizado como adjuvante das CTMs na supressão de linfócitos T. Além disso, investigamos se esse peptídeo exerce efeito sobre a proliferação e potencial migratório das dessas células. Por fim, investigamos a influência de LL-37 na expressão do receptor tipo toll-3 (RTT3) e no perfil de expressão gênica das CTMs. O condicionamento das CTMs com peptídeo LL-37 não influenciou o potencial supressivo dessas células, entretanto, quando usado como adjuvante, LL-37 aumenta o efeito supressor das CTMs. A catelicidina LL-37 não influenciou a capacidade proliferativa das CTMs, mas quando usada como adjuvante realçou o potencial migratório dessas células. Por fim, investigamos mecanismos moleculares que poderiam sustentar os efeitos funcionais observados. LL-37 induziu aumento da expressão do RTT3 nas CTMs e a hiperexpressão de genes anti-inflamatórios, o que, em parte, pode explicar os efeitos observados. De modo geral, os resultados obtidos nesse estudo, podem servir de base para o desenvolvimento de abordagens terapêuticas inovadoras que tenham por objetivo realçar propriedades fundamentais das CTMs, como o efeito imunossupressivo e o potencial migratório, garantindo, ao mesmo tempo, efeito contra microrganismos oportunistas. __________________________________________________________________________________________________ ABSTRACT
Mesenchymal stem cells (MSCs) play an important immunoregulatory function, to escape immune recognition and suppress the immune response. Infusion of MSCs is useful for treatment of conditions where the immune system has exacerbated response, as in graftversus- host disease (GVHD). However, the large number of cells needed to achieve the desired therapeutic effect is a major limitation to the clinical use of these cells. To enhance the suppressive potential of MSCs, two strategies have been carried out: the conditioning (licensing) and combination MSCs therapy with immunosuppressants. The cathelicidin (LL- 37) is an antimicrobial peptide that exerts various modulatory functions on the immune response, stimulating, in some contexts the production of anti-inflammatory factors, such as IL-10, and others, stimulating proinflammatory mediators. Within this context, evaluated in vitro as MSCs conditioning the LL-37 peptide enhances the suppressive potential of these cells and this peptide could be used as adjuvant of MSCs in suppression of T lymphocytes. Furthermore, we investigated whether this peptide has an effect on the proliferation and migration potential of these cells. Finally, we investigated the influence of LL-37 in the toll-like receptor type 3 expression (TLR3) and gene expression profile of MSCs. The conditioning of MSCs with LL-37 peptide did not affect the suppressive potential of these cells, however, when used as adjuvant, LL-37 increases the suppressive effect of MSCs. The LL-37 cathelicidin did not affect the proliferative capacity of MSCs, but when used as an adjuvant enhanced the migration potential of these cells. Finally, we investigate molecular mechanisms that could sustain the observed functional effects. LL-37 induced increased expression in MSCs TLR3 and overexpression of anti-inflammatory genes, which in part may explain the observed effects. In general, the results obtained in this study could serve as the basis for the development of innovative therapeutic approaches which aim to enhance the fundamental properties of MSCs as immunosuppressive effect and migration potential, ensuring at the same time, effect against opportunistic microorganisms.

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Ghannad, Mona. "Design and Synthesis of Collagen-binding Anti-microbial Proteins." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/19981.

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The Herpes simplex virus (HSV) is a virus that commonly infects the skin, and mucous membrane of the mouth, genitalia, and the eye. HSV-1 is the strain that is most commonly associated with corneal infections, and it is the most frequent cause of corneal blindness in North America [1]. Currently no cure is available, and many limitations are characterized by the currently available synthetic antiviral drugs, which suggest the need for other potential drug alternatives and delivery strategies. Anti-microbial peptides are naturally occurring peptides that are potent killers of a broad range of micro-organisms, including bacteria, fungi, and viruses [2]. AMPs are known to be a key component of the innate immune response at the human ocular surface. The human cathelicidin-derived AMP, LL-37, expressed in human corneal epithelial cells provides a wide range of protection against viral pathogens such as HSV-1 [3]. My thesis research addressed the design and recombinant production of hybrid AMP sequences containing LL-37 with the potential ability to form chemical or physical associations with a Collagen scaffold material, such as those used in current artificial cornea constructs to address the need for alternative anti-viral drugs. Three fusion proteins were tested, and compared for feasible design anti-microbial peptide expression and purification in E. coli. It was illustrated that the thioredoxin and SUMO fusion systems are good candidates for successful recombinant production of active designed peptides. The point-mutated LL-37 sequence was successfully expressed and purified using the thioredoxin fusion system. It was demonstrated that this modified LL-37 was effective against HSV-1 infection. The SUMO system was used to express the bio-functional LL-37 containing a collagen-binding sequence. Further work is required to address issues regarding recombinant AMP production, such as increasing enzymatic cleavage efficacy, and minimizing proteolytic degradation or modification.

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Li, Hsin-Ni. "Impact of cationic host defence peptide LL-37 on human neutrophil death and inflammatory responses." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5597.

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Cathelicidins are cationic host defence peptides (CHDP) with essential roles in the innate defence system. These peptides have antimicrobial potential and the capacity to modulate innate immunity and inflammatory processes. Neutrophils (PMN) are the main reservoir of cathelicidins and play key roles in first line defence against infection. The appropriate regulation of PMN function, death, and clearance is critical to innate immunity, and the efferocytosis of apoptotic PMN, in contrast to necrotic cells, is proposed to promote the resolution of inflammation. In this thesis I demonstrate that the human cathelicidin LL-37 rapidly induced secondary necrosis of apoptotic human PMN and identify the essential C-terminal region of LL-37 required for this activity. In addition to the induction of secondary necrosis, higher concentrations of LL-37 also promoted PMN granule contents release. LL-37-induced secondary necrosis did not affect PMN ingestion by human monocyte-derived macrophages and, in contrast to expectation, was not proinflammatory. Interestingly, the anti-inflammatory effects of apoptotic PMN on activated macrophages were retained and even potentiated where LL-37-mediated secondary necrosis induced anti-inflammatory granule content release. Consistent with the results of in vitro studies, in vivo murine sterile peritonitis model revealed the same phenomenon: LL-37-induced secondary necrosis diminished inflammatory responses with decreased PMN influx. I also present data on LL-37- mediated modulation of innate immune effector cell cytokines responses to inflammatory signals. I propose that during acute inflammation LL-37 can modulate innate immune responses through its activity on cytokine production, and that LL-37-mediated secondary necrosis of apoptotic PMN has anti-inflammatory effects, but may also mediate host damage by promoting the release of potentially harmful intracellular contents under chronic or dysregulated conditions.

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18

Jackson, Alexander Rodney. "Pharmacological Evaluation of Cyanoguanidine P2X7 Receptor Antagonists." Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/17186.

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ABSTRACT BACKGROUND AND AIMS: The P2X7 receptor (P2X7R) is an ATP-gated, non-selective cation channel highly expressed on monocytes, macrophages and microglia. Prolonged activation of the P2X7R by ATP leads to cytolytic pore formation and the release of inflammatory mediators including interleukin-1β and prostaglandin E2. Accumulating evidence suggests a role for the P2X7R in neuroinflammation and thus P2X7R antagonists might be useful in diseases including chronic pain, depression and Alzheimer’s disease. Both negative allosteric modulators of the P2X7R, such as the adamantyl benzamides, and orthosteric antagonists, such as the aryl cyanoguanidines, inhibit the ATP-induced release of IL-1β from immune cells. This shared ability to inhibit IL-1β release may explain why no attempts have been made to determine the features which promote binding to the allosteric or orthosteric site. An advantage of targeting the allosteric or orthosteric site might emerge however, if a different agonist of the P2X7R is used. An antimicrobial peptide produced within the human body, LL-37, is also able to activate the P2X7R and yet LL-37 is never used in the characterisation of new series of P2X7R antagonists. The aims of this project were to characterise a novel series of P2X7R antagonists, the adamantyl cyanoguanidines, which have a hybrid structure derived from the adamantyl benzamides and aryl cyanoguanidines. Characterisation of the adamantyl cyanoguanidines should allow determination of the features which promote binding to the orthosteric or allosteric site of the P2X7R, which was one of the primary aims of this project. A second aim was to evaluate the potential of the hybrid series for further development as P2X7R antagonists by considering their potency and physicochemical P a g e | 13 properties. The final aim was to determine if there was any advantage of targeting the orthosteric or allosteric site of the P2X7R particularly with regard to inhibiting LL-37-mediated activation of the P2X7R. METHODS: The potency of adamantyl cyanoguanidines and reference P2X7R antagonists were determined in YO-PRO-1 dye uptake assays and interleukin-1β release assays to develop structure-activity relationships. A potent member of the adamantyl cyanoguanidines and several reference P2X7R antagonists were pharmacologically characterised in Schild assays, washout studies and receptor protection studies. The ability of several negative allosteric modulators and an orthosteric antagonist to inhibit LL-37-induced dye uptake was also examined. RESULTS: More compact adamantyl cyanoguanidines including those with a methylene linker between the adamantyl and cyanoguanidine groups and no linker between the cyanoguanidine group and phenyl ring were more potent than analogues with longer linkers. Ortho-substitution of the phenyl ring or substitution of the ring with 5-quinoline led to increased potency. The potency seen in the dye uptake assay was also seen in the interleukin-1β release assay. A potent member of the series 3-19 was determined to be a slowly reversible negative allosteric modulator as was 1-17 an adamantyl benzamide. The parent aryl cyanoguanidine, A-804598, was confirmed to be an orthosteric antagonist. None of the compounds were able to inhibit LL-37-induced dye uptake. DISCUSSION AND CONCLUSIONS: The determined structure-activity relationships for the adamantyl cyanoguanidines confirm their potential for further development since the series was highly amenable to modification and several potent analogues have favourable physicochemical properties including lower molecular weight. Since 3-19 P a g e | 14 was a negative allosteric modulator despite its structural similarity to A-804598 this suggests the adamantyl group promotes binding to the allosteric site and that cyanoguanidine is a tolerated bioisostere for the acetamide group at the allosteric site. The 5-quinolinyl group, in the appropriate position, facilitates binding to either site. The failure of multiple P2X7R antagonists to inhibit LL-37-induced dye uptake is concerning since LL-37 alone has been shown to induce the release of interleukin-1β from human monocytes. Future research must determine if LL-37 is responsible for cytokine release in vivo and develop small molecule antagonists of the action of LL-37.

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19

Yu, Jie. "Immunomoduatory properties of host defence peptide LL-37 during infection and inflammation in human blood cells." Thesis, University of British Columbia, 2006. http://hdl.handle.net/2429/31500.

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The human cathelicidin, LL-37, is a cationic host defence peptide and serves as an essential component of innate immunity. In addition to its modest antimicrobial activity, LL-37 has been demonstrated to be a multifunctional modulator of innate immune responses, although the mechanism(s) of which have not been elucidated. The present study demonstrated that LL-37 could synergistically enhance IL-1β-induced production of cytokines (IL-6, IL-10) and chemokines (MCP-3) in primary human PBMCs. In contrast to the neutralization of LPS-induced secretion of pro-inflammatory cytokines, LL-37 dramatically augmented LPS-stimulated MCP-3 production. LL-37 by itself induced transient phosphorylation of IkB-α. and the subsequent nuclear translocation of NF - kB subunits p50 and p65, which could be further enhanced in the presence of IL-1β. Similar effects of LL-37 and I L-1β were also observed oh activation of Akt and CREB. Therefore, we propose that, in addition to its well-known anti-inflammatory activity, the human host defence peptide LL-37 also plays an important role in boosting the innate immune responses in combination with inflammatory mediator (IL-1β), which provides a new mechanism for LL-37 in modulating the inflammatory responses in innate immunity.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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20

Zreika, Sami. "Etude de l'impact de la protéine antimicrobienne humaine hCAP18/LL-37 sur le cancer du sein." Thesis, Tours, 2011. http://www.theses.fr/2011TOUR4052.

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Le peptide hCAP18/LL-37, une partie de la défense immunitaire innée, a maintenant été reconnu comme multifonctionnelle pour les cellules eucaryotes. Nos études démontrent sa contribution au développement du cancer, montrant qu'il est surexprimé dans la plupart des tumeurs mammaires humaines, active la signalisation la famille de ERBB et augmente le potentiel métastatique des cellules cancéreuses du sein. Notre comparaison des deux lignées du cancer du sein n'a pas révélé de récepteurs communs, mais une structure peptidique identiques mais de chiralité différente est pré requis pour le peptide dans toutes ses activités. Nous émettons l'hypothèse que LL-37 active indirectement des récepteurs transmembranaires en se liant à la membrane cellulaire. Des peptides tronqués dérivés de LL-37 inhibent ses activités et peuvent aider à concevoir une future thérapie anticancéreuse
The peptide hCAP18/LL-37, part of the innate immune defense, has now been recognized as multifunctional for eukaryotic cells. Our studies demonstrate its contribution to cancer development, showing that it is overexpressed in most human breast tumors, activates ERBB signaling and increases the metastatic potential of breast cancer cells. Our comparison on two breast cancer lines did not reveal any common receptors but identical structural prerequisites for the peptide in all its activities. We hypothesize that LL-37 indirectly activates transmembrane receptors by attaching to the cellular membrane. Truncated derivatives inhibit its activities and may help to design a future anticancer therapy

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Andrault, Pierre-Marie. "Rôle des cathepsines à cystéine dans la régulation du peptide antimicrobien LL-37 lors de pathologies inflammatoire chroniques pulmonaires." Thesis, Tours, 2015. http://www.theses.fr/2015TOUR4035/document.

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Lors de pathologies pulmonaires inflammatoires chroniques comme la mucoviscidose ou la BPCO, le déséquilibre de la balance protéases/antiprotéases aboutit à la dégradation du tissu pulmonaire et à l’inactivation des défenses antimicrobiennes. Les cathepsines à cystéine participent à l’inactivation protéolytique de peptides et protéines antimicrobiens (PAMs) pulmonaires comme le SLPI, la lactoferrine, et les β-défensines HBD-2 et -3 lors de l’emphysème ou de la mucoviscidose. Lors de cette thèse, nous avons étudié la capacité des cathepsines à cystéine B, K, L et S à hydrolyser le peptide LL-37, qui est un PAM important dans l’immunité innée pulmonaire. Seules les cathepsines K et S clivent le LL-37 et inactivent efficacement son activité antimicrobienne. A l’inverse, le LL-37 est un inhibiteur compétitif de la cathepsine L. D’autre part, l’expression pulmonaire de la cathepsine S est fortement augmentée chez les individus fumeurs atteints ou non de BPCO. La fumée de cigarette qui est une source importante de stress oxydatif induit une augmentation significative de l'expression et l'activité de la cathepsine S. Malgré un environnement oxydatif non favorable à l'activité des cathepsines, la cathepsine S parvient à hydrolyser le peptide LL-37 et pourrait ainsi augmenter le risque d’exacerbation lors de la BPCO
During chronic inflammatory lung diseases like cystic fibrosis or COPD, proteases/antiproteases imbalance leads to pulmonary tissue degradation and compromise antimicrobial barrier. Cysteine cathepsins are involved in the proteolytic inactivation of several lung antimicrobial peptides (AMPs) such as SLPI, lactoferrin and β- defensins -2 and -3 during emphysema or cystic fibrosis. During this thesis, we studied the ability of cathepsins B, K, L and S to degrade LL-37, which is an important AMP in lung immunity. Only cathepsins K and S degrade readily LL-37 and inactivate its antimicrobial property. Conversely, LL-37 is a competitive inhibitor of cathepsin L. Beside, lung expression of human cathepsin S is significantly increased in smokers with or without COPD compared to non-smokers. Cigarette smoke that is a major source of oxidative stress significantly increases the expression and activity of cathepsin S. Despite an unfavorable oxidative environment, cathepsin S retains its proteolytic activity toward LL-37 and thus could participate to COPD exacerbation

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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.

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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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Дудник, В. М., К. В. Хромих, and О. П. Федчишен. "Діагностичне значення вмісту протимікробного пептиду С-кінцевого hCAP18 кателіцидину LL-37 у дітей, хворих на бронхіальну астму." Thesis, Сумський державний університет, 2017. http://essuir.sumdu.edu.ua/handle/123456789/64839.

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Бронхіальна астма (БА) - хронічне алергічне захворювання дихальних шляхів, що проявляється нападами задишки або ядухи, сухим кашлем у відповідь на вплив алергену або на тлі простудного захворювання, фізичного навантаження, емоційного стресу [GINA, 2016]. Відомі два сімейства антимікробних пептидів - кателіцидини та дефензини. Кателіцидини - група антимікробних білків, що володіють N- термінальним сигнальним білком, незмінною кателіновою частиною і структурно постійно змінюваних ділянок. Мають широкий спектр антимікробної та імумономодулюючої активності [Абатуров А. Е., 2011]. Поряд з прямою антимікробної здатністю цей білок взаємодіє з іншими білками, викликає міграцію нейтрофілів, моноцитів і Т-клітин, стимулює проліферацію ендотелію. У новонароджених вміст LL-37 підвищено, що компенсує недостатність адаптаційного імунітету[Bals R., 2010].
This article studied the diagnostic value content antimicrobial peptide C-terminal hCAP18 cathelitcidin LL-37 in children with asthma. To achieve this goal we examined 200 children with asthma aged 6 to 18 years. The content of cathelitcidin LL-37 in patients with asthma was significantly higher (p ≥ 0,001), than in the group of healthy children. Cathelitcidin LL-37 content is higher at nonatopic and mixed versions of asthma than in atopic. Also associated with the severity and adequate control of asthma.

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Gambade, Audrey. "Rôle du peptide LL-37 dans le cancer du sein : son interaction avec la membrane plasmique stimule l'entrée de calcium et la migration cellulaire par l'activation des canaux ioniques TRPV2 et BKCa." Thesis, Tours, 2015. http://www.theses.fr/2015TOUR3312/document.

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Le peptide antimicrobien LL-37 a été retrouvé surexprimé dans différents types de cancer et plus particulièrement dans le cancer du sein dans lequel il est associé au développement des métastases. Nous avons observé, in vitro, que la migration de trois lignées cancéreuses mammaires est augmentée par le peptide LL-37 et son énantiomère (D)-LL-37, excluant la fixation du peptide à un récepteur protéique. Sur les cellules cancéreuses mammaires MDA-MB-435s, le peptide se fixe à la membrane plasmique et diminue sa fluidité. La microscopie électronique localise LL-37 dans les cavéoles et à la surface de structures impliquées dans la migration cellulaire, les pseudopodes. LL-37 induit une entrée de calcium via le canal TRPV2 dont l’activité est augmentée par son recrutement dans les pseudopodes. Ce recrutement est dépendant de l’activation de la voie de signalisation PI3K/AKT induite par LL-37. L’entrée de calcium via TRPV2 est potentialisée par l’activation du canal potassique BKCa, localisé aussi dans les pseudopodes. Des ARN interférents contre TRPV2 inhibent à 70% la migration induite par LL-37, donnant un rôle prépondérant à ce canal dans les effets pro-migratoire du peptide. La fixation du peptide LL-37 aux membranes des cellules cancéreuses et l’activation de canaux ioniques constituent un nouvel axe de recherche pour comprendre le rôle du peptide dans la progression tumorale
The antimicrobial peptide LL-37 is overexpressed in several types of cancer, among which breast cancer were it is associated with metastasis development. Our experiments on three mammary cancer cell lines have shown that LL-37 increases cell migration. Both its natural (L)-form and its (D)-enantiomer are equally active, excluding a specific binding to a protein receptor. On the MDA-MB-435s cell line, LL-37 attaches to plasma membrane and reduces its fluidity. Electron microscopy localized LL-37 on the surface of pseudopodia, structures implicated in cell migration, and in caveolae. LL-37 induces calcium entry via the TRPV2 channel, which is recruited to pseudopodia. Recruitment depends on activation of PI3K/AKT signaling induced by LL-37. Calcium entry via TRPV2 is potentiated by activation of the BKCa potassium channel also located in pseudopodia. TRPV2 suppression by RNA interference results in 70% reduction of cell migration induced by LL-37, attributing a crucial role of this channel to the promigratory effects of the peptide. Binding of LL-37 to cancer cell membranes and in consequence the activation of ion channels constitutes a novel research field to understand its role in tumor progression

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Habes, Chahrazed. "Stimulation du signal calcique et de la migration des cellules cancéreuses mammaires par le peptide LL-37 : un mécanisme d’attachement membranaire impliquant les glycosaminoglycanes et les syndécanes." Thesis, Tours, 2019. http://www.theses.fr/2019TOUR3807.

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Le peptide LL-37, peptide antimicrobien de l’immunité innée, est associé au développement tumoral. Sur des lignées cancéreuses mammaires. Il augmente le calcium intracellulaire et la migration. via l’activation de la voie PI3K/AKT et de canaux calciques. L’énantiomère (D)-LL-37 induit des effets similaires, excluant une interaction classique ligand-récepteur. Avec sa structure en hélice α amphipathique et sa charge nette +6, l’hypothèse était que le peptide utilise les charges négatives de glycanes sulfatés ou sialylés pour sa fixation membranaire avant d’induire ses activités. Des lectines végétales reconnaissant des structures glycaniques sialylées ou sulfatées inhibent la migration et le signal calcique induits par LL-37 mais l’implication de sialyltransférases n’a pas été démontrée. Des glycoaminoglycanes (GAGs) tels que les chondroïtine et héparine sulfatées, utilisés en tant que compétiteurs, ou une digestion enzymatique des GAGs (Chondroïtinase et héparinase) conduisent à une inhibition de 50 à 100% des activités migratoire et calcique induites par LL-37. L’inhibition de synthèse des GAGs par le Methylumbelliferyl β-D-xyloside ainsi qu’une diminution de la sulfatation par le chlorate de sodium confirment l’implication de ces glycanes sulfatés. Dans la perceptive d’identifier la ou les protéines glycosylées membranaires susceptibles de transduire les effets de LL-37, nous avons utilisé une approche ciblée en invalidant par siRNA la synthèse des protéines arborant des GAGs. Le syndécane 4 est impliqué dans les activités de LL-37. En conclusion, ces résultats soulignent l’implication de GAGs sulfatés portés par le syndécane 4 pour orienter la fixation de LL-37 à la membrane des cellules cancéreuses et médier ses activités pro-tumorales
Initially characterized by its antimicrobial activities, LL-37 has also been shown to significantly contribute to tumor development. On breast cancer cell lines, LL-37 increases intracellular calcium and their migration via the activation of PI3K/AKT signaling. Its all-D enantiomer (D)-LL-37 induces similar effects, which excludes an protein-protein interaction of LL-37 in a classic ligand-receptor manner. Its structure of an amphipathic a-helix with a net charge of +6 gave rise to the hypothesis that the peptide uses the negative charges of sulfoglycans or sialic acids to facilitate its attachment to the cell membrane and to induce its activities. Whereas several lectins, specifically attaching to sialylated or sulfated structures, blocked the activities of LL-37 on both calcium increase and cell migration, the suppression of several sialyltransferases had no effect. However, the competitive use of glycoaminoglycans (GAG) and chrondroitin and sulfated heparin, or treatment of the cell surface with chondroitinase and heparinase resulted in an activity loss of 50-100%. Similar results were obtained by confirmed by blocking the synthesis of GAGs with Methylumbelliferyl β-D-xyloside, and by suppression of glycan sulfurylation by sodium chlorate. Using a candidate approach by suppressing proteoglycan synthesis by RNA interference, syndecan 4 was shown to be involved in the activities of LL-37. This leads to the conclusion that sulfated GAGs linked to syndecans 4 guides the association of LL-37 to the membrane of cancer cells, thus being a mediator of its activities

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Dannehl, Claudia [Verfasser], and Gerald [Akademischer Betreuer] Brezesinski. "Fragments of the human antimicrobial LL-37 and their interaction with model membranes / Claudia Dannehl. Betreuer: Gerald Brezesinski." Potsdam : Universitätsbibliothek der Universität Potsdam, 2013. http://d-nb.info/104363312X/34.

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Evers, Daniela [Verfasser], and Ursula [Akademischer Betreuer] Bilitewski. "Der Einfluss des antimikrobiellen Peptides LL-37 auf Candida albicans und den Infektionsprozess in vitro / Daniela Evers ; Betreuer: Ursula Bilitewski." Braunschweig : Technische Universität Braunschweig, 2012. http://d-nb.info/117589091X/34.

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Schwab, Julia [Verfasser], and W. [Gutachter] Scheppach. "Antimikrobielle Aktivität humaner Kolonepithelzellen gegenüber E. coli Nissle unter besonderer Berücksichtigung des Cathelicidins LL-37 / Julia Schwab. Gutachter: W. Scheppach." Würzburg : Universität Würzburg, 2014. http://d-nb.info/1102828556/34.

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Xhindoli, Daniela. "Insights into structural features that affect the biological activities and mode of action of the human antimicrobial cathelicidin LL-37." Doctoral thesis, Università degli studi di Trieste, 2014. http://hdl.handle.net/10077/10381.

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2012/2013
The human peptide LL-37 is an important innate immune effector that contributes to defending the organism against infection in different scenarios, ranging from direct bacterial killing to the modulation of immune responses and acting as a signal molecule for different cell types involved in defence or healing. All these functions are possible despite its relatively simple structure, due to its capacity to interact with different types of biological membranes. This cationic peptide is able to switch from a random coil in aqueous solutions into an amphipathic helical structure in presence of salt ions or in the presence of membranes. Intra- and inter-molecular hydrophobic interactions in the hydrophobic sector of the amphipathic helix, together with ionic interactions in the polar sector, make LL-37 prone to self-association, which strongly affects its multiple biological roles, and these therefore need to be explored in detail. In this thesis I have investigated how folding occurs when LL-37 passes from bulk solution to the surface of membranes, and which structural features could be relevant for its in vitro antimicrobial and host cell modulating activities. For this reason comparative biophysical studies were performed on LL-37 and its structurally diverse orthologue in macaque, RL-37. Fluorescent analogues of LL-37 and RL-37, obtained from the systematic substitution of Phe residues with the non-invasive fluorescent probe, p-cyanophenylalanine, contributed to elucidate differential conformational variations in these peptides. Steady-state and time-resolved fluorescence studies confirmed that unlike RL-37, which is monomeric in physiological solution or in presence of neutral model membranes, LL-37 is in an oligomeric form in solution, with a hydrophobic core likely forming near the central region. Furthermore LL-37 appears to interact with both anionic and neutral membranes in an oligomeric form. Cytofluorimetric studies, performed on bacterial cells treated at toxic peptide concentrations, confirmed that the different conformations of LL- and RL-37 in solution give rise to quite different modes of bacterial permeabilization. Lesions of a larger size were observed for LL-37, consistent with a toroidal pore-forming mechanism, while smaller lesions were detected for RL-37 and may indicate an incipient detergent-like mechanism of bacterial inactivation. I further investigated how many molecules are involved in the self-associated form of LL-37, either in solution or in presence of membranes, replacing the Phe5 residue with an analogue bearing Benzoyl-phenylalanine on the side-chain, which is able to crosslink to adjacent molecules when irradiated with UV light. Photo-crosslinking studies revealed that LL-37 is in equilibrium between forms ranging from monomeric to hexameric in physiological solution, and dissociates to lower order oligomers in the presence of membranes, but does not become completely monomeric. The effects of oligomerization on the biological activities of LL-37 were further explored through testing its antiparallel and parallel covalently bound dimers, linked at either end, designed to mimic an obligatory dimeric form of the peptide. Both types of dimerization reduced the antibacterial potency of LL-37 and its capacity to permeabilize the bacterial membrane. Cytotoxic effect against the host cells was instead different for the three forms of dimers tested, and was related to the degree of freedom of the C- or N-terminal region. From the information obtained on the role of oligomerization on the antibacterial and cytotoxic activity of LL-37, I then rationally designed four different analogues of LL-37, which bear minimal primary structure alterations from the native peptide, such as the replacement of one, or exchange of two adjacent residues, in order to modify inter- and intra-molecular salt-bridging. I attempted in this way to transfer some of the RL-37 characteristics to LL-37 in order to improve its antimicrobial properties through reducing its propensity to self-assemble. I confirmed that variations in the equilibrium between attractions or repulsions in LL-37 significantly affect its antibacterial activity and mode of action.
Il peptide antimicrobico dell’uomo LL-37 è un componente importante del sistema immunitario che contribuisce a difendere l’organismo dalle infezioni, per via di diversi meccanismi, che possono variare dall'azione diretta battericida, alla modulazione della risposta immunitaria, agendo da molecola segnale per diversi tipi di cellule coinvolte nella difesa oppure nella guarigione dell'organismo ospite. Tutte queste attività sono dovute alla sua struttura relativamente semplice, ma capace di interagire con diversi tipi di membrane biologiche. Questo peptide carico positivamente è in grado di assumere una conformazione elicoidale in presenza di soluzioni saline oppure in presenza delle membrane. La presenza di interazioni idrofobiche, inter- e intra-molecolari, nel settore idrofobico dell’elica amfipatica insieme alle forze ioniche presenti nella regione polare causano aggregazione di LL-37. Questa caratteristica a sua volta influenza fortemente le diverse attività biologiche di LL-37 e perciò è un aspetto importante che necessita di essere studiato in modo approfondito. In questa tesi io ho investigato come avviene la strutturazione del peptide quando esso passa dalla soluzione alla superficie della membrana, e quali sono gli aspetti strutturali rilevante per l'attività antimicrobica in vitro e per la modulazione della risposta delle cellule dell’ospite. Per questo motivo sono stati usati vari metodi biofisici per confrontare la struttura di LL-37 con il suo ortologo nella scimmia RL-37. Sostituendo un residuo di fenilalanina con la sonda fluorescente non invasiva p-cyano-fenilalanina è stato possibile ottenere vari analoghi fluorescenti dei due peptide nativi, disegnati per ottenere maggior informazione a riguardo le variazioni conformazionali e per investigare il meccanismo d’azione di LL- e RL-37. Gli studi con gli analoghi fluorescenti hanno confermato che diversamente da RL-37, che è monomerico in soluzioni fisiologiche o in presenza di membrane neutre, LL-37 forma un oligomero in soluzione, con un nucleo idrofobico attorno alla regione centrale dell’elica. Inoltre, LL-37 interagisce con le membrane sia cariche positivamente che neutre nella forma oligomerica. Dagli studi fatti con il cyto-fluorimetro, trattando le cellule batteriche con concentrazioni tossiche di peptide, si è notato che i due peptidi con conformazione diversa in soluzione interagiscono e permeabilizzano le membrane in modo diverso. Le lesioni causate nei batteri dopo il trattamento con LL-37 erano regolari e di dimensioni tali da suggerire un meccanismo battericida per via di formazione dei pori di membrana, invece RL-37 causa danni di piccole dimensioni alla membrana batterica, compatibile con un meccanismo d’azione simile a quello di un detergente. Inoltre io ho indagato sul numero di molecole che partecipano alla formazione della forma oligomerica di LL-37, sia in soluzione che in presenza delle membrane. Questo è stato possible tramite la sostituzione di Phe5 con un residuo analogo benzoyl-fenilalanina, il quale permette di legare covalentemente molecole adiacenti, quando viene irradiato con raggi UV. Questi studi hanno dimostrato che LL-37 è in equilibrio tra la forma monomerica ed oligmerica, con estensione fino ad esamero in soluzioni fisiologiche, per poi dissociarsi a forme inferiori oligomeriche in presenza delle membrane. Gli effetti dell’oligomerizzazione del peptide LL-37 sulle sue attività biologiche sono stati ulteriormente investigati tramite lo studio svolto con i dimeri covalenti di LL-37, legati con un ponte di solfuro nella regione C-terminale oppure N-terminale. Questi dimeri, paralleli o antiparalelli sono stati ideati per mimare la fomra oligomerica di LL-37. Entrambi i tipi di dimerizzazione hanno mostrato che l’aggregazione di LL-37 porta ad una riduzione dell’attività antimicrobica e dell’efficienza del peptide a permeabilizzare le membrane batteriche. Invece la cito-tossicità verso le cellule dell’ospite è diversa per tutte e tre forme dimeriche ed è legata al grado di libertà della regione N-terminale del peptide. Date le informazioni ottenute sul ruolo dell’oligomerizzazione sull'attività antibatterica e citotossica di LL-37, io ho disegnato 4 analoghi di LL-37 ottenuti da una minima alterazione della struttura primaria rispetto al peptide nativo, come per esempio lo scambio della posizione di uno o più residui adiacenti in modo da alterare la rete di ponti saline tra le molecole e interamente la struttura del peptide. In questo modo si è voluto attribuire alcune caratteristiche di RL-37 al peptide dell’uomo LL-37, in modo da modificare la sua propensione ad assemblarsi per formare oligomeri. Con questi studi si è confermato che variazioni nel equilibrio delle forze attrattive o repulsive nella catena di LL-37 possono alterare significativamente la sua attività antibatterica e il modo d’azione.
XXVI Ciclo
1985

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Vandewalle-Capo, Marine. "Étude de la sensibilité aux antibiotiques et aux peptides antimicrobiens humains de Legionella pneumophila." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1291/document.

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Legionella pneumophila (Lp) est un pathogène accidentel de l'homme capable d'infecter les macrophages alvéolaires et les pneumocytes. Au cours de l'infection, Legionella se confronte à différents types d'agents antibactériens, dont les peptides antimicrobiens (PAMs) produit par l'hôte et les antibiotiques à activité intracellulaire administrés aux patients. Le mécanisme d'action des PAMs humains à l'encontre de Legionella, ainsi que le niveau de résistance aux antibiotiques de la bactérie sont à ce jour encore peu documentés. Mes travaux ont pour but de contribuer à une meilleure connaissance de l'activité anti-Legionella de ces molécules. La première partie de cette étude a consisté à évaluer la sensibilité d'isolats cliniques de Lp sg 1 à 8 antibiotiques, afin de déterminer le seuil épidémiologique de sensibilité de la bactérie à ces différentes molécules. Nous avons démontré que l'ensemble des isolats cliniques sont sensibles aux antibiotiques testés. Les résultats ont révélé l'existence d'une sous-population présentant une sensibilité réduite aux macrolides. L'analyse des génomes a permis de corréler cette sensibilité diminuée à la présence de la pompe à efflux LpeAB spécifique des macrolides. Cette pompe est présente uniquement dans trois complexes clonaux centrés sur le ST1, le ST701 et le ST1335.La seconde partie de cette étude a été consacrée à la caractérisation de l'activité antibactérienne des PAMs humains LL-37 et HBD-3, ainsi qu'à l'identification de leur(s) mécanisme(s) d'action contre Legionella. L'ensemble des tests réalisés montre que LL-37 et HBD-3 induisent une perte de cultivabilité des légionelles par des modes d'action différents. Les résultats suggèrent que LL-37 agit par perméabilisation des membranes de L. pneumophila. Nos résultats ont également montré que les deux peptides exercent une activité inhibitrice sur la réplication intracellulaire des légionelles, au moins en partie grâce à une collaboration avec la cellule hôte
Legionella pneumophila (Lp) is an accidental human pathogen which can infect alveolar macrophages and pneumocytes. During infection, Legionella have to deal with to various types of antibacterial agents, such as antimicrobial peptides (AMPs) produced by the host, and antibiotics with intracellular activity administered to patients. The mechanism of action of human AMPs against Legionella, and the resistance level to antibiotics of the bacterium are still poorly described. Our work aimed to contribute to a better understanding of the anti-Legionella activity of these molecules. The first part of this study consisted in the evaluation of the susceptibility of clinical Lp sg1 isolates to 8 antibiotics, to determine the epidemiological cut-off values of these different molecules. We demonstrated that all clinical isolates are susceptible to the tested antibiotics. The results revealed the presence of a subpopulation displaying a reduced susceptibility to macrolides. The analysis of the genomes allowed us to correlate this reduced susceptibility to le presence of the LpeAB macrolides efflux pump, found specifically in the sequence types ST1, ST701 and ST1335.The second part of this study was dedicated to the characterization of the antibacterial activity of the human AMPs LL-37 and HBD-3, and to the identification of their mechanism(s) of action against Legionella. All of the experiments show that LL-37 and HBD-3 induce a loss of cultivability by different mode of action. The results suggest that LL-37 is able to permeabilize the membrane of the L. pneumophila cells. Our findings also show that both peptides inhibit the intracellular replication of L. pneumophila, in part through collaboration with the host cell

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McQuade, Rebecca. "Clostridium difficile Responds to Antimicrobial Peptides and Oxidative Stress." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/578613.

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Clostridium difficile (CD) is the leading cause of bacterial hospital-associated infection in North America. How CD colonizes the human host, including its response to the innate immune system and other stresses, is poorly understood. This work considers CD's defenses against two stresses found in the host - the antimicrobial peptide LL-37 and reactive oxygen species (ROS). LL-37 had bactericidal activity against CD. CD strains varied in their sensitivity to the peptide, and epidemic-associated strains were more resistant to LL-37 than others. CD became more resistant to LL-37 following exposure to sub-lethal concentrations of the peptide, suggesting the presence of inducible resistance mechanisms. A quantitative proteomics analysis revealed definite alterations in CD protein expression caused by LL-37. Specific changes included increased expression of DltB, a protein previously reported to confer resistance against other antimicrobial peptides. Notably, disruption of individual LL-37-induced genes did not sensitize CD to the peptide. This suggests functional redundancy, and that LL-37 may cause global changes in protein expression, not limited to antimicrobial peptide resistance determinants. One of the proteins most strongly induced by LL-37 was a predicted superoxide reductase (SOR). As CD is considered a strict anaerobe, expression of a predicted antioxidant protein was an interesting finding. Heterologous expression of CD SOR in a superoxide dismutase-deficient E. coli strain confirmed its action as a superoxide scavenger. Insertional inactivation of SOR rendered CD more sensitive to oxygen and ROS-generating compounds, indicating that SOR contributes to antioxidant defense in CD. SOR mutants were impaired in their ability to cause disease in hamsters, indicating a role for this protein in infection.

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Bedran, Telma Blanca Lombardo [UNESP]. "Efeito antimicrobiano e modulador da resposta imune dos peptídeos hBD-3 e LL-37 e dos polifenóis o chá verde e do cranberry." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/124090.

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The antimicrobial peptides LL-37, hBD-1, hBD-2 and hBD-3 are considered an endogenous antibiotic, with important role in the prevention of periodontal diseases due to their ability to regulate the immune response. However those peptides could be degraded by periodontal pathogens. Therefore, therapies able to up regulate the secretion of those peptides by human cells, and the association of antimicrobial peptides with natural compounds, which may act in synergism to modulate the immune response, may be a novel approach for effectively controlling periodontal diseases. The aim of this in vitro study were: i) investigate the ability of green tea extract and EGCG to induce hBD-1 and hBD-2 secretion and gene expression by gingival epithelial cells (B11) and to protect hBDs from degradation by P. gingivalis, ii) A 3D co-culture model of gingival epithelial cells and fibroblasts stimulated with A. actinomycetemcomitans LPS (1 μg/ml) were used to investigated the anti-inflammatory properties of the hBD-3, LL-37, ACPACs and EGCG and to determine whether LL-37 acts in synergy with AC-PACs, EGCG and hBD-3. Gingival epithelial cells were stimulated with green tea extract or EGCG in the presence and absence of specific inhibitors. The secretion and gene expression of hBD-1 and hBD-2 was respectively measured by ELISA and qPCR. The ability of green tea extract and EGCG to prevent hBDs degradation by P. gingivalis present in a bacterial culture supernatant was evaluated by ELISA. A 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to A. actinomycetemcomitans LPS stimulation compared to fibroblasts and epithelial cells individually. The 3D co-culture model was stimulated with noncytotoxic concentrations of: i) hBD-3 (10 and 20 μM) ...(Complete abstract electronic access below)

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33

Collins, Declan. "Factors predicting patient outcomes in a UK Burn's Unit : the influence of Acinetobacter baumannii and the antimicrobial peptide LL-37 in burn wounds." Thesis, Queen Mary, University of London, 2011. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8716.

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Sepsis and multi-organ failure are the most frequently reported causes of death in burn injuries. Their early identification allows therapies and resources to be targeted in a more effective and efficient way. Due to its frequent antibiotic drug resistance Acinetobacter baumannii (MRAB) is increasingly causing a problem in burns units. New strategies need to be found to combat infection and sepsis in the burn ICU. This study examines the potential of the Albumin Creatinine Ratio, a marker of systemic endothelial dysfunction in predicting outcomes, sepsis and multi-organ failure; the role of Acinetobacter in causing organ failure; and explores for the presence of the cathelicidin, LL-37 in the burn wound and examines it potential utility for treating infection and sepsis. It was found that ACR on admission and at 48 hours is predictive of patient outcomes and the development of sepsis, and may be of use predicting multi-organ failure. Multi-organ failure occurs more frequently in MRAB patients compared to those patients with drug sensitive Acinetobacter baumannii. The number of agency nursing staff and work intensity are possible contributing factors in MRAB acquisition. LL-37 has been found in both acute burn wounds as well as in the grafted healing burn wound and is active against drug resistant Acinetobacter baumannii. ACR can therefore identify those patients at risk of sepsis and may have a role in predicting multi-organ failure. MRAB acquisition in the burns intensive care unit is a significant cause for concern as patients are more likely to suffer from multi-organ failure as well as prolonging their hospital stay and resulting in poorer outcomes. LL-37 has many functions and importantly plays a role in the body’s innate immune system. In the era of increasing antibiotic resistance it may provide a novel therapeutic role in treating MRAB infection.

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Bedran, Telma Blanca Lombardo. "Efeito antimicrobiano e modulador da resposta imune dos peptídeos hBD-3 e LL-37 e dos polifenóis o chá verde e do cranberry /." Araraquara, 2014. http://hdl.handle.net/11449/124090.

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Orientador: Denise Madalena Palomari Spolidório
Banca:Juliana Rico Pires
Banca: Shelon Cristina Souza Pinto
Banca: Luciene Cristina Souza Pinto
Banca: Joni Augusto Cirelli
Resumo:Os peptídeos antimicrobianos como por exemplo a catelicina (LL-37) e as defensinas humanas (hBD-1, hBD-2 e a hBD-3) são considerados antibióticos endógenos com importante papel na prevenção das doenças periodontais, devido a sua capacidade de regulação da resposta imune, sendo que os mesmos podem ser degradados pelos periodontopatógenos. Terapias que aumentem a produção destes peptídeos pelas próprias células do organismo, assim como a associação destes peptídeos com compostos naturais os quais possam agir em sinergismo na regulação da resposta imune, podem ser considerados novas estratégias para o melhor controle das doenças periodontais. Portanto os objetivos deste estudo in vitro foram: i) Avaliar a capacidade do extrato do chá verde (Camellia sinensis) e do seu polifenol, o EGCG, sobre a expressão gênica de hBD-1 e hBD-2 pelas células epiteliais gengivais (B11), sobre a degradação das mesmas frente ao P. gingivalis, ii) Através da utilização do modelo 3D de co-cultura celular, avaliar a capacidade antiinflamatória dos peptídeos hBD-3 e LL-37 quando em associação sobre a produção de citocinas, quimiocinas e fatores de crescimento, iii) Avaliar a capacidade anti-inflamatória da associação do EGCG e do polifenol proveniente do cranberry, o AC-PACs, com o peptídeo LL-37 sobre a produção de citocinas, quimiocinas e fatores de crescimento em modelo de co-cultura celular. As células epiteliais gengivais (B11) foram estimuladas com o extrato do chá verde e com o EGCG na presença e ausência de inibidores específicos. A produção e expressão gênica de hBD-1 e hBD-2 foram quantificados respectivamente pelas técnica de ELISA e qPCR. A capacidade do extrato do chá verde e do EGCG em proteger a degradação de hBDs pelo P. gingivalis foi mensurado através da técnica de ELISA. Foi desenvolvido um modelo em 3D de co-cultura de fibroblastos gengivais embebidos em....(Resumo completo, clicar acesso eletrôni
Abstract: The antimicrobial peptides LL-37, hBD-1, hBD-2 and hBD-3 are considered an endogenous antibiotic, with important role in the prevention of periodontal diseases due to their ability to regulate the immune response. However those peptides could be degraded by periodontal pathogens. Therefore, therapies able to up regulate the secretion of those peptides by human cells, and the association of antimicrobial peptides with natural compounds, which may act in synergism to modulate the immune response, may be a novel approach for effectively controlling periodontal diseases. The aim of this in vitro study were: i) investigate the ability of green tea extract and EGCG to induce hBD-1 and hBD-2 secretion and gene expression by gingival epithelial cells (B11) and to protect hBDs from degradation by P. gingivalis, ii) A 3D co-culture model of gingival epithelial cells and fibroblasts stimulated with A. actinomycetemcomitans LPS (1 μg/ml) were used to investigated the anti-inflammatory properties of the hBD-3, LL-37, ACPACs and EGCG and to determine whether LL-37 acts in synergy with AC-PACs, EGCG and hBD-3. Gingival epithelial cells were stimulated with green tea extract or EGCG in the presence and absence of specific inhibitors. The secretion and gene expression of hBD-1 and hBD-2 was respectively measured by ELISA and qPCR. The ability of green tea extract and EGCG to prevent hBDs degradation by P. gingivalis present in a bacterial culture supernatant was evaluated by ELISA. A 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to A. actinomycetemcomitans LPS stimulation compared to fibroblasts and epithelial cells individually. The 3D co-culture model was stimulated with noncytotoxic concentrations of: i) hBD-3 (10 and 20 μM) ...(Complete abstract electronic access below)
Doutor

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Malhotra, Sankalp. "Immune evasion tactics and immunopathology of mixed mucoid and nonmucoid Pseudomonas aeruginosa populations in cystic fibrosis." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1524156292309518.

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36

Thienhaus, Maike Luisa [Verfasser]. "Die Rolle der antimikrobiellen Peptide humanes beta-Defensin 3 (hBD-3) und LL-37 bei chronisch polypöser Rhinosinusitis und nasaler Besiedelung mit Staphylococcus aureus / Maike Luisa Thienhaus." Kiel : Universitätsbibliothek Kiel, 2014. http://d-nb.info/1046563300/34.

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Bociek, Karol. "Use of Tn5-transposon mutagenesis for the identification of potential novel interactors of antimicrobial peptides in E.coli." Doctoral thesis, Università degli studi di Trieste, 2010. http://hdl.handle.net/10077/3619.

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2008/2009
Antimicrobial peptides (AMPs) participate in the immunity of both animals and plants. In mammals, the most important AMP families are defensins and cathelicidins. The only human cathelicidin, LL-37, is a 37-residue, α-helical, cationic peptide with a direct membranolytic antibacterial activity and various immunomodulatory activities. The aim of our research was to establish a procedure based on an E. coli knock-out mutant library in the search for mutations conferring altered susceptibility to AMPs. The library was created by random insertions of Tn5 transposon into the bacterial genome. The selection of low-susceptibility mutants and their identification was first tested with a Bac7-derived peptide, for which at least one interactor had been found. The procedure was then applied for the identification of mutations that modulate the susceptibility of E. coli cells to LL-37 with the aim of dissecting the steps of the peptide's mode of action. The differences between the wild-type and the mutant phenotypes were characterised using assays determining the bacterial culture growth inhibition and killing, as well as cell binding and membrane permeabilisation. In the majority of the isolated Bac7-resistant mutants, Tn5 was inserted in the sbmA gene, confirming previous findings that showed an essential role for the SbmA protein in the internalisation of Bac7, allowing it to exert its antimicrobial activity. Tn5 insertion in the gene waaY, identified in 15 out of 20 resistant mutants selected with LL-37, lowered the peptide’s ability to inhibit bacterial growth and to kill cells, decreased peptide binding to the bacterial surface and membrane permeabilisation. However, it did not alter the susceptibility to other AMPs. WaaY encodes a specific kinase that phosphorylates the Hep II residue in the core region of bacterial lipopolysaccharide. The inactivation of this enzyme determines a decreased negative charge of the outer membrane, which in turn causes a decrease in the peptide’s binding to the cell surface and in its antibacterial activity. The results reveal a putative LPS-binding site for LL-37 and stress the importance of its initial binding to the cell surface for antimicrobial efficacy. The established procedure utilising Tn5 mutants in the search for bacterial mutations conferring resistance to AMPs, proved efficient and opened a previously unreported branch of research on the mode of action of the human cathelicidin.
I peptidi antimicrobici (AMP) fanno parte dell'immunità innata sia degli animali che delle piante. Le famiglie di AMP più importanti nei mammiferi sono le defensine e le catelicidine. L'unica catelicidina presente nell'uomo, LL-37, è un peptide lineare e cationico di 37 residui con una conformazione ad α-elica, che esercita un’azione microbicida diretta tramite permeabilizzazione di membrana. In aggiunta, il peptide è anche dotato di una serie di funzioni immunomodulatorie. Lo scopo del lavoro è stato quello di mettere a punto una procedura che utilizza una libreria di mutanti knock-out di E. coli, ottenuta mediante inserzioni casuali del trasposone Tn5 nel genoma batterico, per cercare mutazioni che rendano i batteri resistenti agli AMP. La bontà del metodo è stata prima verificata selezionando e identificando mutanti resistenti al frammento 1-16 del peptide Bac7. Una volta ottenuti i risultati attesi, la procedura è stata applicata all'identificazione di mutazioni che modulino la suscettibilità di E. coli a LL-37, con lo scopo di meglio comprenderne il meccanismo d'azione. I fenotipi wild-type e mutante sono stati caratterizzati usando saggi d'inibizione della crescita batterica, di killing e di capacità del peptide di legarsi ai batteri e di permeabilizzarli. Nella maggioranza dei mutanti resistenti al peptide Bac7 isolati, Tn5 ha inattivato il gene sbmA, confermando risultati precedenti che avevano portato a dimostrare l'importanza della proteina SbmA per l'internalizzazione di Bac7. Nel caso di LL-37, in 15 dei 20 mutanti resistenti selezionati il trasposone ha inattivato il gene waaY, che codifica per una chinasi specifica per la fosforilazione del residuo Hep II della regione core del lipopolisaccaride batterico. La mancata fosforilazione nei mutanti selezionati riduce la carica negativa della superficie batterica, che, a sua volta, diminuisce l'inibizione della crescita, il killing, il legame e la permeabilizzazione delle cellule da LL-37, ma non da altri peptidi, suggerendo che la modificazione abbia alterato specificamente l'attività di LL-37, rivelando un suo putativo sito di legame all’LPS e sottolineando l'importanza del legame iniziale alla superficie cellulare per l'efficacia battericida del peptide. La procedura messa a punto per la ricerca di mutazioni che inducono resistenza agli AMP è risultata efficiente ed ha indicato una nuova via per far luce sulla sequenza di eventi responsabile del meccanismo d'azione della catelicidina umana.
XXII Ciclo
1982

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38

Lofton, Tomenius Hava. "Mechanisms and Biological Costs of Bacterial Resistance to Antimicrobial Peptides." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-284119.

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The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

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KO, Yen-Chieh, and 葛彥頡. "Production of recombinant LL-37 and preparing SAW-MIP Biosensor for detection of LL-37." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/70840641425718170863.

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碩士
大同大學
生物工程學系(所)
98
It’s well known that bacteria have ability to develop antibiotic-resistance in short time after treatment. To find therapeutic agents with different mode of action than conventional antibiotics is a necessity. To meet the above requirement, antimicrobial peptides (AMPs) represent a good alternative for infective antibiotics. AMP LL-37 with positive charges belongs to an immune system that has a capacity to distinguish membrane composed by different lipids in human. This unique property enables LL-37 to destroy the negative charged bacteria membranes. In consequence the above mechanism is effective to bacteria those have developed resistance to antibiotics. Neither bacteria nor LL-37 exists in healthy individuals, except suffering urinary tract infection (UTI). The DNA sequence encoding full-length LL-37 was chemically synthesized and cloned into the pET-32a(+) vector for protein expression in BL21(DE3) Escherichia coli. The expressed LL-37-containing fragment was purified by metal affinity chromatography. LL-37 peptide was released by chemical cleavage in 50% formic acid at 50 ℃ for 32 h. and separated by reverse-phase HPLC. We applied the Surface Acoustic Wave–Molecularly Imprinted Polymers principle - a molecular templating polymer aggregation on the Surface Acoustic Wave, to form imprints of the cathelicidin LL-37. LL-37 was polymerized together with functional monomer Methacrylic acid ( MAA )/Poly (ethylene glycol) dimethacrylate（PEG400DMA, Mn=550） (5:95 by volume), in 10 minutes at ambient temperature. The polymerization was caused by bonding protein and polymer together by MAA. The template with identifiable hole of protein was formed. Using this characteristics to screen the structure of LL-37, and detect the frequency variations of surface acoustic wave by network analyzer. We polymerized the concentration 0.02 mg/ml, 0.08 mg/ml, and 0.14 mg/ml of LL-37 on the SAW–MAP sensor area. Removed the target molecule after washing the sensor by NaOH and then measured the frequency as start frequency. The star frequency of concentration 0.02 mg/ml is 106,437,500 Hz, then the frequency decrease to 106,421,875 Hz after rebinding the LL-37. The star frequency of concentration 0.08 mg/ml is 105,125,000 Hz, then the frequency decrease to 105,093,750 Hz after rebinding the LL-37. The star frequency of concentration 0.14 mg/ml is 106,250,000 Hz, then the frequency decrease to 106,202,750 Hz after rebinding the LL-37. So we can ascertain that the SAW–MIP binding higher concentration protein then the frequency would decrease more. Besides, the interference HSA didn’t influence the change of SAW–MIP frequency.

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Cheng, An-Ting, and 鄭安婷. "Interaction of LL-37 with Model Membrane Systems and Preparing Lipid Based Biosensor for the Detection of LL-37." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/76077186069867131972.

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碩士
大同大學
生物工程學系(所)
97
It’s well known that bacteria have ability to develop antibiotic-resistance in short time after treatment. To find therapeutic agents with different mode of action than conventional antibiotics is a necessity. To meet the above requirement, antimicrobial peptides (AMPs) represent a good alternative for infective antibiotics. AMP LL-37 with positive charges belongs to an immune system that has a capacity to distinguish membrane from different lipids in human. This special property makes LL-37 destroy the negative charged bacteria membranes. Therefore the above mechanism is effective to drug resistance bacteria. Normally bacteria and LL-37 will not exist in urine, except for those patients who suffer urinary tract infection (UTI). Hopefully, a screening biosensor can be made by using the above character. DSC (Differential Scanning Calorimeter) was used to measure interaction between LL-37 and liposome that plays a biomembrane role. The stability of the complex can be affected by adding interference salt and urea. In the meantime, the change of liposome structure can be observed by a fluorescence probe on it. Finally, the biosensor was made by immobilizing liposomes on silane coated surface. Testing was made by mixing the sample containing LL-37 on the biosensor plate. The content of LL-37 can be detected and determined by impedance and phase angle variations.

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Bowdish, Dawn Marie Edith. "LL-37, a human host defense peptide with immunomodulatory properties." Thesis, 2005. http://hdl.handle.net/2429/17332.

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Human cationic antimicrobial protein-18 (hCAP-18) is the sole human cathelicidin. It is found at high concentrations in the specific granules of neutrophils, is modestly expressed by epithelial and other cells and can be induced during the course of infection and/or inflammation. The mature, extracellular form of hCAP-18 is termed LL-37, which is a positively-charged, 37-amino acid peptide. Neutrophil-derived host defence peptides were initially discovered as components of the non-oxidative killing mechanisms of neutrophils; however, it is unclear whether LL-37 kills bacteria directly at mucosal surfaces where it is found at lower concentrations. I demonstrated that although LL-37 is antimicrobial in vitro under low salt conditions, it has little or no antimicrobial activity in media containing physiologically relevant cation concentrations. Thus I hypothesised that direct antimicrobial activity was probably not its primary function in vivo at mucosal surfaces. The immunomodulatory properties of LL-37 were investigated in tissue culture media which contains physiological concentrations of cations. When monocytes were treated with LL-37, lipopolysaccharide (LPS)-induced production of proinflammatory cytokines was blocked but TNF-α- induced cytokine production was unaffected and IL-1β-induced cytokine production was enhanced. Consistent with this observation, LL-37 was able to block LPS-induced translocation of the p65 subunit of the pro-inflammatory transcription factor, NF-KB. Other early signalling events mediated by LL-37 included activation of two mitogen-activated protein kinases, p38 and extracellular-regulated kinase. The activation of these kinases was required for IL-8 transcription and release as well as transcription of the chemokines MIP-1α, MIP-1β, and MCP-1. The activation of these kinases and subsequent production and release of IL-8 could be synergistically increased by the presence of granulocyte macrophage-colony stimulating factor, but not related cytokines, indicating that the composition of the inflammatory milieu may affect monocyte responses to LL-37. The data presented here demonstrate that LL-37 is a multi-functional immunomodulator with both pro- and antiinflammatory properties which are maintained at physiological cation concentrations. The observation that LL-37 induced chemokines production suggests that it may recruit leukocytes to sites of infection or inflammation thus providing a possible explanation for the observed antimicrobial properties of this peptide in vivo.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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42

Lee, Jia-yi, and 李佳怡. "Functionalized interdigitated electrodes for sensing LL-37 using impedimetric measurement." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/91636841693677247539.

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碩士
大同大學
生物工程學系(所)
98
The secretion of positively charged antimicrobial peptides, AMP LL-37, is an innate defense mechanism in human and mouse when their urinary tracts are infected by bacteria. They can interact and destroy the negatively charged bacteria membranes, and are ignorant of any possible bacterial resistance derived from drug treatments. Thus LL-37 is considered a good marker of urinary tract infections. We design an interdigitated type sensing electrode on a printed-circuit board (PCB) to measure the electrical characteristics associated with the change of LL-37 concentration. The change in the concentration of LL-37 is reflected in the corresponding resistance and capacitance changes measured by the probe. The advantage of our probe design lies in the fact that, due to the array arrangement, the geometric area of the total sensitive area of the interdigitated electrode is larger than the traditional electrodes in a limited space. Thus the signal and sensitivity are both improved. 　　An optimum frequency is selected by measuring under different frequencies. Measurements are also performed under various interfering conditions, including high salt concentration, buffer solutions, and different pH’s. In order to reduce the effect of interference, we try a gold electrode with SAMs to bind the antibody IgG of LL-37. After LL-37 has be bound by antibody IgG, we use DI water instead of the interferences.

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Doho, Gregory Hyojun. "Global computational regulatory analysis of the anti-endotoxin effect of LL-37." Thesis, 2006. http://hdl.handle.net/2429/17651.

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LL-37, a human cationic peptide, selectively modulates the innate immune response by interacting with the effector cells of innate immunity. It has been demonstrated to stimulate chemokine production and protect against infection, as well as suppress the endotoxin/lipopolysaccharide (LPS)-mediated production of pro-inflammatory cytokines and endotoxic shock in mouse model experiments. Microarray experiments under these conditions have indicated that the expression of numerous genes is altered in the presence of LL-37 and/or LPS, but the mechanisms underlying these transcriptional changes and the immunomodulatory effects of LL-37 are poorly understood. Therefore a computational approach was applied to time course microarray data comprising gene sets upregulated in monocytic cells upon treatment with LPS, LL-37 or LPS and LL-37. Sets of co-expressed genes observed in microarray experiments were clustered by a variety of methods into related temporal gene expression patterns. Subsequently, the promoter regions of the genes in these clusters were analyzed to predict potential transcription factor binding sites (TFBS) and the application of rigorous statistics revealed over-represented TFBS. This then permitted the generation of hypotheses concerning the transcription factors and signaling pathways involved in the anti-endotoxin effect(s) of LL-37. These analyses indicated that LL-37 selectively neutralized the LPS-induced expression of genes with promoters containing the signatures of both nuclear factor (NF)-κB and transcription factors downstream of mitogen-activated protein kinase (MAPK) pathways. Other genes that remained upregulated in the presence of both LPS and LL-37 tended to be deficient in TFBS for NF-κB but did contain the MAPK-related and other TFBS. LL-37 alone tended to induce expression of genes with promoters that bound transcription factors downstream of ERK1/2 and p38, MAPK that are known to be activated by LL-37, but did not induce the expression of most pro-inflammatory genes. Thus these data extend previous hypotheses that selective suppression of the NF-κB pathway is one of the anti-endotoxin mechanisms of LL-37, and indicate that residual gene expression is controlled in part by MAPK pathways.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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44

Tsai, Pei-Wen, and 蔡裴雯. "Molecular Mechanisms of Human Antimicrobial Peptide LL-37 Inhibiting Candida albicans Adhesion." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/25975125409956462305.

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45

Strack, Christina Elisabeth. "Modulation der Genexpression des antimikrobiellen Peptids LL-37 in Abhängigkeit von exogenen Faktoren." Doctoral thesis, 2009. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-38956.

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In ständigem Kontakt mit zahlreichen Mikroorganismen benötigt unser Körper eine Vielzahl an Mechanismen zur Abwehr krankheitserregender Keime. Antimikrobielle Peptide unterstützen als Effektormoleküle die einzellige Epithelschicht des Darms, die als Mukosabarriere unseren Körper vor dem Eindringen pathogener Keime bewahrt. Die Expression des antimikrobiellen Peptids LL-37, auch Cathelicidin genannt, stellt eine Strategie des angeborenen Immunsystems zur Abwehr von Mikroorganismen im Kolon dar. Die Expression des Cathelicidin Gens camp kann, wie in der vorliegenden Arbeit untersucht wurde, durch exogene Faktoren, wie die biologisch aktive Form des Vitamin D3, das 1,25(OH)2D3, die kurzkettige Fettsäure Butyrat und das Ernährungssupplement Intestamin®, angeregt werden. Die Stimulation der Zellen mit den genannten Substanzen führte in allen gestesteten Zelllinien zeit- und dosisabhängig zu einer Steigerung der Expression des Cathelicidin Gens. Die Induktion der camp-Expression durch 1,25(OH)2D3 wird durch die Existenz eines Vitamin D Response Elements (VDRE) in der Promoterrregion des camp-Gens begründet, an das der Vitamin D-Rezeptor Komplex als ligandenabhängiger Transkriptionsfaktor bindet und die Genexpression vermittelt. Die Auswirkungen von Butyrat auf die Genexpression des Cathelicidins werden auf die Modifikation des Acetylierungsstatus der Histonproteine zurückgeführt. Butyrat bewirkt durch eine reversible Hemmung der Histondeacetylase die Hyperacetylierung bestimmter Kernhistone und greift auf diese Weise in die Regulation der Gentranskription ein. Das enterale Ernährungssupplement Intestamin®, das als Pharmakonutrition speziell für schwerkranke Patienten entwickelt wurde, ist reich an Glutamin-Dipeptiden, Tributyrin und Antioxidantien. Der Effekt, den Intestamin® auf die Expression des Cathelicidin Gens ausübt, ist wahrscheinlich auf Tributyrin zurückzuführen. Tributyrin, der Ester aus Glycerin und Butyrat, wird durch Hydrolyse zu Butyrat umgesetzt und bewirkt vermutlich die Steigerung der camp-Expression. Eine Co-Stimulation von GEKI 02 Zellen mit 1,25(OH)2D3 plus Butyrat erzielte nach 48 Stunden eine weitere Steigerung der Expression des Cathelicidin Gens gegenüber beiden Einzelsubstanzen. In allen anderen getesteten Zelllinien zeigte sich keine synergetische Wirkung der beiden Substanzen. Auch eine Co-Stimulation mit Intestamin® plus Butyrat konnte nicht zu einer stärkeren Zunahme der camp-Expression führen als eine Behandlung mit beiden Einzelsubstanzen. Eine synergetische Wirkung der Substanzen 1,25(OH)2D3 und Butyrat in GEKI 02 Zellen könnte durch die Acetylierung der Histone bedingt sein, die eine Auflockerung der Chromatinstruktur bewirkt, was wiederum die Bindung von Transkriptionsfaktoren wie dem Vitamin D-Rezeptor Komplex erleichtert. Die fehlende synergetische Wirkung in allen anderen getesteten Zelllinien könnte mit der Tatsache in Zusammenhang stehen, dass die Induktion der camp-Expression zeitabhängig ist: Vitamin D3 erzielte nach 24 Stunden, Butyrat nach 48 Stunden die deutlichsten Auswirkungen auf die Genexpression des Cathelicidins. Der Einfluss des MEK/ERK Signalweges auf die durch 1,25(OH)2D3, Butyrat und Intestamin® induzierte camp-Expression wurde in den durchgeführten Versuchen mittels des spezifischen MEK 1/2 Inhibitors U0126 untersucht. U0126 blockierte die Induktion des Cathelicidin Gens durch Intestamin® und Butyrat, was die Beteiligung des Signalweges MEK/ERK belegt. Vitamin D3 dagegen übt seinen Einfluss auf LL-37 nicht über den Signalweg MEK/ERK aus. Obwohl Vitamin D3 und Butyrat die Expression des Cathelicidin Gens camp induzieren, konnte die Inkubation der Zellen mit beiden Substanzen die antimikrobielle Aktivität der Kolonepithelzellen gegenüber E.coli im Vergleich zu unbehandelten Kontrollzellen nicht steigern. Ursächlich hierfür könnte neben der Wahl eines apathogenen Bakterienstammes das Mikromilieu der Umgebung sein. Außer der Konzentration des Peptids spielen insbesondere der pH-Wert und die Salzkonzentration des Kulturmediums eine wichtige Rolle, da sie die Ausbildung der Sekundärstruktur, nämlich der α-helikalen Konformation, des LL-37 beeinflussen, die für die Interaktion mit Biomembranen erforderlich ist. Aufgrund der zunehmenden Resistenzentwicklung von Bakterien gegenüber herkömmlichen Antibiotika, wächst das Interesse an der Erforschung antimikrobieller Peptide. Exogene Faktoren wie 1,25(OH)2D3, Butyrat und Intestamin® können eine Steigerung der Expression des Cathelicidin Gens erzielen. Ob sich dieser Effekt allerdings auch in-vivo zeigt und eventuell therapeutischen Einsatz finden könnte, müssen weitere Studien klären
Defence of mucosal and epithelial surfaces against microbial pathogens involves the innate and adaptive immune response. The single cell layer of the colonic epithelium produces an array of immune modulators, including antimicrobial peptides, such as cathelicidins, that participate in the innate immune system. The aim of the present study was to analyse the effect of calcitriol, the histon-deacetylase inhibitor butyrate and the enteral pharmaconutrition supplement Intestamin® on the expression of the cathlicidin gene camp. After exposure to these stimuli a time and dose dependent induction of the expression of the cathelicidin gene was found in all investigated colorectal cell lines. The effect of calcitriol, 1,25(OH)2D3, is mediated by the vitamin D response element (VDRE) in the camp promoter that was bound by the vitamin D receptor (VDR). The induction of cathelicidin expression after treatment with butyrate is attributed to a reversible inhibition of histone deacetylases resulting in modulation of core histone and non-histone proteins and subsequent regulation of the gene transcription. The enteral pharmaconutrition supplement Intestamin® contains glutamine, antioxidant vitamins and tributyrin. The increased cathelicidin level after treatment with Intestamin® are ascribed to tributyrin because tributyrin is a novel structured lipid composed of three molecules of butyrate esterified with glycerol that induces the cathelicidin gene camp after hydrolysis to butyrate. When colorectal cells GEKI 02 were stimulated with 1,25(OH)2D3 plus butyrate, a further increase of cathelicidin expression was seen after 48 hours. This effect was not seen for the incubation with Intestamin® plus butyrate. Changes in the acetylation status of core histone and non-histone proteins caused by butyrate that enhance binding of the vitamin D receptor complex as a transcription factor might be responsible for the synergistic effect of butyrate and calcitriol. To determine the influence of the MEK/ERK signalling pathway in the investigated cell lines the specific MEK/ERK inhibitor U0126 was utilized. Inhibition of the MEK/ERK pathway blocked butyrate- and Intestamin®-induced cathelicidin expression while no effect was observed for calcitriol. Although incubation with vitamin D3 and butyrate increased cathelicidin expression, the antimicrobial activity against E.coli could not be enhanced in the investigated colorectal cells. This might be caused by selection of a commensal bacterium and the absence of relevant microenvironmental stimuli because the antibacterial activity correlates with the extent of α-helicity, which is influenced by ion composition, pH, and peptide concentration. Due to the fast development of bacterial resistance to traditional antibiotics, there is increasing interest in the research on antimicrobial peptides. Exogenic factors like 1,25(OH)2D3, butyrate and Intestamin® can enhance an expression of the cathelicidin gene. Further in vivo studies, however, are necessary to verify this effect for the potential future therapeutic application

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46

Wong, Alicia. "Citrullination of LL-37 as a mechanism that selectively controls immunostimulatory potential of DNA." Praca doktorska, 2019. https://ruj.uj.edu.pl/xmlui/handle/item/73404.

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LL-37, jedyna ludzka katelicydyna, jest amfipatycznym, kationowym peptydem bakteriobójczym, który zawiera 37 reszt aminokwasowych. Dojrzały peptyd uwalniany jest z 18 kDa białka prekursorowego (katelicydyny) przez proteazę-3 w neutrofilach lub przez proteazy serynowe z rodziny kallikrein w keratynocytach. Oprócz zdolności bezpośredniego zabijania mikroorganizmów, LL-37 pełni funkcję immunomodulacyjną. Jedną z nich jest tworzenie kompleksów z ujemnie naładowanymi oligonukleotydami pochodzącymi zarówno z obumierających komórek gospodarza, jak i patogenów. Interakcja ta ułatwia rozpoznawanie DNA przez receptory wewnątrzkomórkowe, takie jak TLR-9 prowadząc do indukcji odpowiedzi immunologicznej. W ostatnim czasie udokumentowano, że zdolność peptydu LL-37 do pełnienia funkcji bakteriobójczych i immunomodulacyjnych zależy w dużym stopniu od jego modyfikacji potranslacyjnych, wśród których deiminacja odgrywa wyjątkową rolę. Struktura pierwszorzędowa LL-37 zawiera pięć reszt argininy (Arg), które są wydajnie cytrulinowane przez deiminazy peptydylo-argininowe (PADs), enzymy, które są zależnymi od wapnia hydrolazami katalizującymi konwersję dodatnio naładowanej argininy do neutralnej cytruliny. W związku z powyższym w niniejszej pracy skupiono się na roli deiminacji peptydu LL-37 w regulacji odpowiedzi zapalnej na kwasy nukleinowe. Uzyskane wyniki dowiodły, że cytrulinacja LL-37 katalizowana przez PAD2 ogranicza zależne od peptydu pochłanianie DNA przez fagocyty. Co ciekawe, karbamylacja peptydu (homocytrulinacja reszt lizyny) nie ma wpływu na jego interakcję z DNA, tak długo jak karbamylowany peptyd nie zostanie poddany działaniu PAD2. W badaniach zastosowano również syntetyczny peptyd LL-37 z resztami Arg zastąpionymi homoargininą (hArg-LL-37), która jest niewrażliwa na proces enzymatycznej deiminacji przez PADy. Dane te wykazały, że wiązanie peptydu LL-37 z DNA, a tym samym jego działanie immunoregulacyjne względem fagocytów nie zależy wyłącznie od ładunku dodatniego peptydu, ale od prawidłowego rozmieszczenia guanidynowych łańcuchów bocznych argininy w natywnej sekwencji peptydu. Aby zbadać, czy powyższe zjawisko odgrywa rolę w stanie zapalnym przeprowadzono badania z wykorzystaniem zewnątrzkomórkowych pułapek neutrofilowych (NETs, ang. neutrophil extracellular traps). Tworzenie tych struktur, zbudowanych z nici genomowego DNA udekorowanych białkami pochodzącymi z ziaren azurofilnych, w tym LL-37, zależy od aktywności PAD4. Szczegółowa analiza składu NETs i ich wpływu na profesjonalne fagocyty sugeruje, że katelicydyna/LL-37 jest cytrulinowana przez PAD4 podczas tworzenia pułapek neutrofilowych. Proces ten odgrywa istotną rolę w kontroli potencjału zapalnego NETs, zwłaszcza w regulacji odpowiedzi immunologicznej na uwolnione z obumierających komórek DNA, które jest rozpoznawane przez receptor TLR9. Przebieg procesu cytrulinacji w warunkach in vivo potwierdzają wyniki analizy serologicznej surowic pochodzących od pacjentów cierpiących na przewlekłe choroby zapalne, takie jak: paradontozę i reumatoidalne zapalenie stawów, w których wykazano podwyższony poziom przeciwciał rozpoznających cytrulinowaną formę peptydu LL-37. Ostatecznie, dowiedziono, że cytrulinowana forma peptydu LL-37 zwiększa powstawanie NETs, co sugeruje mechanizm pozytywnego sprzężenia zwrotnego. Podsumowując, cytrulinacja peptydu LL-37 odgrywa kluczową rolę w jego immunomodulacyjnym działaniu przeciwdziałając utracie tolerancji gospodarza na własne cząsteczki, w tym DNA.
LL-37, the only human cathelicidin, is an amphipathic, cationic antimicrobial peptide that contains 37 amino acids residues. The mature peptide is released from an 18-kDa precursor protein (cathelicidin) by protease-3 in neutrophils or by serine proteases of the kallikrein family in keratinocytes. Besides playing antimicrobial role, LL-37 exerts several immunomodulatory functions that regulate host responses to pathogens. Among them is formation of complexes with negatively charged oligonucleotides derived from both dying host cells and pathogens. Such interaction facilitates the recognition of DNA by intracellular receptors such as TLR9, thereby stimulating the immune response. It was recently documented that the ability of peptide to execute its bactericidal and immunomodulatory functions strongly depends on posttranslational modifications, among which enzymatic deimination (citrullination) of Arg residues plays a unique role. The primary structure of LL-37 contains five arginine (Arg) residues that are effectively citrullinated by peptidyl-arginine deiminases (PADs), enzymes that are calcium-dependent hydrolases catalyzing the conversion of positively charged arginine to neutral citrulline. Therefore, presented study focused on the role of LL-37 deimination in the regulation of the host immune response to nucleic acids. Presented data revealed that citrullination of LL-37 by PADs hindered peptide-dependent DNA uptake and sensing by pDCs. In contrast, carbamylation of the peptide (homocitrullination of lysine residues) had no effect on its interaction with DNA. This activity was abolished by citrullination of Arg residues in the peptide by PAD2. We implemented in the study the synthetic peptide LL-37 with Arg residues substituted by homoarginine (hArg-LL-37), which is insensitive to enzymatic deimination. Using such peptide, we observed that regardless of PAD2 treatment hArg-LL-37 preserved full biological activity. These data have showed that peptide binding to DNA and its immunoregulatory effects on myeloid cells do not depend entirely on the positive charge of LL-37 but on the proper distribution of guanidium side chains of Arg (or homo-Arg) in the native peptide sequence. To explore if above phenomenon plays a role in inflammatory conditions, we introduced in the study neutrophil extracellular traps (NETs). Formation of those structures, composed of genomic DNA and decorated with azurophilic proteins including LL-37, depends on PAD4 activity. Detailed analysis of NETs composition and their influence on myeloid cells suggest that cathelicidin/LL-37 is citrullinated by PADs during NETs formation. Such process makes a marked contribution to the inflammatory potential of NET structures especially in regulating the immunostimulatory response of cell-free DNA, which is mediated by TLR9. Such conclusion was additionally supported by an elevated antibody response against citrullinated LL-37 in blood serum obtained from patients suffering from chronic inflammatory diseases (periodontitis, rheumatoid arthritis). Finally, obtained data revealed that citrullinated LL-37 itself enhances the generation of NETs formation suggesting the mechanism of positive feedback. Taken together, by preventing the breakdown of tolerance to self-molecules, citrullination of LL-37 plays a critical role in immunomodulation of the host response.

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47

Lau, Yee-Lar Elaine. "Characterizing the interactions between the host defence peptide, LL-37, and lung epithelial cells." Thesis, 2004. http://hdl.handle.net/2429/15607.

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LL-37 is a human cationic host defense peptide that is a component of innate immunity. In addition to its modest antimicrobial activity, LL-37 affects the behavior of effector cells involved in the innate immune response, including modification of transcriptional responses. However, its mode of interaction with eukaryotic cells remains unclear. In this research study, the interaction of LL-37 with epithelial cells was characterized in tissue culture by using biotinylated LL-37 (LL-37B) and confocal microscopy. LL-37 was actively taken up into A549 human epithelial cells and eventually localized to the perinuclear region. Specific inhibitors were used to demonstrate that the uptake process was not mediated by actin but required elements normally involved in endocytosis, and that trafficking to the perinuclear region was dependent upon microtubules. Using Scatchard analysis, it was revealed that A549 epithelial cells have two receptors for LL-37B, with high and low affinity for LL-37. Further studies, consistent with other publications, that, at higher concentrations, LL-37 demonstrated some cytotoxicity. Therefore the cytotoxic nature of LL-37 was examined in the presence of different sources of serum. LL-37 was cytotoxic in the presence of 10% fetal bovine serum but not 10% human serum and that cytotoxicity led to apoptosis, as assessed by TUNEL assay and Western blot for pro-caspase-3. High density lipoproteins present in human serum, but not the major constituent in HDL, apolipoprotein A-1, inhibited LL-37-induced cytotoxicity. The localization of LL-37B differed according to the type of serum present during incubation. These results suggest that LL-37 interacts directly with epithelial cells and further our understanding of its role in modulating the innate immune response.

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48

Chen, Yun-Ju, and 陳韻茹. "Study of Molecular Mechanisms of Candida albicans Response to Human Antimicrobial Peptide LL-37." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/50441681136576470916.

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碩士
國立清華大學
分子與細胞生物研究所
97
Candida albicans is one of the most important fungal pathogens in humans. C. albicans is a commensal in healthy individuals and can become invasive and pathogenic in the immunocompromised patients. LL-37 is a human cationic antimicrobial peptide that has been reported to exert its antifungal activity against C. albicans. Membrane-disruptive effects of LL-37 have been well investigated. However, several studies indicated that membrane disruption may not reflect the complex processes involved in the killing of microorganisms by LL-37. The main goal of this study is to explore mechanisms, other than membrane disruption that may involve in killing of C. albicans by LL-37. First, killing ability of LL-37 to C. albicans was determined by viable cell counting and FUN-1 staining. Next, oxidative and osmotic stresses stimuli were correlated to cell death, especially the accumulation of reactive oxygen species (ROS). A MAP kinase Hog1 was indicated to be involved in C. albicans stress response that the hog1 homozygous mutant cells were more sensitive to LL-37 treatment compared to wild type cells. Moreover, activation of Hog1 was detected by Western blot after LL-37 treatment. Finally, chromatin fragmentation and the presence of caspase activity were examined. Results showed that LL-37 lead to C. albicans cell death in a non-apoptotic manner. Together, these findings help us to further understand the mechanisms of C. albicans stress-adaptation and C. albicans cell death induced by antimicrobial peptides.

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49

陳正益. "Studying the mode of action of human antimicrobial peptide LL-37 on Acinetobacter baumannii." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/06836331108733189876.

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碩士
國立清華大學
分子與細胞生物研究所
102
Acinetobacter baumannii is a Gram-negative coccobacillus and is a leading nosocomial pathogen worldwide. Recently, emergence of multidrug resistant A. baumannii has become a great threat to healthcare. Strains resist to the last line of anti-mocrobial agents, tigecycline and colistin, were appeared. Therefore, developing new drugs to treat A. baumannii infections is urgently needed. In this work, a human antimicrobial peptide LL-37 was evaluated for its effects on A. baumannii. We found that LL-37 kills A. baumannii efficiently and reduces cell adhesion and motility. Lipopolysaccharides extracted from A. baumannii cell surface can rescue LL37-mediated inhibition of cell adhesion. Moreover, far-western analysis indicated that LL-37 binds to outer membrane OmpA protein of A. baumannii (AbOmpA), but this binding seems not to correlate with the inhibitory effect on cell adhesion. However, both LPS and AbOmpA were related to sensitivity of A. baumannii to LL-37. Together, this study suggested that LL-37 may be a potential agent in the future treatment of A. baumannii infections.

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50

Iffland, Konrad. "Expression und Regulation des antimikrobiellen Cathelicidin-Peptids LL-37 in humanen Kolonepithelzellen, Monozyten und PBMC." Doctoral thesis, 2004. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-11683.

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Butyrat ist die wichtigste kurzkettige Fettsäure im Kolon und dient der normalen Schleimhaut als trophischer Faktor. Butyrat hat paradoxe Effekte auf Epithelzellen des Kolons: Hauptenergieträger und Wachstumsstimulator normaler Mukosa einerseits, Proliferationshemmer und Apoptoseinduktor kolorektaler Karzinomzellen in vitro andererseits. Butyrat kann zudem die Immunfunktionen der Schleimhaut modulieren. Die einzellige Schicht des Dickdarmepithels ist eine aktive Barriere gegen die intestinalen Bakterien im Kolonlumen. Zusätzlich zur Bildung einer physischen Barriere ist das Epithel mit verschiedenen Effektormolekülen ausgestattet, zu welchen auch antimikrobielle Peptide zählen. Antimikrobielle Peptide spielen eine wichtige Rolle als endogene Antibiotika in der angeborenen Immunabwehr. Es sind kleine kationische Peptide von weniger als 100 Aminosäuren Länge. Sie kommen konserviert bei Insekten, Tieren, Pflanzen und dem Menschen vor. Diese Peptide werden in Zellen des Immunsystems, aber auch in Epithelzellen exprimiert und sezerniert. Sie wirken gegen gram-positive und –negative Bakterien, Viren und Pilze. Zusätzlich zu Ihrem antimikrobiellen Effekt üben diese Peptide einen chemotaktischen Reiz auf unterschiedlichste Immunzellen aus, darunter neutrophile Granulozyten, dendritische Zellen und T-Zellen, stimulieren die Chemokinfreisetzung durch Monozyten, induzieren die Mastzelldegranulation und können das Komplementsystem aktivieren. Beim Menschen sind bisher zehn Defensine und das humane Cathelicidinpeptid LL-37 beschrieben worden. Letzteres war auch Gegenstand unserer Forschungen. LL-37 wird in den Granula von neutrophilen Granulozyten gespeichert und in Knochenmark und Hoden, sowie in Hautkeratinozyten, Lungenepithel, und Plattenepithel der Zunge, des Ösophagus, der Zervix und Vagina exprimiert. In dieser Arbeit wurde die Expression und Modulation des einzigen humanen antimikrobiellen Cathelicidins LL-37 durch Entzündungsmediatoren oder Ernährungsfaktoren untersucht. Die untersuchten Zytokine, darunter TNFa und verschiedene proinflammatorische Interleukine zeigen keinen Einfluss auf die LL-37 Expression in Kolonepithelzellen. Die Expression des antimikrobiellen Peptids scheint dagegen stark mit Zelldifferenzierung verbunden zu sein. Nur differenzierte Epithelzellen im menschlichen Kolon und Ileum exprimieren LL-37 in vivo. Faktoren im Kolonlumen können dagegen einen Einfluss auf die Expression von LL-37 Expression in Kolonepithelzellen ausüben. Insbesondere kurzkettige Fettsäuren, die bei der bakteriellen Fermentation unverdauter Kohlenhydrate im Kolonlumen entstehen und die eine Zelldifferenzierung herbeiführen, induzieren in vitro die Expression des antimikrobiellen LL-37 in verschiedenen Kolonepithelzellen. Gleichzeitig steigert Butyrat die Differenzierung in den untersuchten Zellen. In Primärkulturen kolorektaler Karzinome und normaler Kolonschleimhautepithelzellen induzierte Butyrat die LL-37 Expression nur in undifferenzierten Tumorzellen. Als nächstes wurden Signalwege gesucht, die an der Regulation von LL-37 und der Differenzierung eine Rolle spielen. In Kolonepithelzellen verhindert eine MEK-ERK Blockade eine LL-37 Induktion – ohne die Differenzierung zu beeinträchtigen. Bei einer Blockade des p38/MAP-Kinase Weges stellte es sich genau andersherum dar: Die Differenzierung wurde gehemmt, aber die LL-37 Expression wurde nicht beeinflusst. Somit wird die LL-37 mRNA Transkription in Kolonepithelzellen über den MEK/ERK Signalweg und die Differenzierung in denselben Zellen über den p38/MAP-Kinase Signalweg reguliert. In undifferenzierten Monozyten kann wie schon in Kolonepithelzellen eine Induktion der LL-37 Expression nach Inkubation mit SCFAs beobachtet werden. Bei reifen PBMC jedoch inhibiert Butyrat eine LL-37 Transkription. In nicht differenzierten Monozyten blockt ein MEK/ERK-Hemmer die LL-37 Expression wie in Kolonepithelzellen, dagegen hat diese Blockade in reifen PBMC keine Auswirkung auf die LL-37 Konzentration. Deshalb können noch weitere unbekannte Mechanismen an der LL-37 Regulation in Darmepithelzellen und den LL-37 exprimierenden Immunzellen beteiligt sein. Diese Arbeit bietet neue Einblicke in die Regulation des antimikrobiellen Cathelicidins LL-37 in der menschlichen Darmschleimhaut und kann vielleicht die Basis für eine therapeutische Manipulation der LL-37 Expression liefern. Es muss jedoch noch geklärt werden, ob Butyrat oder andere Ernährungsfaktoren die Schleimhautbarriere dadurch stärken können, indem das Peptid LL-37 und oder andere Effektormoleküle der angeborenen Immunabwehr in vivo hochreguliert werden
The single cell layer of the colonic epithelium is an active barrier against the external environment and the enormous load of intestinal bacteria. In addition to forming a physical barrier, the epithelium is armed with an array of effector molecules including antimicrobial peptides. These peptides can be considered as endogenous antibiotics and are widespread in nature as immediate defense effectors. They have been found in invertebrates, vertebrates, plants as well as bacteria and several human antimicrobial peptides have been characterized. They are mainly stored in vacuoles of granulocytes ready for activation upon stimuli or secreted directly onto mucosal surfaces by epithelial cells. The cathelicidins constitute a family of precursor proteins with a well conserved cathelin pro-region, followed by a highly variable C-terminal antimicrobial domain. The only human cathelicidin gives rise to LL-37, a 37-residue mature antimicrobial peptide, after cleavage from the cathelin propart. LL-37 is present in neutrophils and lymphocytes. In addition, LL-37 is synthesized by bone marrow, keratinocytes of inflamed skin, lung epithelium, and squamous epithelia of human mouth, tongue, esophagus, cervix and vagina. Both purified and chemically synthesized LL-37 peptides exhibit potent and comparable antimicrobial activities in vitro. Alterations of the colonic epithelial barrier may occur in response to dietary changes, medical treatment or disease. A lack of dietary fibre can facilitate bacterial translocation from the gut. Short-chain fatty acids (SCFA), namely acetate, propionate and butyrate, are derived from bacterial fermentation of undigested dietary fibres in the colon. Butyrate and other SCFA exert profound effects on colonic physiology as they affect fluid absorption, colonocyte metabolism, proliferation and differentiation, gut motility and mucosal inflammation. In this study we analyzed the expression and modulation of the single human antimicrobial cathelicidin peptid LL-37 modulated by inflammatoric mediators or dietary fibres. Zytocines or different proinflammatoric interleukins indeed have no effect on expression of LL-37 in colon epithelial cells. Cell differentiation probably is the key determinant of LL-37 expression in colon epithelial cells. Only differentiated epithelial cells express LL-37 in vivo in human colon and ileum. We discovered that distinct pathways are for the induction of genes involved in differentiation on one hand and the expression of the gene encoding LL-37 on the other hand. The expression pattern of LL-37 in the colon crypt implies that these pathways are activated simultaneously in vivo. In summary, this study provides new insights into the regulation of the antimicrobial cathelicidin LL-37 in human colon mucosa and might provide the basis for a therapeutic manipulation of LL-37 expression. However, it remains to be elucidated if butyrate and other dietary substrates can strengthen the epithelial defense barrier by upregulating LL-37 and other effectors of innate immunity in vivo

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