Dissertations / Theses on the topic 'LL37'

As society draws closer to the post-antibiotic era and the pipeline for alternatives dries, there is an urgent need for the development of novel antimicrobial therapies that do not promote bacterial resistance, particularly for immunocompromised chronic wound patients. Antimicrobial peptides (AMPs), including human-derived LL37, show considerable promise as broad spectrum alternatives that also have wound healing properties; however, few have been clinically implemented as novel antimicrobials due to their cytotoxicity stemming from a poor understanding of their mechanisms and low stability in vivo. It has been suggested that tethering, or attaching AMPs onto surfaces, is a viable strategy of delivering bioactive AMPs to surfaces while reducing cytotoxicity and improving stability. Thus, we designed new chimeric versions of LL37 with collagen-binding domains (CBD), derived from collagenase (cCBD-LL37) and fibronectin (fCBD-LL37) for non-covalent tethering onto collagen, a prevalent biopolymer in commercially available wound dressings and scaffolds. Our overall hypothesis was that CBDs would mediate stable tethering of broadly active, non-cytotoxic CBD-LL37 onto collagen-based scaffolds. We first studied the loading, release and bioactivities (e.g. antimicrobial activity and cytotoxicity) of each CBD-LL37 on commercially available 100% collagen type I PURACOL® wound scaffolds. We found that both cCBD-LL37 and fCBD-LL37 bound highly to collagen, were active against relevant wound pathogens, demonstrated stable activity after 14 days of release, and were not cytotoxic to human fibroblasts. The addition of different CBDs onto LL37 also markedly altered their soluble bioactivities. Using similar methods, we then studied the loading, release and bioactivity of each CBD-LL37 on a commercially available FIBRACOL® wound scaffolds, comprised of 90% collagen type I and 10% calcium alginate biopolymers. We found that both cCBD-LL37 and fCBD-LL37 also bound highly to and retained on collagen for 14 days, but were only active against Gram-negative P. aeruginosa. This suggested that the presence other biopolymers in addition to collagen, which is common among commercial wound dressings, could cause significant differences in binding, retention and bioactivities of CBD-LL37. To better understand how CBD modification affected CBD-LL37 structure leading to different bioactivities, we studied the CBD sequence-, peptide structure-, concentration-, time-, and bilayer composition-dependent interactions of soluble CBD-LL37 and compared these findings with the properties of unmodified LL37. Using Molecular Dynamics (MD) simulations, circular dichroism (CD) spectroscopy, quartz crystal microbalance with dissipation (QCM-D), and fluorescent bilayer imaging we determined the structural basis behind CBD alterations in bioactivities. MD and CD, in addition to other intrinsic CBD properties (helicity, amphiphilicity, charge) we hypothesized that cCBD-LL37 utilized similar mechanisms as unmodified LL37 while fCBD-LL37 demonstrated based primarily on surface adsorption. We used QCM-D and Voigt-Kelvin viscoelastic modeling to determine the time- and concentration-dependent interactions of unmodified LL37 with model mammalian lipid bilayers, the mechanisms of which are still controversial in literature despite being widely studied. These results were used to propose a model for the interaction mechanism of LL37 with zwitterionic bilayers that aligned with its bioactive concentrations. LL37 adsorbed at concentrations where it is immunomodulatory until reaching a threshold which corresponded with its antimicrobial concentrations. The threshold was correlated to lipid bilayer saturation, after which LL37 formed transmembrane pores. We observed collapse of the bilayer into a rigid proteolipid film at concentrations higher than the reported cytotoxic threshold of LL37. The mechanistic and structural information for each CBD-LL37 and unmodified LL37 provided a baseline for QCM-D and Voigt-Kelvin viscoelastic modeling to further elucidate the time-, concentration-, lipid composition- and CBD sequence-dependent basis behind the observed bioactivities of cCBD-LL37 and fCBD-LL37. We found that similar to LL37, cCBD-LL37 demonstrated pore formation mechanisms likely due to their similar charges, structural content and amphiphilicity. fCBD-LL37 demonstrated time-dependent, adsorption-based mechanism likely due to its anchoring aromatic residues, low charge, and low amphiphilicity. Knowledge gained from this study allowed mechanistic predictions of two newly designed hypothetical CBD-LL37 peptides. Results from this study contribute to a better understanding of a new class of antimicrobial, non-cytotoxic therapies based on collagen-tethered CBD-LL37, bringing it closer to clinical implementation in chronic wound applications and demonstrate the viability of biopolymer tethering as a platform toward using AMPs to quench the resistance crisis.

As a direct consequence of the misuse of antibiotics since its first discovery, bacteria have developed extensive resistance mechanisms making them once again potential lethal threats. This eventuality has triggered a vast research effort from scientists worldwide to find solutions to mitigate antimicrobial resistance. One such option is the identification of new potential antimicrobial agents, like for example antimicrobial peptides (AMPs). Various methods have been applied to evaluate the properties and determine the complex mechanism of AMPs. However, the details of the mechanism remain unknown and hence the work in this project seeks to examine the suitability of using TIR Raman, a vibrational spectroscopy technique which is sufficiently surface sensitive to study the interaction of AMPs in contact with lipid bilayers, which are just a few nanometres thick. In order to evaluate the information that could be extracted from TIR Raman, measurement of solid supported lipid bilayers in the absence of peptides were first carried out. In particular, the lipid DMPC with a phase transition close to room temperature, was measured at various temperatures to determine spectral changes associated with the transition. For the peptide-membrane interactions, the AMP LL37 was put into contact with solid supported lipid bilayers modelling the cell membranes of bacteria (DOPE, DOPG) or humans (DOPC) respectively. The data clearly indicates that the membrane composition, and specifically the lipid head group charge, play an important role in the peptide-membrane interactions. In the bilayers mimicking bacteria cell membranes, the peptide either absorbed onto or inserted into the bilayer. In contrast, for the bilayer modelling a human cell membrane, no significant variations were detected, indicating no interaction with LL37. The findings presented in this work generally coincide with similar research of LL37 using other techniques. At hand of the herein presented data, TIR Raman has proven suitable and effective in detecting effects of antimicrobial peptides in contact with model lipid bilayers, and hence can be recommended for further studies.
Som en direkt följd av missbruket av antibiotika sedan det först upptäcktes, har bakterier utvecklat omfattande resistensmekanismer vilket föranlett dem att återigen utgöra potentiellt dödlig hot. Denna situation har manat fram en väsentlig forskningsinsats från forskare världen över att hitta lösningar för att minska antimikrobiell resistens. Ett sådant alternativ har varit identifieringen av nya potentiella antimikrobiella substanser, så som till exempel antimikrobiella peptider. Ett flertal metoder har använts för att både evaluera peptiders egenskaper och fastställa deras komplexa mekanism. Detta till trots förblir de exakta detaljerna i mekanismen okända, vilket föranlett arbetet i detta projekt att undersöka lämpligheten i att använda TIR Raman, en vibrational-spektroskopisk metod som är tillräckligt ytkänslig för att studera interaktionen hos antimikrobiella peptider i kontakt med lipidmembran, vilka endast är några få nanometer tjocka. För att evaluera informationen som kan utvinnas med TIR Raman, utfördes först mätningar av lipidmembran, skapade på ett solitt underlag, utan tillägg av peptider. Mer noggrant, har lipiden DMPC med en fasövergång nära vid rumstemperatur, mätts vid olika temperaturer för att fastställa spektrala variationer associerade till övergången. För peptid-membran interaktionerna, sattes den antimikrobiella peptiden LL37 i kontakt med lipidmembran som modellerar cellmembranet hos bakterier (DOPE, DOPG) respektive människor (DOPC). Mätdatan indikerar tydligt att membrankompositionen, och där specifikt laddningen av lipidens huvudgrupp, spelar en viktig roll i membran-peptid interaktionerna. För lipidmembranen som imiterar bakteriella cellmembran, adsorberade peptiden till membranet eller integrerades in i det. Till skillnad från detta, kunde inga signifikanta variationer detekteras för lipidmembranet som modellerade ett mänskligt membran vilket indikerar att det inte interagerar med peptiden LL37. Upptäckterna som presenteras i detta arbete sammanfaller generellt med andra, liknande studier där andra instrument använts för att undersöka LL37. Det kan ur materialet som presenterats här utläsas att TIR Raman visat sig lämpligt och effektivt i detekteringen av antimikrobiella peptider i kontakt med modeller av lipidmembran, och kan därav rekommenderas för fortsatta studier.

Cette thèse présente la conception d'un accélérateur matériel dédié à la simulation de circuits intégrés. Sur cet accélérateur sont développés un ensemble de logiciels constituant un environnement intégré de simulation. Nous y discutons tout d'abord des concepts de base de la modélisation des circuits intégrés, de la simulation logico-fonctionnelle, de la simulation de pannes, des langages de description du matériel, ainsi que des techniques d'accélération de la simulation de circuits intégrés. Nous présentons ensuite la structure générale de l'accélérateur. Il est basé sur une architecture parallèle : un réseau en anneau sur lequel sont disposées des unités de simulation, où chaque unité de simulation est composée de trois microprocesseurs exécutant trois tâches respectivement. l'ensemble des logiciels implémentés sur cet accélérateur est présenté. Le simulateur réalise ainsi la simulation multi-niveaux (porte logique, fonctionnel et interrupteur) et la simulation de pannes. Des outils de compilation permettent l'utilisation des langages de description du matériel pour modéliser les circuits intégrés de manière structurelle et fonctionnelle. Enfin, différentes stratégies de parallélisation de la simulation ainsi que plusieurs algorithmes de simulation adaptés aux différents niveaux d'abstraction sont étudiés

碩士
國立陽明大學
口腔生物研究所
101
Dental caries is one of the most common infectious diseases which may cause demineralization and destruction of the hard tissues and pulp inflammation. With the discovery of regenerative potential of the dental pulp, there is a shift on the treatment of pulpal inflammation to a more conservative approach. The antimicrobial peptide LL37 (cathelicidin), which is part of the innate immune system, has been suggested for the regenerative procedures due to the ability to defense against pathogens and regulate wound healing. However, little was known about the expression of LL37 in dental pulp tissue. In this study, the expressions and functions of LL37 in human dental pulp were studied. The results showed that the expression levels of LL37 were up-regulated in carious dental pulps comparing to the healthy controls. In addition, LL37 enhanced cell proliferation and the expression of collagen in human dental pulp cells. On the other hand, the Escherichia coli (E. coli) lipopolysaccharide (LPS) - induced expressions of IL-1β (interleukin-1β), IL-6 (interleukin-6), IL-8 (interleukin-8), TNF-α (tumor necrosis factor-α) and MCP-1 (monocyte chemoattractant protein-1) were suppressed by the treatment of LL-37. LL37 was shown to neutralize LPS and therefore inhibited LPS-induced NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) translocation. The results of the present study showed the future potential of LL37, which could be used in vital pulp therapy to facilitate pulp healing.

Tese de doutoramento em Biociências na especialidade de Biotecnologia, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Problemas de cicatrização de feridas e suas complicações médicas continuam a ser um dos problemas de saúde mais prevalentes e economicamente onerosos do mundo. Nos Estados Unidos, as feridas crónicas afectam 6.5 milhões de pacientes. Mais de 25 bilhões de dólares são gastos anualmente em tratamento de feridas crónicas e a despesa irá crescer rapidamente devido ao aumento dos custos de saúde, o envelhecimento da população e um aumento acentuado na incidência de diabetes e obesidade em todo o mundo. Dados recentes indicam que 11.7% da população Portuguesa é diabética. Uma das complicações mais graves e debilitantes da diabetes é o desenvolvimento de feridas crónicas e não cicatrizantes. Nos últimos anos, tem havido esforços para se desenvolver terapias avançadas para a cicatrização de feridas crónicas, incluindo o uso tópico de factores de crescimentos ou terapias celulares. Infelizmente, em muitos casos, a eficácia terapêutica é baixa, as terapias são caras, requerem aplicação clínica e não possuem actividade antimicrobiana. Portanto, é imperativo o desenvolvimento de novas terapêuticas para responder a esta necessidade clínica. Os péptidos antimicrobianos (AMPs) sintetizados na pele podem constituir futuras terapias já que são moléculas que formam uma barreira à infecção e alguns deles são muito importantes para a regeneração da pele (4). O péptido LL37 é o AMP mais importante na pele, actuando como primeira linha de defesa contra bactérias, vírus, fungos e desempenha um papel importante na imunomodulação, angiogénese e cicatrização de feridas. Recentemente, foram publicados os resultados de um ensaio clínico fase I / II com o péptido LL37 que demonstram a sua eficácia terapêutica em feridas crónicas; no entanto, foram necessárias múltiplas administrações tópicas. O objectivo principal desta tese foi desenvolver uma formulação de nanopartículas conjugadas com LL37 de forma a possuir maior actividade de cicatrização da pele que o péptido livre (não imobilizado). A formulação de nanopartículas foi preparada por uma nova metodologia na qual foi utilizado o péptido LL37 com um terminal tiol, actuando o péptido como agente de revestimento durante a formação da nanopartícula. Esta formulação tem um núcleo de ouro (Au) e um escudo formado pelo péptido catiónico LL37. Durante esta tese seleccionámos nanopartículas de ouro porque é relativamente fácil a imobilização de altas concentrações de LL37 por unidade de superfície, a modificação das suas propriedades (incluindo tamanho, carga e morfologia), e pelo facto destas nanopartículas terem sido utilizadas na prática clínica há muitos anos. Estas nanopartículas possuem um tamanho controlado (21 nm), baixa polidispersão, um potencial zeta positivo (15 mV), alta densidade de LL37 (250 μg de péptido por mg de nanopartícula) e alta estabilidade em soluções aquosas e meios de cultura celular. As nanopartículas mostraram actividade antimicrobiana contra E. coli e elevada actividade biológica contra células de mamíferos, ou seja, actividade pró-angiogénica em células endoteliais e actividade pró-migratória em queratinócitos. Surpreendentemente, as nanopartículas conjugadas com LL37 possuem menor citotoxicidade que o péptido LL37 livre. Os nossos resultados mostram ainda que as nanopartículas são internalizadas pelos queratinócitos e células endoteliais, e no caso de queratinócitos, o processo de internalização é mediado principalmente pelo receptor scavenger. As nanopartículas internalizadas tendem a se acumular no compartimento endolisomal dos queratinócitos, mas conseguem escapar o compartimento endolisomal em células endoteliais. Em ambos os modelos celulares, as nanopartículas induzem a transactivação do EGFR. No caso dos queratinócitos, o processo é iniciado pela activação de receptores P2X que levam a uma prolongada fosforilação de EGFR e de ERK. Isto leva a que os queratinócitos possuam maiores propriedades migratórias. Os resultados in vivo mostram que nanopartículas conjugadas com LL37 têm melhores propriedades cicatrizantes de feridas comparativamente ao péptido livre. No caso de células endoteliais, o péptido LL37, mas não as nanopartículas conjugadas com LL37, levam à activação do receptor FPRL-1, à acumulação de cálcio intracelular e à secreção de VEGF165. No entanto, tanto o péptido LL37 como as nanopartículas conjugadas com LL37 têm elevada actividade pró-angiogénica, demonstrado num ensaio de membrana corioalantóica (CAM). É provável que a actividade pró-angiogénica das nanopartículas conjugadas com LL37 seja mediada pela transactivação de EGFR, mas mais estudos são necessários para responder a esta questão. Os resultados apresentados nesta tese são um primeiro passo para o desenvolvimento de terapias baseadas em nanomedicina à base de péptidos antimicrobianos. Esta é a primeira terapia que combina propriedades de cicatrização de feridas e actividade antimicrobiana na mesma formulação, é mais barata do que as terapias avançadas actuais, e pode ser administrada uma única vez. Impaired wound healing and its medical complications remain one of the most prevalent and economically burdensome healthcare issues in the world. In the United States alone, chronic wounds affect 6.5 million patients. More than US$25 billion is spent annually on treatment of chronic wounds and the burden is growing rapidly due to increasing health care costs, an aging population and a sharp rise in the incidence of diabetes and obesity worldwide. Recent data indicate that 11.7% of the Portuguese population is diabetic. One of the most serious and debilitating complications of diabetes is the development of chronic non-healing wounds. In recent years, there have been efforts to develop new advanced methodologies to heal chronic wounds including the use of topic growth factors or cell-based thera- pies. Unfortunately, in many cases, the therapeutic efficacy is low, the therapies are expensive and require application in a clinical facility, and they have no antimicro- bial activity. Therefore, development of new therapeutics is absolutely necessary and important to satisfy the unmet clinical need. Antimicrobial peptides (AMPs) synthesized in the skin provide a barrier to infection and some of them are very im- portant for the regeneration of skin. LL37 is the most important AMP in skin, acts as first line of defense against bacteria, virus, fungi and plays an important role in im- munomodulation, angiogenesis and wound healing properties. Recently, phase I/II clinical trials have demonstrated its therapeutic efficacy in chronic wounds; how- ever, multiple topical administrations were required. The main goal of this the- sis was to develop a LL37 nanoparticulate formulation with higher wound healing properties than the free peptide. The nanoparticulate formulation was prepared by a novel methodology in which thiol-terminated LL37 acts as capping agent during NP formation. This formulation has a gold (Au) core and a hydrophilic cationic LL37 peptide shell. We have selected Au NPs because it is relatively easy the immo- bilization of high concentrations of LL37 per surface area, the modification of their properties (including size, charge and morphology), and they have been used in the clinic for many years. LL37-Au NPs have controlled size (21 nm), low polydisper- sity, positive zeta potential (15 mV), high density of LL37 (250 μ g of peptide per mg of NP) and high stability in aqueous solutions and cell culture media. They showed antimicrobial activity against E . coli and high biological activity against mammalian cells, namely pro-angiogenic activity against endothelial cells and pro-migratory activity against keratinocytes. Remarkably, LL37-Au NPs have lower cytotoxicity than LL37 peptide. Our results further show that LL37-Au NPs are internalized by keratinocytes and endothelial cells, and in case of keratinocytes, the internalization process is mostly mediated by scavenger receptors. The internalized LL37-Au NPs tend to accumulate in the endolysomal compartment in keratinocytes while escape the endolysomal compartment in endothelial cells. In both cell models, LL37-Au NPs induce the transactivation of EGFR. In case of keratinocytes, the process is ini- tiated by the activation of P2X receptors that leads to a long-lasting phosphorylation of EGFR and ERK. This results in enhanced migratory properties of keratinocytes. In vivo results show that LL37-Au NPs have superior wound healing properties than LL37 peptide in a splinted mouse full thickness excisional model. In case of endothelial cells, LL37 peptide but not LL37-Au NPs activate FPRL-1, intracellular accumulation of Ca 2 + and secretion of VEGF165. Yet, both LL37 and LL37-Au NPs have high pro-angiogenic activity as demonstrated in a chorioallantoic membrane (CAM) assay. It is likely that the pro-angiogenic activity of LL37-Au NPs is medi- ated by the transactivation of EGFR but further studies are needed to address this issue. The results presented here are an exciting first step toward the development of antimicrobial peptide-based nanotherapeutics for skin disorders paving the way for additional studies in more complex animal models. It is the first skin therapy that combines wound-healing efficacy and antimicrobial activity in the same for- mulation, it is cheaper than the actual advanced therapies, and may be administered only one time.

Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
Despite an increasing awareness of the pathophysiologic events that takes place in the development of wounds of various underlying etiologies, impaired wound healing and its medical complications remain one of the most prevalent and economically burdensome healthcare issues in the world. Approximately 6.5 US million patients are affected by chronic wounds and more than US$25 billion are spent annually for the treatment of this condition. Chronic wounds have many psychosocial consequences, ranging from social isolation to mental malaise. In addition, impaired wound healing and its medical complications remains one of the most prevalent and economically burdensome healthcare issues in the world, due to an increase in aging population and a rise in diabetes and obesity incidence around the world, impaired wound healing will continue to increase. Given this, research into tissue regeneration in chronic wound repair is vital, as well as the development of new therapeutics is absolutely necessary and important to satisfy the unmet clinical need.In this thesis, we studied the therapeutic effects of LL37 conjugated gold nanoparticles coated polyurethane (LL37-AuNP-PU) dressing, which is a novel technology developed by our lab. We performed biocompatibility assays and showed that the viability of the human keratinocytes cells (HaCat cells) was not compromised, indicating that LL37-AuNP-PU dressing is not toxic to the cells. In addition, we also showed that this treatment induces wound closure trough expression of more keratinocytes in the proliferative edges of the wound, as well as in the wound gap. Furthermore, LL37-AuNP-PU dressing regulates the activation of macrophages and improves the progression of the inflammatory stages of wound healing, which makes this environment more prone to the beginning of proliferative and remodeling stages of wound healing.Overall, the results presented in this thesis demonstrated that LL-37-AuNP-PU dressings could potentially be used for the treatment of chronic wounds, diabetic wounds and burns, while preventing bacterial infection.
Apesar da crescente consciencialização dos eventos fisiopatológicos que ocorrem no desenvolvimento de feridas de várias etiologias, a ineficiente cicatrização e as complicações médicas associadas continuam a ser um dos problemas de saúde mais prevalentes e economicamente onerosos no mundo. Aproximadamente 6.5 milhões de doentes dos Estados Unidos são afetados por feridas crónicas e mais de 25 mil milhões de dólares são gastos anualmente no tratamento desta condição. As feridas crónicas têm muitas consequências psicossociais, que vão desde o isolamento social ao mal-estar mental. Além disso, devido ao aumento da população idosa no mundo e a um aumento na incidência de diabetes e obesidade, a ineficaz cicatrização de feridas irá continuar a aumentar. Dado isto, a investigação sobre a regeneração de tecidos nas feridas crónicas tornou-se vital, assim como o desenvolvimento de novas terapêuticas que se tornou absolutamente necessário e importante para satisfazer as necessidades clínicas existentes.Nesta tese, estudámos os efeitos terapêuticos do patch LL37-AuNP-PU, uma nova tecnologia desenvolvida pelo nosso laboratório. Realizámos ensaios de biocompatibilidade e demonstrámos que a viabilidade de queratinócitos humanos não foi comprometida, indicando que o patch LL37-AuNP-PU não é tóxico para as células humanas. Em adição, também demonstrámos que este tratamento induz a cicatrização da ferida através da expressão de mais queratinócitos nas extremidades proliferativas da ferida, assim como na ferida. Por conseguinte, o LL37-AuNP-PU regula a ativação de macrófagos e melhora a resposta inflamatória, assim como a sua progressão no processo de cicatrização de feridas, o que torna esse ambiente mais propício ao início dos estágios proliferativos e de remodelação da cicatrização.No geral, os resultados apresentados nesta tese demonstraram que o patch LL-37-AuNP-PU pode ser potencialmente utilizado para o tratamento de feridas crónicas, diabéticas e queimaduras, ao mesmo tempo que previne infeções bacterianas.
H2020
Outro - Centro 2020

HINTERGRUND: Chronisch entzündliche Darmerkrankungen (CED) zeigen eine Fehlfunktion der Epithelbarriere. Antimikrobielle Peptide wie Cathelicidine sind Effektormoleküle des angeborenen Immunsystems auf Epitheloberflächen. Cathelicidine beeinflussen das Bakterienwachstum und die Entzündungsreaktion, und können eine Rolle in der Pathogenes der CED spielen. In dieser Studie wurde die Expression des einzigem humanen Cathelicidin LL37 in Schleimhautbiopsien von Patienten mit Colitis ulcerosa und Morbus Crohn untersucht. METHODEN: Biopsiematerial aus Colon- und Ileumschleimhaut von insgesamt 61 Patienten (21 Morbus Crohn, 15 Colitis ulcerosa und 25 Kontrollen) wurde mittels Real-Time reverse Transkriptase Polymerasekettenreaktion und Immunhistochemie untersucht. ERGEBNISSE: Die Cathelicidin-Expression ist in Biopsien von Colitis ulcerosa Patienten signifikant erhöht, sowohl aus entzündeten als auch aus nicht entzündeten Darmabschnitten, verglichen mit gesunden Kontrollpatienten. In Biposien von Morbus Crohn Patienten hingegen zeigten die Cathelicidin-Expressionslevel keinen Unterschied beim Vergleich von entzündeten oder nicht entzündeten Darmabschitten mit gesunden Kontrollpatienten. Diese Ergebnisse waren unabhängig vom NOD2-Satus der Patienten. Auch bei Vergleich von Ileumbiopsien konnte kein signifikanter Unterschied in der Cathelicidin-Expression festgestellt werden. Die Biopsien die mittels Immunhistochemie analysiert wurden zeigten Cathelicidin Expression in den oberen Krypten, die in Kontrollen diffus, in Biopsien von Patienten mit CED basal betont ist. DISKUSSION: Bei CED ist die Cathelicidin-Expression im Colon verändert. Bei Patienten mit Colitis ulcerosa ist die Expression erhöht, bei Patienten mit Morbus Crohn nicht. Dies kann die Barrierefunktion des Epithels bei Patienten mit Morbus Crohn beeinträchtigen
BACKGROUND: Inflammatory bowel diseases (IBDs) are characterized by a breakdown of colon epithelial barrier function. Antimicrobial peptides like cathelicidins are molecules of the innate immune system located at epithelial surfaces. Cathelicidins influence microbial growth and inflammation and may play a role in IBD. In this study, the expression of human cathelicidin LL-37 was investigated in the intestinal mucosa from patients suffering from ulcerative colitis or Crohn's disease. METHODS: Biopsy material from colon and ileal mucosa of a total of 61 patients (21 with Crohn's disease, 15 with ulcerative colitis, 25 control patients) was evaluated for cathelicidin expression by real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. RESULTS: Cathelicidin expression was significantly increased in inflamed and non-inflamed colon mucosa from ulcerative colitis patients compared to non-inflamed control mucosa. In patients with Crohn's disease cathelicidin expression was not changed in inflamed or non-inflamed colon or ileal mucosa independent of NOD2 status. Biopsies evaluated by immunohistochemistry showed epithelial cathelicidin expression in the upper crypt that was diffuse in controls and only basal in IBD patients. CONCLUSIONS: In IBD the colonic expression of human cathelicidin is altered: cathelicidin expression is increased in inflamed and non-inflamed mucosa in patients suffering from ulcerative colitis but not in Crohn's disease. This deficiency may further compromise the antimicrobial barrier in Crohn's disease

碩士
大同大學
生物工程學系(所)
98
The aim of this work was to prepare cathelicidin, LL-37, loaded in biopolymers encapsulation. LL-37 is a unique peptide found in human body. The peptide composes of 37 amino acids and folds into α-helix secondary structure with net positive charges. The positive charges on LL-37 interact strongly with the membrane of Gram positive and Gram negative bacteria. LL-37 may disturb and penetrate into the cell membrane, resulting in the death of bacteria. It could be a very useful weapon to kill bacteria, including those with antibiotic resistance. To extend the half-life of LL-37 in human body, we first encapsulated LL-37 with sodium alginate. The alginate beads were collected and further encapsulated with chitosan and Poly (lactide-co- glycolide ) (PLGA). The particle size of beads containing LL-37 ranged from 0.74 to 1.03 ?慆, depending on the formulations. The release profile of LL-37 from chitosan-alginate beads were measured with protein assay and the antibiotic capabilities were surveyed with Pseudomonas aeruginosa culture.