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Clonagem, expressão heteróloga e caracterização do gene LmjF24.0320 que codifica a enzima fumarato hidratase em Leishmania major / Cloning, heterologous expression and characterization of the gene LmjF24.0320 that encodes the enzyme fumarate hydratase in Leishmania major.Patrícia Rosa Feliciano 11 August 2009 (has links)
Leishmania é um protozoário parasito flagelado responsável pela Leishmaniose, doença que afeta 88 países, distribuídos em 4 continentes, e que causa um risco a aproximadamente 350 milhões de pessoas. Estudos recentes em tripanosomatídeos, utilizando Trypanosoma brucei como modelo, sugerem que as enzimas fumarato hidratase, enzimas que catalisam a hidratação reversível da molécula de fumarato em S-malato, são essenciais para sobrevivência de tripanossomatídeos. O presente projeto visou a clonagem, expressão, purificação e caracterização cinética e biofísica da enzima fumarato hidratase codificada pelo gene LmjF24.0320 de Leishmania major. A proteína foi expressa em bactéria e purificada por cromatografia de afinidade. Os ensaios de cinética enzimática mostram que a enzima segue o modelo de cinética de Michaelis-Menten com Km e Vmax de 2,7 ± 0,5 mM e 35,2 ± 5,8 micromol/min/mg para fumarato e 5,2 ± 0,4 mM e 11,8 ± 0,6 micromol/min/mg para S-malato, respectivamente. Para os estudos de localização celular foram produzidos e purificados anticorpos policlonais para as isoformas LmFH-1 e LmFH-2 de Leishmania major através de imunização de coelhos. A combinação de técnicas de imunofluorescência por microscopia confocal, western blotting e controle da atividade enzimática através fracionamento celular com digitonina nos permitiu concluir que a isoforma LmFH-1 se encontra localizada na mitocôndria do parasito, enquanto que a isoforma LmFH-2 possue dupla localização, sendo encontrada tanto no citosol quanto no glicossomo. A presença das isoformas LmFH-1 e LmFH-2 nos três compartimentos celulares: citosol, glicossomo e mitocôndria reforçam a importância da molécula de fumarato em diferentes processos celulares, e fortalecem a idéia de que a enzima fumarato hidratase pode ser considerada um potencial alvo para planejamento de fármacos anti-leishmaniose. / Leishmania parasites are the casual agent of leishamaniasis, a group of disease that affects 88 countries, distributed in four continents, with 350 million people at risk of infection. Recent studies in trypanosomatids, using Trypanosoma brucei as a model suggest that the fumarate hydratase enzymes, which catalyze the stereospecific hydration of fumatare to malate, are essential for trypanosomatid survival. The present project focused the cloning, expression, purification and both kinetic and biophysical characterization of fumarate hydratase encoded by gene LmjF24.0320 of Leishmania major. The protein has been expressed in bacteria and purified by affinity chromatography. The kinetic experiments reveal that the enzyme follow the Michaelis-Menten classic model with Km and Vmax of 2,7 ± 0,5 mM and 35,2 ± 5,8 micromol/min/mg for fumarate and 5,2 ± 0,4 mM and 11,8 ± 0,6 micromol/min/mg for S-malate, respectively. For the subcellular localization studies, polyclonal antisera against both isoforms LmFH-1 e LmFH-2 of Leishmania major were obtained after immunization of rabbits. The combination of confocal microscopy, western blotting and enzymatic activity through cell fractionation by selective permeabilization of membranes with digitonin suggest that LmFH-1 isoform is localized to mitochondria, while the LmFH-2 isoform is found to both cytosol and glycossome. The presence of LmFH-1 and LmFH-2 isoforms at the three cell compartments: cytosol, glycosome and mitochondria strongly supports the premise that fumarate is important for many cell processes and suggests that fumarate hydratase enzyme can be considered an attractive target for the development of anti-leishmaniasis drugs.
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Identificação e caracterização cromossomal de 9 loci de Leishmania (L.) major relacionados com resistência a inibidores da via de biossíntese do ergosterol / Identification and chromosomal localization of 9 Leishmania (L.) major loci related to resistance against two inhibitors of ergosterol biosynthesis pathwayLuciana Aparecida Camizotti 17 December 2008 (has links)
O ergosterol é um componente responsável por manter a integridade e a fluidez das membranas de Leishmania spp. A partir de uma metodologia que consiste em seleção por superexpressão gênica, foram isolados nove diferentes loci de L. (L.) major relacionados com a resistência a dois inibidores da via de biossíntese do ergosterol: Terbinafina (TBF) e Itraconazol (ITZ). Análises funcionais individuais desses nove loci na presença de TBF e ITZ (ou do análogo Cetoconazol - CTZ) apresentaram níveis significantes de resistência após transfecção em células selvagens de L. (L.) major. Nesse trabalho apresentamos a metodologia de isolamento de um desses loci (cItz2), bem como a análise in silico das regiões cromossômicas correspondentes aos insertos dos nove cosmídios no genoma de L. (L.) major / Ergosterol is an important compound responsible to maintain integrity and fluidity of Leishmania spp. membranes. Starting from an overexpression/selection method, our group has isolated nine different loci of L. (L.) major related to resistance against two inhibitors of the ergosterol biosynthesis pathway, Terbinafine (TBF) and Itraconazole (ITZ). Individual functional analysis of these nine loci in the presence of TBF and/or ITZ (or the ITZ analog Ketoconazole, CTZ), have showed significant levels of resistance after transfection into L. (L.) major wild-type cells, followed by over expression induction. In this work, we show the insert mapping and chromosomal identification of one of these loci (cItz2), as well as discuss the in silico chromosomal analysis of the nine correspondent inserts in the L. (L.) major genome
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The role of dendritic cells in the immunoregulation of leishmaniasis - transfection of dendritic cells with mRNA encoding a molecularly defined parasitic antigen / Die Rolle dendritischer Zellen in der Immunregulation der Leishmaniose - Transfektion dendritischer Zellen mit mRNA eines molekular definierten ParasitenantigensKeller, Christian January 2007 (has links) (PDF)
Die kutane Leishmaniose ist eine Infektionskrankheit, die besonders in tropischen und Wüstenregionen endemisch ist, mit einer Inzidenz von 1,5 Millionen Fällen im Jahr und einer Prävalenz von 12 Millionen Infizierten weltweit. Die Infektion kann durch den intrazellulären Parasiten Leishmania major hervorgerufen werden. Am Mausmodell ist die Krankheit ausführlich untersucht. Wie dabei deutlich wurde, ist für die Immunität gegen den Erreger die Induktion einer Klasse von Interferon (IFN)--produzierenden CD4+ T-Helfer-Zellen (TH1-Zellen) entscheidend, welche Makrophagen dazu aktivieren, die von ihnen beherbergten Parasiten abzutöten. Die Umlenkung der Immunantwort in Richtung einer schützenden TH1-Antwort wird auch der Schlüssel zu einem effektiven Impfstoff sein. Ex vivo mit Leishmanienantigenen beladene dendritische Zellen sind vor einiger Zeit als Vakzine gegen L. major-Infektionen beschrieben worden. Ein einzelnes rekombinantes Antigen, LeIF (Leishmania homologue of eukaryotic ribosomal initiation factor 4a), ein parasitäres Protein, das die IL-12-Produktion durch dendritische Zellen stimuliert und das als mikrobiell konserviertes Strukturmolekül (pattern-associated molecular pattern; PAMP) diskutiert wird, vermittelte dabei, zum Pulsen von dendritischen Zellen verwendet, einen schützenden TH1-abhängigen Effekt. Der Einsatz rekombinanter Proteine ist jedoch mit etlichen Nachteilen verbunden, weshalb andere Methoden zur Verabreichung von Antigenen entwickelt wurden. Aus der Tumorforschung ist unlängst die RNA-Elektroporation dendritischer Zellen als eine sichere und vielseitige Methode hervorgegangen, bei der eine große Anzahl von RNA-Molekülen, die für ein bestimmtes Antigen kodieren, durch einen elektrischen Impuls in das Cytosol dendritischer Zellen gelangt. Die vorliegende Arbeit beschreibt zum ersten Mal die Transfektion dendritischer Zellen mit RNA eines molekular definierten Parasitenantigens. Zunächst erfolgte die Etablierung eines standardisierten Protokolls für die RNA-Transfektion mit dem enhanced green fluorescent protein (EGFP) als Reporterantigen. EGFP-RNA war gut translatierbar in einem In-vitro-Translationssystem, und es konnten sowohl eine Zellinie (fetal skin-derived dendritic cells; FSDC) als auch primäre, aus Knochenmarkkulturen der Maus gewonnene dendritische Zellen (bone marrow-derived dendritic cells; BMDC) mit einem Anteil von bis zu 90% bzw. 75% effizient EGFP-transfiziert werden. In beiden Zelltypen wurde die maximale Transfektionseffizienz mit 20 µg RNA erreicht, die mit größeren Mengen an RNA nicht weiter zu steigern war. Die Höhe der Antigenexpression, gemessen als mittlere Fluoreszenzintensität (MFI) in der Durchflußzytometrie, war direkt proportional zur verwendeten RNA-Menge. In FSDC waren die Transfektionseffizienz und die MFI generell höher als in BMDC bei gleicher RNA-Menge. Zudem konnte gezeigt werden, daß eine Behandlung mit LPS die Kinetik beeinflußt: Die maximale Expression war höher und wurde auch eher erreicht, worauf zudem ein schnellerer Abfall folgte. In den Transfektionsexperimenten mit LeIF wurden zwei Varianten von LeIF-RNA verwendet: eine für die gesamte LeIF-Sequenz kodierende LeIF(fl)-RNA, und eine nur für die aminoterminale Hälfte der LeIF-Sequenz (226 Aminosäuren), dem immunogenen Teil des LeIF-Moleküls, kodierende LeIF(226)-RNA. Im Western Blot von Ganzzellysaten dendritischer Zellen war nur LeIF(fl) nach Transfektion nachzuweisen, wohingegen LeIF(226) in LeIF(226)-transfizierten BMDC nie nachzuweisen war. Da beide Konstrukte aber gut im zellfreien System translatierbar waren, stellte der fehlgeschlagene Nachweis von LeIF(226) kein Fehlschlagen der RNA-Translation, sondern vielmehr einen raschen Antigenabbau dar. Es bestand daher die Erwartung, daß LeIF(226)-transfizierte BMDC trotzdem in der Lage sein müßten, von LeIF(226) abgeleitete antigene Peptide an T-Zellen von mit rekombinantem LeIF (rLeIF) immunisierten BALB/c-Mäusen zu präsentieren. Diese Vermutung wurde durch Messung von IFN- in Stimulationsversuchen mit BMDC und T-Zellen bestätigt, die zeigten, daß am Tag 7 der Kultur mit rLeIF gepulste, LeIF(226)- und LeIF(fl)-transfizierte BMDC in der Tat antigenspezifisch T-Zellen aus LeIF-immunisierten Mäusen aktivierten. IL-4 hingegen wurde nicht produziert, was mit der Tatsache vereinbar ist, daß in Lymphknoten LeIF-vakzinierter Mäusen hauptsächlich T-Zellen vom TH1-Typ zu finden sind. In den Überständen LeIF-transfizierter BMDC-Kulturen, im Gegensatz zu rLeIF-gepulsten BMDC, waren die proinflammatorischen Zytokine IL-1β, IL-6, IL-10 und IL-12 nicht nachzuweisen. Dieser Effekt lag nicht am Elektroporationsvorgang, da die Zytokinproduktion von mit rekombinantem LeIF elektroporierten BMDC nur teilweise beeinträchtigt war. Die Expression von CD86 war nach LeIF-Transfektion zudem geringer als nach Pulsen mit rLeIF. LeIF-Transfektion führte mithin nicht zur Reifung dendritischer Zellen. LeIF-transfizierte BMDC könnten im Ergebnis als antigenspezifische Toleranzinduktoren fungiert haben, mit regulatorischen T-Zellen als Respondern. Der Effekt der Transfektion mit LeIF-RNA auf die immunstimulatorische Wirkung von BMDC war nicht signifikant erhöht, wenn BMDC am Tag 8 oder 9 der Kultur verwendet wurden. BMDC, die am Tag 8, und mehr noch am Tag 9 mit rLeIF gepulst wurden, induzierten hingegen eine energische T-Zell-Antwort. BMDC vom Tag 9 waren sogar in der Lage, naive T-Zellen zu aktivieren. Bevor eine starke, gegen LeIF gerichtete T-Zell-Antwort eingeleitet werden kann, müssen dendritische Zellen also letztlich – neben Präsentation des Antigens und Expression kostimulatorischer Moleküle – eine gewisse „Empfindlichkeit“ gegenüber dem Strukturmolekül LeIF besitzen, die mit ihrem Reifungsalter in Zusammenhang steht. Dieses dritte Signal wird nicht durch intrazelluläres LeIF nach Transfektion mit LeIF-RNA übermittelt, oder es wird unterdrückt. Darüber hinaus war nach Elektroporation von rLeIF die IL-12-Produktion von BMDC gänzlich aufgehoben, die Produktion von IL-1 bei höheren Antigendosen reduziert und die Produktion von IL-10 teilweise erhöht. Die Produktion von IL-6 war unbeeinflußt. Dieses veränderte Zytokinprofil legt eine Doppelnatur von LeIF als PAMP nahe: Neben der bei extrazellulärem Vorliegen von LeIF erwiesenen Eigenschaft, die Produktion von IL-12 zu stimulieren, welches die Resistenz des Wirtes gegen L. major steigert, könnte LeIF bei intrazellulärem Vorliegen auch zu Evasionsmechanismen des Parasiten vor dem Immunsystem des Wirtes beitragen, möglicherweise durch Wechselwirkung mit MAP (mitogen-activated protein)-Kinase-Signalwegen. Die Eigenschaften von LeIF als Adjuvans hängen also sowohl von der Verabreichungsmethode (Transfektion mit RNA bzw. Pulsen mit dem rekombinanten Protein) als auch vom Zielkompartiment (extra- bzw. intrazellulär) ab. Zusammenfassend konnte also in dieser Arbeit gezeigt werden, daß BMDC mit einem Parasitenantigen transfizierbar sind. Das Antigen wird dabei prozessiert und präsentiert, aber von dendritischen Zellen nicht als PAMP erkannt. Durch Transfektion mit antigenkodierender mRNA alleine werden mithin nicht alle notwendigen Signale für die Induktion einer potenten Immunantwort übermittelt. / Cutaneous leishmaniasis is an infectious disease that is endemic especially in tropical and desert regions with an incidence of 1.5 million cases per year and a prevalence of 12 million people infected worldwide. The infection can be caused by the intracellular parasite Leishmania major. The disease has been studied extensively in the murine model. It has become apparent that the induction of a class of interferon (IFN)--producing CD4+ T helper cells (TH1 cells) that activate macrophages to kill the parasites they harbor is desicive for the establishment of immunity. The redirection of the host’s immune response towards a protective TH1 phenotype will also be the key to an effective vaccine. Dendritic cells (DC) loaded with leishmanial antigens ex vivo were lately described as vaccines against L. major infections. One single recombinant Leishmania antigen, LeIF (Leishmania homologue of eukaryotic ribosomal initiation factor 4a), which was identified as a protein that stimulates DC to secrete interleukin (IL)-12 and discussed as a pattern-associated molecular pattern (PAMP), was found to mediate a protective TH1-dependent effect when used for pulsing of DC. The application of recombinant proteins is tied to many disadvantages, which is why other methods of antigen administration have been developed. RNA electroporation of DC has recently emerged from tumor research as a safe and versatile method of antigen delivery, by which a large number of RNA molecules encoding a specific antigen gains access to the cytosol of DC by an electrical impulse. The present study describes, for the first time, transfection of DC with RNA encoding a molecularly defined parasite antigen. Initially, a standardized protocol for RNA transfection was established, using the enhanced green fluorescent protein (EGFP) as reporter antigen. EGFP-RNA was well translatable in an in vitro translation system, and both a DC cell line (fetal skin-derived DC; FSDC) and murine primary bone marrow-derived DC (BMDC) could be transfected efficiently, with a yield of up to 90% and 75%, respectively. In both cell types, maximal transfection efficiency was attained with 20 µg RNA and could not be further increased with larger amounts of RNA. The level of antigen expression, measured as the mean fluorescence intensity (MFI) by flow cytometry, was directly proportional to the amount of RNA used for transfection. In FSDC, transfection efficiency and MFI were generally higher than in BMDC when the same amounts of RNA were used. Furthermore, the kinetics was shown to be sensitive to treatment with lipopolysaccharide (LPS): the expression peak was higher and was reached sooner, followed by a more rapid decline. In transfection experiments with LeIF, two variants of LeIF-RNA were used: LeIF(fl)-RNA, encoding the complete LeIF sequence, and LeIF(226)-RNA, encoding only the aminoterminal half of the LeIF sequence (226 amino acids), the immunogenic part of LeIF. Only LeIF(fl) was detectable by Western Blot in whole cell lysates of BMDC after LeIF(fl)-RNA transfection, whereas LeIF(226) could never be detected in LeIF(226)-transfected BMDC. However, as both constructs were well translatable in a cell-free system, the failure to detect LeIF(226) in BMDC lysates did not represent a failure in RNA translation, but rather a rapid antigen degradation. It was therefore expected that LeIF(226)-transfected BMDC should nevertheless be able to present LeIF(226)-derived antigenic peptides to T cells from BALB/c mice primed with recombinant LeIF (rLeIF). This hypothesis was confirmed by measuring IFN- production in BMDC-T cell co-incubation assays, showing that rLeIF-pulsed, LeIF(226)- and LeIF(fl)-transfected day 7 BMDC did indeed activate T cells from LeIF-immunized mice in an antigen-specific manner. In contrast, IL-4 was not produced, which was consistent with the fact that T cells found in lymph nodes from LeIF-primed mice are primarily of the TH1 type. In the supernatants of LeIF-transfected BMDC cultures, in contrast to rLeIF-pulsed BMDC, the proinflammatory cytokines IL-1β, IL-6, IL-10 and IL-12 were not detected. This effect was not due to the electroporation procedure, as cytokine production by BMDC electroporated with rLeIF was only partially impaired. Also, the expression levels of CD86 were lower upon LeIF transfection than after pulsing with rLeIF. Thus, LeIF transfection did not induce maturation of DC. In conclusion, LeIF-transfected BMDC may have acted as semi-mature antigen-specific tolerance inducers, with regulatory T cells as responders. The effect of LeIF transfection on the immunostimulatory capacity of BMDC was not significantly increased when day 8 or 9 BMDC were used. However, day 8, and even more day 9 BMDC pulsed with rLeIF mounted a vigorous T cell response. Day 9 BMDC were able to activate naïve T cells. In conclusion, before a strong T cell response against LeIF can be induced, DC need to – besides presenting antigen and expressing co-stimulatory molecules – exhibit a susceptibility to the innate signaling molecule LeIF which is linked to their maturation age. This third signal is provided by extracellular rLeIF, but it is not conveyed – or is suppressed – by intracellular LeIF after LeIF-RNA transfection. Furthermore, electroporation of rLeIF abrogated IL-12 production by BMDC completely, the production of IL-1 was reduced with higher antigen doses, and the production of IL-10 was partially increased. The IL-6 production was unaffected. This altered cytokine profile suggests that LeIF as a PAMP might have a bipartite nature: besides exhibiting the capacity to stimulate IL-12 production upon extracellular presence, thereby enhancing host resistance against L. major, LeIF could also contribute to parasitic host evasion mechanisms from intracellular compartments of DC, possibly by interfering with mitogen-activated protein (MAP) kinase signaling pathways. Thus, the adjuvant properties of LeIF depend both on its mode of delivery (transfection with RNA vs. pulsing with the recombinant protein) and the targeted compartment (extra- vs. intracellular). From this work, it can be summarized that BMDC are well transfectable with a parasite antigen. The antigen is processed and presented, but it is not recognized as a PAMP by DC. Hence, transfection with antigen-encoding mRNA by itself does not convey all necessary signals for the elicitation of a potent immune response.
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Search for Surrogate Marker(s) of Immunity Following Vaccination with Experimental Vaccine (Autoclaved Leishmania Major + Bacille Calmette-Guérin) in Human VolunteersMahmoodi, Majid 12 1900 (has links)
Cutaneous leishmaniasis (CL) is usually a self-limiting lesion on the skin while visceral leishmaniasis is a progressive, systemic disease with high mortality even if treated. The problem associated with treatment and vector control justifies a search for an effective vaccine which seems to be the only practical means to control the disease. The aim of this study is to identify immunological surrogate marker(s) associated with protection against Leishmania infection. The results indicate that a single dose of ALM+BCG induced Thl-like response but the level of such response is not sufficient for full protection. Accordingly, further evaluation of the vaccine is necessary other strategies multiple injections or changing the adjutant.
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Caracterização molecular de RNAs teloméricos não codantes em Leishmania majorMorea, Edna Gicela Ortiz. January 2018 (has links)
Orientador: Maria Isabel Nogueira Cano / Resumo: Os protozoários do gênero Leishmania causam leishmaniose, uma doença tropical negligenciada, que se apresenta de diferentes formas clínicas e que acomete milhões de pessoas no Brasil e no mundo. Até o momento não existe nenhuma vacina ou tratamento eficientes para leishmaniose e por isso, estudos que contribuam para o entendimento da biologia e fisiologia do parasito podem fornecer uma possibilidade de encontrar novos alvos terapêuticos. Este é o caso dos telômeros, cuja função principal é manter a estabilidade do genoma que se perturbada pode afetar diretamente a proliferação ou multiplicação dos parasitos. Os telômeros são estruturas nucleoprotéicas localizadas nas extremidades dos cromossomos, mantidos pela enzima telomerase. Eles são regulados por diversos processos entre os quais se encontram alguns longos RNAs não codificadores (lncRNA). Entre os lncRNAs com função telomérica está o TER (RNA Telomerase) o qual é um dos principais componentes do complexo telomerase, pois contém uma pequena sequência molde a qual é copiada pela telomerase durante a replicação dos telômeros, o TER foi recentemente identificado em Leishmania spp., porém se desconhece a sua função e biogênese. Outro RNA não codificante com função telomérica é o TERRA (Repetições Teloméricas contendo RNA), os quais são essenciais para a manutenção dos telômeros. Até o momento, identificamos a presença do TERRA expressos a partir das extremidades cromossômicas nas diferentes fases de desenvolvimento (amastigot... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Protozoan from Leishmania genus are the etiologic agents of leishmaniasis, a tropical disease that presents different clinical manifestations and affect million people in Brazil and in the world. There are no eficiente vacines nor treatment protocols against leishmaniasis, therefore, it is crucial to improve the knowledge about Leishmania molecular biology and physiology for the development of new therapies. Actually, telomeres have been considered potential molecular targets as they are responsible to maintain genome stability. Leishmania telomeres similarly to other eukaryotes, are nucleoprotein structures found at the end of the chromosomes maintained by telomerase. They can be regulated by different mechanisms such as the action of long non-coding telomeric RNAs (lncRNAs). Among the telomeric lncRNAs are the telomerase RNAcomponent (TER) which is crucial for telomerase activity because it contains the template sequence that is copied by telomerase during telomere elongation. TER was recently identified in Leishmania although there is no information about its function and biogenesis.TERRA, the Telomere Repeat containing RNA is other telomeric RNA that it is involved with telomere length regulation telomere damage response and alterations in the composition of telomeric chromatin and is essential for telomere maintenance. In this work, we identified TERRA transcripts in the three Leishmania developmental stages (amastigote, promastigote e metacyclic). We interrogate three i... (Complete abstract click electronic access below) / Doutor
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Envolvimento de células ER-MP58+ na produção de IL-12 em linfonodos drenantes da infecção inicial por leishmania major em camundongos BALB/cCardoso, Ludimila Paula Vaz January 2006 (has links)
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Previous issue date: 2006 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The development of a Th1 immune response is the key event to prevent Leishmania
infection and is linked with the IL-12 production by monocytes, dendritic cells,
macrophages and neutrophils. The IL-12 signal induces the increase of IFN-γ
production by T cells, favoring the profile of the Th1 immune response and the
resistance phenotype to the infection. In the vertebrate host, the leishmania inhabits in
cells of the mononuclear phagocyte system (MPS), which is also responsible for the
elimination of the parasite. The participation of immature cells of MPS in the initial
production of IL-12 has been discussed. It was demonstrated recently that a
population of mononuclear phagocytes, derived from bone marrow culture and
expressing the marker ER-HR3 produced great amount of IL-12p40 after stimulation in
vitro with procyclic or metacyclic promastigotes of L. (L.) major. The initial production
of IL-12 by MPS cells is under investigation. It is unclear the role of mononuclear
phagocytes, in different stages of maturation, in the production of
IL-12p40 in vivo. In this work, we evaluated the involvement of MPS cells, in different
stage of maturation, with the IL-12p40 production in vivo in draining lymph nodes of
BALB/c mice after 48 h of infection with L. (L.) major. Our results showed that CD31+
cells (mononuclear phagocytes precursors) were absent in the draining lymph nodes
of the subcutaneously footpad injection and that the number of ER-MP58+
(immature mononuclear phagocytes), ER-HR3+ cells (mononuclear phagocytes in the intermediate stage of maturation) and 33D1+
(mature phagocytes) increase in the period of 48 h after infection. We showed, also, that ER-MP58+
responsible for great part of the draining lymph nodes IL-12 production 48 h after
infection with stationary-phase promastigotes of L. (L.) major in mice BALB/c. We
suggested that, in vivo, the ER-MP58+ population, together with other cellular
populations, known as IL-12 producers, ER-MP58+ cell has an important role in the inflammation induced by the parasitic inoculum. / O desenvolvimento de uma resposta imune do tipo Th1 é o evento chave para
prevenir a infecção murina por Leishmania e está interligada à produção de IL-12 por
monócitos, células dendríticas, macrófagos e neutrófilos. A sinalização via IL-12
aumenta a produção de IFN-γ por células T, favorecendo o perfil Th1 da resposta
imune e o fenótipo de resistência à infecção. A leishmania, no hospedeiro vertebrado,
reside em células do sistema fagocítico mononuclear (SFM), o qual também é
responsável pela eliminação do parasito. A participação de células indiferenciadas do
SFM na produção de IL-12 vem sendo discutida. Foi demonstrado recentemente que
uma população de fagócitos mononucleares, derivados de cultura de medula óssea e
expressando o marcador ER-HR3 produzia grande quantidade de IL-12p40 após
estimulação in vitro com formas procíclicas ou metacíclicas de L. (L.) major. A
produção de IL-12 na infecção por L. (L.) major por fagócitos em diferentes estágios
de maturação in vivo ainda não está claro. Neste trabalho, avaliamos o envolvimento
de células em diferentes estágios de maturação em linfonodos drenantes após 48 h
de infecção por L. (L.) major em camundongos BALB/c, quanto à produção de IL-
12p40. Verificamos que células CD31+ (precursores de fagócitos mononucleares)
estão ausentes nos linfonodos drenantes do sítio do inóculo e que o número de células ER-HR3+ (fagócitos mononucleares em estágio intermediário de maturação), células ER-MP58+ (fagócitos imaturos) e 33D1+ (subpopulações de células dendríticas) aumenta no período de 48 h após a infecção. Mostramos também que,
células ER-MP58+ são as principais responsáveis por grande parte da produção de
IL-12 nos linfonodos drenantes 48 h após a infecção por L. (L.) major em
camundongos BALB/c. Sugerimos que, in vivo, a população ER-MP58+
com as outras populações celulares, conhecidas como produtoras de IL-12, têm um
papel relevante na inflamação induzida pelo inóculo parasitário.
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Mechanism of dendritic cell-based vaccination against Leishmania major / Mechanismus der auf dendritischen Zellen beruhenden Impfung gegen Leishmania majorSchnitzer, Johannes K. January 2012 (has links) (PDF)
Die Impfung mittels Antigen-beladener dendritischer Zellen [DZ] ist mittlerweile eine gut etablierte Technik, die dann zum Einsatz kommt, wenn Standard-Impftechniken versagen, vor Krankheiten zu schützen beziehungsweise diese zu heilen. Die Effizienz dieser Technik konnte bereits für diverse Infektionskrankheiten und Krebserkrankungen in experimentellen Tiermodellen sowie am Menschen gezeigt werden. Hierbei ist die Möglichkeit zur wohldefinierten Manipulation und Antigenbeladung der DZ ein großer Vorteil gegenüber den konventionellen Ansätzen. Jedoch ist vor allem bei der Anwendung im klinischen Bereich die Präparation, Herstellung und Manipulation dieser autologen DZ mit einem erheblichen technischen, zeitlichen sowie finanziellen Aufwand verbunden. Hinsichtlich einer Präventivimpfung gegen eine pandemische Infektionskrankheit, die in hauptsächlich unterentwickelten Ländern vorkommt, wird dieser Aufwand sicherlich ein Hindernis darstellen. Daher muss für solche Fälle ein maßgeschneiderter Impfstoff entwickelt werden, der sich am Vorbild des effektiven DZ-basierten Impfstoffs orientiert. Für die Impfung gegen die Leishmania Parasiten besteht so ein DZ-basierter Impfstoff bereits. Dessen Wirkung, eine T-Zell Antwort vom Typ Th1 zu induzieren, wurde bereits in mehreren Veröffentlichungen demonstriert. Zusätzlich hat aber eine unserer Studien gezeigt, dass das typische Th1-bezogene Zytokin IL-12 zur Differenzierung naiver T-Zellen nicht von den injizierten DZ bereitgestellt werden muss, sondern von der geimpften Maus. Dies gab erste Hinweise auf eine stärkere Beteiligung des Wirts-Immunsystems als zuvor angenommen. Daher sollte hier vertieft der Mechanismus dieser DZ-basierten Impfung untersucht werden, wobei modifizierte Impfstoff-Ansätze zum Einsatz kommen sollten. Dabei wurden die Fragen nach der vom Impfstoff transportierten Information und dem Empfänger dieser Information berücksichtigt. Das aktuelle Paradigma zur DZ-basierten Impfung besagt, dass transferierte DZ im direkten Kontakt mittels dreier Signale T-Zellen stimulieren und aktivieren. Dafür müssen diese DZ mit dem entsprechenden Antigen beladen und aktiviert worden sein um das Antigen-Peptide mittels MHC Molekül im Kontext der Co-Stimulation präsentieren zu können. Jedoch zeigt diese Studie hier, dass weder eine Aktivierung der DZ noch die Präsentation des Antigens mittels passender MHC Moleküle notwendig ist für die Induktion einer protektiven Immunantwort gegen Leishmania Parasiten. Aufgeschlossene, mit Antigen beladene DZ müssen nicht vor dem Transfer mit CpG ODN aktiviert worden sein, um entsprechende Immunität zu verleihen. Ebenso hat der MHC Typ in diesem Falle auch keinen Einfluss auf die Effektivität des Impfstoffs. Da im Weiteren aufgeschlossene mit Leishmania-Antigen beladene Makrophagen nach Impfung die gleiche Wirkung erzielen, wie vorangegangene DZ-basierte Impfstoffe, können keine DZ spezifischen Mechanismen Schlüsselkomponenten der Induktion einer protektiven Immunität sein. Darüber hinaus konnte gezeigt werden, dass die DZ der geimpften Mäuse, eine maßgebliche Rolle bei der Verarbeitung transferierter Signale spielen. Suspensionen aufgeschlossener DZ stellen eine Kombination aus freigesetzten löslichen Molekülen sowie Membranvesikeln dar, die sich nach dem Aufschluss gebildet haben. Nach Auftrennung dieser beiden Fraktionen konnte gezeigt werden, dass ausschließlich die Membran-Fraktion nach Verimpfung eine geeignete Immunantwort zum Schutz vor Leishmania Parasiten induzieren kann. Als Vorteil dieser Aufreinigung erweist sich zudem die stabile Lagermöglichkeit bei -80°C. Somit ist klar gezeigt, dass die Immunität-verleihende Einheit dieser Impfstoffvarianten in der Membran-Fraktion liegt. Verfolgt man die Induktion Th1-zugehöriger Zytokine in in vivo Experimenten so ergibt sich im Falle der Gesamtsuspension aufgeschlossener, mit Leishmania-Antigen beladener DZ ein klares Bild. Diese Suspension erzeugt das volle Spektrum der DZ-basierten Impfung gegen Leishmania Parasiten. Es kann sowohl Produktion von IL-12 und IL-2 als auch eine antigenspezifische T-Zell Proliferation nach Stimulation von Splenozyten mit der entsprechenden Suspension verzeichnet werden. Außerdem produzieren Splenozyten von entsprechend geimpften Mäusen nach Stimulation mit Leishmania-Antigen erhebliche Mengen des entscheidenden Zytokins IFNγ. Obwohl jedoch die Verimpfung aufgereinigter Membranvesikel dieses Ansatzes im Tierversuch zu biologisch sowie statistisch signifikanten Ergebnissen führt, lassen sich die entsprechend Th1-bezogenen Zytokine im in vivo Ansatz nur in geringen Maße nachweisen. Ob dies jedoch für einen in vivo unbemerkten Aktivitätsverlust des Vakzins oder für andere lymphatische Organe als Ort der T-Zell Instruktion spricht, ist noch unbekannt und muss noch geklärt werden. / Dendritic cell-based vaccination is a well established technique for preventive and therapeutic instruction of the immune system where conservative vaccine formulations fail to cure or prevent diseases, respectively. Efficiency of this technique already was demonstrated in infectious diseases as well as for cancer in animal or human studies. Well controlled manipulation and antigen-loading of immature DC is most beneficial to this technique. But, time-consuming and cost-extensive procedures for preparation of DC precursors, expansion and stimulation of DC and inpatient administration are big disadvantages regarding vaccine development for pandemic infectious diseases that occur mainly in underdeveloped countries. Therefore vaccines are needed that are pathogen-tailored and able to induce equal immune responses as their DC-based vaccine models. For vaccination against Leishmania parasites such a DC-based vaccine is feasible and its efficacy to induce protective Th1-based immune responses was already demonstrated in several animal studies. But, one of our own studies indicated supportive activity of host cells exceeding the allocation of T cells to become activated by transferred DC. IL-12, an important cytokine for the induction of Th1-related immune responses, has to be produced by host cells. Therefore, the aim of this study was to investigate the mechanism of BMDC-based vaccination with regard to simplification of the vaccine formulation. Key questions that have been addressed are: Which cells process the information that is transferred by the injected DC and what are the key components of this information? Further more, it was looked at whether altered vaccine formulations are able to induce protective immunity and whether they share equal molecular mechanisms. The current paradigm of BMDC-based vaccination proposes direct interaction of transferred BMDC with host T cells. These BMDC have to be antigen-loaded for stimulation via antigen-peptide-MHC molecule-complexes and they have to be activated for proper co-stimulation of T cells. Here, this study demonstrates that neither activation for co-stimulation nor direct interaction with adequate MHC molecules is needed for the induction of protective immunity against infection with Leishmania-parasites. Disrupted antigen-loaded BMDC are able to induce protective immunity in BALB/c mice without pre-stimulation via CpG ODN. Beyond, if BMDC were used with a different MHC-background than recipient mice then the vaccine still would be efficient in terms of reduction of footpad swelling and parasite load in draining lymph nodes. Even more, DC-specific features are no key component that leads to protective immunity as vaccination with disrupted antigen-loaded MΦ shows equal properties than before mentioned vaccine formulations. Further more, it was found that host DC play a major role in transforming the incoming signal, received from transferred antigen-loaded DC, into Th1-related stimuli and Leishmania-antigen-specific T cell activation. Suspensions of disrupted antigen-loaded DC resemble a combination of laid off soluble molecules together with exosome-like vesicles that formed after disruption of membranes. Here it was shown that separation of the membranous and soluble fractions and subsequent transfer into BALB/c mice will lead to protection of these mice against infection with L. major promastigotes only if the membranous fraction is used as vaccine. More, this vaccine formulation takes advantage of easy storage at -80°C with no need of fresh production. This clearly demonstrates that the immunity-inducing principle of disrupted DC-based vaccination lies within the membrane enclosed fraction. On a molecular level, disrupted antigen-loaded DC induce Th1-related cytokines during vaccination and as response on pathogen encounter. In vivo assays revealed IL-12 production and antigen-specific T cell proliferation among splenocytes that were stimulated with disrupted antigen-loaded DC. Splenocytes of accordingly vaccinated mice produce tremendous amounts of IFNγ after stimulation with Leishmania parasites. In summary, disrupted antigen-loaded BMDC fulfil all characteristics of DC-based vaccination against Leishmania major. But, while purification of membranes of antigen-loaded DC and subsequent transfer to BALB/c mice leads to control of the disease in the animal model, only slight levels of Th1-related cytokines are seen in the in vivo assays. Whether this points towards a loss of vaccine activity on unseen levels or unknown sites where Th1-related immunity is induced by both, complete solution and purified membranes, still has to be determined.
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A role for NK cells in innate immunity against human leishmaniasis /Nylén, Susanne, January 2003 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.
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Der Oligosaccharyltransferase-Komplex aus Saccharomyces cerevisiae : Funktionelle Charakterisierung von Stt3 aus Hefe und seinen Homologen aus Campylobacter, Leishmania und MenschHese, Katrin January 2008 (has links)
Regensburg, Univ., Diss., 2008.
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Análise dos efeitos da superexpressão do componente RNA da telomerase de Leishmania major (LeishTER)Vassilievitch, Alessandro Cabral. January 2018 (has links)
Orientador: Maria Isabel Nogueira Cano / Resumo: Parasitos do gênero Leishmania pertencem à família Trypanosomatidae, os quais causam a leishmaniose, doença tropical negligenciada, que pode se apresentar em três formas clínicas: cutânea, mucocutânea e visceral. O Brasil é um dos países mais afetados pela doença, devido principalmente às condições socioeconômicas, às mudanças climáticas e ambientais. Pesquisas relacionadas à biologia da Leishmania contribuem para o entendimento dos mecanismos fisiológicos do parasito, e assim fornecem a possibilidade de encontrar novos alvos terapêuticos. O estudo dos telômeros de Leishmania se mostram promissores, já que estão relacionados com a estabilidade do genoma. Os telômeros estão localizados nas extremidades dos cromossomos e são responsáveis por proteger os cromossomos assegurando que a informação genética seja corretamente copiada durante a duplicação celular. Os telômeros são elongados por uma transcritase reversa especializada denominada telomerase. A telomerase é uma ribonucleoproteína, constituída por duas subunidades, uma proteína com função de transcriptase reversa denominada TERT, e um componente RNA (TER) que contém a sequência do molde da repetição telomérica copiado pela TERT. Estudos recentes mostram que o TER possui outras funções além de conter apenas um molde para elongamento dos telômeros. Sua estrutura secundária possui domínios com funções de controle da inserção de nucleotídeos pelo TERT, reconhecimento da sequência e ligação de proteínas acessórias. Recentemente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Parasites of the Leishmania genus belong to the Trypanosomatide family, which present peculiar and particular characteristics. Among them are the species that cause leishmaniasis, a neglected tropical disease that can be expressed in three different clinical forms: cutaneous, mucocutaneous and visceral. Brazil is one of the most affected countries, due mainly to socioeconomic conditions, climate change and environmental alterations. Research related to the biology of Leishmania contributes to the understanding of the important physiological mechanisms of the parasite, and thus provide new therapeutic targets against the disease. The study of Leishmania telomeres appears promising since they related are to the genome stability. Telomeres are nucleoprotein structures located at the ends of the chromosomes and are responsible for protecting the chromosomes ensuring that the genetic information copied is correctly during cell duplication. DNA polymerase does not elongate telomeres as the rest of the genetic material, and thus maintained are by the action of a specialized reverse transcriptase named telomerase. Telomerase is a ribonucleoprotein minimally composed by two subunits, a protein with reverse transcriptase function TERT, and an RNA component (TER) that contains the telomeric repeat template sequence copied by TERT. Recent studies shown that TER has other functions besides being just a template for telomeres elongation. Its secondary structure has domains with control fun... (Complete abstract click electronic access below) / Mestre
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