Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Made available in DSpace on 2014-06-12T23:03:08Z (GMT). No. of bitstreams: 2 arquivo8782_1.pdf: 273291 bytes, checksum: 2a96750488e896524818e83f5a20ad79 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2002 / A infecção pelo Tripanosoma cruzi resulta no desenvolvimento de intensa produção de anticorpos e resposta imune celular durante as fases aguda e crônica da doença. O presente trabalho investigou o grau de estimulação da resposta imune, humoral e celular em camundongos BALB/c imunizados com o Ag-Rec CRA de T. cruzi, visando sua utilização em ensaios de imunoproteção. Foram avaliados o perfil isotípico das imunoglobulinas IgG, a reação de hipersensibilidade cutânea, a resposta proliferativa de linfócitos esplênicos e a produção de citocinas intracitoplasmáticas. Os resultados mostraram que o Ag-Rec CRA induziu: 1) um aumento significativo na produção de anticorpos de isotipos IgG2a e IgG3: 2) a produção de IFN-γ por células CD4+ indicando que o CRA induz uma resposta celular tipo Th1 e 3) a produção de TNF-α por células CD8+. Não foi observada diferença significativa na resposta proliferativa associada aos linfócitos T esplênicos frente ao Ag-Rec CRA, quando comparado ao grupo controle sem estimulação. Os animais imunizados com Ag-Rec CRA manifestaram reações de hipersensibilidade imediata, alcançando um pico máximo e, 2 h após a injeção do antígeno, diminuindo lentamente em 24 h. Estes resultados mostraram que o Ag-Rec CRA ativa mecanismos imunes envolvidos na eliminação do parasita e poderá ser importante em induzir a imunidade protetora

No presente trabalho verificamos o papel dos Leucotrienos na modulação da resposta imune durante a fase aguda da infecção experimental pelo Trypanosoma cruzi, usando como modelo camundongos deficientes da enzima 5-lipoxigenase (5-LOko). Os nossos dados demonstram que camundongos infectados pelo T. cruzi produzem metabólitos do ácido aracdônico como PGE2, LTB4 e LTC4. Comparados aos animais controles, os animais 5-LOko apresenta parasitemia mais tardia e menor, tem menor parasitismo tissular, menor infiltrado de células inflamatórias no coração e musculatura esquelética e apresenta menor taxa de mortalidade durante a fase aguda, indicando que animais deficientes de leucotrienos são mais resistentes a infecção pelo parasito. Animais 5-LOko está relacionado com a manutenção de números elevados de células F4/80+ e redução de células CD11b+ durante a infecção e menor número de células T ativadas expressando os marcadores CD4+CD69+, CD4+CD25+, CD4+CD44+ e CD8+CD69+, números inalterados de células T regulatórias CD4+CD25+GITR+ e menor produção de anticorpos parasito-específicos do isotipo IgG2a. O controle eficiente de parasitas por animais 5-LOko está associado ao aumento de células Gr-1+ e CD11c+GR-1+, produção aumentada IL-12, IFN-g, e produzirem menos PGE2, IL-10, ao contrario, animais controles, incapazes de controlar parasitas circulantes, produzem mais PGE2 e IL-10 e menos IL-12 e IFN-g. A baixa mortalidade de animais 5-LOko correlaciona com a produção de PGE2 e IL-10, produzir muita IL-12 e menos IFN-g e NO e baixíssima parasitemia. A mortalidade maior de animais controles envolve a produção IFN-g e altos níveis de LTB4, LTC4, NO e ausência de IL-10, IL-1b, PGE2 e números elevados de parasitas circulantes. Ainda macrófagos de animais 5-LOko apresentam maior capacidade de adesão/internalização de tripomastigotas e alta atividade tripanocida por mecanismo independente da geração de NO. Estes dados em conjunto demonstram que mediadores lipídicos produzidos pela enzima 5-lipoxigenase como LTB4 e LTC4 modulam negativamente a capacidade dos camundongos para geração de uma resposta imune capaz de controlar os parasitos durante a fase aguda da infecção pelo T. cruzi. / Accumulating studies have indicated that 5-lipoxigenase (5-LO) converted lipid mediators as leukotrienes acts modulating the host immune response against infectious diseases. The precise role of leukotrienes during the protozoan infection is unknown. In this work we evaluate the role of leukotrienes during the acute phase of Trypanosoma cruzi infection using as model the 5-lypoxigenase deficient mice (5-LOko). Our results show that PGE2, LTB4 and LTC4 are produced during the Trypanosoma cruzi infection. 5-LOko infected mice are more resistant than control mice as judge by the lower parasitemia, decreased number of parasite nest and inflammatory cells in the heart and skeletal muscle and low rate of mortality. The resistance of 5-LOko mice is associated with the increased capacity of spleen cells to produce cytokines as IL-12 and IFN-g; sustained capacity to produce detectable levels of IL-10 and PGEe and low NO serum levels than control mice. In contrast, the wild type mice are extremely susceptible and are unable to control parasites efficiently. The susceptibility is associated with increased levels of IL-10 and PGE2 and low IL-12 and IFN-g production. The high mortality rate in wild type mice is related with high LTB4, LTC4 and NO levels and bias to produce only type 1 cytokines. Also we shown that resistant 5-LOko mice present increased number of spleen cells expressing GR-1+, GR-1+CD11c+, F4/80+ and lower numbers o spleen cells expressing CD4+CD69+, CD4+CD25+, CD4+CD44+, CD8+CD69+ and CD11b+ and low serum levels of parasite-specific IgG2a than wild type mice and do not present alteration in TREG expressing CD4+CD25+GITR+. Importantly, IFN-g and- LPS activated macrophage from 5-LOko mice but not from wild type mice, present high capacity to recognize typomastigotes, internalize them and strong capacity to kill intracellular parasite as NO independent pathway. The results implicate that high levels leukotrienes, NO and pure type 1 cytokines production is associated to susceptibility to parasite. In contrast, leukotrienes deficiency led to an balanced immune response with relative high levels of type 1 cytokines and relative low levels of NO, type 2 cytokines and PGE2 that efficiently control the parasites. Also indicate that 5-lipoxigenase converted lipid mediators contribute negatively to generation of an effective immune response during the acute phase of T. cruzi infection.

African trypanosomes are unicellular parasites that cause nagana and sleeping sickness in livestock and man, respectively. The major pathogens for the animal disease include Trypanosoma vivax, T. congolense, and T. brucei brucei, whereas T. b. gambiense and T. b. rhodesiense are responsible for human infections. Given that the bloodstream form (BSF) of African trypanosomes is exclusively extracellular, its cell surface forms a critical boundary with the host environment. The cell surface of the BSF African trypanosomes is covered by a dense coat of immunogenic variant surface glycoproteins (VSGs). This surface protein acts as an impenetrable shield that protects the cells from host immune factors and is also involved in antibody clearance and antigenic variation, which collectively ensure that the parasite stays ahead of the host immune system. Gene expression in T. brucei is markedly different from other eukaryotes: most genes are transcribed as long polycistronic units, processed by trans-splicing a 39-nucleotide mini exon at the 5′ and polyadenylation at the 3′ ends of individual genes to generate the mature mRNA. Therefore, gene expression in T. brucei is regulated post-transcriptionally, mainly by the action of RNA binding proteins (RBPs) and conserved elements in the 3′ untranslated regions (UTR) of transcripts. The expression of VSGs is highly regulated, and only a single VSG gene is expressed at a time from one of the ~15 subtelomeric domains termed bloodstream expression sites (BES). When cells are engineered to simultaneously express two VSGs, the total VSG mRNA do not exceed the wild type amounts. This suggests that a robust VSG mRNA balancing mechanism exists in T. brucei. The present study uses inducible and constitutive expression of ectopic VSG genes to show that the endogenous VSG mRNA is regulated only if the second VSG is properly targeted to the ER. Additionally, the endogenous VSG mRNA response is triggered when high amounts of the GFP reporter with a VSG 3′UTR is targeted to the ER. Further evidence that non-VSG ER import signals can efficiently target VSGs to the ER is presented. This study suggests that a robust trans-regulation of the VSG mRNA is elicited at the ER through a feedback loop to keep the VSG transcripts in check and avoid overshooting the secretory pathway capacity. Further, it was shown that induction of expression of the T. vivax VSG ILDat1.2 in T. brucei causes a dual cell cycle arrest, with concomitant upregulation of the protein associated with differentiation (PAD1) expression. It could be shown that T. vivax VSG ILDat1.2 can only be sufficiently expressed in T. brucei after replacing its native GPI signal peptide with that of a T. brucei VSG. Taken together, these data indicate that inefficient VSG GPI anchoring and expression of low levels of the VSG protein can trigger differentiation from slender BSF to stumpy forms. However, a second T. vivax VSG, ILDat2.1, is not expressed in T. brucei even after similar modifications to its GPI signals. An X-ray crystallography approach was utilized to solve the N-terminal domain (NTD) structure of VSG ILDat1.2. This is first structure of a non-T. brucei VSG, and the first of a surface protein of T. vivax to be solved. VSG ILDat1.2 NTD maintains the three-helical bundle scaffold conserved in T. brucei surface proteins. However, it is likely that there are variations in the architecture of the membrane proximal region of the ILDat1.2 NTD and its CTD from T. brucei VSGs. The tractable T. brucei system is presented as a model that can be used to study surface proteins of related trypanosome species, thus creating avenues for further characterization of trypanosome surface coats. / Afrikanische Trypanosomen sind einzellige Parasiten, die Nagana in Nutzvieh und die Schlafkrankheit im Menschen verursachen. Zu den Hauptverursachern der Tierkrankheit gehören Trypanosoma vivax, T. congolense und T. brucei brucei, während T. b. gambiense und T. b. rhodesiense für Infektionen im Menschen verantwortlich sind. Da die Blutstromform (BSF) der afrikanischen Trypanosomen rein extrazellulär vorkommt, bildet die Zelloberfläche eine kritische Grenzregion mit der Wirtsumgebung. Die Zelloberoberfläche der BSF afrikanischer Trypanosomen ist mit einem dichten Mantel an immunogenen variablen Oberflächenglykoproteinen (variant surface glycoprotein, VSG) umgeben. Dieses Oberflächenprotein dient als Barriere zum Schutz gegen Faktoren des Wirtsimmunsystems und spielt ebenfalls eine Rolle in Antikörper-Clearance und antigener Variation, welche gemeinsam dafür sorgen, dass der Parasit dem Wirtsimmunsystem stets einen Schritt voraus bleibt. Die Genexpression von T. brucei weist dezidierte Unterschiede im Vergleich zu anderen Eukaryoten auf: Die meisten Gene werden als lange polyzystronische Einheiten transkribiert, die durch trans-Splicing eines Miniexons aus 39 Nukleotiden am 5′ und Polyadenylierung am 3′ Ende der individuellen Gene prozessiert wird. Daher wird die Genexpression in T. brucei posttranskriptionell reguliert, zumeist durch RNA Bindeproteine (RBPs) und konservierte Elemente in der 3′ untranslatierten Region (UTR). Die Expression der VSGs ist stark reguliert, so wird zu einer gegebenen Zeit stets nur ein VSG Gen aus einer von ~15 Subtelomerregionen, die Blutstrom Expressionsorte (bloodstream expression sites, BES) genannt werden, exprimiert. Zellen, die gentechnisch manipuliert wurden um zwei VSGs zu exprimieren, produzieren die gleiche Menge an VSG mRNA wie Wildtyp Zellen. Dies deutet auf die Existenz eines robusten Mechanismus zur Regulierung der Gesamt-VSG mRNA Menge in T. brucei hin. Diese Arbeit verwendet induzierbare sowie konstitutive Expression eines ektopischen VSG Gens um zu zeigen, dass die endogene VSG mRNA nur reguliert wird, wenn das zweite VSG zum ER gelangt. Außerdem wird die endogene VSG mRNA Antwort auch ausgelöst, wenn hohe Mengen eines GFP Reporters, der eine VSG 3′UTR enthält, zum ER geleitet wird. Weiterhin, wird gezeigt, dass ER Importsignale anderer Proteine VSGs effizient zum ER dirigieren können. Das Ergebnis dieser Studie deutet darauf hin, dass eine Rückkopplungsschleife am ER eine robuste trans-Regulation der VSG mRNA auslöst, die die VSG Transkripte limitiert und somit eine Überlastung des sekretorischen Wegs verhindert. Weiterhin konnte gezeigt werden, dass es nach Induktion der Expression des T. vivax VSGs ILDat1.2 in T. brucei zu einem doppelten Zellzyklusarrest mit gleichzeitiger Hochregulation der Expression des protein associated with differentation (PAD1) kam und dass dieses T. vivax VSG nur nach Austausch des GPI Signalpeptids durch das eines T. brucei VSGs effizient exprimiert werden konnte. Zusammengenommen suggerieren diese Daten, dass eine ineffiziente GPI-Verankerung und wenig abundante Expression des VSGs die Differenzierung der sogenannten slender BSF zur sogenannten stumpy Form einleiten kann. Ein zweites T. vivax VSG, ILDat2.1, konnte hingegen auch nach Austausch des GPI Signals nicht in T. brucei exprimiert werden. Mit Hilfe der Röntgenstrukturanalyse wurde die Struktur der N-terminalen Domäne (NTD) des ILDat1.2 VSGs gelöst. Es handelt sich hierbei um die erste Proteinstruktur eines VSGs, welches nicht aus T. brucei stammt und die erste Struktur eines Oberflächenproteins von T. vivax. Das in T. brucei Oberflächenproteinen konservierte drei-Helix Grundgerüst ist auch in der NTD des ILDat1.2 VSGs enthalten. Die Architektur der Membranproximalen Gegend der IlDat1.2 NTD und CTD unterscheiden sich aber vermutlich von der der T. brucei VSGs. Das leicht handhabbare T. brucei System bietet somit ein geeignetes Modell um die Oberflächenproteine anderer afrikanischer Trypanosomen Spezies zu untersuchen und eröffnet neue Wege zur Charakterisierung ihrer Oberflächenmäntel.

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No description available.

Trypanosoma brucei undergoes developmentally regulated morphological and biochemical changes during its life cycle, being transmitted between the mammalian host and the tsetse fly. It is generally recognized that cellular responses to environmental changes are mediated through signalling pathways, but our understanding of trypanosome signal transduction during differentiation is limited. Protein Tyrosine Phosphatase 1 (TbPTP1) is the one of the few factors identified to be responsible for differentiation from stumpy to procyclic form parasite, whereby TbPTP1 inhibition stimulates transition to insect-form cells (Szoor et al., 2006). In order to characterize the TbPTP1 signalling pathway, a substratetrapping approach was used, which identified a phosphatase TbPIP39 as substrate of TbPTP1. TbPIP39 interacts with, and is dephosphorylated by TbPTP1 in stumpy form cells. Additionally, it has been shown that upon citrate/cis-aconitate (CCA) treatment, phosphorylated TbPIP39 localizes to the parasite glycosomes, the organelles responsible for bloodstream forms metabolism, thereby promoting cellular differentiation to procyclic forms (Szoor et al., 2010). With the aim of further dissecting the TbPTP1 signalling pathway, the substrate-trapping approach was used, which identified one novel TbPTP1 substrate candidate, potentially involved in regulation of differentiation. In addition, the effect of other differentiation triggers, namely protease treatment or mild acid exposure, on the level of TbPIP39 phosphorylation was analyzed, to determine whether these stimuli operate via the same TbPIP39–dependent pathway as CCA signalling. Specifically, changes in the phosphorylation status of TbPIP39 were visualized and quantitated by the use of antibodies detecting either TbPIP39 or the Y278 phosphorylated form of TbPIP39 generated during CCA-dependent differentiation. Both protease treatment and mild acid exposure generated a different pattern of TbPIP39 phosphorylation, thus suggesting a different mechanism of action than CCA. Finally, the possibility of using piggyback strategies targeting TbPTP1 was investigated, as a means to decrease the number of the fly-transmissible stumpy form cells in the bloodstream, thereby controlling parasite transmission. For this purpose, natural and synthetic inhibitors of human PTP1B were tested against the parasite enzyme, since they are being developed by pharmaceutical companies for the treatment of diabetes and obesity. The compounds tested showed a moderate in vitro inhibitory activity against recombinant TbPTP1 and mainly a non-competitive type of inhibition, similarly to that observed for human PTP1B. However, none of the compounds showed in vivo specificity for TbPTP1, indicating that further studies will be needed to identify more specific inhibitors.

No description available.

The purpose of this study was to examine the ultrastructure of both young (dividing form) and adult (stationary form) of Trypanosoma lewisi. Changes observed between the two forms of the organism were related directly to data obtained from previous comparative biochemical studies conducted on these forms of Trypanosoma lewisi.

Memoria para la obtención del Título de Químico Farmacéutico / La enfermedad de Chagas es una patología endémica en América Latina y sin un tratamiento eficaz, causada por el protozoo hemoflagelado Trypanosoma cruzi (T. cruzi). Es un problema mayor de salud identificándose actualmente entre 8 a 9 millones de infectados, de los cuales aproximadamente un 30% desarrolla sintomatología. Sin embargo, la infección se ha globalizado durante los últimos años, confirmándose sobre 300.000 sero-positivos en Estados Unidos y varias decenas de miles en Europa, Asia y Oceanía. Esto se debe a que, aunque T. cruzi es normalmente transmitido por insectos triatominios infectados, se ha descrito la transmisión por transfusión sanguínea, trasplante de órganos, infección congénita y consumo de alimentos contaminados con el parásito. En el ciclo de vida de T. cruzi se pueden identificar 3 etapas principales: amastigote y epimastigote, que cumplen funciones replicativas en el hospedero mamífero y en el insecto vector respectivamente, y la forma tripomastigote, encargada de infectar células eucariontes. Calreticulina de T. cruzi (TcCRT), es una proteína de 45 kDa, aislada, clonada y caracterizada en el Laboratorio de Inmunología de la Agresión Microbiana (LIAM), de la Facultad de Medicina de nuestra Universidad. Se ha demostrado que TcCRT es translocada a la zona de emergencia flagelar de los tripomastigotes, donde captura C1, primer componente de la vía clásica del sistema del complemento. En el humano, esta señal es característica de células apoptóticas. Es por esto que al capturar C1, se reclutan macrófagos para fagocitar a los tripomastigotes. Una vez dentro de estas células, el parásito escapa de la vacuola parasitófora, y comienza a diferenciarse, iniciando el proceso de infección del huésped. Junto a esto, la interacción entre TcCRT y C1 impide la activación del sistema del complemento del hospedero, disminuyendo la lisis del parásito por este medio. Resultados obtenidos previamente en LIAM muestran que TcCRT se expresa marginalmente en la membrana plasmática de la forma epimastigote del clon Dm28c. En base a esto, se propuso como Hipótesis de Trabajo de esta Memoria de Título, que, al incubar epimastigotes con rTcCRT, se promueve su penetración a las células hospederas mamíferas. Los principales resultados obtenidos señalan que: i) epimastigotes unen rTcCRT en su membrana, posiblemente mediante alguna proteína que sirva de anclaje, ii) el tratamiento con rTcCRT en presencia o ausencia de C1q conlleva a un aumento en la penetración del parásito en fibroblastos que expresan CRT, iii) la ausencia de CRT en la membrana de la célula hospedera lleva a una disminución en la penetración de los parásitos. En el contexto del epimastigote, puede que la falta de translocación de TcCRT sea el factor principal para que esta forma presente menor resistencia a lisis por el complemento y falle en desarrollar el ciclo infectivo en el mamífero / Chagas’ disease is an endemic pathology in Latin America, caused by the hemoflagelated protozoan Trypanosoma cruzi (T. cruzi), and still with no available effective treatment. It has become a major health problem, with 8 - 9 million seropositive people, with 30% of them developing symptoms. In the last few years, the infection has globalized, with 300.000 people infected just in the USA, and tens of thousands in Europe, Asia and Oceania. Although T. cruzi is usually transmitted through the bite of Triatominae insects, blood transfusion, organ transplants or congenital infection have also been described. In the parasites life cycle, 3 principal different stages are identified: Replicative amastigotes and epimastigotes, present in the mammal host and Triatominae vector respectively, and trypomastigotes, which infect eukaryotic cells. T. cruzi Calreticulin (TcCRT) is a 45 kDa protein, isolated, cloned and characterized in the Immunology of Microbial Aggression Laboratory (LIAM), Faculty of Medicine, University of Chile. Later, it was demonstrated that TcCRT is translocated from the ER to the flagella emergence zone where it captures C1, the first component of the classical pathway of the complement system. In humans, this interaction is characteristic of apoptotic cells. Once C1 is captured, macrophages are recruited and phagocytose the trypomastigotes. Once inside the cell, the parasites escape the parasitophorous vacuole and start dedifferentiation, leading to the amastigote stages, which will finally differentiate into infective trypomastigotes. Upon TcCRT binding to C1, inhibition of the complement system activation occurs, with a decrease in parasite lysis. According to previous observations in our laboratory, TcCRT is expressed marginally on the plasmatic membrane of the studied epimastigote form. Taken all together, these facts allow us to propose the following Working Hypothesis: Incorporation of exogenously added rTcCRT onto epimastigotes will promote their penetration into mammal hosts cells. The main results obtained show that: i) epimastigotes bind rTcCRT to their cellular membrane, by a still unidentified membrane protein that works as an anchor, ii) rTcCRT-treated epimastigotes, in the presence or absence of C1q, promote an increase in parasite penetration into CRT expressing fibroblasts and iii) the absence of CRT in the host cell correlates with a decrease in parasite penetration. In the epimastigote context, it could be proposed that the lack of TcCRT translocation is an important reason why this form is more easily destroyed by the complement system, and for their incapacity to infect host cells / Fondecyt