CARACTERIZAÇÃO CELULAR DA HEMOLINFA DE FÊMEAS INGURGITADAS DE Amblyomma sculptum

O carrapato Ambyomma sculptum, encontrado as várias regiões do Brasil, é conhecido como “carrapato estrela” ou “carrapato do cavalo” e parasita animais domésticos, silvestres e humanos, sendo o principal transmissor de Rickettsia rickettsii, agente etiológico da Febre Maculosa Brasileira. Possui grande importância veterinária por causar diversos prejuízos aos seus hospedeiros. Estudos sobre sistema imunológico de carrapatos buscam avaliar alterações na resposta celular hemolinfática que faça entender a imunologia e fisiologia envolvida nos mecanismos de controle e resistência. O objetivo deste trabalho foi caracterizar quantitativamente e morfologicamente os hemócitos da hemolinfa de fêmeas ingurgitadas de A. sculptum. A hemolinfa foi coletada por uma perfuração da cutícula na parte dorsal do carrapato e depositada em lâmina de vidro para confecção dos esfregaços que foram corados por solução de Giemsa. Foi realizada a contagem diferencial de hemócitos com base na morfologia observada em microscópio óptico em aumento de 1000 vezes, através da identificação das 100 primeiras células encontradas. A avaliação dos tamanhos de cada célula e de seus componentes foi realizada por leitura das lâminas confeccionadas para contagem diferencial dos hemócitos com o auxílio de um microscópio óptico com luz polarizada. A contagem total de hemócitos foi realizada com auxílio de microscópio óptico e Câmara de Neubauer. A caracterização celular revelou que o carrapato A. sculptum apresenta média de 1024 céls/μL e seis tipos celulares que se distribuem de forma distinta na hemolinfa. Os granulócitos foram os hemócitos mais frequentes (78%), seguidos de plasmatócitos (10%), prohemócitos (6%), esferulócitos (5%), oenocitóide (1%) e adipohemócito com menos de 1% de frequência. De acordo com as características morfológicas os granulócitos foram descritos com tamanho médio de 22x19 μm, sendo células de tamanhos variados e de formas arredondadas a elípticas, com citoplasma coberto de grânulos. Os plasmatócitos se apresentaram polimórficos com citoplasma vacuolizado ou granular e foi o grupo com maior variação de forma e tamanho com medidas médias de 24x10 μm. Os prohemócitos foram o menor tipo celular observado medindo em média 13x11 μm, se apresentando em formato arredondado, com núcleo central e escasso citoplasma. Os esferulócitos caracterizaram-se como células arredondadas (tamanho médio de 22x20μm) com estruturas presentes de forma abundante no citoplasma, as esférulas. Os oenocitóides apresentaram tamanho intermediário (média: 23x19μm), com forma arredondada, margens irregulares e citoplasma com granulações refráteis e aspecto esponjoso. Os adipohemócitos, raramente encontrados, são células de tamanho variável, margens citoplasmáticas irregulares, vesículas refringentes no citoplasma e a quantidade observada não foi suficiente para realização da conferência das medidas com segurança estatística. Conclui-se que a partir da microscopia óptica foi possível determinar o valor médio do total de hemócitos circulantes, classificados morfologicamente como granulócitos, plasmatócitos, prohemócitos, esferulócitos, oenocitóides e adipohemócitos, e determinar a frequência média de cada hemócito na hemolinfa de A. sculptum, onde os granulócitos são o tipo mais frequentes

Urochordate Histoincompatible Interactions Activate Vertebrate-Like Coagulation System Components

The colonial ascidian Botryllus schlosseri expresses a unique allorecognition system. When two histoincompatible Botryllus colonies come into direct contact, they develop an inflammatory-like rejection response. A surprising high number of vertebrates' coagulation genes and coagulation-related domains were disclosed in a cDNA library of differentially expressed sequence tags (ESTs), prepared for this allorejection process. Serine proteases, especially from the trypsin family, were highly represented among Botryllus library ortholgues and its “molecular function” gene ontology analysis. These, together with the built-up clot-like lesions in the interaction area, led us to further test whether a vertebrate-like clotting system participates in Botryllus innate immunity. Three morphologically distinct clot types (points of rejection; POR) were followed. We demonstrated the specific expression of nine coagulation orthologue transcripts in Botryllus rejection processes and effects of the anti-coagulant heparin on POR formation and heartbeats. In situ hybridization of fibrinogen and von Willebrand factor orthologues elucidated enhanced expression patterns specific to histoincompatible reactions as well as common expressions not augmented by innate immunity. Immunohistochemistry for fibrinogen revealed, in naïve and immune challenged colonies alike, specific antibody binding to a small population of Botryllus compartment cells. Altogether, molecular, physiological and morphological outcomes suggest the involvement of vertebrates-like coagulation elements in urochordate immunity, not assigned with vasculature injury

Interleukin-13 protects from atherosclerosis and modulates plaque composition by inducing a regression phenotype

Arteriosklerotische Plaques sind durch die Ansammlung von oxidiertem LDL (OxLDL) sowie von Makrophagen und T-Zellen gekennzeichnet. Die Entwicklung der Läsionen wird zudem stark durch die lokale Expression von Zytokinen beeinflusst. Im Gegensatz zu den von T-Helfer (Th) 1 produzierten Zytokinen, wie zum Beispiel IFN-g, deren pro- atherogener Effekt als gesichert gilt, ist die Rolle der Th2-Zytokine nach wie vor umstritten. In dieser Dissertation wird berichtet, dass ein Mangel an dem Th2-Zytokin IL-13 die Entwicklung von Arteriosklerose beschleunigt, ohne den Cholesterinspiegel im Plasma zu beeinflussen. Die Verabreichung von IL-13 verursachte ein Ansteigen des Kollagengehalts in bereits etablierten Läsionen bei gleichzeitiger Reduktion der durch das vaskuläre Zelladhäsionsmolekül 1 (VCAM-1) hervorgerufenen Monozytenadhäsion. Letzteres führte einerseits zu einer geringeren Anzahl von Makrophagen in den Läsionen und andererseits zu einem vermehrten Auftreten von alternativ aktivierten (M2) Makrophagen, die OxLDL mit einer größeren Effizienz als IFN-g aktivierte (M1) Makrophagen unschädlich machen. IL-13 schützt daher vor Arteriosklerose und verursacht, teilweise durch die Induktion von M2 Makrophagen, eine Regression atherosklerotischer Läsionen.Atherosclerotic lesions are characterized by the accumulation of oxidized LDL (OxLDL) and the infiltration of macrophages and T-cells. Cytokine expression in the microenvironment of evolving lesions can profoundly contribute to plaque development. While the pro-atherogenic effect of T helper (Th) 1 cytokines, such as Interferon (IFN)-g is well established, the role of Th2 cytokines is less clear. For example, interleukin (IL)-5 has been shown to mediate atheroprotection, while IL-4 has been reported to have no or even pro-atherogenic properties. Therefore, we characterized the role of the Th2 cytokine IL-13 in murine atherosclerosis. Here we report that IL-13 administration favourably modulated the morphology of already established atherosclerotic lesions by increasing lesional collagen content and reducing vascular cell adhesion molecule-1 (VCAM-1)-dependent monocyte recruitment, resulting in decreased plaque macrophages. This was accompanied by the induction of alternatively activated (M2) macrophages, which exhibited increased clearance of OxLDL compared to IFN-g-activated (M1) macrophages in vitro. Importantly, deficiency of IL-13 results in accelerated atherosclerosis in LDLR-/- mice without affecting plasma cholesterol levels. Thus, IL-13 protects from atherosclerosis and promotes favourable plaque morphology, in part through the induction of alternatively activated macrophages.Larissa Cardilo dos Reis WeismannAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2012OeBB(VLID)171547

In vivo antithrombotic properties of a heparin from the oocyte test cells of the sea squirt Styela plicata(Chordata-Tunicata)

In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10% of mammalian heparin and about 5% as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing ~300 g, N = 4 in each group) at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 ± 13.5% (mean ± SD) without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 ± 1.4%, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 ± 13% without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding

Cytokines in Atherosclerosis

Cardiovascular disease, such as heart attacks and strokes, are still the world’s leading cause of morbidity and mortality worldwide. Atherosclerosis, an inflammatory disease of the medium and larger arteries in response to various risk factors such as hypertension and a build-up of modified low-density lipoproteins, is the underlying cause of cardiovascular disease. Cytokines are the key orchestrators of the chronic inflammatory response during atherosclerosis disease progression. There is therefore a need to develop a better understanding of the role of cytokines during disease progression in order to potentially discover novel drug targets. This chapter will focus on the role of pro- and anti-inflammatory cytokines during atherosclerosis plaque formation. Therapeutic avenues for targeting cytokine actions will also be discussed

Immunomodulation by splenectomy or by FTY720 protects the heart against ischemia reperfusion injury

The pathogenesis of myocardial ischemia-reperfusion injury (MI/R) involves an inflammatory response in the myocardium undergoing reperfusion. Modulation of this response by splenectomy constitutes an option to protect the heart from MI/R. To mimic the effect of splenectomy in a pharmacological approach, the sphingosine-1-phosphate agonist FTY720 was applied at the onset of reperfusion. In a closed chest model of MI/R, infarct size was assessed by triphenyltetrazolium chloride staining after 1h of ischemia and 24h of reperfusion, and by Masson trichrome staining 21days after reperfusion in splenectomised mice, mice post-conditioned with FTY720 IP (1mg/kg), and controls. In addition, hemodynamic parameters were recorded after 24h and 21days by catheterization. Infarct size, and immune cell invasion of phagocytic monocytes investigated by FACS after 24h of reperfusion were significantly reduced by both splenectomy, and FTY720 treatment. Evaluation after 21days of reperfusion revealed that FTY720 treated animals had an improved hemodynamic outcome compared to placebo treated as well as splenectomised animals. FTY720 treatment reduced cell injury as effectively as splenectomy by lowering the number of phagocytic monocytes invading the myocardium and ameliorated hemodynamic outcome within the first 21days