Complement Interaction with Trypanosomatid Promastigotes in Normal Human Serum

In normal human serum (NHS), axenic promastigotes of Crithidia, Phytomonas, and Leishmania trigger complement activation, and from 1.2 to 1.8 × 105 C3 molecules are deposited per promastigote within 2.5 min. In Leishmania, promastigote C3 binding capacity remains constant during in vitro metacyclogenesis. C3 deposition on promastigotes activated through the classical complement pathway reaches a 50% maximum after ∼50 s, and represents >85% of total C3 bound. In C1q- and C2-deficient human sera, promastigotes cannot activate the classical pathway (CP) unless purified C1q or C2 factors, respectively, are supplemented, demonstrating a requirement for CP factor in promastigote C3 opsonization. NHS depleted of natural anti-Leishmania antibodies cannot trigger promastigote CP activation, but IgM addition restores C3 binding. Furthermore, Leishmania binds natural antibodies in ethylenediaminetetracetic acid (EDTA)-treated NHS; after EDTA removal, promastigote-bound IgM triggers C3 deposition in natural antibody-depleted NHS. Serum collectins and pentraxins thus do not participate significantly in NHS promastigote C3 opsonization. Real-time kinetic analysis of promastigote CP-mediated lysis indicates that between 85–95% of parasites are killed within 2.5 min of serum contact. These data indicate that successful Leishmania infection in man must immediately follow promastigote transmission, and that Leishmania evasion strategies are shaped by the selective pressure exerted by complement

Nuevos datos sobre la estructura general del Carbonífero en el Horst de La Camocha (Gijón, Asturias).

Una reciente campaña de investigación geológico-minera, mediante la realización de transversales de reconocimiento, sondeos de superficie e interior y perfiles sísmicos de reflexión, ha permitido profundizar en el conocimiento de la estructura del Carbonífero en el entorno de Mina La Camocha. En esta zona, al sur de Gijón, los materiales carboníferos, cubiertos por una cobertera permotriásica y jurásica, no afloran. Se precisa el estilo de plegamiento, analizándose y datándose las principales familias de fracturación. El estilo tectónico de los materiales carboníferos productivos no aflorantes, se ilustra mediante varios de los nuevos cortes obtenidos

Caracterización de los parámetros hidráulicos de la cobertera pérmica en el Horst del yacimiento carbonífero de Mina «La Camocha».

En la cobertera pérmica existente sobre el yacimiento carbonífero actualmente en explotación por Mina La Camocha, S. A., se localiza un acuífero multicapa con varios niveles claramente diferenciados. Por medio de diversas técnicas de estudio, se ha tratado de determinar los parámetros hidráulicos de estos materiales en varias localidades próximas a Gijón, con el fin de elaborar un esquema de funcionamiento hidráulico y estimar la posible influencia de este acuífero en las labores de explotación

Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum

<p>Abstract</p> <p>Background</p> <p>The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite <it>Leishmania</it>. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom <it>L. infantum </it>genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome.</p> <p>Results</p> <p>Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings.</p> <p>Conclusions</p> <p>The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into <it>Leishmania </it>physiology.</p

Evidence of Leishmania infantum infection in rabbits (Oryctolagus cuniculus) in a natural area in Madrid, Spain

Leishmaniasis is one of most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world-wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified. Recently, hares have been pointed out as competent reservoirs of Leishmania infantum in Spain, but the role of other lagomorphs has not been clarified. Here, 69 rabbits (Oryctolagus cuniculus) from a natural area in Madrid in which a high density was present were analyzed using indirect (immunofluorescence antibody test, IFAT) and direct (PCR, culture) techniques. Fifty-seven (82.6%) of the animals were positive to at least one technique, with IFAT yielding the highest proportion of positive samples. L. infantum was isolated in 13% animals demonstrating the occurrence of infection in this setting. Our results suggest that rabbits could play a role of competent reservoir of L. infantum and demonstrate that the prevalence of infection is high in the analyzed area

Immune adherence-mediated opsonophagocytosis: the mechanism of Leishmania infection

To mimic the sandfly pool feeding process and characterize the cellular and biochemical events that occur during the early stages of promastigote-host interaction, we developed an ex vivo model of human blood infection with Leishmania promastigotes. Within 30 s of blood contact, Leishmania promastigotes bind natural anti-Leishmania antibodies, which then activate the classical complement pathway and opsonization by the third component of complement. The opsonized promastigotes undergo an immune adherence reaction and bind quantitatively to erythrocyte CR1 receptors; opsonized Leishmania amastigotes also bind to erythrocytes. Progression of infection implies promastigote transfer from erythrocytes to acceptor blood leukocytes. After 10 min of ex vivo infection, 25% of all leukocytes contain intracellular parasites, indicating that blood cells are the early targets for the invading promastigotes. We propose that adaptation to the immune adherence mechanism aids Leishmania survival, promoting rapid promastigote phagocytosis by leukocytes. This facilitates host colonization and may represent the parasite's earliest survival strategy. In light of this mechanism, it is unlikely that infection-blocking vaccines can be developed.This work was supported by grant FIS 94/215 from the Fondo de Investigaciones Sanitarias (Spain)

Kinetic analysis of ex vivo human blood infection by Leishmania

The leishmanioses, vector-borne diseases caused by the trypanosomatid protozoan Leishmania, are transmitted to susceptible mammals by infected phlebotomine sand flies that inoculate promastigotes into hemorrhagic pools created in host skin. We assumed that promastigotes are delivered to a blood pool, and analyzed early promastigote interactions (0-5 min) with host components, which lead to parasite endocytosis by blood leukocytes, and to host infection. Promastigotes were incubated with NHS or with heparinized blood in near-physiological conditions, and we used cell radioimmunoassay and flow cytometry to measure the on-rate constants (k(+1)) of promastigote interactions with natural opsonins and erythrocytes. We obtained quantitative data for parasitized cells to determine the time-course of promastigote binding and internalization by blood leukocytes. In these reactions, promastigotes bind natural opsonins, immune adhere to erythrocytes and activate complement cytolysis, which kills approximately 95% of promastigotes by 2 min post-infection. C3-promastigote binding is a key step in opsonization; nascent C3-promastigotes are the substrate for two simultaneous reactions, C3-promastigote immune adherence (IA) to erythrocytes and complement-mediated promastigote killing. The k(+1) for IA was 75-fold greater than that for promastigote killing, showing that IA facilitates promastigote endocytosis and circumvents lysis. At 5 min post-infection, when reaction velocity is still linear and promastigote concentration is not limiting, 17.4% of granulocytes and 10.7% of monocytes had bound promastigotes, of which approximately 50% and approximately 25%, respectively, carried surface-bound (live) or internalized (live and dead) leishmanias. Of other leukocyte types, 8.5% of B cells bound but did not internalize promastigotes, and T cells, NK cells and CD209(+) dendritic cells did not bind parasites. These data show that, once in contact with blood, promastigote invasion of human leukocytes is an extremely rapid and efficient reaction, and suggest that the IA reaction constitutes a central strategy for this parasite in subverting host innate immune defenses.This work was supported by grants from the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Ministerio de Educación y Ciencia) SAF2004-03094, the Instituto de Salud Carlos III (MPY 1198/02), the Comunidad de Madrid (P2009/AGR-1489) and the Fondo de Investigación Sanitaria (Ministerio de Sanidad y Consumo; PI040814). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Acquiring values of Fricis Barda's literary work in form 10.

Diplomdarba „Friča Bārdas dzejas vērtīborientējoša apguve 10. klasē” mērķis ir izstrādāt metodiskās sistēmas variantu, kas rosinātu 10. klases skolēnus atklāt nozīmīgas vērtības Friča Bārdas daiļradē un kas pilnīgotu jauniešu vērtību sistēmu. Darbam ir piecas nodaļas. 1.Vērtīborientējošas sadarbības nozīmīgums literatūras mācību procesā. 2.Friča Bārdas daiļradē apliecinātās vērtības. 3.Skolotāja sagatavošanās darbam klasē. 4.Friča Bārdas daiļradei veltīto stundu norise vērtīborientējošā aspektā 10. klasei. 5.Skolotāja un skolēnu sadarbības rezultātu analīze un izvērtējums. Pirmajā nodaļā teorētiski pamatoti autores uzskati par vērtīborientējošas sadarbības nozīmīgumu literatūras mācību procesā. Otrā nodaļa veltīta Friča Bārdas personības un daiļrades izpētei vērtīborientējošā aspektā. Trešā nodaļa veltīta skolēnu izpētei un mācību materiālu sagatavošanai. Ceturtajā nodaļā atklāta F. Bārdas daiļrades vērtīborientējošu apguves stundu organizācija. Piektajā nodaļā autore veikusi skolotāja un skolēnu sadarbības rezultātu analīzi un izvērtējumu. Diplomdarba autore, veidojot mācību materiālus, galvenokārt balstījusies uz vērtīborientējošu literatūras mācību procesu. Mācību materiālu efektivitāte pārbaudīta pedagoģiskā izmēģinājuma laikā. Darbs iesakāms gan studentiem, gan vidusskolas latviešu valodas un literatūras skolotājiem.The aim of the Diploma paper „Asquiring values of Fricis Barda’s literary work in From 10” is to work out a version of methodical system, which would arouse the pupils of form 10 to reveal significant values in Fricis Barda’s works and which would improve young people’s values’ perception. The Diploma paper consists of 5 chapters. 1.The significance of value – oriented coorporation in the learning process of literature. 2.The values assumed in Fricis Barda’s works. 3.The teacher’s preparation for the class work. 4.The process of the lesson’s devoted to Fricis Barda’s works in values – oriented aspect for form 10. 5.The evaluation and analysis of the results of the teacher and pupils’ coorporation. In the first chapter the author theoretically basen opinions in significance of values – oriented coorporation in the learning process of literature. The second chapter deals with the study of Fricis Barda’s personality and works in values – oriented aspect. The third chapter deals with pupil’s study and the preparation of teaching materials. In the fourth chapter the author reveals lesson organization of values – oriented acquisition in Fricis Barda’s work. In the fifth chapter the author has carried out the evaluation and the analysis of results of the teacher and pupils’ corporation. The author admits that nowadays the educational values – oriented leading idea is productive and its availability in teaching materials allows to check its effectiveness practically. The author of the Diploma paper has based herself on values – oriented learning process of literature. The Diploma paper is recommended both students, and Latvian language and literature teachers in secondary school