Intravital microscopy technique to study parasite dynamics in the labyrinth layer of the mouse placenta

Intravital imaging techniques are the best approach to investigate in situ cellular behavior under physiological conditions. Many techniques have emerged during these last few years for this purpose. We recently described an intravital imaging technique that allows for the observation of placenta physiological responses at the labyrinth layer of this tissue. This technique will be very useful to study many placental opportunistic infections and in this article we reinforce its usefulness by analyzing placental physiological entrapment of beads and parasites. In particular, our results show that small beads (1.0 μm) or Plasmodium chabaudi-GFP-infected-Red Blood Cells (Pc-GFP-iRBCs) cannot get trapped inside small or large blood vessels of popliteal lymph nodes (PLNs). Inside the placenta, clusters of beads could only be found inside the maternal blood vessels. However, Pc-GFP-iRBCs were found inside and outside the maternal blood vessels. We observed that trophoblasts can ingest infected-Red Blood Cells (iRBCs) in vitro and immunofluorescence of placenta revealed Pc-GFP-iRBCs inside and outside the maternal blood vessels. Taken together, we conclude that fast deposition of particles inside blood vessels seems to be an intrinsic characteristic of placenta blood flow, but iRBCs could be internalized by trophoblast cells. Thus these results represent one of the many possible uses of our intravital imaging technique to address important questions inside the parasitological field.

MyD88 signaling is directly involved in the development of murine placental Malaria

Malaria is a widespread infectious disease caused by the parasite Plasmodium. During pregnancy, malaria infection leads to a range of complications that can affect both the mother and fetus, including stillbirth, infant mortality, and low birth weight. In this study, we utilized a mouse model of placental malaria (PM) infection to determine the importance of the protein MyD88 in the host immune response to Plasmodium during pregnancy. Initially, we demonstrated that Plasmodium berghei NK65GFP adhered to placental tissue via chondroitin sulfate A and induced PM in mice with a C57BL/6 genetic background. To evaluate the involvement of MyD88 in the pathology of PM, we performed a histopathological analysis of placentas obtained from MyD88(-/-) and wild-type (WT) mice following infection on the 19th gestational day. Our data demonstrated that the detrimental placental alterations observed in the infected mice were correlated with the expression of MyD88. Moreover, in the absence of this protein, production of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) was significantly reduced in the infected mice. More importantly, in contrast to fetuses from infected WT mice, which exhibited a reduction in body weight, the fetuses from infected MyD88(-/-) mice did not display significant weight loss compared to their noninfected littermates. In addition, we observed a decrement of maternal care associated with malaria infection, which was attenuated in the MyD88-deficient mice. Collectively, the results of this study illustrate the pivotal importance of the MyD88 signaling pathway in the pathogenesis of placental malaria, thus presenting new possibilities for targeting MyD88 in therapeutic interventions.

Viagem sem volta. [Depoimento a Rodrigo de Oliveira Andrade]

Fêmeas de pinguins-de-magalhães morrem mais que machos durante migração anual.

Bioinformatische Identifizierung und Charakterisierung von Zyklin-abhängigen Kinasen und Zyklinen des Parasiten Eimeria tenella und deren Nutzung im rationalen Wirkstoffdesign

Parasiten der Apicomplexa umfassen sowohl humanpathogene, als auch tierpathogene Protozoen. Beispiele für wichtige Vertreter human- und tierpathogener Parasiten sind Plasmodium falciparum und Eimeria tenella. E. tenella verursacht die Kokzidiose des Hühnchens, eine Darmerkrankung die weltweit für Verluste in einer geschätzten Höhe von bis zu 3 Milliarden US$ verantwortlich zeichnet. Eine prophylaktische Vakzinierung gegen diese Krankheit ist ökonomisch meist ineffizient, und eine Behandlung mit Kokzidiostatika wird durch häufige Resistenzbildung gegen bekannte Wirkstoffe erschwert. Diese Situation erfordert die Entwicklung neuer kostengünstiger Alternativen. Geeignete Zielproteine für die Entwicklung neuartiger Arzneistoffe zur Behandlung der Kokzidiose sind die Zyklin-abhängigen Kinasen (CDKs), zu denen auch die CDK-related Kinase 2 (EtCRK2) aus E. tenella gehört. Diese Proteine sind maßgeblich an der Regulation des Zellzyklus beteiligt. Durch chemische Validierung mit dem CDK Inhibitor Flavopiridol konnte nachgewiesen werden, dass ein Funktionsverlust von CDKs in E. tenella die Vermehrung des Parasiten in Zellkultur inhibiert. E. tenella CDKs sind daher als Zielproteine für die Entwicklung einer Chemotherapie der Kokzidiose geeignet. Mittels bioinformatischer Tiefenanalysen sollten CDK Proteine im Parasiten E. tenella identifiziert werden. Das Genom von E. tenella liegt in Rohfassung vor [ftp://ftp.sanger.ac.uk]. Jedoch waren zum Zeitpunkt dieser Arbeiten viele Sequenzen des Genoms noch nicht annotiert. Homologe CDK Proteine von E. tenella konnten durch den Vergleich von Sequenzinformationen mit anderen Organismen der Apicomplexa identifiziert und analysiert werden. Durch diese Analysen konnten neben der bereits bekannten EtCRK2, drei weitere, bislang nicht annotierte CDKs in E. tenella identifiziert werden (EtCRK1, EtCRK3 sowie EtMRK). Darüber hinaus wurde eine Analyse der entsprechenden Zykline – der Aktivatoren der CDKs – bezüglich Funktion und Struktur, sowie eine Datenbanksuche nach bisher nicht beschriebenen Zyklinen in E. tenella durchgeführt. Diese Suchen ergaben vier neue potentielle Zykline für E. tenella, wovon EtCYC3a als Aktivator der EtCRK2 von María L. Suárez Fernández (Intervet Innovation GmbH, Schwabenheim) bestätigt werden konnte. Sequenzvergleiche lassen vermuten, dass auch EtCYC1 und EtCYC3b in der Lage sind, EtCRK2 zu aktivieren. Außerdem ist anzunehmen, dass EtCYC4 als Aktivator der EtCRK1 fungiert. Ein weiterer Schwerpunkt der vorliegenden Arbeit war die Suche und Optimierung nach neuen Inhibitoren von CDKs aus E. tenella. In vorangegangenen Arbeiten konnten bereits Inhibitoren der EtCRK2 gefunden werden [BEYER, 2007]. Mittels Substruktur- und Ähnlichkeitssuchen konnten im Rahmen dieser Arbeit weitere Inhibitoren der EtCRK2 identifiziert werden. Vier dieser Strukturklassen erfüllen die Kriterien einer Leitstruktur. Eine dieser Leitstrukturen gehört zur Strukturklasse der Benzimidazol-Carbonitrile und ist bislang nicht als Inhibitor anderer Kinasen beschrieben. Diese neu identifizierte Leitstruktur konnte in silico weiter optimiert werden. Im Rahmen dieser Arbeit wurden Bindungsenergien von Vertretern dieser Strukturklasse berechnet, um einen wahrscheinlichen Bindemodus vorherzusagen. Für die weiterführende in silico Optimierung wurde eine virtuelle kombinatorische Substanzbibliothek dieser Klasse erstellt. Die Auswahl geeigneter Verbindungen für eine chemische Synthese erfolgte durch molekulares Docking unter Nutzung von Homologiemodellen der EtCRK2. Darüber hinaus wurde ein in silico Screening nach potentiellen Inhibitoren der PfMRK und EtMRK durchgeführt. Dabei konnten weitere interessante virtuelle Hit-Strukturen aus einer Substanzdatenbank kommerziell erhältlicher Verbindungen gefunden werden. Durch dieses virtuelle Screening konnten jeweils sieben Verbindungen als virtuelle Hits der PfMRK sowie der EtMRK identifiziert werden. Die Häufung von Strukturklassen mit bekannter CDK Aktivität deutet darauf hin, dass während des virtuellen Screenings eine Anreicherung von CDK Inhibitoren stattgefunden hat. Diese Ergebnisse lassen auf eine Weiterentwicklung neuer Wirkstoffe gegen Kokzidiose und Malaria hoffen.

Biochemische Charakterisierung der Zyklin-abhängigen Kinase 2,Aktivator-Komplexe des Parasiten Eimeria tenella sowie deren Nutzung in der Wirkstoffforschung

Der Stamm der Apicomplexa ist eine artenreiche Gruppe, der einzellige, meist obligat intrazelluläre Parasiten angehören, darunter auch erstzunehmende Krankheitserreger wie Plasmodium sp. sowie tierpathogene Vertreter wie Eimeria sp. und Theileria sp. Eimeria sp. verursacht die Kokzidiose beim Huhn. Diese Krankheit bedingt weltweite Verluste in der Geflügelindustrie von etwa 3 Milliarden US$ pro Jahr [DALLOUL & LILLEHOJ, 2006; SHIRLEY et al., 2007; LUCIUS & LOOS-FRANK, 2008]. Die Parasiten weisen eine hohe Resistenzbildungsrate gegen vorhandene Wirkstoffe auf. Zudem ist der Einsatz von Vakzinen mit Nebenwirkungen verbunden und für hohe Produktionskosten verantwortlich. Daher ist die Entwicklung von neuen, kostengünstigen und effektiven Kokzidiostatika eine dringend notwendige Herausforderung [KINNAIRD et al., 2004]. rnAuf Grund ihrer essentiellen, regulatorischen Funktion im eukaryotischen Zellzyklus sind Zyklin-abhängige Kinasen (CDKs) validierte Zielproteine [LEHNINGER et al., 2005]. Auch Eimeria tenella CDC2-related kinase 2 (EtCRK2) wurde bereits mittels des bekannten CDK-Inhibitors Flavopiridol als Zielprotein chemisch validiert [ENGELS et al., 2010]. Wie bei allen CDKs ist die Aktivität von EtCRK2 abhängig von der Bindung eines Aktivators, der zur Zyklin-Proteinfamilie gehört. Dieser natürliche EtCRK2-Aktivator war jedoch bislang nicht bekannt. Deshalb war ein Teil dieser Arbeit die Identifizierung des natürlichen EtCRK2-Aktivators. Bioinformatische Analysen identifizierten vier E. tenella Zyklin-ähnliche Proteine (EtCYC1, EtCYC3a, EtCYC3b und EtCYC4), die nah verwandt zu den Plasmodium falciparum-Zyklinen sind [ENGELS et al., 2010; SUÁREZ FERNÁNDEZ et al., bislang unveröffentlichte Daten]. Im Rahmen dieser Arbeit konnten zwei neue Aktivatoren identifiziert und biochemisch charakterisiert werden: der bekannte CDK-Aktivator XlRINGO und das neue E. tenella-Zyklin EtCYC3a. Nachdem der nicht-radioaktive TR-FRET-Assay für die EtCRK2 etabliert und optimiert wurde, konnte die EtCRK2-Aktivität im Komplex mit beiden Aktivatoren und weitere wichtige kinetische Parameter bestimmt werden.rnZusätzlich wurde dieser Assay zum in vitro Screening einer kommerziellen Chemikalienbibliothek auf die EtCRK2 eingesetzt, um potentielle Inhibitoren für EtCRK2 zu identifizieren. Dieses in vitro Screening gefolgt von einer in silico Hit-Anreicherung identifizierte 19 aktive Verbindungen für die durch EtCYC3a und XlRINGO aktivierte EtCRK2. Zudem wurden drei Struktur-Cluster definiert: Naphthoquinone, 8-Hydroxyquinoline und 2-Pyrimidinyl-aminopiperidin-propan-2-ole. rnDie aktivsten Vertreter von jedem Cluster wurden als Leitstrukturen ausgewählt und auf EtCRK2 und HsCDK2 getestet. Aufgrund ihrer inhibierenden Wirkung auf EtCRK2 stellen diese Verbindungen viel versprechende Leitstrukturen für die Entwicklung eines neuen Antikokzidiums dar. Hiermit konnte auch gezeigt werden, dass BES124764, der Vertreter des 2-Pyrimidinyl-aminopiperidin-propan-2-ol-Clusters, in der Lage ist, die EtCRK2 selektiv zu inhibieren. rnDaher wird BES124764 sowie einige Derivate in den Leitstruktur-Optimierungsprozess für die Auffindung eines neuen Arzneimittelkandidaten gegen Kokzidiose eingehen.rn

Structure-activity relationship and mechanism of action studies of manzamine analogues for the control of neuroinflammation and cerebral infections

Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3beta (GSK-3beta), which is a putative target of manzamines. On the basis of the results presented here, it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.

A research agenda for malaria eradication: basic science and enabling technologies

Today's malaria control efforts are limited by our incomplete understanding of the biology of Plasmodium and of the complex relationships between human populations and the multiple species of mosquito and parasite. Research priorities include the development of in vitro culture systems for the complete life cycle of P. falciparum and P. vivax and the development of an appropriate liver culture system to study hepatic stages. In addition, genetic technologies for the manipulation of Plasmodium need to be improved, the entire parasite metabolome needs to be characterized to identify new druggable targets, and improved information systems for monitoring the changes in epidemiology, pathology, and host-parasite-vector interactions as a result of intensified control need to be established to bridge the gap between bench, preclinical, clinical, and population-based sciences.

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.

Frequency of Malaria and Glucose-6-phosphate Dehydrogenase Deficiency in Tajikistan

Background During the Soviet era, malaria was close to eradication in Tajikistan. Since the early 1990s, the disease has been on the rise and has become endemic in large areas of southern and western Tajikistan. The standard national treatment for Plasmodium vivax is based on primaquine. This entails the risk of severe haemolysis for patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Seasonal and geographical distribution patterns as well as G6PD deficiency frequency were analysed with a view to improve understanding of the current malaria situation in Tajikistan. Methods Spatial and seasonal distribution was analysed, applying a risk model that included key environmental factors such as temperature and the availability of mosquito breeding sites. The frequency of G6PD deficiency was studied at the health service level, including a cross-sectional sample of 382 adult men. Results Analysis revealed high rates of malaria transmission in most districts of the southern province of Khatlon, as well as in some zones in the northern province of Sughd. Three categories of risk areas were identified: (i) zones at relatively high malaria risk with high current incidence rates, where malaria control and prevention measures should be taken at all stages of the transmission cycle; (ii) zones at relatively high malaria risk with low current incidence rates, where malaria prevention measures are recommended; and (iii) zones at intermediate or low malaria risk with low current incidence rates where no particular measures appear necessary. The average prevalence of G6PD deficiency was 2.1% with apparent differences between ethnic groups and geographical regions. Conclusion The study clearly indicates that malaria is a serious health issue in specific regions of Tajikistan. Transmission is mainly determined by temperature. Consequently, locations at lower altitude are more malaria-prone. G6PD deficiency frequency is too moderate to require fundamental changes in standard national treatment of cases of P. vivax.

Argemone mexicana decoction for the treatment of uncomplicated falciparum malaria

A prospective, dose-escalating, quasi-experimental clinical trial was conducted with a traditional healer using a decoction of Argemone mexicana for the treatment of malaria in Mali. The remedy was prescribed in three regimens: once daily for 3 days (Group A; n=23); twice daily for 7 days (Group B; n=40); and four times daily for the first 4 days followed by twice daily for 3 days (Group C; n=17). Thus, 80 patients were included, of whom 80% were aged<5 years and 25% were aged<1 year. All presented to the traditional healer with symptoms of malaria and had a Plasmodium falciparum parasitaemia>2000/microl but no signs of severe malaria. The proportions of adequate clinical response (ACR) at Day 14 were 35%, 73% and 65% in Groups A, B and C, respectively (P=0.011). At Day 14, overall proportions of ACR were lower in children aged<1 year (45%) and higher in patients aged>5 years (81%) (P=0.027). Very few patients had complete parasite clearance, but at Day 14, 67% of patients with ACR had a parasitaemia<2000/microl. No patient needed referral for severe disease. Only minor side effects were observed. Further research should determine whether this local resource could represent a first-aid home treatment in remote areas.

Imported malaria in children in industrialized countries, 1992-2002

Children account for an appreciable proportion of total imported malaria cases, yet few studies have quantified these cases, identified trends, or suggested evidence-based prevention strategies for this group of travelers. We therefore sought to identify numbers of cases and deaths, Plasmodium species, place of malaria acquisition, preventive measures used, and national origin of malaria in children. We analyzed retrospective data from Australia, Denmark, France, Germany, Italy, Japan, the Netherlands, Sweden, Switzerland, the United Kingdom, and the United States and data provided by the United Nations World Tourism Organization. During 1992-2002, >17,000 cases of imported malaria in children were reported in 11 countries where malaria is not endemic; most (>70%) had been acquired in Africa. Returning to country of origin to visit friends and relatives was a risk factor. Malaria prevention for children should be a responsibility of healthcare providers and should be subsidized for low-income travelers to high-risk areas.

Major surface glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse

Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.

Primaquine-induced differential gene expression analysis in mice liver using DNA microarrays

Primaquine (PQ). a clinically important derivative of 8-aminoquinoline used against the hepatic stages (hypnozoites) of Plasmodium vivax and Plasmodium ova Ie. was studied to evaluate and compare between mRNA expression. and biochemical and histological parameters of hepatic stress in adult Swiss mice (Mus musculus). Following single oral dose of PQ (40 mglkg. bw). alanine aminotransferase (ALT) and aspartate aminotransferase (AST) along with hematoxylin and eosin stained liver sections did not show any signs of hepatic stress at 6. 12 and 24 h except for ALT activity at 6 h. However. analysis at RNA transcript level revealed consistent and significant deregulation (p<0.01 and twofold) of 16 probes corresponding to important cellular processes such as protein transportation. transcription regulation. intracellular signaling. protein synthesis, hematopoiesis, cell adhesion and cell proliferation. Pathway analysis identified large number of affected genes corresponding to 40 Gene Ontology terms having a z score greaibr than 2. These results indicate that PQ at high doses may affect gene expression in liver and may produce undesirable outcomes if consumed for longer durations.

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.