A Putative Non-Canonical Ras-Like GTPase from P. falciparum: Chemical Properties and Characterization of the Protein.

During its development the malaria parasite P. falciparum has to adapt to various different environmental contexts. Key cellular mechanisms involving G-protein coupled signal transduction chains are assumed to act at these interfaces. Heterotrimeric G-proteins are absent in Plasmodium. We here describe the first cloning and expression of a putative, non-canonical Ras-like G protein (acronym PfG) from Plasmodium. PfG reveals an open reading frame of 2736 bp encoding a protein of 912 amino acids with a theoretical pI of 8.68 and a molecular weight of 108.57 kDa. Transcript levels and expression are significantly increased in the erythrocytic phase in particular during schizont and gametocyte formation. Most notably, PfG has GTP binding capacity and GTPase activity due to an EngA2 domain present in small Ras-like GTPases in a variety of Bacillus species and Mycobacteria. By contrast, plasmodial PfG is divergent from any human alpha-subunit. PfG was expressed in E. coli as a histidine-tagged fusion protein and was stable only for 3.5 hours. Purification was only possible under native conditions by Nickel-chelate chromatography and subsequent separation by Blue Native PAGE. Binding of a fluorescent GTP analogue BODIPY® FL guanosine 5'O-(thiotriphosphate) was determined by fluorescence emission. Mastoparan stimulated GTP binding in the presence of Mg2+. GTPase activity was determined colorimetrically. Activity expressed as absolute fluorescence was 50% higher for the human paralogue than the activity of the parasitic enzyme. The PfG protein is expressed in the erythrocytic stages and binds GTP after immunoprecipitation. Immunofluorescence using specific antiserum suggests that PfG localizes to the parasite cytosol. The current data suggest that the putitative, Ras-like G-protein might be involved in a non-canonical signaling pathway in Plasmodium. Research on the function of PfG with respect to pathogenesis and antimalarial chemotherapy is currently under way.

[Cell-based therapies - an innovative therapeutic option in ophthalmology: Treating corneal diseases with stem cells]. / Zelltherapeutika - eine innovative Therapieoption in der Ophthalmologie : Stammzellen zur Behandlung von Hornhauterkrankungen.

Pathological changes and disorders of the cornea are a major cause of severe visual impairment and blindness. Replacement of a pathologically altered cornea with healthy corneal tissue from the eye of a suitable donor is among the most common and successful transplantation procedures in medicine. In Germany, approximately 5000-6000 corneal transplantations are performed each year, but the total demand per year is estimated to be twice as high. With a success rate of 90%, the outcome of cornea transplantation is very favourable. However, long-term maintenance and regeneration of a healthy new cornea requires tissue-specific corneal stem cells residing at the basal layer of the limbus, which is the annular transition zone between the cornea and sclera. When this important limbal stem cell population is destroyed or dysfunctional, a pathological condition known as limbal stem cell deficiency (LSCD) manifests. Limbal stem cell deficiency describes conditions associated with impaired corneal wound healing and regeneration. In this situation, transplantation of healthy limbal stem cells is the only curative treatment approach for restoration of an intact and functional ocular surface. To date, treatment of LSCD presents a great challenge for ophthalmologists. However, innovative, cell-therapeutic approaches may open new, promising treatment perspectives. In February 2015, the European Commission granted marketing authorization to the first stem cell-based treatment in the European Union. The product named Holoclar® is an advanced therapy medicinal product (ATMP) for the treatment of moderate to severe LSCD due to physical and chemical burns in adults. Further cell-based treatment approaches are in clinical development.

Analytical evaluation of a BNP assay on the new point-of-care platform respons®IQ.

a) Objectives: respons®IQ is a new point-of-care (POC) immunoassay platform utilizing evanescent field total internal reflection fluorescence (TIRF) detection and active microfluidics controlled by optical sensors. A B-type natriuretic peptide (BNP) assay was developed on this system. The objective was to show that the BNP test fulfils the basic requirements regarding analytical performance, storage stability of cartridges and correlation to reference systems to be used as a POC test. b) Design and methods: Analytical sensitivity and imprecision were determined in 10 separate experiments over a period of one year. Cartridge storage stability at 4-7 °C and 37 °C was tested. The correlation of responsIQ whole blood measurements to a POC reference device and a laboratory analyzer was determined using 100 patient samples. c) Results: Limit of detection (LOD) was 2.3±1.0 pg/ml BNP and within-run coefficient of variation (within-run CV) was 4.8±1.4% down to a concentration of <40 pg/ml BNP. Cartridge storage stability at 4-7 °C was greater than 50 weeks and at 37 °C, stability was three weeks. The correlation of responsIQ results with both reference methods was high (r≥0.972). d) Conclusions: The developed BNP test fulfils the basic requirements for the performance parameters defined above. The test׳s sensitivity was in the performance range of laboratory analyzer BNP tests. This is the first extensive proof of concept of the responsIQ system.

Deoxyhypusine hydroxylase from Plasmodium vivax, the neglected human malaria parasite: molecular cloning, expression and specific inhibition by the 5-LOX inhibitor zileuton.

Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH) completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A) which is the only cellular protein known to contain the unusual amino acid hypusine. Modified EIF-5A is important for proliferation of the malaria parasite. Here, we present the first successful cloning and expression of DOHH from P. vivax causing tertiary malaria. The nucleic acid sequence of 1041 bp encodes an open reading frame of 346 amino acids. Histidine tagged expression of P. vivax DOHH detected a protein of 39.01 kDa in E. coli. The DOHH protein from P. vivax shares significant amino acid identity to the simian orthologues from P. knowlesi and P. yoelii strain H. In contrast to P. falciparum only four E-Z-type HEAT-like repeats are present in P. vivax DOHH with different homology to phycocyanin lyase subunits from cyanobacteria and in proteins participating in energy metabolism of Archaea and Halobacteria. However, phycocyanin lyase activity is absent in P. vivax DOHH. The dohh gene is present as a single copy gene and transcribed throughout the whole erythrocytic cycle. Specific inhibition of recombinant P. vivax DOHH is possible by complexing the ferrous iron with zileuton, an inhibitor of mammalian 5-lipoxygenase (5-LOX). Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine(673). Zileuton inhibited the P. vivax DOHH protein with an IC50 of 12,5 nmol determined by a relative quantification by GC/MS. By contrast, the human orthologue is only less affected with an IC50 of 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme.

Safety assessment of biolistic DNA vaccination.

DNA-based vector systems have been widely studied as new modalities for the prevention and treatment of human diseases. As for all other medicinal products, safety is an important aspect in the evaluation of such products. In this chapter we reflect on the basic safety issues which have been raised with respect to preventive and therapeutic DNA vaccines, including insertional mutagenesis in case of chromosomal integration, possible formation of anti-DNA antibodies, induction of autoimmune responses and/or immunological tolerance. In addition, local reactions at the site of administration and adverse effects resulting from plasmid DNA spread to nontarget tissues are discussed. Most importantly, however, the benefit-risk profile of a medicinal product is crucial for a decision on providing marketing authorization or not. A product has an acceptable benefit-risk profile if the benefits of the product outweigh its risks for the treated patient.

Hepatitis B vaccination in a school age population: a feasibility study.

There remains no consensus on whether to adopt a universal hepatitis B vaccination strategy in the United Kingdom, where the endemicity of hepatitis B virus (HBV) is considered to be very low in the general population. To assess the feasibility and acceptance of a school-based adolescent vaccination approach, 11-13 years old pupils in local secondary schools in the London Borough of Camden and Islington were contacted and offered a three-dose hepatitis B vaccination course using a 0, 1, and 12 months schedule. The adult dose of hepatitis B vaccine (Engerix B GlaxoSmithKline) containing 20 mug recombinant hepatitis B surface antigen (HBsAg) in 1 ml suspension was administered. This dosage is normally intended for adults and children older than 15 years of age, but can be administered in 10-15 years old children when compliance may be low, since a higher proportion of those vaccinated develop protective antibody levels following administration of only two doses of vaccine. Overall, a total of 528 pupils were contacted, with 122 (23%) consenting to be vaccinated. Of these, 117 (96%) received the complete three-dose regimen according to the schedule (four did not receive vaccine: three were non-attendees and one was previously vaccinated; one withdrew following a flu-like adverse event). The results of this study show that it is feasible and practical to administer hepatitis B vaccination to adolescents in a school setting, and that it is possible to achieve high rates of uptake for the complete three-dose course among adolescents. However, in order to attain and sustain high coverage rates among pupils, this would require additional general health promotion, including health education and provision of information, targeting of teachers, pupils, and parents in order to increase participation in a school-based hepatitis B vaccination programme. A further requirement includes the availability of good local health support within schools so as to allow for an efficient vaccine delivery system to maximize vaccination in this setting.

Changes in hypervariable region 1 of the envelope 2 glycoprotein of hepatitis C virus in children and adults with humoral immune defects.

The N-terminal end of the hepatitis C virus (HCV) envelope glycoprotein E2 contains a stretch of 27 amino acids that exhibit increased variability. This hypervariable region 1 (HVR-1), as it is normally referred to, is thought to contain epitopes that come under humoral immune attack. In the present study, 10 patients (5 children and 5 adults) with humoral immune defects and chronic HCV infection were investigated, to see how HVR-1 sequences behave over time in these patients who are unable to produce antibodies. Amplicons of this region showed little or no variation at all over time, indicating that quasispecies variation in this region is driven by the host's humoral immune response.