Integrated dataset of screening hits against multiple neglected disease pathogens

New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach.     

Characterization of sheep hemopexin glycovariants

The hemopexin phenotype HpxB1 isolated from sheep serum, yields three major bands when subjected to starch gel and/or polyacrylamide gel electrophoresis which are here designated as subcomponents HpxB1-I, HpxB1-II and HpxB1-III. Electrospray mass spectrometric analysis of samples of the isolated subcomponents prepared by ion exchange chromatography showed that each was composed of three glycoproteins and that the major difference between the subcomponents was due to their constituent glycoproteins possessing different numbers of sialic acid residues. Combined analysis of the ESI-MS data and of the overall carbohydrate compositional data obtained by colorimetric procedures, leads to the composition of the glycan of each glycoprotein, and a combined methylation and 400 MHz H-NMR analysis of the alkaline cleaved glycans identified them as being of only the biantennaryN-acetyllactosamine type. Taking into account the molecular mass, the carbohydrate content and structure it may be concluded that each of the constituent glycoproteins contain fiveN-glycosidically linked glycans.Abbreviations HpxB1 hemopexin phenotype B1 - Man mannose - Gal galactose - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - GlcNAc-ol N-acetylglucosaminitol     

Simultaneous in situ hybridization and TUNEL to identify cells undergoing apoptosis

Apoptotic cells in tissue sections can be localized by in situ labelling of partly degraded DNA. In a heterogeneous population of cells, however, the specific identity of cell types undergoing apoptosis often cannot be reliably achieved at the light microscope level because of the marked alterations in cellular morphology that characterize apoptosis. In order to clearly specify cell types undergoing apoptosis, in situ end labelling has been coupled to immunohistochemistry. This method is limited by the availability of antibodies that bind to cell-specific protein markers in tissue sections. In contrast, we describe a method that combines in situ end labelling with in situ hybridization, a technique that specifies cell types based on mRNA expression. Taking advantage of the specific expression of surfactant protein C mRNA in type II alveolar epithelial cells, we demonstrate that this technique has the ability to localize alveolar type II cells undergoing apoptosis in vivo after the intratracheal instillation of an antibody that activates the cell surface Fas protein. The wide availability of cell-specific gene markers suggests that this method can be adapted to define cell types that undergo apoptosis during various physiological and pathological states in vivo. This revised version was published online in November 2006 with corrections to the Cover Date.     

Redressing the sex imbalance in knowledge of vector biology

The recent development of transgenic mosquitoes that are resistant to infection by the Plasmodium malarial parasite is a promising new tool in the fight against malaria. However, results of large-scale field releases of alternatively modified mosquitoes carried out during the 1970s and 1980s suggest that this approach could be difficult to implement in the field. These past attempts to control mosquito populations largely floundered as a result of our insufficient understanding of the behavioural ecology of released males. In spite of this, contemporary research on genetic control strategies has concentrated predominantly on molecular aspects, with little progress being made toward resolving key ecological uncertainties, male mosquito ecology being the most important. Here, we review the state of knowledge of male mosquito ecology, and highlight priorities for further research. Case studies of two crop pests, the Mediterranean fruit fly and melon fly, are given as examples of how knowledge of male ecology facilitates successful control in other species. Unless similar information becomes available for mosquitoes, any future genetic control strategy will risk failure.     

Viral protein VP4 is a target of human antibodies enhancing coxsackievirus B4- and B3-induced synthesis of alpha interferon

Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-alpha) by peripheral blood mononuclear cells (PBMC) is enhanced in vitro by nonneutralizing anti-CVB4 antibodies from healthy subjects and, to a higher extent, from patients with insulin-dependent diabetes mellitus. In this study, we focused on identification of the viral target of these antibodies in CVB systems. High levels of IFN-alpha were obtained in supernatants of PBMC incubated with CVB4E2 or CVB3 and plasma from healthy subjects and, to a higher extent, from patients. The VP4 capsid proteins dissociated by heating at 56 degrees C from CVB4E2 (VP4(CVB4)) and CVB3 (VP4(CVB3)) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-alpha synthesis. There was no cross-reaction between VP4(CVB4) and VP4(CVB3) in the inhibiting effect. IFN-alpha levels in culture supernatants showed dose-dependent correlation with anti-VP4 antibodies eluted from plasma specimens using VP4-coated plates. There were higher index values for anti-VP4 antibodies detected by enzyme-linked immunosorbent assay (ELISA) and higher proportions of positive detection in 40 patients than in 40 healthy subjects (80% versus 15% for anti-VP4(CVB4)). There was no relationship between the levels of anti-CVB neutralizing antibodies and the detection of anti-VP4 antibodies by ELISA. The CVB plasma-induced IFN-alpha levels obtained in PBMC cultures in the anti-VP4 antibody-positive groups were significantly higher than those obtained in the anti-VP4 antibody-negative groups regardless of the titers of anti-CVB neutralizing antibodies. These results show that VP4 is the target of antibodies involved in the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-alpha synthesis by PBMC.     

Biocatalysed synthesis of beta-O-glucosides from 9-fluorenon-2-carbohydroxyesters. Part 3: IFN-inducing and anti-HSV-2 properties

In pursuing research on the antiviral, interferon (IFN)-inducing tilorone congeners, a new series of fluoren-carboxyhydroxyesters has been prepared and biologically explored. These esters have subsequently been used as sugar acceptors in the enzymatic transglycosylation reaction using the 'retaining' beta-glycosidase from the archaeon Sulfolobus solfataricus (Ssbeta-Gly). Both aglycones (1-6) and corresponding beta-glucosides (beta-glu 1-beta-glu 6) have been screened for cytotoxicity, interferon-stimulating and antiviral properties against HSV-2. It was found that the addition of compounds beta-glu 5, beta-glu 6 and beta-glu 4 to HSV-2 infected U937 cells downregulates viral replication and triggers cells to release IFN-alpha/beta. Taken together, the results showed improved pharmacological profiles as a consequence of glycosylation. A molecular modelling study carried out on this series of compounds completed the structural characterisation of the novel compounds.     

Glycosylphosphatidylinositol-anchored fungal polysaccharide in Aspergillus fumigatus

Galactomannan is a characteristic polysaccharide of the human filamentous fungal pathogen Aspergillus fumigatus that can be used to diagnose invasive aspergillosis. In this study, we report the isolation of a galactomannan fraction associated to membrane preparations from A. fumigatus mycelium by a lipid anchor. Specific chemical and enzymatic degradations and mass spectrometry analysis showed that the lipid anchor is a glycosylphosphatidylinositol (GPI). The lipid part is an inositol phosphoceramide containing mainly C18-phytosphingosine and monohydroxylated lignoceric acid (2OH-C(24:0) fatty acid). GPI glycan is a tetramannose structure linked to a glucosamine residue: Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN. The galactomannan polymer is linked to the GPI structure through the mannan chain. The GPI structure is a type 1, closely related to the one previously described for the GPI-anchored proteins of A. fumigatus. This is the first time that a fungal polysaccharide is shown to be GPI-anchored.