Vaccination against trypanosomiasis: Can it be done or is the trypanosome truly the ultimate immune destroyer and escape artist?

To date, human African trypanosomiasis (HAT) still threatens millions of people throughout sub-Sahara Africa, and new approaches to disease prevention and treatment remain a priority. It is commonly accepted that HAT is fatal unless treatment is provided. However, despite the well-described general symptoms of disease progression during distinct stages of the infection, leading to encephalitic complications, coma and death, a substantial body of evidence has been reported suggesting that natural acquired immunity could occur. Hence, if under favorable conditions natural infections can lead to correct immune activation and immune protection against HAT, the development of an effective anti-HAT vaccine should remain a central goal in the fight against this disease.<br /> In this review, we will (1) discuss the vaccine candidates that have been proposed over the past years, (2) highlight the main obstacles that an efficient anti-trypanosomiasis vaccine needs to overcome and (3) critically reflect on the validity of the widely used murine model for HAT

Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by Loop-Mediated Isothermal Amplification (LAMP)

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methodology/Principal Findings:&lt;/b&gt; For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusions/Significance:&lt;/b&gt; This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.&lt;/p&gt

Trypanosoma brucei Glycogen Synthase Kinase-3, A Target for Anti-Trypanosomal Drug Development: A Public-Private Partnership to Identify Novel Leads

Over 60 million people in sub-Saharan Africa are at risk of infection with the parasite Trypanosoma brucei which causes Human African Trypanosomiasis (HAT), also known as sleeping sickness. The disease results in systemic and neurological disability to its victims. At present, only four drugs are available for treatment of HAT. However, these drugs are expensive, limited in efficacy and are severely toxic, hence the need to develop new therapies. Previously, the short TbruGSK-3 short has been validated as a potential target for developing new drugs against HAT. Because this enzyme has also been pursued as a drug target for other diseases, several inhibitors are available for screening against the parasite enzyme. Here we present the results of screening over 16,000 inhibitors of human GSK-3β (HsGSK-3) from the Pfizer compound collection against TbruGSK-3 short. The resulting active compounds were tested for selectivity versus HsGSK-3β and a panel of human kinases, as well as their ability to inhibit proliferation of the parasite in vitro. We have identified attractive compounds that now form potential starting points for drug discovery against HAT. This is an example of how a tripartite partnership involving pharmaceutical industries, academic institutions and non-government organisations such as WHO TDR, can stimulate research for neglected diseases

The AVOID programme’s new simulations of the global benefits of stringent climate change mitigation

Quantitative simulations of the global-scale benefits of climate change mitigation are presented, using a harmonised, self-consistent approach based on a single set of climate change scenarios. The approach draws on a synthesis of output from both physically-based and economics-based models, and incorporates uncertainty analyses. Previous studies have projected global and regional climate change and its impacts over the 21st century but have generally focused on analysis of business-as-usual scenarios, with no explicit mitigation policy included. This study finds that both the economics-based and physically-based models indicate that early, stringent mitigation would avoid a large proportion of the impacts of climate change projected for the 2080s. However, it also shows that not all the impacts can now be avoided, so that adaptation would also therefore be needed to avoid some of the potential damage. Delay in mitigation substantially reduces the percentage of impacts that can be avoided, providing strong new quantitative evidence for the need for stringent and prompt global mitigation action on greenhouse gas emissions, combined with effective adaptation, if large, widespread climate change impacts are to be avoided. Energy technology models suggest that such stringent and prompt mitigation action is technologically feasible, although the estimated costs vary depending on the specific modelling approach and assumptions

Evidence for maintenance of sex determinants but not of sexual stages in red yeasts, a group of early diverged basidiomycetes

<p>Abstract</p> <p>Background</p> <p>The red yeasts are an early diverged group of basidiomycetes comprising sexual and asexual species. Sexuality is based on two compatible mating types and sexual identity is determined by <it>MAT </it>loci that encode homeodomain transcription factors, peptide pheromones and their receptors. The objective of the present study was to investigate the presence and integrity of <it>MAT </it>genes throughout the phylogenetic diversity of red yeasts belonging to the order Sporidiobolales.</p> <p>Results</p> <p>We surveyed 18 sexual heterothallic and self-fertile species and 16 asexual species. Functional pheromone receptor homologues (<it>STE3.A1 </it>and <it>STE3.A2</it>) were found in multiple isolates of most of the sexual and asexual species. For each of the two mating types, sequence comparisons with whole-genome data indicated that synteny tended to be conserved along the pheromone receptor region. For the homeodomain transcription factor, likelihood methods suggested that diversifying selection acting on the self/non-self recognition region promotes diversity in sexual species, while rapid evolution seems to be due to relaxed selection in asexual strains.</p> <p>Conclusions</p> <p>The majority of both sexual and asexual species of red yeasts have functional pheromone receptors and homeodomain homologues. This and the frequent existence of asexual strains within sexual species, makes the separation between sexual and asexual species imprecise. Events of loss of sexuality seem to be recent and frequent, but not uniformly distributed within the Sporidiobolales. Loss of sex could promote speciation by fostering the emergence of asexual lineages from an ancestral sexual stock, but does not seem to contribute to the generation of exclusively asexual lineages that persist for a long time.</p

A Deviation from the Bipolar-Tetrapolar Mating Paradigm in an Early Diverged Basidiomycete

In fungi, sexual identity is determined by specialized genomic regions called MAT loci which are the equivalent to sex chromosomes in some animals and plants. Usually, only two sexes or mating types exist, which are determined by two alternate sets of genes (or alleles) at the MAT locus (bipolar system). However, in the phylum Basidiomycota, a unique tetrapolar system emerged in which four different mating types are generated per meiosis. This occurs because two functionally distinct molecular recognition systems, each encoded by one MAT region, constrain the selection of sexual partners. Heterozygosity at both MAT regions is a pre-requisite for mating in both bipolar and tetrapolar basidiomycetes. Tetrapolar mating behaviour results from the absence of genetic linkage between the two regions bringing forth up to thousands of mating types. The subphylum Pucciniomycotina, an early diverged lineage of basidiomycetes encompassing important plant pathogens such as the rusts and saprobes like Rhodosporidium and Sporidiobolus, has been so far poorly explored concerning the content and organization of MAT loci. Here we show that the red yeast Sporidiobolus salmonicolor has a mating system unlike any previously described because occasional disruptions of the genetic cohesion of the bipolar MAT locus originate new mating types. We confirmed that mating is normally bipolar and that heterozygosity at both MAT regions is required for mating. However, a laboratory cross showed that meiotic recombination may occur within the bipolar MAT locus, explaining tetrapolar features like increased allele number and evolution rates of some MAT genes. This pseudo-bipolar system deviates from the classical bipolar–tetrapolar paradigm and, to our knowledge, has never been observed before. We propose a model for MAT evolution in the Basidiomycota in which the pseudo-bipolar system may represent a hitherto unforeseen gradual form of transition from an ancestral tetrapolar system to bipolarity

Gene Conversion Transfers the GAF-A Domain of Phosphodiesterase TbrPDEB1 to One Allele of TbrPDEB2 of Trypanosoma brucei

Cyclic nucleotide specific phosphodiesterases are important regulators of cyclic nucleotide signalling in eukaryotes. In many organisms, including humans and trypanosomes, some of these enzymes contain specific domains (GAF domains) that bind cyclic nucleotides, and that are involved in the regulation of the catalytic domain. In the parasitic protozoon that causes human sleeping sickness, Trypanosoma brucei, two closely related phosphodiesterases each contain two such GAF domains, GAF-A and GAF-B. Their genes are tandemly located on chromosome 9, spaced by only a few thousand nucleotides. We here show that a gene conversion event has exchanged the region that codes for the GAF-A domain of the downstream gene by the closely similar corresponding sequence of the upstream gene. This domain exchange has no effect on intracellular localization of the two enzymes. The gene conversion event has occurred in one particular strain of trypanosomes (Lister427) and is found in all its derivatives, but not in any other strain or isolate. The presence or absence of this gene conversion represents a useful analytical marker for the stringent discrimination of Lister427 derivatives from other trypanosome strains

<i>Trypanosoma brucei</i> DHRF-TS revisited:characterisation of a bifunctional and highly unstable recombinant dihydrofolate reductase-thymidylate synthase

<div><p>Bifunctional dihydrofolate reductase–thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA^- <i>Escherichia coli</i>, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (<i>K</i>_i 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (<i>K</i>_i 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.</p></div

Sema3E/Plexin-D1 Mediated Epithelial-to-Mesenchymal Transition in Ovarian Endometrioid Cancer

Cancer cells often employ developmental cues for advantageous growth and metastasis. Here, we report that an axon guidance molecule, Sema3E, is highly expressed in human high-grade ovarian endometrioid carcinoma, but not low-grade or other ovarian epithelial tumors, and facilitates tumor progression. Unlike its known angiogenic activity, Sema3E acted through Plexin-D1 receptors to augment cell migratory ability and concomitant epithelial-to-mesenchymal transition (EMT). Sema3E-induced EMT in ovarian endometrioid cancer cells was dependent on nuclear localization of Snail1 through activation of phosphatidylinositol-3-kinase and ERK/MAPK. RNAi-mediated knockdown of Sema3E, Plexin-D1 or Snail1 in Sema3E-expressing tumor cells resulted in compromised cell motility, concurrent reversion of EMT and diminished nuclear localization of Snail1. By contrast, forced retention of Snail1 within the nucleus of Sema3E-negative tumor cells induced EMT and enhanced cell motility. These results show that in addition to the angiogenic effects of Sema3E on tumor vascular endothelium, an EMT strategy could be exploited by Sema3E/Plexin-D1 signaling in tumor cells to promote cellular invasion/migration