Properties of Contextual Memory Formed in the Absence of αCaMKII Autophosphorylation

The alpha-isoform of calcium/calmodulin-dependent kinase II (αCaMKII) is a major synaptic kinase that undergoes autophosphorylation after NMDA receptor activation, switching the kinase into a calcium-independent activity state. This αCaMKII autophosphorylation is essential for NMDA receptor-dependent long-term potentiation (LTP), induced by a single tetanus, in hippocampal area CA1 and in neocortex. Furthermore, the αCaMKII autophosphorylation is essential for contextual long-term memory (LTM) formation after a single training trial but not after a massed training session. Here, we show that in the absence of αCaMKII autophosphorylation contextual fear conditioning is hippocampus dependent and that multi-tetanus-dependent late-LTP cannot be induced in hippocampal area CA1. Furthermore, we show that in the absence of αCaMKII autophosphorylation contextual LTM persists for 30 days, the latest time point tested. Additionally, contextual, but not cued, LTM formation in the absence of αCaMKII autophosphorylation appears to be impaired in 18 month-old mice. Taken together, our findings suggest that αCaMKII autophosphorylation-independent plasticity in the hippocampus is sufficient for contextual LTM formation and that αCaMKII autophosphorylation may be important for delaying age-related impairments in hippocampal memory formation. Furthermore, they propose that NMDA receptor-dependent LTP in hippocampal area CA1 is essential for contextual LTM formation after a single trial but not after massed training. Finally, our results challenge the proposal that NMDA receptor-dependent LTP in neocortex is required for remote contextual LTM

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

<p><b>Background:</b> 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.</p> <p><b>Methodology/Principal Findings:</b> 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.</p> <p><b>Conclusions/Significance:</b> 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.</p&gt

Identification of sVSG117 as an immunodiagnostic antigen and evaluation of a dual-antigen lateral flow test for the diagnosis of human african trypanosomiasis

The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT.Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank.In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use

T. brucei Infection Reduces B Lymphopoiesis in Bone Marrow and Truncates Compensatory Splenic Lymphopoiesis through Transitional B-Cell Apoptosis

African trypanosomes of the Trypanosoma brucei species are extracellular protozoan parasites that cause the deadly disease African trypanosomiasis in humans and contribute to the animal counterpart, Nagana. Trypanosome clearance from the bloodstream is mediated by antibodies specific for their Variant Surface Glycoprotein (VSG) coat antigens. However, T. brucei infection induces polyclonal B cell activation, B cell clonal exhaustion, sustained depletion of mature splenic Marginal Zone B (MZB) and Follicular B (FoB) cells, and destruction of the B-cell memory compartment. To determine how trypanosome infection compromises the humoral immune defense system we used a C57BL/6 T. brucei AnTat 1.1 mouse model and multicolor flow cytometry to document B cell development and maturation during infection. Our results show a more than 95% reduction in B cell precursor numbers from the CLP, pre-pro-B, pro-B, pre-B and immature B cell stages in the bone marrow. In the spleen, T. brucei induces extramedullary B lymphopoiesis as evidenced by significant increases in HSC-LMPP, CLP, pre-pro-B, pro-B and pre-B cell populations. However, final B cell maturation is abrogated by infection-induced apoptosis of transitional B cells of both the T1 and T2 populations which is not uniquely dependent on TNF-, Fas-, or prostaglandin-dependent death pathways. Results obtained from ex vivo co-cultures of living bloodstream form trypanosomes and splenocytes demonstrate that trypanosome surface coat-dependent contact with T1/2 B cells triggers their deletion. We conclude that infection-induced and possibly parasite-contact dependent deletion of transitional B cells prevents replenishment of mature B cell compartments during infection thus contributing to a loss of the host's capacity to sustain antibody responses against recurring parasitemic waves

Kinetoplastids:related protozoan pathogens, different diseases

Kinetoplastids are a group of flagellated protozoans that include the species Trypanosoma and Leishmania, which are human pathogens with devastating health and economic effects. The sequencing of the genomes of some of these species has highlighted their genetic relatedness and underlined differences in the diseases that they cause. As we discuss in this Review, steady progress using a combination of molecular, genetic, immunologic, and clinical approaches has substantially increased understanding of these pathogens and important aspects of the diseases that they cause. Consequently, the paths for developing additional measures to control these “neglected diseases” are becoming increasingly clear, and we believe that the opportunities for developing the drugs, diagnostics, vaccines, and other tools necessary to expand the armamentarium to combat these diseases have never been better

How does Trypanosoma equiperdum fit into the Trypanozoon group? A cluster analysis by RAPD and multiplex-endonuclease genotyping approach.

The pathogenic trypanosomes Trypanosoma equiperdum, T. evansi as well as T. brucei are morphologically identical. In horses, these parasites are considered to cause respectively dourine, surra and nagana. Previous molecular attempts to differentiate these species were not successful for T. evansi and T. equiperdum; only T. b. brucei could be differentiated to a certain extent. In this study we analysed 10 T. equiperdum, 8 T. evansi and 4 T. b. brucei using Random Amplified Polymorphic DNA (RAPD) and multiplex-endonuclease fingerprinting, a modified AFLP technique. The results obtained confirm the homogeneity of the T. evansi group tested. The T. b. brucei clustered out in a heterogenous group. For T. equiperdum the situation is more complex: 8 out of 10 T. equiperdum clustered together with the T. evansi group, while 2 T. equiperdum strains were more related to T. b. brucei. Hence, 2 hypotheses can be formulated: (1) only 2 T. equiperdum strains are genuine T. equiperdum causing dourine; all other T. equiperdum strains actually are T. evansi causing surra or (2) T. equiperdum does not exist at all. In that case, the different clinical outcome of horse infections with T. evansi or T. b. brucei is primarily related to the host immune response

Accuracy of five algorithms to diagnose gambiense human African trypanosomiasis.

Algorithms to diagnose gambiense human African trypanosomiasis (HAT, sleeping sickness) are often complex due to the unsatisfactory sensitivity and/or specificity of available tests, and typically include a screening (serological), confirmation (parasitological) and staging component. There is insufficient evidence on the relative accuracy of these algorithms. This paper presents estimates of the accuracy of five algorithms used by past Médecins Sans Frontières programmes in the Republic of Congo, Southern Sudan and Uganda

The genome sequence of <i>Trypanosoma brucei gambiense</i>, causative agent of chronic Human African Trypanosomiasis

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; &lt;i&gt;Trypanosoma brucei gambiense&lt;/i&gt; is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a &lt;i&gt;T. b. brucei&lt;/i&gt; isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between &lt;i&gt;T. b. gambiense&lt;/i&gt; and the reference genome. We sought to identify features that were uniquely associated with &lt;i&gt;T. b. gambiense&lt;/i&gt; and its ability to infect humans.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methods and findings:&lt;/b&gt; An improved high-quality draft genome sequence for the group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt; DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with &lt;i&gt;T. b. brucei&lt;/i&gt; showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in &lt;i&gt;T. b. gambiense&lt;/i&gt; DAL 972. A comparison of the variant surface glycoproteins (VSG) in &lt;i&gt;T. b. brucei&lt;/i&gt; with all &lt;i&gt;T. b. gambiense&lt;/i&gt; sequence reads showed that the essential structural repertoire of VSG domains is conserved across &lt;i&gt;T. brucei&lt;/i&gt;.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; This study provides the first estimate of intraspecific genomic variation within &lt;i&gt;T. brucei&lt;/i&gt;, and so has important consequences for future population genomics studies. We have shown that the &lt;i&gt;T. b. gambiense&lt;/i&gt; genome corresponds closely with the reference, which should therefore be an effective scaffold for any &lt;i&gt;T. brucei&lt;/i&gt; genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in &lt;i&gt;T. b. brucei&lt;/i&gt;, no &lt;i&gt;T. b. gambiense&lt;/i&gt;-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.&lt;/p&gt

A Non-Cytosolic Protein of Trypanosoma evansi Induces CD45-Dependent Lymphocyte Death

In a recent study dealing with a mouse model of Trypanosoma evansi-associated disease, a remarkable synchrony between the parasitaemia peak and the white-blood-cell count nadir was noticed. The present study was designed to establish whether there is a direct causal link between the parasite load during its exponential phase of growth and the disappearance of peripheral blood leukocytes. In vitro experiments performed with trypanosomes and purified peripheral blood mononucleated cells revealed the existence of a lymphotoxin embedded in the T. evansi membrane: a protein sensitive to serine proteases, with a molecular mass of less than 30 kDa. Lymphocytes death induced by this protein was found to depend on the intervention of a lymphocytic protein tyrosine phosphatase. When lymphocytes were exposed to increasing quantities of a monoclonal antibody raised against the extracellular portion of CD45, a transmembrane protein tyrosine phosphatase covering over 10% of the lymphocyte surface, T. evansi membrane extracts showed a dose-dependent decrease in cytotoxicity. As the regulatory functions of CD45 concern not only the fate of lymphocytes but also the activation threshold of the TCR-dependent signal and the amplitude and nature of cytokinic effects, this demonstration of its involvement in T. evansi-dependent lymphotoxicity suggests that T. evansi might manipulate, via CD45, the host's cytokinic and adaptive responses

Isolation of Trypanosoma brucei gambiense from Cured and Relapsed Sleeping Sickness Patients and Adaptation to Laboratory Mice

Human African trypanosomiasis, or sleeping sickness, is still a major public health problem in central Africa. Melarsoprol is widely used for treatment of patients where the parasite has already reached the brain. In some regions in Angola, Sudan, Uganda and Democratic Republic of the Congo, up to half of the patients cannot be cured with melarsoprol. From previous investigations it is not yet clear what causes these high relapse rates. Therefore we aimed to establish a parasite collection isolated from cured as well as relapsed patients for downstream comparative drug sensitivity profiling. From 360 sleeping sickness patients, blood and cerebrospinal fluid (CSF) was collected before treatment and along the prescribed 24 months follow-up. Blood and CSF were inoculated in thicket rats (Grammomys surdaster), Natal multimammate mice (Mastomys natalensis) and immunodeficient laboratory mice (Mus musculus). Thus, we established a unique collection of Trypanosoma brucei gambiense type I parasites, isolated in the same disease focus and within a limited period, including 12 matched strains isolated from the same patient before treatment and after relapse. This collection is now available for genotypic and phenotypic characterisation to investigate the mechanism behind abnormally high treatment failure rates in Mbuji-Mayi, Democratic Republic of the Congo