Haptoglobin-hemoglobin receptor independent killing of African trypanosomes by human serum and trypanosome lytic factors

The haptoglobin-hemoglobin receptor (HpHbR) of African trypanosomes plays a critical role in human innate immunity against these parasites. Localized to the flagellar pocket of the veterinary pathogen Trypanosoma brucei brucei this receptor binds Trypanosome Lytic Factor-1 (TLF-1), a subclass of human high-density lipoprotein (HDL) facilitating endocytosis, lysosomal trafficking and subsequent killing. Recently, we found that group 1 Trypanosoma brucei gambiense does not express a functional HpHbR. We now show that loss of the TbbHpHbR reduces the susceptibility of T. b. brucei to human serum and TLF-1 by 100- and 10,000-fold, respectively. The relatively high concentrations of human serum and TLF-1 needed to kill trypanosomes lacking the HpHbR indicates that high affinity TbbHpHbR binding enhances the cytotoxicity; however, in the absence of TbbHpHbR, other receptors or fluid phase endocytosis are sufficient to provide some level of susceptibility. Human serum contains a second innate immune factor, TLF-2, that has been suggested to kill trypanosomes independently of the TbbHpHbR. We found that T. b. brucei killing by TLF-2 was reduced in TbbHpHbR-deficient cells but to a lesser extent than TLF-1. This suggests that both TLF-1 and TLF-2 can be taken up via the TbbHpHbR but that alternative pathways exist for the uptake of these toxins. Together the findings reported here extend our previously published studies and suggest that group 1 T. b. gambiense has evolved multiple mechanisms to avoid killing by trypanolytic human serum factors

Parasite Street Science: An Arts and Science Public Engagement Project

Sleeping sickness is a significant health issue in rural Africa. The disease, caused by trypanosome parasites and spread by tsetse flies, is fatal if left untreated. It is most prevalent in resource-poor settings often far from healthcare facilities, and initial presentation is similar to malaria. As a result, many cases go undiagnosed or present late in infection. Furthermore, misinformation and a lack of trust in scientists and healthcare professionals has often resulted in diagnosis and treatment not being sought. The aim of Parasite Street Science was to provide a model for using performance theatre to stimulate discussion about research into sleeping sickness to enhance trust in scientists within affected communities in Malawi. The project created a new interactive street theatre performance which was piloted at a football match at Partick Thistle in Glasgow. After refining and modifying for a Malawi audience using Malawian music and collaborating with a professional theatre director, scientists, health promotion officers, clinicians, nurses, survivors of sleeping sickness, and Life Sciences students, it toured sleeping sickness endemic areas, putting on six performances, where invited audiences from affected communities to actively participate in an enjoyable and fun event. Following performances, local community radio was used to widen the reach of the work, with phone in Q&A sessions taking place with local scientists from Malawi answering questions relating to sleeping sickness. Digital resources, including a web site, videos of the performance and interviews with participants have been developed to ensure a tangible legacy of the work

Equivalence of using a desktop virtual reality science simulation at home and in class

The use of virtual laboratories is growing as companies and educational institutions try to expand their reach, cut costs, increase student understanding, and provide more accessible hands on training for future scientists. Many new higher education initiatives outsource lab activities so students now perform them online in a virtual environment rather than in a classroom setting, thereby saving time and money while increasing accessibility. In this paper we explored whether the learning and motivational outcomes of interacting with a desktop virtual reality (VR) science lab simulation on the internet at home are equivalent to interacting with the same simulation in class with teacher supervision. A sample of 112 (76 female) university biology students participated in a between-subjects experimental design, in which participants learned at home or in class from the same virtual laboratory simulation on the topic of microbiology. The home and classroom groups did not differ significantly on post-test learning outcome scores, or on self-report measures of intrinsic motivation or self-efficacy. Furthermore, these conclusions remained after accounting for prior knowledge or goal orientation. In conclusion, the results indicate that virtual simulations are learning activities that students can engage in just as effectively outside of the classroom environment

Digital gene expression analysis of two life cycle stages of the human-infective parasite, Trypanosoma brucei gambiense reveals differentially expressed clusters of co-regulated genes

<p><b>Background</b></p> <p>The evolutionarily ancient parasite, Trypanosoma brucei, is unusual in that the majority of its genes are regulated post-transcriptionally, leading to the suggestion that transcript abundance of most genes does not vary significantly between different life cycle stages despite the fact that the parasite undergoes substantial cellular remodelling and metabolic changes throughout its complex life cycle. To investigate this in the clinically relevant sub-species, Trypanosoma brucei gambiense, which is the causative agent of the fatal human disease African sleeping sickness, we have compared the transcriptome of two different life cycle stages, the potentially human-infective bloodstream forms with the non-human-infective procyclic stage using digital gene expression (DGE) analysis.</p> <p><b>Results</b></p> <p>Over eleven million unique tags were generated, producing expression data for 7360 genes, covering 81% of the genes in the genome. Compared to microarray analysis of the related T. b. brucei parasite, approximately 10 times more genes with a 2.5-fold change in expression levels were detected. The transcriptome analysis revealed the existence of several differentially expressed gene clusters within the genome, indicating that contiguous genes, presumably from the same polycistronic unit, are co-regulated either at the level of transcription or transcript stability.</p> <p><b>Conclusions</b></p> <p>DGE analysis is extremely sensitive for detecting gene expression differences, revealing firstly that a far greater number of genes are stage-regulated than had previously been identified and secondly and more importantly, this analysis has revealed the existence of several differentially expressed clusters of genes present on what appears to be the same polycistronic units, a phenomenon which had not previously been observed in microarray studies. These differentially regulated clusters of genes are in addition to the previously identified RNA polymerase I polycistronic units of variant surface glycoproteins and procyclin expression sites, which encode the major surface proteins of the parasite. This raises a number of questions regarding the function and regulation of the gene clusters that clearly warrant further study.</p&gt

Embedding 21st Century Employability into Assessment and Feedback Practice through a Student-Staff Partnership

Although commonly thought to be only a measure of academic performance, assessments can also provide students with an opportunity to apply skills and knowledge acquired at university into written literacies that prepare them for the transition into prospective careers. Within a Level 2 Microbiology and Immunology course, students struggled to engage with standalone timetabled careers-related sessions, yet they showed enthusiasm when employability was embedded into assessments. A staff-student partnership project explored these issues, with the overall aim of understanding how to effectively embed employability skills into assessment and feedback, which supports the theme of supporting students to position themselves for the future. Through student-run focus groups, this project investigated the reasons for low student engagement with the timetabled employability session and used student views to develop digital initiatives that could be applied to assessment and feedback practice with an emphasis on developing 21st-century competencies. These initiatives were then implemented as pre-session self-directed activities, which helped students link course feedback with employment skills and future career planning, followed by a newly developed in-class reflective feedback session that allowed students time to consider what skills they have developed and make links with future careers. Project evaluation was conducted using a quantitative survey of students involved. This project aims to make feedback more meaningful and better valued by students. The approaches were designed in partnership with former students from the course, this project possesses the unique ability to deliver a genuine student voice both on employability concerns and the role of assessment

Characterization of 7A7, an anti-mouse EGFR monoclonal antibody proposed to be the mouse equivalent of cetuximab.

The Epidermal Growth Factor Receptor (EGFR) is selectively expressed on the surface of numerous tumours, such as non-small cell lung, ovarian, colorectal and head and neck carcinomas. EGFR has therefore become a target for cancer therapy. Cetuximab is a chimeric human/mouse monoclonal antibody (mAb) that binds to EGFR, where it both inhibits signaling and induces cell death by antibody-dependent cell mediated cytotoxicity (ADCC). Cetuximab has been approved for clinical use in patients with head and neck squamous cell carcinoma (HNSCC) and colorectal cancer. However, only 15-20% patients benefit from this drug, thus new strategies to improve cetuximab efficiency are required. We aimed to develop a reliable and easy preclinical mouse model to evaluate the efficacy of EGFR-targeted antibodies and examine the immune mechanisms involved in tumour regression. We selected an anti-mouse EGFR mAb, 7A7, which has been reported to be "mouse cetuximab" and to exhibit similar properties to its human counterpart. Unfortunately, we were unable to reproduce previous results obtained with the 7A7 mAb. In our hands, 7A7 failed to recognize mouse EGFR, both in native and reducing conditions. Moreover, in vivo administration of 7A7 in an EGFR-expressing HPV38 tumour model did not have any impact on tumour regression or animal survival. We conclude that 7A7 does not recognize mouse EGFR and therefore cannot be used as the mouse equivalent of cetuximab use in humans. As a number of groups have spent effort and resources with similar issues we feel that publication is a responsible approach

A Primate APOL1 Variant That Kills Trypanosoma brucei gambiense

Humans are protected against infection from most African trypanosomes by lipoprotein complexes present in serum that contain the trypanolytic pore-forming protein, Apolipoprotein L1 (APOL1). The human-infective trypanosomes, Trypanosoma brucei rhodesiense in East Africa and T. b. gambiense in West Africa have separately evolved mechanisms that allow them to resist APOL1-mediated lysis and cause human African trypanosomiasis, or sleeping sickness, in man. Recently, APOL1 variants were identified from a subset of Old World monkeys, that are able to lyse East African T. b. rhodesiense, by virtue of C-terminal polymorphisms in the APOL1 protein that hinder that parasite’s resistance mechanism. Such variants have been proposed as candidates for developing therapeutic alternatives to the unsatisfactory anti-trypanosomal drugs currently in use. Here we demonstrate the in vitro lytic ability of serum and purified recombinant protein of an APOL1 ortholog from the West African Guinea baboon (Papio papio), which is able to lyse examples of all sub-species of T. brucei including T. b. gambiense group 1 parasites, the most common agent of human African trypanosomiasis. The identification of a variant of APOL1 with trypanolytic ability for both human-infective T. brucei sub-species could be a candidate for universal APOL1-based therapeutic strategies, targeted against all pathogenic African trypanosomes

The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense

Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense

Differences between <i>Trypanosoma brucei gambiense</i> groups 1 and 2 in their resistance to killing by Trypanolytic factor 1

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; The three sub-species of &lt;i&gt;Trypanosoma brucei&lt;/i&gt; are important pathogens of sub-Saharan Africa. &lt;i&gt;T. b. brucei&lt;/i&gt; is unable to infect humans due to sensitivity to trypanosome lytic factors (TLF) 1 and 2 found in human serum. &lt;i&gt;T. b. rhodesiense&lt;/i&gt; and &lt;i&gt;T. b. gambiense&lt;/i&gt; are able to resist lysis by TLF. There are two distinct sub-groups of &lt;i&gt;T. b. gambiense&lt;/i&gt; that differ genetically and by human serum resistance phenotypes. Group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt; have an invariant phenotype whereas group 2 show variable resistance. Previous data indicated that group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt; are resistant to TLF-1 due in-part to reduced uptake of TLF-1 mediated by reduced expression of the TLF-1 receptor (the haptoglobin-hemoglobin receptor (&lt;i&gt;HpHbR&lt;/i&gt;)) gene. Here we investigate if this is also true in group 2 parasites.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methodology:&lt;/b&gt; Isogenic resistant and sensitive group 2 &lt;i&gt;T. b. gambiense&lt;/i&gt; were derived and compared to other T. brucei parasites. Both resistant and sensitive lines express the &lt;i&gt;HpHbR&lt;/i&gt; gene at similar levels and internalized fluorescently labeled TLF-1 similar fashion to &lt;i&gt;T. b. brucei&lt;/i&gt;. Both resistant and sensitive group 2, as well as group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt;, internalize recombinant APOL1, but only sensitive group 2 parasites are lysed.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Our data indicate that, despite group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt; avoiding TLF-1, it is resistant to the main lytic component, APOL1. Similarly group 2 &lt;i&gt;T. b. gambiense&lt;/i&gt; is innately resistant to APOL1, which could be based on the same mechanism. However, group 2 &lt;i&gt;T. b. gambiense&lt;/i&gt; variably displays this phenotype and expression does not appear to correlate with a change in expression site or expression of &lt;i&gt;HpHbR&lt;/i&gt;. Thus there are differences in the mechanism of human serum resistance between &lt;i&gt;T. b. gambiense&lt;/i&gt; groups 1 and 2.&lt;/p&gt