Is conception by in vitro fertilization associated with altered antenatal and postnatal growth trajectories?

ACKNOWLEDGEMENTS We thank Professor Siladitya Bhattacharya for his comment and suggestions made at the very start of this project. FUNDING The study was funded by the FARR Institute.Peer reviewedPostprin

The relationship of endotoxaemia to peripheral and central nervous system inflammatory responses in Human African Trypanosomiasis

Endotoxaemia has been described in cases of Human African trypanosomiasis (HAT), but it is unclear if this phenomenon influences inflammatory pathology either in the periphery or central nervous system (CNS). We studied endotoxin concentrations in the plasma and cerebrospinal fluid (CSF) of Trypanosoma brucei rhodesiense patients using the chromogenic Limulus Amoebocyte lysate assay. The relationship of endotoxin concentration to the presentation of gross signs of inflammation and the inflammatory/counter-inflammatory cytokine profile of the relevant compartments were analysed. We demonstrate that HAT patients exhibit parasitaemia-independent plasma endotoxaemia, and that this is associated with splenomegaly and lymphadenopathy. Endotoxin concentrations normalize rapidly after treatment. There was no evidence of endotoxin release in the CNS. A rapid normalization of endotoxin levels after treatment and lack of association with parasitaemia suggest that gut leakage is the main source of endotoxin in the circulation. Low CSF endotoxin concentrations and a lack of any association with neuroinflammatory markers or neurological sequelae suggest that endotoxin does not play a role in the pathogenesis of the disease in the CNS

Validation of Trypanosoma cruzi inactivation techniques for laboratory use

Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas’ disease, a parasitic infection responsible for significant morbidity and mortality in Latin America. The current treatments have many serious drawbacks and new drugs are urgently required. In the UK, T. cruzi is classified by the Advisory Committee on Dangerous Pathogens (ACDP) as a Hazard Group 3 organism and strict safety practices must be adhered to when handling this pathogen in the laboratory. Validated inactivation techniques are required for safe T. cruzi waste disposal and removal from Containment Level 3 (CL3) facilities for storage, transportation and experimental analysis. Here we assess three T. cruzi. inactivation methods. These include three freeze-thaw cycles, chemical inactivation with Virkon disinfectant, and air drying on Whatman FTA cards (A, B, C, Elute) and on a Mitra microsampling device. After each treatment parasite growth was monitored for 4–6 weeks by microscopic examination. Three freeze-thaw cycles were sufficient to inactivate all T. cruzi CLBrener Luc life cycle stages and Silvio x10/7 A1 large epimastigote cell pellets up to two grams wet weight. Virkon treatment for one hour inactivated T. cruzi Silvio x10/7 subclone A1 and CLBrener Luc both in whole blood and cell culture medium when incubated at a final concentration of 2.5% Virkon, or at ≥1% Virkon when in tenfold excess of sample volume. Air drying also inactivated T. cruzi CLBrener Luc spiked blood when dried on FTA A, B or Elute cards for ≥30 minutes and on a Mitra Microsampler for two hours. However, T. cruzi CLBrener Luc were not inactivated on FTA C cards when dried for up to two hours. These experimentally confirmed conditions provide three validated T. cruzi inactivation methods which can be applied to other related ACDP Hazard Group 2–3 kinetoplastid parasites.</p

Stage progression and neurological symptoms in Trypanosoma brucei rhodesiense sleeping sickness: role of the CNS inflammatory response

Background: Human African trypanosomiasis progresses from an early (hemolymphatic) stage, through CNS invasion to the late (meningoencephalitic) stage. In experimental infections disease progression is associated with neuroinflammatory responses and neurological symptoms, but this concept requires evaluation in African trypanosomiasis patients, where correct diagnosis of the disease stage is of critical therapeutic importance. Methodology/Principal Findings: This was a retrospective study on a cohort of 115 T.b.rhodesiense HAT patients recruited in Eastern Uganda. Paired plasma and CSF samples allowed the measurement of peripheral and CNS immunoglobulin and of CSF cytokine synthesis. Cytokine and immunoglobulin expression were evaluated in relation to disease duration, stage progression and neurological symptoms. Neurological symptoms were not related to stage progression (with the exception of moderate coma). Increases in CNS immunoglobulin, IL-10 and TNF-α synthesis were associated with stage progression and were mirrored by a reduction in TGF-β levels in the CSF. There were no significant associations between CNS immunoglobulin and cytokine production and neurological signs of disease with the exception of moderate coma cases. Within the study group we identified diagnostically early stage cases with no CSF pleocytosis but intrathecal immunoglobulin synthesis and diagnostically late stage cases with marginal CSF pleocytosis and no detectable trypanosomes in the CSF. Conclusions: Our results demonstrate that there is not a direct linkage between stage progression, neurological signs of infection and neuroinflammatory responses in rhodesiense HAT. Neurological signs are observed in both early and late stages, and while intrathecal immunoglobulin synthesis is associated with neurological signs, these are also observed in cases lacking a CNS inflammatory response. While there is an increase in inflammatory cytokine production with stage progression, this is paralleled by increases in CSF IL-10. As stage diagnostics, the CSF immunoglobulins and cytokines studied do not have sufficient sensitivity to be of clinical value

RES-Seq – a barcoded library of drug-resistant <i>Leishmania donovani</i> allowing rapid assessment of cross-resistance and relative fitness

Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. New therapeutic options with diverse mechanisms of actions are required to consolidate progress towards control of this disease and combat drug resistance. Here, we describe the development of a scalable resistance library screen (RES-Seq) as a tool to facilitate the identification and prioritisation of antileishmanial compounds acting via novel mechanisms of action (MoA). We have amassed a large collection of L. donovani cell lines resistant to frontline drugs and compounds in the VL pipeline, with resistance-conferring mutations fully characterised. New phenotypic hits screened against this highly curated panel of resistant lines can determine cross-resistance and potentially shared MoA. The ability to efficiently identify compounds acting via previously established MoA is vital to maintain diversity within drug development portfolios. To expedite screening, short identifier DNA barcodes were introduced into resistant clones enabling pooling and simultaneous screening of multiple cell lines. Illumina sequencing of barcodes enables the growth kinetics and relative fitness of multiple cell lines under compound selection to be tracked. Optimal conditions allowing discrimination of resistant and sensitive clones were established (3× and 10× EC50 for 3 days) and applied to screening of a complex library with VL pre-clinical and clinical drug candidates. RES-Seq is set to play an important role in ensuring that anti-leishmanial compounds exploiting diverse mechanisms of action are developed, ultimately providing options for future drug combination strategies

Spatially and genetically distinct African trypanosome virulence variants defined by host interferon-g response

We describe 2 spatially distinct foci of human African trypansomiasis in eastern Uganda. The Tororo and Soroti foci of &lt;i&gt;Trypanosoma brucei rhodesiense&lt;/i&gt; infection were genetically distinct as characterized by 6 microsatellite and 1 minisatellite polymorphic markers and were characterized by differences in disease progression and host-immune response. In particular, infections with the Tororo genotype exhibited an increased frequency of progression to and severity of the meningoencephalitic stage and higher plasma interferon (IFN)–γ concentration, compared with those with the Soroti genotype. We propose that the magnitude of the systemic IFN-γ response determines the time at which infected individuals develop central nervous system infection and that this is consistent with the recently described role of IFN-γ in facilitating blood-brain barrier transmigration of trypanosomes in an experimental model of infection. The identification of trypanosome isolates with differing disease progression phenotypes provides the first field-based genetic evidence for virulence variants in T. &lt;i&gt;brucei rhodesiense&lt;/i&gt;

The Orthologue of Sjögren's Syndrome Nuclear Autoantigen 1 (SSNA1) in Trypanosoma brucei Is an Immunogenic Self-Assembling Molecule

Primary Sjögren's Syndrome (PSS) is a highly prevalent autoimmune disease, typically manifesting as lymphocytic infiltration of the exocrine glands leading to chronically impaired lacrimal and salivary secretion. Sjögren's Syndrome nuclear autoantigen 1 (SSNA1 or NA14) is a major specific target for autoantibodies in PSS but the precise function and clinical relevance of this protein are largely unknown. Orthologues of the gene are absent from many of the commonly used model organisms but are present in Chlamyodomonas reinhardtii (in which it has been termed DIP13) and most protozoa. We report the functional characterisation of the orthologue of SSNA1 in the kinetoplastid parasite, Trypanosoma brucei. Both TbDIP13 and human SSNA1 are small coiled-coil proteins which are predicted to be remote homologues of the actin-binding protein tropomyosin. We use comparative proteomic methods to identify potential interacting partners of TbDIP13. We also show evidence that TbDIP13 is able to self-assemble into fibril-like structures both in vitro and in vivo, a property which may contribute to its immunogenicity. Endogenous TbDIP13 partially co-localises with acetylated α-tubulin in the insect procyclic stage of the parasite. However, deletion of the DIP13 gene in cultured bloodstream and procyclic stages of T. brucei has little effect on parasite growth or morphology, indicating either a degree of functional redundancy or a function in an alternative stage of the parasite life cycle

Population genetics of trypanosoma brucei rhodesiense: clonality and diversity within and between foci

African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda) and Southern (Malawi) Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics

Identification of a proteasome-targeting arylsulfonamide with potential for the treatment of Chagas' disease

Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas’ disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between β4 and β5 subunits that catalyze chymotrypsin-like activity. A mutation in the β5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced susceptibility to compound 1. Further genetically engineered and in vitro-selected clones resistant to proteasome inhibitors known to bind at the β4/β5 interface were cross-resistant to compound 1. Ubiquitinated proteins were additionally found to accumulate in compound 1-treated epimastigotes. Finally, thermal proteome profiling identified malic enzyme as a secondary target of compound 1, although malic enzyme inhibition was not found to drive potency. These studies identify a novel pharmacophore capable of inhibiting the T. cruzi proteasome that may be exploitable for anti-chagasic drug discovery