A heterotrimeric complex of Toxoplasma proteins promotes parasite survival in interferon gamma-stimulated human cells.

Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here, we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFNγ-activated human cells. Independent screens were carried out using 2 Toxoplasma strains that differ in virulence in mice, leading to the identification of effectors required for survival in IFNγ-activated human cells. We identify the secreted protein GRA57 and 2 other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFNγ-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFNγ in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70, and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that this is likely a secondary consequence of deletion of the complex, unlinked to the IFNγ resistance mediated by these effectors.

Toxoplasma gondii virulence factor ROP1 reduces parasite susceptibility to murine and human innate immune restriction.

Toxoplasma gondii is an intracellular parasite that can infect many host species and is a cause of significant human morbidity worldwide. T. gondii secretes a diverse array of effector proteins into the host cell which are critical for infection. The vast majority of these secreted proteins have no predicted functional domains and remain uncharacterised. Here, we carried out a pooled CRISPR knockout screen in the T. gondii Prugniaud strain in vivo to identify secreted proteins that contribute to parasite immune evasion in the host. We demonstrate that ROP1, the first-identified rhoptry protein of T. gondii, is essential for virulence and has a previously unrecognised role in parasite resistance to interferon gamma-mediated innate immune restriction. This function is conserved in the highly virulent RH strain of T. gondii and contributes to parasite growth in both murine and human macrophages. While ROP1 affects the morphology of rhoptries, from where the protein is secreted, it does not affect rhoptry secretion. Finally, we show that ROP1 co-immunoprecipitates with the host cell protein C1QBP, an emerging regulator of innate immune signaling. In summary, we identify putative in vivo virulence factors in the T. gondii Prugniaud strain and show that ROP1 is an important and previously overlooked effector protein that counteracts both murine and human innate immunity.

UK General Population Utility Values for the SIDECAR-D Instrument Measuring the Impact of Caring for People With Dementia.

OBJECTIVES: Dementia affects many people, with numbers expected to grow as populations age. Many people with dementia receive informal/family/unpaid care, for example, from a spouse or child, which may affect carer quality of life. Measuring the effectiveness of health/social care interventions for carers requires a value measure of the quality-of-life impact of caring. This motivated development of the Scales Measuring the Impact of Dementia on Carers-D (SIDECAR-D) instrument. This study aimed to obtain general population values for SIDECAR-D to aid incorporating the impact of caring in economic evaluation. METHODS: Members of the UK general public completed a best-worst scaling object case survey, which included the 18 SIDECAR-D items and EQ-5D-3L descriptions. Responses were analyzed using scale-adjusted finite mixture models. Relative importance scores (RISs) for the 18 SIDECAR-D items formed the SIDECAR-D relative scale measuring the relative impact of caring. The SIDECAR-D tariff, on the full health = 1, dead = 0 scale, was derived by rescaling EQ-5D-3L and SIDECAR-D RISs so the EQ-5D-3L RISs equaled anchored valuations of the EQ-5D-3L pits state from a visual analog scale task. RESULTS: Five hundred ten respondents completed the survey. The model had 2 parameter and 3 scale classes. Additive utility decrements of SIDECAR-D items ranged from -0.05 to -0.162. Utility scores range from 0.95 for someone affirming 1 item to -0.297 for someone affirming all 18. CONCLUSION: SIDECAR-D is a needs-based scale of the impact on quality of life of caring for someone with dementia, with a valuation tariff to support its use in economic evaluation.

The budget impact of introducing the OMNI® surgical system to a United States health plan for managing mild-to-moderate primary open-angle glaucoma.

OBJECTIVES: Minimally invasive glaucoma surgery devices fill an unmet need in the treatment paradigm between topical intraocular pressure medicines and more invasive filtration procedures. This study evaluated the adoption of The OMNI® Surgical System with or without cataract surgery in primary open-angle glaucoma patients. METHODS: A budget impact analysis estimated costs before and after adoption of OMNI® to a hypothetical US health plan with 1 million Medicare-covered lives over two years. Model input data were derived from published sources and development of the model included primary research with key opinion leaders and payers. The model compared total annual direct costs for OMNI® versus other treatment options (medications, other minimally invasive surgical procedures, selective laser trabeculoplasty) to calculate budget impact. A one-way sensitivity analysis was conducted to assess parameter uncertainty. RESULTS: Increased adoption of OMNI® resulted in budget neutrality over the two years with a decrease in total costs of $35,362. Per member per month incremental costs were $0.00 when used without cataract surgery and yielded cost savings of -$0.01 when used with cataract surgery. Sensitivity analysis confirmed model robustness and identified surgical center fee variability as a key driver of costs. CONCLUSION: OMNI® is budgetary efficient from a US payer perspective.

Systematic Literature Review of Clinical, Economic, and Humanistic Outcomes Following Minimally Invasive Glaucoma Surgery or Selective Laser Trabeculoplasty for the Treatment of Open-Angle Glaucoma with or Without Cataract Extraction.

Introduction: Selective laser trabeculoplasty (SLT) and minimally invasive glaucoma surgery (MIGS) are increasingly used options for mild-to-moderate open-angle glaucoma (OAG) care. While most MIGS devices are indicated for use in combination with cataract surgery only, with phacoemulsification playing a role in lowering IOP, newer technologies can also be used as standalone glaucoma surgery. Methods: This systematic literature review (SLR) aimed to assess the clinical, economic, and humanistic outcomes of MIGS and SLT for the treatment of OAG and was conducted according to PRISMA guidelines. Studies that assessed MIGS or SLT in at least one treatment arm versus any other glaucoma treatment in adults with mild-to-moderate OAG were included. Clinical, humanistic (health-related quality of life [HRQoL] and patient burden), and economic data were extracted, and the methodological quality of included studies was evaluated. Results: A total of 2720 articles were screened, and 81 publications were included. Fifty-eight reported clinical outcomes. The majority assessed iStent or iStent inject (n=41), followed by OMNI (n=9), gonioscopy-assisted transluminal trabeculotomy (GATT) or the Kahook Dual Blade (KDB) (n=7), Hydrus (n=6), SLT (n=5), Xen Gel Stent (n=2), PreserFlo (n=1), and iTrack (n=1). IOP reduction was observed across prospective studies, varying from -31% to -13.7% at month 6 and from -39% to -11.4% at year 1 versus baseline. Most adverse events were transient and non-serious. Limited humanistic and economic data were identified. Conclusion: Given their established efficacy and safety, there is a rationale for wider use of MIGS in mild-to-moderate OAG. Of the MIGS devices, iStent and OMNI have the largest clinical evidence base supporting their sustained effectiveness.

High-throughput identification of Toxoplasma gondii effector proteins that target host cell transcription.

Intracellular pathogens and other endosymbionts reprogram host cell transcription to suppress immune responses and recalibrate biosynthetic pathways. This reprogramming is critical in determining the outcome of infection or colonization. We combine pooled CRISPR knockout screening with dual host-microbe single-cell RNA sequencing, a method we term dual perturb-seq, to identify the molecular mediators of these transcriptional interactions. Applying dual perturb-seq to the intracellular pathogen Toxoplasma gondii, we are able to identify previously uncharacterized effector proteins and directly infer their function from the transcriptomic data. We show that TgGRA59 contributes to the export of other effector proteins from the parasite into the host cell and identify an effector, TgSOS1, that is necessary for sustained host STAT6 signaling and thereby contributes to parasite immune evasion and persistence. Together, this work demonstrates a tool that can be broadly adapted to interrogate host-microbe transcriptional interactions and reveal mechanisms of infection and immune evasion.

Evidence of high exposure to Toxoplasma gondii in free-ranging and captive African carnivores.

Toxoplasma gondii is an ubiquitous intracellular protozoan parasite. Mammals and birds are intermediate hosts and felid species are definitive hosts. In most human altered habitats the domestic cat is the predominant definitive host. Current knowledge of T. gondii infection in African ecosystems is limited. This study aimed to assess exposure to T. gondii in wild carnivores in the Serengeti ecosystem in East Africa. Carnivores can be infected by the consumption of tissue cysts when feeding on infected animals and by incidental ingestion of oocysts from environmental contamination. Incidental ingestion should occur regardless of a species' diet whereas the consumption of cysts should increase the chance of infection in carnivorous species. This predicts higher seropositivity in carnivorous than in insectivorous carnivores and lower seropositivity in juvenile carnivores with a long dependency on milk than in adults. We found high seropositivity in carnivorous species: 100% (15 of 15 samples) in adult African lions, 93% (38 of 41 samples) in adult spotted hyenas and one striped hyena sample was positive, whereas all four samples from the insectivorous bat-eared fox were negative. Juvenile hyenas (11 of 19 sera) had significantly lower seropositivity than adults (38 of 41 sera). Long-term monitoring of spotted hyenas revealed no significant difference in seropositivity between two periods (1988-1992 and 2000 to 2016). Identical results were produced in lion and hyena samples by a commercial multi-species ELISA (at serum dilution 1:10) and an in-house ELISA based on a recombinant T. gondii protein (at serum dilution 1:100), making the latter a useful alternative for small amounts of serum. We suggest that diet, age and lifetime range are factors determining seropositivity in carnivores in the Serengeti ecosystem and suggest that the role of small wild felids in the spread of T. gondii in the African ecosystem warrants investigation.

Recombinant IFN-γ from the bank vole Myodes glareolus: a novel tool for research on rodent reservoirs of zoonotic pathogens.

Rodent species like Myodes glareolus and Microtus spp. are natural reservoirs for many zoonotic pathogens causing human diseases and are gaining increasing interest in the field of eco-immunology as candidate animal models. Despite their importance the lack of immunological reagents has hampered research in these animal species. Here we report the recombinant production and functional characterization of IFN-γ, a central mediator of host's innate and adaptive immune responses, from the bank vole M. glareolus. Soluble dimeric recMgIFN-γ was purified in high yield from Escherichia coli. Its activity on M. glareolus and Microtus arvalis kidney cell lines was assessed by immunofluorescent detection of nuclear translocation and phosphorylation of the transcription factor STAT1. RecMgIFN-γ also induced expression of an IFN-γ-regulated innate immunity gene. Inhibition of vesicular stomatitis virus replication in vole cells upon recMgIFN-γ treatment provided further evidence of its biological activity. Finally, we established a recMgIFN-γ-responsive bank vole reporter cell line that allows the sensitive titration of the cytokine activity via a bioluminescence reporter assay. Taken together, we report valuable tools for future investigations on the immune response against zoonotic pathogens in their natural animal hosts, which might foster the development of novel animal models.

Translational Rodent Models for Research on Parasitic Protozoa-A Review of Confounders and Possibilities.

Rodents, in particular Mus musculus, have a long and invaluable history as models for human diseases in biomedical research, although their translational value has been challenged in a number of cases. We provide some examples in which rodents have been suboptimal as models for human biology and discuss confounders which influence experiments and may explain some of the misleading results. Infections of rodents with protozoan parasites are no exception in requiring close consideration upon model choice. We focus on the significant differences between inbred, outbred and wild animals, and the importance of factors such as microbiota, which are gaining attention as crucial variables in infection experiments. Frequently, mouse or rat models are chosen for convenience, e.g., availability in the institution rather than on an unbiased evaluation of whether they provide the answer to a given question. Apart from a general discussion on translational success or failure, we provide examples where infections with single-celled parasites in a chosen lab rodent gave contradictory or misleading results, and when possible discuss the reason for this. We present emerging alternatives to traditional rodent models, such as humanized mice and organoid primary cell cultures. So-called recombinant inbred strains such as the Collaborative Cross collection are also a potential solution for certain challenges. In addition, we emphasize the advantages of using wild rodents for certain immunological, ecological, and/or behavioral questions. The experimental challenges (e.g., availability of species-specific reagents) that come with the use of such non-model systems are also discussed. Our intention is to foster critical judgment of both traditional and newly available translational rodent models for research on parasitic protozoa that can complement the existing mouse and rat models.

Sirtuin 3 interacts with Lon protease and regulates its acetylation status.

Lon is a mitochondrial protease that degrades oxidized damaged proteins, assists protein folding and participates in maintaining mitochondrial DNA levels. Changes in Lon mRNA levels, protein levels and activity are not always directly correlated, suggesting that Lon could be regulated at post translational level. We found that Lon and SIRT3, the most important mitochondrial sirtuin, colocalize and coimmunoprecipitate in breast cancer cells, and silencing or inhibition of Lon did not alter SIRT3 levels. Silencing of SIRT3 increased the levels of Lon protein and of its acetylation, suggesting that Lon is a target of SIRT3, likely at K917.