Intracellular Plasmodium aquaporin 2 is important for sporozoite production in the mosquito vector and malaria transmission.

Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins, which are channels facilitating the movement of water and other solutes across membranes. We identify an aquaporin in malaria parasites and demonstrate that it is important for completion of Plasmodium development in the mosquito vector. Disruption of AQP2 in the human parasite Plasmodium falciparum and the rodent parasite Plasmodium berghei blocks sporozoite production inside oocysts established on mosquito midguts, greatly limiting parasite infection of salivary glands and transmission to a new host. In vivo epitope tagging of AQP2 in P. berghei, combined with immunofluorescence assays, reveals that the protein is localized in vesicle-like organelles found in the cytoplasm of gametocytes, ookinetes, and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endomembrane system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely resembles intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature among known aquaporins. This in conjunction with the importance of AQP2 for malaria transmission suggests that AQP2 may be a fruitful target of antimalarial interventions.

Identification of genes required for Plasmodium gametocyte-to-sporozoite development in the mosquito vector.

Malaria remains one of the most devastating infectious diseases. Reverse genetic screens offer a powerful approach to identify genes and molecular processes governing malaria parasite biology. However, the complex regulation of gene expression and genotype-phenotype associations in the mosquito vector, along with sexual reproduction, have hindered the development of screens in this critical part of the parasite life cycle. To address this, we developed a genetic approach in the rodent parasite Plasmodium berghei that, in combination with barcode sequencing, circumvents the fertilization roadblock and enables screening for gametocyte-expressed genes required for parasite infection of the mosquito Anopheles coluzzii. Our results confirm previous findings, validating our approach for scaling up, and identify genes necessary for mosquito midgut infection, oocyst development, and salivary gland infection. These findings can aid efforts to study malaria transmission biology and to develop interventions for controlling disease transmission.

Differential Trafficking and Expression of PIR Proteins in Acute and Chronic Plasmodium Infections.

Plasmodium multigene families are thought to play important roles in the pathogenesis of malaria. Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium species. However, their expression pattern and localisation remain to be elucidated. Understanding protein subcellular localisation is fundamental to reveal the functional importance and cell-cell interactions of the PIR proteins. Here, we use the rodent malaria parasite, Plasmodium chabaudi chabaudi, as a model to investigate the localisation pattern of this gene family. We found that most PIR proteins are co-expressed in clusters during acute and chronic infection; members of the S7 clade are predominantly expressed during the acute-phase, whereas members of the L1 clade dominate the chronic-phase of infection. Using peptide antisera specific for S7 or L1 PIRS, we show that these PIRs have different localisations within the infected red blood cells. S7 PIRs are exported into the infected red blood cell cytoplasm where they are co-localised with parasite-induced host cell modifications termed Maurer's clefts, whereas L1 PIRs are localised on or close to the parasitophorous vacuolar membrane. This localisation pattern changes following mosquito transmission and during progression from acute- to chronic-phase of infection. The presence of PIRs in Maurer's clefts, as seen for Plasmodium falciparum RIFIN and STEVOR proteins, might suggest trafficking of the PIRs on the surface of the infected erythrocytes. However, neither S7 nor L1 PIR proteins detected by the peptide antisera are localised on the surface of infected red blood cells, suggesting that they are unlikely to be targets of surface variant-specific antibodies or to be directly involved in adhesion of infected red blood cells to host cells, as described for Plasmodium falciparum VAR proteins. The differences in subcellular localisation of the two major clades of Plasmodium chabaudi PIRs across the blood cycle, and the apparent lack of expression on the red cell surface strongly suggest that the function(s) of this gene family may differ from those of other multigene families of Plasmodium, such as the var genes of Plasmodium falciparum.

Tissue macrophages and interferon-gamma signalling control blood-stage Plasmodium chabaudi infections derived from mosquito-transmitted parasites.

Natural infection with Plasmodium parasites, the causative agents of malaria, occurs via mosquito vectors. However, most of our knowledge of the immune response to the blood stages of Plasmodium is from infections initiated by injection of serially blood-passaged infected red blood cells, resulting in an incomplete life cycle in the mammalian host. Vector transmission of the rodent malaria parasite, Plasmodium chabaudi chabaudi AS has been shown to give rise to a more attenuated blood-stage infection in C57Bl/6J mice, when compared to infections initiated with serially blood-passaged P. chabaudi-infected red blood cells. In mouse models, the host immune response induced by parasites derived from natural mosquito transmission is likely to more closely resemble the immune responses to Plasmodium infections in humans. It is therefore important to determine how the host response differs between the two types of infections. As the spleen is considered to be a major contributor to the protective host response to P. chabaudi, we carried out a comparative transcriptomic analysis of the splenic response to recently mosquito-transmitted and serially blood-passaged parasites in C57Bl/6J mice. The attenuated infection arising from recently mosquito-transmitted parasites is characterised by an earlier and stronger myeloid- and IFNγ-related response. Analyses of spleen lysates from the two infections similarly showed stronger or earlier inflammatory cytokine and chemokine production in the recently mosquito-transmitted blood-stage infections. Furthermore, tissue macrophages, including red pulp macrophages, and IFNγ-signalling in myeloid cells, are required for the early control of P. chabaudi recently mosquito-transmitted parasites, thus contributing to the attenuation of mosquito-transmitted infections. The molecules responsible for this early activation response to recently-transmitted blood-stage parasites in mice would be important to identify, as they may help to elucidate the nature of the initial interactions between blood-stage parasites and the host immune system in naturally transmitted malaria.

A qualitative study of stakeholder views on the use of a digital app for supported self-management in early intervention services for psychosis.

BACKGROUND: Digital tools such as Smartphones have the potential to increase access to mental health support including self-management interventions for individuals with psychosis, and ultimately to improve outcomes. Self-management strategies, including relapse prevention and crisis planning and setting personal recovery goals, are intended to assist people with long-term conditions to take an active role in their recovery, with evidence for a range of benefits. However, their implementation is inconsistent, and access and uptake need to be improved. The current study explores the acceptability of a Smartphone app (My Journey 3) that has been developed to facilitate supported self-management in Early Intervention in Psychosis (EIP) services. METHODS: Semi-structured one-to-one interviews were conducted with twenty-one EIP service users who had access to My Journey 3 as part of a feasibility trial, and with thirteen EIP service clinicians who were supporting service users with the app. Interviews focused on the acceptability and usability of My Journey 3. Data was coded to themes based on the Acceptability of Healthcare Interventions framework. RESULTS: Many service user participants found My Journey 3 to be acceptable. The symptom and medication trackers in particular were described as helpful. A smaller number of service users disliked the intervention. Individual-level factors that appeared to influence acceptability and engagement included recovery stage and symptom severity. Clinicians tended to report that My Journey 3 was a potentially positive addition to service users' care, but they often felt unable to provide support due to competing demands in their work, which in turn may have impacted acceptability and usage of the app. CONCLUSIONS: Our findings suggest that the app is perceived as having potential to improve users' capacity to self-manage and work towards recovery goals, but barriers prevented many clinicians providing consistent and effective support as intended. Further evaluation of supported self-management apps in psychosis is warranted but needs to address implementation challenges from the start.

The Community Navigator Study: Results from a feasibility randomised controlled trial of a programme to reduce loneliness for people with complex anxiety or depression.

BACKGROUND: Loneliness is common among people with mental health problems and predicts poorer recovery from depression and anxiety. Needs for support with loneliness and social relationships are often under-addressed in mental health services. The Community Navigator programme was designed to reduce loneliness for adults (aged 18 and above) with complex depression or anxiety who were using secondary mental health services. Acceptability and feasibility of the programme and a trial evaluation were tested in a feasibility randomised controlled trial with qualitative evaluation. METHODS: Forty participants with depression or anxiety using secondary mental health services were recruited from mental health services in two London sites and randomised to receive: the Community Navigator programme over six months in addition to routine care (n = 30); or routine care (n = 10). Measures of loneliness, depression, other clinical and social outcomes and service use were collected at baseline and six-months follow-up. Levels of engagement in the programme and rates of trial recruitment and retention were assessed. Programme delivery was assessed through session logs completed by Community Navigators. The acceptability of the programme was explored through qualitative interviews (n = 32) with intervention group participants, their family and friends, programme providers and other involved staff. RESULTS: Forty participants were recruited in four months from 65 eligible potential participants asked. No one withdrew from the trial. Follow-up interviews were completed with 35 participants (88%). Process records indicated the programme was delivered as intended: there was a median of seven meetings with their Community Navigator (of a maximum ten) per treatment group participant. Qualitative interviews indicated good acceptability of the programme to stakeholders, and potential utility in reducing loneliness and depression and anxiety. CONCLUSIONS: A definitive, multi-site randomised controlled trial is recommended to evaluate the effectiveness and cost-effectiveness of the Community Navigator programme for people with complex anxiety and depression in secondary mental health services.

PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector.

After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of malaria transmission blocking.

Improving the Subcutaneous Mouse Tumor Model by Effective Manipulation of Magnetic Nanoparticles-Treated Implanted Cancer Cells.

Murine tumor models have played a fundamental role in the development of novel therapeutic interventions and are currently widely used in translational research. Specifically, strategies that aim at reducing inter-animal variability of tumor size in transplantable mouse tumor models are of particular importance. In our approach, we used magnetic nanoparticles to label and manipulate colon cancer cells for the improvement of the standard syngeneic subcutaneous mouse tumor model. Following subcutaneous injection on the scruff of the neck, magnetically-tagged implanted cancer cells were manipulated by applying an external magnetic field towards localized tumor formation. Our data provide evidence that this approach can facilitate the formation of localized tumors of similar shape, reducing thereby the tumor size's variability. For validating the proof-of-principle, a low-dose of 5-FU was administered in small animal groups as a representative anticancer therapy. Under these experimental conditions, the 5-FU-induced tumor growth inhibition was statistically significant only after the implementation of the proposed method. The presented approach is a promising strategy for studying accurately therapeutic interventions in subcutaneous experimental solid tumor models allowing for the detection of statistically significant differences between smaller experimental groups.

Plasmodium berghei P47 is essential for ookinete protection from the Anopheles gambiae complement-like response.

Malaria is a mosquito-borne disease affecting millions of people every year. The rodent parasite Plasmodium berghei has served as a model for human malaria transmission studies and played a pivotal role in dissecting the mosquito immune response against infection. The 6-cysteine protein P47, known to be important for P. berghei female gamete fertility, is shown to serve a different function in Plasmodium falciparum, protecting ookinetes from the mosquito immune response. Here, we investigate the function of P. berghei P47 in Anopheles gambiae mosquito infections. We show that P47 is expressed on the surface of both female gametocytes and ookinetes where it serves distinct functions in promoting gametocyte-to-ookinete development and protecting ookinetes from the mosquito complement-like response, respectively. The latter function is essential, as ookinetes lacking P47 are targeted for killing while traversing the mosquito midgut cells and eliminated upon exposure to hemolymph proteins of the complement-like system. Silencing key factors of the complement-like system restores oocyst development and disease transmission to rodent hosts. Our data establish a dual role of P. berghei P47 in vivo and reinforce the use of this parasite to study the impact of the mosquito immune response on human malaria transmission.