Biophysical properties of blood-stage Plasmodium falciparum malaria: from single-cell host-pathogen interactions to human protective polymorphisms

Malaria is a mosquito-borne infectious disease responsible for half a million deaths every year and long-term economic stagnation in many countries where it is endemic. All symptoms and pathology of malaria are caused by Plasmodium falciparum parasite, and are initiated when parasites invade human red blood cells, then mature and multiply inside them in approximately 48 hours. The invasion process is completed in less than a minute and is one of the most crucial, yet least understood, phases of malaria infection. It also represents a brief window in which the parasites are extracellular and hence exposed to the host immune system, therefore representing a potential target for vaccines and treatments. The work described in this thesis firstly includes the optimisation of a real-time live microscopy platform for recording parasite egress-invasion sequences under controlled conditions, and to investigate their morphology and kinetics. This set-up was employed to address the role of calcium in mediating successful invasion by observing the invasion process simultaneously in bright-field and fluorescence. Elevated calcium signal was found to be absent during the early steps of the process, implying that calcium does not trigger invasion, and an alternative invasion mechanism was suggested. To investigate whether parasite and host cell physical parameters were actively involved in invasion, adhesion forces between parasites and red cells were measured with optical tweezers, while the biophysical properties of the red blood cells such as bending modulus, tension, radius, and viscosity were assessed by analysing their plasma membrane fluctuations. In particular, cells from the Dantu blood group, a rare blood variant found mainly in East Africa that provides up to 70% protection against malaria, and from Beta-thalassaemia individuals, were studied. A general correlation between red blood cell membrane tension, invasion efficiency and dynamics was established, determining a protective tension threshold above which cells are less likely to be invaded. Finally, mature parasites have the ability to bind to the endothelium of peripheral blood vessels, causing impair flow that can lead to a range of fatal conditions. To study malaria cytoadherence to endothelial cells, a microfluidic device was designed to produce an in vitro physiologically relevant model of human circulation. Increasing cytoadhesion was experimentally associated with endothelial glycocalyx disruption as initial factor for malaria pathogenesis. Live imaging methods and techniques adopted in this study highlight mechanisms crucial for malaria infection, and represent an innovative and complementary study of this disease with respect to purely biological approaches. These findings show how changes in red blood cell biophysics can be linked to human evolutionary response against malaria with tangible effects on the population

Evidence against a Role of Elevated Intracellular Ca2+ during Plasmodium falciparum Preinvasion.

Severe malaria is primarily caused by Plasmodium falciparum parasites during their asexual reproduction cycle within red blood cells. One of the least understood stages in this cycle is the brief preinvasion period during which merozoite-red cell contacts lead to apical alignment of the merozoite in readiness for penetration, a stage of major relevance in the control of invasion efficiency. Red blood cell deformations associated with this process were suggested to be active plasma membrane responses mediated by transients of elevated intracellular calcium. Few studies have addressed this hypothesis because of technical challenges, and the results remained inconclusive. Here, Fluo-4 was used as a fluorescent calcium indicator with optimized protocols to investigate the distribution of the dye in red blood cell populations used as P. falciparum invasion targets in egress-invasion assays. Preinvasion dynamics was observed simultaneously under bright-field and fluorescence microscopy by recording egress-invasion events. All the egress-invasion sequences showed red blood cell deformations of varied intensities during the preinvasion period and the echinocytic changes that follow during invasion. Intraerythrocytic calcium signals were absent throughout this interval in over half the records and totally absent during the preinvasion period, regardless of deformation strength. When present, calcium signals were of a punctate modality, initiated within merozoites already poised for invasion. These results argue against a role of elevated intracellular calcium during the preinvasion stage. We suggest an alternative mechanism of merozoite-induced preinvasion deformations based on passive red cell responses to transient agonist-receptor interactions associated with the formation of adhesive coat filaments

Red blood cell tension protects against severe malaria in the Dantu blood group.

Malaria has had a major effect on the human genome, with many protective polymorphisms-such as the sickle-cell trait-having been selected to high frequencies in malaria-endemic regions1,2. The blood group variant Dantu provides 74% protection against all forms of severe malaria in homozygous individuals3-5, a similar degree of protection to that afforded by the sickle-cell trait and considerably greater than that offered by the best malaria vaccine. Until now, however, the protective mechanism has been unknown. Here we demonstrate the effect of Dantu on the ability of the merozoite form of the malaria parasite Plasmodium falciparum to invade red blood cells (RBCs). We find that Dantu is associated with extensive changes to the repertoire of proteins found on the RBC surface, but, unexpectedly, inhibition of invasion does not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we find a strong link between RBC tension and merozoite invasion, and identify a tension threshold above which invasion rarely occurs, even in non-Dantu RBCs. Dantu RBCs have higher average tension than non-Dantu RBCs, meaning that a greater proportion resist invasion. These findings provide both an explanation for the protective effect of Dantu, and fresh insight into why the efficiency of P. falciparum invasion might vary across the heterogenous populations of RBCs found both within and between individuals.JCR, AM and DK were supported by the Wellcome Trust (206194/Z/17/Z). MPW is funded by a Wellcome Senior Fellowship (108070). TNW is funded through Fellowships awarded by the Wellcome Trust (091758 and 202800). SNK is supported by the Wellcome Trust-funded Initiative to Develop African Research Leaders (IDeAL) early-career postdoctoral fellowship (107769/Z/10/Z), supported through the DELTAS Africa Initiative (DEL-15-003). The Wellcome Trust provides core support to The KEMRI/Wellcome Trust Research Programme, Kilifi, Kenya (084535), Wellcome Sanger Institute, Cambridge, UK (206194/Z/17/Z) and the Wellcome Centre for Human Genetics, Oxford, UK (090532/Z/09/Z, 203141). PC is supported by the Engineering and Physical Sciences Research Council (EPSRC) (EP/R011443/1), and VI is supported by the EPSRC and the Sackler fellowship

Centripetal nuclear shape fluctuations associate with chromatin condensation in early prophase.

The nucleus plays a central role in several key cellular processes, including chromosome organisation, DNA replication and gene transcription. Recent work suggests an association between nuclear mechanics and cell-cycle progression, but many aspects of this connection remain unexplored. Here, by monitoring nuclear shape fluctuations at different cell cycle stages, we uncover increasing inward fluctuations in late G2 and in early prophase, which are initially transient, but develop into instabilities when approaching the nuclear-envelope breakdown. We demonstrate that such deformations correlate with chromatin condensation by perturbing both the chromatin and the cytoskeletal structures. We propose that the contrasting forces between an extensile stress and centripetal pulling from chromatin condensation could mechanically link chromosome condensation with nuclear-envelope breakdown, two main nuclear processes occurring during mitosis

Biophysical Tools and Concepts Enable Understanding of Asexual Blood Stage Malaria.

Forces and mechanical properties of cells and tissues set constraints on biological functions, and are key determinants of human physiology. Changes in cell mechanics may arise from disease, or directly contribute to pathogenesis. Malaria gives many striking examples. Plasmodium parasites, the causative agents of malaria, are single-celled organisms that cannot survive outside their hosts; thus, thost-pathogen interactions are fundamental for parasite's biological success and to the host response to infection. These interactions are often combinations of biochemical and mechanical factors, but most research focuses on the molecular side. However, Plasmodium infection of human red blood cells leads to changes in their mechanical properties, which has a crucial impact on disease pathogenesis because of the interaction of infected red blood cells with other human tissues through various adhesion mechanisms, which can be probed and modelled with biophysical techniques. Recently, natural polymorphisms affecting red blood cell biomechanics have also been shown to protect human populations, highlighting the potential of understanding biomechanical factors to inform future vaccines and drug development. Here we review biophysical techniques that have revealed new aspects of Plasmodium falciparum invasion of red blood cells and cytoadhesion of infected cells to the host vasculature. These mechanisms occur differently across Plasmodium species and are linked to malaria pathogenesis. We highlight promising techniques from the fields of bioengineering, immunomechanics, and soft matter physics that could be beneficial for studying malaria. Some approaches might also be applied to other phases of the malaria lifecycle and to apicomplexan infections with complex host-pathogen interactions

Cell anomaly localisation using structured uncertainty prediction networks

This paper proposes an unsupervised approach to anomaly detection in bright-field or fluorescence cell microscopy, where our goal is to localise malaria parasites. This is achieved by building a generative model (a variational autoencoder) that describes healthy cell images, where we additionally model the structure of the predicted image uncertainty, rather than assuming pixelwise independence in the likelihood function. This provides a “whitened” residual representation, where the anticipated structured mistakes by the generative model are reduced, but distinctive structures that did not occur in the training distribution, e.g. parasites are highlighted. We employ the recently published Structured Uncertainty Prediction Networks approach to enable tractable learning of the uncertainty structure. Here, the residual covariance matrix is efficiently approximated using a sparse Cholesky parameterisation. We demonstrate that our proposed approach is more effective for detecting real and synthetic structured image perturbations compared to diagonal Gaussian likelihoods

Treatment of Retinal Angiomatous Proliferation with Intravitreal Anti-VEGF Drugs in Real Life Practice

Purpose: To evaluate the outcomes of intravitreal anti-VEGF in the treatment of retinal angiomatous proliferation (RAP) in real life practice. Methods: The design of the study is a retrospective, interventional, multicentre, case series. All the charts of patients affected by RAP, regularly followed up and treated with anti-VEGF drugs over 12 months were examined. All the patients underwent, both at baseline and over the follow-up, a monthly complete ophthalmologic examination, including best corrected visual acuity (BCVA) on ETDRS charts, fluorescein angiography, indocyanine green angiography, and ocular coherence tomography. Both intravitreal injections of ranibizumab and bevacizumab were considered for the study. After an initial loading phase of three consecutive injections, further re-treatments were administered on the basis of the identification of persistence or recurrence of subretinal/intraretinal fluid. The main outcome measure was the change in the mean BCVA at the 12-month examination. Secondary outomes included the proportion of eyes gaining at least 3 ETDRS lines, the mean change in the central retinal thickness (CRT), and number of injections at the end of the follow-up. Results: Sixty-one eyes of 61 patients were considered for the study. Overall, the mean BCVA changed from 0.62, to 0.47 LogMAR (p: 0.004) at the 12-month examination. Seventeen eyes (28%) gained at least 3 ETDRS lines, whereas no eye lost more than 3 ETDRS lines, over the follow-up. Mean CRT passed from 333μm to 222μm (p < 0.001). Twenty-three eyes (37%) showed serous pigment epithelium detachment (PED) at baseline, which was still visible in 10 eyes (16%) at the end of the follow-up. No difference in BCVA gain was registered comparing ranibizumab and bevacizumab. Pigment epithelium detachment was detectable in 5% and 30% of the eyes, treated with ranibizumab and bevacizumab, respectively (p: 0.01). Mean number of injection was 4 and 4.6 in ranibizumab and bevacizumab subgroups, respectively. Conclusions: Intravitreal anti-VEGF therapy can ensure a visual function improvement in about one third of patients affected by RAP, who are treated in the common clinical practice. Ranibizumab treatment requires less injections and more frequently leads to a pigment epithelium detachment resolution

Consensus on the diagnosis,treatment and follow-up of patients with age-related macular degeneration eligible for ranibizumab

Age-related macular degeneration (AMD) is an irreversible pathology that is the principal cause of serious loss of central vision and legal blindness among people over 60 years of age. There are two forms of AMD: the dry, or atrophic form, and the wet, neovascular form. The latter is less frequent but is the cause of approximately 80-90% of cases of serious loss of vision in a short time period. Early diagnosis is therefore essential to permit intervention as promptly as possible. Currently, the most effective therapy for neovascular AMD uses the new class of anti-VEGF drugs, and ranibizumab is todays 'gold standard for this treatment. The Progetto LUCE (LUCE Project) consists of an advisory board of retinal disease specialists in Lombardy, Italy, whose task is to propose a consensus for the diagnosis, treatment and follow-up of neovascular AMD patients treated with ranibizumab on the basis of a review of the scientific evidence and Italian national health service regulations and the clinical experience of the advisory board members