A necroptosis-independent function of RIPK3 promotes immune dysfunction and prevents control of chronic LCMV infection

Necroptosis is a lytic and inflammatory form of cell death that is highly constrained to mitigate detrimental collateral tissue damageand impaired immunity. These constraints make it difficult to define the relevance of necroptosis in diseases such as chronic andpersistent viral infections and within individual organ systems. The role of necroptotic signalling is further complicated becauseproteins essential to this pathway, such as receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like(MLKL), have been implicated in roles outside of necroptotic signalling. We sought to address this issue by individually defining therole of RIPK3 and MLKL in chronic lymphocytic choriomeningitis virus (LCMV) infection. We investigated if necroptosis contributesto the death of LCMV-specific CD8+ T cells or virally infected target cells during infection. We provide evidence showing thatnecroptosis was redundant in the pathogenesis of acute forms of LCMV (Armstrong strain) and the early stages of chronic (Docilestrain) LCMV infection in vivo. The number of immune cells, their specificity and reactivity towards viral antigens and viral loads arenot altered in the absence of either MLKL or RIPK3 during acute and during the early stages of chronic LCMV infection. However, weidentified that RIPK3 promotes immune dysfunction and prevents control of infection at later stages of chronic LCMV disease. Thiswas not phenocopied by the loss of MLKL indicating that the phenotype was driven by a necroptosis-independent function ofRIPK3. We provide evidence that RIPK3 signaling evoked a dysregulated type 1 interferone response which we linked to animpaired antiviral immune response and abrogated clearance of chronic LCMV infectio

Evaluation of a novel magneto-optical method for the detection of malaria parasites

Improving the efficiency of malaria diagnosis is one of the main goals of current malaria research. We have recently developed a magneto-optical (MO) method which allows high-sensitivity detection of malaria pigment (hemozoin crystals) in blood via the magnetically induced rotational motion of the hemozoin crystals. Here, we evaluate this MO technique for the detection of Plasmodium falciparum in infected erythrocytes using in-vitro parasite cultures covering the entire intraerythrocytic life cycle. Our novel method detected parasite densities as low as approximately 40 parasites per microliter of blood (0.0008% parasitemia) at the ring stage and less than 10 parasites/microL (0.0002% parasitemia) in the case of the later stages. These limits of detection, corresponding to approximately 20 pg/microL of hemozoin produced by the parasites, exceed that of rapid diagnostic tests and compete with the threshold achievable by light microscopic observation of blood smears. The MO diagnosis requires no special training of the operator or specific reagents for parasite detection, except for an inexpensive lysis solution to release intracellular hemozoin. The devices can be designed to a portable format for clinical and in-field tests. Besides testing its diagnostic performance, we also applied the MO technique to investigate the change in hemozoin concentration during parasite maturation. Our preliminary data indicate that this method may offer an efficient tool to determine the amount of hemozoin produced by the different parasite stages in synchronized cultures. Hence, it could eventually be used for testing the susceptibility of parasites to antimalarial drugs

Manipulation of host signalling for the characterisation and control of dengue fever

© 2020 Wasan Otis ForsythDengue fever is a mosquito-transmitted disease of the tropics and sub-tropics that is caused by dengue virus (DENV). There are an estimated 60-100 million clinical cases of dengue fever per year, resulting in at least 10,000 deaths. Most clinical cases of dengue are characterised by flu-like symptoms. However, for unknown reasons, a small proportion (1-2%) of clinical cases progress to a life-threatening form of disease referred to as “severe dengue”. Severe dengue is characterised by cytokine storms, heightened endothelial permeability and associated sequelae such as shock and haemorrhage. During the onset of severe dengue, viraemia and viral antigenaemia are sharply declining or absent. Therefore, it is logical to deduce that dysregulated host signalling is the underlying cause of the cytokine storm phenotype and symptoms of severe dengue. However, although many host factors have been characterised in the context of DENV infection, the root cause of this signalling dysregulation is still poorly understood. Furthermore, there are currently no drug treatments available for the treatment of severe dengue, and although there is a licensed dengue vaccine, it confers only moderate protection, and administration of this vaccine to dengue naive individuals is contraindicated by the World Health Organisation. In the first part of this thesis, I characterised how genetic disruption of key host signalling pathways altered the response of macrophages and mice to DENV infection. I found that infection of cells and mice that had a co-deletion of genes encoding cellular inhibitor of apoptosis proteins (cIAPs) resulted in decreased production of virus, and an exaggerated production of inflammatory cytokines. In the second part of this thesis, I determined whether clinical stage cancer therapeutics could be repurposed as treatments for severe dengue. To investigate this, I established an in vivo mouse model of severe dengue and treated these mice with anti-inflammatory compounds. However, these drug treatments did not reduce clinical manifestations of infection or improve the survival of the infected mice. These studies suggest that cIAPs facilitate the efficient replication of DENV. In addition, I hope that the negative results from my therapeutic experiments can inform future experimental plans, and contribute to reducing the worldwide burden of severe dengue

Magneto-optical (MO) detection of parasitemia in synchronized <i>Plasmodium falciparum</i> cultures.

<p>Panel A: Red and blue curves show the frequency dependent MO signal for samples from the ring and schizont stage cultures, respectively, with various levels of parasite density given in <i>µ</i>L⁻¹ units on the right of the respective curves. The green curves shows the signal from uninfected reference samples. Data plotted with triangles and diamonds are the residual signal from freshly hemolyzed uninfected blood and water, respectively. The frequency scale corresponds to the rotation speed of the magnetic field. Panel B: Red and blue squares in panel B are the MO signal values measured at 20 Hz – indicated by a vertical solid line in panel A – for the dilution series prepared from the original ring and schizont stage cultures, respectively. Solid and open squares correspond to the duplicate samples labeled as samples #1 and samples #2. Triangles indicate the results obtained by remeasuring samples #1 with 24 h delay. The solid lines following the trend of the MO signal at higher parasite densities for ring (red line) and schizont (blue line) samples are guides for the eye. For ring and schizont stage samples with parasite densities lower than 10 parasites/<i>µ</i>L and 1 parasites/<i>µ</i>L, respectively, the MO signal does not further decrease. The green horizontal line shows the residual MO signal of uninfected blood, which is the mean detection limit of our method. The 95% confidence levels of this mean detection limit for the ring and schizont stage samples are indicated by red and blue dashed lines, respectively. Correspondingly, for ring and schizont stage samples with parasite density higher than 40 parasites/<i>µ</i>L and 10 parasites/<i>µ</i>L, respectively, the diagnosis is positive with a confidence of at least 95%. The background signal for freshly hemolyzed uninfected blood and water are also shown by dark and light grey lines. All these horizontal indicators are also shown in panel A for reference. The upper horizontal scale shows the corresponding levels of parasitemia.</p

Using the hemozoin conversion rates reported in the literature for the different parasite stages (rings: 3–5%, trophozoites: 15–20%, and schizonts: 50–70%) [20]–[22], [29], [30] and the stage distribution of the parasites (Fig. 1), we estimated the hemozoin content of culture A (ring stage culture) and culture B (schizont stage culture).

<p>The lower and upper values of the hemozoin content correspond to the lower and upper values of the conversion rates quoted above. Note that the cultures have different parasite densities. We also estimated the hemozoin concentration of the two cultures based on MO signal using the conversion factor c_HZ = 1 ng/<i>µ</i>L →  = 1.4% between the hemozoin concentration and the low-frequency (∼1 Hz) MO signal previously determined for artificial hemozoin crystals suspended in blood <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096981#pone.0096981-Butykai1" target="_blank">[15]</a>.</p

Distribution of parasite life cycle stages in the two <i>Plasmodium falciparum</i> cultures used in the present study.

<p>Panel A: The <i>ring stage culture</i> contained early rings, late rings and some early trophozoites of the first generation after synchronization. The <i>schizont stage culture</i> was on the verge of the first and second life cycles where most of the schizont stages have already turned to early ring stages of the second generation following invasion. Therefore, the ring stage culture contained only the hemozoin present in the parasites up to the early trophozoite stage, while the schizont stage culture had the entire hemozoin content formed during one generation of parasites with the largest portion produced by schizonts. Panel B: Light microscopy images of Giemsa stained thin blood films containing infected red blood cells with parasites in different stages of maturity (taken from these two cultures). In both panels the labels ER, LR, ET, LT, ES and LS correspond to early-ring, late-ring, early-trophozoite, late-trophozoite, early-schizont and late-schizont stages, respectively.</p