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Complications from malaria parasite infections still cost the lives of close to half a million people every year. The most severe is cerebral malaria (CM). Employing murine models of CM, autopsy results, experiments, neuroimaging and microscopic techniques, decades of research activity have investigated the development of CM immunopathology in the hope of identifying steps that could be therapeutically targeted. Yet important questions remain. This review summarizes recent findings, primarily mechanistic insights on the essential cellular and molecular players involved gained within the murine experimental cerebral malaria model. It also highlights recent developments in (a) cell-cell communication events mediated through extracellular vesicles (EVs), (b) mounting evidence for innate immune memory, leading to \"trained\" increased or tolerised responses, and (c) modulation of immune cell function through metabolism, that could shed light on why some patients develop this life-threatening condition whilst many do not.

GPR43 receptor as anti-inflammatory target Recent evidence has suggested that the products of 'friendly' intestinal microbes can help counter inflammatory disease and Type 1 diabetes in the host. Commensal bacteria in the colon are known to ferment dietary fibres to produce short-chain fatty acids, and now a study of a mouse model of colitis shows that these fatty acids down-regulate innate and inflammatory responses by stimulating the chemoattractant receptor GPR43 on neutrophils. This identifies interactions between short-chain fatty acids and GPR43 as a possible target for the manipulation of immune responses. Recent evidence indicates that normal intestinal microbiota may positively influence immune responses and protect against the development of inflammatory diseases. One of the potential protective elements in this process are short-chain fatty acids (SCFAs), which are produced by fermentation of dietary fibre by intestinal microbiota and bind the G-protein-coupled receptor 43 (GPR43). Here it is shown that SCFA–GPR43 interactions profoundly affect inflammatory responses in mice. The immune system responds to pathogens by a variety of pattern recognition molecules such as the Toll-like receptors (TLRs), which promote recognition of dangerous foreign pathogens. However, recent evidence indicates that normal intestinal microbiota might also positively influence immune responses, and protect against the development of inflammatory diseases 1 , 2 . One of these elements may be short-chain fatty acids (SCFAs), which are produced by fermentation of dietary fibre by intestinal microbiota. A feature of human ulcerative colitis and other colitic diseases is a change in ‘healthy’ microbiota such as Bifidobacterium and Bacteriodes 3 , and a concurrent reduction in SCFAs 4 . Moreover, increased intake of fermentable dietary fibre, or SCFAs, seems to be clinically beneficial in the treatment of colitis 5 , 6 , 7 , 8 , 9 . SCFAs bind the G-protein-coupled receptor 43 (GPR43, also known as FFAR2) 10 , 11 , and here we show that SCFA–GPR43 interactions profoundly affect inflammatory responses. Stimulation of GPR43 by SCFAs was necessary for the normal resolution of certain inflammatory responses, because GPR43-deficient ( Gpr43 -/- ) mice showed exacerbated or unresolving inflammation in models of colitis, arthritis and asthma. This seemed to relate to increased production of inflammatory mediators by Gpr43 -/- immune cells, and increased immune cell recruitment. Germ-free mice, which are devoid of bacteria and express little or no SCFAs, showed a similar dysregulation of certain inflammatory responses. GPR43 binding of SCFAs potentially provides a molecular link between diet, gastrointestinal bacterial metabolism, and immune and inflammatory responses.

Neutron Capture Therapy (NCT) for cancer treatment is experiencing renewed interest due to advancements in accelerator-based neutron beams, treatment planning software, and patient positioning devices. This study presents the adaptation of an existing neutron radiography beamline (Dingo), at the OPAL research nuclear reactor, for radiobiological research and novel neutron capture agent development. Human glioblastoma cell cultures were irradiated for up to 10 min with a flux of 2.57 × 10 8 n/cm 2 s (± 2.73 × 10 7 ) and the resulting impact was quantified by assessing DNA damage by both immunocytochemistry and flow cytometry. This low cost methodology extends the capability of an existing beamline to allow the development of novel neutron capture agents and study of neutron radiobiological mechanisms. Increasing availability of neutron sources for biological study in this fashion will accelerate the development of NCT for disease specific clinical application.

Here, we describe a protocol for the selection of human antibody fragments using repertoires displayed on filamentous bacteriophage. Antigen-specific clones are enriched by binding to immobilized antigen, followed by elution and repropagation of phage. After multiple rounds of binding selection, specific clones are identified by ELISA. This article provides an overview of phage display and antibody technology, as well as detailed protocols for the immobilization of antigen, the selection of repertoires on purified or complex antigens and the identification of binders.

Chronic kidney disease (CKD) of unknown etiology (CKDu) mostly affects agricultural communities in Central America, South Asia, Africa, but likely also in North America and Australia. One such area with increased CKDu prevalence is the Medawachchiya District Secretariat Division of the Anuradhapura District in the North Central Province of Sri Lanka. Recent research has focused on the presence of various microbial pathogens in drinking water as potential causal or contributing factors to CKDu, yet no study to date has performed a more comprehensive microbial and water chemistry assessment of household wells used for domestic water supply in areas of high CKDu prevalence. In this study, we describe the chemical composition and total microbial content in 30 domestic household wells in the Medawachchiya District Secretariat Division. While the chemical composition in the tested wells mostly lies within standard drinking water limits, except for high levels of fluoride (F), magnesium (Mg), sodium (Na), chloride (Cl) and calcium (Ca) in some samples, we find a frequent presence of cyanotoxin-producing Microcystis , confirming earlier studies in Sri Lanka. Since the total microbial content of drinking water also directly influences the composition of the human gut microbiome, it can be considered an important determinant of health. Several bacterial phyla were previously reported in the gut microbiome of patients with CKD. Using these bacteria phyla to define operational taxonomic units, we found that these bacteria also occur in the microbiome of the sampled well water. Based on available environmental data, our study demonstrates associations between the abundances of these bacteria with geographical distribution, well water temperature and likely fertilizer use in the local surface water catchment area of the individual household wells. Our results reinforce the recommendation that household wells with stagnant or infrequently used water should be purged prior to use for drinking water, bathing and irrigation. The latter is suggested because of the reported potential accumulation of bacterial toxins by agricultural crops. The observation that bacteria previously found in chronic kidney disease patients are also present in household wells requires a more detailed systematic study of both the human gut and drinking water microbiomes in CKDu patients, in relation to disease prevalence and progression.

Disruption of T cell memory during severe immune suppression results in reactivation of chronic viral infections, such as Epstein Barr virus (EBV) and Cytomegalovirus (CMV). How different subsets of memory T cells contribute to the protective immunity against these viruses remains poorly defined. In this study we examined the compartmentalization of virus-specific, tissue resident memory CD8+ T cells in human lymphoid organs. This revealed two distinct populations of memory CD8+ T cells, that were CD69+CD103+ and CD69+CD103-, and were retained within the spleen and tonsils in the absence of recent T cell stimulation. These two types of memory cells were distinct not only in their phenotype and transcriptional profile, but also in their anatomical localization within tonsils and spleen. The EBV-specific, but not CMV-specific, CD8+ memory T cells preferentially accumulated in the tonsils and acquired a phenotype that ensured their retention at the epithelial sites where EBV replicates. In vitro studies revealed that the cytokine IL-15 can potentiate the retention of circulating effector memory CD8+ T cells by down-regulating the expression of sphingosine-1-phosphate receptor, required for T cell exit from tissues, and its transcriptional activator, Kruppel-like factor 2 (KLF2). Within the tonsils the expression of IL-15 was detected in regions where CD8+ T cells localized, further supporting a role for this cytokine in T cell retention. Together this study provides evidence for the compartmentalization of distinct types of resident memory T cells that could contribute to the long-term protection against persisting viral infections.

In this work, we extend our previously published Monte Carlo simulation model of the Dingo thermal neutron beamline at the Australian Centre for Neutron Scattering model by (1) including a sapphire crystal filter in the model, and (2) utilising the NCrystal package to simulate thermal neutron interactions with the crystalline structure. In addition to previous experimental measurements performed in the beamline’s high-resolution mode, the beam was experimentally characterised in its high-intensity mode upstream from the sample stage (at the tertiary shutter wall exit) and these measurements were used as inputs for the model. The planar neutron distributions were optimised at both the sample stage and tertiary shutter wall exit, and model predictions were validated against experimental gold wire activation measurements. For both configurations—with and without the sapphire filter—we measured neutron fluxes, and performed neutron activation analysis using 11 materials to improve the accuracy of the neutron spectrum in the model relative to the original version. Using the optimised spectrum, we simulated out-of-beam neutron spectra that were further used as the initial input in unfolding code to explore the capability of the current solution to accurately reproduce the experimental results. The normalised neutron planar distribution from the simulation was on average within 2% at the centre, and 6% and 24% at the penumbra of the experimental results at the tertiary shutter wall exit and sample stage, respectively. The specific activities predicted by the refined model were within an average of 13% and 5% of the experimentally measured activities with and without the sapphire filter, respectively. We observed a decrease of around 45% in thermal neutron flux when the sapphire filter is used, which has been reproduced by the model. The maximum value of the logarithm of the ratio of simulated to experimental out-of-beam neutron spectra across 8 locations was 0.6 compared to 2.0 in the previous work, resulting in an average normalised root mean squared error between the unfolded spectrum and experimental measurements of 5% and 9% with and without the filter, respectively. Without the sapphire filter, the optimised predicted in-beam neutron spectrum consists of around 59% thermal, 21% epithermal and 20% fast neutrons, while the addition of the filter provides an almost pure (approximately 98%) thermal neutron beam.

Chemotactic cytokines (chemokines) attract immune cells, although their original evolutionary role may relate more closely with embryonic development. We noted differential expression of the chemokine receptor CXCR7 (RDC-1) on marginal zone B cells, a cell type associated with autoimmune diseases. We generated Cxcr7⁻/⁻ mice but found that CXCR7 deficiency had little effect on B cell composition. However, most Cxcr7⁻/⁻ mice died at birth with ventricular septal defects and semilunar heart valve malformation. Conditional deletion of Cxcr7 in endothelium, using Tie2-Cre transgenic mice, recapitulated this phenotype. Gene profiling of Cxcr7⁻/⁻ heart valve leaflets revealed a defect in the expression of factors essential for valve formation, vessel protection, or endothelial cell growth and survival. We confirmed that the principal chemokine ligand for CXCR7 was CXCL12/SDF-1, which also binds CXCR4. CXCL12 did not induce signaling through CXCR7; however, CXCR7 formed functional heterodimers with CXCR4 and enhanced CXCL12-induced signaling. Our results reveal a specialized role for CXCR7 in endothelial biology and valve development and highlight the distinct developmental role of evolutionary conserved chemokine receptors such as CXCR7 and CXCR4.

Enteropathogenic bacteria elicit mucosal innate and adaptive immune responses. We investigated whether gut epithelial cells played a role in triggering an adaptive immune response by recruiting dendritic cells (DCs). Immature DCs are selectively attracted by the CCL20 chemokine. The expression of the CCL20 gene in human intestinal epithelial cell lines was up-regulated by pathogenic bacteria, including Salmonella species, but not by indigenous bacteria of the intestinal flora. The Salmonella machinery for epithelial cell invasion was not required for CCL20 gene activation. Flagellin but not the lipopolysaccharide was found to be the Salmonella factor responsible for stimulation of epithelial CCL20 production. CCL20 in turn triggered a specific migration of immature DCs. Our data show that crosstalk between bacterial flagellin and epithelial cells is essential for the recruitment of DCs, a mechanism that could be instrumental to initiate adaptive immune responses in the gut.

Aims/hypothesisNumerous adaptations of the maternal immune system are necessary during pregnancy to maintain immunological tolerance to the semi-allogeneic fetus. Several complications of pregnancy have been associated with dysregulation of these adaptive mechanisms. While gestational diabetes mellitus (GDM) has been associated with upregulation of circulating inflammatory factors linked to innate immunity, polarisation of the adaptive immune system has not been extensively characterised in this condition. We aimed to characterise pro- and anti-inflammatory CD4+ (T helper [Th]) T cell subsets in women with GDM vs women without GDM (of similar BMI), during and after pregnancy, and examine the relationship between CD4+ subsets and severity of GDM.MethodsThis is a prospective longitudinal case–control study of 55 women with GDM (cases) and 65 women without GDM (controls) at a tertiary maternity hospital. Quantification of proinflammatory (Th17, Th17.1, Th1) and anti-inflammatory (regulatory T cell [Treg]) CD4+ T cell subsets was performed on peripheral blood at 37 weeks gestation and 7 weeks postpartum, and correlated with clinical characteristics and measures of blood glucose.ResultsWomen with GDM had a significantly greater percentage of Th17 (median 2.49% [interquartile range 1.62–4.60] vs 1.85% [1.13–2.98], p = 0.012) and Th17.1 (3.06% [1.30–4.33] vs 1.55% [0.65–3.13], p = 0.006) cells compared with the control group of women without GDM. Women with GDM also had higher proinflammatory cell ratios (Th17:Treg, Th17.1:Treg and Th1:Treg) in pregnancy compared with the control group of women without GDM. In the control group, there was a statistically significant independent association between 1 h glucose levels in the GTT and Th17 cell percentages, and also between 2 h glucose levels and percentage of Th17 cells. The percentage of Th17 cells and the Th17:Treg ratio declined significantly after delivery in women with GDM, whereas this was not the case with the control group of women. Nevertheless, a milder inflammatory phenotype persisted after delivery (higher Th17:Treg ratio) in women with GDM vs women without.Conclusions/interpretationDysregulation of adaptive immunity supports a novel paradigm of GDM that extends beyond hyperglycaemia and altered innate immunity.