In vitro culture of Plasmodium berghei-ANKA maintains infectivity of mouse erythrocytes inducing cerebral malaria

<p>Abstract</p> <p>Background</p> <p>Infection with <it>Plasmodium berghei </it>is a widely used model of murine malaria and a powerful tool for reverse genetic and pathogenesis studies. However, the efficacy of <it>in vitro </it>reinvasion of erythrocytes is generally low, limiting <it>in vitro </it>studies.</p> <p>Methods</p> <p><it>Plasmodium berghei </it>ANKA-infected blood obtained from a susceptible infected mouse was cultured in various conditions and <it>in vitro </it>parasitaemia was measured every day to evaluate the rate of reinvasion.</p> <p>Results</p> <p>High quality culture media were used and reinvasion rates were improved by vigorous orbital shaking of the flask and increasing density of the medium with gelatin.</p> <p>Discussion</p> <p>Using these settings, reinvasion of normal mouse erythrocytes by the parasite was obtained <it>in vitro </it>over two weeks with preservation of the infectivity <it>in vivo</it>.</p

Real-Time Imaging Reveals the Dynamics of Leukocyte Behaviour during Experimental Cerebral Malaria Pathogenesis

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6Chi monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8+ T lymphocytes. A critical number of CD8+ T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8+ T lymphocytes in regulating vascular pathology in this disease. © 2014 Pai et al

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Posttranslational forms of beta 2-glycoprotein I in the pathogenesis of the antiphospholipid syndrome

The antiphospholipid syndrome (APS) is an autoimmune disease characterised by a procoagulant state that predisposes to recurrent thrombosis and miscarriages. Two major discoveries have advanced our understanding of the underlying complex pathogenesis of the APS. The first was the discovery that beta-2 glycoprotein-1 (β2GPI) is the major auto antigen in APS. The second was the discovery in more recent years that β2GPI contains allosteric disulphide bonds susceptible to posttranslational modification that may be involved in the development of autoantibodies in APS. The main allosteric disulphide bond in the fifth domain of β2GPI can exist in two redox states: free thiol or oxidised. It is the conformational transformation of β2GPI from its free thiol form to its more immunogenic oxidised form that exposes neo-epitopes on the first and fifth domains. The purpose of this review is to highlight the recent findings on the posttranslational forms of β2GPI in the pathogenesis of APS. We suggest that novel assays quantitating the different redox forms of β2GPI in plasma or serum may be used to supplement existing clinical and laboratory assays to more accurately stratify risk of thrombosis or miscarriage in APS patients

Change in microbiota profile after vaginal estriol cream in postmenopausal women with stress incontinence

IntroductionVaginal estrogen is a treatment for genitourinary symptoms of menopause (GSM), which comprises vaginal atrophy and urinary dysfunction, including incontinence. Previous studies show that estrogen therapy promotes lactobacilli abundance and is associated with reduced GSM symptoms, including reduction of stress incontinence. However, detailed longitudinal studies that characterize how the microbiome changes in response to estrogen are scarce. We aimed to compare the vaginal microbiota of postmenopausal women, before and 12 weeks after vaginal estrogen cream.MethodsA total of 44 paired samples from 22 postmenopausal women with vaginal atrophy and stress incontinence were collected pre-vaginal estrogens and were compared to 12 weeks post-vaginal estrogen. Microbiota was characterized by 16S rRNA amplicon sequencing and biodiversity was investigated by comparing the alpha- and beta-diversity and potential markers were identified using differential abundance analysis.ResultsVaginal estrogen treatment was associated with a reduction in vaginal pH and corresponded with a significant reduction in alpha diversity of the microbiota. Healthy vaginal community state type was associated with lower mean pH 4.89 (SD = 0.6), in contrast to dysbiotic state which had a higher mean pH 6.4 (SD = 0.74). Women with lactobacilli dominant community pre-treatment, showed stable microbiota and minimal change in their pH. Women with lactobacilli deficient microbiome pre-treatment improved markedly (p = 0.004) with decrease in pH −1.31 and change to heathier community state types.ConclusionIn postmenopausal women with stress incontinence, vaginal estrogen promotes Lactobacillus and Bifidobacterium growth and lowers vaginal pH. Maximum response is seen in those with a dysbiotic vaginal microbiota pre-treatment

Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells.

International audiencePlatelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in EEA1-positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca(2+) chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as ICAM-1 and VCAM-1 and was not increased by TNF stimulation of HBEC. PMP internalization was dramatically increased in the presence of decomplemented serum suggesting a role for PMP opsonin-dependent phagocytosis. PMP uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+) /H(+) exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions

Transferred EMP generated <i>in vitro</i> induce CM-like pathology in healthy mice.

<p>(<b>A</b>) Detection of PKH-67 labelled EMP in the blood of recipient mice following transfer. Purified EMP (400×10³/mouse) from TNF stimulated and resting mouse brain microvascular endothelial cell line B3 harvested <i>in vitro</i>, were transferred into PbA infected and healthy recipient mice. Clearance of fluorescently labeled EMP from the blood was monitored by flow cytometry. Latex beads (400×10³/mouse, control) can be detected in constant circulation up to 30 minutes post transfer. Data represent the mean from <i>n</i> = 3. Closed circle/solid line indicates beads transferred into healthy mice, open circle/interrupted line indicates beads transferred into infected mice, closed square/solid line indicates NS- EMP transferred into healthy mice, closed triangle/solid line indicates NS-EMP transferred into infected mice, open square/interrupted line indicates TNF-EMP transferred into healthy mice and open triangle/interrupted line indicates TNF-EMP transferred into infected mice. (<b>B</b>) Detection of CD54 and CD106 on <i>in vitro</i> generated MP derived from mouse brain microvascular endothelial cells. Graph shows the expression of CD54 (white bar) and CD106 (black bar) on resting and TNF-stimulated EMP. Data are mean ± SD, *p<0.05. The numbers of CD54⁺ and CD106⁺ EMP in the supernatant increased following TNF-stimulation (mean ± SEM MP/µL; CD54: 9.850±1.064 to 38.050±9.925; 74.11% CD54⁺ shift CD106: 86.183±9.981 to 133.250±19.860; 35.32% CD106⁺ shift). (<b>C</b>) Representative haematoxylin-eosin brain and lung sections from healthy and PbA infected mice treated with PBS or EMP (<i>n = </i>3) on day 6 p.i. Morphometric analysis reveals that EMPs induce significant pathology in healthy brain and lung. In the brain, the arrows heads indicate areas of engorged vessels and haemorrhage. In the lung, multifocal lesions, abnormal cellularity in the alveolar septa, plasma in alveoli and haemorrhage. Treatment with EMP in healthy mice induces haemorrhage in the brain (white arrow head) and increased cellularity (white arrow head) and atelectasis (white arrow) in the lung of recipient mice. Such pathology is not observed in the mice treated with PBS or beads alone. Scale bar (bottom left) indicates 40 µm. (<b>D</b>) Effects of <i>in vitro</i> generated EMP on healthy and PbA infected brain and lung in the following treated groups: PBS (white bar), beads (light grey), NS-EMP (dark grey) and TNF-EMP (black). Representative Haematoxylin-Eosin images (<i>n = </i>3) per group were scored for histopathological signs (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003839#ppat-1003839-t003" target="_blank">Table 3</a>).</p