Cryptococcus neoformans is a significant fungal pathogen of immunocompromised patients. Many questions remain regarding the function of macrophages in normal clearance of cryptococcal infection and the defects present in uncontrolled cryptococcosis. Two current limitations are: 1) The difficulties in interpreting studies using isolated macrophages in the context of the progression of infection, and 2) The use of high resolution imaging in understanding immune cell behavior during animal infection. Here we describe a high-content imaging method in a zebrafish model of cryptococcosis that permits the detailed analysis of macrophage interactions with C. neoformans during infection. Using this approach we demonstrate that, while macrophages are critical for control of C. neoformans, a failure of macrophage response is not the limiting defect in fatal infections. We find phagocytosis is restrained very early in infection and that increases in cryptococcal number are driven by intracellular proliferation. We show that macrophages preferentially phagocytose cryptococci with smaller polysaccharide capsules and that capsule size is greatly increased over twenty-four hours of infection, a change that is sufficient to severely limit further phagocytosis. Thus, high-content imaging of cryptococcal infection in vivo demonstrates how very early interactions between macrophages and cryptococci are critical in the outcome of cryptococcosis.
Objective To compare one-year outcomes of women started on antiretroviral therapy (ART) during pregnancy in the pre-Option B+ era to those in the Option B+ era. Methods A retrospective chart review was performed at three sites in Malawi. Women were included in the ‘pre-Option B+’ cohort if they started ART during pregnancy for a CD4 count < 350 cells/mm3 or WHO 3/4 condition and in the ‘Option B+’ cohort if they started ART during pregnancy regardless of CD4 count or clinical stage. One-year outcomes were compared using Fisher's exact and ANOVA F-tests. Results A higher proportion of women in the pre-Option B+ cohort started ART at WHO stage 3/4 (11.9% versus 1.1%, P < 0.001), switched ART regimens (5.9% versus 0%, P = 0.002), or died in the first year after starting treatment (3.9% versus .5%, P = 0.05). While more women in the Option B+ cohort had poor adherence or defaulted, these differences were not significant. Conclusions At our study sites, the transition to Option B+ has been associated with ART initiation in women with less advanced HIV infection, improved medication tolerability, and lower mortality. Further research is needed to better understand outcomes of Option B+.
Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis.
24Meningitis caused by infectious pathogens are associated with vessel damage and infarct 25 formation, however the physiological cause is unknown. Cryptococcus neoformans, is a 26 human fungal pathogen and causative agent of cryptococcal meningitis, where vascular 27 events are observed in up to 30% of cases, predominantly in severe infection. Therefore, we 28 aimed to investigate how infection may lead to vessel damage and associated pathogen 29 dissemination using a zebrafish model for in vivo live imaging. We find that cryptococcal 30 cells become trapped within the vasculature (dependent on there size) and proliferate there 31 resulting in vasodilation. Localised cryptococcal growth, originating from a single or small 32 number of cryptococcal cells in the vasculature was associated with sites of dissemination 33 and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension 34 reporter we identified dissemination from intact blood vessels and where vessel rupture 35 occurred. Finally, we manipulated blood vessel stifness via cell junctions and found 36 increased stiffness resulted in increased dissemination. Therefore, global vascular 37 vasodilation occurs following infection, resulting in increased vessel tension which 38 subsequently increases dissemination events, representing a positive feedback loop. Thus, 39 we identify a mechanism for blood vessel damage during cryptococcal infection that may 40 represent a cause of vascular damage and cortical infarction more generally in infective 41 meningitis. 42 43 44 45 46 47 48 Introduction 49 Life threatening systemic infection commonly results from tissue invasion requiring 50 dissemination of microbes, usually via the blood stream. Blood vessel damage and blockage 51 are commonly associated with blood infection, as exemplified by mycotic (infective) 52 aneurisms or sub-arachnoid haemorrhage (1). Indeed, both bacterial and fungal meningitis 53 are associated with vascular events including vasculitis, aneurisms and infarcts (1-5). 54The mechanisms of dissemination to the brain in meningitis have been extensively studied in 55 vitro and in vivo. Experimental studies suggest three potential mechanisms: passage of the 56 pathogen between cells of the blood brain barrier, polarised endocytosis and exocytosis of 57 the pathogen by brain vascular endothelial cells, and passage through the blood brain 58 barrier inside immune cells. However, we hypothesised that blood vessel blockage and 59 haemorrhagic dissemination might be an alternative mechanism. 60Cryptococcus neoformans is an opportunistic fungal pathogen causing life threatening 61 cryptococcal meningitis in severely immunocompromised patients. C. neoformans is a 62 significant pathogen of HIV/AIDs positive individuals with cryptococcal meningitis ultimately 63 responsible for 15% of all AIDS related deaths worldwide (6). C . neoformans has 64 previsously been suggested to disseminate from the blood stream into the brain through 65 different routes, including transcytosis, and by...
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