In patients with cerebral malaria (CM), higher levels of cell-specific microparticles (MP) correlate with the presence of neurological symptoms. MP are submicron plasma membrane-derived vesicles that ...express antigens of their cell of origin and phosphatidylserine (PS) on their surface, facilitating their role in coagulation, inflammation and cell adhesion. In this study, the in vivo production, fate and pathogenicity of cell-specific MP during Plasmodium berghei infection of mice were evaluated. Using annexin V, a PS ligand, and flow cytometry, analysis of platelet-free plasma from infected mice with cerebral involvement showed a peak of MP levels at the time of the neurological onset. Phenotypic analyses showed that MP from infected mice were predominantly of platelet, endothelial and erythrocytic origins. To determine the in vivo fate of MP, we adoptively transferred fluorescently labelled MP from mice with CM into healthy or infected recipient mice. MP were quickly cleared following intravenous injection, but microscopic examination revealed arrested MP lining the endothelium of brain vessels of infected, but not healthy, recipient mice. To determine the pathogenicity of MP, we transferred MP from activated endothelial cells into healthy recipient mice and this induced CM-like brain and lung pathology. This study supports a pathogenic role for MP in the aggravation of the neurological lesion and suggests a causal relationship between MP and the development of CM.
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. Ly6C(hi) 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.
Endometriosis has been described by many different theories of pathogenesis over the years. It is now also appreciated to be a state of chronic inflammation, and the role of immune dysfunction in its ...development has been proven. There is increasing evidence to support the role of the microbiome in the formation and progression of endometriosis
inflammatory pathways. The dysbiosis seen in endometriosis is thought to be both causative and a consequence of the pathogenesis. Gut, peritoneal fluid and female reproductive tract microbiota has been studied to understand if there are any microbiome signatures specific to endometriosis. New research on how to manipulate the microbiome for better detection and treatment of endometriosis is emerging.
Platelet‐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 early endosome antigen 1 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 Ca2+ chelation by EDTA, suggesting a partial role for receptor‐mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule‐1 and vascular cell adhesion molecule‐1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet‐derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin‐dependent phagocytosis. Platelet‐derived microparticle 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.
Bacterial cells communicate with host cells and other bacteria through the release of membrane vesicles known as bacterial extracellular vesicles (BEV). BEV are established mediators of intracellular ...signaling, stress tolerance, horizontal gene transfer, immune stimulation and pathogenicity. Both Gram-positive and Gram-negative bacteria produce extracellular vesicles through different mechanisms based on cell structure. BEV contain and transfer different types of cargo such as nucleic acids, proteins and lipids, which are used to interact with and affect host cells such as cytotoxicity and immunomodulation. The role of these membranous microvesicles in host communication, intra- and inter-species cell interaction and signaling, and contribution to various diseases have been well demonstrated. Due to their structure, these vesicles can be easily engineered to be utilized for clinical application, as shown with its role in vaccine therapy, and could be used as a diagnostic and cancer drug delivery tool in the future. However, like other novel therapeutic approaches, further investigation and standardization is imperative for BEV to become a routine vector or a conventional treatment method.
Understanding biochemical mechanisms and changes associated with disease conditions and, therefore, development of improved clinical treatments, is relying increasingly on various biochemical mapping ...and imaging techniques on tissue sections. However, it is essential to be able to ascertain whether the sampling used provides the full biochemical information relevant to the disease and is free from artefacts. A multi-modal micro-spectroscopic approach, including FTIR imaging and PIXE elemental mapping, has been used to study the molecular and elemental profile within cryofixed and formalin-fixed murine brain tissue sections. The results provide strong evidence that amino acids, carbohydrates, lipids, phosphates, proteins and ions, such as Cl(-) and K(+), leach from tissue sections into the aqueous fixative medium during formalin fixation of the sections. Large changes in the concentrations and distributions of most of these components are also observed by washing in PBS even for short periods. The most likely source of the chemical species lost during fixation is the extra-cellular and intra-cellular fluid of tissues. The results highlight that, at best, analysis of formalin-fixed tissues gives only part of the complete biochemical "picture" of a tissue sample. Further, this investigation has highlighted that significant lipid peroxidation/oxidation may occur during formalin fixation and that the use of standard histological fixation reagents can result in significant and differential metal contamination of different regions of tissue sections. While a consistent and reproducible fixation method may be suitable for diagnostic purposes, the findings of this study strongly question the use of formalin fixation prior to spectroscopic studies of the molecular and elemental composition of biological samples, if the primary purpose is mechanistic studies of disease pathogenesis.
Vaginal 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.
A 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.
Vaginal 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 (
= 0.004) with decrease in pH -1.31 and change to heathier community state types.
In postmenopausal women with stress incontinence, vaginal estrogen promotes
and
growth and lowers vaginal pH. Maximum response is seen in those with a dysbiotic vaginal microbiota pre-treatment.
Infection with Plasmodium berghei is a widely used model of murine malaria and a powerful tool for reverse genetic and pathogenesis studies. However, the efficacy of in vitro reinvasion of ...erythrocytes is generally low, limiting in vitro studies.
Plasmodium berghei ANKA-infected blood obtained from a susceptible infected mouse was cultured in various conditions and in vitro parasitaemia was measured every day to evaluate the rate of reinvasion.
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.
Using these settings, reinvasion of normal mouse erythrocytes by the parasite was obtained in vitro over two weeks with preservation of the infectivity in vivo.
The immune responses of males and females to bacterial infections display differences. The mechanisms that underlie this sexual dimorphism are multifactorial. Lipopolysaccharide (LPS) contributes to ...the pathogenesis of endotoxaemia. We have previously demonstrated that the plasma protein beta-2 glycoprotein-1 (β2GPI) reduces LPS-induced inflammation in male mice. In the present study using a more robust infection model of septicaemia the role of β2GPI is examined in both male and female wild type (WT) and β2GPI deficient (β2GPI
) mice challenged with Escherichia coli (E. coli) intravenously. β2GPI deficiency led to an increase of E. coli colony forming units (CFU) in the circulation of both male and female mice. In male β2GPI
mice this was associated with a worse clinical severity score. This difference was not observed between female β2GPI
and female WT mice. Male WT mice had decreased levels of total and increased levels of free thiol β2GPI following administration of LPS or E. coli. This pattern of sexual dimorphic response was also observed in our cohort of humans with sepsis. These findings support a role for β2GPI in modulating the sex-specific susceptibility to gram-negative septicaemia.
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