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STAR Protocols Sep 2022This protocol presents an assay for transmigration analysis of human cytotoxic T cells (CTL) under physiological flow in vitro. We describe detailed analysis steps of...
This protocol presents an assay for transmigration analysis of human cytotoxic T cells (CTL) under physiological flow in vitro. We describe detailed analysis steps of human CTL behavior, from adhesion to diapedesis, using live cell imaging which cannot be achieved by in vivo imaging. The flow system is made of 2D plastic surfaces covered by an endothelial monolayer limiting the system but allows for quantitative analysis of CTL behavior with high modifiability. For complete details on the use and execution of this protocol, please refer to Schoppmeyer et al. (2022).
Topics: Humans; T-Lymphocytes; Transendothelial and Transepithelial Migration
PubMed: 35776649
DOI: 10.1016/j.xpro.2022.101509 -
Methods in Molecular Biology (Clifton,... 2022Adhesion between leukocytes and brain endothelial cells, which line cerebral blood vessels, is a key event in both physiological and pathological conditions such as...
Adhesion between leukocytes and brain endothelial cells, which line cerebral blood vessels, is a key event in both physiological and pathological conditions such as neuroinflammatory diseases. Leukocyte recruitment from blood into tissues is described as a multistep process involving leukocyte rolling on endothelial cells, adhesion, crawling, and diapedesis under hemodynamic shear stress. In neuroinflammatory conditions, there is an increase in leukocyte adhesion to the brain endothelial cells, activated by proinflammatory molecules such as cytokines. Here, we describe an in vitro technique to study the interaction between human leukocytes with human brain endothelial cells under shear stress mimicking the blood flow in vivo, coupled to live-cell imaging.
Topics: Blood-Brain Barrier; Brain; Cell Adhesion; Endothelial Cells; Endothelium, Vascular; Humans; Leukocytes
PubMed: 35733054
DOI: 10.1007/978-1-0716-2289-6_19 -
FASEB Journal : Official Publication of... May 2022Current therapies for ischemic stroke focus on reperfusion but do not address the acute inflammatory response that results in significant reperfusion injury. To advance...
BACKGROUND AND PURPOSE
Current therapies for ischemic stroke focus on reperfusion but do not address the acute inflammatory response that results in significant reperfusion injury. To advance future therapies, a thorough understanding of the precise spatiotemporal underpinnings of leukocyte extravasation and infiltration is necessary. We describe the evolution of the inflammatory response in a mouse transient middle cerebral artery occlusion (tMCAO) stroke model at several time points after reperfusion and the modulation of this response with PECAM blockade.
METHODS
The transient Middle Cerebral Artery Occlusion model (90 minutes of ischemia followed by reperfusion) was used to simulate large vessel occlusion stroke and recanalization. We used wide field and confocal immunofluorescence microscopy to examine the exact distribution of neutrophils with close examination of the leukocyte position with regard to the brain vasculature and the perivascular space. Flow cytometry of single cell suspensions was used to confirm cell identity at different time points post-stroke.
RESULTS
Large ischemic strokes involving both the subcortex and cortex (over 20% of the ischemic hemisphere) were induced in mice. At 12 and 24 hours, leukocyte recruitment and extravasation was primarily localized to the cortical surface. This contrasts with other organs where there is considerable migration of neutrophils deep into the inflamed tissue by 24 hours. Flow cytometry showed at 24 hours a majority of leukocytes were neutrophils. Over 48 to 72 hours, leukocytes were increasingly found deeper into the subcortex. Throughout the infarct (determined with triphenyl tetrazolium chloride staining), leukocyte recruitment was not uniform but rather organized in clusters. Disrupting leukocyte diapedesis with PECAM function-blocking monoclonal antibody restricted leukocytes to within 500 microns of the surface when compared to control; and this was still evident at 72 hours (n=3 mice per group, p<0.01, Control 46% ± 4.0 %; PECAM-1 Ab 62% ± 5.0%). High-resolution wide-field microscopy confirmed inhibition of TEM by PECAM-1 blockade at 24 hours. Flow cytometry showed approximately equal numbers of monocytes and neutrophils at 72 hours.
CONCLUSIONS
Our findings demonstrate that leukocyte infiltration into a stroke evolves over several days following reperfusion. The use of PECAM blockade modulates the natural progression of leukocytes into the infarcted stroke bed. A better understanding of leukocyte spatiotemporal infiltration and its regulators could help inform the next generation of therapeutic interventions.
Topics: Animals; Disease Models, Animal; Infarction, Middle Cerebral Artery; Leukocytes; Mice; Neutrophils; Platelet Endothelial Cell Adhesion Molecule-1; Stroke; Transendothelial and Transepithelial Migration
PubMed: 35723884
DOI: 10.1096/fasebj.2022.36.S1.R5646 -
Journal of Thrombosis and Haemostasis :... Oct 2022Breast cancer results in a three- to four-fold increased risk of venous thromboembolism (VTE), which is associated with reduced patient survival. Despite this, the...
BACKGROUND
Breast cancer results in a three- to four-fold increased risk of venous thromboembolism (VTE), which is associated with reduced patient survival. Despite this, the mechanisms underpinning breast cancer-associated thrombosis remain poorly defined. Tumor cells can trigger endothelial cell (EC) activation resulting in increased von Willebrand factor (VWF) secretion. Importantly, elevated plasma VWF levels constitute an independent biomarker for VTE risk. Moreover, in a model of melanoma, treatment with low molecular weight heparin (LMWH) negatively regulated VWF secretion and attenuated tumor metastasis.
OBJECTIVE
To investigate the role of VWF in breast cancer metastasis and examine the effect of LMWH in modulating EC activation and breast tumor transmigration.
METHODS
von Willebrand factor levels were measured by ELISA. Primary ECs were used to assess tumor-induced activation, angiogenesis, tumor adhesion, and transendothelial migration.
RESULTS AND CONCLUSION
Patients with metastatic breast cancer have markedly elevated plasma VWF:Ag levels that also correlate with poorer survival. MDA-MB-231 and MCF-7 breast cancer cells induce secretion of VWF, angiopoietin-2, and osteoprotegerin from ECs, which is further enhanced by the presence of platelets. Vascular endothelial growth factor-A (VEGF-A) plays an important role in modulating breast cancer-induced VWF release. Moreover, VEGF-A from breast tumor cells also contributes to a pro-angiogenic effect on ECs. VWF multimers secreted from ECs, in response to tumor-VEGF-A, mediate adhesion of breast tumor cells along the endothelium. LMWH inhibits VWF-breast tumor adhesion and transendothelial migration. Our findings highlight the significant crosstalk between tumor cells and the endothelium including increased VWF secretion which may contribute to tumor metastasis.
Topics: Angiopoietin-2; Breast Neoplasms; Endothelial Cells; Female; Heparin, Low-Molecular-Weight; Humans; Osteoprotegerin; Transendothelial and Transepithelial Migration; Vascular Endothelial Growth Factor A; Venous Thromboembolism; von Willebrand Factor
PubMed: 35722954
DOI: 10.1111/jth.15794 -
International Journal of Molecular... May 2022A growing body of work suggests epigenetic dysregulation contributes to endometriosis pathophysiology and female infertility. The chromatin remodeling complex subunit...
A growing body of work suggests epigenetic dysregulation contributes to endometriosis pathophysiology and female infertility. The chromatin remodeling complex subunit AT-rich interaction domain 1A (ARID1A) must be properly expressed to maintain normal uterine function. Endometrial epithelial ARID1A is indispensable for pregnancy establishment in mice through regulation of endometrial gland function; however, ARID1A expression is decreased in infertile women with endometriosis. We hypothesized that ARID1A performs critical operations in the endometrial epithelium necessary for fertility besides maintaining gland function. To identify alterations in uterine gene expression resulting from loss of epithelial ARID1A, we performed RNA-sequencing analysis on pre-implantation uteri from and control mice. Differential expression analysis identified 4181 differentially expressed genes enriched for immune-related ingenuity canonical pathways including agranulocyte adhesion and diapedesis and natural killer cell signaling. RT-qPCR confirmed an increase in pro-inflammatory cytokine and macrophage-related gene expression but a decrease in natural killer cell signaling. Immunostaining confirmed a uterus-specific increase in macrophage infiltration. Flow cytometry delineated an increase in inflammatory macrophages and a decrease in uterine dendritic cells in uteri. These findings demonstrate a role for endometrial epithelial ARID1A in suppressing inflammation and maintaining uterine immune homeostasis, which are required for successful pregnancy and gynecological health.
Topics: Animals; DNA-Binding Proteins; Endometriosis; Endometrium; Female; Homeostasis; Humans; Infertility, Female; Mice; Nuclear Proteins; Pregnancy; Transcription Factors; Uterus
PubMed: 35682747
DOI: 10.3390/ijms23116067 -
Biological Trace Element Research Apr 2023Zinc is structurally and functionally essential for more than 300 enzymes and 2000 transcription factors in human body. Intracellular labile zinc is the metabolically... (Review)
Review
Zinc is structurally and functionally essential for more than 300 enzymes and 2000 transcription factors in human body. Intracellular labile zinc is the metabolically effective zinc and tiny changes in its concentrations significantly affect the intracellular signaling and enzymatic responses. Zinc is crucial for the embrionic and fetal development of heart. Therefore, it is shown to be related with a variety of congenital heart defects. It is involved in epithelial-to-mesenchymal transformation including endocardial cushion development, which is necessary for atrioventricular septation as well as the morphogenesis of heart valves. In atherosclerosis, monocyte endothelial adhesion, and diapedesis, activation and transformation into macrophages and forming foam cells by the ingestion of oxidized LDL are monocyte related steps which need zinc. Intracellular zinc increases intracellular calcium through a variety of pathways and furthermore, zinc itself can work as a second messenger as calcium. These demonstrate the significance of intracellular zinc in heart failure and arterial hypertension. However, extracellular zinc has an opposite effect by blocking calcium channels, explaining decreased serum zinc levels, contrary to the increased cardiomyocyte and erythrocyte zinc levels in hypertensive subjects. These and other data in the literature demonstrate that zinc has important roles in healthy and diseased cardiovascular system but zinc-cardiovascular system relationship is so complex that, it has not been explained in all means. In this article, we try to review some of the available knowledge about this complex relationship.
Topics: Humans; Cardiovascular Diseases; Zinc; Calcium; Heart Valves; Heart Defects, Congenital
PubMed: 35672544
DOI: 10.1007/s12011-022-03292-6 -
Inhalation Toxicology 2022To investigate the molecular mechanisms underlying the pulmonary toxicity induced by exposure to one form of multi-walled carbon nanotubes (MWCNT-7). Rats were exposed,...
To investigate the molecular mechanisms underlying the pulmonary toxicity induced by exposure to one form of multi-walled carbon nanotubes (MWCNT-7). Rats were exposed, by whole-body inhalation, to air or an aerosol containing MWCNT-7 particles at target cumulative doses (concentration x time) ranging from 22.5 to 180 (mg/m)h over a three-day (6 hours/day) period and toxicity and global gene expression profiles were determined in the lungs. MWCNT-7 particles, associated with alveolar macrophages (AMs), were detected in rat lungs following the exposure. Mild to moderate lung pathological changes consisting of increased cellularity, thickening of the alveolar wall, alveolitis, fibrosis, and granuloma formation were detected. Bronchoalveolar lavage (BAL) toxicity parameters such as lactate dehydrogenase activity, number of AMs and polymorphonuclear leukocytes (PMNs), intracellular oxidant generation by phagocytes, and levels of cytokines were significantly ( < 0.05) increased in response to exposure to MWCNT-7. Global gene expression profiling identified several significantly differentially expressed genes (fold change >1.5 and FDR value <0.05) in all the MWCNT-7 exposed rats. Bioinformatic analysis of the gene expression data identified significant enrichment of several diseases/biological function categories (for example, cancer, leukocyte migration, inflammatory response, mitosis, and movement of phagocytes) and canonical pathways (for example, kinetochore metaphase signaling pathway, granulocyte and agranulocyte adhesion and diapedesis, acute phase response, and LXR/RXR activation). The alterations in the lung toxicity parameters and gene expression changes exhibited a dose-response to the MWCNT exposure. Taken together, the data provided insights into the molecular mechanisms underlying the pulmonary toxicity induced by inhalation exposure of rats to MWCNT-7.
Topics: Animals; Bronchoalveolar Lavage Fluid; Gene Expression; Inhalation Exposure; Lung; Nanotubes, Carbon; Rats
PubMed: 35648795
DOI: 10.1080/08958378.2022.2081386 -
Biomedicine & Pharmacotherapy =... Jul 2022Adaptive immunity relies on the efficient recruitment of T cells from the blood into peripheral tissues. However, the current understanding of factor(s) coordinating...
Adaptive immunity relies on the efficient recruitment of T cells from the blood into peripheral tissues. However, the current understanding of factor(s) coordinating these events is incomplete. Previous studies on galectin-9 (Gal-9), have proposed a functionally significant role for this lectin in mediating leukocyte adhesion and transmigration. However, very little is known about its function in T cell migration. Here, we have investigated the role of the Gal-9 on the migration behaviour of both human primary CD4 and CD8 T cells. Our data indicate that Gal-9 supports both CD4 and CD8 T cell adhesion and transmigration in a glycan dependent manner, inducing L-selectin shedding and upregulation of LFA-1 and CXCR4 expression. Additionally, when immobilized, Gal-9 promoted capture and firm adhesion of T cells under flow, in a glycan and integrin-dependent manner. Using an in vivo model, dorsal air pouch, we found that Gal-9 deficient mice display impaired leukocyte trafficking, with a reduction in pro-inflammatory cytokines/chemokines generated locally. Furthermore, we also demonstrate that Gal-9 inhibits the chemotactic function of CXCL12 through direct binding. In conclusion, our study characterises, for the first time, the capture, adhesion, and migration behaviour of CD4 and CD8 T cells to immobilised /endothelial presented Gal-9, under static and physiological flow conditions. We also demonstrate the differential binding characteristics of Gal-9 to T cell subtypes, which could be of potential therapeutic significance, particularly in the treatment of inflammatory-based diseases, given Gal-9 ability to promote apoptosis in pathogenic T cell subsets.
Topics: Animals; CD8-Positive T-Lymphocytes; Galectins; Integrins; Mice; Polysaccharides; Transendothelial and Transepithelial Migration
PubMed: 35643073
DOI: 10.1016/j.biopha.2022.113171 -
Medicina (Kaunas, Lithuania) Apr 2022: It has been demonstrated that Egfl7 promotes tumor cell escape from immunity by downregulating the activation of tumor blood vessels. : to analyze mRNA expression of...
: It has been demonstrated that Egfl7 promotes tumor cell escape from immunity by downregulating the activation of tumor blood vessels. : to analyze mRNA expression of within the tumor microenvironment of high-grade ovarian serous carcinoma and its association with a number of intraepithelial CD4+/CD8+ lymphocytes and ICAM-1 expression. qPCR analysis of mRNA in cancer cells and adjacent stromal endothelium microdissected from formalin-fixed paraffin-embedded tumors of 59 high-grade ovarian serous carcinoma patients, was performed. Infiltration of intraepithelial lymphocytes (CD4+/CD8+) and expression of ICAM-1 were evaluated by immunohistochemistry and compared between tumors with different statuses of expression. : was expressed in cancer cells (9/59, 15.25%), endothelium (8/59, 13.56%), or both cancer cells and adjacent endothelium (4/59, 6.78%). ICAM-1 was expressed on cancer cells (47/59, 79.66%), stromal endothelium (46/59, 77.97%), or both epithelium and endothelium (40 of 59, 67.8%). -positivity of cancer cells and endothelium was associated with lower intraepithelial inflow of CD4+ ( = 0.022 and = 0.029, respectively) and CD8+ lymphocytes ( = 0.004 and = 0.031, respectively) but impact neither epithelial nor endothelial ICAM-1 expression ( = 0.098 and = 0.119, respectively). The patients' median follow-up was 23.83 months (range 1.07-78.07). Lack of prognostic significance of -status and ICAM-1 expression was notified. : is activated in the cancer cells as frequently as in the endothelium of human high-grade ovarian serous carcinoma. Activation of in cancer cells and/or endothelial cells could negatively impact diapedesis regardless of localization.
Topics: CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Calcium-Binding Proteins; Cystadenocarcinoma, Serous; EGF Family of Proteins; Endothelial Cells; Female; Humans; Intercellular Adhesion Molecule-1; Intercellular Signaling Peptides and Proteins; Ovarian Neoplasms; RNA, Messenger; Tumor Microenvironment
PubMed: 35630004
DOI: 10.3390/medicina58050588 -
Biomedicines Apr 2022Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into...
Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into peripheral organs. Neutrophil extravasation into tissues depends on actin remodeling and actin-binding proteins such as cortactin, which is expressed ubiquitously, except for neutrophils. Endothelial cortactin is necessary for proper regulation of neutrophil transendothelial migration and recruitment to sites of infection. We therefore hypothesised that cortactin plays a crucial role in sepsis development by regulating neutrophil trafficking. Using a murine model of sepsis induced by cecal ligation and puncture (CLP), we showed that cortactin-deficient (KO) mice survive better due to reduced lung injury. Histopathological analysis of lungs from septic KO mice revealed absence of oedema, reduced vascular congestion and mucus deposition, and better-preserved alveoli compared to septic wild-type (WT) mice. Additionally, sepsis-induced cytokine storm, excessive neutrophil infiltration into the lung and oxidative stress were significantly reduced in KO mice. Neutrophil depletion 12 h after sepsis improved survival in WT mice by averting lung injury, similar to both neutrophil-depleted and non-depleted KO mice. Our findings highlight a critical role of cortactin for lung neutrophil infiltration and sepsis severity.
PubMed: 35625756
DOI: 10.3390/biomedicines10051019