Anti-Inflammatory Activity of Ferula assafoetida Oleo-Gum-Resin (Asafoetida) against TNF-α-Stimulated Human Umbilical Vein Endothelial Cells (HUVECs).

Inflammation is the body's biological reaction to endogenous and exogenous stimuli. Recent studies have demonstrated several anti-inflammatory properties of Ferula species. In this paper, we decided to study the anti-inflammatory effect of ethanolic extract of Ferula assafoetida oleo-gum-resin (asafoetida) against TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in a flat-bottom plate and then treated with ethanolic extract of asafoetida (EEA, 0-500 µg/ml) and TNF-α (0-100 ng/ml) for 24 h. We used the MTT test to assess cell survival. In addition, the LC-MS analysis was performed to determine the active substances. HUVECs were pretreated with EEA and then induced by TNF-α. Intracellular reactive oxygen species (ROS) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs were evaluated with DCFH-DA and CFSE fluorescent probes, respectively. Gene expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin and surface expression of ICAM-1 protein were measured using real-time PCR and flow cytometry methods, respectively. While TNF-α significantly increased intracellular ROS formation and PBMC adhesion to TNF-α-induced HUVECs, the pretreatment of HUVECs with EEA (125 and 250 µg/ml) significantly reduced the parameters. In addition, EEA pretreatment decreased TNF-α-induced mRNA expression of VCAM-1 and surface protein expression of ICAM-1 in the target cells. Taken together, the results indicated that EEA prevented ROS generation, triggered by TNF-α, and inhibited the expression of VCAM-1 and ICAM-1, leading to reduced PBMC adhesion. These findings suggest that EEA can probably have anti-inflammatory properties.

Blocking of inflammatory heparan sulfate domains by specific antibodies is not protective in experimental glomerulonephritis.

Glomerulonephritis is an acquired serious glomerular disease, which involves the interplay of many factors such as cytokines, chemokines, inflammatory cells, and heparan sulfate (HS). We previously showed that blocking of inflammatory heparan sulfate domains on cultured glomerular endothelium by specific anti-HS single chain antibodies reduced polymorphonuclear cell (PMN) adhesion and chemokine binding. We hypothesized that injection of anti-HS antibodies in PMN-driven experimental glomerulonephritis should reduce glomerular influx of PMNs and thereby lead to a better renal outcome. In contrast to our hypothesis, co-injection of anti-HS antibodies did not alter the final outcome of anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis. Glomerular PMN influx, normally peaking 2 hours after induction of glomerulonephritis with anti-GBM IgG was not reduced by co-injection of anti-HS antibodies. Four days after induction of glomerulonephritis, albuminuria, renal function, glomerular hyalinosis and fibrin deposition were similar in mice treated and not treated with anti-HS antibodies. Interestingly, we observed transient effects in mice co-injected with anti-HS antibodies compared to mice that did not receive anti-HS antibodies: (i) a decreased renal function 2 hours and 1 day after induction of glomerulonephritis; (ii) an increased albuminuria after 2 hours and 1 day; (iii) an increased glomerular fibrin deposition after 1 day; (iv) a reduced glomerular macrophage influx after 1 day; (v) a sustained glomerular presence of PMNs at day 1 and 4, accompanied by an increased renal expression of IL-6, CXCL1, ICAM-1, L-selectin, CD11b and NF-κB. The mechanism underlying these observations induced by anti-HS antibodies remains unclear, but may be explained by a temporarily altered glycocalyx and/or altered function of PMNs due to the binding of anti-HS antibodies. Nevertheless, the evaluated anti-HS antibodies do not show therapeutic potential in anti-GBM-induced glomerulonephritis. Future research should evaluate other strategies to target HS domains involved in inflammatory processes during glomerulonephritis.

Characterization, isolation, and in vitro culture of leptomeningeal fibroblasts.

Meninges, or the membranous coverings of the brain and spinal cord, play host to dozens of morbid pathologies. In this study we provide a method to isolate the leptomeningeal cell layer, identify leptomeninges in histologic slides, and maintain leptomeningeal fibroblasts in in vitro culture. Using an array of transcriptomic, histological, and cytometric analyses, we identified ICAM1 and SLC38A2 as two novel markers of leptomeningeal cells in vivo and in vitro. Our results confirm the fibroblastoid nature of leptomeningeal cells and their ability to form a sheet-like layer that covers the brain and spine parenchyma. These findings will enable researchers in central nervous system barriers to describe leptomeningeal cell functions in health and disease.

Dendrobium catenatum Lindl. Water Extracts Attenuate Atherosclerosis.

OBJECTIVES: Dendrobium catenatum Lindl. (DH) is a Chinese herbal medicine, which is often used to make tea to improve immunity in China. Rumor has it that DH has a protective effect against cardiovascular disease. However, it is not clear how DH can prevent cardiovascular disease, such as atherosclerosis (AS). Therefore, the purpose of this study is to study whether DH can prevent AS and the underlying mechanisms. METHODS: Zebrafish larvae were fed with high-cholesterol diet (HCD) to establish a zebrafish AS model. Then, we used DH water extracts (DHWE) to pretreat AS zebrafish. The plaque formation was detected by HE, EVG, and oil red O staining. Neutrophil and macrophage counts were calculated to evaluate the inflammation level. Reactive oxygen species (ROS) activity, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity in zebrafish were measured to reflect oxidative stress. The cholesterol accumulation and the levels of lipid, triglyceride (TG), and total cholesterol (TC) were measured to reflect lipid metabolism disorder. Then, parallel flow chamber was utilized to establish a low shear stress- (LSS-) induced endothelial cell (EC) dysfunction model. EA.hy926 cells were exposed to LSS (3 dyn/cm2) for 30 min and treated with DHWE. The levels of ROS, SOD, MDA, glutathione (GSH), and glutathiol (GSSG) in EA.hy926 cells were analysed to determine oxidative stress. The release of nitric oxide (NO), endothelin-1 (ET-1), and epoprostenol (PGI2) in EA.hy926 cells was measured to reflect EC dysfunction. The mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in EA.hy926 cells was detected to reflect EC dysfunction inflammation. RESULTS: The results showed that DHWE significantly reduced cholesterol accumulation and macrophage infiltration in early AS. Finally, DHWE significantly alleviate the lipid metabolism disorder, oxidative stress, and inflammation to reduce the plaque formation of AS zebrafish larval model. Meanwhile, we also found that DHWE significantly improved LSS-induced EC dysfunction and oxidative stress in vitro. CONCLUSION: Our results indicate that DHWE could be used as a prevention method to prevent AS.

The zinc finger transcription factor, KLF2, protects against COVID-19 associated endothelial dysfunction.

Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its patho-mechanism being incompletely understood. Emerging evidence has demonstrated that endothelial dysfunction precipitates COVID-19 and its accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. The objective of the present study is to evaluate whether kruppel-like factor 2 (KLF2), a master regulator of vascular homeostasis, represents a therapeutic target for COVID-19-induced endothelial dysfunction. Here, we demonstrate that the expression of KLF2 was reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1ß and TNF-α, two cytokines elevated in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Pharmacologic (atorvastatin and tannic acid) and genetic (adenoviral overexpression) approaches to augment KLF2 levels attenuated COVID-19-serum-induced increase in endothelial inflammation and monocyte adhesion. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis, and reduced angiogenesis). Finally, knockdown of KLF2 partially reversed the ameliorative effect of atorvastatin on COVID-19-serum-induced endothelial inflammation and monocyte adhesion. Collectively, the present study implicates loss of KLF2 as an important molecular event in the development of COVID-19-induced vascular disease and suggests that efforts to augment KLF2 levels may be therapeutically beneficial.

Studying dynamic stress effects on the behaviour of THP-1 cells by microfluidic channels.

Atherosclerosis is a long-term disease process of the vascular system that is characterized by the formation of atherosclerotic plaques, which are inflammatory regions on medium and large-sized arteries. There are many factors contributing to plaque formation, such as changes in shear stress levels, rupture of endothelial cells, accumulation of lipids, and recruitment of leukocytes. Shear stress is one of the main factors that regulates the homeostasis of the circulatory system; therefore, sudden and chronic changes in shear stress may cause severe pathological conditions. In this study, microfluidic channels with cavitations were designed to mimic the shape of the atherosclerotic blood vessel, where the shear stress and pressure difference depend on design of the microchannels. Changes in the inflammatory-related molecules ICAM-1 and IL-8 were investigated in THP-1 cells in response to applied shear stresses in an continuous cycling system through microfluidic channels with periodic cavitations. ICAM-1 mRNA expression and IL-8 release were analyzed by qRT-PCR and ELISA, respectively. Additionally, the adhesion behavior of sheared THP-1 cells to endothelial cells was examined by fluorescence microscopy. The results showed that 15 Pa shear stress significantly increases expression of ICAM-1 gene and IL-8 release in THP-1 cells, whereas it decreases the adhesion between THP-1 cells and endothelial cells.

Protective Effects of Thymoquinone, an Active Compound of Nigella sativa, on Rats with Benzo(a)pyrene-Induced Lung Injury through Regulation of Oxidative Stress and Inflammation.

Benzopyrene [B(a)P] is a well-recognized environmental carcinogen, which promotes oxidative stress, inflammation, and other metabolic complications. In the current study, the therapeutic effects of thymoquinone (TQ) against B(a)P-induced lung injury in experimental rats were examined. B(a)P used at 50 mg/kg b.w. induced lung injury that was investigated via the evaluation of lipid profile, inflammatory markers, nitric oxide (NO), and malondialdehyde (MDA) levels. B(a)P also led to a decrease in superoxide dismutase (SOD) (34.3 vs. 58.5 U/mg protein), glutathione peroxidase (GPx) (42.4 vs. 72.8 U/mg protein), catalase (CAT) (21.2 vs. 30.5 U/mg protein), and total antioxidant capacity compared to normal animals. Treatment with TQ, used at 50 mg/kg b.w., led to a significant reduction in triglycerides (TG) (196.2 vs. 233.7 mg/dL), total cholesterol (TC) (107.2 vs. 129.3 mg/dL), and inflammatory markers and increased the antioxidant enzyme level in comparison with the group that was administered B(a)P only (p < 0.05). B(a)P administration led to the thickening of lung epithelium, increased inflammatory cell infiltration, damaged lung tissue architecture, and led to accumulation of collagen fibres as studied through haematoxylin and eosin (H&E), Sirius red, and Masson's trichrome staining. Moreover, the recognition of apoptotic nuclei and expression pattern of NF-κB were evaluated through the TUNEL assay and immunohistochemistry, respectively. The histopathological changes were found to be considerably low in the TQ-treated animal group. The TUNEL-positive cells increased significantly in the B(a)P-induced group, whereas the TQ-treated group showed a decreased apoptosis rate. Significantly high cytoplasmic expression of NF-κB in the B(a)P-induced group was seen, and this expression was prominently reduced in the TQ-treated group. Our results suggest that TQ can be used in the protection against benzopyrene-caused lung injury.

Deoxycholic acid enhancement of lymphocyte migration through direct interaction with the intestinal vascular endothelium.

BACKGROUND AND AIM: The small intestine plays a central role in gut immunity, and enhanced lymphocyte migration is involved in the pathophysiology of various enteropathy. Bile acid (BA) is closely related to lipid metabolism and gut microbiota and essential for gut homeostasis. However, the effects of BA on gut immunity have not been studied in detail, especially on the small intestine and lymphocyte migration. Therefore, we aimed to investigate the effect of BA on small intestinal lymphocyte microcirculation. METHODS: The effect of deoxycholic acid (DCA), taurocholic acid (tCA), or cholic acid (CA) on the indomethacin (IND)-induced small intestinal enteropathy in mice was investigated. Lymphocyte movements were evaluated after exposure to BA using intravital microscopy. The effects of BA on surface expression of adhesion molecules on the vascular endothelium and lymphocytes through BA receptors were examined in vitro. RESULTS: IND-induced small intestinal enteropathy was histologically aggravated by DCA treatment alone. The expression of adhesion molecules ICAM-1 and VCAM-1 was significantly enhanced by DCA. Exposure to DCA increased lymphocyte adhesion in the microvessels of the ileum, which was partially blocked by anti-α4ß1 integrin antibody in vivo. The expression of ICAM-1 and VCAM-1 was significantly enhanced by DCA in vitro, which was partially suppressed by the sphingosine-1-phosphate receptor 2 (S1PR2) antagonist. The S1PR2 antagonist significantly ameliorated IND-induced and DCA-exaggerated small intestinal injury. CONCLUSION: DCA exacerbated IND-induced small intestinal enteropathy. DCA directly acts on the vascular endothelium and enhances the expression levels of adhesion molecules partially via S1PR2, leading to enhanced small intestinal lymphocyte migration.

α-Conidendrin inhibits the expression of intercellular adhesion molecule-1 induced by tumor necrosis factor-α in human lung adenocarcinoma A549 cells.

α-Conidendrin is a lignan isolated from Taxus wallichiana and other species. In the present study, we demonstrated that α-conidendrin inhibited the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-α (TNF-α) at an IC50 value of 40-60 µM in human lung adenocarcinoma A549 cells. α-Conidendrin decreased ICAM-1 protein and mRNA expression levels at concentrations of 40-100 µM in TNF-α-stimulated A549 cells. The TNF-α-induced mRNA expression of vascular cell adhesion molecule-1, E-selectin, and cyclooxygenase-2 was also reduced by α-conidendrin. In the TNF-α-induced nuclear factor κB (NF-κB) signaling pathway, α-conidendrin did not influence the translocation of the NF-κB subunit RelA from the cytoplasm to the nucleus at concentrations up to 100 µM. A chromatin immunoprecipitation assay revealed that α-conidendrin at 100 µM reduced the binding of RelA to the ICAM-1 promoter in response to a stimulation with TNF-α. Collectively, these results indicated that α-conidendrin interfered with the DNA binding of RelA to the ICAM-1 promoter, thereby reducing ICAM-1 transcription.

Effect of Tribulus Terrestris L. on Expression of ICAM-1, VCAM-1, E-Selectin and Proteome Profile of Human Endothelial Cells In-Vitro.

BACKGROUND: Atherosclerosis is a chronic inflammation that interferes with blood arteries functions due to the accumulation of low density lipids and cholesterol. OBJECTIVE: To investigate the effect of aqueous extract and saponin fraction of Tribulus terrestris L. (TT) on the proteome and expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in the human umbilical vein endothelial cell (HUVEC) and human bone marrow endothelial cell (HBMEC) lines. METHODS: Two cell lines were cultured and induced with lipopolysaccharide (LPS). The primed cells were then treated with aqueous extract and saponin fraction of TT. The protein profile of the endothelial cells was assessed under normal and LPS-induced conditions using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 2D gel electrophoresis (2-DE). The levels of VCAM-1, ICAM-1, and E-selectin were estimated by use of western blotting. RESULTS: LPS-induced HUVECs and HBMECs were shown to significantly increase the expression of ICAM-1, VCAM-1, and E-selectin in comparison to control groups. Our findings revealed that TT extract resulted in significantly more reduced levels of proteome (80 spots) as well as all the three mentioned proteins compared with the effect of saponin fraction alone. CONCLUSION: TT extract and its saponin fraction exerted anti-inflammatory effects on HUVEC and HBMEC lines and reduced the expression of ICAM-1, VCAM-1, and E-selectin. However, the anti-inflammatory effect of aqueous extract was greater than that of saponin fraction. Therefore, TT could be considered as a potential candidate for the treatment or prevention of atherosclerosis.

The Roles of Lipoprotein in Psoriasis.

The association between psoriasis and cardiovascular disease risk has been supported by recent epidemiological data. Patients with psoriasis have an increased adjusted relative risk for myocardial infarction. As such, the cardiovascular risk conferred by severe psoriasis may be comparable to what is seen with other well-established risk factors, such as diabetes mellitus. Previous studies demonstrated that low-density lipoprotein (LDL) plays critical roles during atherogenesis. It may be caused by the accumulation of macrophages and lipoprotein in the vessel wall. Oxidized LDL (ox-LDL) stimulates the expression of adhesion molecules, such as ICAM-1 and VCAM-1, on endothelial cells and increases the attachment of mononuclear cells and the endothelium. Even though previous evidence demonstrated that psoriasis patients have tortuous and dilated blood vessels in the dermis, which results in the leakage of ox-LDL, the leaked ox-LDL may increase the expression of adhesion molecules and cytokines, and disturb the static balance of osmosis. Therefore, exploration of the relationship between hyperlipidemia and psoriasis may be another novel treatment option for psoriasis and may represent the most promising strategy.

Gliclazide attenuates acetic acid-induced colitis via the modulation of PPARγ, NF-κB and MAPK signaling pathways.

Ulcerative Colitis is a universal autoimmune disease with high incidence rates worldwide. It is characterized by the existence of many other concurrent immune-associated ailments, including diabetes. The used strategies for the management of this highly costing and complicated disease face great challenges. Therefore, the urge for new medication with fewer side effects and high efficacy is growing. The peroxisome proliferator-activated receptor-gamma (PPARγ) and nuclear factor Kappa-B (NF-κB) can be considered as crucial targets for the treatment of ulcerative colitis. Several studies reported the antioxidants, anti-inflammatory, and antiapoptotic actions of gliclazide and evaluated its cardioprotective and renoprotective effects. However, its impact on ulcerative colitis has never been investigated. This study delineated the effect of gliclazide administration on ulcerative colitis induced by acetic acid in rats and the underlying molecular mechanisms. Gliclazide (10 mg/kg; p.o) prominently decreased colon tissue injury as assessed by the histopathological analysis as well as myeloperoxidase, and intercellular adhesion molecule-1 levels. Gliclazide significantly alleviated the proinflammatory mediator, IL-6, promoted the anti-inflammatory cytokine, IL-10 and, withheld oxidative stress in the injured colon tissues. The protective effect of gliclazide was mediated through the upregulation of PPARγ and downregulation of NF-κB expression. The diminution of ulcerative colitis was also accompanied by an inhibition of the elevated activity and expression of mitogen-activated protein kinases and caspase-3 as assessed by Western blot and immunohistochemistry, respectively. Our findings spotlight, for the first time, the potential of the antidiabetic agent, gliclazide, to attenuate the experimentally induced ulcerative colitis. Therefore, gliclazide might be a propitious agent for the management of ulcerative colitis in diabetic patients.

A Novel Subset of CD95+ Pro-Inflammatory Macrophages Overcome miR155 Deficiency and May Serve as a Switch From Metabolically Healthy Obesity to Metabolically Unhealthy Obesity.

Metabolically healthy obesity (MHO) accounts for roughly 35% of all obese patients. There is no clear consensus that has been reached on whether MHO is a stable condition or merely a transitory period between metabolically healthy lean and metabolically unhealthy obesity (MUO). Additionally, the mechanisms underlying MHO and any transition to MUO are not clear. Macrophages are the most common immune cells in adipose tissues and have a significant presence in atherosclerosis. Fas (or CD95), which is highly expressed on macrophages, is classically recognized as a pro-apoptotic cell surface receptor. However, Fas also plays a significant role as a pro-inflammatory molecule. Previously, we established a mouse model (ApoE-/-/miR155-/-; DKO mouse) of MHO, based on the criteria of not having metabolic syndrome (MetS) and insulin resistance (IR). In our current study, we hypothesized that MHO is a transition phase toward MUO, and that inflammation driven by our newly classified CD95+CD86- macrophages is a novel mechanism for this transition. We found that, with extended (24 weeks) high-fat diet feeding (HFD), MHO mice became MUO, shown by increased atherosclerosis. Mechanistically, we found the following: 1) at the MHO stage, DKO mice exhibited increased pro-inflammatory markers in adipose tissue, including CD95, and serum; 2) total adipose tissue macrophages (ATMs) increased; 3) CD95+CD86- subset of ATMs also increased; and 4) human aortic endothelial cells (HAECs) were activated (as determined by upregulated ICAM1 expression) when incubated with conditioned media from CD95+-containing DKO ATMs and human peripheral blood mononuclear cells-derived macrophages in comparison to respective controls. These results suggest that extended HFD in MHO mice promotes vascular inflammation and atherosclerosis via increasing CD95+ pro-inflammatory ATMs. In conclusion, we have identified a novel molecular mechanism underlying MHO transition to MUO with HFD. We have also found a previously unappreciated role of CD95+ macrophages as a potentially novel subset that may be utilized to assess pro-inflammatory characteristics of macrophages, specifically in adipose tissue in the absence of pro-inflammatory miR-155. These findings have provided novel insights on MHO transition to MUO and new therapeutic targets for the future treatment of MUO, MetS, other obese diseases, and type II diabetes.

Changes in microparticle profiles by vitamin D receptor activation in chronic kidney disease - a randomized trial.

BACKGROUND: Microparticles (MPs) are biomarkers and mediators of disease through their expression of surface receptors, reflecting activation or stress in their parent cells. Endothelial markers, ICAM-1 and VCAM-1, are implicated in atherosclerosis and associated with cardiovascular risk. Chronic kidney disease (CKD) patients have endothelial dysfunction and high levels of endothelial derived MPs. Vitamin D treatment has been reported to ameliorate endothelial function in CKD patients. We aimed to examine cell specific MP profiles and concentrations of MPs expressing the atherosclerotic markers ICAM-1 and VCAM-1 after treatment with paricalcitol in patients with CKD stage 3-4. METHODS: Sub-study of the previously reported SOLID trial where 36 patients were randomly assigned to placebo, 1 or 2 µg paricalcitol, for 12 weeks. MPs were measured by flow cytometry after labelling with antibodies against endothelial (CD62E), platelet (CD62P, CD41, CD154) leukocyte (CD45) and vascular (CD54, CD106) markers. RESULTS: Patients had a mean age of 65 years with a mean eGFR of 40 mL/min/1.73m2. Concentrations of ICAM-1 positive MPs were significantly reduced by treatment (repeated measures ANOVA p = 0.04). Repeated measures MANOVA of concentrations of endothelial, platelet and leukocyte MPs showed sustained levels in the 2 µg treatment group (p = 0.85) but a decline in the 1 µg (p = 0.04) and placebo groups (p = 0.005). CONCLUSIONS: Treatment with paricalcitol reduces concentrations of ICAM-1 positive MPs. This is accompanied by sustained concentrations of all cell specific MPs in the 2 µg group, and decreasing concentrations in the other groups, possibly due to a more healthy and reactive endothelium with paricalcitol treatment.

Human T-Cell Leukemia Virus Type 1 (HTLV-1) bZIP Factor Upregulates the Expression of ICAM-1 To Facilitate HTLV-1 Infection.

Human T-cell leukemia virus type 1 (HTLV-1) causes multiple pathological effects, ranging from a form of leukemia to a spectrum of inflammation-mediated diseases. These diseases arise from one or several infected CD4+ T cells among thousands acquiring proliferation and survival advantages and ultimately becoming pathogenic. Given the low incidence of HTLV-1-associated diseases among carriers, such cellular evolutionary processes appear to occur rarely. Therefore, infectious spread of HTLV-1 within the T-cell population may be one underlying factor influencing disease development. Free HTLV-1 virions are poorly infectious, so infection of T cells relies on direct contact between infected and target cells. Following contact, virions pass to target cells through a virological synapse or cellular conduits or are transferred to target cells within an extracellular matrix. Lymphocyte functioning antigen 1 (LFA-1) on the surface of the target cell engaging with its ligand, ICAM-1, on the surface of the infected cell (effector cell) initiates and stabilizes cell-cell contact for infection. We found that stable expression of an HTLV-1 accessory protein, HTLV-1 bZIP factor (HBZ), in Jurkat T cells increases homotypic aggregation. This phenotype was attributed to elevated ICAM-1 expression in the presence of HBZ. Using a single-cycle replication-dependent luciferase assay, we found that HBZ expression in Jurkat cells (used as effector cells) increases HTLV-1 infection. Despite this effect, HBZ could not replace the critical infection-related functions of the HTLV-1 regulatory protein Tax. However, in HTLV-1-infected T cells, knockdown of HBZ expression did lead to a decrease in infection efficiency. These overall results suggest that HBZ contributes to HTLV-1 infectivity.IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) causes a variety of diseases, ranging from a fatal form of leukemia to immune-mediated inflammatory diseases. These diseases occur rarely, arising from one or a small subset of virally infected cells infrequently evolving into a pathogenic state. Thus, the process of HTLV-1 cell-to-cell transmission within the host helps influence the probability of disease development. HTLV-1 primarily infects T cells and initially spreads within this cell population when virally infected T cells dock to uninfected target T cells and then transfer HTLV-1 virus particles to the target cells. Here we found that the viral protein HTLV-1 bZIP factor (HBZ) promotes infectivity. HBZ accomplishes this task by increasing the surface abundance of a cellular adhesion protein known as intercellular adhesion molecule 1 (ICAM-1), which helps initiate and stabilize contact (docking) between infected and target T cells. These results define a novel and unexpected function of HBZ, diverging from its defined functions in cellular survival and proliferation.

ICAM-1 promotes the abnormal endothelial cell phenotype in chronic thromboembolic pulmonary hypertension.

BACKGROUND: Pulmonary endothelial cells play a key role in the pathogenesis of Chronic Thromboembolic Pulmonary Hypertension (CTEPH). Increased synthesis and/or the release of intercellular adhesion molecule-1 (ICAM-1) by pulmonary endothelial cells of patients with CTEPH has been recently reported, suggesting a potential role for ICAM-1 in CTEPH. METHODS: We studied pulmonary endarterectomy specimens from 172 patients with CTEPH and pulmonary artery specimens from 97 controls undergoing lobectomy for low-stage cancer without metastasis. RESULTS: ICAM-1 was overexpressed in vitro in isolated and cultured endothelial cells from endarterectomy specimens. Endothelial cell growth and apoptosis resistance were significantly higher in CTEPH specimens than in the controls (p < 0.001). Both abnormalities were abolished by pharmacological inhibition of ICAM-1 synthesis or activity. The overexpression of ICAM-1 contributed to the acquisition and maintenance of abnormal EC growth and apoptosis resistance via the phosphorylation of SRC, p38 and ERK1/2 and the overproduction of survivin. Regarding the ICAM-1 E469K polymorphism, the KE heterozygote genotype was significantly more frequent in CTEPH than in the controls, but it was not associated with disease severity among patients with CTEPH. CONCLUSIONS: ICAM-1 contributes to maintaining the abnormal endothelial cell phenotype in CTEPH.

The HaCaT/THP-1 Cocultured Activation Test (COCAT) for skin sensitization: a study of intra-lab reproducibility and predictivity.

The Cocultured Activation Test (COCAT) consists of cocultured HaCaT (human keratinocyte cell line) and THP-1 cells (surrogate of antigen presenting cells). Individually, these cell lines are used to address key event 2 and 3 of the skin sensitization Adverse Outcome Pathway (AOP). Their exposure in coculture was found to have the potential to increase their response to sensitizing chemicals, enable the detection of pro-haptens and support the identification of skin sensitization potency. The present study was undertaken to assess the predictive capacity of COCAT to both skin sensitization hazard and potency and to assess the intra-laboratory reproducibility of COCAT based on the blind testing of chemicals. Results showed a reproducibility between runs of 80 % for 15 coded chemicals. 100 % sensitivity (9/9), 75 % specificity (3/4) and 92.3 % accuracy (12/13) was found for skin sensitization hazard prediction, while the tests of two chemicals were inconclusive. Including additional chemicals tested during the optimization phase in addition to the blind tested chemicals, the skin sensitization UN GHS sub-categories were correctly predicted for 85.7 % (12/14) Sub-category 1A chemicals, 83.3 % (10/12) Sub-category 1B chemicals and 92.3 % (12/13) 'No Category' chemicals, resulting in an overall accuracy of 87.4 % (34/39). The present study shows the COCAT to be a promising method for the identification of skin sensitization hazard and potency sub-categorization according to the UN GHS classification.

Meningitic Escherichia coli-induced upregulation of PDGF-B and ICAM-1 aggravates blood-brain barrier disruption and neuroinflammatory response.

BACKGROUND: Blood-brain barrier (BBB) disruption and neuroinflammation are considered key mechanisms of pathogenic Escherichia coli invasion of the brain. However, the specific molecules involved in meningitic E. coli-induced BBB breakdown and neuroinflammatory response remain unclear. Our previous RNA-sequencing data from human brain microvascular endothelial cells (hBMECs) revealed two important host factors: platelet-derived growth factor-B (PDGF-B) and intercellular adhesion molecule-1 (ICAM-1), which were significantly upregulated in hBMECs after meningitic E. coli infection. Whether and how PDGF-B and ICAM-1 contribute to the development of E. coli meningitis are still unclear. METHODS: The western blot, real-time PCR, enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence were applied to verify the significant induction of PDGF-B and ICAM-1 by meningitic E. coli in vivo and in vitro. Evan's blue assay and electric cell-substrate impedance sensing assay were combined to identify the effects of PDGF-B on BBB permeability. The CRISPR/Cas9 technology, cell-cell adhesion assay, and electrochemiluminescence assay were used to investigate the role of ICAM-1 in neuroinflammation subversion. RESULTS: We verified the significant induction of PDGF-B and ICAM-1 by meningitic E. coli in mouse as well as monolayer hBMECs models. Functionally, we showed that the increase of PDGF-B may directly enhance the BBB permeability by decreasing the expression of tight junction proteins, and the upregulation of ICAM-1 contributed to neutrophils or monocytes recruitment as well as neuroinflammation subversion in response to meningitic E. coli infection. CONCLUSIONS: Our findings demonstrated the roles of PDGF-B and ICAM-1 in mediating bacterial-induced BBB damage as well as neuroinflammation, providing new concepts and potential targets for future prevention and treatment of bacterial meningitis.

The bacterial protein CNF1 as a new strategy against Plasmodium falciparum cytoadherence.

Plasmodium falciparum severe malaria causes more than 400,000 deaths every year. One feature of P. falciparum-parasitized erythrocytes (pRBC) leading to cerebral malaria (CM), the most dangerous form of severe malaria, is cytoadherence to endothelium and blockage of the brain microvasculature. Preventing ligand-receptor interactions involved in this process could inhibit pRBC sequestration and insurgence of severe disease whilst reversing existing cytoadherence could be a saving life adjunct therapy. Increasing evidence indicate the endothelial Rho signaling as a crucial player in malaria parasite cytoadherence. Therefore, we have used the cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli protein able to modulate the activity of Cdc42, Rac, and Rho, three subfamilies of the Rho GTPases family, to study interactions between infected erythrocytes and cerebral endothelium in co-culture models. The main results are that CNF1 not only prevents cytoadherence but, more importantly, induces the detachment of pRBCs from endothelia monolayers. We first observed that CNF1 does affect neither parasite growth, nor the morphology and concentration of knobs that characterize the parasitized erythrocyte surface, as viewed by scanning electron microscopy. On the other hand, flow cytometry experiments show that cytoadherence reversion induced by CNF1 occurs in parallel with a decreased ICAM-1 receptor expression on the cell surface, suggesting the involvement of a toxin-promoted endocytic activity in such a response. Furthermore, since the endothelial barrier functionality is compromised by P. falciparum, we conducted a permeability assay on endothelial cells, revealing the CNF1 capacity to restore the brain endothelial barrier integrity. Then, using pull-down assays and inhibitory studies, we demonstrated, for the first time, that CNF1 is able not only to prevent but also to cause the parasite detachment by simultaneously activating Rho, Rac and Cdc42 in endothelial cells. All in all our findings indicate that CNF1 may represent a potential novel therapeutic strategy for preventing neurological complications of CM.

In vitro pulmonary and vascular effects induced by different diesel exhaust particles.

Diesel exhaust particles (DEP) are responsible for both respiratory and cardiovascular effects. However many questions are still unravelled and the mechanisms behind the health effects induced by the exposure to ultrafine particles (UFP) need further investigations. Furthermore, different emission sources can lead to diverse biological responses. In this perspective, here we have compared the effects of three DEPs, two standard reference materials (SRM 1650b and 2975) and one DEP directly sampled from a EuroIV vehicle without Diesel Particulate Filter (DPF). For the biological investigations, different in vitro lung models involving both epithelial and vascular endothelial cells, were used. Cell viability, oxidative stress, inflammation, DNA damage and endothelial activation markers were investigated at sub-cytotoxic DEP doses. The data obtained have shown that only DEP EuroIV, which had the major content of polycyclic aromatic hydrocarbons (PAHs) and metals, was able to induce oxidative stress, inflammation and consequent endothelial activation, as demonstrated by the expression of adhesion molecules (ICAM-1 and VCAM-1) and the release of inflammatory markers (IL-8) from endothelial cells. Standard reference materials were not effective under our experimental conditions. These data suggest that oxidative stress, endothelial activation and systemic inflammatory cytokines release are crucial events after DEP exposure and that the source of DEP emission, responsible of the particle chemical fingerprint, may have a key role in the resulting adverse biological outcomes.