Fractalkine Elicits Chemotactic, Phenotypic, and Functional Effects on CX3CR1+CD27- NK Cells in Obesity-Associated Cancer.

Esophagogastric adenocarcinomas (EAC) are obesity-associated malignancies underpinned by severe immune dysregulation and inflammation. Our previous work indicates that NK cells migrate to EAC omentum, where they undergo phenotypic and functional alterations and apoptosis. In this study, we investigate whether such erroneous chemotaxis to omentum is paralleled by compromised NK cell infiltration of EAC patient tumor and examine the role of the inflammatory chemokine fractalkine in shaping the NK cell-mediated response. Our data show diminished NK cell frequencies in EAC tumor compared with those in the circulation and reveal that intratumoral NK cell frequencies decline as visceral obesity increases in EAC patients. Our in vitro findings demonstrate that antagonism of fractalkine receptor CX3CR1 significantly reduces NK cell migration to EAC patient-derived, omental adipose tissue-conditioned media, but not toward tumor-conditioned media. These data suggest fractalkine is a key driver of NK cell chemotaxis to omentum but has a lesser role in NK cell homing to tumor in EAC. We propose that this may offer a novel therapeutic strategy to limit NK cell depletion in the omentum of obese EAC patients, and our data suggest the optimal timing for CX3CR1 antagonism is after neoadjuvant chemoradiotherapy. Our functional studies demonstrate that fractalkine induces the conversion from CX3CR1+CD27- to CX3CR1-CD27+ NK cells and increases their IFN-γ and TNF-α production, indicative of its role in shaping the dominant NK cell phenotype in EAC omentum. This study uncovers crucial and potentially druggable pathways underpinning NK cell dysfunction in obesity-associated cancer and provides compelling insights into fractalkine's diverse biological functions.

A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

The introduction of immune checkpoint inhibitors has demonstrated significant improvements in survival for subsets of cancer patients. However, they carry significant and sometimes life-threatening toxicities. Prompt prediction and monitoring of immune toxicities have the potential to maximise the benefits of immune checkpoint therapy. Herein, we develop a digital nanopillar SERS platform that achieves real-time single cytokine counting and enables dynamic tracking of immune toxicities in cancer patients receiving immune checkpoint inhibitor treatment - broader applications are anticipated in other disease indications. By analysing four prospective cytokine biomarkers that initiate inflammatory responses, the digital nanopillar SERS assay achieves both highly specific and highly sensitive cytokine detection down to attomolar level. Significantly, we report the capability of the assay to longitudinally monitor 10 melanoma patients during immune inhibitor blockade treatment. Here, we show that elevated cytokine concentrations predict for higher risk of developing severe immune toxicities in our pilot cohort of patients.

Immunogenicity and inflammatory properties of respiratory syncytial virus attachment G protein in cotton rats.

Human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and young children worldwide. The attachment (G) protein of RSV is synthesized by infected cells in both a membrane bound (mG) and secreted form (sG) and uses a CX3C motif for binding to its cellular receptor. Cell culture and mouse studies suggest that the G protein mimics the cytokine CX3CL1 by binding to CX3CR1 on immune cells, which is thought to cause increased pulmonary inflammation in vivo. However, because these studies have used RSV lacking its G protein gene or blockade of the G protein with a G protein specific monoclonal antibody, the observed reduction in inflammation may be due to reduced virus replication and spread, and not to a direct role for G protein as a viral chemokine. In order to more directly determine the influence of the soluble and the membrane-bound forms of G protein on the immune system independent of its attachment function for the virion, we expressed the G protein in cotton rat lungs using adeno-associated virus (AAV), a vector system which does not itself induce inflammation. We found no increase in pulmonary inflammation as determined by histology and bronchoalveolar lavage after inoculation of AAVs expressing the membrane bound G protein, the secreted G protein or the complete G protein gene which expresses both forms. The long-term low-level expression of AAV-G did, however, result in the induction of non-neutralizing antibodies, CD8 T cells and partial protection from challenge with RSV. Complete protection was accomplished through co-immunization with AAV-G and an AAV expressing cotton rat interferon α.

Metabolic mediators determine the association of antinuclear antibody subtypes with specific clinical symptoms in systemic sclerosis.

PURPOSE: The aim of this study was to investigate the possible link between different types of systemic sclerosis-specific antinuclear antibodies, adipokines and endothelial molecules which were recently found to have a pathogenic significance in systemic sclerosis. MATERIALS/METHODS: Serum concentration of adiponectin, resistin, leptin, endothelin-1, fractalkine and galectin-3 were determined in the sera of patients with systemic sclerosis (n â€‹= â€‹100) and healthy controls (n â€‹= â€‹20) using ELISA. RESULTS: The following associations between antinuclear antibodies and increased serum concentrations were identified: anticentromere antibodies with endothelin-1 (p â€‹< â€‹0.0001; mean level in patients 2.21 vs control group 1.31 â€‹pg/ml), anti-topoisomerase I antibodies with fractalkine (p â€‹< â€‹0.0001; 3.68 vs 1.68 â€‹ng/ml) and galectin-3 (p â€‹= â€‹0.0010, 6.39 vs 3.26 â€‹ng/ml). Anti-RNA polymerase III antibodies were associated with increased resistin (p â€‹< â€‹0.0001; 15.13 vs 8.54 â€‹ng/ml) and decreased adiponectin (p â€‹< â€‹0.0001; 2894 vs 8847 â€‹ng/ml). CONCLUSION: In systemic sclerosis metabolic and vascular factors may serve as mediators between immunological abnormalities and non-immune driven clinical symptoms.

Evaluation of Proteoforms of the Transmembrane Chemokines CXCL16 and CX3CL1, Their Receptors, and Their Processing Metalloproteinases ADAM10 and ADAM17 in Proliferative Diabetic Retinopathy.

The transmembrane chemokine pathways CXCL16/CXCR6 and CX3CL1/CX3CR1 are strongly implicated in inflammation and angiogenesis. We investigated the involvement of these chemokine pathways and their processing metalloproteinases ADAM10 and ADAM17 in the pathophysiology of proliferative diabetic retinopathy (PDR). Vitreous samples from 32 PDR and 24 non-diabetic patients, epiretinal membranes from 18 patients with PDR, rat retinas, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to CXCL16-stimulated HRMECs was assessed. CXCL16, CX3CL1, ADAM10, ADAM17 and vascular endothelial growth factor (VEGF) levels were significantly increased in vitreous samples from PDR patients. The levels of CXCL16 were 417-fold higher than those of CX3CL1 in PDR vitreous samples. Significant positive correlations were found between the levels of VEGF and the levels of CXCL16, CX3CL1, ADAM10 and ADAM17. Significant positive correlations were detected between the numbers of blood vessels expressing CD31, reflecting the angiogenic activity of PDR epiretinal membranes, and the numbers of blood vessels and stromal cells expressing CXCL16, CXCR6, ADAM10 and ADAM17. CXCL16 induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB and VEGF in cultured Müller cells and tumor necrosis factor-α induced upregulation of soluble CXCL16 and ADAM17 in Müller cells. Treatment of HRMECs with CXCL16 resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and increased leukocyte adhesion to HRMECs. CXCL16 induced HRMEC proliferation, formation of sprouts from HRMEC spheroids and phosphorylation of ERK1/2. Intravitreal administration of CXCL16 in normal rats induced significant upregulation of the p65 subunit of NF-κB, VEGF and ICAM-1 in the retina. Our findings suggest that the chemokine axis CXCL16/CXCR6 and the processing metalloproteinases ADAM10 and ADAM17 might serve a role in the initiation and progression of PDR.

The miR-561-5p/CX3CL1 Signaling Axis Regulates Pulmonary Metastasis in Hepatocellular Carcinoma Involving CX3CR1+ Natural Killer Cells Infiltration.

Natural killer (NK) cell can inhibit tumor initiation and regulates metastatic dissemination, acting as key mediators of the innate immune response. Intrinsic factors modulating NK cells infiltration and its anticancer activity remain poorly characterized. We investigated the roles of dysregulation of micro(mi)RNAs and NK cells in progression of hepatocellular carcinoma (HCC). Methods: Small RNA sequencing were used to detect the miRNA profiles of tumor tissues from HCC patients with (n=14) or without (n=13) pulmonary metastasis and HCC cell lines with different pulmonary metastatic potentials. Chemokine expression profiling and bioinformatics were used to detect the downstream target of candidate target. In gain- and loss-of-function assays were used to investigate the role of miRNA in HCC progression. Different subsets of NK cells were isolated and used for chemotaxis and functional assays in vivo and in vitro. In situ hybridization and immunohistochemical analyses were performed to detect the expression of miRNA in tumor tissues from 242 HCC patients undergoing curative resection from 2010. Results: Three miRNAs (miR-137, miR-149-5p, and miR-561-5p) were identified to be associated with pulmonary metastasis in patients with HCC. miR-561-5p was most highly overexpressed in metastatic HCC tissues and high-metastatic-potential HCC cell lines. In gain- and loss-of-function assays in a murine model, miR-561-5p promoted tumor growth and spread to the lungs. Yet, miR-561-5p did not appear to affect cellular proliferation and migration in vitro. Bioinformatics and chemokine expression profiling identified chemokine (C-X3-C motif) ligand 1 (CX3CL1) as a potential target of miR-561-5p. Furthermore, miR-561-5p promoted tumorigenesis and metastasis via CX3CL1-dependent regulation of CX3CR1+ NK cell infiltration and function. CX3CR1+ NK cells demonstrated stronger in vivo anti-metastatic activity relative to CX3CR1- NK cells. CX3CL1 stimulated chemotactic migration and cytotoxicity in CX3CR1+ NK cells via STAT3 signaling. Blockade of CX3CL1, CX3CR1, or of pSTAT3 signaling pathways attenuated the antitumor responses. Clinical samples exhibited a negative correlation between miR-561-5p expression and levels of CX3CL1 and CX3CR1+ NK cells. High miR-561-5p abundance, low CX3CL1 levels, and low numbers of CX3CR1+ NK cells were associated with adverse prognosis. Conclusion: We delineated a miR-561-5p/CX3CL1/NK cell axis that drives HCC metastasis and demonstrated that CX3CR1+ NK cells serve as potent antitumor therapeutic effectors.

Inhibition of the Progression of Skin Inflammation, Fibrosis, and Vascular Injury by Blockade of the CX3 CL1/CX3 CR1 Pathway in Experimental Mouse Models of Systemic Sclerosis.

OBJECTIVE: To assess the preclinical efficacy and mechanism of action of an anti-CX3 CL1 monoclonal antibody (mAb) in systemic sclerosis (SSc). METHODS: Cultured human dermal fibroblasts were used to evaluate the direct effect of anti-CX3 CL1 mAb on fibroblasts. In addition, bleomycin-induced and growth factor-induced models of SSc were used to investigate the effect of anti-CX3 CL1 mAb on leukocyte infiltration, collagen deposition, and vascular damage in the skin. RESULTS: Anti-CX3 CL1 mAb treatment significantly inhibited Smad3 phosphorylation (P < 0.05) and expression of type I collagen and fibronectin 1 (P < 0.01) in dermal fibroblasts stimulated with transforming growth factor ß1 (TGFß1). In the bleomycin model, daily subcutaneous bleomycin injection increased serum CX3 CL1 levels (P < 0.05) and augmented lesional CX3 CL1 expression. Simultaneous administration of anti-CX3 CL1 mAb or CX3 CR1 deficiency significantly suppressed the dermal thickness, collagen content, and capillary loss caused by bleomycin (P < 0.05). Injection of bleomycin induced expression of pSmad3 and TGFß1 in the skin, which was inhibited by anti-CX3 CL1 mAb. Further, the dermal infiltration of CX3 CR1+ cells, macrophages (inflammatory and alternatively activated [M2-like] subsets), and CD3+ cells significantly decreased following anti-CX3 CL1 mAb therapy (P < 0.05), as did the enhanced skin expression of fibrogenic molecules, such as thymic stromal lymphopoietin and secreted phosphoprotein 1 (P < 0.05). However, the treatment did not significantly reduce established skin fibrosis. In the second model, simultaneous anti-mCX3 CL1 mAb therapy significantly diminished the skin fibrosis induced by serial subcutaneous injection of TGFß and connective tissue growth factor (P < 0.01). CONCLUSION: Anti-CX3 CL1 mAb therapy may be a novel approach for treating early skin fibrosis in inflammation-driven fibrotic skin disorders such as SSc.

FKN Facilitates HK-2 Cell EMT and Tubulointerstitial Lesions via the Wnt/ß-Catenin Pathway in a Murine Model of Lupus Nephritis.

Fractalkine (FKN), also known as chemokine (C-X3-C motif) ligand 1, constitutes an intriguing chemokine with a documented role in the development of numerous inflammatory diseases including autoimmune disease. Specifically, it has been reported that FKN is involved in the disease progression of lupus nephritis (LN). The epithelial-mesenchymal transition (EMT) plays a significant role in the formation of tubulointerstitial lesions (TIL), which are increasingly recognized as a hallmark of tissue fibrogenesis after injury. However, the correlation between FKN and EMT or TIL in LN has not been determined. To investigate the potential role of FKN in EMT and TIL, MRL lymphoproliferation (MRL/lpr) strain mice were treated with an anti-FKN antibody, recombinant-FKN chemokine domain, or isotype antibody. Our results revealed that treatment with the anti-FKN antibody improved EMT, TIL, and renal function in MRL/lpr mice, along with inhibiting activation of the Wnt/ß-catenin signaling pathway. In contrast, administration of the recombinant-FKN chemokine domain had the opposite effect. Furthermore, to further explore the roles of FKN in EMT, we assessed the levels of EMT markers in FKN-depleted or overexpressing human proximal tubule epithelial HK-2 cells. Our results provide the first evidence that the E-cadherin level was upregulated, whereas α-SMA and vimentin expression was downregulated in FKN-depleted HK-2 cells. In contrast, overexpression of FKN in HK-2 cells enhanced EMT. In addition, inhibition of the Wnt/ß-catenin pathway by XAV939 negated the effect of FKN overexpression, whereas activation of the Wnt/ß-catenin pathway by Ang II impaired the effect of the FKN knockout on EMT in HK-2 cells. Together, our data indicate that FKN plays essential roles in the EMT progression and development of TIL in MRL/lpr mice, most likely through activation of the Wnt/ß-catenin signaling pathway.

Circulating CXCR3-CCR6-CXCR5+CD4+ T cells are associated with acute allograft rejection in liver transplantation.

Circulating T follicular helper (cTFH) cells have been demonstrated to be involved in B-cell-mediated alloreactive responses in kidney and liver transplantation; however, whether these cells are involved in acute liver allograft rejection after liver transplantation, and which subsets are involved, remains to be clarified. The present study aimed to investigate the profiles of cTFH cells in acute liver allograft rejection, including the CXC motif receptor 3 (CXCR3)+ chemokine receptor 6 (CCR6)- subset, the CXCR3-CCR6- subset, and the CXCR3-CCR6+ subset. Twelve liver transplant patients with acute rejection (AR) and 20 with no acute rejection (NAR) were enrolled in the study. The results showed that the proportion of CXCR3-CCR6-CXCR5+CD4+ T cells was significantly increased and the proportion of CXCR3-CCR6+CXCR5+CD4+ T cells was significantly decreased in patients with AR compared with patients with NAR. In addition, the proportion of CXCR3-CCR6-CXCR5+CD4+ T cells was positively correlated with the proportion of B cells in patients with AR. The level of serum interleukin (IL)-21 was higher in the AR group than in the NAR groups. Furthermore, the proportion of CXCR3-CCR6-CXCR5+CD4+ T cells was positively correlated with alanine amino transferase (ALT), whereas the proportion of CXCR3-CCR6+ CXCR5+CD4+ T cells was negatively correlated with ALT. B cells and TFH cells were detected in follicular-like structures in liver allograft tissues from patients with AR. These results suggest that CXCR3-CCR6-CXCR5+CD4+ T cells may be involved in acute allograft rejection after liver transplantation.

The R132H mutation in IDH1 promotes the recruitment of NK cells through CX3CL1/CX3CR1 chemotaxis and is correlated with a better prognosis in gliomas.

Mutations in the isocitrate dehydrogenase (IDH) 1 gene, especially the R132H mutation, have been reported to be associated with a better prognosis in glioma patients. However, the underlying molecular mechanisms are not yet well understood. Many factors may contribute to differences in the survival of IDH1 wild-type and IDH1 mutant glioma patients, in which immune components play a potentially important role. In this study, we analyzed The Cancer Genome Atlas (TCGA), and the Chinese Glioma Genome Atlas (CGGA) databases, as well as glioma patient-derived tumor samples. We found that there was a higher infiltration of natural killer (NK) cells in IDH1 mutant glioma patients, and this was correlated with a better prognosis. We also showed that IDH1-R132 tumor cells had higher expression levels of the chemokine CX3CL1. This arises as a result of the conversion of α-ketoglutarate to R(-)-2-hydroxyglutarate by the IDH1 mutant and the resultant phosphorylation of nuclear factor kappa B. Knockdown of CX3CL1 decreased the migration of NK cells. In addition, the high levels of expression of CX3CL1 were positively correlated with glioma patient survival in the TCGA and CGGA databases, and in the clinical samples. Overall, our data have identified a novel mechanism in which R132H mutation of the IDH1 gene serves as a tumor suppressor by promoting the recruitment of NK cells through CX3CL1/CX3CR1 chemotaxis.

Pharmacokinetics, Pharmacodynamics, and Safety of E6011, a Novel Humanized Antifractalkine (CX3CL1) Monoclonal Antibody: A Randomized, Double-Blind, Placebo-Controlled Single-Ascending-Dose Study.

E6011 is a novel humanized antifractalkine (FKN) monoclonal antibody being developed as a therapeutic target for Crohn's disease, rheumatoid arthritis, and primary biliary cholangitis. This study was a randomized, double-blind, placebo-controlled single-ascending-dose study of intravenous administration of E6011 (0.0006-10 mg/kg) in healthy Japanese adult men (n = 64). The starting dose was the minimum anticipated biological effect level (MABEL). MABEL was estimated by extrapolating results of a pharmacokinetic/pharmacodynamic (PK/PD) model relating E6011 exposure and suppression of free soluble FKN using data obtained from cynomolgus monkeys. Safety assessments consisted of monitoring and recording adverse events, laboratory tests, vital signs, intensive electrocardiograms, and chest x-rays. Blood samples to determine PK, PD (serum total FKN concentration), and serum anti-E6011 antibody were collected. Noncompartmental analysis was used to derive PK parameters. Single intravenous infusions of E6011 were safe and well tolerated in healthy subjects. Serum E6011 concentrations showed triphasic elimination. An increase in serum total FKN concentration was observed, confirming target engagement. The dose strategy for patient studies is to select regimens that will attain a minimum serum E6011 exposure of 10 µg/mL, identified as the minimum concentration needed to saturate the target-mediated elimination pathway. Model-based drug development from preclinical stage was successful in identifying dose regimens for clinical testing.

TRAIL/NF-κB/CX3CL1 Mediated Onco-Immuno Crosstalk Leading to TRAIL Resistance of Pancreatic Cancer Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant neoplasms and registers rising death rates in western countries. Due to its late detection in advanced stages, its extremely aggressive nature and the minimal effectiveness of currently available therapies, PDAC is a challenging problem in the clinical field. One characteristic of PDAC is a distinct desmoplasia consisting of fibroblasts, endothelial and immune cells as well as non-cellular components, contributing to therapy resistance. It is well established that the NF-κB signaling pathway controls inflammation, cancer progression and apoptosis resistance in PDAC. This study attempts to identify NF-κB target genes mediating therapy resistance of humane PDAC cell lines towards death ligand induced apoptosis. By using a genome wide unbiased approach the chemokine CX3CL1 was established as a central NF-κB target gene mediating therapy resistance. While no direct impact of CX3CL1 expression on cancer cell apoptosis was identified in co-culture assays it became apparent that CX3CL1 is acting in a paracrine fashion, leading to an increased recruitment of inflammatory cells. These inflammatory cells in turn mediate apoptosis resistance of PDAC cells. Therefore, our data dissect a bifunctional cross-signaling pathway in PDAC between tumor and immune cells giving rise to therapy resistance.

Structures of respiratory syncytial virus G antigen bound to broadly neutralizing antibodies.

Respiratory syncytial virus (RSV) is a top cause of severe lower respiratory tract disease and mortality in young children and the elderly. The viral envelope G glycoprotein contributes to pathogenesis through its roles in host cell attachment and modulation of host immunity. Although the G glycoprotein is a target of protective RSV-neutralizing antibodies, its development as a vaccine antigen has been hindered by its heterogeneous glycosylation and sequence variability outside a conserved central domain (CCD). We describe the cocrystal structures of two high-affinity broadly neutralizing human monoclonal antibodies bound to the RSV G CCD. The antibodies bind to neighboring conformational epitopes, which we named antigenic sites γ1 and γ2, that span a highly conserved surface, illuminating an important region of vulnerability. We further show that isolated RSV G CCD activates the chemokine receptor CX3CR1 and that antibodies block this activity. These studies provide a template for rational vaccine design targeting this key contributor to RSV disease.

Angiogenic factor-driven inflammation promotes extravasation of human proangiogenic monocytes to tumours.

Recruitment of circulating monocytes is critical for tumour angiogenesis. However, how human monocyte subpopulations extravasate to tumours is unclear. Here we show mechanisms of extravasation of human CD14dimCD16+ patrolling and CD14+CD16+ intermediate proangiogenic monocytes (HPMo), using human tumour xenograft models and live imaging of transmigration. IFNγ promotes an increase of the chemokine CX3CL1 on vessel lumen, imposing continuous crawling to HPMo and making these monocytes insensitive to chemokines required for their extravasation. Expression of the angiogenic factor VEGF and the inflammatory cytokine TNF by tumour cells enables HPMo extravasation by inducing GATA3-mediated repression of CX3CL1 expression. Recruited HPMo boosts angiogenesis by secreting MMP9 leading to release of matrix-bound VEGF-A, which amplifies the entry of more HPMo into tumours. Uncovering the extravasation cascade of HPMo sets the stage for future tumour therapies.

Fractalkine and placental growth factor: A duet of inflammation and angiogenesis in cardiovascular disorders.

Inflammation and angiogenesis are two interdependent processes underlying pathogenesis of cardiovascular disorders. The initiation and progression of atherosclerosis strongly depends on specific patterns of cytokine expression. In this review, we analyze correlation between expression of two members of the cytokine family and the processes of inflammation and angiogenesis related to atherosclerosis. Placental growth factor and chemokine CX3XL1 (fractalkine) promote inflammatory cell infiltration, angiogenesis and plaque rupture. Because these cytokines share similar roles during atherosclerotic development, their combined value as a predictor or indicator of inflammation and vascular healing may be extremely useful.

Safety, pharmacokinetics, and efficacy of E6011, an antifractalkine monoclonal antibody, in a first-in-patient phase 1/2 study on rheumatoid arthritis.

OBJECTIVE: Fractalkine (CX3CL1/FKN) is a chemokine that regulates chemotaxis and adhesion of CX3C chemokine receptor 1 (CX3CR1)-expressing inflammatory cells. We conducted the first phase 1/2, open-label, multiple ascending dose study of E6011, a humanized anti-FKN monoclonal antibody, in Japanese rheumatoid arthritis (RA) patients (clinicaltrial.gov identifier: NCT02196558). METHODS: Active RA patients with an inadequate response or intolerance to methotrexate or tumor necrosis factor (TNF) inhibitor received E6011 at week 0, 1, 2, and thereafter every 2 weeks for 12 weeks. RESULTS: Twelve, 15, and 10 subjects were enrolled in the 100, 200, and 400 mg cohorts, respectively. No severe adverse events (AEs) or deaths occurred, and no major differences were observed in the incidence or severity of AEs across the cohorts. Serum E6011 concentrations increased dose dependently. American College of Rheumatology (ACR) 20, 50, and 70 responses at week 12 were 75.0%, 33.3%, and 8.3% in the 100 mg cohort; 66.7%, 20.0%, and 13.3% in the 200 mg cohort; and 60.0%, 30.0%, and 20.0% in the 400 mg cohort, respectively. CONCLUSIONS: E6011 appeared to be safe and well tolerated in RA patients during this 12-week treatment period, suggesting that E6011 has an effective clinical response in active RA patients.

High dengue virus load differentially modulates human microvascular endothelial barrier function during early infection.

Plasma leakage is the main pathophysiological feature in severe dengue, resulting from altered vascular barrier function associated with an inappropriate immune response triggered upon infection. The present study investigated functional changes using an electric cell-substrate impedance sensing system in four (brain, dermal, pulmonary and retinal) human microvascular endothelial cell (MEC) lines infected with purified dengue virus, followed by assessment of cytokine profiles and the expression of inter-endothelial junctional proteins. Modelling of changes in electrical impedance suggests that vascular leakage in dengue-infected MECs is mostly due to the modulation of cell-to-cell interactions, while this loss of vascular barrier function observed in the infected MECs varied between cell lines and DENV serotypes. High levels of inflammatory cytokines (IL-6 and TNF-α), chemokines (CXCL1, CXCL5, CXCL11, CX3CL1, CCL2 and CCL20) and adhesion molecules (VCAM-1) were differentially produced in the four infected MECs. Further, the tight junctional protein, ZO-1, was down-regulated in both the DENV-1-infected brain and pulmonary MECs, while claudin-1, PECAM-1 and VE-cadherin were differentially expressed in these two MECs after infection. Non-purified virus stock was also studied to investigate the impact of virus stock purity on dengue-specific immune responses, and the results suggest that virus stock propagated through cell culture may include factors that mask or alter the DENV-specific immune responses of the MECs. The findings of the present study show that high DENV load differentially modulates human microvascular endothelial barrier function and disrupts the function of inter-endothelial junctional proteins during early infection with organ-specific cytokine production.

Influence of aspirin on the CX3CL1/CX3CR1 signaling pathway in acute pulmonary embolism.

The present study aimed to explore the influence of aspirin on the CX3CL1/CX3CR1 signaling pathway in acute pulmonary embolism (APE) in rats. Our previous study found that CX3CL1/CX3CR1 was increased in APE. However, the effect of this signaling pathway on APE remains unclear. CX3CL1-shRNA adenovirus and CX3CL1-overexpression vector were constructed. Male Sprague-Dawley rats were randomly divided into 9 groups (n=10): normal group (group N), sham operation group (group Sham), sham operation + aspirin group (group ASP), model group (group M), model + ASP group (group M+A), model + shRNA group (group M+SH), sham operation + CX3CL1-overexpression vector group (group Sham+Cx3), model + ASP + shRNA group (group M+A+SH), and model + ASP + CX3CL1-overexpression vector group (group M+A+CX3). Arterial pressure detection, hematoxylin and eosin staining, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and laser confocal scanning microscopy were applied. Aspirin significantly decreased pulmonary artery pressure, improve pathological changes in the embolism, and decreased the expression of CX3CL1/CX3CR1 and CX3CL1/NF-κB. Moreover, the adenovirus-overexpression CX3CL1 vector aggravated the inflammatory changes in APE, which were improved by aspirin. However, the intervention of the adenovirus CX3CL1 vector reduced the change, while its combination with aspirin significantly improved the change. In conclusion, aspirin improved pathological changes in rats with APE via the CX3CL1/CX3CR1 signaling pathway.

Fractalkine/CX3CL1 in rheumatoid arthritis.

Fractalkine is a CX3C chemokine that exists in both membrane-bound and soluble forms. Interaction between fractalkine and its unique receptor (CX3CR1) induces cell adhesion, chemotaxis, crawling, "accessory cell" activity, and survival. The serum level of fractalkine is elevated in patients with rheumatoid arthritis (RA) and is correlated with disease activity. Peripheral blood CD16+ monocytes and a subset of T cells express CX3CR1, while fractalkine is expressed on fibroblast-like synoviocytes and endothelial cells in the synovial tissue of patients with RA. Fractalkine expression is enhanced by tumor necrosis factor-α and interferon-γ, and it promotes the migration of monocytes, T cells, and osteoclast precursors into RA synovial tissue. Fractalkine also induces the production of inflammatory mediators by macrophages, T cells, and fibroblast-like synoviocytes. Moreover, fractalkine promotes angiogenesis and osteoclastogenesis. In an animal model of RA, arthritis was improved by the abrogation of fractalkine. Recently, a clinical trial of an anti-fractalkine monoclonal antibody for the treatment of RA commenced in Japan. We review the multiple roles of fractalkine in the pathogenesis of RA and its potential as a therapeutic target for this disease.