CCL2 Concentrations Research Articles

Prognostic and predictive biomarkers for patients with idiopathic pulmonary fibrosis treated with pirfenidone: post-hoc assessment of the CAPACITY and ASCEND trials

Heterogeneity in the progression of idiopathic pulmonary fibrosis (IPF) might reflect diversity in underlying pathobiology, and represents a major challenge in the prediction of clinical progression and treatment benefit. Previous studies have found peripheral blood concentrations of several protein biomarkers to be prognostic for overall survival duration in patients with IPF, but these findings have generally not been directly compared and replicated between cohorts. We aimed to use the pivotal trials for pirfenidone to evaluate prognostic and predictive properties of biomarkers across multiple endpoints, and whether they are modulated by pirfenidone treatment. We did post-hoc analyses of test and replication cohorts from CAPACITY 004 (NCT00287716), CAPACITY 006 (NCT00287729), and ASCEND (NCT01366209) trials for the plasma proteins CCL13, CCL17, CCL18, CXCL13, CXCL14, COMP, interleukin 13, MMP3, MMP7, osteopontin, periostin, and YKL40. Eligible participants had IPF and received pirfenidone 2403 mg/day or placebo in CAPACITY (test cohort) or ASCEND (replication cohort), were aged 40-80 years, and without missing biomarker data at baseline. To identify biomarkers that were consistently prognostic for clinical outcome measures, the primary analysis was the association between biomarker concentrations at baseline and absolute change in percentage of predicted forced vital capacity (FVC%pred) at 12 months (CAPACITY week 48, ASCEND week 52) in the placebo group. Biomarkers within the test cohort that met predefined success criteria of a prognostic p value less than 0·10 from multivariate analysis were further assessed in the replication cohort. Furthermore, the predictive effect size (ie, biomarkers that were predictive for benefit from pirfenidone) was calculated as the difference in FVC%pred treatment effect (pirfenidone in relation to placebo) between high versus low biomarker subgroups at week 48 (test cohort) or week 52 (replication cohort). Several baseline biomarkers (CCL13, CCL18, COMP, CXCL13, CXCL14, periostin, and YKL40) were prognostic for progression outcomes in the placebo groups of the test cohort. However, only CCL18 was consistently prognostic for absolute change in percentage of FVC%pred in both the test (p=0·032) and replication (p=0·004) cohorts. Pirfenidone treatment benefit was consistent regardless of baseline biomarker concentration. Blood CCL18 concentrations were the most consistent predictor of disease progression across IPF cohorts with potential to inform new target discovery and clinical trial design. Future validation of these findings in prospective studies is warranted. Genentech Inc.

Modulating Macrophage Polarization through CCR2 Inhibition and Multivalent Engagement.

Excessive or prolonged recruitment of inflammatory monocytes to damaged tissue can significantly worsen patient outcomes. Monocytes migrate to sites of tissue inflammation in response to high local concentrations of CCL2, a chemokine that binds to and signals through the CCR2 receptor. While the role of CCR2 in cellular migration is well studied, it is unclear how CCR2 inhibition affects macrophage polarization and if multivalency can increase downstream signaling effects. Using affinity selection with a phage library, we identified a novel single-chain variable fragment (scFv) (58C) that binds specifically and with high affinity to the N-terminal domain of CCR2 ( KD = 59.8 nM). The newly identified 58C-scFv bound to native CCR2 expressed on macrophages and MDA-MB-231 cells, inhibited migration, and induced a pro-inflammatory M1-phenotype in macrophages. The M1/M2 macrophage phenotype ratio for monomeric 58C-scFv was significantly increased over the negative control by 1.0 × 104-fold (iNOS/Arg-1), 5.1 × 104-fold (iNOS/Mgl2), 3.4 × 105-fold (IL-6/Arg-1), and 1.7 × 106-fold (IL-6/Mgl2). The multivalent display of 58C-scFv on liposomes further reduced migration of both cell types by 25-40% and enhanced M1 polarization by 200% over monomeric 58C-scFv. These studies demonstrate that CCR2 inhibition polarizes macrophages toward an inflammatory M1 phenotype, and that multivalent engagement of CCR2 increases the effects of 58C-scFv on polarization and migration. These data provide important insights into the role of multivalency in modulating binding, downstream signaling, and cellular fate.

Type 2 macrophages and Th2 CD4+ cells in interstitial lung diseases (ILDs): an overview.

Interstitial lung diseases (ILDs) are a heterogeneous group characterized mainly by damage to pulmonary parenchyma, through histopathological processes such as granulomatous pneumopathy, inflammation and fibrosis. Factors that generate susceptibility to ILDs include age, exposure to occupational and environmental compounds, genetic, family history, radiation and chemotherapy/immunomodulatory and cigarette smoke. IFN-γ, IL-1β, and LPS are necessary to induce a classical activation of macrophages, whereas cytokines as IL-4 and IL-13 can induce an alternative activation in macrophages, through the JAK-STAT mediated signal transduction. M2 macrophages are identified based on the gene transcription or protein expression of a set of M2 markers. These markers include transmembrane glycoproteins, scavenger receptors, enzymes, growth factors, hormones, cytokines, and cytokine receptors with diverse and often yet unexplored functions. Fibrotic lung disorders may have a M2 polarization background. The Th2 pathway with an elevated CCL-18 (marker of M2) concentration in the bronchoalveolar lavage fluid (BALF) is linked to fibrosis in ILDs. Besides the role of M2 in tissue repair and ECM remodeling, activated fibroblasts summon and stimulate macrophages by producing MCP-1, M-CSF and other chemokines, as well as activated macrophages secrete cytokines that attract and stimulate proliferation, survival and migration of fibroblast mediated by platelet-derived growth factor (PDGF). (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 98-108).

Differential Effect of Viable Versus Necrotic Neutrophils on Mycobacterium tuberculosis Growth and Cytokine Induction in Whole Blood.

Neutrophils exert both positive and negative influences on the host response to tuberculosis, but the mechanisms by which these differential effects are mediated are unknown. We studied the impact of live and dead neutrophils on the control of Mycobacterium tuberculosis using a whole blood bioluminescence-based assay, and assayed supernatant cytokine concentrations using Luminex™ technology and ELISA. CD15+ granulocyte depletion from blood prior to infection with M. tuberculosis-lux impaired control of mycobacteria by 96 h, with a greater effect than depletion of CD4+, CD8+, or CD14+ cells (p < 0.001). Augmentation of blood with viable granulocytes significantly improved control of mycobacteria by 96 h (p = 0.001), but augmentation with necrotic granulocytes had the opposite effect (p = 0.01). Both augmentations decreased supernatant concentrations of tumor necrosis factor and interleukin (IL)-12 p40/p70, but necrotic granulocyte augmentation also increased concentrations of IL-10, G-CSF, GM-CSF, and CCL2. Necrotic neutrophil augmentation reduced phagocytosis of FITC-labeled M. bovis BCG by all phagocytes, whereas viable neutrophil augmentation specifically reduced early uptake by CD14+ cells. The immunosuppressive effect of dead neutrophils required necrotic debris rather than supernatant. We conclude that viable neutrophils enhance control of M. tuberculosis in blood, but necrotic neutrophils have the opposite effect—the latter associated with induction of IL-10, growth factors, and chemoattractants. Our findings suggest a mechanism by which necrotic neutrophils may exert detrimental effects on the host response in active tuberculosis.

Genetic Polymorphism at CCL5 Is Associated With Protection in Chagas' Heart Disease: Antagonistic Participation of CCR1+ and CCR5+ Cells in Chronic Chagasic Cardiomyopathy.

Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8+ and CD4+ T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n = 110) or cardiopathic (mild, B1, n = 163; severe, C, n = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 −403 (rs2107538) CT heterozygotes (OR = 0.5, P-value = 0.04) and T carriers (OR = 0.5, P-value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5–CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1+ CD8+ T cells and CD14+ macrophages were decreased, while frequencies of CCR5+ cells were increased. Importantly, CCR1+CD14+ macrophages were mainly IL-10+, while CCR5+ cells were mostly TNF+. CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy) in infected Ccr5−/− mice supported a protective role for CCR1 in CCC. Furthermore, parasite antigen stimulation of CD patient blood cells increased the frequency of CCR1+CD8+ T cells and CCL5 production. Collectively, our data support that a genetic variant of CCL5 and CCR1+ cells confer protection against Chagas heart disease, identifying the CCL5-CCR1 axis as a target for immunostimulation.

Plasma levels of innate immune mediators are associated with liver fibrosis in low parasite burden Schistosoma mansoni-infected individuals.

In the murine model, it was demonstrated that pro-inflammatory cytokines and chemokines are essential to the formation and modulation of Schistosoma-induced granulomatous inflammation. However, the relationship of these immune mediators and disease severity is hard to be established in naturally infected individuals. The current study evaluates the association between plasma concentrations of MIF, sTNF-R1, CCL3, CCL7 and CCL24 and schistosomiasis morbidity in Schistosoma mansoni-infected patients with a low parasite burden. For this propose, 97 S.mansoni-infected individuals were subjected to abdominal ultrasound analysis and clinical examination. Among them, 88 had plasma concentration of immune mediators estimated by ELISA assay. Multivariate linear regression models were used to evaluate the relationship between the plasma concentration of immune mediators and the variables investigated. Although most individuals presented low parasite burden, over 30% of them showed signs of fibrosis defined by ultrasound measurements and 2 patients had a severe form of schistosomiasis. No association between parasite burden and the plasma levels of chemokine/cytokines or disease severity was observed. There was a positive association between plasma concentration of CCL4, sTNF-R1, CCL3 and MIF with gall bladder thickness and/or with portal vein thickness that are liver fibrosis markers. In contrast, no association was found between CCL7 plasma concentrations with any of the schistosomiasis morbidity parameters evaluated. The data showed that CCL24, sTNFR1, MIF and CCL3 can be detected in plasma of S.mansoni-infected individuals and their concentration would be used as prognostic makers of Schistosoma-induced liver fibrosis, even in individuals with low parasite burden.

CCL2 Protein Regulates Migration and Invasion of THP-1 cells by Autosecreting Inflammatory Chemokines

To investigate the effects and mechanism of CCL2 on the migration and invasion of human leukemia cell THP-1. The CCL2 gene was recombined with the transfer plasmid-PLVX and transfected into THP-1 cells. The CCL2 expression at RNA level was detected by RT-PCR, the CCL2 expression at protein level was determined by Western blot and ELISA, the influence of overexpression of CCL2 recombinant protein and THP-1 cells on the migration and invasion ability of THP-1 cells was analyzed by transwell migration and invasion tests, the PCR-array of migration-related cytokines was used to clarify the patential mechanism. With the Trans-Matrigel assay, the concentration of CCL2 in THP-1 transfected with CCL2 in the upper cells was higher than that in the lower cells, meanwhile, the invasion ability of CCL2-transfected THP-1 cells decreased. Increasing recombinant protein of CCL2 (rpCCL2) in the lower cells promoted migration of THP-1 cells. Migration RT2 profiler PCR array showed that the cells treated with rpCCL2 had higher levels of expression of CCL2, EPX, SPP1, CX3CL1 and CXCL13, as compared with control group. CCL2 affects the migration and invasion of THP-1 by autosecreting a series of inflammatory chemokines.

Surfen, a proteoglycan binding agent, reduces inflammation but inhibits remyelination in murine models of Multiple Sclerosis

Proteoglycans are promising therapeutic targets in Multiple Sclerosis (MS), because they regulate many aspects of the immune response. This was studied using surfen, an agent that binds both heparan sulphate proteoglycans (HSPGs) and chondroitin sulphate proteoglycans (CSPGs). Initial cell culture work on bone marrow derived macrophages (BMDMs) found that surfen reduced concentrations of the chemokines CCL2, CCL4 and CCL5, with reduced messenger (m)RNA expression for Tumor Necrosis Factor, IL-6, IL-1β and inducible nitric oxide synthase. These data were further explored using Experimental Autoimmune Encephalomyelitis (EAE) in mice. Surfen reduced clinical signs during EAE when administered from disease onset, and reduced infiltration by CD4 positive T cells and macrophages into the central nervous system. These mice also showed reduced mRNA expression for the chemokines CCL3 and CCL5, with reduced concentrations of CCL2, CCL3 and CCL5. During EAE, surfen treatment induced a persistent increase in Interleukin (IL)-4 concentrations which may enhance T helper 2 responses. During EAE, surfen treatment reduced mRNA expression for HSPGs (NDST1, agrin, syndecan-4, perlecan, serglycin, syndecan-1) and the CSPG versican. By contrast, surfen increased mRNA expression for the CSPG aggrecan, with no effect on neurocan. During EAE, significant positive correlations were found between mRNA expression and clinical score for syndecan-4, serglycin and syndecan-1 and a significant negative correlation for aggrecan. These correlations were absent in surfen treated mice. Repair in the later stages of MS involves remyelination, which was modeled by injecting lysolecithin (lysophosphatidylcholine, LPC) into mouse corpus callosum to create regions of demyelination. When surfen was injected 2 days after LPC, it delayed remyelination of the lesions, but had no effect when injected 7 days after LPC. The delayed remyelination was associated with local increases in CSPG expression. Therefore surfen suppresses inflammation but inhibits remyelination in these models. A mechanism in common may be increased CSPG expression.

Iloprost Affects Macrophage Activation and CCL2 Concentrations in a Microdialysis Model in Rats.

The hypothesis that locally-released iloprost, a synthetic prostacyclin analog, affects macrophage phenotype at a microdialysis implant in the subcutaneous space of rats was tested. Macrophage activation towards alternatively-activated phenotypes using pharmaceutical release is of interest to improve integration of implants and direct the foreign body reaction toward a successful outcome. Macrophage cell culture was used to test iloprost macrophage activation in vitro. Microdialysis sampling probes were implanted into the subcutaneous space of Sprague-Dawley rats to locally deliver iloprost in awake- and freely-moving rats. Monocyte chemoattractant protein -1 (CCL2) was quantified from collected dialysates using ELISA. Immunohistochemical staining was used to determine the presence of CD163+ macrophages in explanted tissues. Iloprost reduced CCL2 concentrations in NR8383 macrophages stimulated with lipopolysaccharide. CCL2 concentrations in collected dialysates were similarly reduced in the presence of iloprost. Iloprost caused an increase in CD163+ cells in explanted tissue surrounding implanted microdialysis probes at two days post probe implantation. Localized delivery of iloprost caused macrophage activation at the tissue interface of a microdialysis subcutaneous implant in rat. This model system may be useful for testing other potential macrophage modulators in vivo.

Serum Cytokine Profiles in Patients with Dengue Fever at the Acute Infection Phase.

Background Dengue virus (DENV) is transmitted by mosquito and has been circulating in Guangdong, China, for over 30 years. Dengue infection causes mild to severe disease symptoms in human. Cytokine profiles were suggested to be crucial especially during the acute stage in the dengue infection. Aim To determine the cytokine profiles at the acute stage in patients with primary or secondary dengue infection in Guangzhou city in the 2014 outbreak. Methods We investigated 23 inflammatory cytokines in serum collected from dengue-infected patients and analyzed their correlations with their clinical indexes. Results The concentrations of CXCL9, IP-10, CXCL11, IL-8, IL-10, and CCL2 in serum were significantly higher in the groups of DENV-infected patients during the first two weeks than those of control group while CCL17 and CXCL5 showed lower expression level in the patients. Among these cytokines, CXCL9, CCL17, and CXCL5 showed statistical difference between the groups of primary and secondary infections. The platelet count and lactate dehydrogenase were correlated with the level of CCL17 and MIP-1α/CXCL5, respectively, in the group of secondary infection. Conclusions We determined the cytokine profiles in serum of the patients during the 2014 dengue outbreak. The expression of specific cytokines was associated with the secondary infection.

Prominent Levels of the Profibrotic Chemokine CCL18 during Peritonitis: In Vitro Downregulation by Vitamin D Receptor Agonists.

Peritoneal dialysis (PD) is used as a renal replacement therapy, which can be limited by peritoneal membrane ultrafiltration failure (UFF) secondary to fibrotic processes. Peritonitis, a frequent complication of PD, is a major risk factor for peritoneal membrane fibrosis and UFF. Low peritoneal levels of the chemokine CCL18 are associated with preservation of peritoneal membrane function in PD. Given that CCL18 is involved in fibrotic processes and recurrent peritonitis, it is a risk factor for peritoneal membrane failure; thus, we evaluated CCL18 concentrations in peritoneal effluents from patients undergoing peritonitis episodes. Pharmacological interventions aimed at diminishing the production of CCL18 were also explored. Fivefold higher CCL18 peritoneal concentrations were found during acute bacterial peritonitis, in parallel with the increased infiltration of macrophages. Unexpectedly, CCL18 was also highly (50-fold) increased during sterile eosinophilic peritonitis, and peritoneal eosinophils were found to express CCL18. In vitro treatment of peritoneal macrophages with the vitamin D receptor agonist paricalcitol was able to reduce the secretion and the expression of CCL18 in isolated peritoneal macrophages. In conclusion, our study suggests that the chemokine CCL18 can be a mediator of peritoneal membrane failure associated with peritonitis episodes as well as providing a new potential therapeutic target.

Fibroblasts Promote Inflammation and Pain via IL-1α Induction of the Monocyte Chemoattractant Chemokine (C-C Motif) Ligand 2

Fibroblasts persist within fibrotic scar tissue and exhibit considerable phenotypic and functional plasticity. Herein, we hypothesized that scar-associated fibroblasts may be a source of stress-induced inflammatory exacerbations and pain. To test this idea, we used a human model of surgery-induced fibrosis, total knee arthroplasty (TKA). Using a combination of tissue protein expression profiling and bioinformatics, we discovered that many months after TKA, the fibrotic joint exists in a state of unresolved chronic inflammation. Moreover, the infrapatellar fat pad, a soft tissue that becomes highly fibrotic in the post-TKA joint, expresses multiple inflammatory mediators, including the monocyte chemoattractant, chemokine (C-C motif) ligand (CCL) 2, and the innate immune trigger, IL-1α. Fibroblasts isolated from the post-TKA fibrotic infrapatellar fat pad express the IL-1 receptor and on exposure to IL-1α polarize to a highly inflammatory state that enables them to stimulate the recruitment of monocytes. Blockade of fibroblast CCL2 or its transcriptional regulator NF-κB prevented IL-1α–induced monocyte recruitment. Clinical investigations discovered that levels of patient-reported pain in the post-TKA joint correlated with concentrations of CCL2 in the joint tissue, such that the chemokine is effectively a pain biomarker in the TKA patient. We propose that an IL-1α–NF-κB–CCL2 signaling pathway, operating within scar-associated fibroblasts, may be therapeutically manipulated for alleviating inflammation and pain in fibrotic joints and other tissues.

Cytokines and chemokines measured in dried SLA-stimulated whole blood spots for asymptomatic Leishmania infantum and Leishmania donovani infection

Whole blood stimulation with soluble Leishmania antigen (SLA), followed by plasma cytokine and chemokine determination, provides means of detecting subjects with asymptomatic Leishmania infection. This work examines the potential of Protein Saver 903 cards for the storage and transport of SLA-stimulated dried plasma spot samples. Blood was collected from asymptomatic and negative control subjects living in a Leishmania infantum- (Spain) and Leishmania donovani-endemic area (Bangladesh). After SLA-stimulation, three types of sample were prepared: frozen liquid plasma (−20 °C), and plasma dropped onto Protein Saver cards kept at −20 °C (DPS-FZ), and at ambient temperature (DPS-AT). The concentrations of IFN-γ, IL-2, CXCL10, CXCL9, CCL2 and CXCL8 in the thawed liquid plasma (TLP), DPS-FZ and DPS-AT samples were then determined. Strong correlations were seen between the TLP and DPS-FZ/AT samples for all the studied cytokines/chemokines in both the L. infantum and L. donovani areas. Protein Saver 903 cards would therefore appear to allow for the transport of SLA-stimulated plasma samples by courier at ambient temperature. The CXCL10 and CXCL9 detectable in these plasma spots provided robust markers for identifying asymptomatic subjects from both endemic areas. This easy procedure opens up new possibilities for field studies in resource-limited settings, which could help in Leishmania control.

Cerebrospinal fluid and serum IL-8, CCL2, and ICAM-1 concentrations in astrocytic brain tumor patients

BackgroundThe aim of the study was the evaluation of serum and CSF concentrations of CCL2, IL-8, and sICAM-1 in patients with astrocytic tumors as compared to a group of non-tumoral patients.MethodsChemokine concentrations were measured using the ELISA method.ResultsRegardless of the parameter tested and the patient group (brain tumor or non-tumoral patients), statistical differences (P < 0.05) were found between concentrations obtained in CSF compared to values obtained in serum for all proteins tested. CSF IL-8 concentrations were significantly elevated in CNS tumor patients as compared to non-tumoral individuals (P = 0.000); serum CCL2 and sICAM-1 concentrations were significantly decreased in CNS tumors in comparison with the comparative group (P = 0.002 and P = 0.026, respectively). Among proteins tested in the serum, a higher area under the ROC curve (AUC) revealed CCL2 compared to sICAM-1 in differentiating subjects with CNS brain tumors from non-tumoral subjects. AUC for CSF IL-8 was higher than for its index (CSF IL-8/serum IL-8).ConclusionsFor individual biomarkers (IL-8 and CCL2, sICAM-1), measured in CNS brain tumor patients, the appropriate material, respectively CSF or serum, should be chosen and quantitatively tested. Increased cerebrospinal fluid IL-8 with decreased serum CCL2 create a pattern of biomarkers, which may be helpful in the management of CNS astrocytic brain tumors.

Serum CCL20 and its association with SIRT1 activity in multiple sclerosis patients

CCL20 is a potentially important component in the pathogenesis of multiple sclerosis (MS). SIRT1 exhibits a negative regulatory effect on a variety of inflammatory cytokines and can relieve experimental autoimmune encephalomyelitis. The association between the level of CCL20 and SIRT1 activity in MS patients has not been investigated. In the present study, blood samples were collected from 38 RRMS patients and 40 healthy controls. The serum CCL20 levels were measured by ELISA. SIRT1 activity was evaluated by fluorometric assay. We revealed elevated serum CCL20 concentrations in MS, and discovered an inverse correlation between CCL20 and SIRT1 activity in MS patients.

Borrelia burgdorferi basic membrane protein A could induce chemokine production in murine microglia cell line BV2

Lyme neuroborreliosis is a nervous system infectious disease caused by Borrelia burgdorferi (B. burgdorferi). It has been demonstrated that cytokines induced by B. burgdorferi are related to Lyme neuroborreliosis. Microglia is known as a key player in the immune responses that occur within the central nervous system. In response to inflammation, it will be activated and generate cytokines and chemokines. Experiments in vitro cells have showed that B. Burgdorferi membrane protein A (BmpA), a major immunogen of B. Burgdorferi, could induce Lyme arthritis and stimulate human and murine lymphocytes to produce inflammatory cytokines. In our study, the murine microglia BV2 cell line was used as a cell model to explore the stimulating effects of recombinant BmpA (rBmpA); Chemokine chip, ELISA and QPCR technology were used to measure the production of chemokines from microglial cells stimulated by rBmpA. Compared with the negative control group, CXCL2, CCL22, and CCL5 concentrations in the cell supernatant increased significantly after the rBmpA stimulation; the concentration of these chemokines increased with rBmpA concentration increasing; the mRNA expression levels of chemokines (CXCL2, CCL22, and CCL5) in murine BV2 cells increased significantly with 10 μg/mL and 20 μg/mL rBmpA stimulation; CXCL13 was not change after the rBmpA stimulation. Our study shows that chemokines, such as CXCL2, CCL22, and CCL5 were up-regulated by the rBmpA in the BV2 cells. The production of chemokines in Lyme neuroborreliosis may be mainly from microglia cells and the rBmpA may be closely related with the development of Lyme neuroborreliosis.

Abstract 951: Induction of COX-2 and NFκB activation represent TLR3-independent undesirable aspects of poly-I:C activity

Abstract Background: Infiltration of cytotoxic T-cells (CTL) in tumors is known to be associated with improved patient’s clinical outcomes. In contrast, local infiltration with regulatory T-cells (Treg) predicts accelerated progression and shorter overall survival. The above observations highlight the need for new measures to promote selective accumulation of CTLs but not Tregs in tumor microenvironments (TME). Here we compare the TME-modulating impact of poly-I:C with a selective TLR3 ligand, rintatolimod showing an induction of CCL22, CXCL12 and expression of COX-2 and downstream suppressive factors, selectively by poly-I:C. Finally, we report on the optimized combinatorial adjuvant to reprogram tumor microenvironment. Materials and Methods: Twelve ovarian cancer specimens were cultured in the presence of IFNα, indometacin (COX-1/2 inhibitor) or/and one of two synthetic TLR3 ligands, poly-I:C (non-selective activator of TLR3 and helicases) or rintatolimod (selective TLR3 ligand). Biopsies were harvested for mRNA measurements and culture supernatants were analyzed for CCL5, CXCL10 and CCL22 concentrations. Alternatively, established ovarian cancer cell lines, monocyte-derived macrophages, THP1 cells or adult fibroblasts were used in analogous experiments. Chemotaxis assays were performed using pre-activated CD8+ T-cells (top chamber) and supernatants from the differentially treated ovarian cancer specimens. Western blot experiments were used to evaluate the impact of NFκB. Blocking experiments using soluble TNFα receptor inhibitor were performed. Results: Both, Poly-I:C and rintatolimod, induced CTL attractant CXCL10. Unexpectedly, poly-I:C but not rintatolimod promoted the expression of the MDSC/Treg attractant CXCL12, which was reversed by addition of indometacin. Furthermore, poly-I:C induced expression of COX-2 and COX-2-dependent suppressive factors (IDO, IL-10). Evaluating the COX-2 cascade, TNFα was found to be increased in the presence of poly-I:C but not rintatolimod. Additionally, Western Blot analysis showed I-κB degradation (as a marker of NF-κB activation), selectively in response to poly-I:C. We observed that the combination of both TLR-3 ligands with IFNα and indomethacin selectively induced the desirable chemokines CCL5 and CXCL10 and suppressed CCL22 in tumor samples with similar results in the macrophages and fibroblasts. The ovarian cancer cell lines showed only minimal expression of chemokines. Conclusion: We demonstrate the feasibility of selective modulation of TME, using different clinically applicable factors and their combinations. We show for the first time, that poly-I:C, but not a selective TLR3 ligand, induces NFκB and undesirable COX2-dependent suppressive factors, which though can be eliminated through addition of a COX-1/2 inhibitor. These effects may be avoided using a selective TLR3 ligand. Citation Format: Marie-Nicole Theodoraki, Ravi Muthuswamy, Jamie Voyten, Francesmary Modugno, Natasa Obermajer, Robert P. Edwards, Pawel Kalinski. Induction of COX-2 and NFκB activation represent TLR3-independent undesirable aspects of poly-I:C activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 951. doi:10.1158/1538-7445.AM2017-951

Abstract 3908: The mechanism of premetastatic niche formation through FBXW7/NOTCH/CCL2 pathway and clinical trial using CCL2 inhibitor “SK-818” for breast cancer patients

Abstract Background Metastasis is a major cause of death in cancer patients, &amp; elucidation of the mechanisms is expected to provide a basis for the development of new cancer treatment. Premetastatic niche whereby tumors prepare defined organs for metastasized cells is considered to be crucial for the development of metastasis, which consists of host cells such as monocytes, macrophages, &amp; other inflammatory cells. However, the mechanism of how tumor cells communicate with host cells to develop a premetastatic niche remains unclear. FBXW7 is the F-box protein component of an SCF-type ubiquitin ligase, in which it functions as a receptor responsible for substrate recognition followed by the degradation of the substrate. Some part of the substrates of FBXW7 regulates the recruitment of monocytes, macrophages, &amp; other inflammatory cells. Therefore, we hypothesized that expression of FBXW7 in the host cells is a key determinant of the formation of premetastatic niches. Materials &amp; Methods To investigate the role of FBXW7 in the formation of premetastatic niche, we developed bone marrow-specific FBXW7-deficient mice. 1. We transferred B16F10 melanoma cells or E0771 mouse breast cancer cells into the tail vein or mammary fat pad of the mice &amp; the wild type mice to evaluate the metastatic potential to lung. 2. We examined the serum concentrations of various cytokines or chemokines in the mice before &amp; after E0771 cells transplantation using cytokine array. 3. To examine whether the enhanced metastasis apparent in FBXW7-deficient mice is dependent on the CCL2/CCR2 pathway, we treated FBXW7-deficient mice with SK-818, a CCR2 antagonist. 4. We measured the expression of FBXW7 mRNA in peripheral blood of breast cancer patients by qRT-PCR &amp; examined the relationship between FBXW7 mRNA expression &amp; prognosis. Results 1. The extent of lung metastasis of B16F10 or E0771 cell was markedly enhanced in FBXW7-deficient mice. 2. FBXW7-deficient mice exhibited increased serum levels of CCL2. Moreover, accumulation of NOTCH &amp; consequent transcriptional activation of CCL2 was observed in bone marrow-derived stromal cells of FBXW7-deficient mice. 3. Administration of SK-818 blocked the enhancement of lung metastasis in FBXW7-deficient mice. 4. In human breast cancer patients, low FBXW7 mRNA expression in peripheral blood showed high concentration of CCL2 in serum &amp; poor prognosis. Conclusions We found that the FBXW7/NOTCH/CCL2 pathway play a key role in the regulation of cancer metastasis through the formation of premetastatic niches, &amp; that SK-818 inhibit cancer metastasis in mice. SK-818 is currently administered clinically for the treatment of patients with hepatitis B virus infection in Japan. Based on these findings, we have been conducting Phase I clinical trial to assess the safety of SK-818 for breast cancer patients. Citation Format: Miwa Noda, Takaaki Masuda, Naoki Hayashi, Takahiro Kogawa, Shinji Ohno, Keiichi Nakayama, Yohsuke Kuroda, Hidetoshi Eguchi, Shuhei Ito, Masafumi Inomata, Koshi Mimori. The mechanism of premetastatic niche formation through FBXW7/NOTCH/CCL2 pathway and clinical trial using CCL2 inhibitor “SK-818” for breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3908. doi:10.1158/1538-7445.AM2017-3908

Conductive polymer-based bioelectrochemical assembly for in vitro cytotoxicity evaluation: Renoprotective assessment of Salvia officinalis against carbon tetrachloride induced nephrotoxicity

BackgroundThe rise of organic electronics represents one of the most prominent technological developments of the last two decades, with its interface with biological systems highlighting new directions of research. The “soft” nature of conducting polymers renders them unique platforms for cell-based microdevices, allowing their implementation in drug discovery, pharmaceutical effect analysis, environmental pollutant testing etc. MethodsCellular adhesion, proliferation and viability experiments were carried out to verify the biocompatibility of a PEDOT conductive polymer surface. Cyclic voltammetry was employed for estimating the electrocatalytic activity of the renal cell/electrode interface. The nephrotoxicity agent CCl4 and the medicinal plant Salvia officinalis were used on the proposed assembly. Renal cell viability was also assayed through the MTT assay. ResultsRenal cells were able to adhere and proliferate on the conducting polymer surface. Electrochemical responses of the polymer exhibited good correlation with cell number and CCl4 concentration. Amelioration of the CCl4-induced renotoxicity by co-incubation with Salvia officinalis extract was demonstrated by both the MTT assay and the electrode's capacitance. ConclusionsA conducting polymer-based bioelectrochemical assembly was established for in vitro mammalian cytotoxicity/cytoprotection assessment, employing renal cell monolayers as the primary transducers for signal generation and biological sensing. General significanceThe knowledge on PEDOT mammalian cell biocompatibility and possible applications was expanded. The proposed interdisciplinary approach connects soft electronics with biology and could provide a useful tool for preliminary crude drug screening and bioactivity studies of natural products or plant extracts in vitro.

CCL-2 as a possible early marker for remission after traumatic spinal cord injury.

Prospective observational study. To describe the correlation between CCL-2, CCL-3, CCL-4 and CXCL-5 serum levels and remission after traumatic spinal cord injury (SCI) in a human protocol compared with animal studies. Germany, Rhineland-Palatinate (Rheinland-Pfalz). We examined the serum levels of CCL-2, CCL-3, CCL-4 and CXCL-5 over a 12-week period; in particular, at admission and 4, 9 and 12 h, 1 and 3 days and 1, 2, 4, 8 and 12 weeks after trauma. According to our study design, we matched 10 patients with TSCI and neurological remission with 10 patients with an initial ASIA A grade and no neurological remission. In all, 10 patients with vertebral fracture without neurological deficits served as control. Our analysis was performed using a Luminex Cytokine Panel. Multivariate logistic regression models were used to examine the predictive value with respect to neurological remission vs no neurological remission. The results of our study showed differences in the serum expression patterns of CCL-2 in association with the neurological remission (CCL-2 at admission P=0.013). Serum levels of CCL-2 and CCL-4 were significantly different in patients with and without neurological remission. The favored predictive model resulted in an area under the curve (AUC) of 93.1% in the receiver operating characteristic (ROC) analysis. Our results indicate that peripheral serum analysis is a suitable concept for predicting the patient's potential for neurological remission after TSCI. Furthermore, the initial CCL-2 concentration provides an additional predictive value compared with the NLI (neurological level of injury). Therefore, the present study introduces a promising approach for future monitoring concepts and tracking techniques for current therapies. The results indicate that future investigations with an enlarged sample size are needed in order to develop monitoring, prognostic and scoring systems.