Epithelial Neutrophil-activating Protein Research Articles

Abstract PO-061: The role of Cathepsin E expression in pancreatic ductal adenocarcinoma tumorigenesis

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate of 10% due to a lack of early detection and effective treatments. Aspartic protease Cathepsin E (CTSE) expression is increased early in PDAC, but its role in tumorigenesis is unknown. We hypothesize that high CTSE expression promotes proliferation, invasion, inhibition of apoptosis, and the secretion of cancer-associated cytokines in PDAC. Methods: Human PDAC cell lines (Mpanc96, Panc-1, and Miapaca2) were engineered to express high levels of CTSE. Cell proliferation, invasion, and apoptosis were analyzed and compared to control cell lines expressing low levels of CTSE. Cells were treated with aspartic protease inhibitors (Pepstatin A and Ritonavir) and monitored for proliferation (24 - 72 hours) and invasion (48 hours). Apoptosis was measured using propidium iodide and analyzed via flow cytometry. Conditioned media from cells was collected (72 hours) after treatment to assess differential secretion of cancer-associated cytokines using a cytokine array. Results: Proliferation was not affected by the overexpression of CTSE; however, inhibition of CTSE slowed proliferation in the Panc-1 and MiaPaca2, but not Mpanc96 cells. CTSE inhibitors promoted invasion of Mpanc96 and Panc-1, but not Miapaca2 cells, regardless of CTSE levels. Apoptosis was not significantly different between the cell lines treated with the aspartic protease inhibitors compared to controls. Increased expression of CTSE induced the secretion of various cytokines such as epithelial neutrophil-activating protein 78 (ENA-78), a chemokine expressed in inflammatory pancreatic disorders. Treatment with the inhibitor Pepstatin A decreased ENA-78 secretion. Conclusions: CTSE levels does not appear to affect proliferation or apoptosis; however, inhibition of CTSE increased cell invasion and resulted in altered cytokine secretion. Further studies examining how CTSE influences invasiveness, and cytokine secretion may create new opportunities for developing effective therapies for PDAC. Citation Format: Ericka Velez-Bonet, Sabrina Kaul, Corbin Pontious, Marcus Hong, Kelly Dubai, Zobeida Cruz-Monserrate. The role of Cathepsin E expression in pancreatic ductal adenocarcinoma tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-061.

The nano-TiO2 exposure can induce hepatic inflammation involving in a JAK–STAT signalling pathway

TiO2 nanoparticles (TiO2 NPs) have unique physiochemical properties and thus are widely used in daily life. However, these nanoparticles also have potential toxic effects in humans and animals, and the issue of the security TiO2 NPs has also gained prominence. In this article, mice were administered a gavage instillation of 2.5, 5, or 10 mg/kg body weight TiO2 NPs (5–6 nm) for 90 days. We investigated whether TiO2 NPs activate the JAK–STAT signalling pathway, causing nano-TiO2-induced hepatic toxicity. The results demonstrated that with increasing doses of TiO2 NPs the body weights of the mice body decreased, and the liver index, liver dysfunction, infiltration of inflammatory cells, and hepatocyte apoptosis and necrosis increased. Moreover, liver inflammation was accompanied by increased expression of Janus kinase 2, the signal transducers and activators of transcription 3, interleukin-6, cyclooxygenase-2, neutrophil gelatinase-associated lipocalin, purinergic receptor-7, and epithelial neutrophil-activating protein-78 and decreased expression of suppressors of cytokine signalling-1, peroxisome proliferator-activated receptor-γ, and peroxisome proliferator-activated receptor gamma coactivator-1 alpha. In summary, the activation of the JAK–STAT pathway may be involved in the hepatic inflammation induced by chronic nano-TiO2 toxicity.

Chemokine Expression Profiles of Human Hepatoma Cell Lines Mediated by Hepatitis B Virus X Protein.

The hepatitis B virus X protein (HBx), which is encoded by hepatitis B virus (HBV), plays crucial roles in the tumorigenesis of HBV associated hepatocellular carcinoma (HCC). Recent studies suggest that the HBx is involved in regulation of host immune cytokines and chemokines in HBV-associated HCC patients. However, effects of the HBx on autocrine chemokine expression profiles of hepatoma cells, which were shown in modulation of tumor-immune cell interactions, have not been investigated comprehensively. In the present study, human hepatoma cell lines SMMC-7721 and HepG2 were transfected with HBx-expressing plasmid. Human chemokine antibody array 1 (RayBio®), which simultaneously detects 38 chemokine factors, was used to determine chemokine expression profiles. Real-time polymerase chain reaction (real-time PCR) was used to further confirm the differential expression of chemokines. Chemokine antibody array revealed that all 38 chomekines were found to be expressed by SMMC-7721 and HepG2 cell lines. Interleukin-8 (IL-8) was obviously up-regulated, and epithelial neutrophil-activating protein 78 (ENA78), eosinophil chemotactic protein-1 (Eotaxin-1), monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3 and macrophage inflammatory protein-3β (MIP-3β) were significantly declined in both cell lines following transfection of HBx-expressing plasmid. Other chemokines showed little or no significant changes. HBx-induced differential chemokine expression levels were validated by real-time PCR. Hierarchical cluster analysis identified a distinction of chomekine expression profiles between HBX-expressing hepatoma cell lines and controls. Our findings provide new evidence that HBx is able to selectively regulate chomekines in hepatoma cells that may be involved in the regulation of tumor-immune cell interactions.

Fenofibrate Attenuates Neutrophilic Inflammation in Airway Epithelia: Potential Drug Repurposing for Cystic Fibrosis.

A hallmark of cystic fibrosis (CF) lung disease is neutrophilic airway inflammation. Elevated neutrophil counts have been associated with decreased forced expiratory volume in 1 second and poor clinical measures in patients with CF. Interleukin 8 (IL-8), epithelial neutrophil activating protein 78 (ENA-78), tumor necrosis factor alpha (TNF-α), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) contribute to neutrophil activation and disease pathogenesis in the airways of patients with CF. Drugs that modify the production of these chemokines in the airways could potentially benefit CF patients. Thus, we determined the effects of fenofibrate on their production in cell populations obtained from the airways. Human small airway epithelial cells and CF bronchial epithelial cells were treated with IL-1β to induce inflammation. We cotreated the cells with fenofibrate at concentrations ranging from 10 to 50 μM to determine if this drug could attenuate the inflammation. IL-8, ENA-78, TNF-α, GM-CSF, and G-CSF production were measured from the cell culture supernates by ELISA. ANOVA statistical testing was conducted using SPSS 17.0. IL-1β increased the production of each of the chemokines by several fold. Fenofibrate reduced IL-1β induced production of each of these neutrophilic chemokines at the concentrations used. IL-1β increases the production of neutrophilic chemokines in airway epithelial cells. Cotreatment with fenofibrate blunts these processes. Fenofibrate should be explored as a therapeutic option to modulate the abundant neutrophilic inflammation observed in CF.

Effect of G-CSF on induction of ENA-78 and IL-8 in the patients with malignant lymphoma.

Granulocyte colony stimulating factor (G-CSF) restores neutrophil count in patients with chemotherapy-induced neutropenia. G-CSF can also induce production of epithelial neutrophil activating protein-78 (ENA-78) and interleukin-8 (IL-8), chemotactic factors from neutrophils in vitro. This study was purposed to investigate whether this effect is also observed in vivo. 10 lymphoma patients were selected who received chemotherapy and G-CSF (nartograstim) administration. Blood was obtained before chemotherapy [Time Point 1 (TP1)], at neutropenic phase before G-CSF administration (TP2), and at neutrophil recovery phase after G-CSF (TP3). ENA-78 and IL-8 mRNA in neutrophils were quantified by real-time PCR. Phagocytosis and reactive oxygen species (ROS) generation were examined by flow cytometry. The results showed that ENA-78 and IL-8 mRNA expression at TP2 increased in 5 and 8 patients, respectively. The ENA-78 mRNA expression at TP3 was increased in 3 and decreased in 6 patients, and IL-8 mRNA expression at TP3 decreased in 7 patients. G-CSF did not affect phagocytosis and normalized ROS generation in all of the patient. It is concluded that increase of ENA-78 and IL-8 expression in neutrophils is common in chemotherapy-induced neutropenic patients. G-CSF administration does not significantly increase ENA-78 and IL-8 expression.

Bone marrow mast cell density correlates with serum levels of VEGF and CXC chemokines ENA-78 and GRO-α in multiple myeloma

Angiogenesis is a crucial process in growth and progression of multiple myeloma (MM). Mast cells (MCs) play an important role in MM angiogenesis. Various angiogenic mediators secreted by MCs regulate endothelial cell proliferation and function. Among them, ELR(+) CXC chemokines, such as growth-related oncogen-alpha (GRO-α) and epithelial neutrophil activating protein-78 (ENA-78), have been described as potential mediators in regulation of angiogenesis. The purpose of the study was to quantify MCs in bone marrow (BM) biopsies of MM patients, expressed as MC density (MCD), and correlate it with serum concentrations of vascular endothelial factor (VEGF), GRO-α, ENA-78. Fifty-four newly diagnosed MM patients and 22 healthy controls were studied. Tryptase was used for the immunohistochemical stain of MCs. VEGF, GRO-α, and ENA-78 were measured in sera by ELISA. MCD and serum levels of GRO-α, ENA-78, and VEGF were significantly higher in MM patients compared to controls (p<0.001 in all cases). MCD was significantly increasing with increased stage of the disease (p<0.001). Furthermore, significant correlations were found between MCD with VEGF, GRO-α, and ENA-78. These findings support that MCs participate in the pathophysiology of MM and is implicated in the angiogenic process and disease progression.

Cytochrome P450-derived eicosanoids and vascular dysfunction in coronary artery disease patients

ObjectiveAccumulating preclinical and epidemiologic evidence has emerged to suggest that modulation of cytochrome P450 (CYP)-mediated eicosanoid metabolism may be a viable vascular protective therapeutic strategy for the secondary prevention of coronary artery disease (CAD). The functional relationship between CYP-derived eicosanoid metabolite levels and vascular dysfunction in humans with established CAD, however, has not been evaluated. Therefore, we characterized the relationship between inter-individual variation in soluble epoxide hydrolase (sEH) and CYP ω-hydroxylase metabolism and established vascular function phenotypes predictive of prognosis in a cohort of patients with atherosclerotic cardiovascular disease. MethodsPlasma epoxyeicosatrienoic acid (EET), dihydroxyeicosatrienoic acid (DHET), and 20-hydroxyeicosatetraenoic acid (20-HETE) levels were quantified by HPLC-MS/MS in 106 patients with stable, angiographically-confirmed CAD. Relationships between biomarkers of CYP-mediated eicosanoid metabolism and vascular function phenotypes were evaluated by Pearson's correlation. ResultsA significant inverse association was observed between 20-HETE levels (a biomarker of CYP ω-hydroxylase metabolism) and brachial artery flow-mediated dilation (r = −0.255, p = 0.010). An inverse association was also observed between 14,15-EET:DHET ratios (a biomarker of sEH metabolism) and both monocyte chemoattractant protein-1 levels (r = −0.252, p = 0.009) and a consolidated cellular adhesion molecule ‘score’ reflecting the levels of E-selectin and P-selectin (r = −0.216, p = 0.027). No associations with C-reactive protein or epithelial neutrophil-activating protein-78 levels were observed. ConclusionsCollectively, these findings demonstrate that enhanced CYP ω-hydroxylase and sEH metabolic function are associated with more advanced endothelial dysfunction and vascular inflammation, respectively, in patients with established atherosclerotic cardiovascular disease. These findings lay the foundation for future clinical research in this area.

The levels of serum chemokines in patients with Behçet's disease

As chemokines are involved in the regulation of immune responses and inflammatory reactions, this study aimed to investigate the serum levels of some chemokines to determine whether they play roles in the pathogenesis of Behçet's disease (BD). Materials and methods: The study comprised 64 cases, allocated to active or inactive patient groups. The levels of serum macrophage inflammatory protein-1\alpha (MIP-1\alpha), epithelial neutrophil-activating protein-78 (ENA-78), and neutrophil-activating peptide-2 (NAP-2) were measured. Results: The serum levels of MIP-1\alpha and ENA-78 were statistically higher in BD patients than controls (P = 0.032 and P = 0.023, respectively). However, NAP-2 levels did not differ significantly between patients and controls (P > 0.05). A significantly higher mean serum level of ENA-78 was found in patients with active disease compared to patients with inactive disease and controls (P = 0.029 and P = 0.003, respectively). However, mean serum MIP-1\alpha and NAP-2 levels did not differ significantly between active and inactive patients (P > 0.05). Conclusion: In this study, there was a significant elevation of MIP-1\alpha and ENA-78 supporting the role of neutrophil hyperactivity in BD. However, because chemokines insert their effects in the microenvironment and serum concentrations reflect a developing inflammatory process indirectly, we suggest that more comprehensive studies are needed to reveal the role of chemokines in the pathogenesis of the disease.

Monitoring serum levels ELR+ CXC chemokines and the relationship between microvessel density and angiogenic growth factors in multiple myeloma

BackgroundThe ELR+ CXC chemokines are important mediators of tumorigenesis, related to their angiogenic properties. Angiogenesis appears to be a prominent feature in the progression of multiple myeloma (MM). CXC chemokines have four highly conserved cysteine amino acid residues, with the first two cysteine molecules separated by a single amino acid. The angiogenic potential of this group is determined by the presence of three amino acid residues (Glu-Leu-Arg: the ELR motif) preceding the first cysteine amino acid, in the NH2 terminus. AimsThe purpose of this study was to determine serum concentrations of angiogenesis-related chemokines ELR+ motif, such as interleukin-8 (IL-8), epithelial neutrophil activating protein-78 (ENA-78) and growth-related gene alpha (GRO-α), as well the bone marrow microvascular density (MVD) in patients with MM at diagnosis and after treatment, in plateau phase. We also evaluated the relationship among them with other known growth factors involved in angiogenesis. MethodsSerum levels of the ELR+ CXC chemokines: IL-8, ENA-78 and GRO-α as well as of the angiogenic factors: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α) were determined in 63 newly diagnosed MM patients, in 30 in plateau phase and in 20 healthy controls. Serum measurements of them were performed with commercially available kits for ELISA. Bone marrow biopsies were performed before and after treatment, in plateau phase, in order to determine MVD by staining vessels with anti-CD31. ResultsSerum concentrations of IL-8, ENA-78, GRO-α and TNF-α were significantly higher in the group of MM patients (44.5±25.3, 765±572.1, 186.5±129.1 and 4.2±2.8pg/ml, respectively) in comparison to control group (27.3±6.4, 335.1±268.6, 112.5±76.1 and 1.3±0.8pg/ml) (p<0.02 for GRO-α, p<0.001 for other cases). We also found that untreated patients had higher levels of IL-8, ENA-78, GRO-α than post treatment patients, but statistical significant difference was found only for IL-8 (48.36±30.93pg/ml vs. 35.05±19.77pg/ml, p<0.001). Furthermore IL-8, GRO-α, TNF-α, HGF and VEGF were significantly higher with increasing disease stage (p<0.001 in all cases). ENA-78 serum levels were higher in stage III than in stage I and II, but without statistical significance. Additionally we correlated each proinflammatory cytokine with well known angiogenic factors such as HGF, VEGF and TNF-α. A positive correlation was found between serum HGF and IL-8 and GRO-α (r=0.316 p<0.01, r=0.297 p<0.02, respectively). Similarly serum VEGF correlated with ENA-78 and GRO-α (r=0.323 p<0.01, r=0.469 p<0.001, respectively). In the pretreatment group of patients a positive correlation between bone marrow MVD and serum levels of GRO-α was found (r=0.304 p<0.01). There was a difference in survival times between patients with higher than median versus low IL-8, ENA-78 and GRO-α levels, but the differences could not reach statistical significance in either case. ConclusionsThese findings support the hypothesis that ELR+ motif CXC chemokines, such as IL-8, ENA-78 and GRO-α correlate with angiogenic growth factors and may play a role in the progression of MM. Further studies are needed to determine their prognostic and predictive significance.

Neutrophil chemotaxis in granulomatosis with polyangiitis (Wegener's) and idiopathic pulmonary fibrosis

The presence of antineutrophil cytoplasmic antibodies in granulomatosis with polyangiitis (Wegener's) (GPA) implicates the neutrophil as a key effector cell. Previous studies have reported elevated neutrophil counts in the lung, although the determinants of neutrophil chemotaxis in the GPA lung are unknown. Bronchoalveolar lavage fluid (BALF) cell counts, myeloperoxidase (MPO) and chemokines were measured in 27 patients with GPA, 20 disease controls with idiopathic pulmonary fibrosis (IPF) and six healthy controls. CXC chemokine ligand (CXCL)8, interleukin (IL)-1β, epithelial neutrophil-activating protein 78, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor were measured by ELISA. The neutrophil chemotactic potential of BALF was investigated using the under-agarose method, and specific antibodies were used to examine the role of CXCL8 and IL-1β. GPA BALF had an increased neutrophil percentage, and elevated MPO, CXCL8 and G-CSF concentrations compared with healthy controls. Chemotaxis of control neutrophils towards BALF from patients with active (p=0.006) and remission (p=0.077) GPA, and IPF (p=0.001) patients was increased compared with normal controls. BALF-induced chemotaxis correlated with BALF IL-1β (r=0.761, p=0.001) and CXCL8 (r=0.640, p=0.012) in GPA, and was inhibited by anti-CXCL8 (85%; p<0.001) and anti-IL-1β (69%; p<0.001). Our study confirms a neutrophilia and pro-inflammatory alveolar milieu that persists in clinical remission. CXCL8 and IL-1β appear to play important roles in the neutrophil chemotactic response to BALF.

Expressions and significance of CXC chemotactic factors about GROα, ENA-78 and NAP-2 at rat asthma

Objective To observe the expressions of grouth-related oncogen (GRO)α, epithelial neutrophil activating protein-78 (ENA-78) and neutrophil-activating peptide-2 (NAP-2) of rat asthma. And to investigate the role of neutrophil in the pathogenesis of asthma exacerbation. Methods In this experi-ment, the rat model of asthma were randomly divided into two groups on average, including asthma group and control group. Levels of ENA-78 at blood neutrophil were detected by flow cytometry method. The ex-pressions of GROα protein at bronchial wall and NAP-2 protein at blood neutrophil were detected by immuno-histochemieal method. Results Levels of GROα, ENA-78 and NAP-2 proteins in asthma group [0.138 ±0.009(A value), 97.65±13.99(MFI), 0.198±0.016(A value), respectively]were significantly higher than those in control group[0.077±0.010(A value), 50.79±8.66(MFI), 0.079±0.015(A value), re-spectively], all P < 0.01. Conclusion Levels of GROα, ENA-78 and NAP-2 were increased at rat asth-ma. They may be participate in inflammation of asthma exacerbation. Neutrophil may promote inflammatory cells influxing into airway wall via increasing synthesis of CXC chemotactic factors. Key words: Asthma; GROα; ENA-78; NAP-2; Chemotactic factor

Bronchoalveolar Lavage Fluid Cellular Characteristics, Functional Parameters and Cytokine and Chemokine Levels in Interstitial Lung Diseases

Idiopathic pulmonary fibrosis (IPF), hypersensitivity pneumonitis (HP) and sarcoidosis belong to interstitial lung diseases (ILD) where an imbalance of regulatory, profibrotic and antifibrotic cytokines is hypothesized. The relationship of bronchoalveolar lavage (BAL) fluid (BALF) cytokines, BALF cell profile and ILD course is supposed. The aim of our study was to correlate BALF cytokine and chemokine levels with BALF cellular characteristics and lung function parameters in different ILD. Twenty-two sarcoidosis, seven IPF and 11 HP patients underwent lung function tests and BAL. The BALF differential cell counts and superficial cell markers were characterized, and MCP-1, MIP-1alpha, MIP-1beta, RANTES, epithelial neutrophil-activating protein (ENA)-78, FGF, G-CSF, GM-CSF, IFN-gamma, interleukin (IL)-1alpha, IL-1RA, IL-1beta, -2beta, -4beta, -5beta, -6beta, -8beta, -10beta, -17beta, tumour necrosis factor (TNF)-alpha, thromobopoietin (Tpo) and vascular endothelial growth factor (VEGF) values measured. The BALF VEGF values were highest in sarcoidosis (P = 0.0526). IL-1RA values were higher in IPF and HP compared with sarcoidosis (P = 0.0334). IL-8/ENA-78 ratio positively correlated with BALF neutrophil counts in IPF (r = 0.89, P = 0.04). Vital capacity and TL(CO) values positively correlated with VEGF and negatively with IL-8 BALF levels in all ILDs but the correlations were most significant in sarcoidosis group. We suppose that VEGF plays a role in ILDs' early phases and has rather angiogenic than profibrotic effect. On the contrary, IL-8 is probably upregulated in advanced ILDs with prominent fibrosis and marked lung functions decline. We state that BALF VEGF, IL-8 and ENA-78 levels and IL-8/ENA-78 ratio could become useful markers of ILDs' phase, activity and prognosis. They might also be helpful in treatment modality choice.

Influences of aflatoxin B1 on reactive oxygen species generation and chemotaxis of human polymorphonuclear leukocytes

The effects of aflatoxin (AF), a hepatotoxic agent and the strongest carcinogen in nature, on polymorphonuclear leukocyte (PMN) chemotaxis and chemiluminescence (CL) were studied. Luminol-dependent CL activity, which reflects the production of reactive oxygen species (ROS) from PMNs, was up-regulated to approximately 150% when PMNs were treated with 0.05 ng/ml of AFB1 upon stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP) or zymosan. On the other hand, PMN CL activity was down-regulated to approximately 25% of the control when PMNs were treated with 50 ng/ml of AFB1 upon stimulation with zymosan. The effect of AFB1 on PMN chemotaxis was also investigated using the Boyden chamber method. The chemotactic ability of PMNs when interleukin (IL)-8 was used as a chemoattractant was inhibited in a dose-dependent manner at AFB1 concentrations of >10 ng/ml. In reverse transcriptase (RT)-PCR analysis, gene expression levels of CXC chemokine receptor (CXCR)1/2, whose ligands are IL-8, granulocyte chemotactic protein (GCP)-2, neutrophil attractant-activation protein (NAP)-2, and epithelial neutrophil-activating protein (ENA)-78, which regulate PMN chemotaxis, were also down-regulated in a dose-dependent manner by 50 ng/ml AFB1. mRNA expression levels of CXCR1/2 were down-regulated to approximately 85% of that in the controls when PMNs were treated with 100 ng/ml AFB1. These results suggest that a high concentration of AFB1 reduces the chemotactic ability of PMNs via the CXCR1/2 cascade indirectly.

Long‐term bone marrow culture and its clinical potential in chronic wound healing

Bone marrow-derived cells have long been regarded to play a crucial role in the homeostasis of skin. We have previously described the clinical benefit of directly applying autologous bone marrow aspirate and cultured bone marrow cells to recalcitrant chronic skin wounds. The initial response to treatment appears to be vascular in nature with the formation of new blood vessels. The difficulty in consistently growing adequate numbers of cells for delivery to patients was, however, a limiting factor. Here, in a subsequent protocol, we describe an improved bone marrow culture system yielding a reliable growth of bone marrow cells and leading to a greater clinical response. Cells expressing markers of endothelial progenitors including CD133, CD146, and particularly CD14 are enhanced in these cultures. CD14-isolated cells produced colonies in endothelial cell assays and sprouting in matrigel assays. Angiogenic cytokines, including angiogenin, epithelial neutrophil-activating protein-78, growth-regulated oncogene, growth-regulated oncogene-alpha, Interleukin-8, CXC16, and monocyte chemoattractant protein-1, were found to be elevated in these cultures. Administration of improved culture cells to patients with chronic wounds present for >1 year lead to an enhanced clinical response.

Molecular characterization of post-thrombotic syndrome

The post-thrombotic syndrome represents a poorly understood and significant vascular health problem. This review focuses on our current understanding of the pathogenesis of post-thrombotic syndrome. We emphasize the cellular and molecular mechanisms that are responsible for the critical components of post-thrombotic syndrome. These include the initiation of deep venous thrombosis, the pathogenesis of elevated venous pressure, and the factors responsible for nonhealing of venous stasis ulcers.

Targeting adenosine receptors in the treatment of allergic rhinitis: a randomized, double‐blind, placebo‐controlled study

There is evidence that adenosine plays a role in the pathogenesis of asthma and rhinitis; however, it is currently unclear whether adenosine receptors are useful therapeutic targets in the treatment of allergic airway diseases. The study evaluated the efficacy of intranasal treatment with an adenosine A(2A) receptor agonist/adenosine A(3) receptor antagonist (50 micro g), administered twice daily for 7 days, to reduce nasal symptoms and release of inflammatory mediators following intranasal allergen challenge in patients with allergic rhinitis (AR). The compound was compared with twice-daily treatment with intranasal fluticasone proprionate nasal spray (FPANS) for 7 days. A randomized, double-blind, double-dummy, placebo-controlled, three-way balanced, incomplete block, crossover study was conducted on 48 males with verified AR. Following intranasal challenge with either an extract from the house dust mite (HDM), Dermatophagoides pteronyssinus, rye grass or cat dander, nasal responses and the concentrations of albumin, tryptase, myeloperoxidase, eosinophilic cationic protein, epithelial neutrophil-activating protein-78 (ENA-78), IL-5 and IL-8 in nasal secretions were measured and treatment groups were compared. Drug improved nasal blockage but had no significant effect on rhinorrhoea, number of sneezes or peak nasal inspiratory flow measurements when compared with placebo. Drug reduced tryptase release after EAR but did not significantly reduce the levels of other mediators. A novel agonist/antagonist of adenosine A(2A) and A(3) receptors appears to have limited clinical benefit in both the early-phase and the late-phase response to intranasal allergen challenge. However, reduction of some pro-inflammatory mediators suggests that comparable, more selective compounds may have additional benefits meriting further investigation.

Different Angiogenic Activity in Pulmonary Sarcoidosis and Idiopathic Pulmonary Fibrosis

Recent evidence has shown that several chemokines--including those involved in angiogenesis--have been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and sarcoidosis. We speculated that these differences could be attributed to distinct angiogenic and angiostatic profiles. This hypothesis was investigated by estimating the levels of three angiogenic chemokines (growth-related gene [GRO]-alpha, epithelial neutrophil-activating protein [ENA]-78, and interleukin [IL]-8), and three angiostatic chemokines (monokine induced by interferon (IFN)-gamma [MIG], IFN-gamma-inducible protein [IP]-10, and IFN-gamma-inducible T-cell alpha chemoattractant) in serum and BAL fluid (BALF). We studied prospectively 20 patients with sarcoidosis (median age, 46 years; range, 25 to 65 years), 20 patients with IPF (median age, 68 years; range, 40 to 75 years), and 10 normal subjects (median age, 39 years; range, 26 to 60 years). The GRO-a serum and BALF levels of IPF patients were found significantly increased in comparison with healthy subjects (799 pg/mL vs 294 pg/mL [p = 0.022] and 1,827 pg/mL vs 94 pg/mL [p < 0.001], respectively) and sarcoidosis patients (799 pg/mL vs 44 pg/mL [p < 0.001] and 1,827 pg/mL vs 214 pg/mL [p < 0.001], respectively). Moreover, ENA-78 and IL-8 BALF levels in IPF patients were significantly higher compared with sarcoidosis patients (191 pg/mL vs 30 pg/mL [p < 0.001] and 640 pg/mL vs 94 pg/mL [p = 0.03], respectively). MIG serum levels in IPF patients were found significantly upregulated in comparison with sarcoidosis patients and healthy control subjects. However, MIG and IP-10 BALF levels (1,136 pg/mL vs 66 pg/mL [p < 0.001] and 112 pg/mL vs 56 pg/mL [p = 0.037], respectively) were significantly higher in sarcoidosis patients compared with IPF patients. Our data suggest distinct angiogenic profiles between IPF and sarcoidosis, indicating a potential different role of CXC chemokines in the local immunologic response in IPF and pulmonary sarcoidosis.

Mesothelioma environment comprises cytokines and T-regulatory cells that suppress immune responses

Malignant mesothelioma is a cancer with dismal prognosis. The objective of the present study was to address the role of the immune system, tumour micro-environment and potential immunosuppression in mesothelioma. Expression profiles of 80 cytokines were determined in the supernatant of mesothelioma cell lines and the original patient's pleural effusion. Influx of immune effector cells was detected by immunohistochemistry. Angiogenin, vascular endothelial growth factor, transforming growth factor-beta, epithelial neutrophil-activating protein-78 and several other proteins involved in immune suppression, angiogenesis and plasma extravasation could be detected in both supernatant and pleural effusion. Surrounding stroma and/or infiltrating cells were the most likely source of hepatocyte growth factor, macrophage inflammatory protein (MIP)-1delta, MIP-3alpha, neutrophil-activating peptide-2, and pulmonary and activation-regulated chemokine that can cause leukocyte infiltration and activation. There was a massive influx of CD4+ and CD8+ T-lymphocytes and macrophages, but not of dendritic cells, in human mesothelioma biopsies. It was further demonstrated that human mesothelioma tissue contained significant amounts of Foxp3+CD4+CD25+ regulatory T-cells. When these CD25+ regulatory T-cells were depleted in an in vivo mouse model, survival increased. Mesothelioma is infiltrated by immune effector cells but also contains cytokines and regulatory T-cells that suppress an efficient immune response. Immunotherapy of mesothelioma might be more effective when combined with drugs that eliminate or control regulatory T-cells.

Differential and additive effects of platelet-derived chemokines on monocyte arrest on inflamed endothelium under flow conditions

Platelet-derived chemokines, such as regulated on activation, normal T expressed and secreted (RANTES; CC chemokine ligand 5), platelet factor 4 [PF4; CXC chemokine ligand 4 (CXCL4)], and epithelial neutrophil-activating protein 78 (ENA-78; CXCL5), or precursors, such as beta-thromboglobulin, which can be processed to neutrophil-activating protein-2 (NAP-2; CXCL7), may play an important role in monocyte recruitment during atherogenesis. Platelets can deposit chemokines on inflamed endothelium; however, little is known about differential or additive effects of platelet chemokines on monocyte arrest. Here, we demonstrate that preincubation of activated human microvascular endothelial cells (HMVECs) with RANTES, PF4, or NAP-2 but not ENA-78 dose-dependently increased surface immobilization and subsequent monocyte arrest in flow. RANTES was the most potent and efficient arrest chemokine. Pretreatment of HMVECs with beta-thromboglobulin enhanced monocyte arrest in the presence of cathepsin G generating NAP-2. Combined pretreatment of HMVECs with RANTES and PF4 at suboptimal concentrations synergistically increased arrest, and preincubation with chondroitinase ABC abrogated RANTES- and PF4-induced monocyte arrest. This was associated with reduced expression of chondroitin sulfate, RANTES, and PF4 on the HMVEC surface. Perfusion of HMVECs with platelets known to deposit RANTES and PF4 on the endothelial surface enhanced monocyte arrest, which was inhibited by Met-RANTES, chondroitinase, or a blocking antibody to PF4 but not to ENA-78. The relevance of platelet-derived chemokines was confirmed in adhesion assays with activated whole blood, where Met-RANTES and to a lesser extent, antibodies to PF4 and NAP-2 inhibited arrest of CD14-positive monocytes. Thus, multiple platelet-derived chemokines and processable precursors, which can be presented by specific endothelial proteoglycans, may contribute and cooperate differentially to induce monocyte recruitment.

Membrane Array Characterization of 80 Chemokines, Cytokines, and Growth Factors in Open- and Closed-Eye Tears: Angiogenin and Other Defense System Constituents

To adapt membrane-bound antibody array (MA) technology to characterize the distribution of a wide range of bioactive trace proteins in reflex (RTF) and open-eye (OTF) and closed-eye (CTF) tear samples. Tears were collected by capillary tube and centrifuged. A commercially available standard MA and a custom array were modified to maximize the sensitivity of detection and the signal-to-noise ratio, to assay RTF and individually pooled CTF and OTF samples for 80 chemokines, growth factors, cytokines, and angiogenic modulators. The reliability of data was assessed by Western blot and other methods. Coupling an ultrasensitive chemiluminescence substrate system to an MA and optimizing conditions enhanced the sensitivity several hundredfold, allowing the detection of approximately 40 of the 79 probed proteins on the standard array, most of which were shown to be elevated in CTF. Identified entities include the known constituents epidermal growth factor (EGF), monocyte chemoattractant protein (MCP)-1, IL-8, tissue inhibitor of metalloproteinase (TIMP)-1 and -2, and numerous previously undetected tear components, such as angiogenin (ANG), growth factors, and the CXC and CC chemokines IFN-gamma inducible protein (IP)-10, growth-related oncogene (GRO), epithelial neutrophil-activating protein (ENA)-78, and macrophage inflammatory protein (MIP)-3alpha. Identification of other proteins was hindered by high background on the negative control array. Using a less complex custom array dramatically reduced background and allowed the visualization in CTF of proteins, such as VEGF, that were not detected with the standard array. MAs are powerful tools for differential screening of tears for large numbers of trace proteins. Analysis allowed the identification of previously undetected proteins that may participate in the host defense system as well as demonstrated the profound change in tear composition associated with prolonged eye closure in a manner reflective of physiological function.