The "two-signal paradigm" in T cell activation predicts that the cooperation of "signal 1," provided by the T cell receptor (TCR) through engagement of major histocompatility complex (MHC)-presented peptide, with "signal 2″ provided by costimulatory molecules, the prototype of which is CD28, is required to induce T cell effector functions. While the individual signalling pathways are well understood, little is known about global changes in the proteome pattern during TCR/CD28-mediated activation. Therefore, comparative 2-DE-based proteome analyses of CD3(+) CD69(-) resting T cells versus cells incubated with (i) the agonistic anti-CD3 antibody OKT3 mimicking signal 1 in absence or presence of IL-2 and/or with (ii) the agonistic antibody 15E8 triggering CD28-mediated signaling were performed. Differentially regulated spots were defined leading to the identification of proteins involved in the regulation of the metabolism, shaping and maintenance of the cytoskeleton and signal transduction. Representative members of the differentially expressed protein families, such as calmodulin (CALM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L-lactate dehydrogenase (LDH), Rho GDP-dissociation inhibitor 2 (GDIR2), and platelet basic protein (CXCL7), were independently verified by flow cytometry. Data provide a detailed map of individual protein alterations at the global proteome level in response to TCR/CD28-mediated T cell activation.

Diabetic nephropathy (DN) is the leading cause of renal failure worldwide and its complications have become a public health problem. Inflammation, oxidative stress and fibrosis play central roles in the progression of DN that lead to renal failure. Potential deleterious effect of inflammation in early evolution of DN is not fully disclosed. Therefore, it is relevant to explore therapies that might modulate this process in order to reduce DN progression. We explored the beneficial effect of all-trans retinoic acid (ATRA) in early inflammation in glomeruli, proximal and distal tubules in streptozotocin (STZ)-induced diabetes. ATRA was administered (1 mg/kg daily by gavage) on days 3 to 21 after STZ administration. It was found that 21 days after STZ injection, diabetic rats exhibited proteinuria, increased natriuresis and loss of body weight. Besides, diabetes induced an increase in interleukins [IL-1β, IL-1α, IL-16, IL-13, IL-2; tumor necrosis factor alpha (TNF-α)] and transforming growth factor-beta 1 (TGF-β1), chemokines (CCL2, CCL20, CXCL5 and CXCL7), adhesion molecules (ICAM-1 and L-selectin) and growth factors (GM-CSF, VEGF, PDGF) in glomeruli and proximal tubules, whereas ATRA treatment remarkably ameliorated these alterations. To further explore the mechanisms through which ATRA decreased inflammatory response, the NF-κB/p65 signaling mediated by TLR4 was studied. We found that ATRA administration attenuates the TLR4/NF-κB inflammatory signaling and prevents NF-κB nuclear translocation in glomeruli and proximal tubules.

Although originally identified as mediators of inflammation, it is now apparent that chemokines play a fundamental role in tissue development. In this study, ELR(+)-CXC chemokine family members CXCL2 and CXCL7, along with their preferred receptor CXCR2, were expressed at the earliest stages of metanephric development in the rat, and signaling through this receptor was required for the survival and maintenance of the undifferentiated metanephric mesenchyme (MM). A specific antagonist of the CXCR2 receptor SB225002 induced apoptosis in this population but did not affect more mature structures or cells in the ureteric bud. CXCL7 treatment of isolated MM elicited an angiogenic response by upregulation of matrix metalloprotease 9 and endothelial and mesangial markers (platelet-endothelial cell adhesion molecule, Megsin, Thy-1, PDGF receptor alpha, and vascular alpha-actin) and induced SB225002-sensitive cell invasion through a matrix. Because Wilms' tumor cells may similarly depend on CXCR2 signaling for survival, primary tumor samples were analyzed, and 15 of 16 Wilms' tumors were found to be CXCR2 positive, whereas grossly normal kidney tissues from tumor patients or renal cell carcinomas were CXCR2 negative. Furthermore, cell lines derived from Wilms' tumors but not those from renal cell carcinomas were sensitive to SB225002-induced apoptosis. These data provide evidence for a prosurvival and proangiogenic role of ELR(+)-CXC chemokines and their receptor CXCR2 during metanephric development and suggest a novel mechanism for chemotherapeutic intervention in Wilms' tumor.

We previously reported that hypoxic stress enhanced osteoclast differentiation via increasing insulin-like growth factor 2 (IGF2) production. However, the mechanisms underlying IGF2 stimulation remains unknown. In this study, we investigated the molecular mechanisms of osteoclastogenesis by IGF2 treatment. Primary mouse bone marrow cells were cultured with IGF2. Total RNAs were applied to a DNA microarray analysis, and quantitative RT-PCR was then used to confirm the microarray data and clarify which cells expressed the relative genes. The most interesting findings were the upregulations of CXC chemokine ligand 7 (CXCL7) expression in stromal cells and stromal cell-derived factor 1 (SDF1) expression in osteoblastic cells with IGF2 treatment. The addition of exogenous SDF1 to CXCL7 increased the number of osteoclasts and promoted the formation of giant osteoclasts. These results suggest that IGF2 modulates the microenvironment around osteoclast precursor cells. SDF1 together with CXCL7 may promote the formation of giant osteoclasts.

BACKGROUND

Several Echinacea species have been used as nutraceuticals or botanical drugs for "immunostimulation", but scientific evidence supporting their therapeutic use is still controversial. In this study, a phytocompound mixture extracted from the butanol fraction (BF) of a stem and leaf (S+L) extract of E. purpurea ([BF/S+L/Ep]) containing stringently defined bioactive phytocompounds was obtained using standardized and published procedures. The transcriptomic and proteomic effects of this phytoextract on mouse bone marrow-derived dendritic cells (BMDCs) were analyzed using primary cultures.

RESULTS

Treatment of BMDCs with [BF/S+L/Ep] did not significantly influence the phenotypic maturation activity of dendritic cells (DCs). Affymetrix DNA microarray and bioinformatics analyses of genes differentially expressed in DCs treated with [BF/S+L/Ep] for 4 or 12 h revealed that the majority of responsive genes were related to cell adhesion or motility (Cdh10, Itga6, Cdh1, Gja1 and Mmp8), or were chemokines (Cxcl2, Cxcl7) or signaling molecules (Nrxn1, Pkce and Acss1). TRANSPATH database analyses of gene expression and related signaling pathways in treated-DCs predicted the JNK, PP2C-α, AKT, ERK1/2 or MAPKAPK pathways as the putative targets of [BF/S+L/Ep]. In parallel, proteomic analysis showed that the expressions of metabolic-, cytoskeleton- or NF-κB signaling-related proteins were regulated by treatment with [BF/S+L/Ep]. In vitro flow cytometry analysis of chemotaxis-related receptors and in vivo cell trafficking assay further showed that DCs treated with [BF/S+L/Ep] were able to migrate more effectively to peripheral lymph node and spleen tissues than DCs treated as control groups.

CONCLUSIONS

Results from this study suggest that [BF/S+L/Ep] modulates DC mobility and related cellular physiology in the mouse immune system. Moreover, the signaling networks and molecules highlighted here are potential targets for nutritional or clinical application of Echinacea or other candidate medicinal plants.

CXCL7 is an important chemoattractant cytokine, which signals through binding to its receptor CXCR2. Recent studies have demonstrated that the CXCL7/CXCR2 signaling plays a promoting role in several common malignancies, including lung, renal, colon, and breast cancer. However, the regulatory role of CXCL7, in cholangiocarcinoma, as well as the underlying mechanism, has not been previously reported. Herein, we found more positive expression of CXCL7 in cholangiocarcinoma tissues compared to adjacent non-tumor tissues. High CXCL7 expression was significantly correlated with poor differentiation, lymph node metastasis, vascular invasion and advanced clinical stage, but was not associated with age, gender, or tumor size. Besides, the expression of CXCL7 was significantly associated with the Ki67 expression, but not associated with CA199, AFP, or P53 expression in cholangiocarcinoma. Moreover, the overall survival of cholangiocarcinoma patients with high CXCL7 expression was significantly shorter than those with low CXCL7 expression. In vitro study indicated that CXCL7 and CXCR2 were also positively expressed in several common cholangiocarcinoma cell lines, including HuCCT1, HuH28, QBC939, EGI-1, OZ and WITT. SiRNA-induced inhibition of CXCL7 significantly reduced the proliferation and invasion of QBC939 cells. On the contrary, overexpression of CXCL7 markedly promoted these malignant phenotypes of QBC939 cells. Of note, the conditioned medium of CXCL7-overexpresing human hepatic stellate cells could also promote the proliferation and invasion of QBC939 cells, suggesting that CXCL7 may also play an oncogenic role in cholangiocarcinoma in a paracrine-dependent manner, not only in an autocrine-dependent manner. Molecular assay data suggested that the AKT signaling pathway was involved in the CXCL7-mediated malignant phenotypes of QBC939 cells. In summary, our study suggests that CXCL7 plays a promoting role in regulating the growth and metastasis of cholangiocarcinoma.

Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.

Identification of effective biomarkers is crucial for monitoring the treatment and remission of colorectal cancer (CRC) and improving survival. It is particularly important to diagnose CRC before the tumor metastasizes (stage I-II disease) where possible, to provide the greatest opportunity for patient recovery. Here, we evaluated the clinical value of serum chemokine (C-X-C) ligand 7 (CXCL7) concentration as a biomarker for CRC diagnosis. An enzyme-linked immunosorbent assay was used to measure CXCL7 concentration in 560 serum samples from patients with CRC and controls. Logistic regression and receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic efficacy and build mathematical diagnostic models. The concentration of CXCL7 in the CRC group was significantly higher than that in the control group (P < 0.001), with an area under the ROC curve (AUC) value of 0.862 [95% confidence interval (CI): 0.831-0.890]. Further, the AUC of a regression model including the markers carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and carbohydrate antigen 125 (CA125), along with CXCL7, was 0.933 (95% CI: 0.909-0.952). For stage I-II tumors, CXCL7 had the highest AUC (0.823, 95% CI: 0.783-0.858) among the four individual biomarkers. The AUC value for combination model analysis of samples from patients with stage I-II tumors was 0.904 (95% CI: 0.872-0.930), with a sensitivity of 82.76% and a specificity of 87.14%, and an optimal cut-off value of 2.66. AUC values for application of the regression model in subgroup analysis were 0.947 (0.917-0.968) and 0.919 (0.874-0.951) for males and females, respectively. These results suggest that CXCL7 has potential as a serum diagnostic biomarker for detection of CRC. Importantly, the combination of CXCL7, CEA, CA125, and CA19-9 may facilitate diagnosis of CRC with relatively high sensitivity and specificity. Clinical Trial Registration Number: LS2017001.

Although human amniotic epithelial cells (AMEs) are an attractive source of stem cells, their therapeutic potential in wound healing has not been fully investigated. We evaluated the therapeutic potential of AMEs for wound healing. Real-time PCR showed that the epithelialization growth factors epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-B and chemotactic factors interleukin-8 (IL-8 or CXCL8) and neutrophil-activating protein-2 (NAP-2 or CXCL7) were upregulated in AMEs compared with adipose-derived mesenchymal stem cells (ADMs). In vitro scratch wound assays revealed that AME-derived conditioned medium substantially accelerated wound closure. Wounds in NOD/SCID mice were created by skin excision, followed by AME transplantation. AMEs implantation significantly accelerated wound healing and increased cellularity and re-epithelialization. Transplanted AMEs exhibited high engraftment rates and expressed keratinocyte-specific proteins and cytokeratin in the wound area, suggesting direct benefits for cutaneous closure. Taken together, these data indicate that AMEs possess therapeutic capability for wound healing through the secretion of epithelialization growth factors and enhanced engraftment properties. Copyright © 2015 John Wiley & Sons, Ltd.

Platelets and neutrophils contribute to the development of acute lung injury (ALI). However, the mechanism by which platelets make this contribution is incompletely understood. We investigated whether the two most abundant platelet chemokines, CXCL7, which induces neutrophil chemotaxis and activation, and CXCL4, which does neither, mediate ALI through complementary pathogenic pathways. To examine the role of platelet-derived chemokines in the pathogenesis of ALI using Cxcl7-/- and Cxcl4-/- knockout mice and mice that express human CXCL7 or CXCL4, we measured levels of chemokines in these mice. ALI was then induced by acid aspiration, and the severity of injury was evaluated by histology and by the presence of neutrophils and protein in the bronchoalveolar lavage fluid. Pulmonary vascular permeability was studied in vivo by measuring extravasation of fluorescently labeled dextran. Murine CXCL7, both recombinant and native protein released from platelets, can be N-terminally processed by cathepsin G to yield a biologically active CXCL7 fragment. Although Cxcl7-/- mice are protected from lung injury through the preservation of endothelial/epithelial barrier function combined with impaired neutrophils transmigration, Cxcl4-/- mice are protected through improved barrier function without affecting neutrophils transmigration to the airways. Sensitivity to ALI is restored by transgenic expression of CXCL7 or CXCL4. Platelet-derived CXCL7 and CXCL4 contribute to the pathogenesis of ALI through complementary effects on neutrophil chemotaxis and through activation and vascular permeability.

Increased circulating chemokines have been reported during acute myocardial infarction and might give prognostic information about future ischemic events. However, data on the chemokine network in relation to infarct size and measures of left ventricular remodeling after successful percutaneous coronary intervention (PCI) are lacking. A total of 42 patients with first-time ST-segment elevation acute myocardial infarction with a single occluded vessel were recruited, and cardiac magnetic resonance was used for serial assessment (2, 7, and 60 days) of infarct size and left ventricular remodeling. The chemokines were analyzed before and after PCI. After PCI, high levels of CCL4, CXCL16, CXCL10, and, in particular, CXCL8 within the first week after PCI correlated positively with the degree of myocardial damage, as reflected by correlations with the maximum troponin T levels and infarct size after 2 months, as assessed by cardiac magnetic resonance, and with impaired myocardial function after 2 months as assessed by cardiac magnetic resonance and neurohormonal methods. In contrast, the plasma levels of CCL3 and CXCL7 during the first week correlated negatively with myocardial dysfunction after 2 months. In conclusion, our findings suggest a role for chemokines in both adaptive and maladaptive responses after myocardial infarction and might support a role for CCL4, CXCL16, CXCL10, and, in particular, CXCL8 in postmyocardial infarction reperfusion and remodeling.

METHODS

Human annulus fibrosus tissue and cells were analyzed for the presence of chemokine receptors and the migratory effect of selected chemokines.

OBJECTIVE

To investigate spontaneous repair mechanisms and underlying cell recruitment in response to annular tears and degenerative defects.

BACKGROUND

Resorption of herniated disc tissue and the attempt to close annulus tears with repair tissue occur spontaneously. Although chemokines are suggested to play a role in resorption of herniated disc tissue, the role of chemokines in annulus fibrosus homeostasis and repair remains unclear.

METHODS

Cells were isolated from annulus fibrosus tissue and expanded in the presence of human serum. Multiwell chemotaxis assays were used to analyze the migratory effect of human serum and 0 to 1000 nM concentrations of the chemokines CXCL7, CXCL10, CXCL12, CCL25, and XCL1 on annulus fibrosus cells (AFCs) (n = 9 per chemokine and dose). Presence of corresponding chemokine receptors in AFCs was determined by real-time polymerase chain reaction analysis and immunohistochemistry.

RESULTS

Serum (0.1%-10%) significantly (P < 0.01) stimulates the migration of AFCs. Compared with untreated cells, the migration of cells was significantly (P < 0.01) enhanced upon stimulation with 100 to 1000 nM CXCL10 and 1000 nM XCL1. Chemokine receptors showed low expression levels in expanded AFCs as assessed by polymerase chain reaction. Immunohistochemical staining of the CXCL10 receptor CXCR3 and the XCL1 receptor XCR1 showed that the presence of the particular receptors in AFCs expanded under conventional cell culture conditions. In native annulus fibrosus tissue, CXCR3 was evident, whereas XCR1 could not be detected.

CONCLUSIONS

The findings suggest that chemokines, in particular CXCL10, effectively recruit isolated AFCs. This suggests that chemokines are involved in annulus fibrosus homeostasis and potentially in spontaneous annulus repair attempts. This might have important implications for biological annulus-sealing strategies.

It is desirable to have a biomarker which can facilitate low-dose CT in diagnosis of early stage lung cancer. CTAPIII/CXCL7 is reported to be a potential biomarker for diagnosis of early lung cancer. In this study, we investigated the serum level of CTAPIII/CXCL7 in patients at different stage of lung cancer and the diagnostic efficacy of CTAPIII/CXCL7 in NSCLC. The plasma level of CTAPIII/CXCL7 was assayed by ELISA. CEA, SCCAg, and Cyfra211 were measured using a commercial chemiluminescent microparticle immunoassay. A total of 419 subjects were recruited, including 265 NSCLC patients and 154 healthy individuals. The subjects were randomly assigned to a training set and a test set. Receiver operating characteristic (ROC) and binary logistic regression analyses were conducted to evaluate the diagnostic efficacy and establish diagnostic mathematical model. Plasma CTAPIII/CXCL7 levels were significantly higher in NSCLC patients than in controls, which was independent of the stage of NSCLC. The diagnostic efficiency of CTAPIII/CXCL7 in NSCLC (training set: area under ROC curve (AUC) 0.806, 95% CI: 0.748-0.863; test set: AUC 0.773, 95% CI: 0.711-0.835) was greater than that of SCCAg, Cyfra21-1, or CEA. The model combining CTAPIII/CXCL7 with CEA, SCCAg, and Cyfra21-1 was more effective for NSCLC diagnosis than CTAPIII/CXCL7 alone. In addition, plasma level of CTAPIII/CXCL7 may contribute to the early diagnosis of NSCLC. CTAPIII/CXCL7 can be used as a plasma biomarker for the diagnosis of NSCLCs, particularly early stage lung cancer, with relatively high sensitivity and specificity.

Vitamin A plays an essential role in the maintenance of gut homeostasis but its interplay with chemokines has not been explored so far. Using an in vitro model system we studied the effects of human colonic epithelial cells (Caco2, HT-29, and HCT116) derived inflammatory stimuli on monocyte-derived dendritic cells and macrophages. Unstimulated Caco2 and HT-29 cells secreted CCL19, CCL21, and CCL22 chemokines, which could attract dendritic cells and macrophages and induced CCR7 receptor up-regulation by retinoic-acid resulting in dendritic cell migration. The chemokines Mk, CXCL16, and CXCL7 were secreted by all the 3 cell lines tested, and upon stimulation by IL-1β or TNF-α this effect was inhibited by ATRA but had no impact on CXCL1, CXCL8, and CCL20 secretion in response to IL-1β. In the presence of ATRA the supernatants of these cells induced CD103 expression on monocyte-derived dendritic cells and when conditioned by ATRA and cocultured with CD4(+) T-lymphocytes they reduced the proportion of Th17 T-cells. However, in the macrophage-T-cell cocultures the number of these effector T-cells was increased. Thus cytokine-activated colonic epithelial cells trigger the secretion of distinct combinations of chemokines depending on the proinflammatory stimulus and are controlled by retinoic acid, which also governs dendritic cell and macrophage responses.

Chemokines mediate diverse fundamental biological processes, including combating infection. Multiple chemokines are expressed at the site of infection; thus chemokine synergy by heterodimer formation may play a role in determining function. Chemokine function involves interactions with G-protein-coupled receptors and sulfated glycosaminoglycans (GAG). However, very little is known regarding heterodimer structural features and receptor and GAG interactions. Solution nuclear magnetic resonance (NMR) and molecular dynamics characterization of platelet-derived chemokine CXCL7 heterodimerization with chemokines CXCL1, CXCL4, and CXCL8 indicated that packing interactions promote CXCL7-CXCL1 and CXCL7-CXCL4 heterodimers, and electrostatic repulsive interactions disfavor the CXCL7-CXCL8 heterodimer. As characterizing the native heterodimer is challenging due to interference from monomers and homodimers, we engineered a "trapped" disulfide-linked CXCL7-CXCL1 heterodimer. NMR and modeling studies indicated that GAG heparin binding to the heterodimer is distinctly different from the CXCL7 monomer and that the GAG-bound heterodimer is unlikely to bind the receptor. Interestingly, the trapped heterodimer was highly active in a Ca2+ release assay. These data collectively suggest that GAG interactions play a prominent role in determining heterodimer function in vivo. Further, this study provides proof-of-concept that the disulfide trapping strategy can serve as a valuable tool for characterizing the structural and functional features of a chemokine heterodimer.

Chemokines are best known as signaling proteins in the immune system. Recently however, a large number of human chemokines have been shown to exert direct antimicrobial activity. This moonlighting activity appears to be related to the net high positive charge of these immune signaling proteins. Chemokines can be divided into distinct structural elements and some of these have been studied as isolated peptide fragments that can have their own antimicrobial activity. Such peptides often encompass the α-helical region found at the C-terminal end of the parent chemokines, which, similar to other antimicrobial peptides, adopt a well-defined membrane-bound amphipathic structure. Because of their relatively small size, intact chemokines can be studied effectively by NMR spectroscopy to examine their structures in solution. In addition, NMR relaxation experiments of intact chemokines can provide detailed information about the intrinsic dynamic behavior; such analyses have helped for example to understand the activity of TC-1, an antimicrobial variant of CXCL7/NAP-2. With chemokine dimerization and oligomerization influencing their functional properties, the use of NMR diffusion experiments can provide information about monomer-dimer equilibria in solution. Furthermore, NMR chemical shift perturbation experiments can be used to map out the interface between self-associating subunits. Moreover, the unusual case of XCL1/lymphotactin presents a chemokine that can interconvert between two distinct folds in solution, both of which have been elucidated. Finally, recent advances have allowed for the determination of the structures of chemokines in complex with glycosaminoglycans, a process that could interfere with their antimicrobial activity. Taken together, these studies highlight several different structural facets that contribute to the way in which chemokines exert their direct microbicidal actions.

BACKGROUND

Sunitinib is one of the first-line standard treatments for metastatic clear cell renal cell carcinoma (ccRCC) with a median time to progression shorter than 1 year. The objective is to discover predictive markers of response to adapt the treatment at diagnosis.

METHODS

Prospective phase 2 multi-centre trials were conducted in ccRCC patients initiating sunitinib (54 patients) or bevacizumab (45 patients) in the first-line metastatic setting (SUVEGIL and TORAVA trials). The plasmatic level of CXCL7 at baseline was correlated with progression-free survival (PFS).

RESULTS

The cut-off value of CXCL7 for PFS was 250 ng ml-1. Patients with CXCL7 plasmatic levels above the cut-off at baseline (250 ng ml-1) had a significantly longer PFS (hazard ratio 0.323 (95% confidence interval 0.147-0.707), P=0.001). These results were confirmed in a retrospective validation cohort. The levels of CXCL7 did not influence PFS of the bevacizumab-treated patients.

CONCLUSIONS

CXCL7 may be considered as a predictive marker of sunitinib efficacy for ccRCC patients.

Chemokines form a family of signaling proteins mainly responsible for directing the traffic of leukocytes, where their biological activity can be modulated by their oligomerization state. We characterize the dynamics and thermodynamic stability of monomer and homodimer structures of CXCL7, one of the most abundant platelet chemokines, using experimental methods that include circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, and computational methods that include the anisotropic network model (ANM), molecular dynamics (MD) simulations and the distance constraint model (DCM). A consistent picture emerges for the effects of dimerization and Cys5-Cys31 and Cys7-Cys47 disulfide bonds formation. The presence of disulfide bonds is not critical for maintaining structural stability in the monomer or dimer, but the monomer is destabilized more than the dimer upon removal of disulfide bonds. Disulfide bonds play a key role in shaping the characteristics of native state dynamics. The combined analysis shows that upon dimerization flexibly correlated motions are induced between the 30s and 50s loop within each monomer and across the dimer interface. Interestingly, the greatest gain in flexibility upon dimerization occurs when both disulfide bonds are present, and the homodimer is least stable relative to its two monomers. These results suggest that the highly conserved disulfide bonds in chemokines facilitate a structural mechanism that is tuned to optimally distinguish functional characteristics between monomer and dimer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced human monocyte-derived macrophage (GM-Mphi) or macrophage CSF (M-CSF)-induced human monocyte-derived Mphi (M-Mphi) are distinct in terms of the resistance to Mycobacterium tuberculosis. To elucidate the role of molecules involved in the functional differences between these Mphis, we investigated the gene expression profiles using microarray. After culture of CD14+ monocytes with CSFs, Mphis were cultured with or without bacillus Calmette-Guérin (BCG) (GM-Mphi-BCG and M-Mphi-BCG). The gene expression profiles from these cells were compared. Chemokines highly expressed in M-Mphis were selected and evaluated for anti-mycobacterial activity and superoxide production. FN1 and FCGR2B were the most up-regulated genes in GM-Mphi and M-Mphi, respectively. After stimulation with BCG, three chemokine genes (Osteopontin (SPP1), CXC chemokine ligand 7 (CXCL7) and CC chemokine ligand 11 (CCL11)) were highly expressed in M-Mphi-BCG when compared to those in GM-Mphi-BCG. A significantly increased resistance to M. tuberculosis H37Ra was observed after the stimulation of GM-Mphi with SPP1 or CXCL7. Superoxide production levels of SPP1- or CXCL7-stimulated GM-Mphis were higher than those of GM-Mphis without stimulation. These results indicate that both SPP1 and CXCL7 might have a role in the resistance against mycobacteria, at least in part, through augmenting reactive oxygen intermediate production in Mphis.

BACKGROUND

Platelets are an underappreciated factor in the classification of the bleeding tendency of patients with hemophilia. In this cross-sectional study, we investigated platelet activation status and responsiveness in relation to residual factor VIII activity and, within the group with severe hemophilia (<1% residual factor VIII activity), to annual factor VIII consumption.

METHODS

Twenty-one patients with mild-moderate hemophilia A, 13 with severe hemophilia A and 21 healthy controls were studied. The basal level of platelet activation and platelet responsiveness to activation and inhibition were determined by the measurement of platelet P-selectin expression and soluble platelet activation markers.

RESULTS

Patients with severe hemophilia A had a higher percentage of activated platelets at baseline (15.9%) when compared to patients with mild-moderate hemophilia A (8.2%, P=0.014) and controls (6.4%, P<0.001). Both patients with mild-moderate hemophilia A and those with severe hemophilia A had higher levels of the soluble platelet activation markers platelet factor 4 (1.4 and 1.8 pg/10(6) platelets), CXCL7 (65.8 and 48.2 pg/10(6) platelets) and RANTES (12.8 and 9.5 pg/10(6) platelets), compared to controls (platelet factor 4: 0.3 pg/10(6) platelets, P<0.001 and <0.001; CXCL7 20.0 pg/10(6) platelets, P<0.001 and <0.001; RANTES 4.5 pg/10(6) platelets, P<0.001 and =0.003, respectively). In support of these observations, we found clinical evidence that higher platelet P-selectin expression correlates with lower factor VIII consumption in patients with severe hemophilia (Spearman's r -0.65, P=0.043).

CONCLUSIONS

This study indicates that platelets from patients with severe hemophilia A are in a pre-activated state and that this pre-activated state is associated with factor VIII consumption.

BACKGROUND

Nowadays it is thought that the main cause of premature birth is subclinical infection. However, none of the currently used methods provide effective prevention to preterm labor. The aim of the study was to determine the concentration of selected chemokines in sera of patients with premature birth without clinical signs of infection (n = 62), threatened preterm labor (n = 47), and term births (n = 28).

METHODS

To assess the concentration of chemokines in the blood serum, we used a multiplex method, which allows the simultaneous determination of 40 chemokines per sample. The sets consist of the following chemokines: 6Ckine/CCL21, Axl, BTC, CCL28, CTACK/CCL27, CXCL16, ENA-78/CXCL5, Eotaxin-3/CCL26, GCP-2/CXC, GRO (GRO α /CXCL1, GRO β /CXCL2 and GRO γ /CXCL3), HCC-1/CCL14, HCC-4/CCL16, IL-9, IL-17F, IL18-BPa, IL-28A, IL-29, IL-31, IP-10/CXCL10, I-TAC/CXCL11, LIF, LIGHT/TNFSF14, Lymphotactin/XCL1, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, MDC/CCL22, MIF, MIP-3 α /CCL20, MIP-3- β /CCL19, MPIF-1/CCL23, NAP-2/CXCL7, MSP α , OPN, PARC/CCL18, PF4, SDF-1/CXCL12, TARC/CCL17, TECK/CCL25, and TSLP.

RESULTS

We showed possible implication of 4 chemokines, that is, HCC-4, I-TAC, MIP-3 α , and TARC in women with symptoms of preterm delivery.

CONCLUSIONS

On the basis of our findings, it seems that the chemokines may play role in the pathogenesis of preterm labor. Defining their potential as biochemical markers of preterm birth requires further investigation on larger group of patients.

We have investigated the role of CXCL7 in the immune response of human phagocytes against the intracellular bacteria Mycobacterium tuberculosis and Legionella pneumophila. We have observed that polymorphonuclear neutrophil (PMN) chemotaxis induced by the supernatants of infected monocyte derived macrophages (MDM) may be attributed to CXCL8 rather than CXCL7, although both chemokines are present in large quantities. We have also found that CXCL7 is present not only in the supernatants of MDM, but also in the supernatants of PMN of some, but not all, individuals. Western blot analysis revealed that, in both MDM and PMN supernatants appeared two bands with molecular weights consistent with the platelet basic protein (PBP) and the neutrophil activating protein-2 (NAP-2) sizes. Regarding the influence on infected cells, recombinant NAP-2 enhanced the antimicrobial activity of IFNγ activated MDM against L. pneumophila, but not against M. tuberculosis. In addition, U937 cells transfected with a NAP-2 construct inhibited the intracellular multiplication of L. pneumophila, supporting its role in the modulation of the antimicrobial activity. Finally, U937 cells transfected with the NAP-2 construct showed an adherence that was dramatically enhanced when the substrate was fibronectin. We conclude that human phagocytes produce CXCL7 variants that may have a significant influence on the immune response against bacterial pathogens.

The chemokine (C-X-C motif) ligand (CXCL) family plays an important role in inflammation. In order to understand the role of CXC chemokine family in carcinogenesis, this study explored a group of early gastric cancer (GC) patients, and assessed the level of CXC chemokine ligand (CXCL) in blood samples of patients representing systemic circulation and tumor microenvironment, detected the expression of CXC chemokine receptor (CXCR) in tumor tissues, and measured tumor infiltrating immune cell subsets. 69 patients with GC were included in a single center prospective study and were followed up for 6 years. The level of CXCL1-14 was determined by ELISA and the concentration gradient of chemokine was calculated. Western blot was used to detect the expression of CXCR1, CXCR2, CXCR3, and CXCR4 in tumor tissue. CXCL1-14 expression was inhibited by siRNA in HGC27 cells and then the migration ability of HGC27 cells was detected by cell scratch test. The results of this study showed that the chemokine concentrations of CXCL1, CXCL2, CXCL5, CXCL8, CXCL11, and CXCL13 in peripheral blood and tumor drainage blood of patients without recurrence after treatment were significantly lower than those before treatment. The concentrations of CXCL1, CXCL2, CXCL4, CXCL5, CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, CXCL13, and CXCL14 in peripheral blood and tumor drainage blood were significantly higher than those in patients without recurrence. Patients with low expression of CXCR1 and CXCR3 had lower AFP (alpha fetoprotein), smaller tumor volume, and lower TNM tumor stage. Patients with lower expression of CXCR2 and CXCR4 had higher AFP (alpha fetoprotein) level, larger tumor volume, and higher TNM tumor stage. After down-regulation of CXCLs expression, the migration ability of most cell lines was significantly inhibited. This study suggests that CXCL chemokine family plays an important role in the pathogenesis of GC and can be used as a marker for the development of GC.