We have previously shown that fibroblast expression of α11β1 integrin stimulates A549 carcinoma cell growth in a xenograft tumor model. To understand the molecular mechanisms whereby a collagen receptor on fibroblast can regulate tumor growth we have used a 3D heterospheroid system composed of A549 tumor cells and fibroblasts without (α11+/+) or with a deletion (α11-/-) in integrin α11 gene. Our data show that α11-/-/A549 spheroids are larger than α11+/+/A549 spheroids, and that A549 cell number, cell migration and cell invasion in a collagen I gel are decreased in α11-/-/A549 spheroids. Gene expression profiling of differentially expressed genes in fibroblast/A549 spheroids identified CXCL5 as one molecule down-regulated in A549 cells in the absence of α11 on the fibroblasts. Blocking CXCL5 function with the CXCR2 inhibitor SB225002 reduced cell proliferation and cell migration of A549 cells within spheroids, demonstrating that the fibroblast integrin α11β1 in a 3D heterospheroid context affects carcinoma cell growth and invasion by stimulating autocrine secretion of CXCL5. We furthermore suggest that fibroblast α11β1 in fibroblast/A549 spheroids regulates interstitial fluid pressure by compacting the collagen matrix, in turn implying a role for stromal collagen receptors in regulating tensional hemostasis in tumors. In summary, blocking stromal α11β1 integrin function might thus be a stroma-targeted therapeutic strategy to increase the efficacy of chemotherapy.

Bone marrow mesenchymal stem cells (BMSCs) are multipotent stromal cells that can differentiate into a variety of cell types. BMSCs are chemotactically guided towards the cancer cells and contribute to the formation of a cancer microenvironment. The homing of BMSCs was affected by various factors. Disseminated tumour cells (DTCs) in distant organs, especially in the bone marrow, are the source of cancer metastasis and cancer relapse. DTC survival is also determined by the microenvironment. Here we aim to elucidate how cancer-educated BMSCs promote the survival of cancer cells at primary tumour sites and distant sites. We highlight the dynamic change by identifying different gene expression signatures in intratumoral BMSCs and in BMSCs that move back in the bone marrow. Intratumoral BMSCs acquire high mobility and displayed immunosuppressive effects. Intratumoral BMSCs that ultimately home to the bone marrow exhibit a strong immunosuppressive function. Cancer-educated BMSCs promote the survival of lung cancer cells via expansion of MDSCs in bone marrow, primary tumour sites and metastatic sites. These Ly6G⁺ MDSCs suppress proliferation of T cells. CXCL5, nitric oxide and GM-CSF produced by cancer-educated BMSCs contribute to the formation of malignant microenvironments. Treatment with CXCL5 antibody, the iNOS inhibitor 1400w and GM-CSF antibody reduced MDSC expansion in the bone marrow, primary tumour sites and metastatic sites, and promoted the efficiency of PD-L1 antibody. Our study reveals that cancer-educated BMSCs are the component of the niche for primary lung cancer cells and DTCs, and that they can be the target for immunotherapy.

BACKGROUND

Genome-wide gene expression profiling allows for identification of genes involved in the defense response of the host against pathogens. As presented here, transcriptomic analysis and bioinformatics tools were applied in order to identify genes expressed in the mammary gland parenchyma of cows naturally infected with coagulase-positive and coagulase-negative Staphylococci.

RESULTS

In cows infected with coagulase-positive Staphylococci, being in 1st or 2nd lactation, 1700 differentially expressed genes (DEGs) were identified. However, examination of the 3rd or 4th lactations revealed 2200 DEGs. Gene ontology functional classification showed the molecular functions of the DEGs overrepresented the activity of cytokines, chemokines, and their receptors. In cows infected with coagulase-negative Staphylococci, in the 1st or 2nd lactations 418 DEGs, while in the 3rd or 4th lactations, 1200 DEGs were identified that involved in molecular functions such as protein, calcium ion and lipid binding, chemokine activity, and protein homodimerization. Gene network analysis showed DEGs associated with inflammation, cell migration, and immune response to infection, development of cells and tissues, and humoral responses to infections caused by both types of Staphylococci.

CONCLUSIONS

A coagulase-positive Staphylococci infection caused a markedly stronger host response than that of coagulase-negative, resulting in vastly increased DEGs. A significant increase in the expression of the FOS, TNF, and genes encoding the major histocompatibility complex proteins (MHC) was observed. It suggests these genes play a key role in the synchronization of the immune response of the cow's parenchyma against mastitis-causing bacteria. Moreover, the following genes that belong to several physiological pathways (KEGG pathways) were selected for further studies as candidate genes of mammary gland immune response for use in Marker Assisted Selection (MAS): chemokine signaling pathway (CCL2, CXCL5, HCK, CCR1), cell adhesion molecules (CAMs) pathway (BOLA-DQA2, BOLA-DQA1, F11R, ITGAL, CD86), antigen processing and presentation pathway (CD8A, PDIA3, LGMN, IFI30, HSPA1A), and NOD-like receptor signaling pathway (TNF, IL8, IL18, NFKBIA).

Tumor-targeted antibody therapy has had a major impact on reducing morbidity and mortality in a wide range of cancers. Antibodies mediate their antitumor activity in part by activating immune effector cells; however, the tumor microenvironment (TME) is enriched with cellular and soluble mediators that actively suppress generation of antitumor immunity. Here, we investigate the potential of prospectively identifying and neutralizing an immunomodulatory soluble mediator within the TME to enhance therapeutic efficacy of the HER2-directed antibody trastuzumab. Using the D5-HER2 cell line and an immunocompetent human HER2 transgenic animal (hmHER2Tg) in which human HER2 is a self-antigen, we determined that IL4 was present in the TME and produced by both tumor and stromal cells. A siRNA-based screening approach identified STAT5A as a novel negative regulator of IL4 production by D5-HER2 tumor cells. Furthermore, IL4 neutralization using the anti-IL4 antibody 11B11 enhanced the efficacy of trastuzumab and modulated the TME. For example, IL4 neutralization resulted in reduced levels of myeloid chemoattractants CCL2, CCL11, and CXCL5 in the TME. Combination therapy with 11B11 and trastuzumab resulted in a reduction of tumor-infiltrating CD11b(+)CD206(+) myeloid cells compared with monotherapy. These data suggest that IL4 neutralization enhances the efficacy of trastuzumab by influencing the phenotype of myeloid cells within the TME and provide further rationale for combining tumor-targeted antibody therapy with agents that neutralize factors in the TME that suppress generation of productive antitumor immune responses.

OBJECTIVE

Decision on treatment of systemic sclerosis (SSc) related interstitial lung disease (ILD) largely relies on the findings on high resolution computed tomography (HRCT) and there is a need for improvement in assessment of the fibrotic activity. The objectives of this study were to study biomarkers in bronchoalveolar lavage fluid (BALF) from SSc patients with ILD and to relate the findings to the severity and activity of lung fibrosis.

METHODS

Fifteen patients with early SSc and 12 healthy controls were subjected to BAL. Cell counts and analyses of CXCL5, CXCL8 and S100A8/A9 were performed in BALF and serum. COMP and KL-6 were measured in serum. HRCT of lungs was quantified for ground glass opacities (GGO), reticulation and traction bronchiectases.

RESULTS

BALF concentrations of CXCL8 (p < 0.001), CXCL5 (p = 0.002) and S100A8/A9 (p = 0.016) were higher in patients than controls. Serum KL-6 (p < 0.001) was increased in SSc patients and correlated with BALF concentration of eosinophils (rS = 0.57, p = 0.027). Patients with more widespread GGO on HRCT were characterised in BALF by a higher eosinophil count (p = 0.002) and in serum by higher KL-6 (p = 0.008). Patients with more fibrosis were characterised in BALF by higher eosinophil count (p = 0.014), higher CXCL8 (p = 0.005) and S100A8A/A9 (p = 0.014) concentration and in serum by a higher serum COMP (p = 0.023).

CONCLUSIONS

In SSc related ILD, biomarkers from BALF and serum correlate to findings on HRCT suggesting usefulness as markers of presence and extent of lung fibrosis.

The mesothelium, covered by a continuous monolayer of mesothelial cells, is the first protective barrier against metastatic ovarian cancer. However, mesothelial cells release tumor-promoting factors that accelerate the process of peritoneal metastasis. We identified cancer-associated mesothelial cells (CAMs) that had tumor-promoting potential. Here, we found that plasminogen activator inhibitor-1 (PAI-1) induced the formation of CAMs, after which CAMs increasingly secreted the oncogenic factors interleukin-8 (IL-8) and C-X-C motif chemokine ligand 5 (CXCL5), further promoting the metastasis of ovarian cancer cells in a feedback loop. After the formation of CAMs, PAI-1 activated the nuclear factor kappa B (NFκB) pathway in the CAMs, thus transcriptionally upregulating the expression of the downstream NFκB targets IL-8 and CXCL5. Moreover, PAI-1 correlated with peritoneal metastasis in ovarian cancer patients and indicated a poor prognosis. In both ex vivo and in vivo models, after PAI-1 expression was knocked down, the metastasis of ovarian cancer cells decreased significantly. Therefore, targeting PAI-1 may provide a potential target for future therapeutics to prevent the formation of CAMs and alleviate peritoneal metastasis in ovarian cancer patients.

Rationale: Chemokines contribute to cancer metastasis and have long been regarded as attractive therapeutic targets for cancer. However, controversy exists about whether neutralizing chemokines by antibodies promotes or inhibits tumor metastasis, suggesting that the approach to directly target chemokines needs to be scrutinized. Methods: Transwell assay, mouse metastasis experiments and survival analysis were performed to determine the functional role of S100A14 in breast cancer. RNA-Seq, secreted proteomics, ChIP, Western blot, ELISA, transwell assay and neutralizing antibody experiments were employed to investigate the underlying mechanism of S100A14 in breast cancer metastasis. Immunohistochemistry and ELISA were performed to examine the expression and serum levels of S100A14, CCL2 and CXCL5, respectively. Results: Overexpression of S100A14 significantly enhanced migration, invasion and metastasis of breast cancer cells. In contrast, knockout of S100A14 exhibited the opposite effects. Mechanistic studies demonstrated that S100A14 promotes breast cancer metastasis by upregulating the expression and secretion of CCL2 and CXCL5 via NF-κB mediated transcription. The clinical sample analyses showed that S100A14 expression is strongly associated with CCL2/CXCL5 expression and high expression of these three proteins is correlated with worse clinical outcomes. Notably, the serum levels of S100A14, CCL2/CXCL5 have significant diagnostic value for discerning breast cancer patients from healthy individuals. Conclusions: S100A14 is significantly upregulated in breast cancer, it can promote breast cancer metastasis by increasing the expression and secretion of CCL2/CXCL5 via RAGE-NF-κB pathway. And S100A14 has the potential to serve as a serological marker for diagnosis of breast cancer. Collectively, we identify S100A14 as an upstream regulator of CCL2/CXCL5 signaling and a metastatic driver of breast cancer.

Keywords: Breast cancer; CCL2; CXCL5; Metastasis; S100A14.

BACKGROUND

Stachybotrys chartarum is a damp building mould and a potent toxin producer that has been related to serious cases of respiratory health problems. However, the direct link between exposure and health symptoms has not been established.

OBJECTIVE

To examine the mechanism by which exposure to spores of satratoxin producing and non-producing S. chartarum strains induce inflammatory responses in murine lungs.

METHODS

BALB/c mice were intranasally exposed for 3 weeks to spores of a satratoxin-producing and a non-producing S. chartarum strain. Inflammatory cell infiltration was characterized from bronchoalveolar lavage (BAL) fluid. Cytokine and chemokine mRNA expression in lung tissue was measured with real-time PCR. Bronchial responsiveness to methacholine (MCh) was determined by whole-body plethysmography and serum antibody levels by ELISA.

RESULTS

A dose-dependent increase in monocytes, neutrophils and lymphocytes was observed in BAL fluid after intranasal (i.n.) instillation of S. chartarum spores. There was no difference in the BAL between exposure to the satratoxin-producing and the non-producing strains. Infiltration of inflammatory cells was associated with an induction of pro-inflammatory cytokine (IL-1beta, IL-6 and TNF-alpha) and chemokine (CCL3/MIP-1alpha, CCL4/MIP-1beta and CCL2/MCP-1) mRNA levels in the lungs. Interestingly, CXCL5/LIX was the only chemokine that showed significantly higher mRNA levels after exposure to the satratoxin-producing strain compared with the non-producing strain. MCh-induced bronchial responsiveness was not altered significantly after mould instillation. Moreover, no significant increase in total or specific IgE, IgG2a and IgG1 antibody levels were found after S. chartarum exposure.

CONCLUSIONS

These results indicate that lung inflammation induced by i.n. instillations of S. chartarum spores is regulated by the induction of pro-inflammatory cytokines and leucocyte-attracting chemokines. The data also imply that S. chartarum-derived components, other than satratoxins, are mediating the development of this inflammatory response.

Accumulating data suggest that platelets not only regulate thrombosis and haemostasis but also inflammatory processes. Platelets contain numerous potent pro-inflammatory compounds, including the chemokines CCL5 and CXCL4, although their role in acute colitis remains elusive. The aim of this study is to examine the role of platelets and platelet-derived chemokines in acute colitis. Acute colitis is induced in female Balb/c mice by administration of 5% dextran sodium sulfate (DSS) for 5 days. Animals receive a platelet-depleting, anti-CCL5, anti-CXCL4, or a control antibody prior to DSS challenge. Colonic tissue is collected for quantification of myeloperoxidase (MPO) activity, CXCL5, CXCL2, interleukin-6 (IL-6), and CCL5 levels as well as morphological analyses. Platelet depletion reduce tissue damage and clinical disease activity index in DSS-exposed animals. Platelet depletion not only reduces levels of CXCL2 and CXCL5 but also levels of CCL5 in the inflamed colon. Immunoneutralization of CCL5 but not CXCL4 reduces tissue damage, CXC chemokine expression, and neutrophil recruitment in DSS-treated animals. These findings show that platelets play a key role in acute colitis by regulating CXC chemokine generation, neutrophil infiltration, and tissue damage in the colon. Moreover, our results suggest that platelet-derived CCL5 is an important link between platelet activation and neutrophil recruitment in acute colitis.

As a CXC-type chemokine, ENA78/CXCL5 is an important attractant for granulocytes by binding to its receptor CXCR2. Recent studies proved that CXCL5/CXCR2 axis plays an oncogenic role in many human cancers. However, the exact clinical significance of CXCL5 in lung cancer has not been well defined. Here, we found that the serum protein expression of CXCL5 was significantly increased in non-small cell lung cancer (NSCLC) compared with that in healthy volunteers. Immunohistochemistry staining revealed that CXCL5 protein was higher in various lung cancer tissues compared with normal tissues. Moreover, CXCL5 expression correlated with histological grade, tumor size, and TNM stage in NSCLC. Elevated CXCL5 protein abundance predicted poor overall survival in adenocarcinoma patients. Further meta-analysis demonstrated that CXCL5 mRNA expression was also positively associated with tumor stage, lymph node metastasis, and worse survival. Kaplan-Meier plot analyses indicated high CXCL5 was associated with short overall survival and progression-free survival. Together, these results indicated that CXCL5 may be a potential biomarker for NSCLC.

Gut mucosal healing (MH) is considered a key therapeutic target and prognostic parameter in the management of inflammatory bowel disease (IBD). The dipotassium glycyrrhizate (DPG), a salt of the glycoconjugated triterpene glycyrrhizin, has been shown to inhibit the High Mobility Group Box 1 (HMGB1) protein, an allarmin strongly implicated in the pathogenesis of most inflammatory and auto-immune disorders. Here we discuss new insights on how DPG acts on MH comparing the acute phase and the recovery phase from experimental colitis in mice. We found that DPG strongly accelerates MH by differently regulating pro-inflammatory (CXCL1, CXCL3, CXCL5, PTGS2, IL-1β, IL-6, CCL12, CCL7) and wound healing (COL3A1, MMP9, VTN, PLAUR, SERPINE, CSF3, FGF2, FGF7, PLAT, TIMP1) genes as observed only during the recovery phase of colitis. Relevant issue is the identification of extracellular matrix (ECM) remodeling genes, VTN, and PLAUR, as crucial genes to achieve MH during DPG treatment. Furthermore, a noticeable recovery of intestinal epithelial barrier structural organization, wound repair ability, and functionality is observed in two human colorectal adenocarcinoma cell lines exposed to DPG during inflammation. Thus, our study identifies DPG as a potent tool for controlling intestinal inflammation and improving MH.

Microsporidians are a group of emerging pathogens typically associated with chronic diarrhea in immunocompromised individuals. The number of reports of infections with these organisms and the disseminated pathology is growing as diagnostic tools become more readily available. However, little is known about the innate immune response induced by and generated against these parasites. Using a coculture chemotaxis system, primary human macrophages were infected with Encephalitozoon cuniculi or Encephalitozoon intestinalis, and the recruitment of naïve monocytes was monitored. Encephalitozoon spp. induced an average threefold increase in migration of naïve cells 48 h postinfection, which corresponded to optimal infection of monocyte-derived-macrophages. A limited microarray analysis of infected macrophages revealed several chemokines involved in the inflammatory responses whose expression was upregulated, including CCL1, CCL2, CCL3, CCL4, CCL7, CCL15, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8. The levels of 6 of 11 chemokines also present in the microarray were confirmed to be elevated by protein profiling. Kinetic studies confirmed that secreted CCL2, CCL3, and CCL4 were expressed as early as 6 h postinfection, with peak expression at 12 to 24 h and expression remaining until 48 h postinfection. Neutralization of these chemokines, specifically CCL4, significantly reduced the number of migrating cells in vitro, indicating their role in the induction of monocyte migration. This mechanism of recruitment not only supports the evidence that in vivo cellular infiltration occurs but also provides new hosts for the parasites, which escape macrophages by rupturing the host cell. To our knowledge, this is the first documentation that chemokine production is induced by microsporidian infections in human macrophages.

Acute bacterial pneumonia is a significant public health concern worldwide. Understanding the signals coordinating lung innate immunity may foster the development of therapeutics that limit tissue damage and promote host defense. We have previously shown that lung messenger RNA expression of the IL-6 family cytokine oncostatin-M (OSM) is significantly elevated in response to bacterial stimuli. However, its physiological significance during pneumonia is unknown. Here we demonstrate that OSM is rapidly increased in the airspaces of mice after pulmonary infection with Escherichia coli. Neutralization of OSM caused a substantial decrease in airspace neutrophils and macrophages. OSM blockade also caused a marked reduction in lung chemokine (C-X-C motif) ligand (CXCL) 5 expression, whereas other closely related neutrophil chemokines, CXCL1 and CXCL2, were unaffected. Intratracheal administration of recombinant OSM was sufficient to recapitulate the effect on CXCL5 induction, associated with robust activation of the signal transducer and activator of transcription 3 (STAT3) transcription factor. Cell sorting revealed that OSM effects were specific to lung epithelial cells, including a positive feedback loop in which OSM may facilitate expression of its own receptor. Finally, in vitro studies demonstrated that STAT3 was required for maximal OSM-induced CXCL5 expression. These studies demonstrate a novel role for OSM during pneumonia as an important signal to epithelial cells for chemokine induction mediating neutrophil recruitment.

Abdominal sepsis is associated with significant changes in systemic inflammation and coagulation. The purpose of the present study was to examine the role of peripheral blood monocytes for systemic coagulation, including thrombin generation and consumption of coagulation factors. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Plasma and lung levels of IL-6 and C-X-C motif (CXC) chemokines [chemokine CXC ligand (CXCL)1, CXCL2, and CXCL5], pulmonary activity of myeloperoxidase, thrombin generation, and coagulation factors were determined 6 h after CLP induction. Administration of clodronate liposomes decreased circulating levels of monocytes by 96%. Time to peak thrombin formation was increased and peak and total thrombin generation was decreased in plasma from CLP animals. Monocyte depletion decreased time to peak formation of thrombin and increased peak and total generation of thrombin in septic animals. In addition, monocyte depletion decreased the CLP-induced increase in the levels of thrombin-antithrombin complexes in plasma. Depletion of monocytes increased plasma levels of prothrombin, factor V, factor X, and protein C in septic mice. Moreover, depletion of monocytes decreased CLP-induced levels of IL-6 and CXC chemokines in the plasma and lung by >59% and 20%, respectively. CLP-induced myeloperoxidase activity in the lung was attenuated by 44% in animals depleted of monocytes. Taken together, our findings show, for the first time, that peripheral blood monocytes regulate systemic coagulation. The results of our study improve our understanding of the pathophysiology of sepsis and encourage further attempts to target innate immune cell functions in abdominal sepsis.

Tuberculosis (TB) is a major health issue globally. Although typically the disease can be cured by chemotherapy in all age groups, and prevented in part in newborn by vaccination, general consensus exists that development of novel intervention measures requires better understanding of disease mechanisms. Human TB is characterized by polarity between host resistance as seen in 2 billion individuals with latent TB infection and susceptibility occurring in 9 million individuals who develop active TB disease every year. Experimental animal models often do not reflect this polarity adequately, calling for a reverse translational approach. Gene expression profiling has allowed identification of biomarkers that discriminate between latent infection and active disease. Functional analysis of most relevant markers in experimental animal models can help to better understand mechanisms driving disease progression. We have embarked on in-depth characterization of candidate markers of pathology and protection hereby harnessing mouse mutants with defined gene deficiencies. Analysis of mutants deficient in miR-223 expression and CXCL5 production allowed elucidation of relevant pathogenic mechanisms. Intriguingly, these deficiencies were linked to aberrant neutrophil activities. Our findings point to a detrimental potential of neutrophils in TB. Reciprocally, measures that control neutrophils should be leveraged for amelioration of TB in adjunct to chemotherapy.

Acute myeloid leukaemia (AML) cells show constitutive release of several chemokines that occurs in three major clusters: (I) chemokine (C-C motif) ligand (CCL)2-4/chemokine (C-X-C motif) ligand (CXCL)1/8, (II) CCL5/CXCL9-11 and (III) CCL13/17/22/24/CXCL5. Ingenol-3-angelate (PEP005) is an activator of protein kinase C and has antileukaemic and immunostimulatory effects in AML. We investigated primary AML cells derived from 35 unselected patients and determined that PEP005 caused a dose-dependent increase in the release of chemokines from clusters I and II, including several T cell chemotactic chemokines. The release of granulocyte-macrophage colony-stimulating factor and hepatocyte growth factor was also increased. CCL2-4/CXCL1/8 release correlated with nuclear factor (NF)-kappaB expression in untreated AML cells, and PEP005-induced chemokine production was associated with further increases in the expression of the NF-kappaB subunits p50, p52 and p65. Increased DNA binding of NF-kappaB was observed during exposure to PEP005, and the specific NF-kappaB inhibitor BMS-345541 reduced constitutive chemokine release even in the presence of PEP005. Finally, PEP005 decreased expression of stem cell markers (CD117, CXCR4) and increased lineage-associated CD11b and CD14 expression. To conclude, PEP005 has a unique functional pharmacological profile in human AML. Previous studies have described proapoptotic and T cell stimulatory effects and the present study describes additional T cell chemotactic and differentiation-inducing effects.

OBJECTIVE

To evaluate gene expression by microarray analyses of inflammatory mediators in the sinus mucosa of children with and without chronic rhinosinusitis (CRS).

METHODS

Prospective molecular genetics analysis.

METHODS

Children's National Medical Center, Washington, DC.

METHODS

Eleven patients with CRS who underwent endoscopic sinus surgery and 10 control children who underwent craniofacial resection or neurosurgical procedures.

METHODS

Gene expression levels of sinus tissue from 6 patients with CRS and 6 controls and messenger RNA expression levels of upregulated inflammatory/immune response genes, as well as cytokines of interest, determined by quantitative reverse transcription-polymerase chain reaction.

RESULTS

Gene expression using the Plier algorithm yielded the most consistent grouping of samples: 96 genes were significantly upregulated more than 2-fold, and 123 genes were downregulated by at least 50% in the CRS sinus tissues compared with controls (P < .05). GeneSpring analysis demonstrated significant changes in several ontology categories in the CRS samples, including inflammatory/immune response genes. The chemokines CXCL13 and CXCL5, serum amyloid A, serpin B4, and defensin beta1 were highly upregulated >> or =5-fold). Increased expression of these genes was validated by quantitative reverse transcription-polymerase chain reaction in an independent set of tissues. Expression levels of interleukins 5, 6, and 8 were similar in both cohorts; these results were validated by reverse transcription-polymerase chain reaction.

CONCLUSIONS

Microarray analyses of sinus mucosa in children with CRS showed an increased expression of inflammatory genes involved in innate and adaptive immune systems. This technology can be successfully used to identify genes implicated in the pathogenesis of pediatric CRS.

OBJECTIVE

To clarify the composition of wound fluid (WF) and investigate the impact of WF on breast cancer cell lines.

METHODS

The proliferation and migration of WF-treated breast cancer cells MDA-MB-231 and MCF-7 were assessed with colony formation test, MTT cell proliferation test and scratch wound test. The quantitative profiles of WF were analyzed using Bio-Plex Pro kits.

RESULTS

The proliferation and migration of WF-treated breast cancer cells were significantly higher than that of untreated cells. Fifteen cytokines, 29 chemokines and 9 matrix metalloproteinases (MMPs) were assessed in WF. The concentrations of these factors were influenced by post-surgery days, neoadjuvant chemotherapy (NAC), TNM stage, pathological type and molecular subtype. The WF harvested from patients underwent NAC showed significant higher profiles of interleukin-1β (IL-1β), IL-4, IL-6, IL-17F, IL-21, IL-23, IL-25, IL-31, Interferon γ (IFNγ), CD40 ligand (CD40L), tumor necrosis factor α (TNFα), CXCL1, CXCL2, CXCL5, CCL3, CCL7 and CCL20.

CONCLUSIONS

Surgery-induced WF promotes the proliferation and migration of breast cancer cells. The composition of WF is influenced by various clinical features and provides potential therapeutic targets to control local recurrence and tumor progression.

The rapid development in septic patients of features of marked immunosuppression associated with increased risk of nosocomial infections and mortality represents the rational for the initiation of immune targeted treatments in sepsis. However, as there is no clinical sign of immune dysfunctions, the current challenge is to develop biomarkers that will help clinicians identify the patients that would benefit from immunotherapy and monitor its efficacy. Using an in vitro model of endotoxin tolerance (ET), a pivotal feature of sepsis-induced immunosuppression in monocytes, we identified using gene expression profiling by microarray a panel of transcripts associated with the development of ET which expression was restored after immunostimulation with interferon-gamma (IFN-γ). These results were confirmed by qRT-PCR. Importantly, this short-list of markers was further evaluated in patients. Of these transcripts, six (TNFAIP6, FCN1, CXCL10, GBP1, CXCL5 and PID1) were differentially expressed in septic patients' blood compared to healthy blood upon ex vivo LPS stimulation and were restored by IFN-γ. In this study, by combining a microarray approach in an in vitro model and a validation in clinical samples, we identified a panel of six new transcripts that could be used for the identification of septic patients eligible for IFNg therapy. Along with the previously identified markers TNFa, IL10 and HLA-DRA, the potential value of these markers should now be evaluated in a larger cohort of patients. Upon favorable results, they could serve as stratification tools prior to immunostimulatory treatment and to monitor drug efficacy.

BACKGROUND

Aberrant expression of C-X-C motif chemokine 5 (CXCL5) contributes to the progression of various cancers. This study analyzed the clinical significance of serum CXCL5 (sCXCL5) levels of nasopharyngeal carcinoma (NPC) patients, with the goal of building a novel prognostic score model.

METHODS

Serum samples were collected prior to treatment from 290 NPC patients for the detection of sCXCL5 with ELISA. Half of the patients (n = 145) were randomly assigned to the training set to generate the sCXCL5 cutoff point using receiver operator characteristic (ROC) analysis, while the other half (n = 145) were assigned to the testing set for validation. Associations between sCXCL5 levels and clinical characteristics were analyzed. A prognostic score model was built using independent predictors derived from multivariate analysis. A concordance index (C-Index) was used to evaluate prognostic ability.

RESULTS

The sCXCL5 cutoff point was 0.805 ng/ml. Sex, age, histology, T classification, clinical classification and local recurrence were not associated with sCXCL5 levels. However, sCXCL5 levels were positively associated with N classification, distant metastasis and disease progression (P<0.05). A high sCXCL5 level predicted poor 6-year overall survival (OS), poor 6-year distant metastasis-free survival (DMFS), and poor 6-year progression-free survival (PFS). A prognostic score model was subsequently constructed based on sCXCL5 levels and clinical classification (C-C model), which are independent predictors of OS, DMFS, and PFS, as confirmed by the multivariate analysis. Furthermore, this novel model successfully divided the patients into four risk subgroups in the training set, the testing set and the entire set of patients. The C-Indices were 0.751 and 0.762 for the training set and the testing set, respectively.

CONCLUSIONS

sCXCL5 level was determined to be an independent prognostic factor for NPC patients. The novel statistical C-C model, which includes sCXCL5 levels and clinical classification, could be helpful in predicting the prognosis of NPC patients.

BACKGROUND

Dermokine-β is abundant in stratified epithelia and in differentiating keratinocytes in culture. We have recently shown that treatment of keratinocytes with dermokine-β attenuates phosphorylation of extracellular signal-regulated kinase, however, the roles of dermokine-β in vivo remain unknown.

OBJECTIVE

Dermokine-β is overexpressed in marginal keratinocytes during wound healing. This study was conducted to investigate the roles of dermokine-β in the wound healing process.

METHODS

Recombinant human dermokine-β or its active peptide was topically applied to excisional wounds in mice and the relative wound area was calculated. Histological and chemokine expression analyses in wounds were also performed. The chemokine expression levels as well as the chemotactic activity of dermokine-β in cultured keratinocytes were determined.

RESULTS

Topical application of recombinant dermokine-β as well as its carboxy-terminal domain peptide inhibited mouse wound healing at an early phase, reduced infiltration of neutrophils and macrophages into the wounds, inhibited angiogenesis, and decreased the number of myofibroblasts in the wounds. Treatment with dermokine-β augmented IL-10 expression, but attenuated expression of transforming growth factor-β and tumor necrosis factor-α. In addition, application of dermokine-β to skin wounds reduced the expression of CXCL1 and CXCL5, both of which are chemoattractant for neutrophils into wounds. Both dermokine-β and its active peptide decreased the expression of CXCL1, CXCL6, and CXCL8 in cultured human keratinocytes. Treatment of human keratinocytes with dermokine-β inhibited neutrophil chemotaxis.

CONCLUSIONS

These results suggest that dermokine-β delays early cutaneous wound healing in part by inhibiting expression of CXC chemokines containing the ERL-sequence motif.

Several studies indicate that chemokines play important roles in colorectal mucosal immunity by recruiting leukocytes into and out of the lamina propria adjacent to the epithelium. The chemokine CXCL5 which is expressed by epithelial cells within colorectal mucosa is a chemoattractant for neutrophils and has been implicated in Crohn's disease and ulcerative colitis. In addition, CXCL5 is one chemokine which promote angiogenesis related to cancer. The objective of this study was to determine by ELISA assay whether CXCL5 protein level is altered in colorectal cancer (CRC) tissues (n=80) compared with paired normal mucosa. Furthermore, the plasma CXCL5 levels from CRC patients (n=62) compared with controls (n=71) were also examined. Using a TaqMan system we screened for -156G->> C and +398G->>A CXCL5 gene variants in CRC patients (n=228) and a control group (n=231) to assess the role of CXCL5 genotype in CRC. The analyses showed that CXCL5 protein level in colorectal tumours was significantly (P<0.0001) higher than in normal tissue and was lower in plasma in CRC patients compared with controls (P=0.026). Immunohistochemistry revealed CXCL5 immunoreactivity mainly in epithelial cells of the colorectal carcinoma and in normal epithelial cells. Furthermore, patients who were -156C carriers had higher CXCL5 protein concentration compared with -156G carriers in normal tissue (P=0.027) and CXCL5 protein levels in cancerous tissue tended to be higher for the patient -156C carriers (P=0.059). To our knowledge this is the first report on the influence of CXCL5 gene variants and their relation to expression of CXCL5 protein in human CRC.

Catalpol is the main active ingredient of an extract from Radix rehmanniae, which in a previous study showed a protective effect against various types of tissue injury. However, a protective effect of catalpol on uterine inflammation has not been reported. In this study, to investigate the protective mechanism of catalpol on lipopolysaccharide (LPS)-induced bovine endometrial epithelial cells (bEECs) and mouse endometritis, in vitro and in vivo inflammation models were established. The Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway and its downstream inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), western blot (WB), and immunofluorescence techniques. The results from ELISA and qRT-PCR showed that catalpol dose-dependently reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6, and chemokines such as C-X-C motif chemokine ligand 8 (CXCL8) and CXCL5, both in bEECs and in uterine tissue. From the experimental results of WB, qRT-PCR, and immunofluorescence, the expression of TLR4 and the phosphorylation of NF-κB p65 were markedly inhibited by catalpol compared with the LPS group. The inflammatory damage to the mouse uterus caused by LPS was greatly reduced and was accompanied by a decline in myeloperoxidase (MPO) activity. The results of this study suggest that catalpol can exert an anti-inflammatory impact on LPS-induced bEECs and mouse endometritis by inhibiting inflammation and activation of the TLR4/NF-κB signaling pathway.