Background & aims: Carcinoma-associated fibroblasts (CAFs) are key players in multicellular, stromal-dependent alterations leading to HCC pathogenesis. However, the intricate crosstalk between CAFs and other components in the tumor microenvironment (TME) remains unclear. This study aimed to investigate the novel cellular crosstalk among CAFs, tumor cells, and tumor-associated neutrophils (TANs) during different stages of HCC pathogenesis.

Approach & results: In the HCC-TME, CAF-derived cardiotrophin-like cytokine factor 1 (CLCF1) increased CXCL6 and TGF-β secretion in tumor cells, which subsequently promoted tumor cell stemness in an autocrine manner and TAN infiltration and polarization in a paracrine manner. Moreover, CXCL6 and TGF-β secreted by HCC cells activated ERK1/2 signaling of CAFs to produce more CLCF1, thus forming a positive feedback loop to accelerate HCC progression. Inhibition of ERK1/2 or CLCF1/ciliary neurotrophic factor receptor (CNTFR) signaling efficiently impaired CLCF1-mediated crosstalk among CAFs, tumor cells, and TANs both in vitro and in vivo. In clinical samples, upregulation of the CLCF1-CXCL6/TGF-β axis exhibited a marked correlation with increased cancer stem cells, "N2"-polarized TANs, tumor stage, and poor prognosis.

Conclusions: This study reveals a novel cytokine-mediated cellular crosstalk and clinical network involving the CLCF1-CXCL6/TGF-β axis, which regulates the positive feedback loop among CAFs, tumor stemness, and TANs, HCC progression, and patient prognosis. These results may support the CLCF1 cascade as a potential prognostic biomarker and suggest that selective blockade of CLCF1/CNTFR or ERK1/2 signaling could provide an effective therapeutic target for HCC patients.

Keywords: cancer stem cell; cardiotrophin-like cytokine factor 1; polarization; tumor microenvironment; tumor-associated neutrophils.

BACKGROUND

The observation that angiogenesis, the process of new blood vessel formation, in healthy prostate and early prostate cancer is androgen-dependent gave rise to significant questions on how hypervascularization and increased angiogenesis is also achieved at the molecular level in advanced androgen-independent prostate cancer. The exact paracrine molecular network that is hardwired into the proteome of the endothelial and cancer subpopulations participating in this process remains partially understood.

METHODS

Here, we interrogated the signaling pathways and the molecular functional signatures across the proteome of endothelial cells after interacting with various secretomes produced by androgen-dependent and -independent prostate cancer cells.

RESULTS

We found the significant overexpression (P < 0.05) of prominent markers of angiogenesis, such as vonWillebrand factor (vWF) (∼ 2.5-fold) and CD31 (∼ 2-fold) in HUVECs stimulated with conditioned media from the androgen-independent prostate cancer cell line PC3. By mining the proteome of PC3 conditioned media, we discovered a signature of chemokine CXC motif ligands (i.e., CXCL3, CXCL5, CXCL6 and CXCL8) that could potentially coordinate increased angiogenesis in androgen-independent prostate cancer and verified their increased expression (P < 0.05) in both in vitro and xenograft models of androgen-independence.

CONCLUSIONS

Our findings form the basis for understanding the regulation of crucial metastatic phenomena during the transition of androgen-dependent prostate cancer into the highly aggressive, androgen-independent state and provide further insight on potential therapeutic targets of cancer-related angiogenesis.

OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®), a product made of the water soluble fractions of 21 inactivated bacterial strain patterns responsible for respiratory tract infections, is used for the prevention of recurrent upper respiratory tract infections and acute exacerbations in chronic obstructive pulmonary disease patients. OM-85 is able to potentiate both innate and adaptive immune responses. However, the molecular mechanisms responsible for OM-85 activation are still largely unknown. Purpose of this study was to investigate the impact of OM-85 stimulation on human dendritic cell functions. We show that OM-85 selectively induced NF-kB and MAPK activation in human DC with no detectable action on the interferon regulatory factor (IRF) pathway. As a consequence, chemokines (i.e. CXCL8, CXCL6, CCL3, CCL20, CCL22) and B-cell activating cytokines (i.e. IL-6, BAFF and IL-10) were strongly upregulated. OM-85 also synergized with the action of classical pro-inflammatory stimuli used at suboptimal concentrations. Peripheral blood mononuclear cells from patients with COPD, a pathological condition often associated with altered PRR expression pattern, fully retained the capability to respond to OM-85. These results provide new insights on the molecular mechanisms of OM-85 activation of the immune response and strengthen the rational for its use in clinical settings.

BACKGROUND

Periodontitis is a chronic inflammatory disease triggered by the host immune response. Epigenetic modifications also affect the immune response. We assessed CpG methylation in 22 inflammatory candidate genes (ATF2, CCL25, CXCL14, CXCL3, CXCL5, CXCL6, FADD, GATA3, IL10RA, IL12A, IL12B, IL13, IL13RA1, IL15, IL17C, IL17RA, IL4R, IL6R, IL6ST, IL7, INHA, and TYK2) with respect to the occurrence of aggressive periodontitis (AgP).

METHODS

In this study 15 AgP patients (53.3% males, 41.4±10.5 years) and 10 controls (40.0% males, 36.9±17.5 years) were included. The methylation patterns of gingival biopsies were quantified using EpiTect® Methyl Signature PCR Array Human Inflammatory Response.

RESULTS

In gingival biopsies taken from patients with AgP, CpG methylation of CCL25 (1.73% vs. 2.59%, p=0.015) and IL17C (6.89% vs. 19.27%, p=0.002) was significantly reduced as compared with periodontally healthy tissues.

CONCLUSIONS

We showed for the first time a differential methylation pattern for CCL25 and IL17C in periodontitis. CCL25 plays an important role in T-cell development, whereas IL17C regulates innate epithelial immune responses. The decrease in CpG methylation is presumably accompanied by an increase in gene expression. This could lead to a greater availability of CCL25 and interleukin 17C and support periodontal loss of attachment.

Chemokines affect inflammation and cancer through leukocyte attraction and angiogenesis. Here, we demonstrate that CXCL4L1/platelet factor-4 variant (PF-4var), a highly angiostatic chemokine, is poorly chemotactic for phagocytes and is inducible in monocytes by inflammatory mediators but remained undetectable in macrophages and neutrophils. In addition, CXCL4L1/PF-4var production by mesenchymal tumor cells was evidenced in vitro and in vivo by specific ELISA and immunohistochemistry. CXCL4L1/PF-4var, but not CXCL4/PF-4, was coinduced with the angiogenic chemokine CXCL6/granulocyte chemotactic protein-2 (GCP-2) by cytokines, e.g., IL-1beta and IL-17, in sarcoma cells, but not in diploid fibroblasts. Furthermore, the induction of CXCL6/GCP-2 in endothelial cells by IL-1beta was enhanced synergistically by TNF-alpha but inhibited by IFN-gamma, which synergized with IL-1beta to produce the angiostatic CXCL10/IFN-gamma-induced protein-10. These findings indicate that the equilibrium between angiostatic and angiogenic factors during inflammation and tumor progression is rather complex and differs depending on the chemokine, cell type, and stimulus. Selective intervention in the chemokine network may drastically disturb this delicate balance of angiogenesis and tissue repair. Application of angiostatic CXCL4L1/PF-4var without attraction of protumoral phagocytes may be beneficial in cancer therapy.

Chemokines play an important role in the pathogenesis of non-small cell lung cancer (NSCLC). Although the deregulations of chemokines have been reported to be associated with the development and progression of many human cancers including lung cancer, polymorphisms of chemokine genes have not been examined with the survival of NSCLC. We systematically investigated associations of 23 common potentially functional SNPs in the key chemokine genes (CCL2, CCL5, CCL8, CCL20, CCL22, CXCL1, CXCL6, CXCL9 and CXCL12) with the survival of NSCLC in a case cohort of 568 NSCLC patients in a Chinese population. The results showed that variant genotypes of CCL2 rs3760396 and CCL8 rs3138035 were associated with a significantly decreased risk of death for NSCLC (dominant model: adjusted HR=0.65, 95% CI=0.48-0.89 for rs3760396; dominant model: adjusted HR=0.65, 95% CI=0.49-0.86 for rs3138035), while CXCL12 rs1804429 was associated with an increased risk of death for NSCLC (CC vs AA: adjusted HR=6.03, 95% CI=1.44-25.24). Further stepwise regression analysis suggested that only rs3138035, a SNP located at 5'-flanking region of CCL8, was an independently favorable factor for the prognosis of NSCLC and the protective effect was more evident in smokers (adjusted HR=0.61, 95% CI=0.42-0.87), patients with squamous cell cancer (adjusted HR=0.58, 95% CI=0.35-0.96), patients with early stage (adjusted HR=0.32, 95% CI=0.15-0.67) and patients treated with surgical operation (adjusted HR=0.47, 95% CI=0.31-0.71). In addition, the interaction analysis demonstrated that stage and surgical operation interacted with the genetic effect of rs3138035 in relation to NSCLC survival (adjusted P(interaction)=0.02 and 0.01, respectively). These findings suggest that CCL8 rs3138035 may be one of the candidate biomarkers for NSCLC survival and may modify death risk associated with stage and surgical operation. Larger studies incorporating functional evaluations are warranted to validate our findings.

OBJECTIVE

To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogenesis system with affymetrix oligonucleotide array.

METHODS

A microcarrier-based in vitro angiogenesis system was developed, in which ECs migrated into the matrix, proliferated, and formed capillary sprouts. The sprouts elongated, branched and formed networks. The total RNA samples from the HMVECs at the selected time points (0.5, 24, and 72 h) during the capillary morphogenesis were used for microarray analyses, and the data were processed with the softwares provided by the manufacturers. The expression patterns of some genes were validated and confirmed by semi-quantitative RT-PCR. The regulated genes were grouped based on their molecular functions and expression patterns, and among them the expression of chemokines and chemokine receptors was specially examined and their functional implications were analyzed.

RESULTS

A total of 1 961 genes were up- or down-regulated two-folds or above, and among them, 468 genes were up- or down-regulated three-folds or above. The regulated genes could be grouped into categories based on their molecular functions, and were also clustered into six groups based on their patterns of expression. As for chemokines and chemokine receptors, CXCL1/GRO-alpha, CXCL2/GRO-beta, CXCL5/ENA-78, CXCL6/GCP2, IL-8/CXCL8, CXCL12/SDF-1, CXCL9/Mig, CXC11/ITAC, CX3CL1/fractalkine, CCL2/MCP-1, CCL3, CCL5/RANTES, CCL7, CCL15, CCL21, CCL23, CCL28, and CCR1, CCR9, CXCR4 were identified. Moreover, these genes demonstrated different changing patterns during the capillary morphogenesis, which implied that they might have different roles in the sequential process. Among the chemokines identified, CCL2/MCP-1, CCL5/RANTES and CX3CL1 were specially up-regulated at the 24-h time point when the sprouting characterized the morphological change. It was thus suggested that they might exert crucial roles at the early stage of angiogenesis.

CONCLUSIONS

The present study demonstrates a global profile of gene expression during endothelial capillary morphogenesis, and the results provide us much information about the molecular mechanisms of angiogenesis, with which further evaluation of individual genes can be conducted.

In this study we investigated the regulation of CXCR1 and CXCR2 intracellular trafficking. First, we produced a chimeric CXCR2 receptor that contained the internalization motifs of both CXCR2 and CXCR1 (CXCR2: LLKIL sequence; CXCR1: C-terminal phosphorylation sites). Elevated levels of internalization were induced by different ELR-expressing CXC chemokines on the chimeric receptor, as compared to wild-type CXCR2. Analysis of inter-relationships between CXCR1 and CXCR2 during internalization indicated that the exposure of cells that expressed both CXCR1 and CXCR2 to CXCL8 or CXCL6 resulted in decreased levels of CXCR1 internalization as compared to those in cells that expressed only CXCR1. To characterize the role of actin-related components in CXCR1 and CXCR2 trafficking, wortmannin, a potent inhibitor of phosphatidylinositol kinases, was used. The presence of wortmannin during receptor recycling inhibited CXCR1 and CXCR2 re-expression following CXCL8-induced internalization, and resulted in a marked disruption of the proper organization of actin filaments. The kinase-dependent recycling process required CXCR2 C-terminal phosphorylation sites. Our results suggest that actin-related kinases are required for the proper functionality of actin filaments, which are the instrumental factors needed for receptor recycling. In all, CXCR1 and CXCR2 internalization and recycling are tightly regulated by receptor domains and by actin-related kinases.

Anticytokine auto-vaccination is a powerful tool for the study of cytokine functions in vivo but has remained rather esoteric as a result of numerous technical difficulties. We here describe a two-step procedure based on the use of OVA multimers purified by size exclusion chromatography after incubation with glutaraldehyde at pH 6. When such polymers are incubated with a target protein at pH 8.5 to deprotonate reactive amines, complexes are formed that confer immunogenicity to self-antigens. The chemokine GCP-2/CXCL6, the cytokines GM-CSF, IL-17F, IL-17E/IL-25, IL-27, and TGF-β1, and the MMP-9/gelatinase B are discussed as examples. mAb, derived from such immunized mice, have obvious advantages for in vivo studies of the target proteins. Using a mAb against GCP-2, obtained by the method described here, we provide the first demonstration of the major role played by this chemokine in rapid neutrophil mobilization after Leishmania major infection. Pre-activated OVA multimers reactive with amine residues thus provide an efficient carrier for auto-vaccination against 9-90 kDa autologous proteins.

<AbstractText>Various profibrotic and proinflammatory cytokines have been found upregulated in uncomplicated primary retinal detachment (pRD), but without providing a uniform picture. Here, we compare the cyto- and chemokine profiles in pRD with and without proliferative vitreoretinopathy (PVR) in an attempt to unravel relevant differences not in single cytokines, but in the cytokine profiles at diagnosis.</AbstractText><AbstractText>Undiluted vitreous fluid (VF) was obtained at the beginning of surgery from 174 eyes with pRD without relevant PVR (maximally grade B; group 1; n = 81) and with moderate or advanced PVR requiring a gas tamponade (group 2; n = 49) or silicon oil filling (group 3; n = 44). VF of eyes undergoing macular hole (MH) surgery served as controls (group 4; n = 26). Forty-three cytokines were quantified in parallel using a multiplex cytokine analysis system (Bioplex). For all comparisons we applied Holm's correction to control for multiple comparisons.</AbstractText><AbstractText>44.9% of group 2 eyes presented grade C1 and 55.1% C2-C3, whereas 86.4% of group 3 eyes exhibited a PVR grade of C2-D. CCL19 was the only cytokine that displayed higher concentrations in the vitreous of eyes with PVR C1 compared to lower PVR grades. Eyes with PVR C2-D showed higher levels of CCL27, <em>CXCL6</em>, IL4, IL16, CXCL10, CCL8, CCL22, MIG/CXCL9, CCL15, CCL19, CCL 23 and CXCL12 compared to controls. Interestingly, no difference of cytokine levels was detected between C1 and C2-D PVR.</AbstractText><AbstractText>CCL19 may represent a potential biomarker for early PVR progression that holds promise for future diagnostic and therapeutic applications.</AbstractText>

OBJECTIVE

Hypoxia promotes the progression and metastasis of hepatocellular carcinoma (HCC). Hypoxia inducible factor-α (HIF-1α) regulates the expression of various chemokines involved in tumor growth, angiogenesis and metastasis.

METHODS

The role of HIF-1α in HCC tumor growth and invasion and the prognosis of patients with HCC was investigated in cell lines and patient samples. HIF-1α mRNA and protein levels were assessed by qRT-PCR and western blotting. Silencing of HIF-1α downregulated the expression of granulocyte chemotactic protein-2 (GCP-2)/CXCL6, a CXC ELR+ chemokine, in HCC cells, and a luciferase assay showed that HIF-1α binds to a hypoxia response element in the promoter of CXCL6 and regulates its transcription. Induction of HIF-1α by hypoxia promoted the migration and invasion of HCC cells in vitro, and this effect was suppressed by an anti-CXCL6 antibody.

RESULTS

HIF-1α is upregulated in HCC cell lines and tissues and its effect on promoting invasion and metastasis is mediated by its direct interaction with the pro-angiogenic chemokine CXCL6. CXCL6 expression was associated with poor prognosis of HCC patients.

CONCLUSIONS

HIF-1α promotes HCC progression and metastasis by upregulating CXCL6 transcription in HCC cells, providing a potential novel therapeutic target for the treatment of HCC.

OBJECTIVE

The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix(TM) oligonucleotide array technology to identify genes that are regulated by hCTGF.

METHODS

Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix(TM) oligonucleotide array technology. Results were validated by real time RT-PCR.

RESULTS

hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology.

CONCLUSIONS

This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye.

Postpartum Dysgalactia Syndrome (PDS) represents a considerable health problem of postpartum sows, primarily indicated by mastitis and lactation failure. The poorly understood etiology of this multifactorial disease necessitates the use of the porcine mammary epithelial cell (PMEC) model to identify how and to what extent molecular pathogen defense mechanisms prevent bacterial infections at the first cellular barrier of the gland. PMEC were isolated from three lactating sows and challenged with heat-inactivated potential mastitis-causing pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) for 3 h and 24 h, in vitro. We focused on differential gene expression patterns of PMEC after pathogen challenge in comparison with the untreated control by performing microarray analysis. Our results show that a core innate immune response of PMEC is partly shared by E. coli and S. aureus. But E. coli infection induces much faster and stronger inflammatory response than S. aureus infection. An immediate and strong up-regulation of genes encoding cytokines (IL1A and IL8), chemokines (CCL2, CXCL1, CXCL2, CXCL3, and CXCL6) and cell adhesion molecules (VCAM1, ICAM1, and ITGB3) was explicitly obvious post-challenge with E. coli inducing a rapid recruitment and activation of cells of host defense mediated by IL1B and TNF signaling. In contrast, S. aureus infection rather induces the expression of genes encoding monooxygenases (CYP1A1, CYP3A4, and CYP1B1) initiating processes of detoxification and pathogen elimination. The results indicate that the course of PDS depends on the host recognition of different structural and pathogenic profiles first, which critically determines the extent and effectiveness of cellular immune defense after infection.

Rectal cancer is a common type of colorectal cancer with high mortality and morbidity. The objective of this study was to identify gene signatures and uncover the potential mechanisms during rectal cancer samples. The gene expression profiles of GSE87211 data set were downloaded from GEO (Gene Expression Omnibus) database. The GSE87211 data set contained 2363 samples, including 203 rectal cancer samples and 160 matched mucosa control samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted, and protein-protein interaction network of differentially expressed genes (DEGs) was performed by Cytoscape. Then, Gene Expression Profiling Interactive Analysis (GEPIA) was applied to get the hub genes expression level and survival analysis between rectum adenocarcinoma (READ) tissues and normal tissues. In total, 846 DEGs were identified, including 402 upregulated genes and 444 downregulated genes. GO analysis showed that upregulated DEGs were enriched in inflammatory response, signal transduction, cell adhesion, immune response, and positive regulation of cell proliferation. KEGG pathway analysis showed that upregulated DEGs were enriched in cytokine-cytokine receptor interaction, Pi3K-Akt signaling pathway, and chemokine signaling pathway. The top 20 hub genes contained IL8, CXCR1, SSTR2, SST, CXCR2, GALR1, GAL, CXCL1, SSTR1, NPY1R, NPY, AGT, PPY, PPBP, CXCL2, CXCL6, CXCL11, CXCL3, GNG4, and GNGT1, and only four genes significantly increased expression levels with obvious changes of survival analysis in READ tissues based on GEPIA. Our study indicated that identified DEGs might promote our understanding of molecular mechanisms, which might be used as molecular targets or diagnostic biomarkers for the treatment of rectal cancer.

BACKGROUND

Permixon®, the hexanic lipidosterolic extract of saw palmetto Serenoa repens (LSESr), has shown properties that highlight its benefit in the management of benign prostate hyperplasia (BPH). To address its actual anti-inflammatory potency, we used a unique pro-inflammatory mouse model of prostate hyperplasia involving prostate-specific over-expression of prolactin transgene (Pb-Prl).

METHODS

Six month-old Pb-Prl males were administered with Permixon® per os at the daily dose of 100 mg/kg for 28 days. Body and prostate weights were measured weekly and at sacrifice, respectively. Prostate histology was carefully assessed by a pathologist and detailed quantifications of epithelial and stromal compartments were performed using image analysis software. Luminal cell proliferation index was determined using Ki-67 immunostaining, and apoptosis using Bax/Bcl2 mRNA ratio. Tissue inflammation and fibrosis were assessed by histological analyses then quantified using CD45 immunostaining and picrosirius staining, respectively. Expression profiling of selected pro-inflammatory cytokines, chemokines, and chemokine receptors was performed by quantitative RT-PCR.

RESULTS

In this model, Permixon® significantly decreased tissue weight and proliferation index specifically in the ventral lobe. Although treatment had no noticeable effect on epithelial histology of any lobe, it markedly reduced the histological hallmarks of inflammation in all lobes. This was confirmed by the global down-regulation of prostate pro-inflammatory cytokine profile, with significant reduction of CCR7, CXCL6, IL-6, and IL-17 expression.

CONCLUSIONS

In this mouse model of prostate hyperplasia, Permixon® exerted potent anti-inflammatory properties in the whole prostate while anti-androgenic effects were lobe-specific, suggesting that distinct LSESr components may be involved in these effects. Our results support the beneficial role of Permixon® treatment for BPH. The relevance of CCR7, CXCL6, IL-6, and IL-17 as potential biomarkers to follow up BPH inflammatory status needs to be assessed.

BACKGROUND

Femoroacetabular impingement is a frequent cause of hip pain and may lead to secondary osteoarthritis, yet little is known about the molecular events linking mechanical hip impingement and articular cartilage degeneration. The first goal of this study was to quantify the expression of inflammatory cytokine and chemokine, matrix-degrading, and extracellular matrix genes in articular cartilage harvested from control hips and hips with femoroacetabular impingement and end-stage osteoarthritis. The second goal was to analyze the relative expression of these genes in articular cartilage harvested at various stages of osteoarthritis.

METHODS

Cartilage samples were obtained from thirty-two hips undergoing hip preservation surgery for femoroacetabular impingement or hip arthroplasty. Three control cartilage samples were also analyzed. Specimens were graded intraoperatively with regard to the severity of cartilage damage, the radiographic osteoarthritis grade was recorded, and quantitative RT-PCR (real-time polymerase chain reaction) was performed to determine relative gene expression.

RESULTS

Except for interleukin-1β (IL-1β) and CXCL2, the mRNA (messenger RNA) expression of all other chemokine (IL-8, CXCL1, CXCL3, CXCL6, CCL3, and CCL3L1), matrix-degrading (matrix metalloproteinase [MMP]-13 and ADAMTS-4), and structural matrix (COL2A1 [collagen, type II, alpha] and ACAN [aggregan]) genes was higher overall in cartilage from hips with femoroacetabular impingement compared with hips with osteoarthritis and normal controls. The differences reached significance (p ≤ 0.05) for seven of these ten quantified genes, with CXCL3, CXCL6, and COL2A1 being elevated in the femoroacetabular impingement group compared with only the control group and IL-8, CCL3L1, ADAMTS-4, and ACAN being elevated compared with both the osteoarthritis and control groups. When samples were grouped according to the stage of the degenerative cascade, mRNA expression was relatively higher in one of the two middle stages of femoroacetabular impingement (chondromalacia or cleavage/thinning), with the difference reaching significance for IL-8, CXCL2, CXCL3, CCL3L1, and ACAN. ACAN expression was diminished in hips with osteoarthritis compared with femoroacetabular impingement but elevated compared with the control articular cartilage.

CONCLUSIONS

Articular cartilage from the impingement zone of hips with femoroacetabular impingement (and particularly those hips in the cleavage/thinning stage) expressed higher levels of certain inflammatory, anabolic, and catabolic genes, representing a heightened metabolic state.

CONCLUSIONS

The articular cartilage from the impingement zone of hips with femoroacetabular impingement was metabolically hyperactive, supporting the concept that such impingement is a structural precursor to hip osteoarthritis.

BACKGROUND

Neutrophils have a central role in the inflammatory conditions of the central nervous system (CNS). ELR chemokines direct neutrophil migration, but the source of chemokines in the CNS is unclear. We quantified chemokine production using cell-line models of astrocytic and neuronal cells, specifically NT2.N cells, a human line with characteristics of immature neurons, and NT2.A cells, a line with characteristics of astrocytes.

OBJECTIVE

In NT2.N and NT2.A cells, and their parent cell line NT2, we sought to: (1) quantify ELR chemokines, (2) determine receptor (CXCR-1 and CXCR-2) expression, and (3) measure the function of the chemokines generated from these cells.

METHODS

NT2 cells were differentiated into NT2.N cells and NT2.A cells with all trans retinoic acid and mitosis inhibitors. Chemokine concentrations in culture supernatants were determined by ELISA. Immunofluorescence was used to detect CXCR-1 and CXCR-2. RT-PCR was used to determine chemokine and chemokine receptor mRNA. Chemotaxis assays were used to assess function.

RESULTS

ELR chemokines were not detected in supernatants of NT2 or NT2.N cells, although mRNA for GRO-gamma/CXCL3 was found in both. In contrast, in NT2.A cells, mRNA and protein were present for GCP-2/CXCL6, GRO-alpha/CXCL1, GRO-gamma/CXCL3, and IL-8/CXCL8. CXCR-1 and CXCR-2 were expressed on NT2, NT2.N, and NT2.A cells detected by immunofluorescent staining and RT-PCR. Supernatants of NT2.A cells resulted in neutrophil chemotactic function of 30.5 +/- 3.9%, greater than NT2 cells (12.3 +/- 1.6%, mean +/- SEM, P < 0.01).

CONCLUSIONS

We speculate that astrocytes are a source of ELR chemokines in the human CNS and that neurons and astrocytes can respond to those chemokines.

Adenocarcinomas of the prostate arise as multifocal heterogeneous lesions as the likely result of genetic and epigenetic alterations and deranged cell-cell communication. Notch signaling is an important form of intercellular communication with a role in growth/differentiation control and tumorigenesis. Contrasting reports exist in the literature on the role of this pathway in prostate cancer (PCa) development. We show here that i) compared to normal prostate tissue, Notch1 expression is significantly reduced in a substantial fraction of human PCas while it is unaffected or even increased in others; ii) acute Notch activation both inhibits and induces process networks associated with prostatic neoplasms; iii) down-modulation of Notch1 expression and activity in immortalized normal prostate epithelial cells increases their proliferation potential, while increased Notch1 activity in PCa cells suppresses growth and tumorigenicity through a Smad3-dependent mechanism involving p21WAF1/CIP1; iv) prostate cancer cells resistant to Notch growth inhibitory effects retain Notch1-induced upregulation of pro-oncogenic genes, like EPAS1 and CXCL6, also overexpressed in human PCas with high Notch1 levels. Taken together, these results reconcile conflicting data on the role of Notch1 in prostate cancer.

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.

Diabetic nephropathy (DN) is a primary cause of renal failure. However, studies providing renal gene expression profiles of diabetic tubulointerstitial injury are scarce and its molecular mechanisms still await clarification. To identify vital genes involved in the diabetic tubulointerstitial injury, three microarray data sets from gene expression omnibus (GEO) were downloaded. A total of 127 differentially expressed genes (DEGs) were identified by limma package. Gene set enrichment analysis (GSEA) plots showed that sister chromatid cohesion was the most significant enriched gene set positively correlated with the DN group while retinoid X receptor binding was the most significant enriched gene set positively correlated with the control group. Enriched Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DEGs mostly included extracellular matrix organization, extracellular space, extracellular matrix structural constituent, and Staphylococcus aureus infection. Twenty hub genes from three significant modules were ascertained by Cytoscape. Correlation analysis and subgroup analysis between hub genes and clinical features of DN showed that ALB, ANXA1, APOH, C3, CCL19, COL1A2, COL3A1, COL4A1, COL6A3, CXCL6, DCN, EGF, HRG, KNG1, LUM, SERPINA3, SPARC, SRGN, and TIMP1 may involve in diabetic tubulointerstitial injury. ConnectivityMap analysis indicated the most significant three compounds are 5182598, thapsigargin and 5224221. In conclusion, this study may provide new insights into the molecular mechanisms underlying diabetic tubulointerstitial injury as well as potential targets for diagnosis and therapeutics of DN.

Currently, several assays can diagnose acute dengue infection. However, none of these assays can predict the severity of the disease. Biomarkers that predicts the likelihood that a dengue patient will develop a severe form of the disease could permit more efficient patient triage and allows better supportive care for the individual in need, especially during dengue outbreaks.

We measured 20 plasma markers i.e. IFN-γ, IL-10, granzyme-B, CX3CL1, IP-10, RANTES, CXCL8, CXCL6, VCAM, ICAM, VEGF, HGF, sCD25, IL-18, LBP, sCD14, sCD163, MIF, MCP-1 and MIP-1β in 141 dengue patients in over 230 specimens and correlate the levels of these plasma markers with the development of dengue without warning signs (DWS-), dengue with warning signs (DWS+) and severe dengue (SD).

Our results show that the elevation of plasma levels of IL-18 at both febrile and defervescence phase was significantly associated with DWS+ and SD; whilst increase of sCD14 and LBP at febrile phase were associated with severity of dengue disease. By using receiver operating characteristic (ROC) analysis, the IL-18, LBP and sCD14 were significantly predicted the development of more severe form of dengue disease (DWS+/SD) (AUC = 0.768, P < 0.0001; AUC = 0.819, P < 0.0001 and AUC = 0.647, P = 0.014 respectively). Furthermore, we also found that the levels of VEGF were directly correlated and sCD14 was inversely correlated with platelet count, suggesting that the endothelial activation and microbial translocation may played a role in pathogenesis of dengue disease.

Given that the elevation IL-18, LBP and sCD14 among patients with severe form of dengue disease, our findings suggest a pathogenic role for an aberrant inflammasome and monocyte activation in the development of severe form of dengue disease.

Blood levels of inflammation-related markers may reveal molecular pathways contributing to carcinogenesis. To date, prospective associations with colorectal cancer (CRC) risk have been based on few studies with limited sets of analytes. We conducted a case-cohort study within the Japan Public Health Center-based Prospective Study Cohort II, comparing 457 incident CRC cases during median 18 years follow-up with a random subcohort of 774 individuals. Baseline plasma levels of 62 cytokines, soluble receptors, acute-phase proteins, and growth factor markers were measured using Luminex bead-based assays. We estimated hazard ratios (HRs) associating each marker with CRC risk by Cox proportional hazards models adjusted for potential confounders. Subanalyses compared cases by years after blood draw (<5 vs. ≥5) and anatomical subsite (colon vs. rectum). Linear trends in quantiles of four C-C motif ligand (CCL) chemokines, one C-X-C motif ligand (CXCL) chemokine, and a soluble receptor were nominally associated with CRC risk based on ptrend < 0.05, but none met false discovery rate corrected statistical significance. HRs for the 4th vs. 1st quartile were: 1.69 for CCL2/MCP1, 1.61 for soluble tumor necrosis factor receptor 2, 1.39 for CCL15/MIP1D, 1.35 for CCL27/CTACK, 0.70 for CXCL6/GCP2 and 0.61 for CCL3/MIP1A. Among cases diagnosed 5+ years after enrollment, CCL2/MCP1, CCL3/MIP1A and CXCL6/GCP2 retained nominal statistical significance. There were no significant differences in associations between colon and rectum. Our findings implicate chemokine alterations in colorectal carcinogenesis, but require replication for confirmation. Noninvasive chemokine assays may have potential application in colorectal cancer screening and etiologic research.

OBJECTIVE

To test the hypothesis that endometrial stromal cells (ESCs) in endometriosis exhibit increased cell motility under hypoxia.

METHODS

Prospective case-control study.

METHODS

University research laboratory.

METHODS

Women with endometriosis (n = 18) or benign gynecological disease (n=19).

METHODS

Eutopic ESCs were cultured under normoxia (20% O2) or hypoxia (6.5% O2), and migration and invasion capacity assayed, with pathway-focused polymerase chain reaction (PCR) array and ELISAs performed. CD26/dipeptidyl peptidase IV (DPPIV) expression was determined by flow cytometric analysis and enzymatic activity assay. The ESCs supplemented with Diprotin A (CD26 inhibitor), stromal cell-derived factor-1α, or AMD3100 (C-X-C motif receptor 4; CXCR4 blocker) were assayed for their migratory potential.

METHODS

Endometrial stromal cell migration and invasion under hypoxia.

RESULTS

Endometriotic ESCs showed significantly higher migration and invasion through collagen gels under hypoxia compared with nonendometriotic ESCs. The PCR array revealed down-regulation of the migration inhibitor CD26/DPPIV and up-regulation of angiogenic factors (vascular endothelial growth factor A, C-X-C motif Ligand 6; CXCL6) in endometriotic ESCs under hypoxia. The CD26/DPPIV surface expression and activity as well as angiogenic protein secretions suggested that the molecular mechanisms underlying aberrant migratory and angiogenic behavior in endometriotic ESCs. A combinatorial treatment with diprotin A and stromal cell-derived factor-1α effectively enhanced migration and invasion preferentially in endometriotic ESCs cultured hypoxically.

CONCLUSIONS

Loss of CD26/DPPIV under hypoxia and the subsequent increase in migratory and angiogenic factors may favor conditions for lesion development in endometriosis.

OBJECTIVE

To evaluate the proinflammatory effect and molecular mechanism of IL-17 in the intestinal epithelial cell line HT-29.

METHODS

After culture of HT-29 cells with IL-17 and/or TNF-(α), real-time (RT) PCR and Western blot were used to measure the gene expression level of the neutrophil chemokines CXCL1, CXCL2, CXCL5, CXCL6, IL-8 and the Th-17 chemokine CCL20, the phosphorylation level of P38 and TNF-α, and the expression level of IL-8 after treatment with P38 inhibitor. Act1 stable knockdown HT-29 cell line was established to further test the change of P38 phosphorylation after treatment with IL-17 and TNF-α.

RESULTS

When HT-29 cells were cultured with IL-17 and TNF-α, the expression level of neutrophil chemokines (CXCL1, CXCL2, CXCL5, CXCL6, IL-8) and Th17 chemokine (CCL20) was significantly improved (24.96±2.53, 28.47±2.87, 38.08±2.72, 33.47±2.41, 31.7±2.38, 44.37±2.73, respectively) (p<0.01). The results of Western blot showed that IL-17 obviously enhanced the phosphorylation of P38 induced by TNF-α. Compared with the control group, the expression level of IL-8 declined significantly (9.47±1.36 vs 3.06±0.67) when HT-29 was cultured together with IL-17 and TNF-α (p<0.01). P38 inhibition assay showed that P38 pathway played an essential role in IL-17 induced inflammatory response. The level of P38 phosphorylation could not be changed after treatment with IL-17 and TNF-α in Act1 stable knockdown HT-29 cell line.

CONCLUSIONS

IL-17 significantly promoted the gene expression level of TNF-α-induced neutrophil chemokines and Th17 cells chemokine. IL-17 and TNF-α have an obvious synergistic effect on P38.