BACKGROUND

Fibroblasts are the commonest type of cancer stromal cells. Inflammation occurs in cancer tissue, and the inflammatory process has been suggested to be caused by interactions between immune cells and cancer cells. In this study, we clarified that site-specific fibroblasts regulate the formation of a site-specific inflammatory niche according to the depth of gastric cancer cell invasion.

METHODS

Immunohistochemistry was performed with paraffin-embedded tissues. The numbers of immune cells and the fibroblast area were calculated according to the cancer depth. The gene expression patterns of submucosal fibroblasts and subperitoneal fibroblasts stimulated with HSC44PE-conditioned medium were analyzed with a microarray. To examine the effects on the cancer microenvironment of differences in gene expressions between HSC44PE-stimulated submucosal fibroblasts and subperitoneal fibroblasts, assays of HSC44PE proliferation, T cell migration, and M2-like macrophage differentiation were performed.

RESULTS

The distributions of immune cells differed between the submucosal layer and the subserosal layer. The number of M2 macrophages was significantly higher and the fibroblast area was significantly larger in the subserosal layer compared with the submucosal layer. High expression levels of IL1B, TNFSF15, and CCL13 were observed in HSC44PE-stimulated submucosal fibroblasts, and higher expression levels of TGFB2, CSF1, CCL8, and CXCL5 were found in HSC44PE-stimulated subperitoneal fibroblasts. HSC44PE-stimulated subperitoneal fibroblast medium promoted the differentiation of monocytes into M2-like macrophages, whereas HSC44PE-stimulated submucosal fibroblasts significantly induced the migration of Jurkat cells and the growth of HSC44PE cells.

CONCLUSIONS

The dynamic states of immune cells differ between the submucosal and subserosal layers in cancer tissues. Site-specific fibroblasts regulate site-specific inflammatory niche formation according to the depth of cancer cell invasion.

BACKGROUND

It is becoming increasingly evident that platelet chemokines are involved in distinct aspects of atherosclerosis. The aim of this study was to examine the effects of long-term supplementation with purified anthocyanins on platelet chemokines in hypercholesterolemic individuals and to identify correlations of decreased platelet chemokine levels with serum lipid and inflammatory marker levels.

METHODS

A total of 146 hypercholesterolemic individuals were recruited and treated with 320 mg of purified anthocyanins (n = 73) or a placebo (n = 73) daily for 24 weeks in this randomized, double-blind, placebo-controlled trial.

RESULTS

Anthocyanin supplementation for 24 weeks significantly decreased the plasma CXCL7 (-12.32% vs. 4.22%, P = 0.001), CXCL5 (-9.95% vs. 1.93%, P = 0.011), CXCL8 (-6.07% vs. 0.66%, P = 0.004), CXCL12 (-8.11% vs. 5.43%, P = 0.023) and CCL2 levels (-11.63% vs. 12.84%, P = 0.001) compared with the placebo. Interestingly, the decreases in the CXCL7 and CCL2 levels were both positively correlated with the decreases in the serum low-density lipoprotein-cholesterol (LDL-C), high-sensitivity C-reactive protein (hsCRP) and interleukin-1β (IL-1β) levels after anthocyanin supplementation for 24 weeks. The decrease in the CXCL8 level was negatively correlated with the increase in the how-density lipoprotein-cholesterol (HDL-C) level and was positively correlated with the decrease in the soluble P-selectin (sP-selectin) level in the anthocyanin group. In addition, a positive correlation was observed between the decreases in the CXCL12 and tumornecrosis factor-α (TNF-α) levels after anthocyanin supplementation. However, the plasma CXCL4L1, CXCL1, macrophage migration inhibitory factor (MIF) and human plasminogen activator inhibitor 1 (PAI-1) levels did not significantly change following anthocyanin supplementation.

CONCLUSIONS

The present study supports the notion that platelet chemokines are promising targets of anthocyanins in the prevention of atherosclerosis.

BACKGROUND

ChiCTR-TRC-08000240. Registered: 10 December 2008.

BACKGROUND

Genes specifically expressed in one or a few tissues and upregulated in tumors are potentially good serum biomarkers.

METHODS

By applying a recently developed computational method, called multiple normal tissues corrected differential analysis (MNTDA), we identified genes that are likely to be upregulated in the blood of gastric cancer patients as compared to normal controls.

RESULTS

We identified four genes (MMP-1, MMP-3, MMP-12, and CXCL5) as potential serum biomarkers for gastric cancer. Of these four genes, only MMP-1 was significantly upregulated in the sera of 40 gastric cancer patients, as compared to 40 control sera. The same pattern was observed in the second cohort of 80 gastric cancer patients and 80 controls. In a combined analysis, the level of serum MMP-1 in gastric cancer patients was significantly higher than the level in control samples (P<0.0001). The use of MMP-1 was 62.5% sensitive and 62.5% specific in detecting gastric cancer patients. Patients with high serum levels of MMP-1 had a significantly worse outcome than patients with low serum MMP-1 levels. Finally, we determined that preoperative serum MMP-1 levels were prognostic, independent of tumor stage.

CONCLUSIONS

MMP-1 is a potential prognostic marker for gastric cancer patients after gastrectomy.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system. Most MS patients experience periods of symptom exacerbation (relapses) followed by periods of partial recovery (remission). Interestingly, upper-respiratory viral infections increase the risk for relapse. Here, we used an autoimmune-prone T-cell receptor transgenic mouse (2D2) and a mouse-adapted human influenza virus to test the hypothesis that upper-respiratory viral infection can cause glial activation, promote immune cell trafficking to the CNS, and trigger disease. Specifically, we inoculated 2D2 mice with influenza A virus (Puerto Rico/8/34; PR8) and then monitored them for symptoms of inflammatory demyelination. Clinical and histological experimental autoimmune encephalomyelitis was observed in ∼29% of infected 2D2 mice. To further understand how peripheral infection could contribute to disease onset, we inoculated wild-type C57BL/6 mice and measured transcriptomic alterations occurring in the cerebellum and spinal cord and monitored immune cell surveillance of the CNS by flow cytometry. Infection caused temporal alterations in the transcriptome of both the cerebellum and spinal cord that was consistent with glial activation and increased T-cell, monocyte, and neutrophil trafficking to the brain at day 8 post infection. Finally, Cxcl5 expression was up-regulated in the brains of influenza-infected mice and was elevated in cerebrospinal fluid of MS patients during relapse compared with specimens acquired during remission. Collectively, these data identify a mechanism by which peripheral infection may exacerbate MS as well as other neurological diseases.

Background: Ulcerative colitis (UC) is an inflammatory bowel disease that is difficult to diagnose and treat. To date, the degree of inflammation in patients with UC has mainly been determined by measuring the levels of nonspecific indicators, such as C-reactive protein and the erythrocyte sedimentation rate, but these indicators have an unsatisfactory specificity. In this study, we performed bioinformatics analysis using data from the National Center for Biotechnology Information-Gene Expression Omnibus (NCBI-GEO) databases and verified the selected core genes in a mouse model of dextran sulfate sodium (DSS)-induced colitis.

Aim: To identify UC-related differentially expressed genes (DEGs) using a bioinformatics analysis and verify them in vivo and to identify novel biomarkers and the underlying mechanisms of UC.

Methods: Two microarray datasets from the NCBI-GEO database were used, and DEGs between patients with UC and healthy controls were analyzed using GEO2R and Venn diagrams. We annotated these genes based on their functions and signaling pathways, and then protein-protein interactions (PPIs) were identified using the Search Tool for the Retrieval of Interacting Genes. The data were further analyzed with Cytoscape software and the Molecular Complex Detection (MCODE) app. The core genes were selected and a Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed. Finally, colitis model mice were established by administering DSS, and the top three core genes were verified in colitis mice using real-time polymerase chain reaction (PCR).

Results: One hundred and seventy-seven DEGs, 118 upregulated and 59 downregulated, were initially identified from the GEO2R analysis and predominantly participated in inflammation-related pathways. Seven clusters with close interactions in UC formed: Seventeen core genes were upregulated [C-X-C motif chemokine ligand 13 (CXCL13), C-X-C motif chemokine receptor 2 (CXCR2), CXCL9, CXCL5, C-C motif chemokine ligand 18, interleukin 1 beta, matrix metallopeptidase 9, CXCL3, formyl peptide receptor 1, complement component 3, CXCL8, CXCL1, CXCL10, CXCL2, CXCL6, CXCL11 and hydroxycarboxylic acid receptor 3] and one was downregulated [neuropeptide Y receptor Y1 (NYP1R)] in the top cluster according to the PPI and MCODE analyses. These genes were substantially enriched in the cytokine-cytokine receptor interaction and chemokine signaling pathways. The top three core genes (CXCL13, NYP1R, and CXCR2) were selected and verified in a mouse model of colitis using real-time PCR Increased expression was observed compared with the control mice, but only CXCR2 expression was significantly different.

Conclusion: Core DEGs identified in UC are related to inflammation and immunity inflammation, indicating that these reactions are core features of the pathogenesis of UC. CXCR2 may reflect the degree of inflammation in patients with UC.

Keywords: Bioinformatics analysis; C-X-C motif chemokine ligand 13; C-X-C motif chemokine receptor 2; Colitis model mice; Neuropeptide Y receptor Y1; Ulcerative colitis.

Contraction of cultured myotubes with application of electric pulse stimulation (EPS) has been utilized for investigating cellular responses associated with actual contractile activity. However, cultured myotubes derived from human subjects often exhibit relatively poor EPS-evoked contractile activity, resulting in minimal contraction-inducible responses (i.e. myokine secretion). We herein describe an "in vitro exercise model", using hybrid myotubes comprised of human myoblasts and murine C2C12 myoblasts, exhibiting vigorous contractile activity in response to EPS. Species-specific analyses including RT-PCR and the BioPlex assay allowed us to separately evaluate contraction-inducible gene expressions and myokine secretions from human and mouse constituents of hybrid myotubes. The hybrid myotubes, half of which had arisen from primary human satellite cells obtained from biopsy samples, exhibited remarkable increases in the secretions of human cytokines (myokines) including interleukins (IL-6, IL-8, IL-10, and IL16), CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CC chemokines (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), and IFN-γ in response to EPS-evoked contractile activity. Together, these results indicate that inadequacies arising from human muscle cells are effectively overcome by fusing them with murine C2C12 cells, thereby supporting the development of contractility and the resulting cellular responses of human-origin muscle cells. Our approach, using hybrid myotubes, further expands the usefulness of the "in vitro exercise model".

BACKGROUND

Irritant contact dermatitis (ICD) is a cutaneous inflammatory response to a variety of triggers that requires no sensitization and accounts for up to 80% of occupational dermatitis cases. IL-6 has been alternately associated with both allergic and irritant dermatitis and is closely linked to skin wound healing, therefore making it an ideal candidate to investigate in the mechanism of ICD.

RESULTS

Despite being a well-known pro-inflammatory cytokine, IL-6 deficient (IL-6KO) mice show much more severe ICD than controls. Transcriptome analysis was employed to examine irritant-exposed and control skin samples from C57BL/6 and IL-6KO mice. Over 1900 transcripts were found differentially modulated between C57 (1184 total) and IL-6KO (802 total) mice with the magnitude of expression significantly disparate. Overall gene ontology revealed metabolic and cellular enriched functional processes but numerous pro-inflammatory and immune associated genes (Cxcl2, Cxcl3, Cxcl5, Acod, Hamp, c-Lectins, for example), keratin associated genes (Krt6b and various Krtaps), and members of the Sprr and Lce family, which promote skin barrier integrity and keratinocyte functions, were also differentially modulated.

CONCLUSIONS

The altered expression of these genes may provide a potential mechanism to explain the increased ICD severity in IL-6-deficient mice. Overall, this study offers new insight into the pathogenesis of ICD, indicates new mediators/biomarkers that may influence the variability of responses to irritants and provides potential targets for therapeutic development.

Chemokines play diverse roles in human pathophysiology, ranging from trafficking leukocytes and immunosurveillance to the regulation of metabolism and neural function. Chemokine function is intimately coupled to binding tissue glycosaminoglycans (GAGs) heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS). Currently, very little is known about how the structural features and sequences of a given chemokine, the structure and sulfation pattern of a given GAG, and structural differences among GAGs and among chemokines impact binding interactions. In this study, we used solution NMR spectroscopy to characterize the binding interactions of two related neutrophil-activating chemokines, CXCL1 and CXCL5, with HS, CS, and DS. For both chemokines, the dimer bound all three GAGs with higher affinity than did the monomer, and affinities of the chemokines for CS and DS were lower than for HS. NMR-based structural models reveal diverse binding geometries and that the binding surfaces for each of the three GAGs were different between the two chemokines. However, a given chemokine had similar binding interactions with CS and DS that were different from HS. Considering CXCL1 and CXCL5 activate the same CXCR2 receptor, we conclude that differences in GAG interactions play a role in determining the nature of chemokine gradients, levels of free chemokine available for receptor activation, and how chemokines bind their receptors in a chemokine-specific manner.

Sepsis is a life-threatening organ dysfunction condition caused by a dysregulated host response to an infection. Here we report that the circulating levels of growth and differentiation factor-15 (GDF15) are strongly increased in septic shock patients and correlate with mortality. In mice, we find that peptidoglycan is a potent ligand that signals through the TLR2-Myd88 axis for the secretion of GDF15, and that Gdf15-deficient mice are protected against abdominal sepsis due to increased chemokine CXC ligand 5 (CXCL5)-mediated recruitment of neutrophils into the peritoneum, leading to better local bacterial control. Our results identify GDF15 as a potential target to improve sepsis treatment. Its inhibition should increase neutrophil recruitment to the site of infection and consequently lead to better pathogen control and clearance.

Keywords: CXCL5; GDF15; neutrophils; sepsis.

Leukocytes sense extracellular ATP, a danger-associated molecular pattern, released during cellular stress and death, via activation of cell surface P2X and P2Y receptors. Here, we investigate P2 receptor expression in primary human monocyte-derived macrophages and receptors that mediate ATP-evoked intracellular [Ca2+]i signals and cytokine production in response to ATP concentrations that exclude P2X7 receptor activation. Expression of P2X1, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y13 was confirmed by quantitative RT-PCR and immunocytochemistry. ATP elicited intracellular Ca2+ responses in a concentration-dependent fashion (EC50 = 11.4 ± 2.9 μM, n = 3). P2Y11 and P2Y13 activations mediated the amplitude of [Ca2+]i response, whereas P2X4 activation, but not P2X1 or P2X7, determined the duration of Ca2+ response during a sustained phase. ATP mediated gene induction of CXCL5, a proinflammatory chemokine. P2X4 antagonism (PSB-12062 or BX430) inhibited ATP-mediated induction of CXCL5 gene expression and secretion of CXCL5 by primary macrophage. Inhibition of CXCL5 secretion by P2X4 antagonists was lost in the absence of extracellular Ca2+ Reciprocally, positive allosteric modulation of P2X4 (ivermectin) augmented ATP-mediated CXCL5 secretion. P2X7, P2Y11, or P2Y13 receptor did not contribute to CXCL5 secretion. Together, the data reveals a role for P2X4 in determining the duration of ATP-evoked Ca2+ responses and CXCL5 secretion in human primary macrophage.

IL-4 plays an important role in the pathogenesis of atopic dermatitis (AD), a common chronic inflammatory skin disease. We have generated IL-4 transgenic (Tg) mice by over-expressing IL-4 in the epidermis. These mice spontaneously develop chronic pruritic inflammatory skin lesions, which meet the clinical and histological diagnostic criteria for human AD. Systemic survey of immune-related genes in this mouse model, however, has not been performed. In this study, we utilize PCR array technique to examine hundreds of inflammation-related genes in the IL-4 Tg mice before and after the onset of skin lesions as well as in their wild type (WT) littermates. Only those genes with at least 2-fold up-regulation or down-regulation and with a P-value of less than 0.05 in comparison to WT controls were identified and analyzed. In the skin lesions, many chemokines, pro-inflammatory cytokines, and other AD-related factors are dysregulated compared to the wild type mice. Particularly, CXCL5, IL-1β, IL-24, IL-6, oncostatin M, PTGS2, FPR1 and REG3γ are up-regulated several hundred-fold. In the pre-lesional group that shows no obvious skin abnormality on clinical observation, 30 dysregulated genes are nevertheless identified though the fold changes are much less than that of the lesional group, including CCL6, CCL8, CCL11, CCL17, CXCL13, CXCL14, CXCR3 and IL-12Rβ2. Finally using ELISA, we demonstrate that 4 most dramatically up-regulated factors in the skin are also elevated in the peripheral blood of the IL-4 Tg mice. Taken together, our data have identified hundreds of dysregulated factors in the IL-4 Tg mice before and after the onset of skin lesions. Future detailed examination of these factors will shed light on our understanding of the development and progression of AD and help to discover important biomarkers for clinical AD diagnosis and treatment.

Germ-line stem cells (GSCs) constitute a stem cell population with remarkable stability and proliferative potential in vitro and are a useful model for studying the mechanism of self-renewal and "stemness" function of committed tissue stem cells. To identify GSC-specific genes, we performed subtractive hybridization using cDNA from GSCs, testis, and embryonic stem (ES) cells, and successfully identified 11 genes highly expressed in GSCs. Histological analysis confirmed expression of Cry alpha b, Mcpt8, Cxcl5, Fth1, Ctla2 alpha, and Spp1 in undifferentiated spermatogonia on the basement membrane area of the seminiferous epithelium of the testis, where the GSC niche is thought to be located. Among GSC-specific genes, quantitative PCR analysis showed seven genes-Fth1, Cry alpha b, Spp1, Bcap31, Arhgap1, Ctla2 alpha, and Serpina3g-to be common transcripts highly expressed in hematopoietic stem cells (HSCs). Histological analysis confirmed that Ctla2 alpha-, Serpina3g-, and Spp1-expressing cells were observed in the trabecular bone region of the bone marrow, where the HSC niche is located. Furthermore, histological analysis revealed that only Spp1 was expressed in the hair follicle bulge in the area of the hair follicle stem cell niche. Thus, identifying stemness genes by comparative analysis to GSCs is a powerful tool with which to explore the fundamental commonalities of HSCs and other stem cell types.

Liquid chromatography using a tandem mass spectrometer (LC-MS/MS) is a method of proteomic analysis. A shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolution. The hepatic function of mice with acute hepatitis following the intraperitoneal administration of CCL4 was improved by the tail vein administration of the culture conditional medium (CM) of human mesenchymal stem cells from adipose tissue (hMSC-AT). In this study, a secreted protein expression analysis of hMSC-AT was performed using LC-MS/MS; 128 proteins were identified. LC-MS/MS showed that 106 new functional proteins and 22 proteins (FINC, PAI1, POSTN, PGS2, TIMP1, AMPN, CFAH, VIME, PEDF, SPRC, LEG1, ITGBL, ENOA, CSPG2, CLUS, IBP4, IBP7, PGS1, IBP2, STC2, CTHR1, CD9) were previously reported in hMSC-AT-CMs. In addition, various proteins associated with growth (SAP, SEM7A, PTK7); immune system processes (CO1A2, CO1A1, CATB, TSP1, GAS6, PTX3, C1 S, SEM7A, G3P, PXDN, SRCRL, CD248, SPON2, ENPP2, CD109, CFAB, CATL1, MFAP5, MIF, CXCL5, ADAM9, CATK); and reproduction (MMP2, CATB, FBLN1, SAP, MFGM, GDN, CYTC) were identified in hMSC-AT-CMs. These results indicate that a comprehensive expression analysis of proteins by LC-MS/MS is useful for investigating new factors associated with cellular components, biological processes, and molecular functions.

The molecular changes in gene expression following ablative laser treatment of skin lesions, such as atrophic scars and UV-damaged skin, are not completely understood. A standardized in vitro model of human skin, to study the effects of laser treatment on human skin, has been recently developed. Therefore, the aim of the investigation was to examine morphological and molecular changes caused by fractional ablative erbium:YAG laser treatment on an in vitro full-thickness 3D standardized organotypic model of human skin. A fractional ablative erbium:YAG laser was used to irradiate organotypic human 3D models. Laser treatments were performed at four different settings using a variety of stacked pulses with similar cumulative total energy fluence (60 J/cm2). Specimens were harvested at specified time points and real-time PCR (qRT-PCR) and microarray studies were performed. Frozen sections were examined histologically. Three days after erbium:YAG laser treatment, a significantly increased mRNA expression of matrix metalloproteinases and their inhibitors (MMP1, MMP2, MMP3, TIMP1, and TIMP2), chemokines (CXCL1, CXCL2, CXCL5, and CXCL6), and cytokines such as IL6, IL8, and IL24 could be detected. qRT-PCR studies confirmed the enhanced mRNA expression of IL6, IL8, IL24, CXCLs, and MMPs. In contrast, the mRNA expression of epidermal differentiation markers, such as keratin-associated protein 4, filaggrin, filaggrin 2, and loricrin, and antimicrobial peptides (S100A7A, S100A9, and S100A12) as well as CASP14, DSG2, IL18, and IL36β was reduced. Four different settings with similar cumulative doses have been tested (N10%, C10%, E10%, and W25%). These laser treatments resulted in different morphological changes and effects on gene regulations. Longer pulse durations (1000 μs) especially had the strongest impact on gene expression and resulted in an upregulation of genes, such as collagen-1A2, collagen-5A2, and collagen-6A2, as well as FGF2. Histologically, all treatment settings resulted in a complete regeneration of the epidermis 3 days after irradiation. Fractional ablative erbium:YAG laser treatment with a pulse stacking technique resulted in histological alterations and shifts in the expression of various genes related to epidermal differentiation, inflammation, and dermal remodeling depending on the treatment setting applied. A standardized in vitro 3D model of human skin proved to be a useful tool for exploring the effects of various laser settings both on skin morphology and gene expression during wound healing. It provides novel data on the gene expression and microscopic architecture of the exposed skin. This may enhance our understanding of laser treatment at a molecular level.

Ozone is a common, potent oxidant pollutant in industrialized nations. Ozone exposure causes airway hyperreactivity, lung hyperpermeability, inflammation, and cell damage in humans and laboratory animals, and exposure to ozone has been associated with exacerbation of asthma, altered lung function, and mortality. The mechanisms of ozone-induced lung injury and differential susceptibility are not fully understood. Ozone-induced lung inflammation is mediated, in part, by the innate immune system. We hypothesized that mannose-binding lectin (MBL), an innate immunity serum protein, contributes to the proinflammatory events caused by ozone-mediated activation of the innate immune system. Wild-type (Mbl(+/+)) and MBL-deficient (Mbl(-/-)) mice were exposed to ozone (0.3 ppm) for up to 72 h, and bronchoalveolar lavage fluid was examined for inflammatory markers. Mean numbers of eosinophils and neutrophils and levels of the neutrophil attractants C-X-C motif chemokines 2 [Cxcl2 (major intrinsic protein 2)] and 5 [Cxcl5 (limb expression, LIX)] in the bronchoalveolar lavage fluid were significantly lower in Mbl(-/-) than Mbl(+/+) mice exposed to ozone. Using genome-wide mRNA microarray analyses, we identified significant differences in transcript response profiles and networks at baseline [e.g., nuclear factor erythroid-related factor 2 (NRF2)-mediated oxidative stress response] and after exposure (e.g., humoral immune response) between Mbl(+/+) and Mbl(-/-) mice. The microarray data were further analyzed to discover several informative differential response patterns and subsequent gene sets, including the antimicrobial response and the inflammatory response. We also used the lists of gene transcripts to search the LINCS L1000CDS(2) data sets to identify agents that are predicted to perturb ozone-induced changes in gene transcripts and inflammation. These novel findings demonstrate that targeted deletion of Mbl caused differential levels of inflammation-related gene sets at baseline and after exposure to ozone and significantly reduced pulmonary inflammation, thus indicating an important innate immunomodulatory role of the gene in this model.

BACKGROUND

CXCL5, also known as epithelial cell-derived neutrophil-activating peptide (ENA-78), is a chemokine that has a role in the development of cardiovascular and other diseases. We have previously scanned the full length CXCL5 gene and reported the -156G>C (rs352046) polymorphism in the promoter region of this gene.

OBJECTIVE

The aim of this study was to examine whether there was an association between this polymorphism and type 2 diabetes mellitus or its microvascular complications in an Iranian population.

METHODS

A total of 230 patients with type 2 diabetes were recruited from Rafsanjan, in the south-east of Iran; 102 healthy control subjects were recruited from the same area. The region containing the CXCL5 -156G>C polymorphism was genotyped by PCR amplification and restriction fragment length polymorphism analysis, and allele frequency data were analyzed using STATA 8 software.

RESULTS

We observed that patients with type 2 diabetes had a higher frequency of carrying either the G/C or C/C genotype compared with healthy controls (C/G + C/C vs G/G; p = 0.004; odds ratio [OR] 2.17; 95% CI 1.27, 3.80). In addition, the frequency of allele C was significantly increased in patients with diabetes compared with controls (p = 0.01; OR 1.72; 95% CI 1.07, 2.86). No association was found between this polymorphism and diabetic microvascular complications.

CONCLUSIONS

Our findings suggest a role of CXCL5 in the pathogenesis of diabetes. The mechanism behind this role needs to be investigated further. Moreover, replications in other populations with larger sample sizes are required to confirm these findings.

Steroid receptor coactivator 3 (SRC-3) is a transcriptional coactivator that interacts with nuclear receptors and some other transcription factors to enhance their effects on target gene transcription. We reported previously that SRC-3-deficient (SRC-3-/-) mice are extremely susceptible to Escherichia coli-induced septic peritonitis as a result of uncontrolled inflammation and a defect in bacterial clearance. In this study, we observed significant upregulation of SRC-3 in colonic epithelial cells in response to Citrobacter rodentium infection. Based on these findings, we hypothesized that SRC-3 is involved in host defense against attaching and effacing bacterial infection. We compared the responses of SRC-3-/- and wild-type mice to intestinal C. rodentium infection. We found that SRC-3-/- mice exhibited delayed clearance of C. rodentium and more severe tissue pathology after oral infection with C. rodentium compared with wild-type mice. SRC-3-/- mice expressed normal antimicrobial peptides in the colons but exhibited delayed recruitment of neutrophils into the colonic mucosa. Accordingly, SRC-3-/- mice showed a delayed induction of CXCL2 and CXCL5 in colonic epithelial cells, which are responsible for neutrophil recruitment. At the molecular level, we found that SRC-3 can activate the NF-κB signaling pathway to promote CXCL2 expression at the transcriptional level. Collectively, we show that SRC-3 contributes to host defense against enteric bacteria, at least in part via upregulating CXCL2 expression to recruit neutrophils.

Psoriasis is a chronic inflammatory skin disease with numerous involved factors. miR-146a and miR-146b (miR-146a/b) are anti-inflammatory miRNAs that are increased in psoriatic skin. SERPINB2 has been shown to be upregulated in the inflammation and infections. Here we aimed to study the relationship between miR-146a/b and SERPINB2 and to delineate the role of SERPINB2 in association of plaque psoriasis. We report increased SERPINB2 expression in the skin of psoriasis patients, which was in a positive relationship with psoriasis severity and in a negative relationship with miR-146a/b in psoriatic lesions. In cultured keratinocytes, both cellular and secreted SERPINB2 levels were strongly induced in response to IFN-γ and TNF-α. Interestingly, SERPINB2 mRNA was down-regulated by IL-17A and the combination of TNF-α and IL-17A at timepoints when miR-146a was increased. The predicted binding site for miR-146a/b in 3' untranslated region of SERPINB2 revealed no activity in luciferase assay, while siRNA silencing of miR-146a/b direct targets IRAK1 and CARD10 resulted in reduced expression of SERPINB2, suggesting that miR-146a/b indirectly control SERPINB2 expression in the skin. The siRNA silencing of SERPINB2 increased the expression of IL-8, CXCL5 and CCL5 and migration of neutrophils revealing its anti-inflammatory role in keratinocytes. Our data together suggest that SERPINB2 and miR-146a/b are part of disease-related network of molecules that are coordinately regulated and act in controlling the inflammatory responses in psoriatic skin.

We evaluated an anti-inflammatory effect of topical administration of tofacitinib, janus kinase (JAK) blocker, on corneal inflammation. Topical instillation of either tofacitinib or PBS was applied after wounding BALB/c mice corneas with alkali burn. Topical instillation was performed until day 14 after injury and injured eye was analyzed. The vascularized area in the alkali burned cornea was significantly reduced in the tofacitinib group compared with that in the PBS group. The immunoreactivity of Gr-1, F4/80, IFN-γ, and phosphorylated STAT(signal transducer and activator of transcription)1 in corneal stroma was diminished significantly in the tofacitinib group. Using laser capture microdissection system and quantitative PCR array analysis, the expression levels of CXCL9, CXCL5, CCL7, CCL2, MMP(matrix metalloproteinase)-9, and STAT1 in corneal stroma were down-regulated in the tofacitinib group. In in vitro study, human fibroblast pretreated by IFN-γ showed phosphorylation of STAT1, and this phosphorylation was down-regulated by adding tofacitinib to the culture medium. These results indicate the topical application of JAK inhibitor causes down-regulation of JAK- or IFN-γ-related molecules. Therefore, we deduce that application of JAK inhibitor for topical instillation may contribute to the treatment of corneal inflammation.

BACKGROUND

Previous work reported the anti-arthritic synergy afforded by combining calcitonin (CT) and glucocorticoids (GC). Here we focus on the pairing of elcatonin (eCT) and dexamethasone (Dex), querying whether: i) this was a class-effect action; ii) mechanistic insights could be unveiled; iii) the synergy affected canonical GC adverse effects.

METHODS

Using the rat collagen-induced arthritis model, different combinations of eCT and Dex, were administered from disease onset to peak (day 11 to 18). Macroscopic disease score was monitored throughout, with biochemical and histological analyses conducted on plasma and tissues at day 18. The effect on acute hyperglycaemia and liver enzyme message were also assessed.

RESULTS

Whilst eCT alone was inactive, it synergised at 1 µg/kg with low doses of Dex (7.5 or 15 µg/kg) to yield an anti-arthritic efficacy equivalent to a 4- to 7-fold higher Dex dose. Mechanistically, the anti-arthritic synergy corresponded to a marked attenuation in RA-relevant analytes. CXCL5 expression, in both plasma and joint, was markedly inhibited by the co-therapy. Finally, co-administration of eCT did not exacerbate metrics of GC adverse effects, and rescued some of them.

CONCLUSIONS

We present evidence of a class-effect action for the anti-arthritic synergy of CT/GC combination, underpinned by the powerful inhibition of joint destruction markers. Furthermore, we identify CXCL5 as a marker for the combination therapy with potential diagnostic and prognostic utility. Substantial GC dose reduction, together with the absence of exacerbated adverse effects, indicated a significant clinical potential for this co-therapy in RA and beyond.

BACKGROUND

Epithelial neutrophil activating peptide (ENA-78) is encoded by the polymorphic CXCL5 gene and is a recruiter and activator of neutrophils. Furthermore, ENA-78 may be involved in pathological inflammatory processes and variable drug responses.

METHODS

To facilitate future disease-gene and pharmacogenetic investigation of ENA-78, we developed and cross-validated medium- to high-throughput genotyping assays for 2 commonly occurring CXCL5 polymorphisms (rs352046 and rs425535). Furthermore, we compared allele and genotype frequencies in a U.S. population with those of a previously studied European population.

RESULTS

There was 100% genotype concordance between the 2 methods used (Pyrosequencing and TaqMan). Variant allele frequencies for rs352046 were consistent between the U.S. (16%) and European (16%) populations, while the rs425535 variant allele was more than twice as high in the European cohort (38% vs. 16%). There was complete linkage of genotypes at both loci in our population.

CONCLUSIONS

The distribution of variant alleles for the 2 polymorphisms studied should be further evaluated in other populations. In addition, our data highlight the importance of assay validation using multiple platforms.

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors (statins) have been suggested to attenuate abdominal aortic aneurysm (AAA) growth. However, the effects of statins in human AAA tissues are not fully elucidated. The aim of this study was to investigate the direct effects of statins on proinflammatory molecules in human AAA walls in ex vivo culture. Simvastatin strongly inhibited the activation of nuclear factor (NF)-κB induced by tumor necrosis factor (TNF)-α in human AAA walls, but showed little effect on c-jun N-terminal kinase (JNK) activation. Simvastatin, as well as pitavastatin significantly reduced the secretion of matrix metalloproteinase (MMP)-9, monocyte chemoattractant protein (MCP)-2 and epithelial neutrophil-activating peptide (CXCL5) under both basal and TNF-α-stimulated conditions. Similar to statins, the Rac1 inhibitor NSC23766 significantly inhibited the activation of NF-κB, accompanied by a decreased secretion of MMP-9, MCP-2 and CXCL5. Moreover, the effect of simvastatin and the JNK inhibitor SP600125 was additive in inhibiting the secretion of MMP-9, MCP-2 and CXCL5. These findings indicate that statins preferentially inhibit the Rac1/NF-κB pathway to suppress MMP-9 and chemokine secretion in human AAA, suggesting a mechanism for the potential effect of statins in attenuating AAA progression.

The efficacy of the ACE (angiotensin-converting enzyme) inhibitor perindopril in coronary artery disease [EUROPA (European trial on reduction of cardiac events with perindopril in stable coronary artery disease) study] is associated with the rs12050217 A/G single nucleotide polymorphism in the B1 receptor (bradykinin type 1 receptor) gene. To investigate the underlying mechanism, we examined the effect of this polymorphism on B1-receptor-mediated coronary artery dilation and peripheral blood mononuclear cell activation. Vasorelaxant responses of human coronary microarteries from subjects without coronary disease to des-Arg(9)-bradykinin and to bradykinin were studied in organ bath experiments. Des-Arg9-bradykinin responses were endothelium-dependent and exclusively mediated by B1 receptors, whereas responses to bradykinin were induced through B2 receptors (bradykinin type 2 receptors). The presence of the G allele reduced the response to 3 × 10(-8) mol/l des-Arg(9)-bradykinin by 29% [AA (n=13) compared with AG/GG (n=8); P<0.03], and tended to lower concentration-related responses (P=0.065) to this agonist, whereas the responses to bradykinin were unaffected by the rs12050217 genotype. In freshly obtained human mononuclear cells 1 μmol/l des-Arg(9)-bradykinin increased expression of the pro-inflammatory factors CXCL5 (CXC chemokine ligand 5) and IL6 (interleukin-6). These responses were not affected by genotype and exclusively occurred in blood cells from women, correlating (in the case of CXCL5) with their plasma 17β-oestradiol levels (r(2)=0.32, P=0.02; n=17). IL-1β (interleukin-1β) increased CXCL5 and IL6 expression in both genders, and this response was not associated with 17β-oestradiol levels. The gender difference in responses to B1 receptor stimulation in blood mononuclear cells implies possible gender differences in the response to ACE inhibitor therapy, which needs to be studied more comprehensively. The observed decrease in coronary vasodilator response might contribute to the impaired treatment response to perindopril of G allele carriers found in the EUROPA study.