JAM2, NCAM1, ITGA8 and CNTN1. In the present study, we successfully identified a group of genes that showed methylation and gene expression changes in well-defined colorectal cancer tissues with high purity. The integrated analysis gives additional insight regarding the regulation of colorectal cancer-associated genes and their underlying mechanisms that contribute to colorectal carcinogenesis.Apart from genetic mutations, epigenetic alteration is a common phenomenon that contributes to neoplastic transformation in colorectal cancer. Transcriptional silencing of tumor-suppressor genes without changes in the DNA sequence is explained by the existence of promoter hypermethylation. To test this hypothesis, we integrated the epigenome and transcriptome data from a similar set of colorectal tissue samples. Methylation profiling was performed using the Illumina InfiniumHumanMethylation27 BeadChip on 55 paired cancer and adjacent normal epithelial cells. Fifteen of the 55 paired tissues were used for gene expression profiling using the Affymetrix GeneChip Human Gene 1.0 ST array. Validation was carried out on 150 colorectal tissues using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) technique. PCA and supervised hierarchical clustering in the two microarray datasets showed good separation between cancer and normal samples. Significant genes from the two analyses were obtained based on a ≥2-fold change and a false discovery rate (FDR) P-value of <0.05. We identified 1,081 differentially hypermethylated CpG sites and 36 hypomethylated CpG sites. We also found 709 upregulated and 699 downregulated genes from the gene expression profiling. A comparison of the two datasets revealed 32 overlapping genes with 27 being hypermethylated with downregulated expression and 4 hypermethylated with upregulated expression. One gene was found to be hypomethylated and downregulated. The most enriched molecular pathway identified was cell adhesion molecules that involved 4 overlapped genes,  In recent decades, the incidence of colorectal cancer (CRC) has increased by 2- to 4-fold in many Eastern Asia countries such as China, Japan, South Korea and Singapore ,2. The hEpigenetic markers such as methylation markers in CRC were first reported 10 years ago in DNA from stool and blood samples . DNA metCGI hypermethylation in the promoter region is thought to be linked with the transcriptional inactivation of tumor-suppressor genes ,15. This® methylation array on 28 normal mucosa and 91 CRC samples, 202 CpG sites with 90 hypermethylated and 42 hypomethylated loci that involved 132 genes were identified (DOCK8), visual system homeobox 2 (VSX2), microRNA 34b (miR-34b), glucagon-like peptide 1 receptor (GLP1R1), B-cell translocation gene 4 (BTG4), BEN domain containing 4 (BEND4), neuronal pentraxin I (NPTX1), ALX homeobox 3 (ALX3), zinc finger protein 583 (ZNF583), homer homolog 2 (HOMER2) and gap junction protein, gamma 1 (GJC1)  method, the microarray chip-based study is a widely used approach in exploring methylation markers in CRC ,19,20. Ientified . Using tentified . A more entified . Among t1 (GJC1) .MYC in directing the transcriptional activation and repression in CRC. Additional findings included repetitive mutations in APC membrane recruitment protein 1 (FAM123B) and AT rich interactive domain 1A (ARID1A) as well as a novel mutation in SRY (sex determining region Y)-box 9 (SOX9)  .Studies using a similar integrative approach of analyses on Asian patients are lacking. Therefore, the aim of the present study was to investigate the biological complexity of CRC by integrating DNA methylation and gene expression profiling signatures using paired samples from local patients. The integrated signatures may later serve as potential diagnostic markers for the prognostication of CRC. We also hypothesized that CpG hypermethylation in the promoter region of CRC-associated genes led to a low expression of the respective genes.A total of 55 paired colorectal carcinoma and their corresponding adjacent normal epithelial cells (10 cm away from the tumor) were collected at surgery from patients at the Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia. Patients provided written informed consent to participate in the present study. This study was approved by the UKM Research Ethics Committee (Reference no: UKM 1.5.3.5/244/UMBI-004-2012).μm thickness using a cryostat . The sections were stained with hematoxylin and eosin (H&E) and were examined by the histopathologist. Tissue samples were considered representative when >80% malignant cells were present. Normal cells also contained >80% normal epithelial cells and were free from malignant or inflammatory cells. Patients with chemotherapy or radiotherapy prior to surgery were excluded from the study.The clinical tissues were snap-frozen in liquid nitrogen prior to sectioning. Tissue samples were sectioned into 5–7 Genomic DNA was isolated using the Qiagen DNeasy Tissue kit  according to the manufacturer’s instructions. The quantity and purity of DNA were quantified using the NanoDrop ND-1000 spectrophotometer . Gel electrophoresis was used to check the integrity of the DNA. Only samples with good purity were included in the study. Methylation profiling was performed in 110 samples using the HumanMethylation27 Beadchip to analyze 27,578 CpG sites covering 14,495 genes. Bisulphite conversion was carried out in the methylation assay using the EZ DNA Methylation-Gold kit . The microarray study was carried out according to the Infinium II Methylation Assay manual protocol. All the chips were scanned on a single BeadArray reader to avoid bias.® Genotyping Console™ (Affymetrix) was used to extract the expression data and Partek Genomic Suite 6.6 (version 6.12.0713)  was utilized for subsequent analysis.Total RNA from 15 paired representative tissues were extracted using the Qiagen QIAampMini Plus kit (Qiagen) according to the manufacturer’s instructions. The quantity and purity of RNA were quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). The integrity of RNA was measured using the Agilent Bioanalyzer 2100 . Only samples with an OD 260/280 of 1.8–2.1 and RNA integrity number (RIN) ≥6.0 were included in the gene expression study. The GeneChip Human Gene 1.0 ST array  that has 764,885 distinct probes covering 28,869 well-annotated genes was used. The assay was carried out using the Affymetrix expression protocol. The microarray chips were scanned using the GeneChip Scanner 3000 7G (Affymetrix). The AffymetrixThe control panel from Illumina BeadStudio software 2011 (version 1.9.0)  was used to determine the quality of our methylation microarray assay. β-value was used to determine the methylation status of each sample. This value was derived from each locus from the microarray and range from the lowest methylation value (β=0) to the highest methylation value (β=1). β-value was generated as the intensity of the methylated probe/total of the intensity of the methylated probe and the intensity of the unmethylated probe.The generated β-value was exported to the Partek Genomic Suite 6.6 (version 6.12.0713) (Partek) for subsequent analysis. PCA mapping was used to determine the quality of the samples. Three-way analysis of variance (ANOVA) with ≥2-fold change and P<0.05 with FDR were used to compare the differential methylated CpG loci between the cancer and normal groups. To remove the batch effect, the scanned date of the chips was controlled. A Gene Ontology enrichment analysis was carried out to investigate whether the genes found to be differentially methylated could be classified into a Gene Ontology category more often than expected by chance. A functional group with a high enrichment score was considered the leading group.Differential gene expression analysis was carried out using the Partek Genomic Suite 6.6 software with three-way ANOVA analysis. The expression data were normalized using quantile normalization and robust multi-array analysis (RMA) background correction. The differentially expressed genes were reported to be significant in the cancer group when the fold-change was >2.0 and P-value with FDR was <0.05. The batch effect was removed as a source of variation.The analysis was performed using the Partek Genomic Suite 6.6 software. We used the Pearson (linear) correlation to determine the correlation of our data. The correlated genes were considered significant at P<0.05.Overlapped genes were identified based on significant gene symbols from the methylation and expression datasets. Datasets were imported into the MySQL relational database for downstream data analysis. The MySQL database allows rapid and accurate data filtering across different datasets. The datasets were compared in the pair-wise manner. Unique gene symbols identified between the overlapping comparisons were used in downstream analysis. Along with the unique gene list, methylation and expression values were extracted from the datasets for chromosome mapping, circular map generation and KEGG Pathway mapping. Mapping of the integrated gene list allowed visualization of overlapping genes on chromosome map overview, circular map overview and also KEGG pathway maps.SFRP2, BTG4, APC and GPX7) were selected from the DNA methylation profile for validation purpose. Validation was carried out using the MS-MLPA and followed the manufacturer’s instructions. The primers were designed and customized following the guidelines from MRC Holland . The amplification PCR product was carried out by using the 3500 Genetic Analyzer . The electrophoresis result was assessed by using the Coffalyser software version 1.0.0.43 (MRC-Holland). For the analyses, dataset of MS-MLPA was analyzed by the method described in a previous study  showed that the normal group (indicated by red color) was clustered distinctly from the tumor group (indicated by blue color) from the profiling data . For theThe methylation profiling involved 110 CRC samples together with their neighboring non-cancerous colonic cells. The mean age for all patients was 60.4±12.83 years . The metPRKAR1B), cannabinoid receptor interacting protein 1 (C2orf32), zinc finger protein 542 (ZNF542), KH domain containing, RNA binding, signal transduction-associated 3 (KHDRBS), interferon regulatory factor 4 (IRF4), tissue factor pathway inhibitor 2 (TFPI2), potassium voltage-gated channel, KQT-like subfamily, member 5 (KCNQ5), filamin-binding LIM protein 1 (FBLIM1), eyes absent homolog 4 (EYA4) and spastic paraplegia 20 (SPG20). The top 10 most significantly hypomethylated loci were Fc receptor-like 3 (FCRL3), Granzyme K  (PRSS1), bactericidal/permeability-increasing protein (BPI), v-akt murine thymoma viral oncogene homolog 3 (AKT3), pipecolic acid oxidase (PIPOX), peptidase inhibitor 3 (PI3), bactericidal/permeability-increasing protein-like 3 (BPIL3), solute carrier family 26, member 4 (SLC26A4), long intergenic non-protein coding RNA 152 (MGC4677) and defensin, β 119 (DEFB119).A total of 1,123 loci (845 genes) were found to be differentially methylated in CRC compared with the normal colonic epithelial samples. These loci were further classified into 1,081 hypermethylated loci (804 genes), 36 hypomethylated loci (36 genes) and 6 sex-chromosome methylated loci (5 genes). Sex-chromosomes loci were eliminated for the subsequent analysis to avoid bias to the present study. This is due to the methylation involved in the X-inactivation process that silences one out of the two copies of X chromosome in female in order to compensate for the gene dosage effect. Supervised hierarchical clustering of the significant differentially methylated loci in our study is shown in CLDN1), phosphoribosyl pyrophosphate amidotransferase (PPAT), tumor protein D52-like 2 (TPD52L2), serine hydroxymethyltransferase 2 (SHMT2), cell division cycle associated 7 (CDCA7), chaperonin containing TCP1, subunit 3 (CCT3), eukaryotic translation initiation factor 2, subunit 2 β (EIF2S2), thyroid hormone receptor interactor 13 (TRIP13), inhibin, β A (INHBA) and negative elongation factor complex member C/D (TH1L). The top 10 most significant differentially downregulated genes were chromosome 2 open reading frame 88 (C2orf88), alcohol dehydrogenase 1B (ADH1B), erythrocyte membrane protein band 4.1-like 4A (EPB41L4A), transmembrane and immunoglobulin domain containing 1 (TMIGD1), serum/glucocorticoid regulated kinase 1 (SGK1), histone deacetylase 9 (HDAC9), UDP-glucose pyrophosphorylase 2 (UGP2), sodium channel, voltage-gated, type IX, α subunit (SCN9A), membrane-spanning 4-domains, subfamily A, member 12 (MS4A12) and solute carrier family 4, sodium bicarbonate cotransporter, member 4 (SLC4A4).The data of the genome wide expression profiling have been recently reported . StatistSFRP2), BTG4, adenomatous polyposis coli (APC) and glutathione peroxidase 7 (GPX7), to validate the methylation profiling data. Data analysis of MS-MLPA showed high methylation for all four validated genes in CRC compared to the normal samples  (r= 0.799443) (SLC4A4 (0.77751), family with sequence similarity 5, member C (FAM5C) (0.777301), partner of NOB1 homolog (PNO1) (0.769136) and CD163 molecule-like 1 (CD163L1) (0.766757). The top 5 negatively correlated genes were myosin light chain kinase (MYLK) (−0.756185) (NR5A2) (−0.738724), transforming growth factor, β-induced (TGFBI) (−0.674433), von Willebrand factor A domain containing 5A (VWA5A) (−0.673862) and D-tyrosyl-tRNA deacylase 1 (DTD1) (−0.652264).We correlated the data at P<0.05 and identified 188 significant loci (covered by 136 genes) from the two profiles including 83 negatively (66 genes) and 105 positively correlated loci (70 genes). The top 5 positively correlated genes were heat shock 60 kDa protein 1 (.799443) , SLC4A4 .756185) , nuclearJAM2, NCAM1, ITGA8 and CNTN1.We identified 32 significant overlapping genes from the methylation and gene expression profiles . Most ofWe analyzed the frequency of the methylated loci, the upregulated or downregulated genes and the integrated genes in each chromosome. HIST1H2BB) with histone cluster 1, H3c (HIST1H3C) and histone cluster 1, H3f (HIST1H3F) with histone cluster 1, H3g (HIST1H3G), another in the FOX family (forkhead box F1 (FOXF1), fork-head box F1 (FOXC2) and forkhead box FL (FOXL1)] and two large clusters in the zinc finger (ZNF) family. For the gene expression study, 16 clusters were distributed across the chromosomes including one single cluster which encompassed >10 genes. This cluster included the metallothionein 2A (MT2A), nucleoporin 93 kDa (NUP93) and 8 other genes derived from the metallothionein 1 (MT1) family.We identified four clusters of genes when we examined closely the distribution of methylated genes in each chromosome. These clusters were located in chromosome 6, 16 and 19 and belonged to specific gene families. One cluster was within the HIST1H family [histone cluster 1, H2bb , bol, boule-like (BOLL), cut-like homeobox 2 (CUTL2), KCNQ5, protocadherin γ subfamily C, 4 (PCDHGC4), PRKAR1B, solute carrier family 6 , member 15 (SLC6A15), SPG20, TFPI2 and unc-5 homolog C (UNC5C)  . The ADHquencing .GSTM2, ZNF655, MYH11, HHIP, RSPO3, RNF152, CAV1, SFRP2, JAM2, SPG20, TMEFF2, SST, SLIT2, SCNN1B, ADAMST1, GLI3, CKB and BMP3  and 5 with a positive association (hyper-up and hypo-down). Of the 27 hypermethylated genes with low gene expression, 18 were related to CRC including and BMP3 ,30–38. Oor genes –43. The ectively ,45.SFRP2 is a dominant-negative inhibitor in the Wnt signaling pathway that is involved in regulating cell proliferation, migration, and differentiation (SFRP2 that resulted in the downregulation of SFRP2 was detected via methylation-specific PCR (MSP) and quantitative PCR (qPCR) in colorectal carcinoma as compared to adenoma . Functional lncRNA may act as an activator or repressor for the expression of genes at the post-transcriptional level via mechanisms such as alternative splicing, influencing RNA polymerase binding efficiency and even by modification of epigenetic state of the gene (We also identified the ectively ,52. In tthylated . In a semelanoma . The lowt glioma . The posthe gene –59.JAM2, NCAM1, ITGA8 and CNTN1. CAM plays a vital physical function in defining the multicellular organism’s structure and signaling as well (JAM2. One study reported an inverse association between methylation and the gene expression of JAM2 in pre-malignant and malignant colorectal tissues (JAM2, which is involved in cell-cell adhesion and cell-environment interaction.From the integrative analysis, we found that 11 out of 95 KEGG pathways were associated with colorectal carcinogenesis. The most enriched pathway identified in our data was cell adhesion molecules, covered by the integrated genes  as well ,61. It i as well . CAMs su as well ,64. To t tissues . This obIn conclusion, our integrative analysis which combined DNA methylation and gene expression profiling datasets revealed a potential panel of biomarkers for the diagnosis or prognosis of colorectal cancer. Our integrative analysis also revealed a list of novel genes not previously reported in colorectal cancer.


To identify loci associated with BMI and T2D we undertook a genome-wide association study using 1,075,436 quality-controlled single nucleotide polymorphisms (SNPs) genotyped (Illumina 2.5M Duo Beadchip) in 402 individuals in extended pedigrees from a Western Australian Aboriginal community. Imputation using the thousand genomes (1000G) reference panel extended the analysis to 6,724,284 post quality-control autosomal SNPs. No associations achieved genome-wide significance, commonly accepted as P<5x10-8. Nevertheless, genes/pathways in common with other ethnicities were identified despite the arrival of Aboriginal people in Australia >45,000 years ago. The top hit  for BMI lies 5’ of NTRK2, the type 2 neurotrophic tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF) that regulates energy balance downstream of melanocortin-4 receptor (MC4R). PIK3C2G  was associated with BMI, but not with T2D as reported elsewhere. BMI also associated with CNTNAP2 , previously identified as the strongest gene-by-environment interaction for BMI in African-Americans. The top hit  for T2D lies 5’ of BCL9 that, along with TCF7L2, promotes beta-catenin’s transcriptional activity in the WNT signaling pathway. Additional hits occurred in genes affecting pancreatic  and/or GABA  functions. Notable associations observed for genes previously identified at genome-wide significance in other populations included MC4R (Pgenotyped = 4.49x10-4) for BMI and IGF2BP2 Pimputed_1000G = 2.55x10-6) for T2D. Our results may provide novel functional leads in understanding disease pathogenesis in this Australian Aboriginal population.A body mass index (BMI) >22kg/m Further in-house QC procedures at the level of individuals resulted in one individual being dropped due to a missing data rate >5%, 2 exclusions due to unintentional duplication, and no exclusions due to outlying heterozygosity (using 3 standard deviation limits), discordant sex, or divergent ancestry . This provided a post-QC dataset of 402 family members, including 361 with repeated body mass index measurements and 391  with information on doctor diagnosed T2D as per criteria outlined above. SNPs with minor allele frequency (MAF) <0.05, or with more than 5% missing data, were removed prior to association analysis (cf. below). As family data were employed in our study, complicated by a degree of over-relatedness in the pedigrees, a check of Hardy Weinberg Equilibrium (HWE) was not used as a SNP QC check since it was not clear that we should expect all SNPs to be in HWE. A subset of 70,420 genotyped SNPs with pairwise linkage disequilibrium  to lookr2) for each genotyped SNP. Directly genotyped SNPs were masked then imputed and concordance between the true genotype and the most likely imputed genotype [where probability prediction threshold > 0.9] for each SNP was calculated [r2 between the true (discrete) allele dosage  and the imputed (continuous) allelic dosages. For low frequency SNPs, r2 is the preferred accuracy measure because, unlike concordance, allele frequency is taken into account. We also compared these imputation accuracies for the 195 individuals determined by PCA to represent pure Martu ancestry against the 207 deemed to be of mixed ethnicity by PCA, using the data generated from imputation of the full dataset for the 402 individuals.Pre-phasing of genotyped data was performed using SHAPEIT , and implculated –26, as wP<5×10-8), or a maximum of 97% power at suggestive significance levels (P<1×10-5), for SNPs with MAF>0.3 conferring allelic effects (betas) of magnitude half a unit of standard deviation. For effects (betas) of magnitude 0.25 units of standard deviation, the maximum powers achievable are lowered to 1% and 9% respectively. The 89 T2D cases and 302 unaffected family members provide a maximum of 32% power to detect associations at genome wide significance levels (P<5×10-8), or 71% power at suggestive significance levels (P<1×10-5), for SNPs with MAF>0.3 conferring allelic odds ratios >2.5. For odds ratios of 1.5, the powers are lowered to 1.4% and 0.06% respectively.Heritability for mean BMI was determined from self-reported pedigree structures using the QTDT software package , and sep10P-values were graphed against their chromosomal location. Pairwise LD patterns between all regional SNPs and the top SNP were calculated specifically for this Australian Aboriginal study data using 146 unrelated individuals from the total sample of 402 genotyped individuals. The 146 unrelated individuals were selected by iterative removal of the individuals with the greatest number of estimated relationships with IBD>0.1875.Manhattan plots were generated using the mhtplot function of ‘gap’, a genetic analysis package for use in R (see URLs). Quantile-quantile (Q-Q) plots were generated, and inflation factors (denoted λ) calculated in R version 2.15.0 by dividing the median of the observed chi-squared statistics by the median of the theoretical chi-squared distribution. Regional plots of association were created using LocusZoom  in whichSLC28A3 to NTRK2 from human, cow and mouse was undertaken to locate evolutionarily conserved non-coding sequences (CNS) that might contain regulatory elements and transcription factor binding sites (TFBS). Genomic sequences for the three organisms were exported in FASTA format from ENSEMBL  and associated annotation exported in the form of a General Feature File (GFF) file. The global alignment tool Multi-LAGAN [de novo for CpG island-like elements, we firstly masked repeat regions using RepeatMasker (see URLs) following which putative CpG islands were searched for using CpGIsland Searcher [Global alignment of genomic sequence for the region ti-LAGAN , 37 was ti-LAGAN  was usedti-LAGAN , AliBabati-LAGAN , and Matti-LAGAN  with a mSearcher  with parThe 402 post-QC genotyped individuals used in the GWAS belonged to a small number of inter-related extended pedigrees, as depicted in the radial plot which shows hierarchical clustering of estimated pairwise identity-by-descent allele-sharing .. Princi2 is a significant risk factor for T2D in Australian Aboriginal populations [P = 10-9) than fitting a single curve with a sex-specific displacement. We constructed standardized residuals from these curves by subtracting the observed BMI value from the fitted curve and dividing by the estimated standard deviation . All GWAS analyses of BMI (cf. below) were carried out using these standardized residuals. The extreme female outlier was not used in fitting the female polynomial curve or in any of the GWAS analyses. This analysis of BMI shows (a) that the majority of the adult population have BMI >22 and are therefore at increased risk of diabetes; (b) a proportion of 15–20 year olds also fall into this category; and (c) there are many individuals in the community who can be classified as overweight (BMI 25.00–29.99) or obese  according to Center for Disease Control (CDC) [A BMI >22kg/mulations . Fig. 1 ulations  for all ol (CDC)  and Worlol (CDC)  internatol (CDC) , we estiol (CDC) –48. Estiol (CDC)  gave a lOur dataset for the study population  part B sLongitudinal BMI data available for the 361 post-QC individuals with both BMI measurements and genotyped SNP-chip data were representative of the full range of BMI values in the population  part B. P<5×10-8 [P = 0.05/1,075,436 or 4.65×10-8) used for this analysis of genotyped data. No specific SNPs reported to achieve genome-wide significance for association with BMI in other populations achieved P<10-2 in our study, as determined by interrogation of the NIH NHGRI Catalogue of GWAS studies [P<10-2 in our study population for the genes previously reported to achieve genome-wide significance (i.e. P<5×10-8) for association with BMI in other populations. Notably (cf. below), MC4R  was present in this list. Extending the table to include genes previously reported in GWAS for BMI at P<10-5 in other populations provided evidence for association at CNTNAP2 . SNPs at CNTNAP2 were also among the top 50 SNPs from the BMI analysis in our study population  for BMI in our study population lies in the intergenic region (NTRK2 encoding the type 2 neurotrophic tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF) that regulates energy balance downstream of melanocortin-4 receptor (MC4R). Both BDNF and MC4R have previously been shown to be associated with obesity in other populations (see RBM7 (rs6848632 Pgenotyped = 1.43×10-5) which has been related to pancreatic function [PIK3C2G (rs12816270 Pgenotyped = 8.06×10-6) which has previously been associated with T2D [A Manhattan plot showing the genome-wide results for BMI based on the genotyped data is presented in P<5×10-8  and conc studies . Neverthpulation . Data foions see . Other tions see  occurredfunction , and at with T2D .P<10-2 in our study, as determined by interrogation of the NIH NHGRI Catalogue of GWAS studies [P<10-2 in our study population for the genes previously reported to achieve genome-wide significance (i.e. P<5×10-8) for association with T2D in other populations. Five genes  had genotyped SNPs with P<10-3. Of note, no SNPs at P<10-2 were observed for TCF7L2 [KCNJ11 [Pgenotyped = 5.59×10-6) for T2D in our study population lies 5’ of BCL9 encoding a protein that, along with TCF7L2, promotes beta-catenin’s transcriptional activity in the WNT signaling pathway. Additional hits (1.07×10-4≤ Pgenotyped ≤4.55×10-5) of functional interest occurred in genes involved in pancreatic  and/or  [GABRR1 , KCNA1 [1 [KCNA1 ) functior2, for SNPs at different MAF. As expected, r2 is lower for SNPs with MAF 0.01–0.03 (mean r2 77.2–85.4) compared to SNPs with MAF 0.03–0.05 (mean r2 81.5–88.5%) and MAF 0.05–0.5 (mean r2 85.9–90.7%). When examined across individuals, mean imputation accuracy for the 195 individuals of pure Martu ancestry  were not different to measures for the 207 individuals of mixed ethnicity  or to measures across all 402 individuals .Recent reports have focused on improving tools for imputation of SNPs based on both the 1000 genomes (1000G) and HapMap project data , 65. It NTRK2  and at PIK3C2G  but not at other genes of functional interest (BCL9. Novel findings in the top 100 SNPs for 1000G imputed data included a hit at the previously identified gene for T2D IGF2BP2 (Pimputed_1000G = 2.55×10-6) that had not achieved P<10-2 in the genotyped data, and hits at Pimputed_1000G <10-6 in genes not previously associated genetically or functionally with T2D .In relation to the GWAS analyses for BMI , 1000G iinterest . Similarinterest , top SNPinterest  were gen10P-values for T2D adjusted for the standardized BMI residual were highly correlated with the original unadjusted analysis for both genotyped and imputed data, and (B) conversely,—log10P-values for unadjusted BMI and BMI adjusted for T2D were likewise highly correlated. This suggests that genetic effects on T2D as originally calculated are largely independent of any effects attributable to the mean standardized BMI residual. Specifically, the significance levels for genes of functional interest are of the same order of magnitude in both the adjusted  was initially performed without any covariate adjustment. Since T2D and BMI are highly correlated and have been shown to have shared genetic influences , the queadjusted  and the adjusted . Similaradjusted , S7 Fig.function , and PIKpulation .NTRK2 gene, with little evidence for linkage disequilibrium between SNPs in this peak of association and SNPs within the coding region of the gene. Conditioning on the top genotyped and top imputed SNPs  in relation to CNS upstream of NTRK2  was of equivalent significance to the top SNP rs1347857 (Pgenotyped = 1.5×10-6) and was unique in being the only top SNP located within a CNS peak, with the risk allele causing loss of a NHP1 binding site falling within a half-palindromic estrogen response element (TGAGTagtTGA/GCC) [As with most GWAS for common complex diseases , top SNPted SNPs . reducedof NTRK2 . These 82 2>0.8; . SNP rs1TGA/GCC) , 71. NTRTGA/GCC) . Other iTGA/GCC) , regulatTGA/GCC) . FurtherResults of the discovery GWAS undertaken here provide the first hypothesis-free insights into genetic risk factors for high BMI and T2D in an Australian Aboriginal population. Although estimates suggest the arrival of Aboriginal people in Australia more than 45,000 years ago , 67, we -2) was obtained for a number of genes previously shown to achieve genome wide significance in other populations . Recent [TCF7L2 . Whilst  [TCF7L2 , 80 for BCL9. BCL9 encodes a protein that, along with TCF7L2, promotes beta-catenin’s transcriptional activity in the WNT-signaling pathway [KCNJ6, KCNA1, and GABRR1. KCNJ6 lies adjacent to KCNJ15 on chromosome 21q22.13. KCNJ6 is an ATP-sensitive potassium channel that regulates insulin secretion in pancreatic beta cells in response to nutrients [GABRR1 is a member of the rho subunit family of GABA A receptors. Previous studies in China have demonstrated association between polymorphisms at GABRR1 and diabetic cataract [For T2D, the top association in our study population, supported by both genotyped and imputed data, was in the region of  pathway . WNT-sig pathway . Additioutrients . KCNJ15 utrients . KCNA1 iutrients . It has utrients , and mututrients . GABRR1 cataract . Recentlcataract . GABA-A cataract . Recent cataract .PIK3C2G, a gene previously associated with T2D and with serum insulin levels in Japan [PIK3C2G and hyperlipidemia and myocardial infarction [Amongst our top hits for BMI was in Japan . PIK3C2Gin Japan  related in Japan  of exprefarction .CNTNAP2 that encodes contactin associated protein-like 2 which localizes to the juxtaparanodal region of the nodes of Ranvier in myelinated axons, where it is required for proper localization of the potassium voltage-gated channel KCNA1 [Cntnap2 in mice impair Kv1.1 localization, and can be obesity-promoting or obesity-resistant in diet-induced obesity depending on genetic background [CNTNAP2 was among the strongest gene x environment interactions among African-Americans with BMI as outcome, specifically interacting with dietary energy intake [CNTNAP2 is amongst genes that lie within regions of de novo duplications and deletions recently linked to syndromic obesity in children [A second hit for BMI in our population was el KCNA1 . Mutatiockground . Polymory intake . CNTNAP2children .SLC28A3 and NTRK2 on chromosome 9q21.33. SLC28A3 is a concentrative nucleoside transporter with broad specificity for pyrimidine and purine nucleosides [TRKB or tyrosine kinase receptor B) is the receptor for BDNF and regulates energy balance downstream of MC4R. Associations between BMI and SNPs at both MC4R [BDNF [NTRK2 and BMI has only previously been observed (P = 1.04×10-6) in a gene-centric multi-ethnic meta-analysis of 108,912 individuals genotyped on the ITMAT-Broad-Candidate Gene Association Resource (CARe) array containing 49,320 SNPs across 2100 metabolic and cardiovascular-related loci [Bdnf mutants that express decreased amounts of Ntrk2 show hyperphagia and maturity-onset obesity [BDNF were significantly associated with more food servings in a study of obesity susceptibility loci and dietary intake [Perhaps the most convincing association observed in our study was between BMI and SNPs that lie in the intergenic region between leosides , and is oth MC4R –97 and B4R [BDNF , 99 have obesity , while ry intake . Deficiey intake  of largey intake , highligIn summary—we report here data for the first GWAS of complex disease in an Australian Aboriginal population. Whilst the top hits for BMI and T2D are in novel genes not yet reported for other GWAS, they occur in genes that belong to key pathways strongly supported by previous GWAS in other ethnicities. This means that current international efforts , 105 to The URLs for web resources presented herein are as follows:http://www.R-project.org/R version 2.15.0, http://www.mrc-epid.cam.ac.uk/Personal/jinghua.zhao/r-progs.htmThe mhtplot function of ‘gap’ for R, http://ucscbrowser.genenetwork.org/cgi-bin/hgGatewayUCSC genome-browser, http://www.genome.gov/encode/The Encode Project, http://www.repeatmasker.orgSmit AFA, Hubley R, Green P (1996–2010) RepeatMasker Open-3.0. S1 FigThe genomic kinship matrix was first calculated in GenABEL v1.7–6, and converted to a distance matrix and hierarchical cluster analysis using single linkage on the dissimilarities. The ape package was used to produce radial tree plot.(PDF)Click here for additional data file.S2 Figr2) ≤0.3 and MAF >0.01 was used in PCA (SMARTPCA within EIGENSOFT) to look at population substructure across the 402 genotyped family members. Plots (A) PC1 x PC2, (B) PC1xPC3, and (C) PC2xPC3 show individuals, color coded by age (see key). (D) shows the PC1 x PC2 plot in which individuals are color coded (see key) according to their T2D status. PCA plots (E) PC1 x PC2, (F) PC1xPC3, and (G) PC2xPC3 show individuals color coded by ancestry (see key). Note that the Martu clusters comprise 73 individuals aged < 20, 25 individuals aged 20–30, 57 individuals aged 30–50, and 40 individuals aged > 50. Hence there is no evidence for disproportionate representation in any age class. Reference HapMap populations are not included at the specific request of the Board of the Aboriginal Health Service.A subset of 70,420 genotyped SNPs with pairwise linkage disequilibrium Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(A) results for genotyped data; and (B) results for 1000G imputed data. SNPs in red show the region of the top association in this discovery GWAS.(PDF)Click here for additional data file.S7 FigSLC28A3 to NTRK2 on Chromosome 9; (B) CNTNAP2 on Chromosome 7; (C) RBM7 on Chromosome 4; and (D) PIK3C2G on Chromosome 12. In each plot the −log10 P-values are shown on the upper section, with SNPs colored (see key) based on their r2 with the labeled top hit SNP (purple), calculated in the 146 unrelated genotyped individuals. Red arrows highlight the position of the BMI hit on the T2D plot. The bottom section of each plot shows the genes marked as horizontal lines.(A) (PDF)Click here for additional data file.S8 FigAnalysis undertaken using an additive model in FaST-LMM.(PDF)Click here for additional data file.S9 FigP<5×10-6) genotyped  and 1000G imputed  SNPs, with the top imputed SNP (rs1140653), the top genotyped SNP (rs1347857) and the SNP of functional interest discussed in the main text (rs1866439) shown again on separate rows. These SNPs are shown relative to: (i) the CpG island-like motif identified using CpG Island Searcher; (ii) the various fragments of LINE-1 (L1) elements (with the L1MA3 element in which the top imputed SNP is located shown in blue); and (iii) peaks of histone methylation  or acetylation (H3K27Ac) measured in 7 cell lines from the ENCODE project.The plot shows positions of the top ((PDF)Click here for additional data file.S1 TableThe 391 post-QC genotyped individuals used in the GWAS belonged to a small number of interrelated extended pedigrees, as depicted in the radial plot of pairwise identity-by-descent allele-sharing presented in Characteristics of the 391 genotyped individuals used in the GWAS are provided here.(PDF)Click here for additional data file.S2 TableDICER (P <10-6).Bold indicates a SNP hits observed in the imputed but not the genotyped data, including at (PDF)Click here for additional data file.S3 TableResults for the top 50 hits for “BMI longitudinal” GWAS analysis in GenABEL using each individual BMI reading as a separate observation, modelling the correlation between readings via the estimated kinship and using the genomic control deflation factor to avoid inflation of the overall distribution of test statistics. Results are for allele-wise tests under an additive model of inheritance. Bold indicates SNP associations of functional interest presented in main (PDF)Click here for additional data file.S4 TableIGF2BP2 (P <10-5).Bold indicates SNP hits observed in the imputed but not the genotyped data, including at (PDF)Click here for additional data file.S5 TableResults are for allele-wise tests under an additive model of inheritance. Bold indicates SNP associations of functional interest presented in main (PDF)Click here for additional data file.S6 TableP <10-6) coincide with two top genes of functional interest  presented in main DICER a previously observed GWAS hit for BMI Click here for additional data file.S7 TableP<10-6) not observed in the genotyped data. Bold plus grey shading indicates hits that coincide with top gene of functional interest (BCL9) presented in main IGF2BP2 a previously observed GWAS hit for T2D Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Our results suggest that domestic Korean viruses were transferred directly from wild birds through at least two independent introductions. Our data did not indicate that wild birds carried poultry viruses between Korea and China, but rather, that wild-type H7 viruses were introduced several times into different poultry populations in eastern Asia.During surveillance programs in Korea between January 2006 and March 2011, 31 H7 avian influenza viruses were isolated from wild birds and domestic ducks and genetically characterized using large-scale sequence data. All Korean H7 viruses belonged to the Eurasian lineage, which showed substantial genetic diversity, in particular in the wild birds. The Korean H7 viruses from poultry were closely related to those of wild birds. Interestingly, two viruses originating in domestic ducks in our study had the same gene constellations in all segment genes as viruses originating in wild birds. The Korean H7 isolates contained avian-type receptors (Q226 and G228), no NA stalk deletion (positions 69–73), no C-terminal deletion (positions 218–230) in NS1, and no substitutions in PB2-627, PB1-368, and M2-31, compared with H7N9 viruses. In pathogenicity experiments, none of the Korean H7 isolates tested induced clinical signs in domestic ducks or mice. Furthermore, while they replicated poorly, with low titers (10  H7 influenza A virus (IAV) circulates continuously in wild birds worldwide In the spring of 2013, H7N9 IAVs causing human infections were first identified in China, and by 12 August 2013, this virus had infected 135 people in China and Taiwan, and killed 44 In South Korea, active surveillance has been applied to wild birds and domestic birds since the first H5N1 HPAI outbreak in 2003–2004. A variety of IAVs, including H5 and H7 low pathogenic avian influenza (LPAI) viruses, have been isolated in wild birds In this study, we characterized H7 IAVs (31 isolates) from wild birds and two domestic ducks in Korea between January 2006 and March 2011 through surveillance programs. We attempted to elucidate relationships between H7 viruses from wild birds and poultry from Korea and elsewhere in Asia and Europe by comparing their genetic characteristics. Focusing on the Eurasian-origin viruses, we conducted large-scale genetic analyses to elucidate the origin of the Korean H7 viruses in domestic ducks. In addition, we evaluated the pathogenic potential of these IAVs using animal experiments performed in domestic ducks and in mice.Through active surveillance programs in South Korea between January 2006 and March 2011, 31 H7 IAVs were isolated from duck farms  and more than 30 wild bird habitats  located mainly along the western and southern plain regions of South Korea where migratory birds aggregate. Although it cannot be established with certainty that the birds were migratory rather than resident, the fecal samples were collected from habitats that serve as overwintering locales for migratory wild birds arriving from northern Asia, including Russia and Mongolia, during September to the following March This study did not involve endangered or protected species; only fecal samples from migratory birds were collected. Each sample was suspended in an antibiotic phosphate buffered saline (PBS) solution and centrifuged at 3,500 rpm  for 5 min. The supernatants were inoculated into the allantoic cavities of 9- to 11-day-old embryonated hen eggs and incubated at 37°C for 4–5 days. After incubating the eggs, the allantoic fluid was harvested and centrifuged for purification. Virus presence was determined using a hemagglutination assay, and the detected viruses were subtyped by reverse transcription (RT)-PCR using influenza-specific primers http://www.barcodinglife.org).As fecal samples, rather than captured bird samples, were used for AI surveillance, AI prevalence or subtypes could not be correlated with bird species prior to 2007. However, after 2007, it was possible to identify host species, via the barcoding system, using mitochondrial DNA recovered from fecal samples Viral RNA was extracted from virus-containing allantoic fluid of embryonated eggs using the Viral Gene-spin viral DNA/RNA extraction kit . RT-PCR was performed using the QIAGEN OneStep RT-PCR Kit  with segment-specific primers This study was carried out in strict accordance with the recommendations in the Guidelines of the Institutional Animal Care and Use Committee of the Animal and Plant Quarantine Agency  in Korea . All mice autopsies were performed under anesthesia with Zoletil , and all efforts were made to minimize suffering. This study did not affect endangered or protected species because only fecal samples of migratory birds were collected.To assess pathogenicity in domestic ducks and mice, 2-week-old domestic ducks  and 6-week-old BALB/c mice  were inoculated intranasally with each virus. The following host-virus combinations were included in the experiments: A/duck/Korea/BC10/2007 (H7N3), A/duck/Korea/GJ56/2007 (H7N8), A/wild duck/Korea/MHC35-41/2011 (H7N9), A/wild duck/Korea/CSM27-12/2009 (H7N6), and A/common teal/Korea/MHC5-8/2009 (H7N7). Domestic ducks and mice were observed for clinical signs during the experiments. To determine virus shedding, OP and cloacal swab samples in domestic ducks were collected from each group of nine domestic ducks on days 1, 3, 5, 7, and 10 post-inoculation. To determine virus replication in mice, lung tissue samples were collected on days 1, 3, 5, 7, and 10 post-inoculation, from two euthanized mice per group. To elucidate patterns of virus replication in major tissues of the infected host, two domestic ducks per group were euthanized and tissue samples  were collected at 3 and 7 days post-inoculation (dpi). For virus isolation, each swab sample was suspended in 1 ml of sterilized PBS containing 1% gentamicin and each tissue sample was homogenized in PBS with antibiotics to a final concentration of 10% wt/vol. Samples were then centrifuged at 3,500 rpm for 5 min and each 0.1 ml supernatant was inoculated into the allantoic cavities of 9–11-day-old embryonated hen eggs, which were then incubated at 37°C for 4–5 days. Allantoic fluid from the incubated eggs was harvested and centrifuged for purification. Virus presence was determined by hemagglutination assay.r-value was subsequently calculated as described previously Antigenic analyses were performed by a hemagglutinin inhibition (HI) test using chicken antisera generated against the tested viruses, as described previously Anas platyrhynchos) (27.6%), common teal (Anas crecca) (3.4%), Northern shoveler (Anas clypeata) (3.4%), other wild duck (48.3%), bean goose  (3.4%), magpie (Pica pica serica) (3.4%), and unidentified species (10.3%). The NA genes had various subtypes: N7 (39.7%), N9 (35.5%), N3 (12.9%), N8 (6.5%), N2 (3.2%), and N6 (3.2%)  and H4  were the most abundantly detected HA subtypes, followed by H7 , H9 , H1 , H5 , H3 , H11 , H10 , H2 , H12 , and H8, H13, and mixed virus . Thirty-one H7 viruses were isolated from two domestic ducks (living on farms) without clinical signs and 29 fecal samples from various wild birds, including mallard (6 (3.2%) .The HA genes of H7 viruses can be divided into three main lineages, namely, the Eurasian, Australian, and American lineages , with thMost of the NA genes of the Korean H7 viruses belonged to the Eurasian lineage. However, one virus ) clustered with the American lineage in the N2 gene and fourteen viruses including one virus (A/duck/Korea/GJ56/2007 (H7N8)) in our study clustered with the American lineage in the N8 gene . Like thPhylogenetic analysis of internal genes indicated that the H7 viruses of the Eurasian lineage were composed of various sublineages, which showed genetic relationships between wild birds and poultry, dependent on regions and periods of isolation of the H7 viruses . The intTwo viruses originating in domestic ducks (A/duck/Korea/BC10/2007 and A/duck/Korea/GJ56/2007) in our study had the same gene constellations in all segment genes as viruses originating in wild birds. A/duck/Korea/BC10/2007 and A/duck/Korea/GJ56/2007 were detected from broiler ducks and domesticated mallard ducks of living on farms, respectively. A/duck/Korea/BC10/2007 was closely related to those from bean goose in 2008 (A/bean goose/Korea/SH20-17/2008(H7N3)) with 93.6–98.6%  and 96.8–99.5%  similarity at the nucleotide and amino acid level, respectively. A/duck/Korea/GJ56/2007 was closely related to those from mallard in 2007 ) with 99.7–100%  and 99.7–100%  similarity at the nucleotide and amino acid level, respectively.The Korean H7 isolates analyzed in this study had various motifs, PEIPKGR, PEPPKGR, PETPKGR, PEIPKRR, and PELPKGR, all with one or two amino acid R (arginine) residues at the HA cleavage site, which have been associated with low pathogenic effects in poultry. The H7N9 viruses of China also had the PEIPKGR motif . The Kor6.5 EID50/100 ul) and 6-week-old BALB/c mice (106.5 EID50/50 ul). None of the H7 IAVs tested induced clinical signs in inoculated domestic ducks or mice. In domestic ducks, all five viruses were recovered in OP swabs at low titers (10 0.7–1.3EID50/50 µl). In addition, most of the IAVs were rarely seen in cloacal swabs, up to 10 dpi. Likewise, no IAVs had replicated in any of the tissue samples tested . In the lung samples from mice, however, all five viruses had replicated well, up to 7–10 dpi (10 0.7–4.3EID50/50 µl)  were intranasally inoculated into 2-week-old domestic ducks (100/50 µl) . These rr-value 0.125–0.5), providing evidence of the antigenic similarity among the Korean H7 IAVs ”, and initiate official control measures We isolated 31 H7 subtypes during a nationwide surveillance program between January 2006 and March 2011. The major subtypes of the H7 IAVs were H7N7 (39.7%) and H7N9 (35.5%) in wild birds, similar to previous surveys of low pathogenic avian influenza viruses (LPAIVs) in European and Korean wild waterfowl Phylogenetic analysis of the HA genes of the Korean H7 viruses revealed a genetically diverse population that could be divided into three sublineages . The Korean H7 viruses from poultry belonged to Korea-I, which was highly related to those from wild birds in Korea. Interestingly, two viruses originating in domestic ducks (A/duck/Korea/BC10/2007 and A/duck/Korea/GJ56/2007) in Korea had the same gene constellations in all segment genes as viruses originating in wild birds (data not shown). These results suggest that the two domestic Korean viruses were transferred directly from wild birds through at least two independent introductions.The HA gene of the H7N9 viruses isolated from humans in China originated from poultry sold in live bird markets. These viruses were phylogenetically close to four H7N3 viruses (94.5–95.4%) isolated from ducks in Zhejiang in 2010–2012 . Before Phylogenetic analysis of internal genes revealed that all of the internal genes of H7 Korean viruses, except for the NS gene, showed genetic diversity, with two to four distinct sublineages that were related to the H7 or H9 viruses from various wild birds and poultry in Asia and Europe. The PB2, PA, and NP genes of some Korean H7 viruses clustered with those of the H7N9 or H9N2 viruses from humans and poultry in China in 2013, with 86.1–97.3%, 91.8–95.6%, and 90.0–94.8% similarity, respectively. However, all of the internal genes of H7N9 viruses shared the highest similarity with H9N2 viruses (96.3–99.2%) circulating in poultry in eastern China rather than Korean H7 viruses .Most of the NA genes of the Korean H7 viruses belonged to the Eurasian lineage, whereas one of the N2 viruses and some of the N8 viruses belonged to the American lineage . In our study, none of the H7 viruses tested induced clinical signs in domestic ducks. The H7 viruses showed no efficient replication in the ducks, according to the results from OP swabs (low titers) and cloacal swabs and major tissue samples  had the same gene constellations in all segment genes as viruses originating in wild birds, whereas the H7N9 viruses could have emerged through long-term maintenance of H7 and H9 viruses in poultry in China. This suggests that wild birds did not carry poultry viruses between Korea and China, but rather, that wild-type H7 viruses were independently introduced several times into poultry in eastern Asia, with no role whatsoever for wild bird migrations between Korea and China.Figure S1Phylogenies of seven surface genes: H7 (n = 541) (a), N9 (n = 179) (b), N7 (n = 148) (c), N3 (n = 273) (d), N8 (n = 194) (e), N2 (n = 191) (f), and N6 (n = 381) (g). Tip and branch colors represent host origin  of all of the Korean H7 viruses. Phylogenetic trees were constructed using the maximum likelihood method with general time-reversible model with invariant sites and 4 gamma-distributed heterogeneous substitution rates (GTR+ I + Γ4 model) and 100 bootstrap replications  in PhyML 3.0 (ZIP)Click here for additional data file.Figure S2Phylogenies of six internal genes: PB2 (n = 495) (a), PB1 (n = 509) (b), PA (n = 520) (c), NP (n = 472) (d), M (n = 520) (e), and NS (n = 545) (f). Tip and branch colors represent host origin  of all of the Korean H7 viruses. Phylogenetic trees were constructed using the maximum likelihood method with a general time-reversible model with invariant sites and 4 gamma-distributed heterogeneous substitution rates (GTR+ I + Γ4 model) and 100 bootstrap replications  in PhyML 3.0 . Statistical support for the phylogenies was assessed by the approximate likelihood test using a Shimodaira-Hasegawa-like procedure in PhyML 3.0. The topology of trees was visualized in FigTree 1.4.(ZIP)Click here for additional data file.


Mycobacterium tuberculosis has immunosuppressive properties, inducing a tolerogenic phenotype in dendritic cells and MDSCs, contributing to graft acceptance and tumor growth. However, its role in macrophage polarization remains to be elucidated. We asked whether DnaK was able to modulate macrophage phenotype. Murine macrophages, derived from bone marrow, or from the peritoneum, were incubated with DnaK and their phenotype compared to M1 or M2 polarized macrophages. Treatment with DnaK leads macrophages to present higher arginase I activity, IL-10 production and FIZZ1 and Ym1 expression. Furthermore, DnaK increased surface levels of CD206. Importantly, DnaK-treated macrophages were able to promote tumor growth in an allogeneic melanoma model. Our results suggest that DnaK polarizes macrophages to the M2-like phenotype and could constitute a virulence factor and is an important immunomodulator of macrophage responses.Macrophages are myeloid cells that play an essential role in inflammation and host defense, regulating immune responses and maintaining tissue homeostasis. Depending on the microenvironment, macrophages can polarize to two distinct phenotypes. The M1 phenotype is activated by IFN-γ and bacterial products, and displays an inflammatory profile, while M2 macrophages are activated by IL-4 and tend to be anti-inflammatory or immunosupressive. It was observed that DnaK from  Chil3l3) and FIZZ1  genes Macrophages are myeloid cells which have an important role during inflammation, infection resolution, tissue repair and cancer Mycobacterium tuberculosis can reprogram macrophages to M2 via secretion of IL-10 Both in infections and tumors, a switch from Th1 (or M1) to Th2 (or M2) immunity can occur, leading to the generation of a suppressive environment that abrogates effector immunity The chaperone DnaK is the major bacterial counterpart of heat shock protein 70 (HSP70) Mycobacterium tuberculosisRecently, DnaK was found in vesicles released by in vivo in an allogeneic murine melanoma model, enhancing tumor growth. Our results indicate that macrophages treated with DnaK become functional M2-like cells, with tumor promoting potential.In the present study we investigated whether mycobacterial DnaK polarizes murine macrophages. Macrophages treated with DnaK behaved like M2 macrophages. Furthermore, these cells presented M2 function To verify the effect of DnaK treatment on macrophages polarization, we treated macrophages differentiated from bone-marrow cells (BMMs) of B6 mice with different DnaK concentrations and compared iNOS and arginase activities between cells stimulated with LPS (M1), IL-4 (M2) or untreated cells. iNOS activity was induced in M1 macrophages but not in cells treated with DnaK (30 or 60 µg/mL) or in M2 . HoweverMurine polarized macrophages exhibit a distinct gene signature which can be used as polarization-associated markers To investigate the profile of cytokines released by the BMMs, we analyzed the production of TNF-α, MCP-1, IL-6, IL-10 and TGF-β upon stimulation with LPS, DnaK or IL-4 for 24 h. The treatment of BMMs with LPS led to an increased production of TNF-α when compared to control, IL-4 and DnaK treatments . ProductClassically activated macrophages (M1) express CD80 and CD86 on surface. CD206 - the mannose receptor - is a specific marker of alternatively activated macrophages (M2). To analyze the expression of these surface molecules, we stimulated BMMs with DnaK, LPS or IL-4 and analyzed them for the expression of CD206 and CD80 by flow cytometry. We also asked if DnaK could have modulatory effect in another macrophage population. To answer that, we treated peritoneal macrophages with DnaK and compared them with M1 and M2 macrophages. DnaK-treated peritoneal macrophages presented a lower percentage of CD80+ cells when compared control and M1 macrophages . Also, mb) in BALB/c mice (I-Ad) and followed tumor growth over several days.To test whether the M2-like macrophages generated by DnaK treatment were functional M2s, we tested their ability to promote tumor growth in an allogeneic murine melanoma model. We co-injected BMMs previously treated with DnaK or M1 macrophages, or untreated BMMs, with B16F10 (B16) cells . All mice were housed in individual and standard mini-isolators (Techniplast) in an SPF facility  with free access to water and food. Mice used in experiments have a range of weight between 18–22 g. The method of euthanasia used was a carbon dioxide (CO2) chamber (Beiramar). All procedures were performed in accordance with the guidelines of the Federation of Brazilian Societies for Experimental Biology and approved by the Ethics Committee for the Use of Animals of Pontifícia Universidade Católica do Rio Grande do Sul (CEUA-PUCRS) under protocol ID CEUA 12/00316.Mycobacterium tuberculosis was produced with the construct pET23a(+)/MtbDnaK in XL1-blue Escherichia coli and purified according to Mehlert Recombinant DnaK of 6) were cultured in 24-well plates in serum-free medium AIM-V (Gibco) with 10 ng/mL of GM-CSF (Peprotech). At day 3, medium was collected and cells were cultured for a further 3 days in the presence of fresh AIM-V with 10 ng/mL of GM-CSF. On the seventh day of culture, the non-adherents cells were separated from adherent cells (macrophages) and stimulated as described below. The purity in BMMs cultures was higher than 90% as assessed by staining with anti-F4/80 antibodies (data not shown).Macrophages were derived from bone marrow of C57BL/6 WT mice. Cells . The cells were washed twice with sterile PBS and suspended in AIM-V, transferred to a 24 multi-well plates and allowed to attach for 30 min. Unattached cells were washed out with medium. The adherent cells, mainly peritoneal macrophages, were used for the experiments thereafter. Macrophages were evaluated by microscopic examination with May-Grunwald and Giemsa stains, indicating macrophage purity higher than 80%. Purity was confirmed by flow cytometry, using the F4/80 Ab (data not shown).The obtained macrophages (from bone marrow or peritoneum) were stimulated for 24 h in serum-free AIM-V with 30 or 60 µg/mL of DnaK or left unstimulated. For the generation of classically or alternatively activated macrophages, cells were stimulated with LPS (30 ng/mL) or IL-4 (40 ng/mL) (both purchased from Peprotech) for 24 h, respectively.2 were added and the enzyme was heat-activated for 10 min at 56°C. Similar amounts of samples (40 µL) and 0.5 M L arginine (pH 9.7) were mixed and incubated for 1 h at 37°C. The reaction was stopped by adding 400 µL of H2SO4 (96%), H3PO4 (85%), H2O . The urea concentration was measured at 540 nm after the addition of 8 µL of α-isonitropropiophenone 6%, followed by heating at 95°C for 30 min. Values were compared with a standard curve of urea concentration.Arginase activity in cell lysates was measured based on the conversion of L-arginine to L-ornithine and urea according to the technique described by Corraliza and collaborators Nitrite concentrations were measured using the Greiss reaction The Fc receptors of macrophages were blocked with 24G2 supernatant containing 10% mouse serum and 10% rat serum, and later stained for F4/80 (BM8) from eBioscience; CD80 (16-10A1) from BD Biosciences; and CD206 (MR5D3) from AbDSerotec. Cells were analyzed using FACSCanto II (BD Biosciences) and BD FACSDiva software (BD Biosciences). Data obtained were analyzed using Flowjo software .b, CD86 and viability (Fixable Viability Dye from eBioscience).On Day 16 after the subcutaneous co-injection of polarized BMMs and B16 tumor cells, the mice were euthanized and tumors were excised and digested with collagenase D (Roche). The single cell suspension obtained was filtered to eliminate debri and stained for flow cytometry, using antibodies to F4/80, IATotal RNA was isolated from murine macrophage cultures using RNAeasy kit (Qiagen) according to manufacturer's instructions. The concentration of the purified total RNA samples was measured using a Qubit RNA Assay Kit (Invitrogen) and the Qubit Fluorometer (Invitrogen). We added 50 ng of RNA each cDNA synthesis reaction using the SuperScript-III RT pre-amplification system . cDNA concentrations were measured using Qubit dsDNA HS Assay Kit (Invitrogen) and the Qubit Fluorometer (Invitrogen).Real time PCR was carried out StepOne Real-Time PCR System (Applied Biosystems) using Platinum SYBR Green qPCRSuperMix-UDG (Invitrogen) following the manufacturer's instructions. The thermal cycling conditions included an initial denaturation for 2 min at 95°C and 40 cycles consisting of a denaturation step at 95°C for 15 s, an annealing step at 60°C for 30 s and an extension step for 1 min at 70°C. Samples was analyzed in triplicates. The relative mRNA levels were calculated using the comparative Ct method TCCTGGCTCACACTGAATTC and R: CTGCGTGCATAAATTGTATAGCA; for Fizz1 (Retnla) F: TCCCAGTGAATACTGATGAGA and R: CACTCTGGATCTCCCAAGA; and for Ym1 (Chi3l3) F: GGGCATACCTTTATCCTGAG and R: CCACTGAAGTCATCCATGTCPrimers sequences we used for β-microglobulin were F: Supernatants of cell cultures were analyzed for the presence of TNF, IL-10, MCP-1, IL-6, IL-12p70 and IFN-γ with the CBA Mouse Inflammation kit (BD Biosciences), according to manufacturer's instructions. Samples were analyzed using FACSCanto II (BD Biosciences) and BD FACSDiva software (BD Biosciences). Data obtained were analyzed using FCAP Array software  and expressed in pg/ml. TGF-β measurements were made using a human/mouse TGF-β1 (2nd Gen) ELISA Ready-SET-Go! kit (eBioscience), according to the manufacturer's instructions.2 atmosphere.The murine melanoma cell line B16F10 (ATTC CRL-6475) was cultured with DMEM media (Cultilab) supplemented with 10% of fetal calf serum (FCS) (Cultilab), 1× essentials amino acids (Gibco), 1× vitamins (Gibco) and 55 µM of β-mercaptoethanol at 37°C with 5% of CO5) were co-injected with 104 of BMMs treated with LPS or DnaK as previously described in 100 µL of serum-free RPMI. Injections were performed subcutaneously in the thigh of male BALB/c or C57BL/6 mice, after anesthesia with 83 mg/kg of ketamine and 17 mg/kg of xylazine. Mice were photographed and tumor growth was evaluated using a digital caliper (Mitutoyo) in days 8, 10, 12, 14 and 16 post tumor injections. We used a modified ellipsoid formula 0.52 (Length×Width2) to access the tumor volume B16F10 cells . The one-way ANOVA test was used to determine differences between groups. Multiple comparisons among levels were checked with Tukey post hoc test. Differences between specific points were determined by a t test. The level of significance was set at p<0.05.


IL23R) and autophagy-related 16-like 1 (ATG16L1) genes have been associated with the disease. Our objective was to assess the frequency of ATG16L1 (T300A) and IL23R (L310P) variants in Moroccan IBD (Crohn’s disease and Ulcerative Colitis) patients and to evaluate a possible effect of these variants on disease’s phenotype and clinical course.Inflammatory bowel diseases (IBD) are chronic diseases of the gastrointestinal tract. Although their pathogenesis is unclear, the combination of genetic predisposition and environmental components are believed to be the main cause of these diseases. Recently, many variants in interleukin 23 receptor (ATG16L1 (T300A) and IL23R (L310P) variants by PCR-restriction fragment length polymorphism.96 Moroccan IBD patients and 114 unrelated volunteers were genotyped for ATG16L1 (T300A) and IL23R (L310P) variants in a Moroccan group. We found that IL23R (L310P) variant conferred a protective effect for crohn’s disease (CD) but not ulcerative colitis (UC) patients. The presence of ATG16L1 (T300A) mutated alleles was associated with CD type but not with disease onset. In addition, the carriage of T300A variant alleles conferred a protective effect in UC.This is the first report on the prevalence of ATG16L1 and IL23R variants was not significantly different between patients and controls. However a possible role of ATG16L1 (T300A) on CD phenotype was suggested.Our results showed that the prevalence of The online version of this article (doi:10.1186/1756-0500-7-570) contains supplementary material, which is available to authorized users. Inflammatory bowel disease (IBD) is a chronic and multifactorial disease of the gastrointestinal tract. It includes Crohn’s disease (CD), ulcerative colitis (UC) and undetermined colitis. Their etiologies remain complex and unclear involving an inadequately defined relationship between microbial insult, genetic predisposition and altered intestinal barrier permeability . SeveralIL23R) [ATG16L1) [Genetic variations in genes related to innate and adaptive immunity have been implicated in IBD pathogenesis. Positive correlations were reported for Interleukin 23 receptor (ATG16L1)  genes.23R gene with chronic inflammatory diseases especially IBD (CD and UC). The variant L310P of IL23R gene (more frequent in controls) was reported to confer a strong protection against CD [IL-23 is a heterodimeric cytokine produced by activated macrophages and dendritic cells. It consists of two subunits, a p40 subunit, shared with the IL-12, and a specific IL-23 subunit called p19 , 6. StudATG16L1 and IBD in various populations. The ATG16L1 gene plays a key role in autophagy pathways. It encodes a protein widely expressed in intestinal epithelial cells, lymphocytes and macrophages and mediates resistance to intracellular pathogens such as bacteria and viral particles [Several studies have established an association between articles . Hampe aarticles . SubsequATG16L1 and IL23R variants in the Moroccan population. Hence, we aimed to examine the association between IL23R (L310P) and ATG16L1 (T300A) polymorphisms and inflammatory bowel disease (Crohn’s disease and Ulcerative colitis) in a cohort of Moroccan patients.No data were available on the frequency of the In this study, a group of 96 Moroccan unrelated IBD patients were recruited at the gastroenterology department of Averroes Hospital, Casablanca, Morocco. The control group included 114 unrelated Moroccan volunteers (blood donors) with no discernable symptoms suggestive of IBD. The diagnosis of CD or UC was based on established clinical, radiological, endoscopic, and histopathology criteria.Demographic and clinical characteristics were obtained from the participants through a detailed questionnaire. CD phenotype was stratified by age at diagnosis, location and disease’s behaviour according to the Montreal classification .For UC patients, anatomic location was subgrouped using the Paris classification as being ulcerative proctitis (E1), left-sided UC (E2), and extensive UC (E3) .Differences in the frequency of disease characteristics such as age at diagnosis, gender, extra-intestinal manifestations, similar familial cases, and antecedents like appendectomy and smoking were also assessed. The study was approved by the medical school of Casablanca ethical committee. A written informed consent was obtained from all participants or their guardians. Both IBD patients and control group are originated from the different regions of Morocco and confirmed the Moroccan origin of their parents and grandparents.IL23R and ATG16L1 variants genotyping was performed using polymerase chain reaction (PCR) restriction fragment length polymorphism analysis (RFLP) as described respectively by Lin et al. and Csöngei et al. [Genomic DNA was isolated from whole blood samples by salting-out method . DNA amoi et al. , 14.Reactions were performed in a final volume of 25 μl. PCR products were cleaved with Hph I (L310P) and Lwe I (T300A)  and electrophored on a 3% agarose gel in the presence of a molecular weight marker ladder 100 . After staining with ethidium bromide, Ultraviolet was used on a transilluminator for reading the gel.IL23R (L301P) ATG16L1 (T300A) genotypes was determined by Fisher's exact test  at 95%). The χ2 test or Fisher test was used to correlate the IL23R and ATG16L1 polymorphisms and clinical parameters. The P value (<0.05) was considered statistically significant in all variables.Statistical analysis was performed using MedCalc statistical software version 11.6. The Hardy-Weinberg equilibrium test was performed separately for patients and controls to measure the distribution of polymorphisms. The association between IBD (CD and UC) and ATG16L1 (T300A) and IL23R (L310P) along with 69 Crohn’s disease patients (25 women and 44 men) and 30 UC patients (14 women and 16 men). The average age of diagnosis was 24.17 ± 2.48 for CD patients and 35.37 ± 5 for UC patients. For control group, epidemiological and clinical data are shown in Additional file One hundred fourteen participants from the general population were genotyped for ATG16L1) and L310P (IL23R) in CD patients and controls respectively.Statistical analysis of the distribution of SNPs studied showed that allele frequencies were conformed to Hardy–Weinberg expectations   for T300A (ATG16L1 and IL23R genotypes (Tables ATG16L1 polymorphism (T300A) with P = 0.03 ; 1.22   and 0.74  , located on the 6) Table . In addiATG16L1 (T300A) and IL23R (L310P) polymorphisms with UC. Analysis of distribution of the two polymorphisms showed that allele frequencies were in Hardy-Weinberg equilibrium  for ATG16L1 and IL23R .Additionally, our study assessed the association of For both polymorphisms, no genotype-phenotype correlation was observed in UC Tables  and 6.TaThe genotypic and allelic frequencies did not significantly differ between UC patients and healthy controls for the two polymorphisms Tables  and 8.TaATG16L1 gene have a protective effect for UC, with an odds ratio of 0.90    variant and CD [During a genome-wide survey of 19 779 non-synonymous single nucleotide polymorphisms, the Thr300Ala) variant, located at the N terminus of the WD-repeat domain in ATG16L1, was found to be highly associated with CD by using a haplotype and regression analysis [00Ala vart and CD , 18. Thit and CD .ATG16L1 (T300A) genetic variant with CD and UC in Moroccan patients and controls. Upon association analysis, we were not able to establish a significant effect on CD risk in Moroccan IBD cohort. Our result was in concordance with the lack of association reported in a replication study performed in Japan [ATG16L1 variant is associated with susceptibility to adult CD, but not with early-onset disease in a Scottish cohort.In the present study, we examined the association of in Japan . In addiin Japan  observedATG16L1 T300A variant may confers risk or protection from infection, depending on the nature of the pathogen and the typical duration of infection. The cellular expression of ATG16L1 facilitates bacterial invasion, however the IBD-associated ATG16L1 T300A variant may be protective against bacterial infection.Regarding UC, a protective effect of this polymorphism was identified. At present, it can only be speculated how ATG16L1 T300A variant show protection against invasion by Salmonella [Messer et al. demonstrated that Intestinal epithelial cells somatically targeted to express the lmonella .IL23R gene is another potential candidate gene for CD risk [IL23R gene with CD and UC [The  CD risk . IL-23R  CD risk . The IL- CD risk –39. SimiD and UC , 40–43.IL23R gene variants have a differential effect on Th17 cells with increased Th17 cytokine secretion in patients with CD-associated IL23R variants and decreased cytokine secretion in patients with CD-protective IL23R variants [It was hypothesized that variants .IL23R gene was also shared by UC patients and did not detect a significant association. Our subgroup analyses are likely underpowered for revealing a genotype– phenotype relationship. This result confirms previous studies on Italian [In the present study, carriage of the variant allele was associated with a protective effect for CD patients, similarly to previously reported studies , 46. We  Italian  and Nort Italian .ATG16L1 plays an important role in CD behaviour and confers protection for UC. In addition, IL23R gene showed a protective effect for individuals with the TT genotype and T allele against the development of Crohn’s disease.In summary, the present study seems to indicate that ATG16L1 and IL23R in the pathogenesis of IBD in patients of different ethnic origin, with a limited role in the Moroccan population. Due to small sample size, an association cannot be ruled out. Further studies in larger groups would be required to confirm these findings.Therefore, our results could reinforce the notion of a different relevance of Additional file 1: Table S1: Clinical and epidemiological parameters of control group. (DOCX 11 KB)


S100a8 and S100a9 messenger RNA expression through an extracellular signal-related kinase-dependent mechanism. Suppression of S100A8 and S100A9 in cancer cells using short hairpin RNA significantly diminished migration and invasion in culture. Downregulation of S100A8 and S100A9 had no effect on subcutaneous tumor growth. However, colony size was greatly reduced in liver metastases with decreased invasion into adjacent tissue. In tissue culture and in the liver colonies derived from cancer cells with knockdown of S100A8 and S100A9, MMP2 and MMP9 expression was decreased, consistent with the reduction in migration and invasion. Our findings demonstrate that monocytes/macrophages in the metastatic liver microenvironment induce S100A8 and S100A9 in cancer cells, and that these proteins are essential for tumor cell migration and invasion. S100A8 and S100A9, however, are not responsible for stimulation of proliferation. This study implicates S100A8 and S100A9 as important mediators of tumor cell aggressiveness, and highlights the therapeutic potential of S100A8 and S100A9 for interference of metastasis.Myeloid cells promote the development of distant metastases, but little is known about the molecular mechanisms underlying this process. Here we have begun to uncover the effects of myeloid cells on cancer cells in a mouse model of liver metastasis. Monocytes/macrophages, but not granulocytes, isolated from experimental liver metastases stimulated migration and invasion of MC38 colon and Lewis lung carcinoma cells. In response to conditioned media from tumor-infiltrating monocytes/macrophages, cancer cells upregulated  These myeloid cells are highly heterogeneous with cells of both the monocytic and granulocytic lineages, and have considerable phenotypic plasticity with both positive and negative effects on tumor growth and metastasis.9 Myeloid cells have been shown to affect all of these steps.Distant metastasis remains the main cause of cancer-related death. During the early stages of metastasis, tumor cells acquire migratory and invasive characteristics, allowing movement into surrounding extracellular matrix and tissues, intravasation into blood vessels, and dissemination via the circulation. Following extravasation into target tissues, tumor cells initiate metastatic colonization, in part by evading tumor surveillance and instigating an angiogenic response.+ cells comprising granulocytes and monocytes/macrophages. Depletion of CD11b+ cells led to markedly reduced colony growth. To begin to understand how these effects were mediated, we isolated cancer cells after removal of the CD11b+ myeloid cells. Angiopoietin-like 7 (ANGPTL7) expression was greatly reduced in the cancer cells. Enforced overexpression of ANGPTL7 inhibited growth of liver metastases and subcutaneous tumors. In the same study, we also found that S100A8 and S100A9 expression in cancer cells was altered by removal of the CD11b+ cells.10We previously examined the effects of infiltrating myeloid cells on experimental liver metastases generated by intrasplenic inoculation of MC38 colon and Lewis lung carcinoma (LLC) cells. These metastatic colonies were infiltrated by CD11b11 although some functions are independent of heterocomplex formation.12 These proteins stimulate chemotaxis, cell migration and adhesion,13 but also have anti-inflammatory roles in oxidant scavenging, tissue repair and resolution of inflammation.14 The effects of S100A8 and S100A9 are dependent on concentration, post-translational modifications,16 oligomeric states and/or the microenvironment.12Here we explored the significance of S100A8 and S100A9 induction by the myeloid cells in the tumor microenvironment. S100A8 and S100A9 are calcium-binding proteins that form homo- and heterocomplexes (S100A8/A9) that are important for their biological activity,18 In colorectal cancers, increased S100A8 and S100A9 expression correlated with differentiation, Dukes stage and lymph node metastasis.19 Similarly, in prostate cancer, S100A8 and S100A9 were expressed at increased levels in high-grade adenocarcinomas compared with benign tissues.20 S100A8 and S100A9 expression in breast cancer correlated with HER2 expression and lymph node metastasis.21 These studies indicate that S100A8 and S100A9 levels are elevated in cancer tissues compared with normal and benign tissues, and their increased expression is associated with tumor aggressiveness and metastasis.S100A8 and S100A9 are expressed to a greater extent in colorectal, prostate and breast cancers.23 and myeloid-derived suppressor cells24 to promote pre-metastatic niche formation, tumor growth and metastasis.25 S100A8 and S100A9 are also expressed by tumor cells,26 and although there have been many studies detailing the functions of stromal-derived S100A8 and S100A9, little is known about the effects of tumor-derived S100A8 and S100A9. In this study, we report that monocytes/macrophages induce S100a8 and S100a9 messenger RNA (mRNA) expression in cancer cells in an extracellular signal-related kinase (ERK)-dependent manner. S100A8 and S100A9 expression in cancer cells was critical for invasion by liver metastases. These findings detail a novel molecular mechanism through which tumor-derived expression of S100A8 and S100A9, regulated by infiltrating monocytes/macrophages, dictates a more aggressive phenotype.In the published literature, S100A8 and S100A9 are reported as predominantly expressed within tumors by immune cells, and their expression can stimulate the recruitment of myeloid+ myeloid cells that could be divided into three distinct groups based on their Gr1 expression . The Gr1high cells were predominantly granulocytes, whereas the Gr1mid and Gr1low cells were monocytes/macrophages.27 The latter two groups were interrelated as Gr1mid cells could give rise to Gr1low cells.10 Gr1high granulocytes and Gr1mid/low monocytes/macrophages were isolated from liver metastases by fluorescence-activated cell sorting (FACS), and after 3 days of culture, conditioned media were collected and added to cultures of MC38 and LLC cancer cells. The conditioned medium from tumor-infiltrating monocytes/macrophages significantly increased proliferation of MC38 and LLC cells. Conditioned medium from the granulocytic population increased MC38 cell proliferation to a lesser extent, and had no effect on LLC cell proliferation  in tumor-infiltrating monocyte/macrophage-conditioned media compared with the conditioned media from naive monocytes/macrophages , suggestLC cells . BlockadLC cells .To assess the changes in the cancer cells resulting from exposure to the conditioned media, we examined the signaling pathways using the PathScan Intracellular Signaling Array Kit . Conditioned medium from monocytes/macrophages provoked a pronounced increase in ERK1/2 phosphorylation (Thr202/Tyr204) compared with controls. Granulocyte-conditioned medium activated ERK1/2, but to a lesser extent. Changes in phosphorylation levels of other signaling pathway indicators on the array were not apparent .28 and is activated by TNFα (100 ng/ml) in MC38 and LLC cells  in MC38 and LLC cells suppressed tumor cell proliferation in response to monocyte/macrophage- and granulocyte-conditioned media . Transfection of MC38 and LLC cells with S100A8-shRNA decreased basal ectively . S100A9-LC cells . S100A8 blotting .10 and in keeping with this, S100A8 or S100A9 knockdown did not alter ANGPTL7 mRNA levels  are known to have key roles in tumor cell migration and invasion.er cells . Reductiin vivo, we injected S100A8-shRNA-, S100A9-shRNA- and scrambled control-shRNA-transfected MC38 and LLC cells subcutaneously into syngeneic mice. Subcutaneous tumor growth was not affected, consistent with the absence of the effects on proliferation in tissue culture  and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For all cell lines, mycoplasma testing using the MycoAlert Mycoplasma Detection kit  was performed every 3 months, and cell line DNA typing  was performed in October 2014. Cells were regularly assessed based on their morphology before use. Monocytes/macrophages (CD11b+/Gr1mid/low) and granulocytes (CD11b+/Gr1high) were isolated from single-cell suspensions of tumor-bearing livers by FACS based on CD11b and Gr1 expression. These cells have been previously characterized based on their morphology, and Ly6C, Ly6G and CD11c expression.27 Isolated myeloid cells were cultured in 2% fetal bovine serum in Roswell Park Memorial Institute  media for 72 h before conditioned media was collected. MC38 or LLC cells (1 × 105 cells per well) were co-cultured for 8 h in the myeloid cell-conditioned media or with 100 ng/ml recombinant mouse TNFα  in 12-well plates before gene analysis. The ERK inhibitor U0126  was added to the cell culture in some instances. To generate stable knockdown of S100A8 and S100A9, MC38 and LLC cells were transfected with lentivirus-mediated shRNA targeting S100A8 or S100A9 or the scrambled shRNA control . Transfected cells were selected with 2.5 μg/ml puromycin.MC38 and LLC cells stably expressing green fluorescent protein (GFP) were generated and cultured as described.5 in 100 μl phosphate-buffered saline) were injected into the spleen of anesthetized female C57BL/6 mice, the spleen was removed after 1 min and mice were killed 2–3 weeks after tumor cell injection. Subcutaneous tumors formed after injection of tumor cells  were injected subcutaneously into the left flank of anesthetized C57BL/6 mice. Volumes were determined by caliper measurement of the length, width and height of each tumor. For all animal work, at least five or more mice were randomized and included in each experimental group, and all animals used were included in the analysis. Animal studies were not blinded during data analysis.Animal procedures were carried out in accordance with the UK Animal (Scientific Procedures) Act 1986 and followed local ethics review. Female C57BL/6 mice at 6–8 weeks of age were purchased from Charles River Laboratories . To develop liver metastasis, tumor cells . Antibodies used in FACS included rat anti-mouse CD11b PE-Cy7 , rat anti-mouse Gr1 PE  and isotype controls rat IgG2a PE and rat IgG2b PE-Cy7 (eBioscience). Flow cytometry was performed using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo software version 7.2.5 . Myeloid cells were sorted into granulocyte (CD11b+/Gr1high) or monocyte/macrophage (CD11b+/Gr1mid/low) subsets using a MoFlo XDP High-Speed Cell Sorter  by the antibodies described above.Single-cell suspensions were prepared from tumor-bearing livers as previously described.Cells were lyzed in radioimmunoprecipitation assay buffer and lysates were assayed by western blot analysis for the expression of S100A8 , S100A9 , MMP2  and MMP9 , ERK1/2  or phosphorylated ERK1/2  using the indicated antibodies.44Mouse livers were snap-frozen, sectioned (10–12 μm) and fixed in acetone, before blocking with serum and incubating with primary antibodies at 4 °C overnight. Primary antibodies included anti-mouse Ki67 , anti-mouse CD31 , anti-mouse MMP2  and anti-mouse MMP  or isotype rabbit and rat IgG controls . Fluorescence-conjugated secondary antibodies  were added and tissues were mounted with Vectashield containing 4',6-diamidino-2-phenylindole (Vector Laboratories). Apoptotic cells were stained using the ApopTag Fluorescein In Situ Apoptosis Detection Kit according to the manufacturer's instruction . Images were acquired using an inverted epifluorescence microscope  with a digital camera  or an inverted confocal microscope  and processed using ImageJ , Adobe Photoshop CS4  or ImageScope Version 12.1.0.5029 . Tissue sections were also fixed in acetone before staining with hematoxylin and eosin according to the standard protocol. Invasion index was determined by the ratio of the invasive area to total tumor area, as calculated as 1− from hematoxylin- and eosin-stained images as previously described.3 cells per well in 96-well plates in control or myeloid cell-conditioned media. Cell proliferation was measured according to the manufacturer's instruction  at the indicated times using the water-soluble tetrazolium (WST)-1 reagent (10 μl added to each well at end point). Cell migration and invasion were assayed using the CytoSelect 96-well Cell Migration and Invasion Assay according to the manufacturer's instruction . In brief, MC38 and LLC cells expressing GFP were resuspended in serum-free RPMI containing 0.1% bovine serum albumin (BSA). For the migration assay, granulocyte- or monocyte/macrophage-conditioned media or RPMI media containing 2% fetal bovine serum (control media) was added to the bottom chamber (100 μl per well). Tumor cell suspensions (5 × 105 cells per ml) in serum-free RPMI containing 0.1% BSA were added to the top insert and cells were incubated at 37 °C for 8 h before assessing the number of tumor cells in the bottom chamber using GFP fluorescence. For the invasion assays, tumor cells (5 × 105 cells per ml), alone or mixed with isolated granulocytes or monocytes/macrophages (1 × 105 cells per ml), were added to the top chamber in serum-free RPMI containing 0.1% BSA, whereas RPMI media containing 2% fetal bovine serum was added to the bottom chamber. Cells were incubated at 37 °C for 8 h before assessing the number of tumor cells in the bottom chamber using GFP fluorescence.Cells were cultured in serum-free RPMI for 24 h before seeding at 1 × 104 MC38 and LLC control, S100A8 or S100A9 knockdown cells were plated in 96-well plates and incubated with control or monocyte/macrophage-conditioned media for 8 h. Ionomycin  was added as a positive control. Media was removed and cells incubated with dye loading solution according to the manufacturer's protocol. Plates were read on a fluorescent plate reader at 494/516 nm.Intracellular calcium levels were measured using the FLUO-4 NW Calcium Assay kit . In brief, 3 × 10MC38 or LLC cells were lyzed 24 h after culture in granulocyte- or monocyte/macrophage-conditioned media. Cell lysates were assayed using the PathScan Intracellular Signaling Array Kit according to the manufacturer's protocol . Densitometric measurements were made using ImageJ . MC38 and LLC cells were cultured alone or with 100 ng/ml recombinant mouse TNFα (Invitrogen), lyzed and assayed for total ERK using MEK1/2 antibodies  and for phosphorylated ERK using p44/42 MAPK antibodies .Expression of TNFα in conditioned media was assayed using the TNF alpha mouse enzyme-linked immunosorbent assay kit (Abcam). TNFα activity was neutralized in the myeloid cell-conditioned media using 50 μg/ml mouse TNF-α antibody .RNA was isolated with Trizol (Invitrogen) according to the manufacturer's instructions, and complementary DNA  was synthesized using the SuperScript VILO cDNA synthesis kit (Invitrogen) or the Tetro cDNA synthesis kit . Real-time PCR reaction mixtures were prepared with the Platinum SYBR Green qPCR Supermix-UDG (Invitrogen) and performed on a Stratagene MX3005P thermocycler . Data were analyzed using the ΔCT method and normalized to hypoxanthine–guanine phosphoribosyl transferase (HPRT). Primers used included mouse S100A8 , mouse S100A9 , human S100A9 , mouse HPRT  and human HPRT .t-tests, or one-way analysis of variance with Bonferroni post test for multiple comparisons. A P-value of<0.05 was considered significant.Statistical analyses were performed using GraphPad Prism . For all experiments, data were expressed as mean±s.e.m. and analyzed by unpaired two-sided Student's