Association between C10X polymorphism in the CARD8 gene and inflammatory markers in young healthy individuals in the LBA study.

BACKGROUND: The Caspase activation and recruitment domain 8 (CARD8) protein is a component of innate immunity as a negative regulator of NF- ĸB, and has been associated with regulation of proteins involved in inflammation. Expression of CARD8 mRNA and protein has been identified in human atherosclerotic lesions, and the truncated T30A variant (rs2043211) of CARD8 has been associated with lower C-reactive (CRP) and MCP-1 levels in myocardial infarction patients. The present study examines the role of a genetic variation in the CARD8 gene in relation to a selection of markers of inflammation. METHODS: In a cross-sectional study of young healthy individuals (18.0-25.9 yrs, n = 744) the association between the rs2043211 variant in the CARD8 gene and protein markers of inflammation was assessed. Genotyping of the CARD8 C10X (rs2043211) polymorphism was performed with TaqMan real time PCR on DNA from blood samples. Protein levels were studied via Olink inflammation panel ( https://olink.com/ ). Using linear models, we analyzed men and two groups of women with and without estrogen containing contraceptives separately, due to previous findings indicating differences between estrogen users and non-estrogen using women. Genotypes were analyzed by additive, recessive and dominant models. RESULTS: The minor (A) allele of the rs2043211 polymorphism in the CARD8 gene was associated with lower levels of CCL20 and IL-6 in men (CCL20, Additive model: p = 0.023; Dominant model: p = 0.016. IL-6, Additive model: p = 0.042; Dominant model: p = 0.039). The associations remained significant also after adjustment for age and potential intermediate variables. CONCLUSIONS: Our data indicate that CARD8 may be involved in the regulation of CCL20 and IL-6 in men. No such association was observed in women. These findings strengthen and support previous in vitro data on IL-6 and CCL20 and highlight the importance of CARD8 as a factor in the regulation of inflammatory proteins. The reason to the difference between sexes is however not clear, and the influence of estrogen as a possible factor important for the inflammatory response needs to be further explored.

CRP levels are significantly associated with CRP genotype and estrogen use in The Lifestyle, Biomarker and Atherosclerosis (LBA) study.

BACKGROUND: The C-reactive protein (CRP) is an important biomarker for atherosclerosis and single nucleotide polymorphisms (SNPs) in the CRP locus have been associated with altered CRP levels and associated with risk for cardiovascular disease. However, the association between genetic variations in the CRP gene, estrogen use and CRP levels or early signs of atherosclerosis in young healthy individuals is not fully characterized. We aimed to evaluate the influence of five genetic variants on both plasma CRP levels and carotid intima-media thickness (cIMT) values, including aspects on estrogen containing contraceptive use in females. METHODS: Genotyping was performed with TaqMan real time PCR and compared with high sensitivity CRP serum levels in 780 Swedish young, self-reported healthy individuals. Haplotypes of the SNPs were estimated with the PHASE v 2.1. The cIMT was measured by 12 MHz ultrasound. The contraceptive use was self-reported. RESULTS: Strong associations between CRP and genotype were observed for rs3091244, rs1800947, rs1130864, and rs1205 in women (all p < 0.001). In men, only rs1800947 was associated with CRP (p = 0.029). The independent effect of genotypes on CRP remained significant also after adjustment for established risk factors. Female carriers of the H1/ATGTG haplotype had higher CRP than non-carriers. This was specifically pronounced in the estrogen-using group (p < 0.001), and they had also higher cIMT (p = 0.002) than non-carriers but with a small cIMT difference between the haplotype groups (0.02 mm). In parallel, a significant correlation between CRP and cIMT in the estrogen using group was observed (r = 0.194; p = 0.026). CONCLUSIONS: Estrogen use, genotypes and haplotypes in the CRP locus are significantly associated with CRP levels. Based on an observed interaction effect between sex/estrogen use and the H1/ATGTG haplotype on CRP, and a marginally thicker cIMT in the estrogen using group, our data suggest that both genotypes and estrogen usage could be involved in arterial wall structural differences. The causality between CRP levels and cIMT remains unclear, and the observed difference in cIMT is not clinically relevant in the present state. Future larger and longitudinal studies may shed further light on the role of more long-term estrogen use and early atherosclerosis.

Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.

Crohn's disease and ulcerative colitis, the two common forms of inflammatory bowel disease (IBD), affect over 2.5 million people of European ancestry, with rising prevalence in other populations. Genome-wide association studies and subsequent meta-analyses of these two diseases as separate phenotypes have implicated previously unsuspected mechanisms, such as autophagy, in their pathogenesis and showed that some IBD loci are shared with other inflammatory diseases. Here we expand on the knowledge of relevant pathways by undertaking a meta-analysis of Crohn's disease and ulcerative colitis genome-wide association scans, followed by extensive validation of significant findings, with a combined total of more than 75,000 cases and controls. We identify 71 new associations, for a total of 163 IBD loci, that meet genome-wide significance thresholds. Most loci contribute to both phenotypes, and both directional (consistently favouring one allele over the course of human history) and balancing (favouring the retention of both alleles within populations) selection effects are evident. Many IBD loci are also implicated in other immune-mediated disorders, most notably with ankylosing spondylitis and psoriasis. We also observe considerable overlap between susceptibility loci for IBD and mycobacterial infection. Gene co-expression network analysis emphasizes this relationship, with pathways shared between host responses to mycobacteria and those predisposing to IBD.

CARD8 gene encoding a protein of innate immunity is expressed in human atherosclerosis and associated with markers of inflammation.

Inflammation is a key factor in the development of atherosclerotic coronary artery disease. It is promoted through the inflammasome, a molecular machine that produces IL (interleukin)-1ß in response to cholesterol crystal accumulation in macrophages. The CARD8 (caspase recruitment domain 8) protein modulates this process by suppressing caspase 1 and the transcription factor NF-κB (nuclear factor κB). The expression of CARD8 mRNA was examined in atherosclerotic vascular tissue and the impact on MI (myocardial infarction) of a polymorphism in the CARD8 gene determined. CARD8 mRNA was analysed by microarray of human atherosclerotic tissue and compared with transplant donor arterial tissue. Microarray analysis was performed for proximal genes associated with the rs2043211 locus in plaque. The CARD8 rs2043211 polymorphism was analysed by genotyping of two Swedish MI cohorts, FIA (First Myocardial Infarction in Northern Sweden) and SCARF (Stockholm Coronary Atherosclerosis Risk Factor). The CRP (C-reactive protein) level was measured in both cohorts, but the levels of the pro-inflammatory cytokines IL-1ß, IL-18, TNF (tumour necrosis factor) and MCP-1 (monocyte chemoattractant protein) were measured in sera available from the SCARF cohort. CARD8 mRNA was highly expressed in atherosclerotic plaques compared with the expression in transplant donor vessel (P<0.00001). The minor allele was associated with lower expression of CARD8 in the plaques, suggesting that CARD8 may promote inflammation. Carriers of the minor allele of the rs2043211 polymorphism also displayed lower circulating CRP and lower levels of the pro-atherosclerotic chemokine MCP-1. However, no significant association could be detected between this polymorphism and MI in the two cohorts. Genetic alterations in the CARD8 gene therefore seem to be of limited importance for the development of MI.

Genetic variation and alterations of genes involved in NFκB/TNFAIP3- and NLRP3-inflammasome signaling affect susceptibility and outcome of colorectal cancer.

Colorectal tumors are continuously exposed to an inflammatory environment, which together with mitogenic signals sustain several cancer hallmarks. Nuclear factor-kappa B (NFκB) is a major regulator of inflammation and variation in NFκB-associated genes could potentially be used as biomarkers to identify patients with increased risk of colorectal cancer (CRC) development, and/or a rapidly progressing disease. In this study, 348 CRC cases and 806 randomly selected healthy individuals from southeastern Sweden were examined with regard to seven polymorphisms in NFκB pathway-associated genes. Log-rank-tests and Cox proportional hazard regression analysis examined the association between the polymorphisms and CRC-specific survival, whereas chi-square tests and logistic regression analysis were used to test for associations between the polymorphisms and CRC susceptibility. Gene expression and loss of heterozygosity analyses of TNFAIP3 were carried out in a subset of tumors to assess its role as a tumor suppressor in CRC. Heterozygous and polymorphic TNFAIP3 (rs6920220), heterozygous NLRP3 (Q705K) and polymorphic NFκB -94 ATTG ins/del genotypes were found to be associated with poorer survival in patients diagnosed with invasive CRC (aHR = 5.2, 95% CI: 2.5-10.9, P < 0.001). TNFAIP3 mRNA levels were significantly decreased in tumors compared with adjacent non-neoplastic mucosa (P < 0.0001) and loss of heterozygosity of 6q23.3 (TNFAIP3) was detected in 17% of cases, whereas only 2.5% of the investigated specimens displayed TNFAIP3 gene mutations. We propose that TNFAIP3 (rs6920220), NLRP3 (Q705K) and NFκB -94 ATTG ins/del polymorphisms are associated with poor survival in patients with advanced CRC and may be used as prognostic markers. Experimental results indicate that TNFAIP3 may act as a tumor suppressor in CRC.

Analysis of SNPs with an effect on gene expression identifies UBE2L3 and BCL3 as potential new risk genes for Crohn's disease.

Genome-wide association studies (GWAS) for Crohn's disease (CD) have identified loci explaining approximately 20% of the total genetic risk of CD. Part of the other genetic risk loci is probably partly hidden among signals discarded by the multiple testing correction needed in the analysis of GWAS data. Strategies for finding these hidden loci require large replication cohorts and are costly to perform. We adopted a strategy of selecting SNPs for follow-up that showed a correlation to gene expression [cis-expression quantitative trait loci (eQTLs)] since these have been shown more likely to be trait-associated. First we show that there is an overrepresentation of cis-eQTLs in the known CD-associated loci. Then SNPs were selected for follow-up by screening the top 500 SNP hits from a CD GWAS data set. We identified 10 cis-eQTL SNPs. These 10 SNPs were tested for association with CD in two independent cohorts of Dutch CD patients (1539) and healthy controls (2648). In a combined analysis, we identified two cis-eQTL SNPs that were associated with CD rs2298428 in UBE2L3 (P=5.22x10(-5)) and rs2927488 in BCL3 (P=2.94x10(-4)). After adding additional publicly available data from a previously reported meta-analysis, the association with rs2298428 almost reached genome-wide significance (P=2.40x10(-7)) and the association with rs2927488 was corroborated (P=6.46x10(-4)). We have identified UBE2L3 and BCL3 as likely novel risk genes for CD. UBE2L3 is also associated with other immune-mediated diseases. These results show that eQTL-based pre-selection for follow-up is a useful approach for identifying risk loci from a moderately sized GWAS.

Novel purine analogues regulate IL-1ß release via inhibition of JAK activity in human aortic smooth muscle cells.

Purine analogues bearing a nitrate ester motif were previously discovered as cardioprotective and anti-inflammatory agents, but the anti-inflammatory mechanism remains to be established. We therefore investigated the anti-inflammatory effect of two purine analogues, MK118 bearing a nitrate ester moiety and the methyl-substituted analogue MK196 in Aortic Smooth Muscle Cells (AoSMCs), with emphasis on IL-1ß release. The AoSMCs were stimulated with LPS with or without purine analogue, followed by ELISA, Olink proteomics, Western blot and real time PCR of NLRP3 inflammasome components. Both purine analogues inhibited the release of proteins involved in inflammation, such as TRAIL, CCL4, CSF1 and IL-1ß in AoSMCs, as well as intracellular gene and protein expression of IL-1ß and NLRP3 inflammasome components. MK196, but not MK118, also inhibited the LPS-induced release of IL-7, CXCL10, PD-L1, FLT3L and CCL20. We also showed that MK118 and possibly MK196 act via inhibition of JAKs. In silico studies showed that the purine moiety is a competent hinge binding motif and that the purine-piperazine scaffold is well accommodated in the lipophilic groove of JAK1-3. Both compounds establish interactions with catalytic amino acids in the active site of JAK1-3 and the terminal nitrate ester of MK118 was revealed as a promising pharmacophore. Our data suggest that MK118 and MK196 inhibit the release of proinflammatory proteins in AoSMCs, and targets JAK1-3 activation. Purine analogues also inhibit the expression of NLRP3 inflammasome genes and proteins and may in the future be evaluated for anti-inflammatory aspects on inflammatory diseases.

Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells.

The Caspase activation and recruitment domain 8 (CARD8) protein is a component of innate immunity and overexpression of CARD8 mRNA was previously identified in atherosclerosis. However, very little is known about the regulation of CARD8 in endothelial cells and atherosclerosis. The aim of this study was to investigate CARD8 in the regulation of cytokine and chemokine expression in endothelial cells. Sections of human atherosclerotic lesions and non-atherosclerotic arteries were immunostained for CARD8 protein. Expression of CARD8 was correlated to mediators of inflammation in atherosclerotic lesions using Biobank of Karolinska Endarterectomies microarray data. The CARD8 mRNA was knocked-down in human umbilical vein endothelial cells (HUVECs) in vitro, followed by quantitative RT-PCR analysis and OLINK Proteomics. Endothelial and smooth muscle cells in arterial tissue expressed CARD8 and CARD8 correlated with vWF, CD163 and the expression of inflammatory genes, such as CXCL1, CXCL6 and PDGF-A in plaque. Knock-down of CARD8 in HUVECs significantly altered proteins involved in inflammatory response, such as CXCL1, CXCL6, PDGF-A, MCP-1 and IL-6. The present study suggest that CARD8 regulate the expression of cytokines and chemokines in endothelial cells and atherosclerotic lesions, suggesting that CARD8 plays a significant role in endothelial activation.

A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition.

Natural purines like ATP, ADP and adenosine have crucial roles in platelet physiology. This knowledge has been significant in drug development and today ADP receptor antagonists are widely used for prevention of thrombotic events following myocardial infarction and ischaemic stroke. Recent studies have shown that a purine analogue bearing nitrate ester group (denoted MK128) has anti-inflammatory effects probably due to its ability to donate nitric oxide (NO). However, other pharmacological mechanisms may contribute to the observed effect. The aim of the present study was to establish the anti-platelet activity and elucidate the underlying molecular mechanism(s) of the purine analogue MK128. We found that MK128 reduced aggregation and secretion induced by the thrombin receptor agonist SFLLRN and nearly abolished aggregation and secretion induced by thromboxane A2 (TxA2) and collagen receptor agonists. The inhibition took place despite blockage of the NO/cGMP signalling system. Furthermore, interaction between MK128 and platelet purinergic receptors did not explain the observed inhibition. Instead, we found that MK128 concentration-dependently inhibited Rho-associated kinase (ROCK), which led to decreased ROCK-dependent myosin phosphatase target subunit (MYPT)-1 phosphorylation and suppression of platelet functional responses.

Matrix metalloproteinase (MMP) -1, -2, -3 and -9 promoter polymorphisms in colorectal cancer.

BACKGROUND: Matrix metalloproteinases (MMPs) are a group of matrix-degrading proteins implicated in several pathological processes, e.g., invasion and metastasis in malignant diseases such as colorectal cancer (CRC). MATERIALS AND METHODS: One hundred and twenty-seven CRC patients and 208 controls were genotyped for MMP-1, -2, -3 and -9 promoter polymorphisms. The genotyping was performed with PCR/primer-extension/DHPLC or PCR/RFLP. RESULTS: The MMP-1 2G allele was significantly associated with CRC (p=0.037). No significant association between CRC and MMP-2, -3 or -9 polymorphisms was evident. The analysis of polymorphisms in the clinicopathological subgroups displayed no significant associations. CONCLUSION: The MMP-1 promoter polymorphism seems to affect the susceptibility to CRC, while MMP-2, -3 and -9 polymorphisms appear less likely to have any impact on CRC.

NLRP3 Inflammasome Expression and Activation in Human Atherosclerosis.

BACKGROUND: The NLR family, pyrin domain containing 3 (NLRP3) inflammasome is an interleukin (IL)-1ß and IL-18 cytokine processing complex that is activated in inflammatory conditions. The role of the NLRP3 inflammasome in the pathogenesis of atherosclerosis and myocardial infarction is not fully understood. METHODS AND RESULTS: Atherosclerotic plaques were analyzed for transcripts of the NLRP3 inflammasome, and for IL-1ß release. The Swedish First-ever myocardial Infarction study in Ac-county (FIA) cohort consisting of DNA from 555 myocardial infarction patients and 1016 healthy individuals was used to determine the frequency of 4 single nucleotide polymorphisms (SNPs) from the downstream regulatory region of NLRP3. Expression of NLRP3, Apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 (CASP1), IL1B, and IL18 mRNA was significantly increased in atherosclerotic plaques compared to normal arteries. The expression of NLRP3 mRNA was significantly higher in plaques of symptomatic patients when compared to asymptomatic ones. CD68-positive macrophages were observed in the same areas of atherosclerotic lesions as NLRP3 and ASC expression. Occasionally, expression of NLRP3 and ASC was also present in smooth muscle cells. Cholesterol crystals and ATP induced IL-1ß release from lipopolysaccharide-primed human atherosclerotic lesion plaques. The minor alleles of the variants rs4266924, rs6672995, and rs10733113 were associated with NLRP3 mRNA levels in peripheral blood mononuclear cells but not with the risk of myocardial infarction. CONCLUSIONS: Our results indicate a possible role of the NLRP3 inflammasome and its genetic variants in the pathogenesis of atherosclerosis.

Association analysis of copy numbers of FC-gamma receptor genes for rheumatoid arthritis and other immune-mediated phenotypes.

Segmental duplications (SDs) comprise about 5% of the human genome and are enriched for immune genes. SD loci often show copy numbers variations (CNV), which are difficult to tag with genotyping methods. CNV in the Fcγ receptor region (FCGR) has been suggested to be associated with rheumatic diseases. The objective of this study was to delineate association of FCGR-CNV with rheumatoid arthritis (RA), coeliac disease and Inflammatory bowel disease incidence. We developed a method to accurately quantify CNV in SD loci based on the intensity values from the Immunochip platform and applied it to the FCGR locus. We determined the method's validity using three independent assays: segregation analysis in families, arrayCGH, and whole genome sequencing. Our data showed the presence of two separate CNVs in the FCGR locus. The first region encodes FCGR2A, FCGR3A and part of FCGR2C gene, the second encodes another part of FCGR2C, FCGR3B and FCGR2B. Analysis of CNV status in 4578 individuals with RA and 5457 controls indicated association of duplications in the FCGR3B gene in antibody-negative RA (P=0.002, OR=1.43). Deletion in FCGR3B was associated with increased risk of antibody-positive RA, consistently with previous reports (P=0.023, OR=1.23). A clear genotype-phenotype relationship was observed: CNV polymorphisms of the FCGR3A gene correlated to CD16A expression (encoded by FCGR3A) on CD8 T-cells. In conclusion, our method allows determining the CNV status of the FCGR locus, we identified association of CNV in FCGR3B to RA and showed a functional relationship between CNV in the FCGR3A gene and CD16A expression.

Nitric oxide synthase 2 (NOS2) promoter polymorphisms in colorectal cancer.

Previously, increased expression of nitric oxide synthase 2 (NOS2) in colorectal cancer (CRC) has been identified. The NOS2 gene is transcriptionally regulated, which suggests that polymorphisms in the NOS2 promoter may have a role for CRC development and progression. The genotyping was performed with PCR/RFLP, single strand conformation analysis or MegaBACE genotyping of normal blood DNA from CRC patients and normal healthy controls. However, no significant association between NOS2 polymorphisms and CRC onset or clinical outcome was evident. In conclusion, these results, therefore, suggest that NOS2 promoter polymorphisms have a limited effect on the onset or progression of CRC.

Molecular identification of Helicobacter DNA present in human colorectal adenocarcinomas by 16S rDNA PCR amplification and pyrosequencing analysis.

Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27%). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P = 0.815), nor between Dukes' classes A/B and C/D (P = 0.262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens.

Polymorphism in the NLRP3 inflammasome-associated EIF2AK2 gene and inflammatory bowel disease.

Inflammatory bowel disease (IBD) is the common name for numerous relapsing inflammatory conditions, and is the collective name for Crohn's disease (CD) and ulcerative colitis (UC). The activation of the inflammasome in the pathogenesis of IBD has recently been identified, however the underlying mechanisms remain unclear. An activator of the inflammasome is double-stranded RNA-dependent protein kinase R, also termed EIF2AK2. A genetic alteration in the EIF2AK2 gene has previously been shown to be associated with Alzheimer's disease. The present study genotyped samples from a Swedish cohort of patients with IBD and healthy controls for an EIF2AK2 polymorphism. The rs2254958 polymorphism in the 5'­untranslated region of the EIF2AK2 gene was genotyped by TaqMan® single nucleotide polymorphism genotyping, followed by allelic discrimination. However, no significant association was determined between the rs2254958 polymorphism and the development of IBD, or clinical outcome. In conclusion, the results of the present study suggest that the rs2254958 polymorphism has a limited effect on the onset or progression of IBD.

HER4 tumor expression in breast cancer patients randomized to treatment with or without tamoxifen.

The human epidermal growth factor receptor (HER) 4 is a relative of HER2 and has been associated to endocrine breast cancer and prediction of tamoxifen response. In addition to PI3K/Akt and MAPK pathway activation, ligand binding to HER4 triggers proteolytic cleavage and release of an intracellular receptor domain (4ICD) with signaling properties. The aim of the present study was to analyze HER4 protein expression and intracellular localization in breast cancer tissue from patients randomized to treatment with or without adjuvant tamoxifen. To investigate HER4 expression and localization in response to estradiol (E2) and 4-hydroxytamoxifen (4-OHT) exposure, we also performed in vitro studies. Cytoplasmic, nuclear and membrane expression of HER4 protein was evaluated by immunohistochemical staining in tumor tissue from 912 breast cancer patients. Three different breast epithelia cancer cell lines were exposed to E2 and 4-OHT and mRNA expression was analyzed using qPCR. Further, nuclear and cytoplasmic proteins were separated and analyzed with western blotting. We found an association between nuclear HER4 protein expression and ER-positivity (P=0.004). Furthermore, significant association was found between cytoplasmic HER4 and ER-negativity (P<0.0005), PgR-negativity (P<0.0005), tumor size >20 mm (P=0.001) and HER2-negativity (P=0.008). However, no overall significance of HER4 on recurrence-free survival was found. After E2 exposure, HER4 mRNA and protein expression had decreased in two cell lines in vitro yet no changes in nuclear or cytoplasmic protein fractions were seen. In conclusion, nuclear HER4 seem to be co-located with ER, however, we did not find support for overall HER4 expression in independently predicting response of tamoxifen treatment. The possible influence of separate isoforms was not tested and future studies may further evaluate HER4 significance.

Expression of cytosolic and group X secretory phospholipase A(2) genes in human colorectal adenocarcinomas.

Gene expression of cytosolic phospholipase A(2) (cPLA(2)) and protein level of secretory PLA(2) group X (sPLA(2)-X) are upregulated in human colorectal cancer and provide cyclooxygenase-2 (COX-2) with arachidonic acid, resulting in increased levels of PGE(2). Mutated ras-genes are suggested to be involved in the regulatory pathway of cPLA(2) in lung cancer cells. We analysed the gene expression of cPLA(2) and sPLA(2)-X in 42 and 38 primary colorectal tumours, respectively, with and without K-ras mutations. We found an up-regulation of cPLA(2) mRNA but the induction in tumour tissues does not correlate with Ras-gene mutations. Moreover, our results cannot consistently reflect an overexpression of sPLA(2)-X gene in colorectal cancer tissues.

Nitric oxide synthase 2 mRNA expression in relation to p53 and adenomatous polyposis coli mutations in primary colorectal adenocarcinomas.

BACKGROUND: The inducible nitric (NO) synthase 2 (NOS2) is upregulated in breast, brain, colon, and gynecological tumors, which indicate that NO may have a role in tumorigenesis. However, little is known about the role and regulation of NOS2 in colorectal carcinomas. Recent in vitro experiments have implicated that NOS2 is downregulated by p53 accumulation. Virtual analysis of the NOS2 promoter showed putative TCF-4/Lef-1 response elements, which indicate a potential regulation of NOS2 expression by activation of the adenomatous polyposis coli (APC)/beta-catenin pathway. METHODS: NOS2 mRNA expression was investigated in 59 colorectal carcinomas by reverse transcriptase/real-time polymerase chain reaction and related to mutations in the p53, APC, and beta-catenin genes. Presence of NOS2 protein was studied by Western blot, and the localization was studied by immunohistochemistry. Loss of heterozygosity was studied in the region of the NOS2 gene. RESULTS: The NOS2 mRNA and protein expression were significantly higher in tumors than in control tissue. Immunohistochemistry revealed extensive NOS2 staining in the epithelial cells and, to a minor degree, in leukocytes. Increased NOS2 mRNA expression was found in Dukes' stages A and B compared with the C and D stages. No relationship was found between elevated NOS2 expression and loss of heterozygosity in the later stages according to Dukes' classification or mutations in the p53, APC, or beta-catenin genes. CONCLUSIONS: Inactivating mutations in the p53 and APC pathways are not the main explanation for the increased NOS2 expression found in colorectal tumors.

Correlation of genetic risk and messenger RNA expression in a Th17/IL23 pathway analysis in inflammatory bowel disease.

BACKGROUND: The Th17/IL23 pathway has both genetically and biologically been implicated in the pathogenesis of the inflammatory bowel diseases (IBD), Crohn's disease, and ulcerative colitis. So far, it is unknown whether and how associated risk variants affect expression of the genes encoding for Th17/IL23 pathway proteins. METHODS: Ten IBD-associated SNPs residing near Th17/IL23 genes were used to construct a genetic risk model in 753 Dutch IBD cases and 1045 controls. In an independent cohort of 40 Crohn's disease, 40 ulcerative colitis, and 40 controls, the genetic risk load and presence of IBD were correlated to quantitative PCR-generated messenger RNA (mRNA) expression of 9 representative Th17/IL23 genes in both unstimulated and PMA/CaLo stimulated peripheral blood mononuclear cells. In 1240 individuals with various immunological diseases with whole genome genotype and mRNA-expression data, we also assessed correlation between genetic risk load and differential mRNA expression and sought for SNPs affecting expression of all currently known Th17/IL23 pathway genes (cis-expression quantitative trait locus). RESULTS: The presence of IBD, but not the genetic risk load, was correlated to differential mRNA expression for IL6 in unstimulated peripheral blood mononuclear cells and to IL23A and RORC in response to stimulation. The cis-expression quantitative trait locus analysis showed little evidence for correlation between genetic risk load and mRNA expression of Th17/IL23 genes, because we identified for only 2 of 22 Th17/IL23 genes a cis-expression quantitative trait locus single nucleotide polymorphism that is also associated to IBD (STAT3 and CCR6). CONCLUSIONS: Our results suggest that only the presence of IBD and not the genetic risk load alters mRNA expression levels of IBD-associated Th17/IL23 genes.