Genetic variation in the serotonin receptor gene affects immune responses in rheumatoid arthritis.

Many genetic variants associate with the risk of developing rheumatoid arthritis (RA); however, their functional roles are largely unknown. Here, we aimed to investigate whether the RA-associated serotonin receptor 2A (HTR2A) haplotype affects T-cell and monocyte functions. Patients with established RA (n=379) were genotyped for two single-nucleotide polymorphisms (SNPs) in the HTR2A locus, rs6314 and rs1328674, to define presence of the risk haplotype for each individual. Patients with and without the RA-associated TC haplotype were selected and T-cell and monocyte function was monitored following in vitro stimulations with staphylococcal enterotoxin B and lipopolysaccharide (LPS) using multiparameter flow cytometry. Within the cohort, 44 patients were heterozygous for the TC haplotype (11.6%) while none were homozygous. Upon stimulation, T cells from TC-carrier patients produced more proinflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin-17 (IL-17) and interferon gamma (IFN-γ)) and monocytes produced higher levels of TNF-α compared with patients carrying the non-TC haplotype (P<0.05 and 0.01, respectively). Such cytokine production could be inhibited in the presence of the selective 5-HT2 receptor agonist (2,5-Dimethoxy-4-iodoamphetamine, DOI); interestingly, this effect was more pronounced in TC carriers. Our data demonstrate that association of RA with a distinct serotonin receptor haplotype has functional impact by affecting the immunological phenotype of T cells and monocytes.

Ovarian cancer-associated polymorphisms in the BNC2 gene among women with endometriosis.

BACKGROUND: Endometriosis is a common benign gynaecological disease. Epidemiological studies have demonstrated associations between endometriosis and ovarian cancer. Recent genome-wide association studies of ovarian cancer have identified several single nucleotide polymorphisms (SNPs) in the Basonuclin 2 (BNC2) gene. In this study, we investigated these polymorphism in women with endometriosis. METHODS: Six SNPs in and upstream of the BNC2 gene (rs3814113, rs4445329, rs10962656, rs12379183, rs10756819 and rs1339552) were investigated using TaqMan allelic discrimination analysis in a Caucasian population (cases: 798, controls: 351). Allelic frequencies were used as main outcome measure. RESULTS: No associations were observed between the analysed SNPs and endometriosis. CONCLUSIONS: Our results suggest that the analysed polymorphisms in the BNC2 gene are unlikely to contribute to the previously reported risk of ovarian cancer in women with endometriosis.

Major histocompatibility complex class II transactivator gene polymorphism: associations with Löfgren's syndrome.

The major histocompatibility complex (MHC) class II transactivator (MHC2TA) is known as a master regulator for expression of MHC class II molecules. In the present study, we investigated the influence on the risk for sarcoidosis of two variants of the MHC2TA gene, selected from previous association studies of inflammatory diseases. Seven hundred and twenty-eight sarcoidosis patients and 873 controls matched by ethnicity were included in the study. Patients were classified as with Löfgren's syndrome (or not) as subphenotypes. Individuals were genotyped for two single nucleotide polymorphisms (SNPs) of the MHC2TA gene, rs3087456 A/G and rs11074932 C/T, and were human leukocyte antigen (HLA)-DRB1-typed. After correction for multiple testing, our data showed a significant association with Löfgren's syndrome in allelic model for the rs3087456 SNP, which was not detected in non-Löfgren's patients. A similar trend was noted for the rs11074932 SNP. These risk factors were independent of HLA-DRB1*03, which is known to be associated with Löfgren's syndrome. The finding of a new genetic association between Löfgren's syndrome and MHC2TA gene polymorphisms, which seems independent of HLA-DRB1*03 and relates to the expression of MHC class II molecules, strongly supports the idea that Löfgren's syndrome is a separate disease entity.

Genetic variants of CC chemokine genes in experimental autoimmune encephalomyelitis, multiple sclerosis and rheumatoid arthritis.

Multiple sclerosis (MS) is a complex disorder of the central nervous system, causing inflammation, demyelination and axonal damage. A limited number of genetic risk factors for MS have been identified, but the etiology of the disease remains largely unknown. For the identification of genes regulating neuroinflammation we used a rat model of MS, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), and carried out a linkage analysis in an advanced intercross line (AIL). We thereby redefine the Eae18b locus to a 0.88 Mb region, including a cluster of chemokine genes. Further, we show differential expression of Ccl2, Ccl11 and Ccl11 during EAE in rat strains with opposite susceptibility to EAE, regulated by genotype in Eae18b. The human homologous genes were tested for association to MS in 3841 cases and 4046 controls from four Nordic countries. A haplotype in CCL2 and rs3136682 in CCL1 show a protective association to MS, whereas a haplotype in CCL13 is disease predisposing. In the HLA-DRB1* 15 positive subgroup, we also identified an association to a risk haplotype in CCL2, suggesting an influence from the human leukocyte antigen (HLA) locus. We further identified association to rheumatoid arthritis in CCL2, CCL8 and CCL13, indicating common regulatory mechanisms for complex diseases.

Genetic variations in the serotonin 5-HT2A receptor gene (HTR2A) are associated with rheumatoid arthritis.

OBJECTIVES: To analyse the association between the genetic polymorphisms within the HTR2A gene for the serotonin receptor and rheumatoid arthritis (RA). METHODS: HTR2A gene polymorphisms were analysed in patients with RA and controls from two study populations using PCR based restriction endonuclease mapping or TaqMan allelic discrimination with more than 4000 individuals included in the current study. RESULTS: At the discovery stage we detected significant differences in frequency of rs6313 (T102C polymorphism) between the patients with RA and controls (p = 0.006). Following validation with an extended set of single nucleotide polymorphisms (SNPs) and number of DNA samples, a trend in associations in allelic model for SNPs rs6314, rs1328674, rs6313 and rs6311 (p = 0.006, 0.002, 0.006, 0.009) was seen, although it was lost after correction for multi-comparison for all but rs1328674 (empirical p value = 0.021). However, haplotype frequency analysis based on these four SNPs showed significantly low representation of TCTT combination in patients with RA in comparison with controls (3.6% and 5.6%, p<0.001 on chi(2) test, empirical p = 0.004 after 100 000 permutations) and a significantly higher frequency of CTCC combination in patients with RA in comparison with controls (3.6% and 2.2%, p = 0.002 on chi(2) test, empirical p = 0.022 after 100 000 permutations). CONCLUSIONS: In our study, genetic polymorphisms at the HTR2A gene are associated with susceptibility for RA, suggesting possible links between the serotonergic system and development of the disease.

Low IL-1alpha expression in bladder cancer tissue and survival.

OBJECTIVE: To investigate whether the previously reported association between IL-1alpha mRNA levels and survival in urinary bladder cancer remains in an extended patient material and to search a mechanism behind a possible antitumoral activity of IL-1alpha. PATIENTS AND METHODS: IL-1alpha mRNA levels were determined in 164 tumors with quantitative TaqMan PCR. RESULTS: A large variation was found in mRNA levels of IL-1alpha. We found, by immunohistochemistry, that IL-1alpha is expressed by tumor rather than stromal cells. In a univariate Cox proportional hazards model, low levels (median split) of IL-1alpha mRNA were associated with a relative hazard ratio (RHR) of 1.7 (95% CI: 1.0-2.9) for cancer-specific death (n=157); a restriction to muscle invasive tumors (n=63) resulted in an RHR of 1.8 (0.9-3.3). In bivariate analyses, adjustment for age, stage and grade respectively, decreased the RHR and the association between IL-1alpha expression and cancer-specific survival was not statistically significant. Which factors to regard as confounders remains unclear. CONCLUSIONS: Low levels of IL-1alpha mRNA expression are associated with an increased risk for cancer-specific death in the investigated material. However, confounding is an issue and to determine whether or not the observed association is causal, we need a defined mechanism and data from other studies.

Prognostic importance of the uPa/PAI-1 complex in breast cancer.

The complex between urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 (plasminogen activator inhibitor 1) has been prognostically evaluated in patients with breast cancer. The concentrations of uPA antigen, PAI-1 antigen and the uPA/PAI-1 complex were analysed in extracts from breast cancer tumours from 233 patients (median follow-up of patients: 71months). The uPA/PAI-1 complex typically constituted about 5% of the uPA antigen (total uPA). The concentration of complex was found to correlate more strongly to the concentration of PAI-1 (r = 0.72; p < 0.0001) than to the concentration of uPA (r = 0.55: p < 0.0001). Interestingly, in this material the uPA/PAI-1 complex (using an optimised cutoff level of 0.22 ng microg(-1) DNA) had a stronger prognostic value than optimised cut-off valuesfor uPA or PAI-1. The data suggest that activation of prourokinase within the tumour, which is a prerequisite for the formation of the uPA/PAI-1 complex, is of better prognostic value than the production of prourokinase or PAI-1 in the breast cancer tumour.

Low interleukin-1alpha messenger RNA levels predict decreased overall survival time of patients with urinary bladder carcinoma.

Due to our inability to exactly characterize tumours, many patients with urinary bladder cancer undergo unnecessary surgery or cytostatic therapy. We have here studied the expression of the cytokine interleukin-1alpha (IL-1alpha ) in 73 human bladder carcinomas in relation to patient survival, and examined possible relationships between IL-1alpha and urokinase plasminogen activator (uPA) expression. Expression levels of IL-1alpha and uPA mRNA were determined by RT-PCR using the quantitative TaqMan technique. The levels of IL-1alpha mRNA expression did not differ significantly between tumours of different grade or stage. Calculation of the overall survival rates showed a decreased overall survival time for patients with low levels of IL-1alpha mRNA in their tumours (log rank; P = 0.0002, median follow up: 37 months). Low tumoral IL-1alpha expression predicted decreased survival of patients with poorly differentiated tumours (P< 0.005) and of patients with invasive tumours (P = 0.02). uPA expression was about 4-fold increased in poorly differentiated tumours. High levels of uPA mRNA were associated with decreased overall survival times (log rank; P = 0.032, n = 60). We conclude that IL-1alpha is important for bladder cancer biology, and that measurements of this cytokine may be useful in pre-treatment characterization of urinary bladder cancer.

Inhibition of extracellular signal-regulated kinase 1/2 activity of the breast cancer cell line MDA-MB-231 leads to major alterations in the pattern of protein expression.

Biochemical and genetic strategies have implied that aberrant signaling in the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase pathway contributes significantly to transformed phenotypes. Using PD98059, an inhibitor of the ERK-kinase MEK1, we have here assessed the effects of ERK inhibition on the pattern of protein expression in the metastatic human breast cancer cell line MDA-MB-231. At a concentration of inhibitor which did not significantly affect cell growth, PD98059 induced large changes in the expression of MDA-MB-231 polypeptides. The majority of these changes were due to decreased expression of low-abundance proteins. Decreases of more abundant proteins such as glutathione-S-transferase pi, hsp80 and hsp100 were also recorded. The levels of a few proteins increased, among them cytokeratin 8. We conclude that PD98059 treatment of MDA-MB-231 cells induces large changes in protein expression.

ERK signalling in metastatic human MDA-MB-231 breast carcinoma cells is adapted to obtain high urokinase expression and rapid cell proliferation.

Increased urokinase plasminogen activator (u-PA) production is associated with tumor invasion and metastasis in several malignancies, including breast cancer. The mechanisms underlying constitutive u-PA expression are not well understood. We examined the relationship between the signal strength of the ERK pathway and the level of u-PA expression in the metastatic human breast cancer cell line MDA-MB-231. Treatment with the MEK1 inhibitor PD98059 resulted in decreased ERK1/2 phosphorylation and decreased u-PA mRNA and protein expression. Inhibition of ERK1/2 activity also led to decreased cell proliferation and to decreased cyclin D1 expression. Less than 5% of total ERK1/2 was phosphorylated in exponentially growing MDA-MB-231 cells, and ERK1/2 activity could be stimulated by okadaic acid. Okadaic acid did not stimulate u-PA expression, but induced strong expression of the cdk-inhibitor p21Cip1. These findings suggest that ERK1/2 signaling is tuned to a level which results in high u-PA expression and rapid cell proliferation.