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Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale., Competing Interests: Declaration of competing interest The authors declare no competing, personal financial or other competing interests. Funding or employment sources played no role in the design, presentation or discussion of the presented research., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Allosterism is a common phenomenon in protein biochemistry that allows rapid regulation of protein stability; dynamics and function. However, the mechanisms by which allosterism occurs (by mutations or post-translational modifications (PTMs)) may be complex, particularly due to long-range propagation of the perturbation across protein structures. In this work, we have investigated allosteric communication in the multifunctional, cancer-related and antioxidant protein NQO1 by mutating several fully buried leucine residues (L7, L10 and L30) to smaller residues (V, A and G) at sites in the N-terminal domain. In almost all cases, mutated residues were not close to the FAD or the active site. Mutations L→G strongly compromised conformational stability and solubility, and L30A and L30V also notably decreased solubility. The mutation L10A, closer to the FAD binding site, severely decreased FAD binding affinity (≈20 fold vs. WT) through long-range and context-dependent effects. Using a combination of experimental and computational analyses, we show that most of the effects are found in the apo state of the protein, in contrast to other common polymorphisms and PTMs previously characterized in NQO1. The integrated study presented here is a first step towards a detailed structural-functional mapping of the mutational landscape of NQO1, a multifunctional and redox signaling protein of high biomedical relevance.

HIF-1α is a master regulator of oxygen homeostasis involved in different stages of cancer development. Thus, HIF-1α inhibition represents an interesting target for anti-cancer therapy. It was recently shown that the HIF-1α interaction with NQO1 inhibits proteasomal degradation of the former, thus suggesting that targeting the stability and/or function of NQO1 could lead to the destabilization of HIF-1α as a therapeutic approach. Since the molecular interactions of NQO1 with HIF-1α are beginning to be unraveled, in this review we discuss: (1) Structure-function relationships of HIF-1α; (2) our current knowledge on the intracellular functions and stability of NQO1; (3) the pharmacological modulation of NQO1 by small ligands regarding function and stability; (4) the potential effects of genetic variability of NQO1 in HIF-1α levels and function; (5) the molecular determinants of NQO1 as a chaperone of many different proteins including cancer-associated factors such as HIF-1α, p53 and p73α. This knowledge is then further discussed in the context of potentially targeting the intracellular stability of HIF-1α by acting on its chaperone, NQO1. This could result in novel anti-cancer therapies, always considering that the substantial genetic variability in NQO1 would likely result in different phenotypic responses among individuals.

The multifunctional nature of human flavoproteins is critically linked to their ability to populate multiple conformational states. Ligand binding, post-translational modifications and disease-associated mutations can reshape this functional landscape, although the structure-function relationships of these effects are not well understood. Herein, we characterized the structural and functional consequences of two mutations (the cancer-associated P187S and the phosphomimetic S82D) on different ligation states which are relevant to flavin binding, intracellular stability and catalysis of the disease-associated NQO1 flavoprotein. We found that these mutations affected the stability locally and their effects propagated differently through the protein structure depending both on the nature of the mutation and the ligand bound, showing directional preference from the mutated site and leading to specific phenotypic manifestations in different functional traits (FAD binding, catalysis and inhibition, intracellular stability and pharmacological response to ligands). Our study thus supports that pleitropic effects of disease-causing mutations and phosphorylation events on human flavoproteins may be caused by long-range structural propagation of stability effects to different functional sites that depend on the ligation-state and site-specific perturbations. Our approach can be of general application to investigate these pleiotropic effects at the flavoproteome scale in the absence of high-resolution structural models., (Copyright © 2021. Published by Elsevier B.V.)

Testes of seasonally breeding species experience a severe functional regression before the non-breeding period, which implies a substantial mass reduction due to massive germ-cell depletion. Two alternative mechanisms of seasonal germ-cell depletion have been described in mammals, apoptosis and desquamation (sloughing), but their prevalence has not been determined yet due to reduced number of species studied. We performed a morphological, hormonal, and molecular study of the mechanism of seasonal testicular regression in males of the Egyptian long eared-hedgehog (Hemiechinus auritus). Our results show that live, non-apoptotic, germ cells are massively depleted by desquamation during the testis regression process. This is concomitant with both decreased levels of serum testosterone and irregular distribution of the cell-adhesion molecules in the seminiferous epithelium. The inactive testes maintain some meiotic activity as meiosis onset is not halted and spermatocytes die by apoptosis at the pachytene stage. Our data support the notion that apoptosis is not the major testis regression effector in mammals. Instead, desquamation appears to be a common mechanism in this class., Competing Interests: The authors have declared that no competing interests exist.

Alterations in the extracellular matrix (ECM) production and remodeling of smooth muscle cells (SMCs) have been implicated in processes related to the differentiation in atherosclerosis. Due to the anti-atherosclerotic properties of the tetracyclines, we aimed to investigate whether cholesterol supplementation changes the effect of doxycycline over the ECM proteins synthesis and whether isoprenylated proteins and Rho A protein activation are affected. SMC primary culture isolated from chicks exposed to atherogenic factors in vivo (a cholesterol-rich diet, SMC-Ch), comparing it with control cultures isolated after a standard diet (SMC-C). After treatment with 20 nM doxycycline, [H 3 ]-proline and [H 3 ]-mevalonate incorporation were used to measure the synthesis of collagen and isoprenylated proteins, respectively. Real-time PCR was assessed to determine col1a2, col2a1, col3a1, fibronectin, and mmp2 gene expression and the pull-down technique was applied to determine the Rho A activation state. A higher synthesis of collagens and isoprenylated proteins in SMC-Ch than in SMC-C was determined showing that doxycycline inhibits ECM production and remodeling in both SMC types of cultures. Moreover, preliminary results about the effect of doxycycline on protein isoprenylation and Rho A protein activation led us to discuss the possibility that membrane G-protein activation pathways could mediate the molecular mechanism., (Copyright © 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.)

Cholesterol-lowering effects apart, statins can improve the endothelial function, stabilize the atherosclerotic plaques, decrease the oxidative stress and inflammation and inhibit the thrombogenic response by means of the inhibition of isoprenoids, which serve as lipid attachments for intracellular signaling molecules. We aimed to evaluate whether the effect of statins on RhoA activity mediate extracellular matrix production, particularly affecting collagen type I, in smooth muscle cells (SMCs). Our results showed that lovastatin decreased collagen expression in primary cultured chicken SMCs as determined by incorporation of [H3]-proline, RT-PCR and immunocytochemistry. This fall was parallel to that found in Rho A activity. Similar results were found when GGTI-298, a RhoA inhibitor, was added to the culture medium. Mevalonate or geranylgeranyl pyrophosphate reverted these effects. In order to elucidate the role of Rho A in these events we transfected the cell line A10 (rat SMCs) with constitutively active (G14V) or dominant negative RhoA (T19N) constructs. The last ones showed similar results regarding collagen production that those stated above in lovastatin treated primary SMC cultures. Constitutively active RhoA transfected cells showed the opposite effects. Next we performed a promoter activity assay to exclude post-transcriptional mechanisms implicated in these studies. We found a similar pattern in col1a2 promoter activity to that found in collagen expression. Our results have demonstrated that statins regulate the activation of RhoA through its isoprenylation, which is crucial for the regulation of extracellular matrix synthesis in SMCs.

Objectives: Pancreatic ductal adenocarcinoma is still one of the deadliest solid cancers so the finding of new therapeutic approaches and novel targets are of utmost importance. Glycoprotein nonmetastatic melanoma protein B (GPNMB), initially termed glycoprotein nonmetastatic gene B and also named osteoactivin (OA), is a type 1 transmembrane protein that has been recently found to play a role in cancer cell proliferation, angiogenesis, and invasion. Due to its potential responsibility in cancer aggressiveness, the main objective of this work was to assess the role of GPNMB/OA in human pancreatic cancer., Methods: Using the human pancreatic cancer cell line Panc-1 in vitro, the effects of GPNMB on growth, proliferation, and invasion were tested by BrdU uptake, cell cycle and Annexin V-FITC analysis, RT-PCR, protein expression, and invasion chamber assays., Results: Our results showed that GPNMB/OA protein expression prevents cells from apoptosis-enhancing proliferation and represents a novel modulator of the invasion and metastasis in pancreatic cancer cells., Conclusions: Due to its main membrane localization in cancer cells and its role in the aggressiveness of pancreatic cancer, GPNMB/OA could represent a novel targeted therapy for pancreatic cancer being attractive for antibody-based therapies.

Objective: To assess the influence of the TNFA rs1800629 (G > A) polymorphism in the risk of cardiovascular (CV) disease and subclinical atherosclerosis in patients with rheumatoid arthritis (RA)., Methods: 587 patients fulfilling the 1987 American College of Rheumatology classification criteria for RA were studied. Patients were genotyped for the TNFA rs1800629 polymorphism using predesigned TaqMan single nucleotide polymorphism genotyping assay. Also, HLA-DRB1 genotyping was performed using molecular based methods. Carotid artery intima-media thickness, flow-mediated endothelium-dependent and endothelium independent vasodilatation, used as surrogate markers of subclinical atherosclerosis, were measured in a subgroup of patients., Results: We observed a higher frequency of carriers of the minor allele A among the patients with CV disease (with 37.6% vs. without 27.9%, p = 0.06, OR 1.56 [95% confidence interval-CI 0.95-2.54]). Carriers of the minor allele A exhibited a higher risk of CV events after adjustment for demographic and traditional CV risk factors (p = 0.023, HR 1.72 [95% CI 1.076-2.74]). Also, a significant interaction between this polymorphism and the presence of the rheumatoid shared epitope (SE) was observed (p = 0.024). Due to this, the association between carriers of the minor allele A and CV disease was only present in carriers of the SE, even after adjustment (p = 0.001, HR 2.43 [95% CI 1.41-4.19]). No significant association between the TNFA variant and the surrogate markers of subclinical atherosclerosis was observed., Conclusion: Our results show that TNFA rs1800629 gene polymorphism is associated with predisposition to CV complications in patients with RA. This predisposition is restricted to individuals carrying the rheumatoid SE., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: The Fc receptors II and III (FcgR2a, and FcgR3a) play a crucial role in the regulation of the immune response. The FcgR2a*519GG and FcgR3a*559CC genotypes have been associated with several autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, nephritis, and possibly to type I diabetes, and celiac disease. In a large multicenter, two-stage study of 6570 people, we tested whether the FcgR2a and FcgR3a genes were also involved in inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC)., Methods: We genotyped the FcgR2a*A519G and FcgR3a*A559C functional variants in 4205 IBD patients in six well-phenotyped Caucasian IBD cohorts and 2365 ethnically matched controls recruited from the Netherlands, Spain, and New Zealand., Results: In the initial Dutch study we found a significant association of FcgR2a genotypes with IBD (P-genotype = 0.02); while the FcgR2a*519GG was more common in controls (23%) than in IBD patients (18%; odds ratio [OR] = 0.75; 95% confidence interval [CI] 0.61-0.92; P = 0.004). This association was corroborated by a combined analysis across all the study populations (Mantel-Haenszel [MH] OR = 0.84; 0.74-0.95; P = 0.005) in the next stage. The Fcgr2a*GG genotype was associated with both UC (MH-OR = 0.84; 0.72-0.97; P = 0.01) and CD (MH-OR = 0.84; 0.73-0.97; P = 0.01), suggesting that this genotype confers a protective effect against IBD. There was no association of FcgR3a*A559C genotypes with IBD, CD, or UC in any of the three studied populations., Conclusions: The FcgR2a*519G functional variant was associated with IBD and reduced susceptibility to UC and to CD in Caucasians. There was no association between FcgR3a*5A559C and IBD, CD or UC., (Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.)

Objectives: To determine whether the PTPN22, STAT4 and TRAF1/C5 gene polymorphisms may be implicated in the development of cardiovascular (CV) events and subclinical atherosclerosis manifested by the presence of endothelial dysfunction or increased carotid intima-media thickness (IMT) in a series of Spanish patients with rheumatoid arthritis (RA)., Methods: Six hundred and twelve patients fulfilling the 1987 American College of Rheumatology classification criteria for RA, seen at the rheumatology outpatient clinics of Hospital Xeral-Calde, Lugo, and Hospital San Carlos, Madrid, were studied. Patients were genotyped using predesigned TaqMan single nucleotide polymorphism genotyping assays. Moreover, between March and December 2007, a subgroup of unselected RA patients with no history of CV events was studied for the presence of subclinical atherosclerosis by the assessment of the endothelial function (n=126) and the carotid artery IMT (n=110) by ultrasonography studies., Results: No significant differences in the allele or genotype frequencies for the PTPN22, STAT4 and TRAF1/C5 gene polymorphisms between RA patients with or without CV events were found. It was also the case when we analysed the potential influence of the genotypes in the presence of endothelial dysfunction or increased carotid artery IMT of patients with RA., Conclusions: Our results do not show that the PTPN22, STAT4 and TRAF1/C5 gene polymorphisms may confer a direct risk of CV disease in patients with RA.

Objective: Giant cell arteritis (GCA) is a complex polygenic disease in which more than 1 genetic locus is likely to contribute to disease susceptibility and clinical expression. BANK, an adaptor molecule, has been suggested to participate in the B cell antigen receptors-mediated calcium homeostasis. We assessed for the first time the implication of BANK1 functional variants in susceptibility to GCA., Methods: Two hundred twenty-two patients with biopsy-proven GCA and 534 matched controls were assessed. DNA from patients and controls was obtained from peripheral blood. Samples were genotyped for 3 putative functional BANK1 gene polymorphisms (rs17266594 T/C, rs10516487 G/A, rs3733197 G/A) using a TaqMan allele discrimination assay., Results: No significant differences were observed in genotype distribution between patients with biopsy-proven GCA and controls for these 3 gene polymorphisms. A trend for a decreased risk of having GCA was observed in individuals carrying the BANK1 rs3733197 GG genotype (patients with GCA 43.9% compared to 51.6% in controls; p = 0.06, OR 0.73, 95% CI 0.53-1.02). The frequency of BANK1 rs3733197 allele G was marginally decreased in patients with biopsy-proven GCA compared to controls (p = 0.09, OR 0.82, 95% CI 0.64-1.04). Haplotype analysis of 3-single-nucleotide polymorphisms found no statistically significant differences between patients with GCA and controls. No significant differences for the BANK1 gene polymorphisms were found when patients were stratified according to specific clinical features of the disease., Conclusion: Our results do not support a major implication of the BANK1 locus in susceptibility to GCA.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs that leads to profound disability and premature death. To identify new SSc susceptibility loci, we conducted the first genome-wide association study in a population of European ancestry including a total of 2,296 individuals with SSc and 5,171 controls. Analysis of 279,621 autosomal SNPs followed by replication testing in an independent case-control set of European ancestry (2,753 individuals with SSc (cases) and 4,569 controls) identified a new susceptibility locus for systemic sclerosis at CD247 (1q22-23, rs2056626, P = 2.09 x 10(-7) in the discovery samples, P = 3.39 x 10(-9) in the combined analysis). Additionally, we confirm and firmly establish the role of the MHC (P = 2.31 x 10(-18)), IRF5 (P = 1.86 x 10(-13)) and STAT4 (P = 3.37 x 10(-9)) gene regions as SSc genetic risk factors.

Objective: Recently, 2 independent studies have identified an association between several single-nucleotide polymorphisms (SNP) located in the 6q23 chromosomal region and rheumatoid arthritis (RA). Like RA, giant cell arteritis (GCA) is also a complex polygenic disease in which more than 1 genetic locus is likely to contribute to disease susceptibility and clinical expression. We analyzed the involvement of the rs6920220 (G/A) polymorphism from the 6q23/TNFAIP3 gene region in susceptibility to GCA., Methods: Two hundred twenty patients with biopsy-proven GCA and 490 matched controls were assessed. DNA from patients and controls was obtained from peripheral blood. Samples were geno-typed for the 6q23 region rs6920220 using a TaqMan allele discrimination assay and by polymerase chain reaction (PCR) amplification. After PCR, the genotype of each sample was attributed automatically by allelic-specific fluorescence using the ABI Prism 7900 sequence detection system., Results: No significant differences in the genotype distribution between patients with GCA and controls for the rs6920220 (G/A) polymorphism were found. No significant differences were observed when patients with GCA were stratified according to the presence of specific clinical features of the disease such as polymyalgia rheumatica or severe ischemic manifestations or specific visual ischemic complications., Conclusion: Our results show no involvement of this 6q23/TNFAIP3 gene region SNP in the susceptibility to or clinical expression of GCA.

Giant cell arteritis (GCA) is a complex polygenic disease in which more than one genetic locus is likely to contribute to disease susceptibility and clinical expression. In the present study, we have analyzed for first time the implication of rs13277113 and rs2736340 variants from the C8orf13-BLK gene region in the susceptibility to GCA. A total of 220 biopsy-proven GCA patients and 486 matched controls were assessed. DNA from patients and controls was obtained from peripheral blood. Samples were genotyped for the C8orf13-BLK region rs13277113 and rs2736340 using a predesigned TaqMan allele discrimination assay. No significant differences in the genotype distribution between GCA patients and controls for the rs13277113 and rs2736340 C8orf13-BLK gene variants were found. GCA patients were also stratified according to the presence of specific clinical features of the disease. In this regard, the allele A of the rs13277113 variant was overrepresented in patients with severe ischemic manifestations compared with patients without severe ischemic manifestations (p = 0.04; OR =1.65; 95% CI = 0.99-2.78). In conclusion, our results do not support a major implication of the C8orf13-BLK gene region in susceptibility to GCA. However, a potential implication of the rs13277113 variant in the development of severe ischemic complications may exist., (Copyright 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Apoptosis of vascular smooth muscle cells (SMC) is critically involved in the progression of atherosclerosis. We previously reported that dietary cholesterol intake induces changes in SMC at molecular and gene expression levels. The objectives of the present study were to investigate the differential response to nitric oxide of vascular SMC obtained from chicks after cholesterol and fish oil dietary intake and to examine effects on the main pro-apoptotic and anti-apoptotic genes. Dietary cholesterol intake reduced the Bcl-2/Bax (anti-apoptotic/pro-apoptotic) protein ratio in SMC, making them more susceptible to apoptosis. When cholesterol was withdrawn and replaced with a fish oil-enriched diet, the Bcl-xl/Bax protein ratio significantly increased, reversing the changes induced by cholesterol. The decrease in c-myc gene expression after apoptotic stimuli and the increase in Bcl-xl/Bax ratio indicate that fish oil has a protective role against apoptosis in SMC. Nitroprussiate-like nitric oxide donors exerted an intensive action on vascular SMC cultures. However, SMC-C (isolated from animals fed with control diet) and SMC-Ch (isolated from animals fed with cholesterol-enriched diet) responded differently to nitric oxide, especially in their bcl-2 and bcl-xl gene expression. SMC isolated from animals fed with cholesterol-enriched and then fish oil-enriched diet (SMC-Ch-FO cultures) showed an intermediate apoptosis level (Bcl-2/Bax ratio) between SMC-C and SMC-Ch, induction of c-myc expression and elevated p53 expression. These findings indicate that fish oil protects SMC against apoptosis., (Copyright 2009 Elsevier Inc. All rights reserved.)

Objectives: Since the transcription factor hypoxia-inducible factor 1 (HIF-1) is a key early mediator of the response to ischemia and giant cell arteritis (GCA) is a polygenic disease leading to severe ischemic complications, in the present study we analysed for first time the implication of two HIF-1alpha gene polymorphisms in the susceptibility to and clinical expression of GCA., Methods: Two hundred and fifteen biopsy-proven GCA patients and 470 matched controls were assessed. DNA from patients and controls was obtained from peripheral blood. Samples were genotyped for two single nucleotide polymorphisms, rs11549465 (C/T) and rs11549467 (G/A), using a pre-designed TaqMan allele discrimination assay. Post PCR, the genotype of each sample was attributed automatically by measuring the allelic specific fluorescence on the ABI PRIM 7900 sequence., Results: The HIF-1alpha, rs11549465 TT genotype was extremely uncommon in both GCA patients (2.3%) and controls (2.1%). Although the frequency of individuals carrying the CT or TT genotypes was increased in GCA patients (25.1%) compared to controls (20.4%) the difference was not statistically significant (OR 1.30 [95% CI: 0.89- 1.91]; p=0.17). Also, all GCA patients and most controls (98.9%) were homozygous for the rs11549467 GG genotype. GCA patients carrying the rs11549465 CT or TT genotypes had a slight increased risk of developing visual ischemic complications (33.1%) compared to the remaining GCA patients (22.8%); OR 1.60 (95% CI: 0.81- 3.16); p=0.18., Conclusions: Our results do not confirm an implication of HIF-1alpha gene polymorphisms in the susceptibility to and clinical expression of GCA.

Introduction: We determined the contribution of the methylene tetrahydrofolate reductase (MTHFR) 677 C>T and 1298 A>C gene polymorphisms to the susceptibility to rheumatoid arthritis (RA). We also assessed whether these two MTHFR gene polymorphisms may be implicated in the development of cardiovascular (CV) events and subclinical atherosclerosis manifested by the presence of endothelial dysfunction, in a series of Spanish patients with RA., Methods: Six hundred and twelve patients fulfilling the 1987 American College of Rheumatology classification criteria for RA, seen at the rheumatology outpatient clinics of Hospital Xeral-Calde, Lugo and Hospital San Carlos, Madrid, were studied. Patients and controls (n = 865) were genotyped using predesigned TaqMan SNP genotyping assays., Results: No significant differences in allele or genotype frequencies for the MTHFR gene polymorphisms between RA patients and controls were found. Also, no association between the MTHFR 677 C>T polymorphism and CV events or endothelial dysfunction was observed. However, the MTHFR 1298 allele C frequency was increased in patients with CV events after 5 years (38.7% versus 30.3%; odds ratio = 1.45; 95% confidence interval = 1.00 to 2.10; P = 0.04) and 10 years (42.2% versus 31.0%; odds ratio = 1.62; 95% confidence interval = 1.08 to 2.43; P = 0.01) follow up. Moreover, patients carrying the MTHFR 1298 AC and CC genotypes had a significantly decreased flow-mediated endothelium-dependent vasodilatation (4.3 +/- 3.9%) compared with those carrying the MTHFR 1298 AA genotype (6.5 +/- 4.4%) (P = 0.005)., Conclusions: Our results show that the MTHFR 1298 A>C gene polymorphism confers an increased risk for subclinical atherosclerosis and CV events in patients with RA.

Objective: Interferon (IFN) regulatory factors (IRF) are transcriptional mediators of IFN-induced signaling pathways and are involved in immune response. We have analyzed for the first time the association of 2 IRF5 gene variants in the susceptibility to giant cell arteritis (GCA)., Methods: Two hundred twenty patients with biopsy-proven GCA and 520 matched controls were assessed. DNA from patients and controls was obtained from peripheral blood. Samples were genotyped for the IRF5 rs2004640 and for the IRF5 CGGGG insertion/deletion polymorphism using a predesigned TaqMan allele discrimination assay and by polymerase chain reaction amplification, followed by an ABI3100 sequencer, respectively., Results: A genotyping rate of 96% was achieved in this series of GCA patients. No significant differences were found in the genotype distribution between GCA patients and controls for both IRF5 gene variants. In this regard, similar genotype frequencies were found in GCA patients and controls. No significant differences were observed when GCA patients were stratified according to the presence of specific clinical features of the disease such as polymyalgia rheumatica or severe ischemic complications., Conclusion: Our results showed no association of IRF5 rs2004640 and CGGGG insertion/deletion polymorphisms in the susceptibility to and clinical expression of GCA.

Objectives: To assess whether the polymorphism of the macrophage migration inhibitory factor (MIF) gene at the position -173 is implicated in the disease susceptibility, risk of cardiovascular (CV) events and presence of subclinical atherosclerosis in patients with rheumatoid arthritis (RA)., Patients and Methods: A series of 293 unselected patients fulfilling the 1987 American College of Rheumatology classification criteria for RA seen at the rheumatology outpatient clinic of Hospital Xeral-Calde, Lugo, Spain and 526 matched controls were studied for differences in the MIF-173 G/C gene biallelic polymorphism. A total of 182 consecutive patients that had been periodically followed between March 1996 and September 1996 until patient's death or January 1, 2008 were assessed for the presence of CV events. Moreover, between March and December 2007, a subgroup of unselected RA patients with no history of CV events was studied for the presence of subclinical atherosclerosis by the assessment of the endothelial function (n=107) and the carotid artery intima-media thickness (IMT) (n=91) by ultrasonography studies. Patients and controls were genotyped for the MIF-173 G/C gene polymorphism using a PCR system with pre-developed TaqMan allelic discrimination assay., Results: No significant differences in allele or genotype frequencies for the MIF-173 gene polymorphism between RA patients and controls were found. Forty-four of the 182 patients followed between 1996 and January 2008 experienced CV events. Although the frequency of MIF-173 GG homozygous was increased in those who had CV events (88.6%) compared to those who did not suffer these complication (73.2%), the difference was not statistically significant. It was also the case when we analyzed the potential influence of MIF-173 genotypes in the presence of endothelial dysfunction or increased carotid IMT of patients with RA., Conclusions: Our results do not show that MIF-173 gene polymorphism may infer a direct risk for disease susceptibility or CV disease in patients with RA.

Objective: A novel association with a 100-kb region on chromosome 9 that contains the tumor necrosis factor receptor-associated factor 1 (TRAF1) and C5 genes has been observed in some autoimmune rheumatic diseases, in particular in rheumatoid arthritis. We analyzed the influence of 2 single-nucleotide polymorphisms (SNP) from the TRAF1/C5 region in susceptibility to giant cell arteritis (GCA)., Methods: We assessed 220 patients with biopsy-proven GCA and 410 matched controls. DNA from patients and controls was obtained from peripheral blood. Samples were genotyped for the rs10818488 and rs2900180 TRAF1/C5 gene polymorphisms by polymerase chain reaction, using a predesigned TaqMan allele discrimination assay., Results: A genotyping rate of 95% was achieved in this series of GCA. No significant differences in the genotype distribution between GCA patients and controls were found for the 2 SNP. GCA patients exhibited a reduced frequency of TRAF1/C5 AA homozygosity (7.6%) compared to controls (12.7%) but the difference was only marginally significant (OR 0.58, 95% CI 0.30-1.11, p = 0.07). The frequency of minor allele T of TRAF1/C5 rs2900180 was also slightly reduced in patients (24.3%) compared to controls (27.8%) (p = 0.19). No significant differences were observed when patients were stratified according to the presence of specific clinical disease features., Conclusion: Our results showed no influence of rs10818488 and rs2900180 TRAF1/C5 gene polymorphisms in susceptibility to and clinical expression of GCA.

Objective: To determine whether the interleukin (IL)6 -174 gene polymorphism may influence the development of subclinical atherosclerosis manifested by the presence of endothelial dysfunction in RA patients., Patients and Methods: 311 patients (228 [73.3%] women; 243 [78.1%] rheumatoid factor positive) who fulfilled the 1987 ACR classification criteria for RA seen at the Rheumatology outpatient clinic of Hospital Xeral-Calde, Lugo between March 1996 and December 2006 and 226 matched controls were included in this study. Between March and December 2007, a subgroup of 98 patients randomly selected was assessed for the presence of endothelial dysfunction. Patients and controls were genotyped for a single biallelic (G/C) nucleotide polymorphism (rs1800795) in the promoter region at the position -174 of the IL6 gene using a TaqMan 5' allele discrimination assay., Results: No significant differences in the IL6 -174 allele or genotype frequency between RA patients and controls were found. However, RA patients homozygous for the IL6 -174 GG genotype had more severe endothelial dysfunction (flow-mediated endothelium-dependent vasodilatation-FMD%: 4.2 + or - 6.6) than those carrying the IL6 -174 GC (FMD%: 6.3 + or - 8.1) or IL6 -174 CC (FMD%: 6.0 + or - 3.3) genotypes. In this regard, significant differences were observed when FMD% values in RA patients carrying the IL6 -174 GG genotype were compared with that observed in those carrying the IL6 -174 GC and the IL6 -174 CC genotypes (FMD%: 6.3 + or - 4.6) (p=0.02)., Conclusions: Our results support a role of IL6 -174 gene polymorphism in the development of subclinical atherosclerosis in patients with RA.

Objectives: Genome-wide association studies have reported the role of the interleukin (IL) 2-IL21 chromosomal region at 4q27 in several autoimmune conditions. Mice deficient in IL-2 develop a disease with clinical and histological similarity to ulcerative colitis (UC) in humans. Modest evidence of linkage with UC was tentatively proposed for the IL2 gene more than a decade ago. Therefore, we decide to investigate the association of polymorphisms in the IL-2 axis (IL2, IL2RA, and IL2RB genes) with inflammatory bowel diseases (IBDs)., Methods: Seven hundred and twenty-eight white Spanish unrelated IBD patients (356 Crohn's disease (CD) and 372 UC) and 549 ethnically matched controls were included in a case-control study. In addition, a Spanish replication cohort with 562 CD and 430 UC patients and 1,310 controls were analyzed. Eight single-nucleotide polymorphisms previously associated with different autoimmune diseases were analyzed using TaqMan chemistry., Results: The IL2-rs6822844 polymorphism modified CD predisposition (P=0.002; odds ratio, OR (95% confidence interval, CI)=0.61 (0.44-0.84)); this was replicated in the other Spanish cohort, resulting in a strong protective effect of the minor allele in the merged samples (P=0.0002; OR (95% CI)=0.70 (0.58-0.85)). A similar effect of rs6822844 was detected for UC. Another marker, rs11938795, also showed evidence of an association with CD (P=0.006; OR (95% CI)=0.73 (0.58-0.92))., Conclusions: Polymorphisms within the IL2-IL21 linkage disequilibrium (LD) block show a novel association with IBD, this is concordant with suggestive previous results of whole genome analyses in CD and type 1 diabetes. Our data agree with the effect previously observed for other conditions and delineate a shared underlying mechanism.

Objective: Dendritic cells localized at the adventitia-media border of the normal medium-sized arteries play a pivotal role in the initiation of giant cell arteritis (GCA). These cells express a singular surface receptor profile, including a series of Toll-like receptors (TLR). Ligands of TLR-4 promote activation and differentiation of adventitial dendritic cells and are directly implicated in the pathogenesis of GCA. We aimed to assess the potential implication of the TLR4-(+896 A/G) gene polymorphism in the susceptibility to GCA., Methods: A total of 210 patients diagnosed with biopsy-proven GCA and 678 matched controls were included in our study. DNA from patients and controls was obtained from peripheral blood. Samples were genotyped for the TLR4-(+896 A/G) (rs4986790) gene polymorphism by polymerase chain reaction, using a predesigned TaqMan allele discrimination assay., Results: The TLR4 +896 G allele was significantly increased in biopsy-proven GCA patients compared to controls [p = 0.01; odds ratio (OR) 1.65; 95% confidence interval (CI) 1.08-2.52]. The increase was due to a significantly increased frequency of heterozygosity for the TLR4 -896 A/G genotype in the group of patients with biopsy-proven GCA compared to controls (TLR4 -896 A/G heterozygous in patients with GCA 18.1% compared to 11.4% in controls: p = 0.01; OR 1.72; 95% CI 1.10-2.69). However, no significant differences were observed when patients with GCA were stratified according to the presence of specific clinical features of the disease., Conclusion: Our results show for the first time an association of TLR4-(+896 A/G) gene polymorphism with susceptibility to biopsy-proven GCA.

Objective: To investigate the potential implication of the STAT4 gene polymorphism rs7574865 in the predisposition to or the clinical expression of giant cell arteritis (GCA)., Methods: A total of 212 patients diagnosed with biopsy-proven GCA were studied. DNA from patients and controls matched by age, sex, and ethnicity was obtained from peripheral blood. Samples were genotyped for STAT4 rs7574865 polymorphism., Results: No statistically significant differences in the allele frequencies for the STAT4 rs7574865 polymorphism were observed between patients and controls. Although we observed an increased frequency of the T/T genotype in GCA patients (6.0%) compared to healthy controls (3.9%), this difference did not achieve statistical significance (OR 1.57, 95% CI 0.72-3.41). No statistically significant differences in allele or genotype frequencies were observed when patients were stratified according to the presence of typical disease features such as polymyalgia rheumatica, severe ischemic manifestations, and visual ischemic complications in the setting of this vasculitis., Conclusion: Our results do not support a major role of the STAT4 rs7574865 gene polymorphism in susceptibility to or clinical manifestations of GCA.

Objective: To investigate the potential implication of several polymorphisms of the C-reactive protein (CRP) gene in the predisposition to or clinical expression of giant cell arteritis (GCA)., Methods: A total of 125 patients diagnosed with biopsy-proven GCA and 234 ethnically matched controls from the Lugo region of Northwestern Spain were included in our study. Four functional gene polymorphisms for CRP rs1417938, rs1800947, rs1205, and rs3093059 variants were assessed using a polymerase chain reaction system with predeveloped TaqMan allelic discrimination assay., Results: Although we observed a significant increase in the frequency of heterozygotes for rs1417938 A/T [odds ratio (OR) = 1.70; 95% confidence interval (CI) 1.04-2.80; p = 0.03] and rs1205 C/T (OR 1.73; 95% CI 1.07-2.78; p = 0.02) in patients with GCA, no statistically significant differences in the allelic frequencies of these 2 polymorphisms were found between patients with GCA and controls. A marginal significant increase in the frequency of rs3093059 allele T in patients with GCA compared to controls was observed (OR 1.81; 95% CI 0.97-3.39; p = 0.04). However, the increased frequency of patients with GCA homozygous for rs3093059 T/T in patients with GCA compared to controls was out of the range of significance (OR 1.77; 95% CI 0.92-3.40; p = 0.07). No significant differences were found when we stratified patients with GCA according to the presence of polymyalgia rheumatica or severe ischemic complications of the disease., Conclusion: The functional CRP gene polymorphisms assessed in our study do not seem to play a major role in the pathogenesis of GCA in individuals from Northwestern Spain.

Objective: Complex interactions between environmental and genetic determinants in both the host immune system and the vasculature may operate modifying the vascular risk in rheumatoid arthritis (RA). An increased incidence of cardiovascular (CV) events in RA patients carrying HLA-DRB1 shared epitope alleles, in particular HLA-DRB1*0404, has recently been found. In the present study we have assessed the potential contribution of inducible and endothelial nitric oxide synthase (NOS2A and NOS3) gene polymorphisms to CV events in a cohort of patients with rheumatoid arthritis (RA). Also, interactions between NOS2A or NOS3 gene polymorphisms and HLA-DRB1 alleles for the risk of developing CV events were assessed., Patients and Methods: One hundred and eighty-two consecutive patients fulfilling the 1987 American College of Rheumatology classification criteria for RA seen at the Rheumatology outpatient clinic of Hospital Xeral Calde, Lugo, Northwest Spain, between March and September 1996 were included. Patients were genotyped by PCR based techniques for a multiallelic (CCTTT)n repeat in the promoter region of the NOS2A gene and for a T/C polymorphism at position -786 in the promoter region and a polymorphism in exon 7 (298Glu/Asp or 5557G/T) of the NOS3 gene. They were prospectively followed and clinical records were examined until patient's death or September 1, 2005. At the end of the study 39 (21%) patients had experienced CV events., Results: No significant differences in allele or genotype frequencies for the NOS2A promoter CCTTT repeat microsatellite and NOS3 gene polymorphisms between RA patients with or without CV events were found. However, an increased frequency of CV events was observed in RA patients who carried the HLA-DRB1*0404 allele and were homozygous for the NOS3 (-786) TT genotype (OR: 9.06 [95% CI: 1.29-63.37]; p= 0.03) or for the presence of long NOS2A alleles (OR: 11.7 [95% CI: 1.53-88.4]); p= 0.02)., Conclusions: Our results show that NOS2A or NOS3 gene polymorphisms do not infer a direct risk for CV events in RA. However, some interactions between NOS gene polymorphisms and HLA-DRB1 alleles confer and increased risk of developing CV events in patients with RA.

Smooth muscle cells (SMCs) undergo changes related to proliferation and apoptosis in the physiological remodeling of vessels and in diseases such as atherosclerosis and restenosis. Recent studies also have demonstrated the vascular cell proliferation and programmed cell death contribute to changes in vascular architecture in normal development and in disease. The present study was designed to investigate the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, using an in vivo/in vitro cell model in which SMCs were isolated and culture from chicken exposed to an atherogenic cholesterol-rich diet (SMC-Ch) and/or an antiatherogenic fish oil-rich diet (SMC-Ch-FO). Cells were exposed in vitro to 25-hydroxycholesterol to study levels of apoptosis and apoptotic proteins Bcl-2, Bcl-X(L) and Bax and the expression of bcl-2 and bcl-x(L), genes. The quantitative real-time reverse transcriptase-polymerase chain reaction and the Immunoblotting western blot analysis showed that 25-hydroxycholesterol produces apoptosis in SMCs, mediated by a high increase in Bax protein and Bax gene expression. These changes were more marked in SMC-Ch than in SMC-Ch-FO, indicating that dietary cholesterol produces changes in SMCs that make them more susceptible to 25-hydroxycholesterol-mediated apoptosis. Our results suggest that the replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of cholesterol-induced changes in the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, making SMCs more resistant to apoptosis.