On meta- and mega-analyses for gene-environment interactions.

Gene-by-environment (G × E) interactions are important in explaining the missing heritability and understanding the causation of complex diseases, but a single, moderately sized study often has limited statistical power to detect such interactions. With the increasing need for integrating data and reporting results from multiple collaborative studies or sites, debate over choice between mega- versus meta-analysis continues. In principle, data from different sites can be integrated at the individual level into a "mega" data set, which can be fit by a joint "mega-analysis." Alternatively, analyses can be done at each site, and results across sites can be combined through a "meta-analysis" procedure without integrating individual level data across sites. Although mega-analysis has been advocated in several recent initiatives, meta-analysis has the advantages of simplicity and feasibility, and has recently led to several important findings in identifying main genetic effects. In this paper, we conducted empirical and simulation studies, using data from a G × E study of lung cancer, to compare the mega- and meta-analyses in four commonly used G × E analyses under the scenario that the number of studies is small and sample sizes of individual studies are relatively large. We compared the two data integration approaches in the context of fixed effect models and random effects models separately. Our investigations provide valuable insights in understanding the differences between mega- and meta-analyses in practice of combining small number of studies in identifying G × E interactions.

Peripheral blood leukocyte mitochondrial DNA content and risk of lung cancer.

Background: Previous studies of peripheral blood leukocyte mitochondrial DNA (mtDNA) content and risk of lung cancer have yielded inconsistent results, and no studies have evaluated the association between mtDNA content and post-resection lung cancer outcomes. Methods: Using a case-control study design, we evaluated the association between mtDNA content and risk of lung cancer in 465 cases and 378 controls. We also evaluated the association between mtDNA content and survival in 189 cases with surgically resected non-small cell lung cancer (NSCLC). Relative mtDNA content was measured using a quantitative real-time polymerase chain reaction (PCR) assay in peripheral blood genomic DNA. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using multivariable logistic regression, adjusting for age, gender, race, and smoking history. Results: mtDNA content was lower in cases compared to controls, with medians of 1.26 [interquartile range (IQR), 0.98-1.70)] and 1.79 (IQR, 1.34-2.10; P<0.001), respectively. Compared to the quartile of subjects with the highest mtDNA content, there was significantly higher likelihood of lung cancer in the second lowest quartile (OR 3.44; 95% CI: 2.06-5.75) and the lowest quartile (OR 6.36; 95% CI: 3.86-10.47). In patients with resected NSCLC, there was no association between lower mtDNA content and recurrence-free survival (RFS) [hazard ratio (HR) 0.89; 95% CI: 0.47-1.66] or overall survival (OS) (HR 0.71; 95% CI: 0.35-1.46). Conclusions: Thus, our results counter previous studies and find that lower mtDNA content is associated with lung cancer risk. Our results suggest that mtDNA content could potentially serve as a risk biomarker, but is not associated with survival outcomes in NSCLC.

Quantification of Plasma 8-Isoprostane by High-Performance Liquid Chromatography with Tandem Mass Spectrometry in a Case-Control Study of Lung Cancer.

AIM: 8-iso-prostaglandin F2α is a biomarker of lipid peroxidation, and one of the most commonly used measures of oxidative stress. It is an established biomarker of lung cancer risk. It is commonly measured by enzyme-linked immunosorbent assay. Given its importance, we developed a stable isotope dilution UPLC-tandem mass spectrometric method for the rapid determination of 8-isoprostane in blood. METHODS: We tested the discriminatory capability of the method in 49 lung cancer patients, 55 benign lung nodule patients detected by chest X-ray, and 41 patients with chronic obstructive pulmonary disease (COPD) or asthma. RESULTS: Significant differences were found in mean 8-isoprostane levels between the three groups (p = 0.027), and post-hoc tests found higher levels in the lung cancer patients than in patients with benign nodules (p = 0.032) and COPD/asthma (p = 0.014). The receiving operating characteristic area under the curve (AUC) was 0.69 for differentiating the lung cancer group from the benign nodule group, and 0.7 for differentiating from the COPD/asthma group. CONCLUSIONS: The UPLC-MS/MS-based method is an efficient analytical tool for measuring 8-isoprostane plasma concentrations. The results suggest exploring its utility as a marker for early lung cancer screening.

Genetic and lifestyle variables associated with homocysteine concentrations and the distribution of folate derivatives in healthy premenopausal women.

BACKGROUND: Low folate and high homocysteine (Hcy) concentrations are associated with pregnancy-related pathologies such as spina bifida. Polymorphisms in folate/Hcy metabolic enzymes may contribute to this potentially pathogenic biochemical phenotype. METHODS: The study comprised 26 Caucasian and 23 African-American premenopausal women. Subjects gave fasting blood samples for biochemical phenotyping and genotyping. Total Hcy (tHcy) and both plasma and red blood cell (RBC) folate derivatives (i.e. tetrahydrofolate [THF], 5-methylTHF [5-MTHF], and 5,10-methenylTHF [5,10-MTHF]) were measured using stable isotope dilution liquid chromatography, multiple reaction monitoring, and mass spectrometry. Eleven polymorphisms from nine folate/Hcy pathway genes were genotyped. Tests of association between genetic, lifestyle, and biochemical variables were applied. RESULTS: In African American women, tHcy concentrations were associated (p < 0.05) with total RBC folate, RBC 5-MTHF, B(12), and polymorphisms in methionine synthase (MTR) and thymidylate synthase (TYMS). In Caucasian women, tHcy concentrations were not associated with total folate levels, but were associated (p < 0.05) with RBC THF, ratios of RBC 5-MTHF:THF, and polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR) and MTR. In African Americans, folate derivative levels were associated with smoking, B(12), and polymorphisms in MTR, TYMS, methionine synthase reductase (MTRR), and reduced folate carrier1 (RFC1). In Caucasians, folate derivative levels were associated with vitamin use, B(12), and polymorphisms in MTHFR, TYMS, and RFC1. CONCLUSIONS: Polymorphisms in the folate/Hcy pathway are associated with tHcy and folate derivative levels. In African American and Caucasian women, different factors are associated with folate/Hcy phenotypes and may contribute to race-specific differences in the risks of a range of pregnancy-related pathologies.

The reduced folate carrier (SLC19A1) c.80G>A polymorphism is associated with red cell folate concentrations among women.

Low folate status may be a consequence of suboptimal intake, transport or cellular utilization of folate and, together with elevated homocysteine, is a recognized risk factor or marker for several human pathologies. As folate transport across cell membranes is mediated in part by the reduced folate carrier (RFC1), variants within SLC19A1, the gene that encodes RFC1, may influence disease risk via an effect on folate and/or homocysteine levels. The present study was undertaken to assess the association between the SLC19A1 c.80G>A polymorphism and folate/homocysteine concentrations in healthy young adults from Northern Ireland. The SLC19A1 c.80G>A polymorphism was not strongly associated with either serum folate or homocysteine concentrations in either men or women. However, in women, but not in men, this polymorphism explained 5% of the variation in red blood cell (RBC) folate levels (P= 0.02). Relative to women with the SLC19A1 c.80GG genotype, women with the GA and AA genotypes had higher RBC folate concentrations. Consequently, compared to women with the SLC19A1 c.80GA and AA genotypes, women who are homozygous for the 80G allele may be at increased risk of having a child affected with a neural tube defect and of developing pathologies that have been associated with folate insufficiency, such as cardiovascular disease.

Folate/homocysteine phenotypes and MTHFR 677C>T genotypes are associated with serum levels of monocyte chemoattractant protein-1.

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that recruits monocytes into the subendothelial cell layer in atherosclerotic lesions. Elevated homocysteine (hyperhomocysteinemia), which is usually associated with low-folate status, is a known risk factor for many pathologies with inflammatory etiologies. The present study was undertaken to examine whether there are associations between MCP-1 concentrations and folate/Hcy phenotype or methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype in healthy young adults. In females, MCP-1 concentrations were positively correlated with Hcy and negatively correlated with both serum and red blood cell folate; female smokers and MTHFR 677T carriers had particularly elevated MCP-1 concentrations. Similar relationships were not seen in males. These findings may have implications for understanding the female predominance observed for a range of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis.

Influence of the cystathionine beta-synthase 844ins68 and methylenetetrahydrofolate reductase 677C>T polymorphisms on folate and homocysteine concentrations.

A high homocysteine, low folate phenotype is a feature of many diseases. The effect of the cystathionine beta-synthase (CBS) 844ins68 polymorphism on homocysteine and folate concentrations was examined alone and in the context of the 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism in a Northwestern European male population. The MTHFR 677TT genotype is known to be associated with increased homocysteine and decreased folate relative to CT heterozygotes and CC homozygotes in this and other populations. MTHFR 677TT homozygotes who were also CBS 844ins68 carriers had homocysteine and folate concentrations similar to those of individuals with the MTHFR 677CT and CC genotypes. Homocysteine levels in MTHFR 677TT subjects carrying the CBS 844ins68 allele were 24.1% lower than in non-carriers (6.66 vs 8.77 micromol/l, P=0.045), and serum folate levels were 27.7% higher (11.16 vs 8.74 nmol/l, P=0.034). These findings suggest that the CBS 844ins68 allele 'normalizes' homocysteine and folate levels in MTHFR 677TT individuals.

An insertion/deletion polymorphism of the dihydrofolate reductase (DHFR) gene is associated with serum and red blood cell folate concentrations in women.

A low serum folate and high homocysteine phenotype is associated with an increased risk of neural tube defects (NTDs), cardiovascular diseases and other pathologies. Thus defining both genetic and non-genetic factors that may impact folate/homocysteine metabolism will enhance our understanding of the etiologic mechanisms underlying these conditions and facilitate risk assessment. Dihydrofolate reductase catalyzes the reduction of folic acid to dihydrofolate and thereafter to tetrahydrofolate. The impact of the dihydrofolate reductase (DHFR) c.86 + 60_78 insertion/deletion (ins/del) polymorphism on folate and homocysteine concentrations was analyzed using data from healthy young adults from Northern Ireland, collected as part of visit three of the Young Hearts Project. Among men the DHFR c.86 + 60_78 polymorphism was not significantly associated with serum or red blood cell folate concentrations, or with homocysteine concentrations. Among women the DHFR c.86 + 60_78 polymorphism explained 2% of the variation in RBC folate levels and 5% of the variation in serum folate levels, but did not appear to have an independent effect on homocysteine. Relative to women with the DHFR c.86 + 60_78 ins/ins and ins/del genotypes, del/del homozygotes had increased serum and red blood cell folate concentrations and may therefore be at decreased risk of having offspring affected by NTDs and of other adverse reproductive and health outcomes attributable to low folate.

Evidence for sex differences in the determinants of homocysteine concentrations.

A high homocysteine phenotype, often accompanied by low folate, is associated with several pathologies including cardiovascular disease and birth defects. This phenotype appears to be influenced by both genetic and environmental factors, which may act in a sex-dependent manner. The present analyses were undertaken to identify the determinants of homocysteine concentrations in young men and women, and are based on data from a cohort of young, reproductive age (20-26 years old) individuals in Northern Ireland. Multivariate modeling indicated that homocysteine concentrations are associated with red blood cell (RBC) folate, vitamin B(12), MTHFR 677C>T genotype and smoking status in both males and females. However, the inter-relationships between these variables appear to differ between the sexes. Specifically, homocysteine levels in males were significantly associated with interactions between MTHFR 677C>T genotype and both RBC folate and smoking status. In contrast, homocysteine levels in females were significantly associated with interactions between smoking status and RBC folate. These results suggest that the characteristics of individuals who are at the highest risk for a high homocysteine phenotype differ for males and females. Among males, those with the MTHFR 677TT genotype appear to be at the highest risk and to be the most vulnerable to factors (e.g. smoking, low RBC folate) that are associated with homocysteine raising effects. Among females, smokers (regardless of MTHFR genotype) appear to be at the highest risk, and to be the most vulnerable to a single factor (i.e. RBC folate) that is associated with homocysteine raising effects.

Genetic and biochemical determinants of serum concentrations of monocyte chemoattractant protein-1, a potential neural tube defect risk factor.

BACKGROUND: Women with the AA genotype at the (-2518)A>G promoter polymorphism of CCL-2, which encodes the potent pro-inflammatory chemokine monocyte chemoattractant protein 1 (MCP-1), may be at increased risk for having offspring affected by spina bifida. As the A allele at this locus has been associated with decreased transcription of MCP-1 mRNA relative to the G allele, the observed genetic association suggests that the risk of spina bifida may be increased in the offspring of women with low MCP-1 levels. The present study was undertaken to identify potential determinants of MCP-1 levels in women of reproductive age. METHODS: A small cohort of Caucasian and African-American women of reproductive age was recruited to participate in an exploratory investigation of the determinants of several disease-related, biochemical phenotypes, including MCP-1. Subjects completed a brief questionnaire and provided a fasting blood sample for biochemical and genetic studies. Potential biochemical, genetic, and lifestyle factors were assessed for their association with MCP-1 levels using linear regression analyses. RESULTS: In this cohort, MCP-1 levels were significantly higher in Caucasians as compared to African-Americans. Further, among women of both races, there was evidence that MCP-1 levels were associated with smoking status, MTHFR 677C>T genotype, and red blood cell tetrahydrofolate levels. CONCLUSIONS: The results of these analyses indicate that, if maternal CCL-2 genotype is related to the risk of spina bifida, this relationship is likely to be more complex than initially hypothesized, perhaps depending upon folate intake, MTHFR 677C>T genotype, the distribution of folate derivatives, and immune/inflammatory activity.

Thiol and cardiovascular risk factor status in a male northern Irish population.

OBJECTIVES: Raised plasma homocysteine is a risk factor for cardiovascular disease (CVD). Cysteine has also been associated with CVD risk. In this study, we investigated the association between known CVD risk factors, dietary factors, and total plasma cysteine, cysteinyl-glycine, and homocysteine. METHODS: The study group was 765 male workers aged between 30-49 years. The dietary habits of the subjects were recorded using a food frequency questionnaire. Body mass index (BMI), smoking status, and blood pressure were assessed, and fasting blood samples were taken for analysis of serum concentrations of vitamins, lipids, total plasma cysteine, cysteinyl-glycine, and homocysteine, and genotyping for the methylenetetrahydrofolate reductase (MTHFR) polymorphism. RESULTS: In multivariable analyses, cysteine was significantly positively associated with age and negatively associated with serum vitamin B12 and serum vitamin B6, while cysteinyl-glycine was significantly positively associated with BMI. Homocysteine (tHcy) was significantly negatively associated with serum folate, serum vitamin B12, and fruit and vegetable intake, and also depended on the MTHFR 677C>T genotype. CONCLUSIONS: Our data show a significant relationship between age, serum levels of B-vitamins and cysteine, and BMI and cysteinyl-glycine. In agreement with other studies, we also confirm an association between tHcy, serum folate and vitamin B12, MTHFR genotype, and fruit and vegetable intake. Further investigation into the role of these thiols and their determinants in CVD is required.

Association of methylenetetrahydrofolate reductase polymorphism and the risk of squamous cell carcinoma in renal transplant patients.

The relative risk of developing cutaneous squamous cell carcinoma (SCC) is significantly increased after organ transplantation. We investigated the genetic association of SCC in two pathways associated with cancer risks, with the potential for modification by vitamin supplementation. A total of 367 renal transplant recipients (117 with SCC and 250 without any skin cancer) were genotyped for key polymorphisms in the folate pathway (methylene tetrahydrofolate reductase; MTHFR:C677T), and the vitamin D pathway (vitamin D receptor: Intron8G/T;). Individuals carrying the MTHFR 677T allele had a marked increase in risk of SCC (adjusted odds ratio=2.54, P=0.002, after adjustment for age, ender, skin type, sun exposure score, and immunosuppression duration; lower 95% confidence boundary odds ratio of 1.41). In contrast, vitamin D receptor polymorphisms were not significantly associated. Folate-sensitive pathways may play a critical role in the elevated rate of SCC in renal transplant recipients.

Cytotoxic doses of ketoconazole affect expression of a subset of hepatic genes.

Ketoconazole is a widely prescribed antifungal drug, which has also been investigated as an anticancer therapy in both clinical and pre-clinical settings. However, severe hepatic injuries were reported to be associated with the use of ketoconazole, even in patients routinely monitored for their liver functions. Several questions concerning ketoconazole-induced hepatic injury remain unanswered, including (1) does ketoconazole alter cytochrome P450 expression at the transcriptional level?, (2) what types of gene products responsible for cytotoxicity are induced by ketoconazole?, and (3) what role do the major metabolites of ketoconazole play in this pathophysiologic process? A mouse model was employed to investigate hepatic gene expression following hepatotoxic doses of ketoconazole. Hepatic gene expression was analyzed using a toxicogenomic microarray platform, which is comprised of cDNA probes generated from livers exposed to various hepatoxicants. These hepatoxicants fall into five well-studied toxicological categories: peroxisome proliferators, aryl hydrocarbon receptor agonists, noncoplanar polychlorinated biphenyls, inflammatory agents, and hypoxia-inducing agents. Nine genes encoding enzymes involved in Phase I metabolism and one Phase II enzyme (glutathione S-transferase) were found to be upregulated. Serum amyloid A (SAA1/2) and hepcidin were the only genes that were downregulated among the 2364 genes assessed. In vitro cytotoxicity and transcription analyses revealed that SAA and hepcidin are associated with the general toxicity of ketoconazole, and might be usefully explored as generalized surrogate markers of xenobiotic-induced hepatic injury. Finally, it was shown that the primary metabolite of ketoconazole (de-N-acetyl ketoconazole) is largely responsible for the hepatoxicity and the downregulation of SAA and hepcidin.

Pretreatment Red Blood Cell Total Folate Concentration Is Associated With Response to Pemetrexed in Stage IV Nonsquamous Non-Small-cell Lung Cancer.

INTRODUCTION: Pemetrexed inhibits folate-dependent enzymes involved in pyrimidine and purine synthesis. Previous studies of genetic variation in these enzymes as predictors of pemetrexed efficacy have yielded inconsistent results. We investigated whether red blood cell (RBC) total folate, a phenotypic rather than genotypic, marker of cellular folate status was associated with the response to pemetrexed-based chemotherapy in advanced nonsquamous non-small-cell lung cancer (NSCLC). MATERIALS AND METHODS: We conducted a prospective cohort study of patients with stage IV nonsquamous NSCLC receiving first-line chemotherapy containing pemetrexed. The pretreatment RBC total folate level was quantified using liquid chromatography mass spectrometry. We then compared the objective response rate (ORR) between patients with RBC total folate concentrations greater than and less than an optimal cutoff value determined from the receiver operating characteristic curve. A logistic regression model was used to adjust for age, sex, and the use of bevacizumab. RESULTS: The ORR was 62% (32 of 52 patients). Receiver operating characteristic analysis was used to establish that a RBC total folate cutoff value of 364.6 nM optimally discriminated between pemetrexed responders and nonresponders. Patients with RBC total folate < 364.5 nM had an ORR of 27% compared with 71% for patients with RBC total folate > 364.5 nM (P = .01). This difference persisted after adjusting for age, sex, and the use of bevacizumab (odds ratio, 0.07; 95% confidence interval, 0.01-0.57; P = .01). CONCLUSION: A low pretreatment RBC total folate was associated with an inferior response to pemetrexed-based chemotherapy in stage IV nonsquamous NSCLC. Larger, multicenter studies are needed to validate RBC total folate as a predictive marker of pemetrexed response.

Mild folate deficiency induces a proatherosclerotic phenotype in endothelial cells.

Low folate/high homocysteine (Hcy) is an established risk marker for cardiovascular disease (CVD). Some in vivo studies suggest low folate may independently contribute to CVD. To study the effects of mild folate deficiency on endothelial function, we adapted the EA.hy 926 endothelial cell line to growth in medium containing 23 nM folic acid (LO cells) or 9 microM folic acid (HI cells). Folate derivatives were substantially depleted in LO cells relative to HI cells. No differences were seen in intracellular homocysteine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), the SAM:SAH ratio, or global DNA methylation, and there was no consistent difference in secreted homocysteine. A greater percentage of LO than HI cells were in S phase of the cell cycle; supplementation of LO cells with thymidine/hypoxanthine prevented this. LO cells were more elongated than HI cells and did not form tight monolayers. Stress fibers were very prominent in LO but not HI cells. Treatment of LO cells with rho kinase inhibitors abolished stress fibers and partially normalized cell shape. LO cell monolayers were more permeable than HI cell monolayers at confluence, and MCP-1 mRNA and protein expression was higher in LO than HI cells. Our results suggest that mild folate deficiency is proatherosclerotic.

Genome-wide association study confirms lung cancer susceptibility loci on chromosomes 5p15 and 15q25 in an African-American population.

OBJECTIVES: Genome-wide association studies (GWAS) of lung cancer have identified regions of common genetic variation with lung cancer risk in Europeans who smoke and never-smoking Asian women. This study aimed to conduct a GWAS in African Americans, who have higher rates of lung cancer despite smoking fewer cigarettes per day when compared with Caucasians. This population provides a different genetic architecture based on underlying African ancestry allowing the identification of new regions and exploration of known regions for finer mapping. MATERIALS AND METHODS: We genotyped 1,024,001 SNPs in 1737 cases and 3602 controls in stage 1, followed by a replication phase of 20 SNPs (p<1.51×10(-5)) in an independent set of 866 cases and 796 controls in stage 2. RESULTS AND CONCLUSION: In the combined analysis, we confirmed two loci to be associated with lung cancer that achieved the threshold of genome-wide significance: 15q25.1 marked by rs2036527 (p=1.3×10(-9); OR=1.32; 95% CI=1.20-1.44) near CHRNA5, and 5p15.33 marked by rs2853677 (p=2.8×10(-9); OR=1.28; 95% CI=1.18-1.39) near TERT. The association with rs2853677 is driven by the adenocarcinoma subtype of lung cancer (p=1.3×10(-8); OR=1.37; 95% CI=1.23-1.54). No SNPs reached genome-wide significance for either of the main effect models examining smoking - cigarettes per day and current or former smoker. Our study was powered to identify strong risk loci for lung cancer in African Americans; we confirmed results previously reported in African Americans and other populations for two loci near plausible candidate genes, CHRNA5 and TERT, on 15q25.1 and 5p15.33 respectively, are associated with lung cancer. Additional work is required to map and understand the biological underpinnings of the strong association of these loci with lung cancer risk in African Americans.

Spina bifida.

Spina bifida results from failure of fusion of the caudal neural tube, and is one of the most common malformations of human structure. The causes of this disorder are heterogeneous and include chromosome abnormalities, single gene disorders, and teratogenic exposures. However, the cause is not known in most cases. Up to 70% of spina bifida cases can be prevented by maternal, periconceptional folic acid supplementation. The mechanism underlying this protective effect is unknown, but it is likely to include genes that regulate folate transport and metabolism. Individuals with spina bifida need both surgical and medical management. Although surgical closure of the malformation is generally done in the neonatal period, a randomised clinical trial to assess in utero closure of spina bifida has been initiated in the USA. Medical management is a lifelong necessity for individuals with spina bifida, and should be provided by a multidisciplinary team.

Expression of alternatively spliced interleukin-1 receptor accessory protein mRNAs is differentially regulated during inflammation and apoptosis.

Two alternative splice variants of the interleukin-1 receptor accessory protein (IL-1RAcP) mRNA are known. Membrane-bound IL-1RAcP (mIL-1RAcP) promotes intracellular interleukin-1 (IL-1) signalling whereas soluble IL-1RAcP (sIL-1RAcP) is probably an inhibitor of IL-1 signalling. Here we establish that sIL-1RAcP mRNA levels increase 16-fold in response to phorbol esters in the human hepatoma cell line HepG2 via a mechanism that depends on de novo protein synthesis. Following exposure of cells to UV light, a potent inducer of apoptosis, mIL-1RAcP mRNA is rapidly down-regulated and a new steady-state level established briefly before a gradual return to pretreatment levels. Following treatment with staurosporine, also an inducer of apoptosis, mIL-1RAcP mRNA levels steadily decrease through 72 h, with little change in sIL-1RAcP mRNA levels. A novel alternative splice variant, sIL-1RAcP-beta, was identified. Its sequence indicates that sIL-1RAcP-beta is secreted and has a unique second half of the third immunoglobulin (Ig) domain. The dramatic changes in levels of IL-1RAcP mRNAs suggest important functions in regulating sensitivity to IL-1 during stress and may play a role in oncogenic processes that are engaged during chronic inflammation.

Genetic evidence that nitric oxide modulates homocysteine: the NOS3 894TT genotype is a risk factor for hyperhomocystenemia.

OBJECTIVE: Mild hyperhomocystenemia is an independent, graded risk factor for cardiovascular disease. Genetic determinants of hyperhomocystenemia include functional polymorphisms in several folate/homocysteine metabolic enzymes. Nitric oxide may also modulate plasma homocysteine (tHcy) concentrations, either by direct inhibition of methionine synthase or via an indirect effect on folate catabolism. METHODS AND RESULTS: The hypothesis that the endothelial nitric oxide synthase (NOS3) G894T polymorphism is a genetic determinant of tHcy concentrations was tested in 2 independent healthy adult populations. In both populations, NOS3 genotype was significantly associated with tHcy concentrations in nonsmokers with low folate (P=0.03 for each). Models were constructed to adjust for known determinants of tHcy concentrations and test for interactions between NOS3 genotype and these determinants in nonsmokers from each population. NOS3 genotype remained a significant determinant of tHcy concentrations after adjustment. Interactions between NOS3 genotype and serum folate were significant in both populations, and the interaction between NOS3 genotype and MTHFR C677T genotype was significant in the larger population. CONCLUSIONS: These data indicate that the NOS3 894TT genotype is a risk factor for elevated tHcy in healthy nonsmoking adults with low serum folate and supports the hypothesis that nitric oxide modulates homocysteine through an effect on folate catabolism.

TGF-beta down-regulates IL-1alpha-induced TLR2 expression in murine hepatocytes.

We have previously reported that the proinflammatory cytokine interleukin (IL)-1alpha can up-regulate functional Toll-like receptor 2 (TLR2) expression in primary-cultured murine hepatocytes, and bacterial lipopeptide (BLP) is capable of signaling through TLR2 to induce serum amyloid A (SAA) expression in hepatocytes. In the present study, we investigated the effect of the anti-inflammatory cytokine transforming growth factor-beta (TGF-beta) on TLR2 expression in primary-cultured murine hepatocytes. At the mRNA and protein levels, TGF-beta up-regulated TLR2 expression but inhibited TLR2 expression induced by IL-1alpha at 24 h. BLP-induced SAA promoter activity could be augmented by pretreatment with IL-1alpha but not TGF-beta or the combination of TGF-beta and IL-1alpha. TLR2 promoter activity and nuclear factor (NF)-kappaB activation by IL-1alpha were inhibited by TGF-beta treatment. Pretreatment with TGF-beta strongly suppressed IL-1alpha-induced TLR2 promoter activity and NF-kappaB activation, which was consistent with the down-regulation of type I IL-1 receptor (IL-1RI) mRNA expression. IL-1alpha up-regulated IL-1RI mRNA, but it was inhibited by the treatment with TGF-beta. These results suggest that TGF-beta suppresses the induction of TLR2 expression by IL-1alpha through down-regulation of IL-1RI expression. These results also demonstrate the disparity between IL-1alpha and TGF-beta in regulating TLR2-mediated SAA production in hepatocytes.