BackgroundHigh intake of fruits and vegetables is generally associated with reduced levels of inflammation. In line with this, plasma levels of carotenoids have shown inverse associations with inflammatory markers, in particular C-reactive protein (CRP) and leukocyte counts. However, it remains unclear to what extent carotenoids are associated with specific leukocyte subsets or other inflammatory markers. This study systematically assessed the inter-relationships among total and individual carotenoids, circulating leukocyte subsets, and soluble inflammatory markers in a middle-aged population.MethodsA subcohort of 1078 subjects, aged 50-64, was recruited from the Swedish CArdioPulmonary bioImage Study (SCAPIS) cohort. Leukocyte subsets were determined by whole blood flow cytometry. Five major carotenoids, namely lutein, beta-cryptoxanthin, lycopene, alpha-carotene and beta-carotene, and inflammatory markers including CRP, interleukin (IL)-6 and interleukin-18, matrix metalloproteinase (MMP)-9, and myeloperoxidase (MPO), were measured in plasma. Nutrient intake was estimated by validated food frequency questionnaires.ResultsAmong leukocyte subsets, only granulocytes showed independent and inverse associations with all carotenoids after adjustment. CRP, IL-18, and MMP-9 exhibited similar inverse relationships with most carotenoids. Mediation analysis revealed that the associations of carotenoids with CRP and MMP-9 were mediated by granulocyte counts. Lutein and beta-cryptoxanthin remained independently associated with MMP-9 after accounting for the mediation effects of granulocyte counts. No estimated nutrient intake showed comparable associations with leukocyte subsets or inflammatory markers.ConclusionsTo our knowledge, this is the largest population-based study investigating relationships between plasma carotenoids, leukocyte subsets, and soluble inflammatory markers. It provides evidence that low levels of carotenoids in plasma are linked to low-grade chronic inflammation and, furthermore, that this relationship is mediated by granulocyte numbers.

Background Fatty degeneration of thymus (or thymus involution) has long been considered a normal ageing process. However, there is emerging evidence that thymic involution is linked to T cell aging, chronic inflammation and increased morbidity. Other factors, aside from chronological age, have been proposed to affect the involution rate. In the present study, we investigated the imaging characteristics of thymus on computed tomography (CT) in a Swedish middle-aged population. The major aims were to establish the prevalence of fatty degeneration of thymus and to determine its associations with demographic, lifestyle and clinical factors, as well as inflammation, T cell differentiation and thymic output. Results In total, 1 048 randomly invited individuals (aged 50-64 years, 49% females) were included and thoroughly characterized. CT evaluation of thymus included measurements of attenuation, size and a 4-point scoring system, with scale 0-3 based on the ratio of fat and soft tissue. A majority, 615 (59%) showed complete fatty degeneration, 259 (25%) predominantly fatty attenuation, 105 (10%) half fatty and half soft-tissue attenuation, while 69 (6.6%) presented with a solid thymic gland with predominantly soft-tissue attenuation. Age, male sex, high BMI, abdominal obesity and low dietary intake of fiber were independently associated with complete fatty degeneration of thymus. Also, fatty degeneration of thymus as well as low CT attenuation values were independently related to lower proportion of naive CD8(+) T cells, which in turn was related to lower thymic output, assessed by T- cell receptor excision circle (TREC) levels. Conclusion Among Swedish middle-aged subjects, nearly two-thirds showed complete fatty degeneration of thymus on CT. This was linked to depletion of naive CD8(+) T cells indicating that CT scans of thymus might be used to estimate immunological aging. Furthermore, our findings support the intriguing concept that obesity as well as low fiber intake contribute to immunological aging, thereby raising the possibility of preventive strategies.

OBJECTIVES: To investigate the association between type I collagen α1 chain (COL1α1) levels and coronary artery disease (CAD) by using absolute quantification in plasma. Also, to investigate the correlates of COL1α1 to clinical characteristics and circulating markers of collagen metabolism.

DESIGN: Life conditions, Stress and Health (LSH) study: prospective cohort study, here with a nested case-control design.Assessing Platelet Activity in Coronary Heart Disease (APACHE) study: prospective cohort study.

SETTING: LSH: primary care setting, southeast Sweden.APACHE: cardiology department, university hospital, southeast Sweden.

PARTICIPANTS: LSH: 1007 randomly recruited individuals aged 45-69 (50% women). Exclusion criteria was serious disease. After 13 years of follow-up, 86 cases with primary endpoint were identified and sex-matched/age-matched to 184 controls.

APACHE: 125 patients with myocardial infarction (MI), 73 with ST-elevation MI and 52 with non-ST-elevation MI.

EXCLUSION CRITERIA: Intervention study participation, warfarin treatment and short life expectancy.

PRIMARY AND SECONDARY OUTCOME MEASURES: Primary outcome was the association between baseline COL1α1 and first-time major event of CAD, defined as fatal/non-fatal MI or coronary revascularisation after 13 years. Secondary outcomes were the association between the collagen biomarkers PRO-C1 (N-terminal pro-peptide of type I collagen)/C1M (matrix metalloproteinase-mediated degradation of type I collagen) and CAD; temporal change of COL1α1 after acute MI up to 6 months and lastly, correlates between COL1α1 and patient characteristics along with circulating markers of collagen metabolism.

RESULTS: COL1α1 levels were associated with CAD, both unadjusted (HR=0.69, 95% CI=0.56 to 0.87) and adjusted (HR=0.55, 95% CI=0.41 to 0.75). PRO-C1 was associated with CAD, unadjusted (HR=0.62, 95% CI=0.47 to 0.82) and adjusted (HR=0.61, 95% CI=0.43 to 0.86), while C1M was not. In patients with MI, COL1α1 remained unchanged up to 6 months. COL1α1 was correlated to PRO-C1, but not to C1M.

CONCLUSIONS: Plasma COL1α1 was independently and inversely associated with CAD. Furthermore, COL1α1 appeared to reflect collagen synthesis but not degradation. Future studies are needed to confirm whether COL1α1 is a clinically useful biomarker of CAD.

Lutein is a dietary lipophilic compound with anti-inflammatory properties. We havepreviously shown that dairy fat can improve the lutein content in spinach smoothies. It is, however,unclear whether fat concentrations and fermentation status in dairy products affect lutein liberation insmoothies. Moreover, plant-based milks vary in fat, protein, and fiber content which may affect luteindissolution. This study aimed to provide translatable information to consumers by comparing luteinliberation in spinach smoothies made with different dairy or plant-based liquids in domestic settings.The smoothies were digested in vitro, and liberated lutein was measured by high-performance liquidchromatography (HPLC). High-fat and medium-fat cow’s milk, as well as coconut milk with andwithout additives, were found to significantly improve lutein liberation by 36%, 30%, 25%, and 42%,respectively, compared to blending spinach with water alone. Adjustment models suggested thatthe effects of cow’s milk and coconut milk were derived from fat and protein, respectively. On theother hand, soymilk with and without additives showed significantly reduced lutein liberation by40% and 61%, respectively. To summarize, only 4 out of 14 tested liquids increased lutein liberationin spinach smoothies. The results highlight the importance of testing food companions for lipophilicactive ingredients.

Background: Inflammation and oxidative stress form a vicious circle in atherosclerosis. Oxidative stress can have detrimental effects on T cells. A unique subset of CD4(+) T cells, known as regulatory T (T-reg) cells, has been associated with atheroprotective effects. Reduced numbers of T-reg cells is a consistent finding in patients with chronic coronary syndrome (CCS). However, it is unclear to what extent these cells are sensitive to oxidative stress. In this pilot study, we tested the hypothesis that oxidative stress might be a potential contributor to the T-reg cell deficit in CCS patients. Methods: Thirty patients with CCS and 24 healthy controls were included. T-reg (CD4+CD25+CD127(-)) and conventional T (CD4+CD25(-), T-conv) cells were isolated and treated with increasing doses of H2O2. Intracellular ROS levels and cell death were measured after 2 and 18 h, respectively. The expression of antioxidant genes was measured in freshly isolated T-reg and T-conv cells. Also, total antioxidant capacity (TAC) was measured in fresh peripheral blood mononuclear cells, and oxidized (ox) LDL/LDL ratios were determined in plasma. Results: At all doses of H2O2, T-reg cells accumulated more ROS and exhibited higher rates of death than their T-conv counterparts, p < 0.0001. T-reg cells also expressed higher levels of antioxidant genes, including thioredoxin and thioredoxin reductase-1 (p < 0.0001), though without any differences between CCS patients and controls. T-conv cells from CCS patients were, on the other hand, more sensitive to oxidative stress ex vivo and expressed more thioredoxin reductase-1 than T-conv cells from controls, p < 0.05. Also, TAC levels were lower in patients, 0.97 vs 1.53 UAE/100 mu g, p = 0.001, while oxLDL/LDL ratios were higher, 29 vs 22, p = 0.006. Conclusion: T-reg cells isolated from either CCS patients or healthy controls were all highly sensitive to oxidative stress ex vivo. There were signs of oxidant-antioxidant imbalance in CCS patients and we thus assume that oxidative stress may play a role in the reduction of T-reg cells in vivo.

Identification of biomarkers can help monitor and prevent cardiovascular disease (CVD) risk. We performed an exploratory analysis to identify potential biomarkers for coronary heart disease (CHD) in participants from the Life Conditions, Stress, and Health study. A total of 1,007 participants (50% women), randomly selected from the general population, were followed for incident CHD at 8 and 13 years of follow-up. Plasma levels of 184 CVD-related biomarkers were measured in samples collected at baseline in 86 cases with CHD and 184 age- and sex-matched controls by proximity extension assay. Biomarker levels were presented as normalized protein expression values (log 2 scale). After adjusting for confounding factors, 6 biomarkers showed significant association with incident CHD at 13 years. In a sensitivity analysis, this association remained significant at 8 years for 3 biomarkers; collagen alpha-1(I) chain (COL1A1), bone morphogenetic protein-6 (BMP-6), and interleukin-6 receptor alpha chain (IL-6R alpha). When entering these biomarkers in the full adjustment model simultaneously, their association with incident CHD at 13 years remained significant, hazards ratio being 0.671, 0.335, and 2.854, respectively per unit increase in normalized protein expression values. Subjects with low COL1A1, low BMP-6, and high IL-6R alpha levels had a hazards ratio of 5.097 for incident CHD risk (p = 0.019), compared with those without. In conclusion, we identified COL1A1, BMP-6 and IL-6Ra as biomarkers for incident CHD over a long-term follow-up in this exploratory analysis. For COL1A1 and BMP-6 this has not been previously reported. Further studies are needed to confirm our findings and establish their clinical relevance.

Lutein, abundant in dark leafy vegetables, has been associated with several health promoting effects. Still, to what extent different preparation conditions and practices affect the liberation of lutein from food is not fully understood. Here, we compared a range of domestic methods under realistic conditions to prepare spinach, the most common lutein-rich vegetable. After preparations, samples were processed by in vitro digestion and lutein was quantified by HPLC. Data indicate that short-term and medium-term heating of spinach, independent of heating method, substantially reduced liberated lutein and reduction was most pronounced after long boiling times. Interestingly, the loss of lutein in heated samples was partly compensated when samples were reheated in the microwave. However, the highest yield of liberated lutein was obtained from liquefied spinach. Additional dairy enhanced the liquefaction effect. Thus, for optimal liberation of lutein, liquefaction of raw spinach appears to be the method of choice.

Background Low-grade systemic inflammation is a predictor of recurrent cardiac events in patients with coronary artery disease (CAD). Plasma proteins such as matrix metalloproteinase (MMP)-9 and myeloperoxidase (MPO) have been shown to reflect basal as well as stress-induced inflammation in CAD. Measurements of MMP-9 and MPO in saliva might pose several advantages. Therefore, we investigated whether salivary levels of MMP-9 and MPO corresponded to plasma levels in patients with coronary artery disease (CAD), both at rest and after acute physical exercise. Methods A bicycle ergometer test was used as a model for stress-induced inflammation. Twenty-three CAD patients performed the test on two occasions 3-6 months apart. Whole unstimulated saliva was collected before, directly after and 30 min after exercise while plasma was collected before and after 30 min. MMP-9 and MPO in saliva and plasma were determined by Luminex. Results MMP-9 and MPO levels were 2- to 4-fold higher in saliva than in plasma. Amongst the saliva samples, and also to a great extent amongst the plasma samples, the levels of both types of protein showed strong intercorrelations between the levels at rest and after exercise during the two visits. However, there were no (or weak) correlations between salivary and plasma MMP-9 and none between salivary and plasma MPO. Conclusion We conclude that salivary diagnostics cannot be used to assess systemic levels of MMP-9 and MPO in CAD patients, neither at rest nor after acute physical exercise.

Objective: Mechanisms behind sustained inflammation in patients with coronary artery disease (CAD) are not clarified but hypothalamus-pituitary-adrenal (HPA) axis dysfunction may have a role. Here, we investigated whether inflammatory status of peripheral blood mononuclear cells (PBMCs) was associated with altered glucocorticoid sensitivity in CAD patients. Methods: In 55 CAD patients and 30 controls, mRNA levels of GR-alpha, GR-beta, NF-kappa B, I kappa B alpha, MMP-9 and TIMP-1 were measured in PBMCs. Suppressive effects of dexamethasone on GR-alpha, GR-beta, NF-kappa B, I kappa B alpha, MMP-9 and TIMP-1 mRNA levels were assessed in PBMCs ex vivo. Salivary cortisol was repeatedly measured over 3 days. Results: GR-alpha mRNA levels were higher in CAD patients than in controls, 0.50 (0.38-0.59) versus 0.26 (0.18-0.37), pamp;lt;.001, while GR-beta mRNA levels were equally low in both groups. GR-alpha mRNA expression was associated with inflammatory gene expression and, also, with flatter diurnal cortisol rhythm. In both patients and controls, dexamethasone suppressed gene expression of NF-B, IB, MMP-9 and TIMP-1 (p amp;lt; .001). Dexamethasone also reduced GR-alpha mRNA levels (p amp;lt; .001), while LPS increased it (p amp;lt; .001). Conclusions: PBMCs from CAD patients displayed an inflammatory gene expression profile. This was not explained by reduced glucocorticoid sensitivity. Instead, inflammation was associated with increased expression of GR-alpha mRNA, suggesting a hypocortisolemic state.

Sphingomyelin (SM) levels in the circulation correlate positively with atherosclerosis burden. SM is a ubiquitous component of human diets, but it is unclear if dietary SM increases circulating SM levels. Dietary choline increases atherosclerosis by raising circulating trimethylamine N-oxide (TMAO) levels in mice and humans. As SM has a choline head group, we ask in this study if dietary SM accelerates atherosclerotic lesion development by increasing circulating SM and TMAO levels. Three studies were performed: (Study 1) C57BL/6 mice were maintained on a high fat diet with or without SM supplementation for 4 weeks prior to quantification of serum TMAO and SM levels; (Study 2) atherosclerosis was studied in apoE(-/-) mice after 16 weeks of a high fat diet without or with SM supplementation and (Study 3) apoE(-/-) mice were maintained on a chow diet for 19 weeks without or with SM supplementation and antibiotic treatment prior to quantification of atherosclerotic lesions and serum TMAO and SM levels. SM consumption did not increase circulating SM levels or atherosclerosis in high fat-fed apoE(-/-) mice. Serum TMAO levels in C57BL/6 mice were low and had no effect atherosclerosis lesion development. Dietary SM supplementation significantly reduced atherosclerotic lesion area in the aortic arch of chow-fed apoE(-/-) mice. This study establishes that dietary SM does not affect circulating SM levels or increase atherosclerosis in high fat-fed apoE(-/-) mice, but it is anti-atherogenic in chow-fed apoE(-/-) mice.