Lifestyle and demographic associations with 47 inflammatory and vascular stress biomarkers in 9876 blood donors.

BACKGROUND: The emerging use of biomarkers in research and tailored care introduces a need for information about the association between biomarkers and basic demographics and lifestyle factors revealing expectable concentrations in healthy individuals while considering general demographic differences. METHODS: A selection of 47 biomarkers, including markers of inflammation and vascular stress, were measured in plasma samples from 9876 Danish Blood Donor Study participants. Using regression models, we examined the association between biomarkers and sex, age, Body Mass Index (BMI), and smoking. RESULTS: Here we show that concentrations of inflammation and vascular stress biomarkers generally increase with higher age, BMI, and smoking. Sex-specific effects are observed for multiple biomarkers. CONCLUSION: This study provides comprehensive information on concentrations of 47 plasma biomarkers in healthy individuals. The study emphasizes that knowledge about biomarker concentrations in healthy individuals is critical for improved understanding of disease pathology and for tailored care and decision support tools.

Blood-based biomarkers are circulating molecules that can help to indicate health or disease. Biomarker levels may vary depending on demographic and lifestyle factors such as age, sex, smoking status, and body mass index. Here, we examine the effects of these demographic and lifestyle factors on levels of biomarkers related to activation of the immune system and cardiovascular stress. Measurements of 47 different proteins were performed on blood samples from nearly 10,000 healthy Danish blood donors. Measurement data were linked with questionnaire data to assess effects of lifestyle. We found that immune activation and vascular stress generally increased with age, BMI, and smoking. As these measurements are from healthy blood donors they can serve as a reference for expectable effects and inflammation levels in healthy individuals. Knowledge about the healthy state is important for understanding disease progression and optimizing care.

Symptoms of attention deficit hyperactivity disorder are associated with Hidradenitis suppurativa in Danish blood donors.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease associated with psychiatric comorbidity. Attention deficit hyperactivity disorder (ADHD) is a mental disorder associated with systemic and skin inflammation such as psoriasis and atopic dermatitis. Whether HS symptoms are associated with ADHD symptoms remains unexplored. Thus, the aim of this study was to explore the possible association between HS and ADHD. Participants in the Danish Blood Donor Study (DBDS) were included in this cross-sectional study during 2015-2017. The participants provided questionnaire data on screening items of HS, ADHD symptoms (ASRS-score), and depressive symptoms, smoking and body mass index (BMI). A logistic regression with HS symptoms as a binary outcome predicted by ADHD adjusted for age, sex, smoking, BMI, and depression was conducted to investigate the association between HS and ADHD. A total of 52,909 Danish blood donors were included in the study. Of these were 1004/52,909 (1.9%) considered participants with HS. Of the participants with HS, 74/996 (7.4%) screened positive of ADHD symptoms, while only 1786/51,129 (3.5%) of the participants without HS screened positive of ADHD. Adjusted for confounders, ADHD was positively associated with HS, odds ratio 1.85 (95% confidence interval: 1.43-2.37). Psychiatric comorbidity of HS is not limited to depression and anxiety. This study shows a positive association between HS and ADHD. Further research on the biological mechanisms behind this association is warranted.

Smoking is associated with infection risk in healthy blood donors.

OBJECTIVES: There is a gap in knowledge about the effects of smoking on overall infection risk in otherwise healthy populations, possibly leading to underestimation of the dangers of smoking. The present study aimed to examine the association of smoking with the risk of infections in a large cohort of healthy blood donors. METHODS: This cohort study used questionnaire and health register data from 127 831 Danish blood donors. Multivariable Cox proportional hazards analysis was applied to estimate the association of current smoking with the risk of all-cause infection defined as hospital-based treatment for infection or filled prescriptions of antimicrobials stratified for age and adjusted for relevant confounders. RESULTS: Among 18 272 current smokers, 12 272 filled an antimicrobial prescription and 2035 received hospital-based treatment for infections. Among 101 974 non-smokers, 65 117 filled a prescription and 8501 received hospital-based treatment for infections. Smokers had a higher risk of all-cause infection than non-smokers (hazard ratio estimates were 1.27 in males and 1.33 in females for hospital-based treatment and 1.11 in males and up to 1.20 in females for filled prescriptions). Smoking was most strongly associated with an increased incidence of respiratory tract infection, abscesses, skin infection, and prescriptions for these ailments (hazard ratio up to 2.29). Furthermore, smokers' risk of filled prescriptions of broad-spectrum penicillin was increased (hazard ratio up to 1.96). CONCLUSIONS: Current smoking was strongly associated with the risk of hospital-based treatment of infection and filled prescriptions of antimicrobials in a large cohort of healthy individuals. These findings warrant an increased focus on infectious disease risk among smokers.

Prevalence of major depressive disorder in 51,658 otherwise healthy adult Danes: Sex differences in symptomatology and prediction of future anti-depressive medication.

Major Depressive Disorder (MDD) is a heterogeneous disease, which displays sex differences in symptomatology. This study aimed to assess point prevalence of MDD in undiagnosed, healthy adults as well as sex differences in symptomatology and clarify if specific symptoms increased the later need for anti-depressive medication. The study included 51,658 blood donors. Depressive symptoms were assessed according to ICD-10 using the Major Depression Inventory. Demographics, previous MDD, anti-depressive medication were collected from questionnaires and population registers. Descriptive, Logistic and Cox regression analyses were conducted. In total, 1.15% participants met the criteria for MDD. Women were significantly more likely to experience "increased appetite" and less likely to experience "a feeling of life not worth living", compared to men. MDD significantly associated with an increased hazard of later receiving a prescription for anti-depressive medication. The risk increased proportionally with increasing MDD severity. The two symptoms, "feeling that life is not worth living" and "trouble sleeping" were the strongest individual predictive symptoms of future anti-depressive medication in women and men, respectively. The results confirm findings in MDD patient groups. The diagnostic and prognostic value should be investigated further to address their potential as part of the clinical assessment.

Increased soluble urokinase plasminogen activator levels modulate monocyte function to promote atherosclerosis.

People with kidney disease are disproportionately affected by atherosclerosis for unclear reasons. Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived mediator of kidney disease, levels of which are strongly associated with cardiovascular outcomes. We assessed suPAR's pathogenic involvement in atherosclerosis using epidemiologic, genetic, and experimental approaches. We found serum suPAR levels to be predictive of coronary artery calcification and cardiovascular events in 5,406 participants without known coronary disease. In a genome-wide association meta-analysis including over 25,000 individuals, we identified a missense variant in the plasminogen activator, urokinase receptor (PLAUR) gene (rs4760), confirmed experimentally to lead to higher suPAR levels. Mendelian randomization analysis in the UK Biobank using rs4760 indicated a causal association between genetically predicted suPAR levels and atherosclerotic phenotypes. In an experimental model of atherosclerosis, proprotein convertase subtilisin/kexin-9 (Pcsk9) transfection in mice overexpressing suPAR (suPARTg) led to substantially increased atherosclerotic plaques with necrotic cores and macrophage infiltration compared with those in WT mice, despite similar cholesterol levels. Prior to induction of atherosclerosis, aortas of suPARTg mice excreted higher levels of CCL2 and had higher monocyte counts compared with WT aortas. Aortic and circulating suPARTg monocytes exhibited a proinflammatory profile and enhanced chemotaxis. These findings characterize suPAR as a pathogenic factor for atherosclerosis acting at least partially through modulation of monocyte function.

IL-6 Autoantibodies Predict Lower Platelet Counts and Altered Plasma Cytokine Profiles in Healthy Blood Donors: Results From the Danish Blood Donor Study.

Cytokine-specific autoantibodies (c-aAb) represent a novel type of immune dysfunction. Though they have been detected in both patient cohorts and healthy individuals, and have immunomodulatory properties, the full extent of their influence remains unknown. Based on the critical role of several cytokines in thrombopoiesis, we investigated if there is an association between c-aAb and platelet variables in healthy individuals, with a specific focus on c-aAb against a known thrombopoietic cytokine, IL-6. Using platelet count and mean platelet volume in 3,569 healthy participants of the Danish Blood Donor Study as dependent variables, we performed a series of multivariate regression analyses using five cytokine autoantibodies, including IL-6 c-aAb, as independent variables. In men, high titers of IL-6 c-aAb were negatively associated with platelet counts (ß = -24 *109/l (95% confidence interval -43 to -6), p = 0.008) and positively associated with mean platelet volume (ß = 0.4 fL (95% confidence interval 0.0-0.7) p = 0.043). These associations were exacerbated when adjusting for undetectable C-reactive protein levels, which we used as a proxy for c-aAb mediated IL-6 inhibition in vivo. Furthermore, in a smaller subgroup, individuals with high vs. low titer IL-6 c-aAb had different profiles of plasma IL-6, IL-10, TNFα and TPO, further suggesting a functional inhibition of IL-6 by high titers of circulating IL-6 c-aAb. We therefore speculate that in addition to their immunomodulatory potential IL-6 c-aAb may interfere with thrombopoiesis - directly or indirectly - under normal physiological conditions. This study is the first to suggest an influence of c-aAb on platelets in healthy individuals, beyond their apparent effects on immune competence.

Population-based prevalence of cranial autonomic symptoms in migraine and proposed diagnostic appendix criteria.

BACKGROUND: Migraine with cranial autonomic symptoms is well described in the literature, but its prevalence in previous studies varies enormously. A precise estimate of the prevalence in a population-based material is important because migraine with cranial autonomic symptoms might represent an endophenotype, in which genetic and pathophysiological features differ from those without cranial autonomic features. The aim of the present study, therefore, was to estimate the prevalence in a big population-based sample using both questionnaire-based diagnosis (N = 12,620) and interview-based diagnosis (N = 302). We validate questionnaire-based diagnosis of migraine with cranial autonomic symptoms and develop the first diagnostic criteria for future research of this possible endophenotype. METHODS: The Danish Blood Donor Study included 127,802 persons who all received a migraine diagnostic questionnaire. Participants who had answered the diagnostic questionnaire constituted the Danish Migraine Population Cohort (N = 62,677) of whom 12,620 had migraine. The diagnostic migraine questionnaire included questions about the following cranial autonomic symptoms: Facial/forehead sweating, lacrimation, ptosis, conjunctival injection, rhinorrhea, nasal congestion, and miosis. Validation was performed by a follow-up semi-structured, purpose-built interview of 302 participants with migraine, where detailed questions were asked to ascertain the validity of the symptoms. RESULTS: The questionnaire-based prevalences of one, respectively two cranial autonomic symptoms were 57% and 31%. The semi-structured interview-based prevalences of one, respectively two symptoms were 44% and 22%. The most common symptoms were facial/forehead sweating (39%) and lacrimation (24%). The specificity of the questionnaire was 80% and the sensitivity was 68%. Correlation analysis showed a weak correlation between symptoms ranging from 0.07 - 0.41, and no clear clustering of symptoms was detected. We suggest the first diagnostic appendix criteria for genetic and epidemiological studies and tighter criteria for clinical and pathophysiological studies. We encourage further studies of severity and consistency of symptoms. CONCLUSION: Migraine with cranial autonomic symptoms is prevalent in the general population. Suggested diagnostic appendix criteria are important for future studies of this possible migraine endophenotype.

Chronic inflammation markers and cytokine-specific autoantibodies in Danish blood donors with restless legs syndrome.

Restless Legs Syndrome (RLS) is a neurological sensorimotor disorder negatively impacting sufferers' quality of sleep and health-related quality of life. The pathophysiology of RLS is poorly understood and research focusing on the link between RLS and inflammation has been limited. Our study aimed to investigate whether chronic inflammation markers C-reactive protein (CRP) and soluble urokinase-type plasminogen activator receptor (suPAR), as well plasma levels of five different cytokine-specific autoantibodies (c-aAb), i.e. modulators of inflammation, associate with RLS in otherwise healthy individuals. CRP, suPAR and c-aAb were measured in plasma samples of participants from the Danish Blood Donor Study in 2010. Returning donors between 2015 and 2018 completed the validated Cambridge-Hopkins RLS-questionnaire for RLS assessment, resulting in datasets with RLS assessment and values for CRP (N = 3564), suPAR (N = 2546) and c-aAb (N = 1478). We performed logistic regression models using the CRP, suPAR or c-aAb as the independent variable and RLS status as the dependent variable, adjusted for appropriate covariates. Our study indicates that a high concentration of CRP is associated with RLS, while an increased probability of experiencing frequent RLS symptoms in those with an elevated plasma suPAR level appears to be mediated through lifestyle factors. We additionally report that a high titer of autoantibodies specific against the cytokine interferon-alpha was associated with RLS. Our results support the existence of links between systemic inflammation and RLS, though further RLS studies on CRP, suPAR and c-aAb in larger cohorts are warranted to confirm our findings and further reveal the hitherto underexplored links between RLS and inflammation.

Rare SLC13A1 variants associate with intervertebral disc disorder highlighting role of sulfate in disc pathology.

Back pain is a common and debilitating disorder with largely unknown underlying biology. Here we report a genome-wide association study of back pain using diagnoses assigned in clinical practice; dorsalgia (119,100 cases, 909,847 controls) and intervertebral disc disorder (IDD) (58,854 cases, 922,958 controls). We identify 41 variants at 33 loci. The most significant association (ORIDD = 0.92, P = 1.6 × 10-39; ORdorsalgia = 0.92, P = 7.2 × 10-15) is with a 3'UTR variant (rs1871452-T) in CHST3, encoding a sulfotransferase enzyme expressed in intervertebral discs. The largest effects on IDD are conferred by rare (MAF = 0.07 - 0.32%) loss-of-function (LoF) variants in SLC13A1, encoding a sodium-sulfate co-transporter (LoF burden OR = 1.44, P = 3.1 × 10-11); variants that also associate with reduced serum sulfate. Genes implicated by this study are involved in cartilage and bone biology, as well as neurological and inflammatory processes.

No association between plasma hepcidin levels and restless legs syndrome - results from the Danish Blood Donor Study.

BACKGROUND: Restless Legs Syndrome (RLS) is a neurological sensorimotor disorder that occurs in the evening and night, thereby often impacting quality of sleep in sufferers. The aetiology of RLS is not completely understood although iron dysregulation has been suggested as a likely pathway. The relationship between RLS and the iron regulatory protein hepcidin has not been studied in large cohorts. We aimed to assess whether an association between plasma hepcidin variation and RLS exists in a large cohort of healthy individuals. METHODS: Plasma hepcidin levels were measured in 9708 Danish blood donors from the Danish Blood Donor Study all of whom correctly completed the validated Cambridge-Hopkins RLS-questionnaire for RLS assessment. RESULTS: A total of 466 blood donors were determined as current RLS cases in the sample (4.8%). RLS cases had a significantly higher proportion of females (56.7% vs 46.7%; P < 0.001) and were older (median age [IQR] 40.6 years vs 38.0 years; P = 0.010) than controls. RLS cases were also more frequent smokers (P = 0.004). No significant differences were found in body mass index (BMI), alcohol consumption, time of donation and donation history between cases and controls. No difference in plasma hepcidin levels was observed between RLS cases and controls (median concentration [IQR]: 10.5 ng/ml [6.3-16.4] in RLS cases vs 10.5 ng/ml [6.0-16.5] in controls). Using a logistic regression model, we found that hepcidin levels were not associated with RLS after adjusting for age, sex, alcohol consumption, smoking status, donation time and donation history (OR = 1.00 [0.99-1.02] per 1 ng/ml increase of hepcidin; P = 0.429). CONCLUSION: Our study in Danish blood donors did not find an association between RLS and plasma hepcidin levels. Our findings suggest that plasma hepcidin's role as a potential diagnostic biomarker of RLS is inadequate.

Investigating the inflammation marker neutrophil-to-lymphocyte ratio in Danish blood donors with restless legs syndrome.

BACKGROUND: Restless Legs Syndrome (RLS) is a neurological sensorimotor disorder that occurs in the evening and night, thereby impacting quality of sleep in sufferers. The pathophysiology of RLS is poorly understood but inflammation has been proposed as possibly being involved. The neutrophil-to-lymphocyte ratio (NLR) can be used as an inflammation marker but results from small studies have been inconclusive in determining whether NLR is associated with RLS. We aimed to assess whether an association between NLR and RLS exists in a large cohort of healthy individuals. METHODS: Neutrophils and lymphocytes were measured in blood samples of 13,055 individuals from the Danish Blood Donor Study, all of whom completed the validated Cambridge-Hopkins RLS-questionnaire for RLS assessment. RESULTS: In the sample, 661 individuals were determined as current RLS cases (5.1%). A higher proportion of individuals with RLS were females (62.5% vs 47.5%; P<0.001) and RLS cases were older than controls (P<0.001), but no differences in body mass index (BMI), smoking or alcohol consumption were found between the two groups. An increased NLR was observed in RLS cases compared to controls (median NLR: 1.80 vs 1.72; P = 0.033). In an unadjusted logistic regression model, increased NLR was associated with RLS (OR = 1.10 per NLR unit increase [95%CI:1.01-1.20]; P = 0.032); however, the association was not significant in multivariate models adjusting for sex and age (P = 0.094) or sex, age, alcohol consumption, smoking status and BMI (P = 0.107). CONCLUSION: We found no association between RLS and NLR among Danish blood donors after adjusting for sex, age, alcohol consumption, smoking status and BMI. Further studies are needed to determine whether inflammation is a risk factor for RLS.

Atopic respiratory diseases and IgE sensitization are associated with leukocyte subset concentrations in 14,440 blood donors.

BACKGROUND AND AIMS: Allergic rhinitis (AR), allergic conjunctivitis (AC), and asthma are characterized by activation of the immune system. The aim of this study was to explore the long-term association between AR, AC, asthma, and specific immunoglobulin E (IgE) and blood platelet and leukocyte differential counts. MATERIAL AND METHODS: In the Danish Blood Donor Study, 14,440 participants from Central Denmark Region had platelet and leukocyte differential counts available and completed a questionnaire regarding AR, AC, and asthma. Of these participants, 8485 were tested for IgE to inhalation allergens. RESULTS: The prevalence of AR, AC, asthma, and IgE sensitization was 19%, 15%, 9%, and 29%, respectively. AR, AC, asthma, wheeze, and IgE sensitization was associated with increased blood eosinophil concentration even in IgE sensitized participants who did not report any allergy or asthma. The strongest associations were observed for participants with current disease. We found no differences in eosinophil concentration between months without symptoms and months with symptoms of AR and asthma. CONCLUSION: AR, AC, asthma, wheezing, and IgE sensitization to inhalation allergens are associated with increased eosinophil concentration. This may reflect a persistent inflammation even in periods without symptomatic disease.

The association between water hardness and xerosis-Results from the Danish Blood Donor Study.

BACKGROUND: The pathophysiology of xerosis depends on extrinsic and intrinsic exposures. Residential hard water may constitute such an exposure. OBJECTIVES: To estimate the prevalence of xerosis and to compare water hardness exposure in blood donors with and without xerosis. METHODS: In this retrospective cohort study in 2018-2019, blood donors with self-reported moderately or severely dry skin were compared to blood donors without dry skin. Blood donors with ichthyosis, lichen planus and psoriasis were excluded. Water hardness data was collected from the Geology Survey of Denmark and Greenland. RESULTS: Overall, 4,748 of 30,721 (15.5%; 95% confidence interval 15.1-15.9%) blood donors had xerosis. After excluding blood donors with ichthyosis, lichen planus and psoriasis, 4,416 blood donors (2,559 females; median age 38.4 years [interquartile range 28.0-49.8]; 700 smokers) remained in this study. Water softer than 12-24 degrees Deutsche härte was associated with decreased probability of xerosis (odds ratio 0.83; 95% confidence interval 0.74-0.94) and water harder than 12-24 degrees Deutsche härte was associated with increased probability of xerosis (odds ratio 1.22; 95% confidence interval 1.03-1.45). The association between water hardness and xerosis remained significant after excluding blood donors with dermatitis. CONCLUSIONS: Water hardness is associated with xerosis independent of other dermatoses.

Eleven genomic loci affect plasma levels of chronic inflammation marker soluble urokinase-type plasminogen activator receptor.

Soluble urokinase-type plasminogen activator receptor (suPAR) is a chronic inflammation marker associated with the development of a range of diseases, including cancer and cardiovascular disease. The genetics of suPAR remain unexplored but may shed light on the biology of the marker and its connection to outcomes. We report a heritability estimate of 60% for the variation in suPAR and performed a genome-wide association meta-analysis on suPAR levels measured in Iceland (N = 35,559) and in Denmark (N = 12,177). We identified 13 independently genome-wide significant sequence variants associated with suPAR across 11 distinct loci. Associated variants were found in and around genes encoding uPAR (PLAUR), its ligand uPA (PLAU), the kidney-disease-associated gene PLA2R1 as well as genes with relations to glycosylation, glycoprotein biosynthesis, and the immune response. These findings provide new insight into the causes of variation in suPAR plasma levels, which may clarify suPAR's potential role in associated diseases, as well as the underlying mechanisms that give suPAR its prognostic value as a unique marker of chronic inflammation.

The impact of health-related quality of life and depressive symptoms on blood donor career-Results from the Danish blood donor study.

BACKGROUND: Blood donors report better health-related quality of life (HRQL) than non-donors. Likewise, donors reporting good health are less likely to stop donating and have a higher donation frequency. This is evidence of the healthy donor effect (HDE). This study is the first to investigate the impact of HRQL and depressive symptoms on subsequent donor career. STUDY DESIGN AND METHODS: Prospective cohort study includes 102,065 participants from the Danish Blood Donor Study applying the 12-item short-form health survey (SF-12) measuring a mental (MCS) and a physical component score (PCS) and the Major Depression Inventory (MDI). Poisson and Cox regression models were used to assess the effect of SF-12 and MDI scores on donation frequency and donor cessation. Higher MCS/PCS scores indicate good HRQL, while higher MDI score indicates higher experience of depressive symptoms. RESULTS: For both sexes, MCS was positively correlated with donation frequency for up to 5 years, and similarly for PCS among women. A negative correlation between MDI score and donation frequency in the year following assessment was observed only among men. No correlation was observed among women. An increase in both MCS and PCS was associated with a lower risk of donation cessation in both sexes, while an increase in MDI score was only associated with an increased risk of donation cessation in men. CONCLUSION: MCS, PCS, and MDI score affect donor career. Thus, adjusting for donation frequency may reduce HDE-bias in donor health research. However, because of the small effect sizes, other ways of quantifying HDE may be beneficial.

A genome-wide meta-analysis yields 46 new loci associating with biomarkers of iron homeostasis.

Iron is essential for many biological functions and iron deficiency and overload have major health implications. We performed a meta-analysis of three genome-wide association studies from Iceland, the UK and Denmark of blood levels of ferritin (N = 246,139), total iron binding capacity (N = 135,430), iron (N = 163,511) and transferrin saturation (N = 131,471). We found 62 independent sequence variants associating with iron homeostasis parameters at 56 loci, including 46 novel loci. Variants at DUOX2, F5, SLC11A2 and TMPRSS6 associate with iron deficiency anemia, while variants at TF, HFE, TFR2 and TMPRSS6 associate with iron overload. A HBS1L-MYB intergenic region variant associates both with increased risk of iron overload and reduced risk of iron deficiency anemia. The DUOX2 missense variant is present in 14% of the population, associates with all iron homeostasis biomarkers, and increases the risk of iron deficiency anemia by 29%. The associations implicate proteins contributing to the main physiological processes involved in iron homeostasis: iron sensing and storage, inflammation, absorption of iron from the gut, iron recycling, erythropoiesis and bleeding/menstruation.