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.

Longitudinal changes and variation in human DNA methylation analysed with the Illumina MethylationEPIC BeadChip assay and their implications on forensic age prediction.

DNA methylation, a pivotal epigenetic modification, plays a crucial role in regulating gene expression and is known to undergo dynamic changes with age. The present study investigated epigenome-wide methylation profiles in 64 individuals over two time points, 15 years apart, using the Illumina EPIC850k arrays. A mixed-effects model identified 2821 age-associated differentially methylated CpG positions (aDMPs) with a median rate of change of 0.18% per year, consistent with a 10-15% change during a human lifespan. Significant variation in the baseline DNA methylation levels between individuals of similar ages as well as inconsistent direction of change with time across individuals were observed for all the aDMPs. Twenty-three of the 2821 aDMPs were previously incorporated into forensic age prediction models. These markers displayed larger changes in DNA methylation with age compared to all the aDMPs and less variation among individuals. Nevertheless, the forensic aDMPs also showed inter-individual variations in the direction of DNA methylation changes. Only cg16867657 in ELOVL2 exhibited a uniform direction of the age-related change among the investigated individuals, which supports the current knowledge that CpG sites in ELOVL2 are the best markers for age prediction.

Children and adolescents with attention deficit hyperactivity disorder and autism spectrum disorder share distinct microbiota compositions.

An association has been suggested between altered gut microbiota, and attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD), respectively. Thus, we analyzed the gut microbiota composition in children and adolescents with or without these disorders and evaluated the systemic effects of these bacteria. We recruited study participants diagnosed with ADHD, ASD, and comorbid ADHD/ASD, while the control groups consisted both of siblings and non-related children. The gut microbiota was analyzed by 16S rRNA gene sequencing of the V4 region, while the concentration of lipopolysaccharide-binding protein (LBP), cytokines, and other signaling molecules were measured in plasma. Importantly the gut microbiota compositions of cases with ADHD and ASD were highly similar for both alpha- and beta-diversity while differing from that of non-related controls. Furthermore, a subset of ADHD and ASD cases had an increased LBP concentration compared to non-affected children, which was positively correlated with interleukin (IL)-8, 12, and 13. These observations indicate disruption of the intestinal barrier and immune dysregulation among the subset of children with ADHD or ASD.

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.

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.

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.

Large-scale study of Toxoplasma and Cytomegalovirus shows an association between infection and serious psychiatric disorders.

BACKGROUND: Common infectious pathogens have been associated with psychiatric disorders, self-violence and risk-taking behavior. METHODS: This case-control study reviews register data on 81,912 individuals from the Danish Blood Donor Study to identify individuals who have a psychiatric diagnosis (N = 2591), have attempted or committed suicide (N = 655), or have had traffic accidents (N = 2724). For all cases, controls were frequency matched by age and sex, resulting in 11,546 participants. Plasma samples were analyzed for immunoglobulin G (IgG) antibodies against Toxoplasma gondii and cytomegalovirus (CMV). RESULTS: T. gondii was detected in 25·9% of the population and was associated with schizophrenia (odds ratio [OR], 1·47; 95% confidence interval [CI], 1·03-2·09). Accounting for temporality, with pathogen exposure preceding outcome, the association was even stronger (IRR, 2·78; 95% CI, 1·27-6·09). A very weak association between traffic accident and toxoplasmosis (OR, 1·11; 95% CI, 1·00-1·23, p = 0.054) was found. CMV was detected in 60·8% of the studied population and was associated with any psychiatric disorder (OR, 1·17; 95% CI, 1·06-1·29), but also with a smaller group of neurotic, stress-related, and somatoform disorders (OR, 1·27; 95% CI, 1·12-1·44), and with attempting or committing suicide (OR, 1·31; 95% CI, 1·10-1·56). Accounting for temporality, any psychiatric disorder (IRR, 1·37; 95% CI, 1·08-1·74) and mood disorders (IRR, 1·43; 95% CI, 1·01-2·04) were associated with exposure to CMV. No association between traffic accident and CMV (OR, 1·06; 95% CI, 0·97-1·17) was found. CONCLUSIONS: This large-scale serological study is the first study to examine temporality of pathogen exposure and to provide evidence of a causal relationship between T. gondii and schizophrenia, and between CMV and any psychiatric disorder.

Impact of polymorphisms in the HCP5 and HLA-C, and ZNRD1 genes on HIV viral load.

AIMS: Single nucleotide polymorphisms (SNPs) in the human leucocyte antigen (HLA) complex P5 (HCP5), HLA-C, and near the zinc ribbon domain containing 1 (ZNRD1) have been shown to influence viral load (VL) set point in HIV-infected individuals with a known seroconversion onset. We aimed to determine the influence of HCP5 rs2395029, HLA-C rs9264942, and ZNRD1 rs3869068 on VL in antiretroviral-naïve individuals and on time to the first VL<51 copies/ml and on CD4(+) T-cell recovery after initiation of combination antiretroviral therapy (cART). MATERIAL AND METHODS: We genotyped the rs2395029 (A>C), rs9264942 (T>C), and rs3869068 (C>T) SNPs in 1897 Caucasians from The Danish HIV Cohort Study - a prospective, nationwide, population-based study of HIV-infected individuals in Denmark. General linear models evaluated the effect of SNPs on VL in antiretroviral-naïve individuals 0-18months after diagnosis and on CD4(+) T-cell recovery during cART. Cox proportional hazard regression analysis assessed the association with time to first VL<51 copies/ml. All models were assuming additive genetic effects. RESULTS: The rs2395029, rs9264942, and rs3869068 minor alleles were associated with lower VL in antiretroviral-naïve individuals (rs2395029: [mean VL (copies/ml)], A/A: 70,795 [61,660-79,433], A/C: 33,884 [19,498-58,884], P=0.002; rs9264942: TT: 81,283 [67,608-97,724], T/C: 63,096 [54,954-75,858], CC: 38,905 [25,119-58,884], P<0.0001; rs3869068, CC: 72,444 [63,096-83,176], C/T: 45,709 [33,113-64,565], TT: 58,884 [20,417-169,824], P=0.01). Moreover, the C-alleles of rs2395029 and rs9264942 were associated with shorter time to VL<51 copies/ml: (HR [95% confidence interval], 1.67 [1.09-1.72], P=0.008; 1.16 [1.06-1.28], P=0.002; 1.30 [1.08-1.53], P=0.005, respectively, adjusted for last VL before cART). None of the SNPs predicted CD4(+) T-cell recovery during cART. CONCLUSIONS: The minor alleles of rs2395029, rs9264942, and rs3689068 associate with lower VL among antiretroviral-naïve individuals and with shorter time to first VL<51copies/ml during cART even after adjustment for VL before cART.

Human leukocyte antigen-G and regulatory T cells during specific immunotherapy for pollen allergy.

BACKGROUND: TH2-biased immune responses are important in allergy pathogenesis. Mechanisms of allergen-specific immunotherapy (SIT) might include the induction of regulatory T cells (Tregs) and immunoglobulin (Ig) G4 blocking antibodies, a reduction in the number of effector cells, and skewing of the cytokine profile towards a TH1-polarized immune response. We investigated the effects of SIT on T cells, on immunomodulation of human leukocyte antigen (HLA)-G, which has been associated with allergy, on regulatory cytokine expression, and on serum allergen-specific antibody subclasses (IgE and IgG4). METHODS: Eleven birch and/or grass pollen-allergic patients and 10 healthy nonatopic controls were studied before and during SIT. Tregs, chemokine receptors, soluble HLA-G (sHLA-G), Ig-like transcript (ILT) 2, specific IgE, and IgG4 were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated with pollen extract in vitro and immune factors were evaluated. RESULTS: During SIT, the main changes in the peripheral blood were an increase in CXCR3(+)CD4(+)CD25(+)CD127(low/-) Tregs and a decrease in CCR4(+)CD4(+)CD25(+)CD127(low/-) Tregs, an increase in allergen-specific IgG4, and a decrease in sHLA-G during the first half of the treatment period. In the PBMC in vitro experiments, the following changes were observed upon allergen-stimulation: an increase in CD4(+)CD25(+)CD127(low/-) Tregs and ILT2(+)CD4(+)CD25(+)CD127(low/-) Tregs, an increase in IL-10 and IL-2 levels, and an increase in sHLA-G that was most pronounced at the start of SIT. CONCLUSIONS: The changes in CXCR3(+)CD4(+)CD25(+)CD127(low/-) Treg, IgG4, and sHLA-G levels in the peripheral blood and in ILT2(+) Treg, IL-10, IL-2, and sHLA-G levels upon in vitro allergen stimulation suggest an upregulation in immunomodulatory factors and, to some degree, a shift towards TH1 during SIT.

Genome-Wide Association Study of Genetic Variants in LPS-Stimulated IL-6, IL-8, IL-10, IL-1ra and TNF-α Cytokine Response in a Danish Cohort.

BACKGROUND: Cytokine response plays a vital role in various human lipopolysaccharide (LPS) infectious and inflammatory diseases. This study aimed to find genetic variants that might affect the levels of LPS-induced interleukin (IL)-6, IL-8, IL-10, IL-1ra and tumor necrosis factor (TNF)-α cytokine production. METHODS: We performed an initial genome-wide association study using Affymetrix Human Mapping 500 K GeneChip® to screen 130 healthy individuals of Danish descent. The levels of IL-6, IL-8, IL-10, IL-1ra and TNF-α in 24-hour LPS-stimulated whole blood samples were compared within different genotypes. The 152 most significant SNPs were replicated using Illumina Golden Gate® GeneChip in an independent cohort of 186 Danish individuals. Next, 9 of the most statistical significant SNPs were replicated using PCR-based genotyping in an independent cohort of 400 Danish individuals. All results were analyzed in a combined study among the 716 Danish individuals. RESULTS: Only one marker of the 500 K Gene Chip in the discovery study showed a significant association with LPS-induced IL-1ra cytokine levels after Bonferroni correction (P<10(-7)). However, this SNP was not associated with the IL-1ra cytokine levels in the replication dataset. No SNPs reached genome-wide significance for the five cytokine levels in the combined analysis of all three stages. CONCLUSIONS: The associations between the genetic variants and the LPS-induced IL-6, IL-8, IL-10, IL-1ra and TNF-α cytokine levels were not significant in the meta-analysis. This present study does not support a strong genetic effect of LPS-stimulated cytokine production; however, the potential for type II errors should be considered.

HLA-G 3' untranslated region 14-base pair deletion: association with poor survival in an HIV-1-infected Zimbabwean population.

We aimed to evaluate whether the HLA-G 14-base pair (bp) polymorphism (rs16375) has an impact on human immunodeficiency virus HIV progression and survival in an antiretroviral therapy-naive Zimbabwean cohort (n = 312). Rs16375 was genotyped using a competitive allele-specific polymerase chain reaction system; CD4 cell counts and HIV RNA were measured with flow cytometry and commercially available polymerase chain reaction; survival was followed up for 4.3 years. The homozygous HLA-G -14-bp genotype is associated with higher viral load (P = .004), lower CD4 cell count (P = .01), and increased mortality (hazard ratio, 1.9; 95% confidence interval, 1.033-3.522; P = .04) compared with HLA-G +14-bp carriers.

CCL3L gene copy number and survival in an HIV-1 infected Zimbabwean population.

The C-C motif chemokine ligand 3-like (CCL3L) protein is a potent chemoattractant which by binding to C-C chemokine receptor type 5 (CCR5) inhibits human immunodeficiency virus (HIV) entry. Copy number variation (CNV) of the CCL3L has been shown to be associated with HIV susceptibility and progression to AIDS, but these results have been inconsistent. We examined a Zimbabwean study population for an association of CCL3L CNV with HIV status, progression (CD4 T-cells and viral load), and survival. Another aim was to investigate the possible effects of CCL3L CNV on CCL3 protein concentration. A treatment-naïve cohort, which included 153 HIV infected and 159 HIV uninfected individuals, was followed for up to 4.3 years. The CNV of the CCL3L was determined by duplex real-time polymerase chain reaction. We found no association between four CCL3L CNV strata and HIV status (P=0.7), CD4 T-cell count (P=0.9), viral load (P=0.9), or CCL3 protein levels (P=1.0). Survival among the HIV infected individuals did not differ according to CCL3L copy number. In this cohort, CCL3L CNV did not affect HIV status, pathogenesis, or survival.

Human leukocyte antigen-G in the male reproductive system and in seminal plasma.

One of the non-classical human leukocyte antigen (HLA) class Ib proteins, HLA-G, is believed to exert important immunoregulatory functions, especially during pregnancy. The presence of HLA protein in paternal seminal fluid has been suggested to have an influence on the risk of developing pre-eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system. Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA-G in hyperplastic prostatic tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma.

Soluble human leukocyte antigen-G isoforms in maternal plasma in early and late pregnancy.

PROBLEM: Human Leukocyte Antigen (HLA)-G is a class Ib gene located in the human major histocompatibility complex (MHC). Several lines of investigation indicate that the HLA-G molecule is involved in the maternal acceptance of the semi-allogenic fetus during pregnancy and in the development of tolerance. Expression of soluble HLA-G (sHLA-G) is positively correlated with successful in vitro fertilization (IVF) treatments, and aberrant expression of HLA-G in certain complications of pregnancy, such as pre-eclampsia and spontaneous abortion, has been reported. The main purpose of this study was to investigate the levels of different soluble HLA-G isoforms in maternal plasma in early and late pregnancy. METHOD OF STUDY: Soluble HLA-G (sHLA-G) can be detected in maternal blood, and in this study, two different isoforms of sHLA-G, namely sHLA-G1 generated by shedding of membrane-bound HLA-G1 and HLA-G generated by specific HLA-G transcripts, have been investigated early [median of 16.4 weeks of gestation (GW)] and late (median: 38.9 GW) in pregnancy in an original cohort of 580 pregnant Caucasian women. RESULTS: Lower concentrations of sHLA-G1 were found late in pregnancy (>32 GW) in a group of women with severe pre-eclampsia compared with controls with uncomplicated pregnancies (P = 0.029, P(C) = 0.09; Mann-Whitney; Logistic regression analysis: P = 0.024, OR = 0.920, 95% CI: 0.855-0.989). However, this was not the case with HLA-G5, and significantly more of the cases with severe pre-eclampsia had detectable plasma HLA-G5 compared with that of the control group (P = 0.013, P(C) = 0.04; Mann-Whitney). Similar findings were not observed in women with gestational hypertension or existing hypertension continuing into pregnancy. Furthermore, there was a trend toward lower maternal plasma sHLA-G1 in a group of women with premature birth (<37 GW) compared with that of the control group (P = 0.028, P(C) = 0.17; Mann-Whitney). On the contrary, HLA-G5 was lower in the control group compared with that in the premature group (P = 0.004, P(C) = 0.02; Mann-Whitney). CONCLUSION: This study shows in line with other published studies that a high, detectable soluble HLA-G concentration in maternal plasma or serum is not mandatory for a successful pregnancy. However, complications during pregnancy, such as (severe) pre-eclampsia, spontaneous abortion, IUGR, and premature birth, are associated with a low or undetectable level of soluble HLA-G in the maternal blood circulation. Also, this study indicates that sHLA-G1 is the interesting soluble HLA-G isoform in pre-eclampsia, and that low or undetectable levels of HLA-G5 at the end of pregnancy seem to be associated with an uncomplicated normal pregnancy, whereas in severe pre-eclampsia and possibly other pregnancy complications, such as preterm birth and IUGR, the level of HLA-G5 is higher.

Human leukocyte antigen-G polymorphism in relation to expression, function, and disease.

Human leukocyte antigen-G (HLA-G) is a nonclassical class Ib molecule belonging to the major histocompatibility complex. HLA-G appears to play a role in the suppression of immune responses and contribute to long-term immune escape or tolerance. The focus of this review is polymorphism in the HLA-G gene and protein and its possible importance in expression, function, and disease associations.