Type 1 diabetes (T1D) and celiac disease (CeD) are common autoimmune diseases in children where the pathophysiology is not fully characterized. The autoimmune process involves a complex scenario of both inflammatory and regulatory features. Galectin-1 (GAL-1) has a wide range of biological activities e.g., interaction with immune cells. We examined the relationship between GAL-1 and soluble immune markers and T cell subsets in a cohort of children with T1D and/or CeD relative to healthy children. Galectin-1, together with several soluble immune markers (e g interleukins (IL)), tumor necrosis factor (TNF), acute phase proteins and matrix metalloproteinases (MMP) were measured in sera from children with T1D and/or CeD by fluorochrome (Luminex) technique using children without these diseases as a reference. Subgroups of T cells, including T regulatory (Treg) cells, were analyzed by flow cytometry. Association between GAL-1, pro-inflammatory markers and Treg cells differed depending on which illness combination was present. In children with both T1D and CeD, GAL-1 correlated positively with pro-inflammatory markers (IL-1β, -6 and TNF-α). Composite scores increased the strength of correlation between GAL-1 and pro-inflammatory markers, Th1-associated interferon (IFN)-γ, and T1D-associated visfatin. Contrary, in children diagnosed with exclusively T1D, GAL-1 was positively correlated to CD25hi and CD25hiCD101+ Treg cells. For children with only CeD, no association between GAL-1 and other immune markers was observed. In conclusion, the association observed between GAL-1, soluble immune markers and Treg cells may indicate a role for GAL-1 in the pathophysiology of T1D and, to some extent, also in CeD.

Our purpose was to characterize the pattern of B-cell subsets in children with a combined diagnosis of type 1 diabetes (T1D) and celiac disease (C) since children with single or double diagnosis of these autoimmune diseases may differ in peripheral B-cell subset phenotype patterns.

B-cells were analyzed with flow cytometry for the expression of differentiation/maturation markers to identify transitional, naive and memory B-cells. Transitional (CD24hiCD38hiCD19+) and memory Bregs (CD24hiCD27+CD19+, CD1d+CD27+CD19+, CD5+CD1d+CD19+) were classified as B-cells with regulatory capacity.

Children with a combined diagnosis of T1D and C showed a pattern of diminished peripheral B-cell subsets. The B-cells compartment in children with combined diagnosis had higher percentages of memory B subsets and Bregs, including activated subsets, compared to children with either T1D or C. Children with combined diagnosis had a lower percentage of naive B-cells (CD27-CD19+; IgD+CD19+) and an increased percentage of memory B-cells (CD27+CD19+; IgD-CD19+). A similar alteration was seen among the CD39+ expressing naive and memory B cells. Memory Bregs (CD1d+CD27+CD19+) were more frequent, contrary to the lower percentage of CD5+ transitional Bregs in children with a combined diagnosis. In children with either T1D or C, the peripheral B-cell compartment was dominated by naive cells.

Differences in the pattern of heterogenous peripheral B-cell repertoire subsets reflect a shifting in the B-cell compartment between children with T1D and/or C. This is an immunological challenge of impact for the pathophysiology of these autoimmune diseases.

Numerous studies have shown that lifestyle factors, such as regular physical activity and vitamin D intake, may remarkably improve overall health and mental wellbeing. This is especially important in older adults whose vitamin D deficiency occurs with a high prevalence. This study aimed to examine the influence of lifestyle and vitamin D on global DNA methylation patterns in an elderly cohort in Southwest of Sweden. We also sought to examine the methylation levels of specific genes involved in vitamin D's molecular and metabolic activated pathways. We performed a genome wide methylation analysis, using Illumina Infinium DNA Methylation EPIC 850kBeadChip array, on 277 healthy individuals from Southwest Sweden at the age of 70–95. The study participants also answered queries on lifestyle, vitamin intake, heart medication, and estimated health. Vitamin D intake did not in general affect methylation patterns, which is in concert with other studies. However, when comparing the group of individuals taking vitamin supplements, including vitamin D, with those not taking supplements, a difference in methylation in the solute carrier family 25 (SCL25A24) gene was found. This confirms a previous finding, where changes in expression of SLC25A24 were associated with vitamin D treatment in human monocytes. The combination of vitamin D intake and high physical activity increased methylation of genes linked to regulation of vitamin D receptor pathway, the Wnt pathway and general cancer processes. To our knowledge, this is the first study detecting epigenetic markers associated with the combined effects of vitamin D supplementation and high physical activity. These results deserve to be further investigated in an extended, interventional study cohort, where also the levels of 25(OH)D3 can be monitored.

Nuclear receptors (NRs) are ligand-activated transcription factors that regulate gene expression when bound to specific DNA sequences. Crosstalk between steroid NR systems has been studied for understanding the development of hormone-driven cancers but not to an extent at a genetic level. This study aimed to investigate crosstalk between steroid NRs in conserved intron and exon sequences, with a focus on steroid NRs involved in prostate cancer etiology. For this purpose, we evaluated conserved intron and exon sequences among all 49 members of the NR Superfamily (NRS) and their relevance as regulatory sequences and NR-binding sequences. Sequence conservation was found to be higher in the first intron (35%), when compared with downstream introns. Seventy-nine percent of the conserved regions in the NRS contained putative transcription factor binding sites (TFBS) and a large fraction of these sequences contained splicing sites (SS). Analysis of transcription factors binding to putative intronic and exonic TFBS revealed that 5 and 16%, respectively, were NRs. The present study suggests crosstalk between steroid NRs, e.g., vitamin D, estrogen, progesterone, and retinoic acid endocrine systems, through cis-regulatory elements in conserved sequences of introns and exons. This investigation gives evidence for crosstalk between steroid hormones and contributes to novel targets for steroid NR regulation.

Prostate cancer (PCa) is a highly heterogeneous and unpredictable progressive disease. Sensitivity of PCa cells to androgens play a central role in tumor aggressiveness but biomarkers with high sensitivity and specificity that follow the progression of the disease has not yet been verified. The vitamin D endocrine system and its receptors, the Vitamin D Receptor (VDR) and the Protein Disulfide-Isomerase A3 (PDIA3), are related to anti-tumoral effects as well as carcinogenesis and have therefore been suggested as potential candidates for the prevention and therapy of several cancer forms, including PCa. In this study, we evaluated the mRNA expression of VDR and PDIA3 involved in vitamin D signaling in cell lines representing different stages of PCa (PNT2, P4E6, LNCaP, DU145 and PC3). This study further aimed to evaluate vitamin D receptors and their isoforms as potential markers for clinical diagnosis of PCa. A novel transcript isoform of PDIA3 (PDIA3N) was identified and found to be expressed in all PCa cell lines analyzed. Androgen-independent cell lines showed a higher mRNA expression ratio between PDIA3N/PDIA3 contrary to androgen-dependent cell lines that showed a lower mRNA expression ratio between PDIA3N/PDIA3. The structure of PDIA3N differed from PDIA3. PDIA3N was found to be a N-truncated isoform of PDIA3 and differences in protein structure suggests an altered protein function i.e. cell location, thioredoxin activity and affinity for 1,25(OH)₂D₃. Collectively, PDIA3 transcript isoforms, the ratio between PDIA3N/PDIA3 and especially PDIA3N, are proposed as candidate markers for future studies with different stages of PCa progression.

Neonatal sepsis is common, lethal, and hard to diagnose. In combination with clinical findings and blood culture, biomarkers are crucial to make the correct diagnose. A Swedish national inquiry indicated that neonatologists were not quite satisfied with the available biomarkers. We assessed the kinetics of 15 biomarkers simultaneously: ferritin, fibrinogen, granulocyte colony-stimulating factor (G-CSF), interferon (IFN)-γ, interleukin (IL)-1β, −6, −8, −10, macrophage inflammatory protein (MIP)-1β, procalcitonin, resistin, serum amyloid A (SAA), tumor necrosis factor (TNF)-α, tissue plasminogen activator-3 and visfatin. The goal was to observe how quickly they rise in response to infection, and for how long they remain elevated. From a neonatal intensive care unit, newborns ≥28 weeks gestational age were recruited. Sixty-eight newborns were recruited to the study group (SG), and fifty-one to the control group (CG). The study group subjects were divided into three subgroups depending on clinical findings: confirmed sepsis (CSG), suspected sepsis (SSG) and no sepsis. CSG and SSG were also merged into an entire sepsis group (ESG) for sub-analysis. Blood samples were collected at three time-points; 0 h, 12–24 h and 48–72 h, in order to mimic a “clinical setting”. At 0 h, visfatin was elevated in SSG compared to CG; G-CSF, IFN-γ, IL-1β, −8 and − 10 were elevated in SSG and ESG compared to CG, whereas IL-6 and SAA were elevated in all groups compared to CG. At 12–24 h, IL-8 was elevated in ESG compared to CG, visfatin was elevated in ESG and SSG compared to CG, and SAA was elevated in all three groups compared to CG. At 48–72 h, fibrinogen was elevated in ESG compared to CG, IFN-γ and IL-1β were elevated in SSG and ESG compared to CG, whereas IL-8 and SAA were elevated in all three groups compared to CG. A function of time-formula is introduced as a tool for theoretical prediction of biomarker levels at any time-point. We conclude that SAA has the most favorable kinetics regarding diagnosing neonatal sepsis, of the biomarkers studied. It is also readily available methodologically, making it a prime candidate for clinical use.

Immune cells and their mediators are key players in human cancer progression involving alternation in the number and function of immune cells, both peripheral and at the site of tumor. Through reliable predictive biomarkers, cancer can be predicted, and progression and response to therapy followed. Thereby immune biomarkers, e.g., cytokines and chemokines can serve as intermediate mediators of cancer diagnostics. Multiplex analysis of immune mediators in small blood volumes allows for rapid quantification of large number of circulating analytes. The fluorochrome (Luminex) technique is a bead-based sandwich immunoassay that combines the enzyme-linked immunosorbent assay (ELISA) with flow cytometry. The Luminex technique allows multiple immune mediators to be measured simultaneously in small volumes, and provides a convenient and sensitive tool for the detection of a large number of extracellular secreted cytokines and chemokines to be used in prediction and therapy prognosis of cancer.

The technique is based on so-called microspheres (beads) that serve as a solid phase for molecular detection. These individually dyed microbeads have monoclonal antibodies directed against the cyto- and chemokines of interest and allow a simultaneous detection of up to nearly 100 cyto- and chemokines in a dual-laser flow analyzer. Immune mediators can be detected in serum and plasma samples as well as in cell culture supernatants from in vitro stimulated peripheral blood mononuclear cells (PBMC). This chapter describes the Luminex technique for detection of immune mediators in cancer by using magnetic bead sandwich immunoassay, with focus on some important pre-analytic factors, e.g., cell separation and cryopreservation and thawing of PBMC that may affect the outcome of detection of immune mediators. The Luminex technique thus represents a very suitable method to identify immune mediators in cancer tissues in order to diagnose and improve clinical outcome of cancer.

Children diagnosed with a combination of type 1 diabetes (T1D) and celiac disease (CD) show a dysregulated T helper (Th) 1/Th17 response. Besides the cellular involvement, several soluble immune markers are involved in the autoimmune process of both T1D and CD. Only few studies have examined the peripheral pattern of different cytokines, chemokines, and acute phase proteins (APP) in children with combined T1D and CD. To our knowledge no studies have evaluated the serum levels of adipocytokines and matrix metalloproteinases (MMPs) in this context. The purpose of the present study was to acquire more knowledge and to gain deeper understanding on peripheral immunoregulatory milieu in children with both T1D and CD. The study included children diagnosed with both T1D and CD (n=18), children with T1D (n=27) or CD (n=16), and reference children (n=42). Sera were collected and analysis of twenty-eight immune markers (cytokines, chemokines, APPs, adipocytokines and MMPs) was performed with Luminex technique. The major findings showed that children with double diagnosis had lower serum levels of interleukin (IL)-22, monocyte chemoattractant protein (MIP)-1α, monocyte chemoattractant protein (MCP)-1, procalcitonin, fibrinogen, visfatin and matrix metalloproteinase (MMP)-2. These results indicate a suppressed immune profile in children with combined T1D and CD, including Th17 cytokines, chemokines, acute phase proteins, adipocytokines and MMPs. We conclude that besides cytokines and chemokines, other immune markers, e.g. APPs, adipocytokines and MMPs, are of importance for further investigations to elucidate the heterogeneous immune processes present in patients diagnosed with T1D in combination with CD.

Purpose. The study aimed to investigate resting levels of several selected growth and metabolic hormones in a group of 24 endurance-trained adolescents (aged 13-19 years) compared with 24 untrained age- and sex-matched controls, and to investigate if increased cardiac dimensions were related to these hormones at rest with emphasis on insulin-like growth factor-1 (IGF-1).

Methods. The hormones (cortisol, IGF-1, IGF-2, follicle-stimulating hormone, growth hormone, luteinizing hormone, prolactin, and thyroid-stimulating hormone) were analysed with chemiluminescence microparticle immunoassay (CMIA) or multiplex fluorochrome (Luminex) technique. Cardiac dimensions were assessed by echocardiographic examination at rest. Peak oxygen uptake was obtained by a maximal cardiopulmonary exercise test on a treadmill.

Results. Circulating levels of analysed hormones at rest did not differ between the groups. A correlation was found between increased cardiac dimensions and IGF-1 in the controls, but not in the active group. This correlation declined also among the controls when the cardiac parameters were indexed for body surface area.

Conclusion. Increased cardiac dimensions in endurance-trained adolescents could not be related to resting levels of hormones associated with growth and metabolism, including IGF-1 and GH. In addition, the resting levels of these hormones seem not to be affected by intense regular endurance exercise in adolescents. These findings may contribute to the knowledge about cellular signaling that trigger growth as well as cardiac adaptation to endurance training in young athletes.