Galectin-1 correlates with inflammatory markers and T regulatory cells in children with type 1 diabetes and/or celiac disease.

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. GAL-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 analysed 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-1ß, IL-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.

Shift in the B cell subsets between children with type 1 diabetes and/or celiac disease.

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 (mBregs; CD24hiCD27+CD19+, CD1d+CD27+CD19+, and 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 heterogeneous 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 on the pathophysiology of these autoimmune diseases.

Characterization of methylation patterns associated with lifestyle factors and vitamin D supplementation in a healthy elderly cohort from Southwest Sweden.

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.

Inhibition of CYP27B1 and CYP24 Increases the Anti-proliferative Effects of 25-Hydroxyvitamin D3 in LNCaP Cells.

BACKGROUND/AIM: Growing evidence suggests that vitamin D3 exerts anticancer effects. The present study aimed to evaluate 25-hydroxyvitamin D3 (25(OH)D3) as a potential endocrine factor regulating proliferation and vitamin D receptor expression in LNCaP prostate cancer cells. MATERIALS AND METHODS: Cell counting after treatment was utilized to assess the effect of 25(OH)D3 on cell proliferation. Changes in mRNA expression of the vitamin D receptors, VDR and PDIA3, were evaluated using droplet digital polymerase chain reaction (ddPCR). RESULTS: 25(OH)D3 inhibited cell proliferation in a dose- and time-dependent manner. The inhibitory effect of 25(OH)D3 on cell proliferation was potentiated after inhibition of CYP17B1 and CYP24 by genistein, preventing further metabolism of 25(OH)D3 to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). Expression of PDIA3 and VDR mRNA increased after treatment with 25(OH)D3, whereas the ratio between PDIA3 and VDR mRNA remained unchanged. CONCLUSION: 25(OH)D3 has a direct inhibitory effect on cell proliferation, which is enhanced and accelerated when the metabolism of 25(OH)D3 to 1,25(OH)2D3 and 24,25(OH)2D3 was inhibited by the CYP17B1 and CYP24 inhibitor genistein. Furthermore, treatment with 25(OH)D3 increased receptor transcript expression, suggesting an increased VDR stability and sensibility of the treated cells.

Serum amyloid A - A prime candidate for identification of neonatal sepsis.

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)-1ß, -6, -8, -10, macrophage inflammatory protein (MIP)-1ß, 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-1ß, -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-1ß 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.

Cis-regulatory elements in conserved non-coding sequences of nuclear receptor genes indicate for crosstalk between endocrine systems.

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.

Differential expression of protein disulfide-isomerase A3 isoforms, PDIA3 and PDIA3N, in human prostate cancer cell lines representing different stages of prostate cancer.

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.

A Useful Guide for Analysis of Immune Mediators in Cancer by Fluorochrome (Luminex) Technique.

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.

Resting Level of Insulin-Like Growth Factor-1 Is Not at Play in Cardiac Enlargement in Endurance-Trained Adolescents.

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.

Subsets of CD4+, CD8+, and CD25hi Lymphocytes Are in General Not Influenced by Isolation and Long-Term Cryopreservation.

Several key factors can affect the outcome of immunological studies; isolation/cryopreservation can possibly alter T, B, NK, and T-regulatory (Treg) cell marker expression patterns. Blood samples from 50 blood donors supplemented with Na-heparin or K2EDTA were handled within 4 and 24 h after blood sampling. PBMC were isolated with different density gradients. Flow cytometric analysis of intracellular and extracellular CD markers was performed on blood samples freshly isolated PBMC, and PBMC was thawed 6 and 12 mo post-cryopreservation for the purpose of identifying B, NK, Th, T-cytotoxic, and Treg cells. No differences were observed in the percentages for CD3+, CD3+CD4+, CD3+CD8+, CD19+, or CD56+CD16+ cells within 24 h of sampling regardless of which supplement or isolation techniques were used. Differentiated (diff) CD4+ cells were in general less affected by isolation and cryopreservation than diff CD8+ cells. Terminally diff effector CD4+ and CD8+ cells were not affected by either isolation of lymphocytes or cryopreservation. In contrast, naive and early-diff effector memory CD4+ and CD8+ cells were affected by isolation and cryopreservation. The percentages of Treg cells defined as CD4+CD25hi expressing CD101 or CD129, CD4+CD25hiCD127-, and CD4+CD25hiCD127-FOXP3+, respectively, remained stable after isolation and cryopreservation. Subsets expressing CD127, with or without FOXP3, were not affected by isolation/cryopreservation. Subsets expressing CD39, contrary to CD45RA, on CD4+CD25+CD127- cells with or without FOXP3 were not affected by either isolation or cryopreservation. In conclusion, subsets of CD4+, CD8+, and CD25hi lymphocytes are in general not influenced by isolation and long-term cryopreservation.

Left ventricular diastolic function is enhanced after peak exercise in endurance-trained adolescents as well as in their non-trained controls.

The aims of the study were to explore the temporal change of cardiac function after peak exercise in adolescents, and to investigate how these functional changes relate to maximal oxygen uptake (VO2max ). The cohort consisted of 27 endurance-trained adolescents aged 13-19 years, and 27 controls individually matched by age and gender. Standard echocardiography and colour tissue Doppler were performed at rest, and immediately after as well as 15 min after a maximal cardio pulmonary exercise test (CPET) on a treadmill. The changes in systolic and diastolic parameters after exercise compared to baseline were similar in both groups. The septal E/e'-ratio increased immediately after exercise in both the active and the control groups (from 9·2 to 11·0; P<0·001, and from 8·7 to 10·2; P = 0·008, respectively). In a comparison between the two groups after CPET, the septal E/e'-ratio was higher in the active group both immediately after exercise and 15 min later compared to the control group (P = 0·007 and P = 0·006, respectively). We demonstrated a positive correlation between VO2max and cardiac function including LVEF and E/e' immediately after CPET, but the strongest correlation was found between VO2max and LVEDV (r = 0·67, P<0·001) as well as septal E/e' (r = 0·34, P = 0·013). Enhanced diastolic function was found in both groups, but this was more pronounced in active adolescents. The cardiac functional response to exercise, in terms of LVEF and E/e', correlates with the increase in VO2 uptake. These findings in trained as well as un-trained teenagers have practical implications when assessing cardiac function.

Management of Children with Autism Spectrum Disorder in the Anesthesia and Radiographic Context.

OBJECTIVE: As a primary objective, this study purports to develop guidelines to better care for children with autism spectrum disorder (ASD), particularly regarding these children's preparation for anesthesia and radiologic procedures. METHODS: Using a Delphi method with an online distribution of questionnaire, guidelines for caring for children with ASD were created. Twenty-one participants were included in the expert panel. These participants were working with children with ASD in several anesthesia and radiology departments in Sweden. A list of items was created from a previous survey and the literature. In the first round, the items with <60% agreement were discarded. Items were merged, and a new list was created. Two more similar rounds were performed. In the last 2 rounds, 21 participants responded, and 80% agreement was considered to be consensus. RESULTS: The final guidelines consisted of 14 items and a checklist of 16 factors. The 5 areas covered by the items and the checklist were as follows: planning involving parents/guardians, features in the environment, and use of time, communication, and the health care professionals. The organization was important in making it possible for the health care professional to care for the individual child according to the child's needs. It was important to involve the parents/guardians to obtain knowledge about the functioning of the child. CONCLUSION: A caring encounter involving a child with ASD in the anesthesia and radiology contexts requires advance planning, catered specifically to the individual needs of each child. To accomplish this, general knowledge regarding ASD and ASD's particular manifestation in the child entrusted to their care is required from the health care workers. The organization needs to have structures in place to facilitate this process.

Regular endurance training in adolescents impacts atrial and ventricular size and function.

AIMS: The aims of the study were to explore the effects of long-term endurance exercise on atrial and ventricular size and function in adolescents and to examine whether these changes are related to maximal oxygen uptake (VO2max). METHODS AND RESULTS: Twenty-seven long-term endurance-trained adolescents aged 13-19 years were individually matched by age and gender with 27 controls. All participants, 22 girls and 32 boys, underwent an echocardiographic examination at rest, including standard and colour tissue Doppler investigation. VO2max was assessed during treadmill exercise. All heart dimensions indexed for body size were larger in the physically active group compared with controls: left ventricular end-diastolic volume 60 vs. 50 mL/m2 (P <0.001), left atrial volume 27 vs. 19 mL/m2 (P < 0.001), and right ventricular (RV) and right atrial area 15 vs. 13 and 9 vs. 7 cm2/m2, respectively (P <0.001 for both). There were strong associations between the size of the cardiac chambers and VO2max. Further, we found improved systolic function in the active group compared with controls: left ventricular ejection fraction 61 vs. 59% (P= 0.036), tricuspid annular plane systolic excursion 12 vs. 10 mm/m2 (P= 0.008), and RV early peak systolic velocity s' 11 vs. 10 cm/s (P = 0.031). CONCLUSION: Cardiac remodelling to long-term endurance exercise in adolescents is manifested by an increase in atrial as well as ventricular dimensions. The physically active group also demonstrated functional remodelling with an increase in TAPSE and systolic RV wall velocity. These findings have practical implications when assessing cardiac enlargement and function in physically active youngsters.

Perioperative and Anesthesia Guidelines for Children with Autism: A Nationwide Survey from Sweden.

OBJECTIVE: The overall aim of this study was to describe the current set of guidelines for the preparation and care for children with autism spectrum disorder (ASD) in the perioperative setting across Sweden and explore the content of these guidelines in detail. METHOD: An online questionnaire was distributed to the chairpersons of all anesthesia departments (n = 68) and pediatric departments (n = 38) throughout Sweden. Follow-up phone calls were made to those departments that did not return the questionnaire. The presence of guidelines was analyzed through descriptive statistics. These guidelines and comments on routines used in these departments were analyzed inspired by conventional content analysis. RESULTS: Seven of the 68 anesthesia departments and none of the 38 pediatric departments across Sweden have guidelines for preparing and/or administering care to children with ASD within the perioperative setting. From the guidelines and routines used, 3 categories emerge: "lacking the necessary conditions," "no extra considerations needed," and "care with specific consideration for children with ASD." These 3 categories span a continuum in the care. In the first category, the anesthesia induction could result in the child with ASD being physically restrained. In the last category, the entire encounter with the health care service would be adapted to the specific needs of the child. CONCLUSION: There is a lack of evidence-based guidelines specifically designed to meet the needs of children with ASD in the preoperative period in Sweden. Further research is needed to understand if children with ASD would benefit from evidence-based guidelines.

The Link between Psychological Stress and Autoimmune Response in Children.

Stress is defined as a state of threatened homeostasis or disharmony that is counteracted by a complex repertoire of physiological and behavioral adaptive responses in order to establish homeostasis. Confronted with a stressful condition, the nervous and immune systems initiate a coping process to maintain homeostasis in the body. Psychological stress, recognized as a public health issue in children and young adults, may be one mechanism to induce and maintain autoimmunity in children. It is necessary to increase our understanding of how psychological stress can affect the immune system at a young age because autoimmune diseases, especially type 1 diabetes, are alarmingly common in children. Psychological stress may be involved in other autoimmune diseases, such as celiac disease, systemic lupus erythematosus, and juvenile idiopathic arthritis, that frequently occur in children as well. This review summarizes the studies attempting to evaluate the link between psychological stress and autoimmune response in children. A number of them have observed that the autoimmune disease itself causes psychological stress. We are far from fully understanding how long-term psychological stress is linked to autoimmune response in children with a high risk of, or already diagnosed, autoimmune disease.

The challenge of measuring elusive immune markers by enzyme-linked immuno-spot (ELISPOT) technique.

The enzyme-linked immuno-spot (ELISPOT) technique is a sensitive method used for measurement of elusive immune markers in limited-volume samples. By virtue of the exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses), which was not possible before, is now relatively easy. However, development of a method sensitive enough to pinpoint elusive immune markers at the single-cell level is a challenge since there are a number of demands that have to be fulfilled and traps to avoid, achieving a valuable outcome.To optimize the environment for in vitro culture and analysis of immune spots by ELISPOT, a number of criteria have to be fulfilled: processing of sample and perhaps also cryopreservation of cells before analysis and, for the ELISPOT assay, optimal cell culture, positive and negative controls, antigen concentration, and, finally, development and readout of spots.If these criteria are fulfilled for your ELISPOT assay, you will likely have the opportunity to pinpoint elusive immune markers at the single-cell level. This chapter describes the ELISPOT assay for detection of cytokines (e.g., IFN-γ and IL-4), with focus on the main criteria that affect the assay. However, this method could be easily adapted to measure other immune markers in small volumes of biological samples.

A useful guide for analysis of immune markers by fluorochrome (Luminex) technique.

The fluorochrome Luminex technique is a bead-based sandwich immunoassay that combines the enzyme-linked immuno sorbent assay (ELISA) with flow cytometry. The Luminex technique allows multiple cytokines to be measured simultaneously in small volumes and provides a convenient and sensitive tool for the detection of a large number of, e.g., extracellular secreted cytokines to characterize cytokine profiles.The technique is based on the so-called microspheres (beads) that serve as a solid phase for molecular detection. These individually dyed micro-beads have monoclonal antibodies directed against the cytokines and chemokines of interest and allow simultaneous detection of up to nearly 100 cytokines and chemokines in a dual-laser flow analyzer. Immune markers 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 multiple cytokines by magnetic bead sandwich immunoassay, with a special focus on some important pre-analytical factors, such as cell separation, cryopreservation, and PBMC thawing that may affect the detection outcome of immune markers. This method can also be easily adapted to measuring other biomarkers in biological samples.

Psychological stress in children may alter the immune response.

Psychological stress is a public health issue even in children and has been associated with a number of immunological diseases. The aim of this study was to examine the relationship between psychological stress and immune response in healthy children, with special focus on autoimmunity. In this study, psychological stress was based on a composite measure of stress in the family across the domains: 1) serious life events, 2) parenting stress, 3) lack of social support, and 4) parental worries. PBMCs, collected from 5-y-old high-stressed children (n = 26) and from 5-y-old children without high stress within the family (n = 52), from the All Babies In Southeast Sweden cohort, were stimulated with Ags (tetanus toxoid and ß-lactoglobulin) and diabetes-related autoantigens (glutamic acid decarboxylase 65, insulin, heat shock protein 60, and tyrosine phosphatase). Immune markers (cytokines and chemokines), clinical parameters (C-peptide, proinsulin, glucose), and cortisol, as an indicator of stress, were analyzed. Children from families with high psychological stress showed a low spontaneous immune activity (IL-5, IL-10, IL-13, IL-17, CCL2, CCL3, and CXCL10; p < 0.01) but an increased immune response to tetanus toxoid, ß-lactoglobulin, and the autoantigens glutamic acid decarboxylase 65, heat shock protein 60, and tyrosine phosphatase (IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ, TNF-α, CCL2, CCL3, and CXCL10; p < 0.05). Children within the high-stress group showed high level of cortisol, but low level of C-peptide, compared with the control group (p < 0.05). This supports the hypothesis that psychological stress may contribute to an imbalance in the immune response but also to a pathological effect on the insulin-producing ß cells.

General immune dampening is associated with disturbed metabolism at diagnosis of type 1 diabetes.

BACKGROUND: Type 1 diabetes (T1D) is a serious diagnosis with the prospect of grave short- and long-term complications and even death if poorly managed. An attempt has been made to describe how clinical and immunological deviations might influence each other close to the diagnosis of T1D. METHODS: Sixty-nine newly diagnosed T1D children were studied together with a reference group of 30 healthy children. Cytokines (interleukin (IL)-6, IL-10, IL-13, IL-17, interferon-γ, and tumor necrosis factor-α) were detected in in vitro culture by multiplex fluorochrome technique. Information of clinical status of the patients such as BMI, weight loss, pubertal stage, duration of symptoms, previous and/or ongoing infections, insulin requirement, and ketoacidosis were gathered together with the analysis of C-peptide and glycosylated hemoglobin (HbA1c). RESULTS: In general, low cytokine secretion was found at diagnosis of T1D. However, high C-peptide, short duration of symptoms, or an infection prior to diagnosis was associated with increased immune activity including proinflammatory, Th2-associated, and Tr1-associated cytokines. In contrast, ketoacidosis and later pubertal stage at onset of disease were more related to a Th1-prone response. CONCLUSION: There is a general immune dampening at diagnosis of T1D, which appears to be related to the metabolic state close to diagnosis.

Altered immune profile from pre-diabetes to manifestation of type 1 diabetes.

BACKGROUND: While the mechanisms leading to ß-cell destruction and clinical onset of T1D are still unclear, the composition of the immune profile is probably important for the outcome of immune activity. The aim of this study was to investigate the composition and possible changes of the immunological profile, spontaneously and following stimulation with the autoantigens GAD65, and HSP60, at high-risk and T1D onset and further to 8 months post diagnosis. METHODS: Fifteen first-degree relatives of T1D patients with a high risk of developing the disease within five years, 25 children approximately four days and 8 months after diagnosis of T1D and 16 healthy children were included in the study. Cytokines (IL-1ß, -6, -7, -10, -13, -17, IFN-γ and TNF-α) and chemokines (CCL2, -3, -4, -5 and CXCL10) associated with Th1, Th2, Tr1 and inflammatory cells were detected in cell culture supernatants by Luminex-technique, and markers associated with regulatory T-cells (FOXP3, CTLA-4 and TGF-ß) by real-time RT-PCR. RESULTS: High-risk individuals differed in immunity from that seen in healthy and T1D children. High-risk individuals had a low TNF-α response and fewer responders from mitogen exposure as well as low spontaneous secretions of IL-13 compared to healthy children. High-risk individuals that later developed T1D, had a lower FOXP3 and CTLA-4 mRNA expression, following stimulation with GAD65, in combination with higher secretion of the pro-inflammatory chemokine CCL4. CONCLUSION: Changes in immunity seen in individuals with high risk of developing T1D points to alterations/actions in the immune system already early in the pre-diabetic phase.