Multiplex bead-based immunoassay for the free soluble forms of the HLA-G receptors,ILT2 and ILT4

Human leukocyte antigen (HLA)-G is an immune-inhibitory molecule that exerts its function via interaction with two main inhibitory receptors: ILT2 and ILT4. This interaction is considered to be an immune checkpoint. HLA-G can be found as a soluble molecule, but it is not known if its receptors can also be found as soluble molecules. In this work, we present a multiplex luminex-based assay to measure soluble ILT2 (sILT2) and soluble ILT4 (sILT4) molecules together. It is based on two antibody pairs, GHI/75 and HP-F1-PE for ILT2 and 27D6 and 42D1-PE for ILT4. The characterization of our method reveals that it specifically detects the free soluble forms of sILT2 and sILT4, and not those complexed to HLA Class I molecules such as their ligand of highest affinity HLA-G. A study on two small cohorts of cancer patients demonstrated that soluble ILT2 and ILT4 molecules were of low abundance in the plasma of healthy controls, but that elevated levels of plasmatic sILT2 were present in non-muscle-infiltrating bladder cancer patients. This demonstrated that the titration test is indeed working, and that soluble ILT2 molecules do exist in pathological contexts, which relevance may now be sought on larger cohorts and other pathologies.     

Cytometry-based analysis of HLA-G functions according to ILT2 expression

HLA-G was described as a molecule inhibiting NK and T cells functions through its receptor, ILT2. However, most functional studies of HLA-G were so far performed on heterogeneous immune populations and regardless of ILT2 expression. This may lead to an underestimation of the effect of HLA-G. Thus, considering the immune subpopulations sensitive to HLA-G remained an important issue in the field. Here we present a new cytometry assay to evaluate HLA-G effects on both NK and CD8⁺ T cell cytotoxic functions.Using flow cytometry allows for the comparison of HLA-G function on multiple subsets and multiple functions in the same time. In particular, we sharpen the analysis by specifically studying the immune subpopulations expressing HLA-G receptor ILT2. We focused our work on: IFN-gamma production and cytotoxicity (CD107a expression) by CD8⁺ T cells and NK cells expressing or not ILT2. We compared the expression of these markers in presence of target cells, expressing or not HLA-G1, and added a blocking antibody to reverse HLA-G inhibition.This new method allows for the discrimination of cell subsets responding and non-responding to HLA-G1 in one tube. We confirm that HLA-G-specifically inhibits the ILT2⁺ CD8⁺ T cell and ILT2⁺ NK cell subsets but not ILT2-negative ones. By blocking HLA-G/ILT2 interaction using an anti-ILT2 antibody we restored the cytotoxicity level, corroborating the specific inhibition of HLA-G1. We believe that our methodology enables to investigate HLA-G immune functions easily and finely towards other immune cell lineages or expressing other receptors, and might be applied in several pathological contexts, such as cancer and transplantation.     

Nutritional Status of Vitamin D and the Effect of Vitamin D Supplementation in Korean Breast-fed Infants

We investigated the vitamin D status and the effect of vitamin D supplementation in Korean breast-fed infants. The healthy term newborns were divided into 3 groups; A, formula-fed; B, breast-fed only; S, breast-fed with vitamin D supplementation. We measured serum concentrations of vitamin D (25OHD3), calcium (Ca), phosphorus (P), alkaline phosphatase (AP), intact parathyroid hormone (iPTH) and bone mineral density (BMD) at 6 and 12 months of age. Using questionnaires, average duration of sun-light exposure and dietary intake of vitamin D, Ca and P were obtained. At 6 and 12 months of age, 25OHD3 was significantly higher in group S than in group B (P<0.001). iPTH was significantly lower in group S than in group B at 6 months (P=0.001), but did not differ at 12 months. Regardless of vitamin D supplementation, BMD was lower in group B and S than in group A (P<0.05). Total intake of vitamin D differed among 3 groups (P<0.001, A>S>B), but total intake of Ca and P were higher in group A than in group B and S (P<0.001). In conclusion, breast-fed infants show lower vitamin D status and bone mineralization than formula-fed infants. Vitamin D supplementation (200 IU/day) in breast-fed infants increases serum 25-OH vitamin D₃, but not bone mineral density.     

Vitamin D effects on musculoskeletal health,immunity, autoimmunity,cardiovascular disease,cancer, fertility,pregnancy, dementia and mortality—A review of recent evidence

Background

Optimal vitamin D intake and its status are important not only for bone and calcium-phosphate metabolism, but also for overall health and well-being. Vitamin D deficiency and insufficiency as a global health problem are likely to be a risk for wide spectrum of acute and chronic illnesses.

Methods

A review of randomized controlled trials, meta-analyses, and other evidence of vitamin D action on various health outcomes.

Results

Adequate vitamin D status seems to be protective against musculoskeletal disorders (muscle weakness, falls, fractures), infectious diseases, autoimmune diseases, cardiovascular disease, type 1 and type 2 diabetes mellitus, several types of cancer, neurocognitive dysfunction and mental illness, and other diseases, as well as infertility and adverse pregnancy and birth outcomes. Vitamin D deficiency/insufficiency is associated with all-cause mortality.

Conclusions

Adequate vitamin D supplementation and sensible sunlight exposure to reach optimal vitamin D status are among the front line factors of prophylaxis for the spectrum of disorders. Supplementation guidance and population strategies for the eradication of vitamin D deficiency must be included in the priorities of physicians, medical professionals and healthcare policy-makers.     

Maternal vitamin D intake during pregnancy is inversely associated with asthma and allergic rhinitis in 5-year-old children

Background Vitamin D is known to have a number of immunological effects and it may play a role in preventing allergic diseases.
Objectives To study the effect of maternal intake of vitamin D during pregnancy on the emergence of asthma, allergic rhinitis (AR), and atopic eczema by the age of 5 years in children with HLA-DQB1-conferred susceptibility for type 1 diabetes.
Methods Children (1669) participating in the population-based birth cohort study were followed for asthma, AR, and atopic eczema assessed by validated questionnaire at 5 years. Maternal diet was assessed by a food-frequency questionnaire.
Results The mean maternal intake of vitamin D was 5.1 (SD 2.6) μg from food and 1.4 (2.6) μg from supplements. Only 32% of the women were taking vitamin D supplements. When adjusted for potential confounders, maternal intake of vitamin D from food was negatively related to risk of asthma [hazard ratio (HR) 0.80; 95% confidence interval (CI) 0.64–0.99] and AR [HR 0.85; 95% CI 0.75–0.97]. Vitamin D supplements alone were not associated with any outcome. Adjustment for maternal intake of other dietary factors did not change the results.
Conclusion Maternal vitamin D intake from foods during pregnancy may be negatively associated with risk of asthma and AR in childhood.     

Vitamin D deficiency and depressive symptoms in pregnancy are associated with adverse perinatal outcomes

Prenatal vitamin D deficiency and prenatal depression are both separately associated with adverse perinatal outcomes; however, to our knowledge no studies have investigated the effects of having both risk factors. Our objective was to determine to what extent vitamin D deficiency predicts adverse perinatal outcomes and whether elevated depressive symptoms in pregnancy places women at additional increased risk. This study was a secondary data analysis of prospective data collected from a cohort of pregnant women (N?=?101) in an obstetric clinic of a large medical center. Maternal vitamin D deficiency (serum 25(OH)D?≤?20 ng/ml) and depressive symptoms (Edinburgh Postnatal Depression Scale, EPDS) were assessed in early pregnancy. A composite of four adverse perinatal outcomes (low birth weight, preterm birth, small-for-gestational age, and preeclampsia) were abstracted from medical charts. Nineteen of the 101 women had one or more adverse perinatal outcome and 84% with an adverse outcome (16/19) were not White. Both prenatal and time of delivery vitamin D deficiency were associated with developing an adverse outcome compared to those vitamin D sufficient (prenatal relative risk 3.43; 95% CI 1.60–7.34, p?=?0.004; delivery time relative risk 5.14, 95% CI 2.68–9.86, p?=?0.004). These both remained significant after adjusting for BMI. A higher rate of adverse outcome was found when women had both prenatal vitamin D deficiency and elevated depressive symptoms (EPDS?≥?10). Sixty percent with both risk factors had an adverse perinatal outcome versus 17% with only one or neither risk factor (relative risk 3.60; 95% CI 1.55–8.38, p?=?0.045), worthy of investigation with larger samples. Together, prenatal vitamin D deficiency and elevated depressive symptoms in pregnancy may increase risk for adverse perinatal outcomes, especially in racial minorities. Obstetric providers should consider routine prenatal depression screening. The impact of vitamin D supplementation to reduce risk for adverse perinatal outcomes should be studied in prospective trials. Our results suggest that supplementation early in pregnancy might be especially beneficial for depressed women.     

Vitamin A Supplementation Reduces IL‐17 and RORc Gene Expression in Atherosclerotic Patients

Vitamin A is a potential mediator of T helper cells in atherosclerosis. The purpose of this study was to evaluate the effect of vitamin A supplementation on expression of Th17 cells‐related IL‐17 and RORc genes in atherosclerotic patients. Thirty one atherosclerotic patients and 15 healthy controls were studied for 4 months. Atherosclerotic patients were randomly divided into vitamin A or placebo groups. Healthy controls and patients in vitamin A group received 25,000 IU retinyl palmitate per day. Peripheral blood mononuclear cells were isolated, cultured and divided into three groups including fresh cells, phytohemagglutinin (PHA)‐activated T cells and ox‐LDL‐activated T cells. Gene expressions of T cells were studied by real‐time PCR. In atherosclerotic patients, vitamin A supplementation resulted in significant decrease in IL‐17 gene expression by 0.63‐fold in fresh cell, 0.82‐fold in PHA‐activated cells and 0.65‐fold in ox‐LDL‐activated cells (P < 0.05 for all). RORc gene expression in fresh cells as well as ox‐LDL‐activated cells decreased significantly after vitamin A supplementation in atherosclerotic patients (P = 0.0001 for both). In PHA‐activated cells, vitamin A supplementation significantly decreased RORc gene in both atherosclerotic patients and healthy subjects by 0.87‐fold and 0.72, respectively, while in placebo group, the RORc gene expression significantly increased by 1.17‐fold (P < 0.05 for all). Findings of this study suggest that vitamin A supplementation may be an effective approach to slow progression of atherosclerosis.     

Vitamin D immunoregulation through dendritic cells

Vitamin D (VD3) has been linked to immunological processes, and its supplementation may have a role in treatment or prevention of diseases with underlying autoimmune or pro‐inflammatory states. As initiators of the immune responses, dendritic cells (DC) are a potential target of VD3 to dampen autoimmunity and inflammation, but the role of DC in VD3‐mediated immunomodulation in vivo is not understood. In addition to being targets of VD3, DC can provide a local source of bioactive VD3 for regulation of T‐cell responses. Here we review existing studies that describe the tolerogenic potential of VD3 on DC, and discuss them in the context of current understanding of DC development and function. We speculate on mechanisms that might account for the potent but poorly understood tolerogenic activities of VD3 and the role of DC as both targets and sources of this hormone.     

Maternal diet during pregnancy: an emerging risk factor for childhood asthma

Evaluation of: Willers SM, Wijga AH, Brunekreef B et al. Maternal food consumption during pregnancy and the longitudinal development of childhood asthma. Am. J. Respir. Crit. Care Med. 178, 124–131 (2008).

It has been hypothesized that the recent marked increase in the prevalence of asthma may, in part, be a consequence of changing diet. There is increasing interest in the possibility that childhood asthma may be influenced by maternal diet during pregnancy and an increasing number of studies have highlighted associations between childhood asthma and maternal intake of certain foods (e.g., fish, fruits and vegetables) and nutrients (e.g., vitamin E, vitamin D, zinc and polyunsaturated fatty acids) during pregnancy. Maternal diet during pregnancy has the potential to influence fetal immune and airway development during a critical period of life with long-term irreversible consequences, such as childhood asthma. Further research, particularly intervention studies, needs to be carried out to establish whether dietary intervention during pregnancy can be used as a healthy, low-cost, public-health measure to reduce the prevalence of childhood asthma.     

Vitamin D treatment modulates immune activation in cystic fibrosis

Persistent inflammatory response in cystic fibrosis (CF) airways is believed to play a central role in the progression of lung damage. Anti‐inflammatory treatment may slow lung disease progression, but adverse side effects have limited its use. Vitamin D has immunoregulatory properties. We randomized 16 CF patients to receive vitamin D2, vitamin D3 or to serve as controls, and investigated the effect of vitamin D supplementation on soluble immunological parameters, myeloid dendritic cells (mDCs) and T cell activation. Three months of vitamin D treatment were followed by two washout months. Vitamin D status at baseline was correlated negatively with haptoglobin, erythrocyte sedimentation rate and immunoglobulin A concentration. Total vitamin D dose per kg bodyweight correlated with the down‐modulation of the co‐stimulatory receptor CD86 on mDCs. Vitamin D treatment was associated with reduced CD279 (PD‐1) expression on CD4⁺ and CD8⁺ T cells, as well as decreased frequency of CD8⁺ T cells co‐expressing the activation markers CD38 and human leucocyte antigen D‐related (HLA‐DR) in a dose‐dependent manner. There was a trend towards decreased mucosal‐associated invariant T cells (MAIT) cell frequency in patients receiving vitamin D and free serum 25‐hydroxyvitamin D (free‐s25OHD) correlated positively with CD38 expression by these cells. At the end of intervention, the change in free‐s25OHD was correlated negatively with the change in CD279 (PD‐1) expression on MAIT cells. Collectively, these data indicate that vitamin D has robust pleiotropic immunomodulatory effects in CF. Larger studies are needed to explore the immunomodulatory treatment potential of vitamin D in CF in more detail.     

Is vitamin D deficiency to blame for the asthma epidemic?

In the 1960s, the prevalence of asthma and allergic diseases began to increase worldwide. Currently, the burden of the disease is more than 300 million people affected. We hypothesize that as populations grow more prosperous, more time is spent indoors, and there is less exposure to sunlight, leading to decreased cutaneous vitamin D production. Coupled with inadequate intake from foods and supplements, this then leads to vitamin D deficiency, particularly in pregnant women, resulting in more asthma and allergy in their offspring. Vitamin D has been linked to immune system and lung development in utero, and our epidemiologic studies show that higher vitamin D intake by pregnant mothers reduces asthma risk by as much as 40% in children 3 to 5 years old. Vitamin D deficiency has been associated with obesity, African American race (particularly in urban, inner-city settings), and recent immigrants to westernized countries, thus reflecting the epidemiologic patterns observed in the asthma epidemic. Providing adequate vitamin D supplementation in pregnancy may lead to significant decreases in asthma incidence in young children.