Apolipoprotein E4 Expression Causes Gain of Toxic Function in Isogenic Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

OBJECTIVE: The ApoE (apolipoprotein) allele epsilon 4 is a major genetic risk factor for Alzheimer disease, cardiovascular disorders, and stroke, indicating that it significantly impacts cerebral and vascular systems. However, very little is known about how APOE genotype affects brain endothelial cells, which form a network of tight junctions to regulate communication between the brain and circulating blood factors. Approach and Results: Here, we present a novel model of endothelial dysfunction using isogenic human induced pluripotent stem cell-derived cells harboring different alleles of the APOE gene, specifically ApoE 3/3, 3/4, and 4/4. We show for the first time that ApoE4 expression by endothelial cells is sufficient to cause a toxic gain of cellular dysfunction. Using RNAseq, we found significant effects of ApoE4 on signaling pathways involved in blood coagulation and barrier function. These changes were associated with altered cell function, including increased binding of platelets to ECs with the 3/4 or 4/4 genotype. ApoE4-positive cells exhibited a proinflammatory state and prothrombotic state, evidenced by higher secretion of Aß (amyloid-ß) 40 and 42, increased release of cytokines, and overexpression of the platelet-binding protein VWF (vonWillebrand factor). Immunohistochemistry of human brain Alzheimer disease brains also showed increased VWF expression with the ApoE4/4 genotype. Finally, pharmacological inhibition of inflammation in ECs by celastrol rescued overexpression of VWF in cells expressing ApoE4. CONCLUSIONS: These cells provide novel insight into ApoE4-mediated endothelial dysfunction and provide a new platform to test potential therapies for vascular disorders.

The bioavailability of iron picolinate is comparable to iron sulfate when fortified into a complementary fruit yogurt: a stable iron isotope study in young women.

PURPOSE: A technological gap exists for the iron (Fe) fortification of difficult-to-fortify products, such as wet and acid food products containing polyphenols, with stable and bioavailable Fe. Fe picolinate, a novel food ingredient, was found to be stable over time in this type of matrix. The objective of this study was to measure the Fe bioavailability of Fe picolinate in a complementary fruit yogurt. METHODS: The bioavailability of Fe picolinate was determined using stable iron isotopes in a double blind, randomized cross-over design in non-anemic Swiss women (n = 19; 25.1 ± 4.6 years). Fractional Fe absorption was measured from Fe picolinate (2.5 mg 57Fe per serving in two servings given morning and afternoon) and from Fe sulfate (2.5 mg 54Fe per serving in two servings given morning and afternoon) in a fortified dairy complementary food (i.e. yogurt containing fruits). Fe absorption was determined based on erythrocyte incorporation of isotopic labels 14 days after consumption of the last test meal. RESULTS: Geometric mean (95% CI) fractional iron absorption from Fe picolinate and Fe sulfate were not significantly different: 5.2% (3.8-7.2%) and 5.3% (3.8-7.3%) (N.S.), respectively. Relative bioavailability of Fe picolinate versus Fe sulfate was 0.99 (0.85-1.15). CONCLUSION: Therefore, Fe picolinate is a promising compound for the fortification of difficult-to-fortify foods, to help meet Fe requirements of infants, young children and women of childbearing age.

A Rapid Chemiluminescence Assay for Measurement of Folate in Small Volumes of Breast Milk.

Early life exposure to folate has long lasting effects on development and health. Newborns obtain part of their folate from maternal milk. Studies on health effects of milk folate require rapid, affordable and reliable measurements in large numbers of samples from cohort studies. Recently, a competitive chemiluminescence assay for quantification of folate has become available for automated diagnostic measurement of folate in human serum or plasma. We tested if this method ("FOLA" from Siemens Healthcare) could also be used for human milk. To minimize interference and matrix effects, samples had to be skimmed, diluted seven times with demineralized water, and heated for 5 min at 90 °C. Folate could thus be measured in a linear range between 8.4 and 111.7 nM, with recoveries for the most relevant form, 5-methyltetrahydrofolate (5-MeTHF), of 96%-107%. Results were comparable to those with a recently validated Liquid Chromatography/Mass Spectrometry method (Y = 0.998X - 0.2; R2 = 0.807). The FOLA method was subsequently used for samples from the LIFE Child cohort in Germany, providing first data of breast milk folate in this country (range: 6.2-100.7 nM). This technique could indeed prove useful for large cohorts with multiple samplings.

Estradiol and progesterone strongly inhibit the innate immune response of mononuclear cells in newborns.

Newborns are particularly susceptible to bacterial infections due to qualitative and quantitative deficiencies of the neonatal innate immune system. However, the mechanisms underlying these deficiencies are poorly understood. Given that fetuses are exposed to high concentrations of estradiol and progesterone during gestation and at time of delivery, we analyzed the effects of these hormones on the response of neonatal innate immune cells to endotoxin, bacterial lipopeptide, and Escherichia coli and group B Streptococcus, the two most common causes of early-onset neonatal sepsis. Here we show that at concentrations present in umbilical cord blood, estradiol and progesterone are as powerful as hydrocortisone for inhibition of cytokine production by cord blood mononuclear cells (CBMCs) and newborn monocytes. Interestingly, CBMCs and newborn monocytes are more sensitive to the effects of estradiol and progesterone than adult peripheral blood mononuclear cells and monocytes. This increased sensitivity is associated with higher expression levels of estrogen and membrane progesterone receptors but is independent of a downregulation of Toll-like receptor 2 (TLR2), TLR4, and myeloid differentiation primary response gene 88 in newborn cells. Estradiol and progesterone mediate their anti-inflammatory activity through inhibition of the NF-κB pathway but not the mitogen-activated protein kinase pathway in CBMCs. Altogether, these results suggest that elevated umbilical cord blood concentrations of estradiol and progesterone acting on mononuclear cells expressing high levels of steroid receptors contribute to impair innate immune responses in newborns. Therefore, intrauterine exposure to estradiol and progesterone may participate in increasing susceptibility to infection during the neonatal period.

Strong EBV-specific CD8+ T-cell response in patients with early multiple sclerosis.

Epstein-Barr virus (EBV) has been associated with multiple sclerosis (MS), however, most studies examining the relationship between the virus and the disease have been based on serologies, and if EBV is linked to MS, CD8+ T cells are likely to be involved as they are important both in MS pathogenesis and in controlling viruses. We hypothesized that valuable information on the link between MS and EBV would be ascertained from the study of frequency and activation levels of EBV-specific CD8+ T cells in different categories of MS patients and control subjects. We investigated EBV-specific cellular immune responses using proliferation and enzyme linked immunospot assays, and humoral immune responses by analysis of anti-EBV antibodies, in a cohort of 164 subjects, including 108 patients with different stages of MS, 35 with other neurological diseases and 21 healthy control subjects. Additionally, the cohort were all tested against cytomegalovirus (CMV), another neurotropic herpes virus not convincingly associated with MS, nor thought to be deleterious to the disease. We corrected all data for age using linear regression analysis over the total cohorts of EBV- and CMV-infected subjects. In the whole cohort, the rate of EBV and CMV infections were 99% and 51%, respectively. The frequency of IFN-gamma secreting EBV-specific CD8+ T cells in patients with clinically isolated syndrome (CIS) was significantly higher than that found in patients with relapsing-remitting MS (RR-MS), secondary-progressive MS, primary-progressive MS, patients with other neurological diseases and healthy controls. The shorter the interval between MS onset and our assays, the more intense was the EBV-specific CD8+ T-cell response. Confirming the above results, we found that EBV-specific CD8+ T-cell responses decreased in 12/13 patients with CIS followed prospectively for 1.0 +/- 0.2 years. In contrast, there was no difference between categories for EBV-specific CD4+ T cell, or for CMV-specific CD4+ and CD8+ T-cell responses. Anti-EBV-encoded nuclear antigen-1 (EBNA-1)-specific antibodies correlated with EBV-specific CD8+ T cells in patients with CIS and RR-MS. However, whereas EBV-specific CD8+ T cells were increased the most in early MS, EBNA-1-specific antibodies were increased in early as well as in progressive forms of MS. Our data show high levels of CD8+ T-cell activation against EBV--but not CMV--early in the course of MS, which support the hypothesis that EBV might be associated with the onset of this disease.

Effects of protein quantity and type on diet induced thermogenesis in overweight adults: A randomized controlled trial.

BACKGROUND & AIMS: Protein content of a meal is hypothesized to drive DIT dose-dependently. However, no single meal study exists comparing two different doses of protein on DIT. In addition, the source of protein, particularly whey protein, was shown to have a higher DIT than casein and soy in the acute setting, however the mechanism behind this difference is not yet clear. The aim of the present work is therefore to evaluate the efficacy of two different doses and types of protein (whey protein and casein) on DIT in overweight adults. METHODS: Randomized, double blind crossover including seventeen overweight men and women assigned to four isocaloric study treatments where protein and carbohydrate were exchanged: control, 30 g of whey protein microgels (WPM30), 50 g WPM (WPM50) or 50 g micellar casein (MC50). Energy expenditure was measured by indirect calorimetry. Blood, breath and urine samples were collected in order to measure substrate oxidation, amino acid profile, glucose and insulin, protein turnover and other metabolic parameters. RESULTS: DIT was 6.7 ± 3.7%, 13.0 ± 5.0%, 18.0 ± 5.0% and 16.0 ± 5.0% for control, WPM30, WPM50 and MC50, respectively. There was a significant difference between WPM50 and WPM30 (p < 0.005) and a trend was observed between WPM50 and MC50 (p = 0.06). WPM50 resulted in the highest total, essential, and branched-chain plasma amino acid concentrations when compared with the other study treatments (p < 0.005) and a higher insulin concentration than MC50 (p < 0.005). Protein oxidation was higher for WPM50 than MC50. Protein turnover was significantly correlated with DIT through total leucine oxidation (r = 0.52, p = 0.005). CONCLUSIONS: Our findings show that DIT does increase at a dose beyond 30 g of WPM and that the type of dairy protein may have an effect on DIT with WPM tending towards a higher DIT than casein. Although further research is required to understand the mechanism behind the effect of different protein sources on thermogenesis, we suggest that amongst the components of protein turnover, protein oxidation may be an important driver of thermogenesis at doses higher than 30 g. These results have concrete implications when choosing a dose of protein to optimize its thermogenic effect. CLINICAL TRIAL REGISTRY NUMBER: NCT02303080 www.clinicaltrials.gov.

A 48-Hour Vegan Diet Challenge in Healthy Women and Men Induces a BRANCH-Chain Amino Acid Related, Health Associated, Metabolic Signature.

SCOPE: Research is limited on diet challenges to improve health. A short-term, vegan protein diet regimen nutritionally balanced in macronutrient composition compared to an omnivorous diet is hypothesized to improve metabolic measurements of blood sugar regulation, blood lipids, and amino acid metabolism. METHODS AND RESULTS: This randomized, cross-over, controlled vegan versus animal diet challenge is conducted on 21 (11 female,10 male) healthy participants. Fasting plasma is measured during a 3 d diet intervention for clinical biochemistry and metabonomics. Intervention diet plans meet individual caloric needs. Meals are provided and supervised. Diet compliance is monitored. CONCLUSIONS: The vegan diet lowers triglycerides, insulin and homeostatic model assessment (HOMA-IR), bile acids, elevated magnesium levels, and changed branched-chain amino acids (BCAAs) metabolism (p < 0.05), potentiating insulin and blood sugar control after 48 h. Cholesterol control improves significantly in the vegan versus omnivorous diets. Plasma amino acid and magnesium concentrations positively correlate with dietary amino acids. Polyunsaturated fatty acids and dietary fiber inversely correlate with insulin, HOMA-IR, and triglycerides. Nutritional biochemistries, BCAAs, insulin, and HOMA-IR are impacted by sexual dimorphism. A health-promoting, BCAA-associated metabolic signature is produced from a short-term, healthy, controlled, vegan diet challenge when compared with a healthy, controlled, omnivorous diet.

Immune responses to JC virus in patients with multiple sclerosis treated with natalizumab: a cross-sectional and longitudinal study.

BACKGROUND: Natalizumab is used to prevent relapses and progression of disability in patients with multiple sclerosis but has been associated with progressive multifocal leukoencephalopathy (PML). We aimed to better understand the associations between JC virus, which causes PML, and natalizumab treatment. METHODS: We prospectively assessed patients with multiple sclerosis who started treatment with natalizumab. Blood and urine samples were tested for the presence of JC virus DNA with quantitative real-time PCR before treatment and at regular intervals after treatment onset for up to 18 months. At the same timepoints, by use of proliferation and enzyme-linked immunospot assays, the cellular immune responses against JC virus, Epstein-Barr virus, cytomegalovirus, myelin oligodendrocyte glycoprotein, and myelin oligodendrocyte basic protein (MOBP) were assessed. Humoral immune response specific to JC virus was assessed with an enzyme immunoassay. The same experiments were done on blood samples from patients with multiple sclerosis before and 10 months after the start of interferon beta treatment. FINDINGS: We assessed 24 patients with multiple sclerosis who received natalizumab and 16 who received interferon beta. In patients treated with natalizumab, JC virus DNA was not detected in the blood at any timepoint. However, JC virus DNA was present in the urine of six patients and in most of these patients the concentrations of JC virus DNA were stable over time. Compared with pretreatment values, the cellular immune response was increased to cytomegalovirus at 6 months, to JC virus at 1, 9, and 12 months, and to Epstein-Barr virus and MOBP at 12 months. Humoral responses remained stable. There were no increases in cellular immune responses specific to the viruses or myelin proteins in the 16 patients treated with interferon beta. INTERPRETATION: Natalizumab increases cellular immune responses specific to viruses and myelin proteins in the peripheral blood after 1 year, without evidence of viral reactivation. FUNDING: Swiss National Foundation, Swiss Society for Multiple Sclerosis, and Biogen Dompé.

CSF enrichment of highly differentiated CD8+ T cells in early multiple sclerosis.

CD8+ T cells may play an important role in multiple sclerosis (MS). Whether these cells would be involved in early stages of MS is unclear. We enrolled 52 patients with suspected MS, determined the recruitment of their highly differentiated (CCR7-/CD45RA+ or -) T cells (T(HD)) in the CSF as compared to peripheral blood and followed them for 12+/-7.3 months. A ROC curve showed that a CD8+/CD4+ T(HD) cells ratio of 0.94 helped to distinguish relapsing-remitting (RR-MS) and possible MS (Po-MS) from primary-progressive MS (PP-MS) and other neurological diseases patients (OND) patients (p=0.039), risk ratio of 2.29 (95% CI: 1.13-4.66; p=0.006). The CSF enrichment in CD8+ T(HD) cells was greater than in CD4+ T(HD) cells in RR/Po-MS patients (p=0.024) and than in CD8+ T(HD) cells in PP-MS/OND patients (p=0.006). These data suggest that CD8+ T(HD) cells play a role in the early stages of RR-MS.