Generation and Characterization of a Novel Recombinant Antibody Against 15-Ketocholestane Isolated by Phage-Display

The employment of monoclonal antibodies (Mabs) to identify disease-associated biomarkers in clinical samples represents the underlying principle for many diagnostic tests. To date, these have been principally developed for protein targets with few reported applications for lipids due to their hydrophobicity and poor immunogenicity. Oxysterols represent a family of lipids implicated in diverse human diseases where Mab-based detection assays could have a profound effect on their utility as clinical biomarkers. These are usually identified in patients’ samples by mass- spectrometry based approaches. Here, we describe an antibody phage-library based screening methodology for generating a recombinant monoclonal antibody (RAb) targeting the oxysterol-15-ketocholestane (15-KA), a lipid implicated in multiple sclerosis and Autoimmune Encephalomyelitis (EAE). The antibody is highly specific for 15-KA and shows little or no binding activity for other closely related oxysterols. We employ RAb2E9 to address the controversy over whether 15-KA is a true biomarker for MS/EAE and show that 15-KA is undetectable in serum taken from mice with EAE using antibody based detection methodologies; a finding confirmed by mass-spectrometry analysis. This study demonstrates the technical feasibility of using phage display to isolate highly specific antibodies against poorly immunogenic, small molecule lipids

Placental inositol reduced in gestational diabetes as glucose alters inositol transporters and IMPA1 enzyme expression

ContextPerturbed inositol physiology in insulin-resistant conditions has led to proposals of inositol supplementation for gestational diabetes (GDM) prevention, but placental inositol biology is poorly understood.ObjectiveInvestigate associations of maternal glycemia with placental inositol content, determine glucose effects on placental expression of inositol enzymes and transporters, and examine relations with birthweight.Design and ParticipantsCase-control study of placentae from term singleton pregnancies (GDM n = 24, non-GDM n = 26), and culture of another 9 placentae in different concentrations of glucose and myo-inositol for 48 hours.Main Outcome MeasuresPlacental inositol was quantified by the Megazyme assay. Relative expression of enzymes involved in myo-inositol metabolism and plasma membrane inositol transport was determined by quantitative RT-PCR and immunoblotting. Linear regression analyses were adjusted for maternal age, body mass index, ethnicity, gestational age, and sex.ResultsPlacental inositol content was 17% lower in GDM compared with non-GDM. Higher maternal mid-gestation glycemia were associated with lower placental inositol. Increasing fasting glycemia was associated with lower protein levels of the myo-inositol synthesis enzyme, IMPA1, and the inositol transporters, SLC5A11 and SLC2A13, the expression of which also correlated with placental inositol content. In vitro, higher glucose concentrations reduced IMPA1 and SLC5A11 mRNA expression. Increasing fasting glycemia positively associated with customized birthweight percentile as expected in cases with low placental inositol, but this association was attenuated with high placental inositol.ConclusionGlycemia-induced dysregulation of placental inositol synthesis and transport may be implicated in reduced placental inositol content in GDM, and this may in turn be permissive to accelerated fetal growth

Metabolism of C-13-Labeled Fatty Acids in Term Human Placental Explants by Liquid Chromatography-Mass Spectrometry

10.1210/en.2018-01020ENDOCRINOLOGY16061394-140

Myo-inositol alters C-labelled fatty acid metabolism in human placental explants

We postulate that myo-inositol, a proposed intervention for gestational-diabetes, affects transplacental lipid supply to the fetus. We investigated the effect of myo-inositol on fatty-acid processing in human placental-explants from uncomplicated pregnancies. Explants were incubated with 13C-labeled palmitic-acid, 13C-oleic-acid and 13C-docosahexaenoic-acid across a range of myo-inositol concentrations for 24 h and 48 h. The incorporation of labeled-fatty-acids into individual lipids was quantified by liquid-chromatography-mass-spectrometry. At 24 h, myo-inositol increased the amount of 13C-palmitic-acid and 13C-oleic-acid labeled lipids (median fold-change relative to control=1). Significant effects were seen with 30 µM myo-inositol (physiological) for 13C-palmitic-acid-lysophosphatidylcholines (1.26) and 13C-palmitic-acid-phosphatidylethanolamines (1.17). At 48 h, myo-inositol addition increased 13C-oleic-acid-lipids but decreased 13C-palmitic-acid and 13C-docosahexaenoic-acid lipids. Significant effects were seen with 30 µM myo-inositol for 13C-oleic-acid-phosphatidylcholines (1.25), 13C-oleic-acid-phosphatidylethanolamines (1.37) and 13C-oleic-acid-triacylglycerols (1.32) and with 100 µM myo-inositol for 13C-docosahexaenoic-acid-triacylglycerols (0.78). Lipids labeled with the same 13C-fatty-acid showed similar responses when tested at the same time-point, suggesting myo-inositol alters upstream processes such as fatty-acid uptake or activation. Myo-inositol supplementation may alter placental lipid physiology with unknown clinical consequences

Metabolism of 13C-labeled fatty acids in term human placental explants by liquid chromatography mass spectrometry

Placental lipid transport and metabolism is poorly understood despite its importance for fetal development and life-long health. We aimed to explore fatty acid (FA) processing in human villous placental explants from seven uncomplicated term singleton pregnancies delivered by elective cesarean section. Explants were treated with stable-isotope labeled palmitic acid (13C-PA), oleic acid (13C-OA) or docosahexaenoic acid (13C-DHA) for 3, 24 or 48 hours. Stable isotope labeled lipids synthesised by placental explants from labelled FA were quantified, alongside endogenous unlabeled placental lipids, by liquid-chromatography mass-spectrometry. Labeled phosphatidylcholines (PC), triacylglycerols (TAG), and phosphatidylethanolamines were detected in explants, whilst labeled lysophosphatidylcholines were found in both explants and conditioned-media. 13C-PA was primarily directed into PC synthesis (74% of 13C-PA-labeled lipids), while 13C-OA was directed almost equally into PC and TAG synthesis (45% and 53%, respectively, of 13C-OA-labeled lipids). 13C-DHA was only detectable in TAGs. TAGs demonstrated the highest isotopic enrichment for all 13C-FAs with 13C-OA-TAGs comprising &gt;50% of total OA-TAGs (unlabelled and labeled); consistent with TAGs being a labile and accessible reservoir for FA storage. Variations in lipid incorporation were correlated to maternal glycemia and BMI suggesting that this experimental model could be used to investigate the effect of maternal factors on placental lipid metabolism. We conclude that lipid metabolic partitioning of freshly-imported FAs into labile and less labile lipid reservoirs in placenta is FA-dependent. This process may partly mediate the physiological preferential transplacental transfer of particular FAs to the fetus, but may also be implicated in the fetoplacental pathophysiology of maternal metabolic dysfunction.</p

Interleukin-6 and Neural Stem Cells: More Than Gliogenesis

Besides its wide range of action as a proinflammatory cytokine in the immune system, interleukin-6 (IL-6) has also attracted much attention due to its influence on the nervous system. In the present study we show that the designer fusion protein H-IL-6, consisting of IL-6 and its specific receptor IL-6R-α, but not IL-6 alone, mediates both neuro- as well as gliogenesis. Using immunocytochemistry, Western blot, and patch-clamp recording, we demonstrate that H-IL-6 induces the differentiation of neural stem cells (NSCs) specifically into glutamate-responsive neurons and two morphological distinctive astroglia cell types. H-IL-6–activated neurogenesis seems to be induced by the MAPK/CREB (mitogen-activated protein kinase/cAMP response element-binding protein) cascade, whereas gliogenesis is mediated via the STAT-3 (signal transducers and activators of transcription protein-3) signaling pathway. Our finding that IL-6 mediates both processes depending on its specific soluble receptor sIL-6R-α has implications for the potential treatment of neurodegenerative diseases

ABC transporters, neural stem cells and neurogenesis : a different perspective

Stem cells intrigue. They have the ability to divide exponentially, recreate the stem cell compartment, as well as create differentiated cells to generate tissues. Therefore, they should be natural candidates to provide a renewable source of cells for transplantation applied in regenerative medicine. Stem cells have the capacity to generate specific tissues or even whole organs like the blood, heart, or bones. A subgroup of stem cells, the neural stem cells (NSCs), is characterized as a self-renewing population that generates neurons and glia of the developing brain. They can be isolated, genetically manipulated and differentiated in vitro and reintroduced into a developing, adult or a pathologically altered central nervous system. NSCs have been considered for use in cell replacement therapies in various neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Characterization of genes with tightly controlled expression patterns during differentiation represents an approach to understanding the regulation of stem cell commitment. The regulation of stem cell biology by the ATP-binding cassette (ABC) transporters has emerged as an important new field of investigation. As a major focus of stem cell research is in the manipulation of cells to enable differentiation into a targeted cell population; in this review, we discuss recent literatures on ABC transporters and stem cells, and propose an integrated view on the role of the ABC transporters, especially ABCA2, ABCA3, ABCB1 and ABCG2, in NSCs' proliferation, differentiation and regulation, along with comparisons to that in hematopoietic and other stem cells.Accepted versio