RAGE Expression in Human T Cells: A Link between Environmental Factors and Adaptive Immune Responses

The Receptor for Advanced Glycation Endproducts (RAGE) is a scavenger ligand that binds glycated endproducts as well as molecules released during cell death such as S100b and HMGB1. RAGE is expressed on antigen presenting cells where it may participate in activation of innate immune responses but its role in adaptive human immune responses has not been described. We have found that RAGE is expressed intracellularly in human T cells following TCR activation but constitutively on T cells from patients with diabetes. The levels of RAGE on T cells from patients with diabetes are not related to the level of glucose control. It co-localizes to the endosomes. Its expression increases in activated T cells from healthy control subjects but bystander cells also express RAGE after stimulation of the antigen specific T cells. RAGE ligands enhance RAGE expression. In patients with T1D, the level of RAGE expression decreases with T cell activation. RAGE+ T cells express higher levels of IL-17A, CD107a, and IL-5 than RAGE− cells from the same individual with T1D. Our studies have identified the expression of RAGE on adaptive immune cells and a role for this receptor and its ligands in modulating human immune responses

MS1 induces a spontaneous formation of free-floating insulin-positive PIs.

<p><b><i>A.</i></b> Light and phase contrast microscopy showing the formation of βTC3 PIs over 8 d. <b><i>B.</i></b> IF staining of βTC3 monolayers. Blue- DAPI, Red- Insulin and merge. <b><i>C.</i></b> 3-D reconstruction of z-stack confocal images of a representative PI. Blue- DAPI, Red- Insulin and merge.</p

A Minimally-invasive Blood-derived Biomarker of Oligodendrocyte Cell-loss in Multiple Sclerosis

Multiple sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS). Minimally invasive biomarkers of MS are required for disease diagnosis and treatment. Differentially methylated circulating-free DNA (cfDNA) is a useful biomarker for disease diagnosis and prognosis, and may offer to be a viable approach for understanding MS. Here, methylation-specific primers and quantitative real-time PCR were used to study methylation patterns of the myelin oligodendrocyte glycoprotein (MOG) gene, which is expressed primarily in myelin-producing oligodendrocytes (ODCs). MOG-DNA was demethylated in O4+ ODCs in mice and in DNA from human oligodendrocyte precursor cells (OPCs) when compared with other cell types. In the cuprizone-fed mouse model of demyelination, ODC derived demethylated MOG cfDNA was increased in serum and was associated with tissue-wide demyelination, demonstrating the utility of demethylated MOG cfDNA as a biomarker of ODC death. Collected sera from patients with active (symptomatic) relapsing-remitting MS (RRMS) demonstrated a higher signature of demethylated MOG cfDNA when compared with patients with inactive disease and healthy controls. Taken together, these results offer a minimally invasive approach to measuring ODC death in the blood of MS patients that may be used to monitor disease progression

Col-IV and laminin are detected in and around the PI.

<p><b><i>A.</i></b> IF staining of MS1 cells. Blue-DAPI, Green-CD31, Red-BS1 and merge. White/Yellow represents double positive cells. <b><i>B.</i></b> RT-PCR for laminin β1 and col-IV in MS1, whole murine islet preps, and βTC3 cells. Laminin α1 and α2 were not detected (data not shown). <b><i>C.</i></b> IF staining of MS1 cells. Blue- DAPI, Green- col-IV, White-Laminin, and merge. <b><i>D.</i></b> 3-D reconstruction of z-stack imaging of an 8 d old PI. <b><i>E.</i></b> Non-consecutive z-stack confocal images of a PI. Blue- DAPI, Red- Insulin, Green- col-IV, White- laminin.</p

Baseline and glucose stimulated insulin expression and secretion are enhanced in MS1-induced PIs.

<p><b><i>A.</i></b> FACS staining for proinsulin expression in monolayers (dotted line) and PIs (solid line). Grey histogram- isotype control. MFI: mono = 6579, PIs = 7486 <b><i>B.</i></b> Quantitative RT-PCR analysis of insulin 1 expression in βTC3 monolayers (closed bars) or PIs (opened bars). Data represents an average of 3 independent experiments. *<i>p<0.025</i>. Monolayers and PIs were cultured for 8 d, washed with media lacking glucose, and incubated with escalating glucose levels for 6 h. <b><i>C.</i></b> Insulin ELISA analysis of supernatant from βTC3 monolayers and PIs. Experiment represents three independent repeats. N = 3 per group. Two-way ANOVA analysis<b>.</b> <i>^†p<0.001, *p<0.0001.</i></p

Evaluation of β cell loss in MS1-induced PIs.

<p><b><i>A.</i></b> FACS staining for 7-AAD viability dye of monolayers (dotted line) and PIs (solid line). Grey histogram unstained control. Black arrow show 7-AAD positive cells. <b><i>B.</i></b> Non-consecutive z-stack confocal images of a 14 d old PI. Blue- DAPI, Red- Insulin, Green- cleaved caspase-3. White arrows point to caspase-3 positive cells.</p