Metabolomics based markers predict type 2 diabetes in a 14-year follow-up study

Chemical probes are key components of the bioimaging toolbox, as they label biomolecules in cells and tissues. The new challenge in bioimaging is to design chemical probes for three-dimensional (3D) tissue imaging. In this work, we discovered that light scattering of metal nanoparticles can provide 3D imaging contrast in intact and transparent tissues. The nanoparticles can act as a template for the chemical growth of a metal layer to further enhance the scattering signal. The use of chemically grown nanoparticles in whole tissues can amplify the scattering to produce a 1.4 million-fold greater photon yield than obtained using common fluorophores. These probes are non-photobleaching and can be used alongside fluorophores without interference. We demonstrated three distinct biomedical applications: (a) molecular imaging of blood vessels, (b) tracking of nanodrug carriers in tumors, and (c) mapping of lesions and immune cells in a multiple sclerosis mouse model. Our strategy establishes a distinct yet complementary set of imaging probes for understanding disease mechanisms in three dimensions

Summary of EAE experiments.

<p>Summary of EAE experiments.</p

Secondary B Cell Receptor Diversification Is Necessary for T Cell Mediated Neuro-Inflammation during Experimental Autoimmune Encephalomyelitis

<div><p>Background</p><p>Clinical studies of B cell depletion in Multiple Sclerosis (MS) have revealed that B Lymphocytes are involved in the neuro-inflammatory process, yet it remains unclear how B cells can exert pro- and anti-inflammatory functions during MS. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of MS whereby myelin-specific T cells become activated and subsequently migrate to the Central Nervous System (CNS) where they perform pro-inflammatory functions such as cytokine secretion. Typically EAE is induced by immunization of mice of a susceptible genetic background with peptide antigen emulsified in Complete Freund's Adjuvant. However, novel roles for B-lymphocytes in EAE may also be explored by immunization with full-length myelin oligodendrocyte glycoprotein (MOG) that contains the B cell conformational epitope. Here we show that full length MOG immunization promotes a chronic disease in mice that depends on antigen-driven secondary diversification of the B cell receptor.</p><p>Methods</p><p>Activation-Induced Deaminase (AID) is an enzyme that is essential for antigen-driven secondary diversification of the B cell receptor. We immunized AID^−/− mice with the extracellular domain (amino acids 1–120) of recombinant human MOG protein (rhMOG) and examined the incidence and severity of disease in AID^−/− versus wild type mice. Corresponding with these clinical measurements, we also evaluated parameters of T cell activation in the periphery and the CNS as well as the generation of anti-MOG antibodies (Ab).</p><p>Conclusions</p><p>AID^−/− mice exhibit reduced severity and incidence of EAE. This suggests that the secondary diversification of the B cell receptor is required for B cells to exert their full encephalogenic potential during rhMOG-induced EAE, and possibly also during MS.</p></div

Lymph node cells from WT and AID^−/− mice produce equivalent levels of cytokines in response to immunization with rhMOG.

<p>WT and AID^−/− mice were immunized with recombinant human MOG (rhMOG) and draining axillary and brachial lymph nodes were harvested after 7 days post-immunization. Four million lymph node cells were plated along with 20 µg of rhMOG. Cultures were kept for 48 hours after which the supernatant was harvested and evaluated for cytokine production by ELISA. The limit of detection of the assay is 125 pg/ml for the IFNγ ELISA and 63 pg/ml for the IL-17 ELISA. Five mice per group were assessed.</p

Appearance of class switched B cells in the CNS during EAE.

<p>WT and AID^−/− mice were immunized with recombinant human MOG (rhMOG) and spinal cords were extracted and processed at day 15 (peak of disease). B cells were identified by the expression of CD19 and B220. Class switched B cells were further identified by the absence of IgM/IgD and reported here as a percentage of the total number of B220⁺CD19⁺ B cells. Representative FACS analysis can be seen in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061478#pone.0061478.s002" target="_blank">Figure S2B</a>.</p

Anti-MOG antibodies generated in WT and AID^−/− mice.

<p>WT and AID^−/− mice were immunized with recombinant human MOG (rhMOG) and titres of anti-MOG Ab were evaluated at day 15 (peak of disease). As expected, AID^−/− mice only made anti-MOG Ab of the IgM isotype, whereas WT mice produced anti-MOG IgG1 and IgG2c Ab (and very little IgM at this time point). The average OD for each mouse at a set concentration of serum (half-way point of the curve) is depicted, and raw OD values can be seen in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061478#pone.0061478.s003" target="_blank">Figure S3</a>. In this experiment, 5–6 mice per group were tested and an additional experiment tested 8–12 mice.</p

Impaired accumulation of cytokine producing CD4⁺ T cells in the CNS of AID^−/− mice.

<p>WT and AID^−/− mice were immunized with recombinant human MOG (rhMOG) and spinal cords (<b>A</b>) and brains (<b>B</b>) were extracted and processed at day 15: the peak of disease. Leukocytes were stimulated <i>ex vivo</i> with PMA/Ionomycin, and brefeldin A was added in the last 4 hours. Cells were then subjected to surface and intracellular cytokine staining. This experiment was performed twice with similar results and 6 mice per group were tested in this experiment. Representative FACS analysis can be seen in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061478#pone.0061478.s002" target="_blank">Figure S2A</a>.</p

AID^−/− mice exhibit attenuated EAE in response to rhMOG but not MOG_35-55.

<p>(<b>A</b>) WT and AID^−/− mice were immunized with MOG_35-55 in CFA and examined for clinical symptoms. A representative experiment is shown, and 2 experiments were performed with similar results (shown here are n = 6 WT and n = 5 AID^−/− mice per group). (<b>B</b>) WT and AID^−/− mice were immunized with rhMOG in CFA and examined for clinical symptoms. A representative experiment is shown (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061478#pone-0061478-t001" target="_blank">Table 1</a> for all 4 experiments) with n = 11 WT and n = 7 AID^−/− mice per group. At the termination of the experiment, spinal cords were dissected and stained with H&E and counterstained with Luxol fast blue. A representative example of 6 separate mice is shown (original magnification ×200). See arrows for areas of cellular infiltrates (<b>C</b>). Cell infiltration in the spinal cord tissue was assessed as follows: meningeal infiltrate (score 1), perivascular infiltrate (score 2), parenchymal infiltrate (score 3) (<b>D</b>).</p

HIPK1 is expressed in hematopoietic cells.

<p>A, Analysis of the tissue-specific gene expression profiles of <i>hipk1</i> and <i>hipk2</i> using the BioGPS-Gene Portal Hub web application (Genomics Institute of the Novartis Research Foundation (GNF). For this study, B and T lymphocytes were obtained from spleen. B, Primary B and T lymphocytes were isolated from the spleens of <i>HIPK1^+/+</i> (WT) and <i>HIPK1^−/−</i> (KO) mice. mRNA was isolated and reverse transcribed to cDNA. PCR amplification was used to determine if <i>hipk1</i> was expressed. <i>gapdh</i> was used as a control.</p

Basal serum Ig levels.

<p>A, Basal serum Ig levels from 12 week-old <i>HIPK1^+/+</i> and <i>HIPK1^−/−</i> mice were measured by ELISA. n = 4, except for IgG2c, where n = 8. *p≤0.05, **p≤0.01.</p