Mast cells (MCs) are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Recent work provided evidence that MCs are able to activate B and T lymphocytes through the release of vesicles called exosomes. Here we demonstrate that exosomes, which are located in the endocytic pathway, harbor exogenous Ags that associate with other molecules endowed with immunomodulatory functions, including 60- and 70-kDa heat shock proteins. Administration to naive mice of Ag-containing exosomes in the absence of conventional adjuvants elicits specific Ab responses across the MHC II haplotype barrier. We demonstrate that MC-exosomes induce immature dendritic cells (DCs) to up-regulate MHC class II, CD80, CD86, and CD40 molecules and to acquire potent Ag-presenting capacity to T cells. Uptake and processing of Ag-associated exosomes by endogenous DCs were also demonstrated. Finally, exosome-associated heat shock proteins are critical for the acquisition by DCs of the Ag-presenting function. This work demonstrates a heretofore unrecognized collaborative interaction between MCs and DCs leading to the elicitation of specific immune responses.

BackgroundGlyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor.Methodology/Principal FindingsCultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki = 5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-α, IL-6, IL-8, IL-1β) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1.Conclusions/SignificanceThe results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.

Apolipoprotein E (apoE) plays a role in the pathogenesis of Alzheimer disease (AD). It is involved in the receptor-mediated cellular clearance of the amyloid beta-protein (Abeta) and in the perivascular drainage of the extracellular fluid. Microvascular changes are also associated with AD and have been discussed as a possible reason for altered perivascular drainage. To further clarify the role of apoE in the perivascular and vascular pathology in AD patients, we studied its occurrence and distribution in the perivascular space, the perivascular neuropil, and in the vessel wall of AD and control cases with and without small vessel disease (SVD). Apolipoprotein E was found in the perivascular space and in the neuropil around arteries of the basal ganglia from control and AD cases disclosing no major differences. Western blot analysis of basal ganglia tissue also revealed no significant differences pertaining to the amount of full-length and C-terminal truncated apoE in AD cases compared with controls. In contrast, Abeta occurred in apoE-positive perivascular astrocytes in AD cases but not in controls. In blood vessels, apoE and immunoglobulin G were detected within the SVD-altered vessel wall. The severity of SVD was associated with the occurrence of apoE in the vessel wall and with that of Abeta in perivascular astrocytes. These results point to an important role of apoE in the perivascular clearance of Abeta in the human brain. The occurrence of apoE and immunoglobulin G in SVD lesions and in the perivascular space suggests that the presence of SVD results in plasma-protein leakage into the brain. It is therefore tempting to speculate that apoE represents a pathogenetic link between SVD and AD.

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One unexpected feature of the human genome is the high structural variability across individuals. Frequently, large regions of the genome show structural polymorphisms and many vary in their abundance. However, accurate methods for the characterization and typing of such copy number variations (CNV) are needed. The defensin cluster at the human region 8p23.1 is one of the best studied CNV regions due to its potential clinical relevance for innate immunity, inflammation, and cancer. The region can be divided into two subclusters, which harbor predominantly either alpha- or beta-defensin genes. Previous studies assessing individual copy numbers gave different results regarding whether the complete beta-defensin cluster varies or only particular genes therein. We applied multiplex ligation-dependent probe amplification (MLPA) to measure defensin locus copy numbers in 42 samples. The data show strict copy number concordance of all 10 loci typed within the beta-defensin cluster in each individual, while seven loci within the alpha-defensin cluster are consistently found as single copies per chromosome. The exception is DEFA3, which is located within the alpha-defensin cluster and was found to also differ in copy number interindividually. Absolute copy numbers ranged from two to nine for the beta-defensin cluster and zero to four for DEFA3. The CNV-typed individuals, including HapMap samples, are publicly available and may serve as a universal reference for absolute copy number determination. On this basis, MLPA represents a reliable technique for medium- to high-throughput typing of 8p23.1 defensin CNV in association studies for diverse clinical phenotypes.